WO2022223544A1 - COMBINATION THERAPY OF NUCANT AND mTOR INHIBITORS FOR TREATING CANCER - Google Patents

COMBINATION THERAPY OF NUCANT AND mTOR INHIBITORS FOR TREATING CANCER Download PDF

Info

Publication number
WO2022223544A1
WO2022223544A1 PCT/EP2022/060288 EP2022060288W WO2022223544A1 WO 2022223544 A1 WO2022223544 A1 WO 2022223544A1 EP 2022060288 W EP2022060288 W EP 2022060288W WO 2022223544 A1 WO2022223544 A1 WO 2022223544A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
cell
mtor
cells
bond
Prior art date
Application number
PCT/EP2022/060288
Other languages
French (fr)
Inventor
Mounira CHALABI-DCHAR
Ilaria CASCONE
José COURTY
Philippe Bouvet
Original Assignee
Centre National De La Recherche Scientifique (Cnrs)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Centre National De La Recherche Scientifique (Cnrs) filed Critical Centre National De La Recherche Scientifique (Cnrs)
Publication of WO2022223544A1 publication Critical patent/WO2022223544A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • the description relates to a method or treating cancer, such as pancreatic cancer, with a combination treatment with at least one mechanistic target of rapamycin (mTOR) inhibitor and at least one polyvalent synthetic compound.
  • cancer such as pancreatic cancer
  • mTOR mechanistic target of rapamycin
  • Pancreatic ductal adenocarcinoma is a highly aggressive disease, for which mortality closely parallels incidence with 5-year survival rate less than 6% (Kamisawa et al. 2016). It is predicted to be the second leading cause of cancer-related death by 2030 (Soreide et al. 2019).
  • the biology of PDAC contributes to early recurrence and metastasis, resistance to chemotherapy and radiotherapy, because of its complexity at the genomic, epigenetic and metabolic levels, with multiple activated biological pathways and crosstalk.
  • KRAS oncogene
  • CDKN2A encoding pi 6
  • TP 53 and SMAD4 tumor suppressor genes
  • Mechanistic target of rapamycin (mTOR), or FK506-binding protein 12- rapamycin-associated protein (FRAP) is a kinase that is a member of the phosphatidylinositol 3 -kinase-related kinase family and is encoded by the MTOR gene in humans.
  • mTOR interacts with other proteins to form the mTOR complex 1 (mTORCl) and mTOR complex 2 (mTORC2), which regulate different cellular processes, including cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy, and transcription.
  • mTOR inhibitors inhibit the mTOR serine/threonine protein kinase, and is comprised of two families of inhibitors.
  • the first generation of mTOR inhibitors are allosteric mTORCl inhibitors and include inhibitors, such as, rapamycin (SIROLIMUS), CCI-779 (TEMSIROLIMUS), RAD001 (EVEROLIMUS), AP-23573 (RID AF OROLIMU S), UMIROLIMUS, and ZOTAROLIMUS.
  • the second generation of MTOR inhibitors are ATP- competitive mTOR kinase inhibitors that inhibit both mTORCl and mTORC2, and includes inhibitors, such as, AZD2014 (VISTUSERTIB), INK 128 (SAPANISERTIB), and AZD8055.
  • Nucleolin is an RNA- and protein-binding multifunctional protein that has become the focus of interest in the cancer biology field in recent years for several reasons.
  • NCL expression seems to correlate with the proliferation rate of the cells with an overexpression in several cancer types, including hepatocellular carcinoma (Guo et al. 2014), acute myeloid leukemia (Marcel et al. 2017), non-small-cell lung cancer (Zhao et al. 2013), gastric cancer (Qiu et al. 2013), and PDAC (Gilles et al. 2016).
  • NCL is present on the surface of many cell types, and this extracellular form of NCL is a hallmark of proliferative and cancer cells (Ugrinova et al. 2018; Hovanessian et al. 2000; Koutsioumpa et Papadimitriou 2014; Farin et al. 2011).
  • NCL is a key protein for the regulation of several processes required for the proliferation and division of cells. In particular, it plays an important role in the coordination of cell metabolism with cell division and in the biogenesis of ribosomes which are required to sustain the higher level of translation in cells that have a high proliferation rate.
  • the high expression level of NCL in cancer cells is related to high levels of protein synthesis.
  • W02007/125210 discloses polyvalent or multivalent synthetic peptides made of at least three particular pseudopeptide units grafted to a support.
  • Nucant compounds This family of compounds (which have been named Nucant compounds), has been shown to interact with surface nucleolin RGG domain and to have both anti -proliferative and anti-angiogenic properties and to be useful for the treatment of cancer or inflammatory diseases. They have a fairly broad spectrum of activity against angiogenic factors, a good solubility in aqueous media, improved resistance to in vivo breakdown processes (due to the presence of a modified bond in pseudopeptide units), showed very few side effects, and have a synthesis process that is easily adaptable to an industrial scale. Specific exemplified compounds include compound HB 19 and compounds Nucant 1, 2, 3, 6 and 7. This document is incorporated herein by reference in its entirety.
  • WO2009/141687 discloses improved Nucant compounds, in which lysine residues in the pseudopeptide units are all in the same L or D configuration.
  • This document notably describes compound Nucant 6L (N6L), which corresponds to compound Nucant 6 as disclosed in W02007/125210, in which all lysine residues of the pseudopeptide units are in L configuration.
  • N6L compound Nucant 6L
  • This compound showed improve anti-cancer activity compared to complex compound Nucant 6 in which the lysine residue of each pseudopeptide unit may be in L or D configuration.
  • the compounds were also shown to improve wound healing.
  • WO2009/141687 also discloses compounds Nucant 4, 8 and 9.
  • N6L As discussed above, the pseudo-peptides HB19 and N6L were shown to interact withNCL and to inhibitthe proliferation of cancer cells (Krust et al. 2011; Damien Destouches et al. 2008). In addition, N6L as well as a NCL-blocking antibody impairs both in vitro and in vivo angiogenesis by targeting ECs and tumor vessels (Destouches et al. 2011; Birmpas et al. 2012). Many studies reported the antitumor activities of N6L in several cancer types including, breast cancer (Destouches et al. 2011), glioblastoma (Benedetti et al. 2015)(Dhez et al.
  • NCL targeting by N6L blocks both tumor progression and normalizes tumor vasculature, improving the delivery and efficacy of chemotherapeutic drug (Gilles et al. 2016).
  • WO 2012/045750 discloses compositions comprising a mixture of Nucant multivalent synthetic compound and glycosaminoglycan (GAGs) forming microspheres.
  • GAGs glycosaminoglycan
  • the GAGs mediated and/or enhances the beneficial therapeutic activity of the Nucant compounds within the composition.
  • This document is incorporate herein by reference in its entirety.
  • Pancreatic cancer is an aggressive disease characterized by high invasiveness, rapid progression, and resistance to conventional therapy. There is an urgent need to identify new molecules to improve current cancer therapies, including pancreatic cancer therapies, with better efficacy and less toxicity. Thus, there is an urgent need for improved therapies, with better efficacy and decreased toxicity, for the treatment of cancer, such as pancreatic cancer.
  • the present disclosure describes the surprising and unexpected discovery that the coadministration of a mechanistic target of rapamycin (mTOR) inhibitor and a polyvalent synthetic compound of the present disclosure have a synergistic effect in inhibiting cancer cell growth and cancer cell viability.
  • mTOR mechanistic target of rapamycin
  • an aspect the disclosure provides a method of treating cancer (e.g., pancreatic cancer) in a subject, the method comprising administering or co-administering to a cell or a subject in need thereof an effective amount of at least one (e.g., 1, 2, 3, or 4) mTOR inhibitor and an effective amount of at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound, wherein the mTOR inhibitor and the polyvalent synthetic compound are effective for treating or ameliorating at least one symptom of the cancer (e.g., pancreatic cancer), wherein the polyvalent synthetic compound comprises a support comprising at least 3 pseudopeptides coupled or grafted thereto, wherein the support is a linear peptide support having the formula (la) or (lb):
  • each X is independently any amino acid (e.g. any proteinogenic amino acid);
  • Z is proline or derivative thereof; each Y 1 and Y 2 is independently a basic amino acid (e.g., arginine, lysine, lysine derivative, or lysine analog); n is 0 or 1; m is 0, 1, 2, or 3; and
  • the polyvalent synthetic compound is present/administered in a composition that includes a pharmaceutically acceptable carrier, excipient, or diluent.
  • the support has the formula (la). [0018] In any aspect or embodiment described herein, n and m are each 0.
  • z is an integer from 3 to 10.
  • z is 6.
  • the polyvalent synthetic compound comprises 3 to 15 pseudopeptides.
  • one or more of the pseudopeptides (e.g., each/all of the pseudopeptides) is coupled or grafted directly on the support.
  • the pseudopeptides are grafted or coupled to the support via a lysine of the support.
  • the pseudopeptides are optically pure pseudopeptides.
  • the Y 1 and the Y 2 have an L configuration or D configuration.
  • the Y 1 and the Y 2 have an L configuration.
  • each Y 1 and Y 2 is independently lysine or arginine.
  • each Y 1 and Y 2 is a lysine having an L configuration.
  • each Y 1 and Y 2 is independently selected from ornithine, homolysine, and diaminoheptanoic acid (e.g., 2,7-diaminoheptanoic acid).
  • the Y represents a reduced bond.
  • the m is 0 or 1.
  • one or more X is a proteinogenic amino acid.
  • the polyvalent synthetic compound has the structure:
  • the mTOR inhibitor is an allosteric mTOR inhibitor (such as, one or more (e.g., 1, 2, 3, or 4) of rapamycin (SIROLIMUS), CCI- 779 (TEMSIROLIMUS), RAD001 (EVEROLIMUS), AP-23573 (RID AF OROLIMU S), UMIROLIMUS, and ZOTAROLIMUS).
  • SIROLIMUS rapamycin
  • CCI- 779 CCI- 779
  • RAD001 EVEROLIMUS
  • AP-23573 RID AF OROLIMU S
  • UMIROLIMUS UMIROLIMUS
  • ZOTAROLIMUS ZOTAROLIMUS
  • the mTOR inhibitor is an ATP- competitive mTOR inhibitor (such as, one or more (e.g., 1, 2, 3, or 4) of AZD2014 (VISTUSERTIB), INK128 (SAPANISERTIB), and AZD8055).
  • AZD2014 VISTUSERTIB
  • INK128 SAPANISERTIB
  • AZD8055 AZD8055
  • the mTOR inhibitor is an mTORCl inhibitor, such as an allosteric mTORCl inhibitor or an ATP -competitive mTOR kinase inhibitor.
  • the mTOR inhibitor is an mTORC2 inhibitor, such as an ATP-competitive mTOR kinase inhibitor.
  • the mTOR inhibitor is selected from Rapamycin, AZD2014, and INK 128.
  • the cancer is pancreatic cancer.
  • the cancer or the pancreatic cancer is pancreatic ductile adenocarcinoma.
  • the cancer or the pancreatic cancer is pancreatic ductile adenocarcinoma, pancreatic adenocarcinoma, pancreatic squamous cell carcinoma, pancreatic adenosquamous carcinoma, pancreatic colloid carcinoma, preferably pancreatic ductile adenocarcinoma.
  • the polyvalent synthetic compound and the mTOR inhibitor have a synergistic inhibitory effect on the pancreatic cancer cell growth and/or cell viability.
  • Figure 1 Structure of nucleolin protein. Human nucleolin consists of 707 amino acids. Nucleolin can be broken down into two main parts (Ginisty, H., Sicard, H., Roger, B., and Bouvet, P. Structure and functions of nucleolin. (1999) J. Cell Science 112, 761-772; Srivastava, M., and Pollard, H. B. Molecular dissection of nucleolin's role in growth and cell proliferation: new insights. (1999) FASEB J. 13, 1911-1922): N-terminal (aa 1-308), and C- terminal (aa 309-707).
  • the N-terminal domain consists of 4 long acidic domains, consisting of an uninterrupted repetition of glutamic acid and aspartic acid (Al, A2, A3, A4).
  • the C-terminal domain consists of alternating hydrophobic and hydrophilic regions forming 4 RNA Binding Domains (RBD I, II, III, and IV), and its extremity (aa 644-707 or SEQ ID No. 5) carries the highly basic RGG domain comprised of Arg-Gly-Gly repetitions.
  • RGD I, II, III, and IV RNA Binding Domains
  • a 644-707 or SEQ ID No. 5 carries the highly basic RGG domain comprised of Arg-Gly-Gly repetitions.
  • FIGS 3A, 3B, 3C, and 3D NCL targeting by N6L decreases mPDAC cell proliferation by inducing apoptosis.
  • mPDAC cells were seeded in 96-well plates and allowed to adhere overnight. Cells were treated with increasing concentrations of N6L.
  • A mPDAC cell growth was assessed by time-lapse Incucyte® over 120 hours.
  • B In parallel, mPDAC cell viability was determined by MTS assay periodically over 72 hours.
  • C and (D) Cells were seeded in 96-well places, allowed to adhere overnight. Apoptosis induction assay upon increasing doses of N6L on mPDAC cells.
  • FIGS 4A, 4B, 4C, 4D, and 4E NCL targeting by N6L impacts/ mPDAC cell translation.
  • A Experimental design of translatome analysis procedure. mPDAC cells were treated (T) or not (NT) with 50 mM of N6L for 48 hours. Cytosolic lysates from cells were sedimented on sucrose gradient (15-47%). Following ultracentrifugation, 40S and 60S ribosome subunits, the 80S monosomes, and polysomes are separated. RNAs were extracted from polysome pooled fractions (mRNA-associated polysomes) and cytoplasmic fractions (cytoplasmic mRNA).
  • RNA quality and integrity were verified using a Bioanalyser 2100 before sequencing and bioinformatics analysis.
  • B) and C Polysome profiles of mPDAC cells non-treated (mPDAC NT in (B)) or treated with N6L (mPDAC T in (C)). Curves show absorbance at 254 nm as a function of sedimentation.
  • D) and E mPDAC cells, treated or not treated with N6L, were incubated with puromycin for the measurement of overall protein synthesis by SUnSET method and then subjected to western-blotting (D). Protein synthesis rates were quantified by measuring the signal intensity in each lane and normalizing the values to that of the control lane (E). w/o Puro, without puromycin. Data represented are the means ⁇ SD of at least three experiments. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001 and n.s. for not significant, as determined by Student t-test on final data point.
  • Figures 5A, 5B, 5C, 5D, 5E, and 5F. Bioinformatics analysis of mPDAC cell translatome upon N6L treatment.
  • A Percentage of translationally deregulated mRNAs in response to N6L treatment.
  • C Among these 886 translationally deregulated genes, 24.04% were up-regulated and 75.96% were down- regulated
  • TI Translational Index
  • FIG. 6A, 6B, 6C, 6D, 6E, and 6F NCL targeting by N6L dysregulates mPDAC cell translation. Bioinformatics analysis.
  • A and (B) Venn diagram showing the number of transcriptionally up-regulated, down-regulated or stable genes per Translation Index (TI) DOWN (C) or TI UP (D) category obtained in FIG. 5 A- FIG. 5F.
  • A Translation Index (TI) for eukaryote Initiator Factors (elF) mRNA under N6L treatment.
  • B - (E) mPDAC cells were treated (T) or not treated (NT) with N6L for 48 hours following which EiF3 subunits protein expression was analyzed using western-blotting ((B) and (D)) and quantified in relation to Actin ((C) and (E)).
  • FIGS 8A, 8B, 8C, 8D, 8E, and 8F NCL targeting by N6L increases 5’ TOP and 5’TISU mRNA translation by activating mTOR pathways.
  • A TI for 5’ TOP-containing mRNA under N6L treatment (right).
  • B Venn diagram comparing 5’ TOP-gene overlap between our TI UP mRNAs and 5’TOP gene lists from Gentilella et al. 2017.
  • C Table 2 summarizes the distribution of 5’ TOP and non-5’TOP mRNAs according to their transcriptional expression (up, down or stable).
  • mPDAC cells were treated (T) or not treated (NT) with N6L for 48 hours following which RPL36 and RPS3 expression at protein level was analyzed using western-blotting (upper panel) and quantified in relation to H3 (lower panel).
  • E Illustrates the design of 5’ TOP Firefly-luciferase reporter constructs, with the first C of the TOP motif as the +1 Transcription Start Site (TSS).
  • TSS Transcription Start Site
  • F Fold change of Firefly luciferase activities under 5’TOP motifs of Rpl36 and Rps3 mRNAs upon 48 hours of increasing concentrations of N6L treatment in mPDAC, after normalization to mPDAC NT. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****p ⁇ 0.0001 and n.s. for not statistically significant, as determined by Student t-test on final data point.
  • Figures 9A, 9B, 9C, 9D, 9E, and 9F The impact of mTOR inhibitors treatment on mPDAC and PDX cell growth.
  • A)-(C) mPDAC cell lines were seeded in 96-well plates and allowed to adhere overnight. Cells were treated with 10 or 25nM of Rapamycin (RAPA, (A)), AZD2014 (AZD, (B)) or INK128 (INK, (C)) over 72 hours.
  • RAPA Rapamycin
  • AZD2014 AZD, (B)
  • INK128 INK128
  • PAN014 PDAC PDX
  • Cells were treated with 1, 10 or lOOnM of RAPA (D), AZD (E) or INK (F) over 96 hours. Cell growth was assessed by time-lapse InCucyte ZOOM®. The AUC (Area Under Curves) was calculated by InCucyte ZOOM® software. Growth for treated cells was normalized to untreated cells on the same plate.
  • FIGS 10A, 10B, IOC, and 10D NCL targeting by N6L increases 5’ TOP mRNA translation by activating mTOR pathways.
  • A mTOR pathway activation was analyzed using western-blotting in mPDAC cells, non-treated (NT) or treated (T) with N6L at 50mM for 48 hours (lanes 1,2) or after NCL siRNA depletion (lanes 3,4).
  • g and b represent hyper- phosphorylated and a hypo-phosphorylated forms of 4EBP1.
  • the phosphorylated-to-total protein ratio was quantified for RPS6, AKT and 4EBP1.
  • (B) and (C) mTOR pathway activation was analyzed in mPDAC, non-treated (NT) or treated (T) with N6L at increasing doses using western-blotting (B) and Immunofluorescence staining (C) by anti-phosphorylated RPS6 (red) and DAPI for nuclei (blue).
  • N6L and mTORi combination acts synergistically to decrease PDAC cell and spheroid growth and organoid viability.
  • (A)-(C) mPDAC cell lines were seeded in 96-well plates and allowed to adhere overnight. Cells were treated with 10 or 25nM of Rapamycin (RAPA, (A)), AZD2014 (AZD, (B)) or INK128 (INK, (C)) in combinations with N6L at 10mM over 72 hours.
  • D-F, PDAC PDX (PAN014) cell lines were seeded in 96-well plates and allowed to adhere 2 days.
  • Cells were treated with 1, 10 or lOOnM of RAPA (D), AZD (E) or INK (F) in combinations with N6L at 10mM over 96 hours.
  • Cell growth was assessed by IncuCyte ZOOM® live-imaging.
  • the AUC Ana Under Curves
  • Growth for treated cells was normalized to untreated cells on the same plate.
  • G Synergy/antagonism calculation.
  • the Contour views from LOEWE model were selected as graphical outputs for the synergy distribution.
  • FIGS 12A, 12B, 12C, 12D, and 12E Synergistic effects of N6L/mTORi combination on Pane cells.
  • A Pane cell lines were seeded in 96-well plates and allowed to adhere overnight. Cells were treated with concentrations of N6L alone at 10, 30 or 50mM (A, upper left), Rapamycin (RAPA) alone at 25 or 50nM (A, upper middle), INK128 (INK) alone at 10 or 25nM (A, upper right) or in combinations (lower) over 72 hours. Cell growth was assessed by time-lapse Incucyte®. The AUC (Area Under Curves) was calculated by Incucyte® software.
  • FIGS 13A and 13B N6L combined to mTORi treatment inactivate mTOR pathway by decreasing RPS6 and 4EBP1 phosphorylation.
  • mTOR pathway inhibition was analyzed using western-blotting in mPDAC (A) and PDX PDAC (B) cells non-treated (NT) or treated with N6L, RAPA, INK 128 and AZD2014 alone or in combination (N6L+RAPA, N6L+INK, or N6L+AZD).
  • FIGS 14A, 14B, 14C, 14D, and 14E Synergistic effects of N6L/mTORi combination on MiaPaca cells.
  • A MiaPaca cell lines were seeded in 96-well plates and allowed to adhere overnight. Cells were treated with concentrations of N6L alone at 10, 30 or 50mM (upper left), Rapamycin (RAPA) alone at 25 or 50nM (upper middle), INK128 (INK) alone at 10 or 25nM (upper right) or in combinations (lower) over 72 hours. Cell growth was assessed by time-lapse Incucyte®. The AUC (Area Under Curves) was calculated by Incucyte® software.
  • a reference to "A and/or B", when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B" can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.
  • co-administration and “co-administering” or “combination therapy” refer to both concurrent administration (administration of two or more therapeutic agents at the same time) and time varied administration (administration of one or more therapeutic agents at a time different from that of the administration of an additional therapeutic agent or agents), as long as the therapeutic agents are present in the patient to some extent, preferably at effective amounts, at the same time.
  • one or more of the present compounds described herein are co-administered at least one (e.g., 1, 2, 3, or 4) mTOR inhibitor and at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound.
  • the co-administration of at least one (e.g., 1, 2, 3, or 4) mTOR inhibitor and at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound results in synergistic activity and/or therapy, including anticancer activity, such as inhibiting cell growth and/or decreasing cell viability, or anti-angiogenic activity.
  • nucant can mean but is in no way limited to a nucleolin- binding compound or entity, e.g., nucleolin antagonist, including, for example, a nucleolin binding peptide or pseudopeptide, or a derivative or analog thereof (collectively “Nucant peptide”) or polyvalent synthetic compound as described herein.
  • nucleolin-binding compound or entity e.g., nucleolin antagonist, including, for example, a nucleolin binding peptide or pseudopeptide, or a derivative or analog thereof (collectively “Nucant peptide”) or polyvalent synthetic compound as described herein.
  • compound refers to any specific chemical compound disclosed herein, including the polyvalent synthetic compound described herein, and includes tautomers, regioisomers, geometric isomers, and where applicable, stereoisomers, including optical isomers (enantiomers) and other stereoisomers (diastereomers) thereof, as well as pharmaceutically acceptable salts and derivatives, including prodrug and/or deuterated forms thereof where applicable, in context.
  • Deuterated compounds contemplated herein are those in which one or more of the hydrogen atoms contained in the drug molecule have been replaced by deuterium.
  • the term compound generally refers to a single compound, but also may include other compounds such as stereoisomers, regioisomers and/or optical isomers (including racemic mixtures) as well as specific enantiomers or enantiomerically enriched mixtures of disclosed compounds.
  • the term also refers, in context to prodrug forms of compounds which have been modified to facilitate the administration and delivery of compounds to a site of activity. It is noted that in describing the present compounds, numerous substituents and variables associated with same, among others, are described. It is understood by those of ordinary skill that molecules which are described herein are stable compounds as generally described hereunder. When the bond is shown, both a double bond and single bond are represented or understood within the context of the compound shown and well-known rules for valence interactions.
  • analogs are compositions that have a structure similar to, but not identical to, the native compound.
  • peptides can mean, but is in no way limited to, recombinant polypeptide having at least 4 amino acids connected by peptide bonds. Furthermore, peptides of the present disclosure may include amino acid mimentics, and analogs. Recombinant forms of the peptides can be produced according to standard methods and protocols which are well known to those of skill in the art, including for example, expression of recombinant proteins in prokaryotic and/or eukaryotic cells followed by one or more isolation and purification steps, and/or chemically synthesizing peptides or portions thereof using a peptide synthesizer.
  • biologically active can mean, but is in no way limited to, the ability of an agent, such as the compounds provided by the present disclosure, to effectuate a physiological change or response.
  • the response may be detected, for example, at the cellular level, for example, as a change in growth and/or viability, gene expression, protein quantity, protein modification, protein activity, or combination thereof; at the tissue level; at the systemic level; or at the organism level.
  • Techniques used to monitor these phenotypic changes include, for example, measuring: the binding of a ligand to its receptor in or on a cell, activation of cell signaling pathways, stimulation or activation of a cellular response, secretion or release of bioactive molecules from the cell, cellular proliferation and/or differentiation, or a combination thereof.
  • the biological activity of a peptide provided by the present disclosure can be determined by detecting its ability to inhibit the growth and/or proliferation of a cell.
  • patient or “subject” is used throughout the specification to describe an animal, preferably a human or a domesticated animal, to whom treatment, including prophylactic treatment, with the compositions according to the present disclosure is provided.
  • patient refers to that specific animal, including a domesticated animal such as a dog or cat, or a farm animal such as a horse, cow, sheep, etc.
  • patient refers to a human patient unless otherwise stated or implied from the context of the use of the term.
  • the term "effective amount/dose,” “pharmaceutically effective amount/dose,” “pharmaceutically effective amount/dose” or “therapeutically effective amount/dose” can mean, but is in no way limited to, that amount/dose of the active pharmaceutical ingredient sufficient to prevent, inhibit the occurrence, ameliorate, delay or treat (alleviate a symptom to some extent, preferably all) the symptoms of a condition, disorder or disease state.
  • the effective amount depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize.
  • an amount between 0.1 mg/kg and 1000 mg/kg body weight/day of active ingredients is administered dependent upon potency of the agent.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions can mean, but is in no way limited to, a composition or formulation that allows for the effective distribution of an agent provided by the present disclosure, which is in a form suitable for administration to the physical location most suitable for their desired activity, e.g., systemic administration.
  • Non-limiting examples of agents suitable for formulation with the, e.g., compounds of the present disclosure include: cinnamoyl, PEG, phospholipids or lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into various tissues, for example the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers, such as poly(DL-lactide-coglycolide) microspheres for sustained release delivery after implantation (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc.
  • nanoparticles such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).
  • pharmaceutically acceptable can mean, but is in no way limited to, entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
  • pharmaceutically acceptable carrier or “pharmacologically acceptable carrier” can mean, but is in no way limited to, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.
  • Such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • systemic administration refers to a route of administration that is, e.g., enteral or parenteral, and results in the systemic distribution of an agent leading to systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body.
  • Suitable forms depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to).
  • pharmacological compositions injected into the blood stream should be soluble.
  • Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect.
  • Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.
  • the rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size.
  • the use of a liposome or other drug carrier comprising the compositions of the present disclosure can potentially localize the drug, for example, in certain tissue types, such as the tissues of the pancreas.
  • a liposome formulation which can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful.
  • any amino acid means any natural or synthetic amino acid, possibly modified by the presence of one or more substituents.
  • nucleic acid alterations refers to the substitution, deletion or addition of nucleic acids that alter, add or delete a single amino acid or a small number of amino acids in a coding sequence where the nucleic acid alterations result in the substitution of a chemically similar amino acid.
  • Amino acids that may serve as conservative substitutions for each other include the following: Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q); hydrophilic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Hydrophobic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C).
  • sequences that differ by conservative variations are generally homologous.
  • non-conservative multations refers to substitutions, deletions or addition of nucleic acids that alter, add or delete a single amino acid or a small number of amino acids in a coding sequence where the nucleic acid alterations results in the substitution of an amino acid that is not chemical similar — i.e., an alteration that does not result in a conservative mutation.
  • binding can mean, but is in no way limited to, the physical or chemical interaction, direct or indirect, between two molecules (e.g., compounds, amino acids, nucleotides, polypeptides, or nucleic acids). Binding includes covalent, hydrogen bond, ionic, non-ionic, van der Waals, hydrophobic interactions, and the like.
  • the term "cell”, as used herein, can mean, but is in no way limited to, its usual biological sense, and does not refer to an entire multicellular organism.
  • the cell can, for example, be in vivo , in vitro or ex vivo , e.g., in cell culture, or present in a multicellular organism, including, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats.
  • the cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
  • support refers to any pharmaceutically acceptable molecule, in other words without intrinsic toxicity, on which at least 3 pseudopeptide units can be grafted or coupled.
  • An acceptable support therefore has to be of sufficient size to allow at least 3 pseudopeptide units to be grafted on it.
  • there are 3 to 20 pseudopeptides grafted or coupled to the support preferably 3 to 8 pseudopeptide.
  • Such an acceptable support should also preferably be large enough to allow at least 3, preferably 3 to 8, pseudopeptide units can come together to interact in the RGG domain of one or more nucleolin molecules.
  • the support is not immunogenic.
  • amino acid with a basic side chain means any natural or non-natural amino acid whose side chain R has a pKa value greater than 7 (pKa(R)>7).
  • any amino acid can be used for Yi and Y2, as long as its side chain has a pKa value greater than 7, preferably greater than 7.5, greater than 8, greater than 8.5 or greater than 9.
  • lysine (K, pKa(R) ⁇ 10.5), arginine (R, pKa(R) ⁇ 12.5), ornithine (inferior homologue of lysine, pKa(R) ⁇ 10.8) generally considered to be natural basic amino acids.
  • Yi and Y2 are independently selected from arginine (R), lysine (K) and ornithine. Even more advantageously, Yi is a lysine (K) and Y2 is an arginine (R).
  • other non natural amino acids can be used instead as long as the pKa value of their side chain R is greater than 7, preferably greater than 7.5, greater than 8, greater than 8.5, or greater than 9.
  • grafted or “coupled” as used herein for the pseudopeptide or pseudopeptide units described herein means being bound to the support by means of a covalent bond, either directly or through the intermediate of a spacer compound between the pseudopeptide and support.
  • one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, or even all) of the pseudopeptides are grafted or coupled directly on the support without a spacer compound between them and the support.
  • one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, or even all) of the pseudopeptides are grafted or coupled on the support through an intermediate of a spacer.
  • acceptable spacers include an alkyl group, ethylene glycol, piperazine, or an amino acid of the type aminohexanoic acid or beta-alanine.
  • pharmaceutically acceptable salt is used throughout the specification to describe, where applicable, a salt form of one or more of the compounds described herein which are presented to increase the solubility of the compound in the gastic juices of the patient's gastrointestinal tract in order to promote dissolution and the bioavailability of the compounds.
  • Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids, where applicable. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium, magnesium and ammonium salts, among numerous other acids and bases well known in the pharmaceutical art. Sodium and potassium salts are particularly preferred as neutralization salts of the phosphates according to the present disclosure.
  • pharmaceutically acceptable derivative is used throughout the specification to describe any pharmaceutically acceptable prodrug form (such as an ester, amide other prodrug group), which, upon administration to a patient, provides directly or indirectly the present compound or an active metabolite of the present compound.
  • the present disclosure provides methods for treating and/or preventing a disease or disorder related to the detrimental growth and/or proliferation of a cell, e.g., a cancer cell.
  • the method comprises administering or co-administering a composition comprising an effective amount of at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound of the present disclosure and at least one (e.g., 1, 2, 3, or 4) mTOR inhibitor to a subject or a cell, in vivo , ex vivo , or in vitro , wherein the composition is effective in inhibiting or preventing the growth and/or proliferation and/or viability of a cancer cell.
  • at least one e.g., 1, 2, 3, or 4
  • mTOR inhibitor e.g., 1, 2, 3, or 4
  • the composition is effective in inhibiting or preventing the growth and/or proliferation and/or viability of a cancer cell.
  • an aspect the disclosure provides a method of treating cancer (such as pancreatic cancer) in a subject, the method comprising administering or co-administering to a cell or a subject in need thereof an effective amount of at least one (e.g., 1, 2, 3, or 4) mTOR inhibitor and an effective amount of at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound described herein, wherein the mTOR inhibitor and the polyvalent synthetic compound are effective for treating or ameliorating at least one symptom of the cancer (such as pancreatic cancer).
  • cancer such as pancreatic cancer
  • the polyvalent synthetic compound is present/administered in a composition, such as a therapeutic composition or a pharmaceutical composition, that includes a pharmaceutically acceptable carrier, excipient, or diluent.
  • the cancer is pancreatic cancer.
  • the cancer or the pancreatic cancer is pancreatic ductile adenocarcinoma.
  • the pancreatic cancer is pancreatic ductile adenocarcinoma, pancreatic adenocarcinoma, pancreatic squamous cell carcinoma, pancreatic adenosquamous carcinoma, pancreatic colloid carcinoma, preferably pancreatic ductile adenocarcinoma.
  • the polyvalent synthetic compound and the mTOR inhibitor have a synergistic inhibitory effect on the pancreatic cancer cell growth and/or cell viability.
  • the Nucant or polyvalent synthetic compounds described therein are known to be useful for the treatment of a disease involving deregulation of cell proliferation and/or angiogenesis. Therefore, in an additional aspect, the present disclosure provides methods of treating a disease or disorder involving deregulation of cell proliferation and/or angiogenesis comprising the step of administering or co-administering to a cell, a subject or an individual at least one (e.g., 1, 2, 3, or 4) mTOR inhibitor and an effective amount of a therapeutic composition comprising at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound of the present disclosure.
  • a disease or disorder involving deregulation of cell proliferation and/or angiogenesis comprising the step of administering or co-administering to a cell, a subject or an individual at least one (e.g., 1, 2, 3, or 4) mTOR inhibitor and an effective amount of a therapeutic composition comprising at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound of the present disclosure.
  • disease involving deregulation of cell proliferation and/or angiogenesis means, in the context of the present disclosure, any human or animal disease affecting one or more organs in which one or more abnormal cell proliferation phenomena are observed, as well as groups of cells or tissues and/or abnormal neovascularisation.
  • diseases include all types of cancer, such as adenoma, sarcoma, carcinoma, lymphoma, and especially cancer of the ovary, breast, pancreas, lymphatic ganglion, skin, blood, lung, brain, kidney, liver, nasopharyngeal cavity, thyroid, central nervous system, prostate, colon, rectum, uterine neck, testicles or bladder.
  • composition of the present disclosure as described herein may thus be for use with an mTOR inhibitor in the treatment of disease involving deregulation of cell proliferation and/or angiogenesis.
  • a further aspect of the present disclosure provides methods for treating and/or preventing a disease or disorder related to the growth and/or proliferation of a cancer cell in an individual.
  • the composition of the present disclosure is for use with an mTOR inhibitor in the treatment and/or prevention of a disease or disorder related to the growth and/or proliferation of a cancer cell in an individual, in particular for the treatment and/or prevention of cancer.
  • the methods described herein comprise administering or co-administering a mTOR inhibitor and (1) a composition comprising an effective amount of at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound of the present disclosure or (2) a composition comprising microspheres of at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound of the present disclosure, to a cell, a subject, or an individual, wherein the compound/composition and mTOR inhibitor are effective in inhibiting or preventing the growth and/or proliferation and/or viability of the cancer cell.
  • Nucant compounds have been shown to bind surface nucleolin RGG domain.
  • tumor cells such as tumor cells derived from hepatic carcinoma (Semenkovich, C. F., Ostlund, R. E. T, Olson, M. O., and Yang, J. W.
  • TASP inhibitors of HIV entry bind specifically to a 95-kDa cell surface protein.
  • J. Biol. Chem. 272, 7159-7166 and Callebaut, C., Blanco, J., Benkirane, N., Krust, B., Jacotot, E., Guichard, G., Seddiki, N., Svab, J., Dam, E., Muller, S., Briand, J. P., and Hovanessian, A. G. Identification of V3 loop-binding proteins as potential receptors implicated in the binding of HIV particles to CD4(+) cells. (1998) J. Biol. Chem.
  • Nucleolin expressed at the cell surface is a marker of endothelial cells in angiogenic blood vessels. (2003) J. Cell Biol. 163, 871-878), cells which are involved in the angiogenesis process. Because the binding of the polyvalent synthetic compounds described herein to nucleolin is a generalized effect, the combination therapy provided by the present disclosure would be suitable for treating any known cancer.
  • types of cancer suitable for treatment with the compositions and methods provided by the present disclosure include, Acute Lymphoblastic Leukemia; Acute Myeloid Leukemia; Adrenocortical Carcinoma; Adrenocortical Carcinoma; AIDS-Related Cancers; AIDS-Related Lymphoma; Anal Cancer; Appendix Cancer; Astrocytomas; Atypical Teratoid/Rhabdoid Tumor; Basal Cell Carcinoma; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bone Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma; Brain Stem Glioma; Brain Tumor; Embryonal Tumors; Astrocytomas; Craniopharyngioma; Ependymoblastoma; Medulloepithelioma; Pineal Parenchymal Tumors of Intermediate Differentiation; Supratentorial Primitive Neuroectodermal Tumors and Pineoblastom
  • the polyvalent synthetic compounds described therein form microspheres when admixed with a GAG. Therefore, in an additional aspect, the present disclosure provides methods of treating a disease or disorder, e.g., cancer, comprising the step of administering or co-administering to a cell, a subject, or an individual an effective amount of at least one (e.g., 1, 2, 3, or 4) mTOR inhibitor and an effective amount of a therapeutic composition comprising microspheres of at least one (e.g., 1, 2, 3, or 4) of the polyvalent synthetic compounds described herein and a GAG.
  • the microspheres are formed prior to administration.
  • the microspheres are formed after administration to the subject or patient, e.g., in vivo.
  • the therapeutic composition of the polyvalent synthetic compounds of the present disclosure is any pharmaceutically acceptable form and administered by any pharmaceutically acceptable route, for example, the therapeutic composition can be administered as an oral dosage, either single daily dose or unitary dosage form, for the treatment of a muscle disorder or conditions, e.g., diabetes.
  • Such pharmaceutically acceptable carriers and excipients and methods of administration will be readily apparent to those of skill in the art, and include compositions and methods as described in the USP-NF 2008 (United States Pharmacopeia/National Formulary), which is incorporated herein by reference in its entirety. Therefore, another aspect of the present disclosure provides pharmaceutically acceptable formulations of the polyvalent synthetic compounds of the present disclosure.
  • pharmaceutically acceptable formulations include salts of the polyvalent synthetic compounds described herein (e.g., acid addition salts, such as salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid).
  • acid addition salts such as salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
  • the polyvalent synthetic compounds described herein are formulated for parenteral administration, e.g., formulated for injection via the intravenous, intraarthricular, intrathecal, intramuscular, sub -cutaneous, intra-lesional, or even intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intraarthricular, intrathecal, intramuscular, sub -cutaneous, intra-lesional, or even intraperitoneal routes.
  • an aqueous composition that contains a cancer marker antibody, conjugate, inhibitor or other agent as an active component or ingredient will be known to those of skill in the art in light of the present disclosure.
  • such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
  • a “pharmacological composition”, “pharmacological formulation”, or the like, as used herein, refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or subject, preferably a human.
  • systemic administration in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (e.g., a cell to which the negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations, such as toxicity and forms which prevent the composition or formulation from exerting its effect.
  • Preparations for administration of the therapeutic of the present disclosure include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non- aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Exemplary aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles including fluid and nutrient replenishers, electrolyte replenishers, and the like.
  • Preservatives and other additives may be added such as, for example, antimicrobial agents, anti-oxidants, chelating agents and inert gases and the like.
  • a pharmaceutical composition of the polyvalent synthetic compound of the present disclosure is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral — e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, intraperitoneal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation is enclosed in ampoules, disposable syringes or multiple dose vials, each of which may be made of glass or plastic.
  • Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.
  • the rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size.
  • the use of a liposome or other drug carrier comprising the polyvalent synthetic compounds of the present disclosure can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES).
  • RES reticular endothelial system
  • the composition of the present disclosure includes or is a liposome formulation with the polyvalent synthetic compounds described herein, wherein the liposome formulation facilitates the association of the polyvalent synthetic compounds described herein with the surface of cells, such as, lymphocytes and macrophages.
  • the formulations and the polyvalent synthetic compounds of the present disclosure can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
  • parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like.
  • a pharmaceutical formulation comprising polyvalent synthetic compounds of the present disclosure and a pharmaceutically acceptable carrier.
  • One or more polyvalent synthetic compound of the present disclosure can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients.
  • the pharmaceutical compositions comprising polyvalent synthetic compounds of the present disclosure can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
  • compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, eth
  • compositions may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • Excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.
  • Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan mono
  • the aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or n-propyl p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • sweetening agents such as sucrose or saccharin.
  • Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents and flavoring agents can be added to provide palatable oral preparations.
  • These compositions can be preserved by the addition of an anti -oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.
  • Pharmaceutical compositions having polyvalent synthetic compounds of the present disclosure can also be in the form of oil-in-water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil or mixtures of these.
  • Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions can also contain sweetening and flavoring agents.
  • Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents.
  • the pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a non -toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • Suitable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the polyvalent synthetic compounds for use according to the present disclosure are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethan-e, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethan-e, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • suitable powder base such as lactose or starch.
  • the polyvalent synthetic compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • the polyvalent synthetic compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the polyvalent synthetic compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the polyvalent synthetic compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor.TM.. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • the polyvalent synthetic compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the present disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Sustained-release preparations can be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and gamma-ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT. TM. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
  • polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • the therapeutic compositions having the polyvalent synthetic compounds of the present disclosure can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.
  • the use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.
  • the mTOR inhibitor is an mTORCl inhibitor, such as an allosteric mTORCl inhibitor or an ATP -competitive mTOR kinase inhibitor.
  • the mTOR inhibitor is an mTORC2 inhibitor, such as an ATP-competitive mTOR kinase inhibitor.
  • the mTOR inhibitor is one or more (e.g., 1, 2, 3, or 4) inhibitors selected from allosteric mTOR inhibitors and ATP-competitive mTOR inhibitors. In any aspect or embodiment described herein, the mTOR inhibitor is one or more (e.g., 1, 2, 3, or 4) allosteric mTOR inhibitors. In any aspect or embodiment described herein, the mTOR inhibitor is one or more (e.g., 1, 2, 3, or 4) ATP-competitive mTOR inhibitors.
  • the allosteric mTOR inhibitors is one or more (e.g., 1, 2, 3, or 4) inhibitors selected from rapamycin (SIROLIMUS), CCI-779 (TEMSIROLIMUS), RAD001 (EVEROLIMUS), AP-23573 (RID AF OROLIMU S), UMIROLIMUS, and ZOTAROLIMUS.
  • the ATP-competitive mTOR inhibitor is one or more (e.g., 1, 2, 3, or 4) inhibitors selected from AZD2014 (VISTUSERTIB), INK 128 (SAPANISERTIB), and AZD8055.
  • the mTOR inhibitor is one or more (e.g., 1, 2, 3, or 4) inhibitors selected from Rapamycin, AZD2014, and INK 128.
  • nucleolin Located essentially in the nucleus of normal cells where it is protected, nucleolin is, however, abundant at the surface of the cells that are proliferating and cells that of active endothelial cells where it can be a target for the polyvalent synthetic compounds described herein and their derivatives, which binds with specificity to surface nucleolin and/or glycoaminoglycans (GAGs).
  • GAGs glycoaminoglycans
  • the polyvalent synthetic compound comprises a support comprising at least 3 pseudopeptides coupled or grafted thereto, wherein the support is a linear peptide support having the formula (la) or (lb):
  • each X is independently any amino acid (e.g. any proteinogenic amino acid); each Y 1 and Y 2 is independently a basic amino acid (e.g., arginine, lysine, lysine derivative, or lysine analog); n is 0 or 1; m is 0, 1, 2, or 3; and
  • An advantage of them polyvalent synthetic compounds described herein demonstrate dual activity; the polyvalent synthetic compounds are capable of blocking independently both tumor proliferation and angiogenesis. Another major advantage of the polyvalent synthetic compounds of the present disclosure is that they demonstrate an excellent safety profile. Additionally, because of its small size of the polyvalent synthetic compounds, they are not immunogenic and are easy to manufacture, thereby making the cost of production reasonable.
  • the polyvalent synthetic compounds described herein bind surface nucleolin, which is present in active endothelial cells responsible for angiogenesis as well as in cancer cells, in a quasi irreversible manner.
  • the polyvalent synthetic compounds of the present disclosure have shown a binary effect: direct blockade of the growth of tumor cells and inhibition of the angiogenesis which led to the complete eradication of implanted tumor in animal models.
  • the specific binding occurs with the RGG domain located in the C-terminal region of the protein. After interaction the complex is internalized rapidly through a temperature dependent mechanism.
  • the polyvalent synthetic compounds of the present disclosure can be considered as a stable and irreversible ligand of the surface nucleolin. After internalization, the polyvalent synthetic compounds of the present disclosure remains in the cytoplasm and does not cross the nuclear membrane.
  • nucant and polyvalent synthetic compounds of the present disclosure also encompasses peptides having minor modifications, for example, conservative amino acid modifications, chemical modification to mimic valence properties, and modifications that serve to increase its stability, solubility, biouptake and/or bioavailability; for example, absorption from the gut or penetration through the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • a pharmaceutically acceptable carrier can contain physiologically acceptable compounds that include carbohydrates such as glucose, sucrose or dextrans; antioxidants, such as ascorbic acid or glutathione; chelating agents; and low molecular weight proteins. Additional modifications to a peptide or polyvalent synthetic compounds described herein that can increase its bioavailability include conjugating the peptide to a lipophilic moiety, such as a lipophilic amino acid or compound.
  • polyvalent synthetic compounds of the present disclosure is intended to encompass pseudopeptides as described herein having one or several minor modifications to the sequence.
  • Contemplated modifications include chemical or enzymatic modifications (e.g. acylation, phosphorylation, glycosylation, etc.), and substitutions of one or several amino acids to the sequence.
  • modifications can be desirable in order to enhance the bioactivity, bioavailability or stability of the peptide, or to facilitate its synthesis or purification.
  • the polyvalent synthetic compounds of the present disclosure can be conjugated to one or more of a carrier or a cytotoxic agent, either directly or indirectly (e.g., via a linker moiety).
  • a cytotoxic agent may be covalently bound to a linker moiety, which is in turn covalently bound to the carrier.
  • a linker moiety can be, for example, an amino acid (including mimetics, analogs, and derivatives), a peptide or polypeptide (including mimetics, analogs, and derivatives), a sterically labile compound, lipid, aliphatic group, carbohydrate, glyceride, a nucleotide or nucleic acid, peptide nucleic acid, nucleic acid derivative, and the like.
  • a conjugate in which the compounds of the present disclosure is conjugated to is cytotoxic drug 5-fluoro-uracyl ("5-FU").
  • Contemplated amino acid substitutions to the compounds of the present disclosure and provided for by the polyvalent synthetic compounds of the present disclosure include conservative changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of an apolar amino acid with another apolar amino acid; replacement of a charged amino acid with a similarly charged amino acid, etc.).
  • conservative changes e.g., replacement of an apolar amino acid with another apolar amino acid; replacement of a charged amino acid with a similarly charged amino acid, etc.
  • nonconservative changes e.g., replacement of an uncharged polar amino acid with an apolar amino acid; replacement of a charged amino acid with an uncharged polar amino acid, etc.
  • non-linear variants of the sequence of the polyvalent synthetic compounds described herein including branched sequences and cyclic sequences, and variants that contain one or more D-amino acid residues in place of their L-amino acid counterparts, may be made.
  • the polyvalent synthetic compound(s) of the present disclosure is incorporated into liposomes (Gregoriadis, Liposome Technology, Vols. I to III, 2nd ed. (CRC Press, Boca Raton Fla. (1993)). Liposomes, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • the polyvalent synthetic compounds of the present disclosure can be prepared as nanoparticles.
  • adsorbing polyvalent synthetic compounds onto the surface of nanoparticles has proven effective in delivering peptide drugs to the brain (see Kreuter et al., Brain Res. 674:171-174 (1995)).
  • Exemplary nanoparticles are colloidal polymer particles of poly-butylcyanoacrylate with the polyvalent synthetic compounds of the present disclosure adsorbed onto the surface and then coated with polysorbate 80.
  • the carrier may be modified so that the modified version of the carrier may be more easily conjugated to a diagnostic agent.
  • the polyvalent synthetic compounds described herein may or may not be optically pure, which means that the residues, such as lysine residues, in the pseudopeptide units may either be in random L or D configuration (not optically pure), or be all in D configuration (optically pure) or all in L configuration (optically pure).
  • the polyvalent synthetic compounds described herein are optically pure, e.g. the amino acids residues (such as lysine residues) in the pseudopeptide units are all in D configuration or all in L configuration, preferably all in L configuration.
  • Such optically pure polyvalent synthetic compounds can be obtained by the method described in WO2009/141687.
  • the polyvalent synthetic compound comprises 3 to 15 pseudopeptides.
  • one or more of the pseudopeptides (e.g., each/all of the pseudopeptides) is coupled or grafted directly on the support.
  • the pseudopeptides are grafted or coupled to the support via a lysine of the support.
  • the pseudopeptides are optically pure pseudopeptides.
  • the polyvalent synthetic compound has the structure:
  • each pseudopeptide independently has formula (Ha):
  • each X is independently any amino acid (e.g. any proteinogenic amino acid);
  • Z is proline or derivative thereof; each Y 1 and Y 2 is independently a basic amino acid (e.g., arginine, lysine, lysine derivative, or lysine analog); n is 0 or 1; m is 0, 1, 2, or 3; and
  • each pseudopeptide independently has formula (lib):
  • each X is independently any amino acid (e.g. any proteinogenic amino acid); each Y 1 and Y 2 is independently a basic amino acid (e.g., arginine, lysine, lysine derivative, or lysine analog); n is 0 or 1; m is 0, 1, 2, or 3; and
  • n and m are each 0.
  • the Y 1 and the Y 2 have an L configuration or D configuration.
  • the Y 1 and the Y 2 have an L configuration.
  • each Y 1 and Y 2 is independently lysine or arginine.
  • each Y 1 and Y 2 is a lysine having an L configuration.
  • each Y 1 and Y 2 is independently selected from ornithine, homolysine, and diaminoheptanoic acid (e.g., 2,7-diaminoheptanoic acid).
  • the Y represents a reduced bond.
  • the m is 0 or 1.
  • one or more X is a proteinogenic amino acid.
  • the support is a linear peptide support having the formula (la):
  • the support is a linear peptide support having the formula (lb):
  • z is an integer from 3 to 10.
  • z is 6.
  • the support is selected from a linear peptide, a cyclic peptide, a peptoid (N-substituted glycine oligomer) that is linear or cyclic, a foldamer (oligomer or polymer with a strong tendency to adopt a compact, well-defined and predictable conformation in solution), a linear polymer or a spherical dendrimer (macromolecule consisting or polymers which combine according to a tree like process around a multifunctional central core), a sugar or a nanoparticle.
  • the support is selected from a linear peptide, a cyclic peptide, a linear peptoid, or a cyclic peptoid.
  • the support is a linear peptide having lysine present in an amount of greater than 25%.
  • one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more, or even all) of the pseudopeptides are grafted or coupled to a lysine present on the support.
  • the support linear peptide has a sequence selected from KKKGPKEKGC (SEQ ID No. 7), KKKKGC (SEQ ID No. 8), KKKKGPKKKKGA (SEQ ID No. 9) or KKKGPKEKAhxCONEE (SEQ ID NO: 10), wherein Ahx represents hexanoic amino acid and CONEE represents the fact that the acid group is replaced by an amide group, AhxCONEE, representing (2S)-2-aminohexanamide, or a linear sequence consisting of 2-4 units (KAKPG, SEQ ID No. 11), namely sequence AcKAKPGKAKPGKAKPGCONEE (SEQ ID No.
  • the support linear peptide is peptide KKKGPKEKAhxCONEE (SEQ ID No. 10), wherein Ahx represents hexanoic amino acid and CONEE represents the fact that the acid group is replaced by an amide group, or peptide AcKAKPGKAKPGKAKPGCOME (SEQ ID No. 12) where Ac represents an acetyl group CH 3 -CO- and CONH 2 means that the acid group COOH of glycine is replaced by an amide group CONH 2.
  • the linear peptide support that forms a helicoidal structure comprises at least 3 (e.g., 3 to 20, 3 to 10 or 3 to 8) repetitions of peptide units having the sequence Aib-Lys-Aib-Gly (SEQ ID No. 2) or Lys-Aib-Gly (SEQ ID No. 1), where Aib represents 2-amino-isobutyric acid.
  • at least half of the SEQ ID No. 2 or SEQ ID No. 1 peptide units e.g., each of the peptide units
  • have a pseudopeptide of the present disclosure coupled or grafted thereupon e.g., grafter to the lysine of each peptide unit).
  • a quadrivalent compound with 4 pseudopeptide units of the present disclosure includes a support that is a linear peptide forming a helicoidal structure having the formula Ac-Lys-Aib-Gly-Lys-Aib- Gly-Lys-Aib-Gly-Lys-Aib-Gly-COME (SEQ ID No. 13), where Ac represents a CH3-CO- group and CONH2 means that the acid group COOH of glycine is replaced by an amid group COME.
  • a pentavalent compound with 5 pseudopeptide units of the present disclosure includes a support that is a linear peptide forming a helicoidal structure having the formula Aib-Lys-Aib-Gly-Aib-Lys- Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly (SEQ ID No.
  • the support in a linear peptide forming a helicoidal structure having a formula selected from Ac-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly- Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly- Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly- Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-COME (SEQ ID No.
  • Ac represents an acetyl group CH3-CO- and COME means that the COOH acid group of glycine is replaced by an amide group COME
  • Ac-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib- Gly-COME SEQ ID No. 17 where the Ac group represents an acetyl group CH3-CO- and COME means that the COOH acid group of glycine is replaced by an amide group COME.
  • a hexavalent compound with 6 pseudopeptide units of the present disclosure includes a support that is a linear peptide forming a helicoidal structure having the formula Ac-Aib-Lys-Aib-Gly-Aib- Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-CONH2 (SEQ ID No.
  • Ac represents a CH3-CO- group and CONH2 means that the acid group COOH of glycine is replaced by an amide group CONH2, or Ac-Lys-Aib-Gly-Lys- Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-COME (SEQ ID No.19) where Ac represents a CH3-CO- group and CONH2 means that the acid group COOH of glycine is replaced by an amid group COME.
  • an octavalent compound with 8 pseudopeptide units of the present disclosure includes a support that is a linear peptide forming a helicoidal structure having the formula Ac-Lys-Aib-Gly-Lys-Aib- Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Gly-CONH 2 (SEQ ID No. 20), where Ac represents a CH 3 -CO- group and COME means that the acid group COOH of glycine is replaced by an amid group COME.
  • the support is a cyclic peptide or a cyclic peptoid. This allows the flexibility of the structure of the support to be restricted.
  • the cyclic peptide or a cyclic peptoid support is selected from hexa-cyclic peptide, octa-cyclic peptide, deca-cyclic peptide, dodeca-cyclic peptide, or a chain of N-alkyl Glycine residue (an exemplary cyclic peptoid).
  • the support has amino acid residues in the L (levorotatory) and D (dextrorotatory) configuration in alternation (D,L-cyclopeptide).
  • the support is a cyclic hexapeptide consisting of alternate alanine (A) residues of configuration D and lysine residues (K) of configuration L with 3 KPR units with a Y (-CH2N-) bond between K and P.
  • the support includes or has 5 lysine residues linked by amide bonds at the e amino group of each Lysine residue may also be used.
  • the support is selected from: (i) a cyclic hexapeptide consisting of alternating alkaline (A) residues of configuration D and Lysine (K) residues of configuration L; (ii) 5 lysine residues linked by amide bonds at the e amino group of each Lysine residue; and (iii) a linear peptide of sequence SEQ ID No. 7 through SEQ ID No. 20.
  • the support is a linear peptide or cyclic peptide having the pseudopeptide units directly coupled or grafted to the peptide to lysine residues of the peptide support, at the amino group in the a or e position (e.g., at the amino group in the position (on the side chain) of lysine).
  • the direct grafting or coupling of pseudopeptide units on the peptide support is carried out by means of an amide bond between the acid group COOH of the amino acid in the C-terminal position of the pseudopeptide unit and an amino group of a lysine residue (e.g., the amino group in the e position (on the side chain) of lysine).
  • a lysine residue e.g., the amino group in the e position (on the side chain) of lysine.
  • At least 3 pseudopeptide units are grafted or coupled on or to the support.
  • the inventors have demonstrated previously the importance of binding to the RGG domain of nucleolin for exceptional anti -tumor efficacy of the Nucant peptides, derivatives, and analogues thereof.
  • the binding to the RGG domain of nucleolin is obtained by means of multivalent presentation of several pseudopeptide units, such as those described herein.
  • the compound includes at least 3 pseudopeptide units grafted on or coupled to the support.
  • the compounds has a support is a linear peptide of sequence KKKGPKEKGC, KKKKGC, KKKKGPKKKKGA or KKKGPKEKAhxCOME, and has at least three (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) pseudopeptides directly or indirectly grafted or coupled thereupon.
  • the compound described herein includes 3 to 8 pseudopeptide units (e.g., 4-7, 4-6, 4 or 5, or 5 or 6 pseudopeptide units) grafted on or coupled to the support.
  • amino acid means any natural or synthetic amino acid, possibly modified by the presence of one or more substituents. More precisely the term amino acid means an alpha aminated amino acid with the following general structure: wherein R represents the side chain of the amino acid. In any aspect or embodiment described herein, R represents the side chain of a side or non-side amino acid.
  • natural amino acid means any amino acid which is found naturally in vivo in a living being.
  • Natural amino acids therefore include amino acids coded by mRNA incorporated into proteins during translation but also other amino acids found naturally in vivo which are a product or by-product of a metabolic process, such as for example ornithine, which is generated by the urea production process by arginase from L-arginine.
  • the amino acids used can therefore be a natural amino acid or not a natural amino acid.
  • Natural amino acids generally have an L configuration but also, an amino acid of the present disclosure can have the L or D configuration.
  • R is of course not limited to the side chains of natural amino acid but can be freely chosen.
  • the any amino acid can be a non-proteinogenic amino acid.
  • the compounds of the present disclosure include a modified peptide bond Y, which provides significantly more resistant to at least one protease than a standard peptide.
  • the inventors have also discovered that the presence of the modified bond Y makes it possible to significantly increase the efficacy of binding to nucleolin. This phenomenon may be due to the fact that this allows the compounds of the present disclosure to form an irreversible complex with nucleolin.
  • standard peptide bond means an amide bond of formula (-CONH-) which is normally present between 2 amino acids in a natural protein. Such a bond is sensitive to the action of proteases.
  • modified peptide bonds Y means a chemical bond between 2 amino acids of chemical formula distinct from the standard peptide bond of formula (-CONH-). This modified bond Y is such that it is significantly more resistant to at least one protease than a standard peptide bond of formula (-CONH-).
  • proteease also known as “peptidase” or “proteolytic enzyme” means any enzyme which cleaves the standard peptide bonds in proteins. This process is known as proteolytic cleavage.
  • proteases This involves the use of a water molecule, which is what leads to proteases being classified as hydrolases.
  • the proteases namely include proteases known as N-peptidases which carry out the cleavage of the N-terminal end of proteins. These proteases are particularly inconvenient in terms of the in vivo stability of peptides without modified peptide bonds.
  • pseudopeptide units of the compounds of the present disclosure include a modified bond Y between Y 1 and proline such that the resistance of the sub-unit of formula (I) is significantly increased which is essential for binding to nucleolin, namely to these N-peptidases.
  • Y represents a reduced bond (-CH 2 -NH-), which significantly increase the compounds resistance to at least one protease.
  • the Y bond should therefore make it possible to significantly increase resistance to at least one N-peptidase. This makes it possible to significantly increase the half-life of compounds of the present disclosure in vivo and in vitro.
  • HB19 which has a modified bond Y, has a half-life of more than 24 hours in human serum or fetal calf serum at 37°C, whereas the same compound with a standard peptide bond instead of the Y bond only has a half-life of one hour under these same conditions.
  • additional Y bonds is expected to further increase resistance of the compounds of the present disclosure to proteases.
  • additional Y bonds does further complicate the process of synthesizing compounds of the present disclosure and is thus optional. Therefore, in any aspect or embodiment described herein, further Y may be present on the pseudopeptide, the support, or both.
  • Immunochemical assays useful for practicing methods of the disclosure are well known to those skilled in the art, as described, for example, in Klug, T. L. et al ., Cancer Res., 44:1048 (1984), Herlyn, M. et al, J. Clin. Immunol., 2:135 (1982), Metzgar, R. S. et al., Proc. Natl. Acad. Sci., USA. 81:5242 (1984), Papsidero, L. D. et al. , Cancer Res., 44:4653 (1984), Hayes, D. F. et al, J. Clin. Invest., 75:1671 (1985), Killian, C. S. et al, J.
  • the compounds described herein can be produced using well known recombinant methods or via well-known synthetic methods.
  • Pancreatic cancer is an aggressive disease characterized by high invasiveness, rapid progression, and resistance to conventional therapy. There is an urgent need to identify new molecules to improve current therapies, with better efficacy and less toxicity. It was previously shown that N6L, a synthetic pseudo-peptide that targets nucleolin (NCL) impairs PDAC growth and normalizes tumor vessels in animal models.
  • NCL nucleolin
  • N6L treatment strongly reduces pancreatic cells proliferation, which then undergoes a translational reprogramming through the activation of the mTOR pathway.
  • the inventors made the surprising and unexpected discovery that N6L and mTOR inhibitors act synergistically to inhibit the proliferation of several PDAC and human PDX cell lines. This new combination therapy of N6L and mTOR inhibitors constitutes a new alternative to treat pancreatic cancer.
  • N6L NCL antagonist
  • N6L decreases ribosome biogenesis and protein synthesis in PDAC cells.
  • NCL is a master regulator protein required for rRNA transcription and ribosome biogenesis. Whether the N6L pseudo-peptide had any effect on the production of ribosomes in PDAC cells was first examined. Time course analysis of mPDAC treated with different concentrations of N6L shows that at 50 mM a strong reduction of cell proliferation was observed (FIG. 3A) while metabolic activity which reflected cell viability, assessed by MTS test, was reduced by 56% (FIG. 3B). With N6L treatment impairing cell viability of mPDAC cells, whether the death of the cells was due to caspase dependent apoptosis was examined.
  • Apoptosis induction assay was performed using the IncuCyte ZOOM®.
  • a fluorescent reagent activated when effector caspases 3/7 were activated too, allowed the device to count the number of apoptotic cells per mm 2 every 2 hours until 72 hours.
  • a strong and significantly increase of caspase dependent apoptosis in mPDAC treated cells in a dose dependent manner was observed, as compared to non-treated cells (FIG. 3C).
  • the morphology of the N6L-treated cells appeared different to non-treated cells, more “thorny”.
  • Ribosome biogenesis requires the initial transcription of rDNA genes to a single pre-rRNA (47S) that is subsequently cleaved in several pre-rRNA to ultimately provide 5.8S, 18S and 28 S rRNAs.
  • the level of rDNA transcription was measured by RT-qPCR using 5’ETS probe (before the early cleavage), which is able to detect short-lived rRNA sequence of 47S. It was found that the 47S ribosomal RNA synthesis was repressed by 58% after N6L treatment (FIG. 4B).
  • NCL targeting by N6L impairs protein synthesis and induces a translational reprogramming in mPDAC cells.
  • mRNA translation is a central cellular process that regulates growth and metabolism (Sonenberg et Hinnebusch 2009).
  • N6L treatment we performed polysome profiling analysis on cells treated or not treated with N6L for 48 hours (FIG. 4A-FIG. 4F).
  • RNAs were extracted from an aliquot of the cytoplasmic fractions, while the remaining fraction was analyzed through sucrose gradient to perform polysome profiling. RNAs were then extracted from the polysomal pooled fractions. The extracted RNAs (from cytoplasmic and polysomal pooled fractions) were submitted to deep sequencing (workflow shown in FIG 4A).
  • results indicate a decrease of protein synthesis in N6L-treated cells and agree with the decrease in the mPDAC translation capacity upon N6L treatment.
  • results from the deep sequencing of cytoplasmic and polysomal fractions were analyzed (FIG. 5A-FIG. 5F).
  • the RNA-sequencing analysis using the package DeSeq2 showed that among the 14,384 mRNAs present in mPDAC, 39% (5,610) were significantly expressed on both non-treated cells and N6L-treated cells (p- adj ⁇ 0.05) (FIG. 5A and FIG. 5B).
  • TI translational index
  • Gene Ontology analysis was performed using the functional annotation clustering analytic modules of EnrichR bioinformatics resources that provides a rank classification of enriched functions based on determination of P-v alues and enrichment scores (Kuleshov et al. 2016; Kuleshov et al. 2019).
  • Gene Ontology analysis revealed functional pathways corresponding to cell cycle, Fanconi anemia pathway and RNA transport for the 673 mRNAs translationally repressed (FIG. 5E) and functional pathways corresponding to Ribosome/translation, Alzeimer’s disease, and oxidative phosphorylation for the 213 mRNAs translationally up-regulated (FIG. 5F).
  • N6L induces a global decrease of mRNA translation correlated with a decrease of EiF3 mRNAs translation.
  • FIG. 5E The expression of the mRNAs coding for essential proteins involved in translation regulation that were subjected themselves to a translational regulation as revealed by our Functional Gene Ontology analysis was examined (FIG. 5E).
  • FIG.2E shows several mRNA encode for protein involved in translation initiation (FIG.2E).
  • EiF3e-j mRNA encoding the other members
  • FIG. 7A Using Western blotting analysis, EiF3a and EiF3c protein levels were significantly reduced (FIG. 7B and FIG. 7C) while the protein level of the other EiF3 members (EiF3e, EiF3h, and EiF3k) did not change (FIG. 7D and FIG. 7E) upon N6L treatment.
  • Ribosomal protein transcripts are characterized by the presence of a specific motif on their 5’UTR called 5 terminal oligopyrimidine (TOP) sequence immediately adjacent to the cap structure (Meyuhas et Kahan 2015). This family of TOP mRNAs encode proteins involved in translation initiation, elongation, and termination.
  • TOP 5 terminal oligopyrimidine
  • NCL targeting by N6L promotes a selective recruitment to polysomes of some mRNAs bearing 5’ TOP motif in their 5’UTR. This recruitment maintains translation as seen for RPL36 and RPS3 using wester-blotting analysis (FIG. 8D) despite the decreased accumulation of these mRNAs upon N6L treatment.
  • the 5’TOP motif of Rpl36 and Rps3 mRNAs were inserted upstream of firefly gene reporters and downstream a CMV promoter (FIG. 8E). These plasmids, pFL-5’TOP, were co-transfected with pRL (for normalization) in mPDAC cells treated with increasing doses of N6L. After 24 hours, the luciferase activity was quantified to determine if the 5’UTR was required for the increased translation of the chimeric mRNA.
  • N6L treatment induced a strong phosphorylation of RPS6, 4EBP1 (y and b forms), and ART, as compared to non-treated mPDAC cells (FIG. 10A; lanes 1-2) while expression of NCL protein is decreased. Whether NCL expression modulates the mTOR pathway was examined. Indeed, the activation of mTOR, via the hyper-phosphorylation of RPS6, ART and 4EBP1, was also observed when NCL was down regulated by siRNA (FIG. 10A, lanes 3-4). In addition, the phosphorylation of RPS6 and ART occurred in a N6L dose- dependent manner as revealed by Western blotting (FIG. 10B) and immunofluorescence analysis of phosphorylated RPS6 (FIG. IOC).
  • mTORi Two classes of mTORi were examined, allosteric mTORCl inhibitor Rapamycin (RAPA, Sirolimus) and ATP-competitive mTOR kinase inhibitors AZD2014 (Vistusertib) and INK128 (Sapanisertib), which inhibit both mTORCl and mTORC2 (Pike et al. 2013)(Hsieh et al. 2012).
  • PDX patient-derived xenograft
  • PDX models faithfully reproduce the molecular features of PDAC human tumors, display a high predictive value for clinical efficacy in patients, and are critically needed for the development of new treatments.
  • mTORi alone have no or modest effects on PDX cell growth at the tested concentrations (FIG. 9D-9F), except for INK128 at high concentration of 100 nM, the combination of RAPA, AZD2014 or INK128 with N6L was superior to single agent treatment (FIG. 11D-FIG. 11F) and this effect on PDX growth inhibition was synergistic (FIG. 11G, lower panels).
  • NCL nucleolin
  • N6L nucleolin
  • the nucleolin aptamer N6L shows antitumor effects on breast cancer_(Destouches et al. 2011), glioblastoma (Benedetti et al. 2015)(Dhez et al. 2018), non-small cell lung carcinoma (Ramos et al. 2020), and PDAC (Gilles et al. 2016; Sanhaji et al. 2019).
  • the 5’TOP motif begins with a C nucleotide directly adjacent to the cap structure followed by a series of approximately 4-515 pyrimidines often followed by a G-rich region (Meyuhas, O. & Kahan, T 2015).
  • the 5’ TOP motif is highly conserved and is found in all human ribosomal proteins as well as non-ribosomal proteins involved in translation (Yamashita, R. et al. 2008 and Levy, S., et al. 1991).
  • the shared TOP motif allows cells to quickly modulated the expression of proteins involved in ribosome production and protein synthesis in response to changes in cellular homeostasis. It was hypothesized that the cells activate the translation of these 5’ TOP mRNAs to counteract the negative effect of N6L on cell survival. Mechanistically, the translation of these 5’UTR motifs is regulated by the mTOR pathway.
  • the mTOR pathway is crucial to control cell growth and survival, in physiological as well as in pathological conditions. It acts in two distinct mTOR complexes, mTORCl and mTORC2, which differ in associated proteins and by sensitivity to rapamycin and its derivatives (Sabatini 2006).
  • the major function of mTORCl is the promotion of cellular growth and proliferation via increasing protein synthesis and inhibition of autophagy.
  • Functions of mTORC2 are less well studied and include organization of the actin cytoskeleton, control of cellular metabolism, and anti-apoptotic properties via stimulation of the AKT- FOXO pathway.
  • mTORCl enhance 5 'TOP mRNA translation by two mechanisms: phosphorylation of 4EBP1 and/or LARPl (Thoreen, C. C. et al. 2012, Fonseca, B. D., et al. 2018, and Hsieh, A. C. et al. 2012).
  • 4EBP1 is phosphorylated, which prevents 4EBP1 from disrupting the interaction of eIF4E and the mRNA cap, and has been shown to selectively enhance 5'TOP mRNA translation (Thoreen, C. C. et al. 2012 and Hsieh, A. C. et al. 2012).
  • the role of LARPl in the regulation of TOP mRNAs is, however, controversial. While LARPl was shown to regulate TOP mRNA stability, it has also been described as a positive or negative regulator of TOP mRNA translation, depending on the context and the energy status of the cells (Al-Ashtal, H. A., et al. 2021).
  • LARPl interacts with raptor and is phosphorylated by mTORCl, which is thought to modulate its mRNA-binding activity (Philippe, L., et al. 2020 and Hong, S. et al. 2017).
  • mTORCl phosphorylated by 4EBP1, RPS6 and ART
  • LARPl accumulation of LARPl upon N6L treatment enhanced the 5’TOP mRNA translation, thereby demonstrating that the mTOR pathway is activated and that LARPl could act as a positive regulator.
  • the mTOR pathway is reported to be aberrantly active in several cancers including PDAC (Tian, T., et al. 2019 and Bellizzi, A. M., 2010) in part due to mutations in upstream regulatory molecules, including PTEN, ART and TSCl/2. Most PDAC cancers have RAS mutations leading to activation of the MEK/ERK pathway, which can inactivate TSCl/2, thereby activating TORC1. Therefore, mTOR is a relevant target for the treatment of PDAC (Tian, T., et al. 2019).
  • mTOR inhibitors such as rapamycin, its rapalogs and dual mTORCl/mTORC2 inhibitors — have been examined in various cancer models, including PDAC (Tian, T., et al. 2019 and Hassan, Z. et al. 2018).
  • PDAC Potassium-Coupled Device
  • mTOR inhibitors utilized as monotherapy in clinical trials however, have not shown significant effects and sometimes are dampened by several resistance mechanisms (Guri, Y. & Hall, M. N. 2016).
  • Combined therapies with mTOR inhibitors and other pathway inhibitors or conventional therapies are under investigation in preclinical and clinical trials in different tumor types (Clinical trials. clinical trials.
  • NCL targeting by N6L enhanced the mTOR pathway inducing a reprogramming of the translatome by an increase of specific mRNAs (including 5’TOP mRNAs) in the polysomes, while the translation of most of the mRNAs is strongly down regulated.
  • This specific recruitment of mRNAs coding for ribosomal proteins and for factors required for the regulation of translation could be involved in mechanisms put in place by the cell in reaction to N6L to try to better resist the inhibitory effects of the drugs.
  • Ncl mRNA does not change upon N6L treatment as previously reported in mice PDAC tumor treated with N6L (Gilles, Maione, Cossutta, Carpentier, Caruana, Di Maria, et al. 2016). 2016). Unlike here where RNA was extracted from one cell type (mPDAC), RNA was extracted from mice PDAC treated tumors, which consisted of multiple cell types. However, the level of NCL protein decreases upon N6L treatment. NCL may self-regulate its own transcription and/or translation.
  • N6L affects the synthesis of NCL protein.
  • the consequences of the NCL down- regulation are the disorganization of the nucleolar structure as shown by electronic microscopy images of the mPDAC nucleolus ultrastructure and a decrease of rDNA transcription by decreasing the expression of the 47S pre-ribosomal RNA.
  • NCL is bound to pre-ribosomal RNA, but it is not found in mature cytoplasmic ribosomes suggesting that NCL is only involved in early steps of ribosome biogenesis.
  • NCL plays many functions in ribosome biogenesis, from rDNA chromatin structure, RNA polymerase I transcription regulation to the maturation of pre-rRNA and assembly of pre-ribosomes (for review see (Ugrinova et al. 2018)).
  • the data here demonstrates that N6L affects the nucleolar main function of NCL probably very important to explain vital function of NCL for cell proliferation (Ugrinova et al. 2007).
  • NCL down-regulation by N6L induces a decrease of protein synthesis and global translation rate in PDAC cells.
  • This result highlights another important cytoplasmic function of nucleolin, although the cytoplasmic fraction of NCL probably does not represent more than a few % of the total protein.
  • NCL binds and regulates mRNA involved in cell proliferation and apoptosis, such as BCL-2 (Sengupta et al. 2004), AKT (Abdelmohsen et al. 2011), or TP53 mRNA (Chen, Guo, et Kastan 2012) thanks to its RNA binding properties.
  • NCL By regulating their stability and translation rate, NCL possesses both anti-apoptotic and proto-oncogenic properties. Indeed, it was found that down-regulation ofNCL by N6L induces a decrease of PD AC cell proliferation and an increase of apoptosis, as previously demonstrated.
  • NCL targeting by N6L induces a translational reprogramming revealing that a subset of genes remains unexpectedly translationally active when global translation is inhibited. These genes are implicated in translation machinery and cell metabolism. It is believed that the cells activate this subset of genes to counteract the negative effect of N6L on cell survival, establishing a cell resistant mechanism to this N6L treatment.
  • the strategy adopted by a subset of these genes to counteract the energy-stress-mediated inhibition involves their translational control by the 5’ TOP. The majority of these are well-expressed, supporting a hypothesis whereby TOP motifs permit rapid and reversible regulation of these mRNAs without the metabolic cost of degrading and resynthesizing the transcripts.
  • the 5’TOP motif is defined as a +1 C directly adjacent to the 5' cap structure and followed by a series of 4 to 14 pyrimidine nucleotides, which renders translation of the mRNA hypersensitive to a variety of growth signals, including those transmitted by the mTORCl pathway (Meyuhas et Kahan 2015). How mTOR regulates TOP mRNA translation has been a persistent mystery.
  • LARPl interacts with raptor and is phosphorylated by mTORCl, which is thought to modulate its mRNA-binding activity (Hong et al. 2017; Philippe et al. 2020).
  • mTORCl phosphorylated by mTORCl
  • LARPl could act as positive regulator (interaction of LARPl with other protein/complex in PD AC cells).
  • the translation of these 5’UTR motifs is under the mTOR pathway.
  • the mTOR pathway is crucial to control cell growth and survival in physiological and pathological conditions. It acts in two distinct mTOR complexes, mTORCl and mTORC2, which differ in associated proteins and by sensitivity to rapamycin and its derivatives (Sabatini 2006).
  • the major function of mTORCl is promotion of cellular growth and proliferation via increasing protein synthesis and inhibiting autophagy. Functions of mTORC2 are less well studied and include organization of the actin cytoskeleton, control of cellular metabolism, and anti-apoptotic properties via stimulation of the AKT-FOXO pathway.
  • the mTOR pathway is reported to be significantly active in several cancers including PDAC (Tian, Li, et Zhang 2019; Bellizzi et al. 2010).
  • mTOR is implicated in drug resistance (Guri et Hall 2016).
  • NCL targeting by N6L induced an activation of the mTOR pathway in the data above, allowing PDAC cells to escape N6L treatment.
  • mTOR inhibitors were examined in combination with N6L in different preclinical models of human and murine PDAC cells.
  • mTOR inhibitors should be considered as a valuable addition to chemotherapy or targeted cancer therapy, either as an option for relapsed patients or as a frontline combination therapy to prevent or delay the development of resistance due to sustained mTOR signaling (Rodrik-Outmezguine et al. 2016).
  • mTOR inhibitors utilized as monotherapy in cancer are sometimes dampened by several resistance mechanisms.
  • combined therapies with mTOR inhibitors and other pathway inhibitors or conventional therapies are under investigation in preclinical and clinical trials in different tumor types. Hence, novel therapeutic strategies based on mTOR inhibition still need to be developed to treat PDAC cancers.
  • the nucleolin antagonist N6L (NUCANT®) was obtained from UREKA Pharma-ImmuPharma compagny.
  • PDAC cell lines and RNAi transfection were maintained in DMEM medium and HeLa cells were maintained in aMEM medium containing Glutamax (PAA) 1% non-essential amino acids, supplemented with 10% FBS and 1% Penicillin/Streptomycin.
  • PDAC PDX cell lines were purchased from CTIBIOTECH and maintained in CTIM.Cancer2 medium. All cells were incubated at 37°C in a humidified incubator with 5% C02. Routine Mycoplasma testing was performed by MycoAlert Mycoplasma Detection Kit (catalog no. LT07-118).
  • siRNAs (Eurogentec) specific for nucleolin was used as previously described (Kumar et al. 2017).
  • siRNA control we used stealth high GC siRNA (Invitrogen). Cells were transfected in a 6-well dish using siRNA at 2 nM final concentration.
  • siRNAs were diluted in 200 m ⁇ of OptiMEM (Gibco) and plated in a well.
  • PD AC cell lines were cultured in 96-well ultra -low attachment (ULA) plate and allowed to form spheroid overnight. Next day, PD AC spheroids were treated or not treated with different concentration of N6L alone or in combination with mTORi, RAPA and INK128 in 4 replicates. Spheroid growth was monitored by IncuCyte ZOOM® live-cell imaging. Over 96h, the spheroid area was calculated using ImageJ software and data were normalized to control.
  • mPDAC cells (lxlO 3 cells/mouse in 50 pL PBS) were orthotopically injected into the pancrease of FVB/n syngeneic mice, as previously described (Gilles, M.-E., et al. 2016). Mice were sacrificed after 4 weeks and tumor-derived organoids were established by following Tuveson’s laboratory protocol (Boj, S. F. et al. 2015). Briefly, tumors were digested with 0.012% (w/v) collagenase XI (Sigma) and 0.012% (w/v) dispase (Gibco) in DMEM media containing 1% FBS. The resulting cell suspension was incorporated into growth factor-reduced Matrigel (Coming) to obtain a 3D culture.
  • MTS Viability assay
  • apoptosis analysis Cell viability was assessed by MTS assay (CellTiter 96 AQueous MTS Reagent, Promega). Briefly, cell medium was supplemented with MTS reagent 20 m ⁇ /well, incubated for 2 h, and then the absorbance at 450nm was recorded on TECAN microplate Reader (Sunrise®). Viability for treated cells was normalized to non-treated cells on the same plate.
  • Apoptosis analysis was performed on the IncuCyte ZOOM® with Caspase-3/7 Green Reagent for Apoptosis (Essen BioScience). Cells were treated or not treated with increasing concentrations of N6L in 96-well plates in triplicate and Caspase-3/7 reagent reagent was added to the cells. Phase-contrast and fluorescent images were acquired 72 hours later. IncuCyte ZOOM® live-imaging software was used to count the fluorescent object number per mm 3 in each well which reflects the caspase 3/7 activation. Staurosporine (ImM) was used as a positive control for inducing apoptosis.
  • ImM Staurosporine
  • mPDAC cells were seeded onto cover slips in 24-well plate (BD Falcon) to adhere overnight. The next day, cells were treated or not treated with the increasing concentrations of N6L. After 48 hours of treatment, cells were rinsed with cold PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature followed by permeabilization with 0.1% Triton X-100. The cells were subjected to immunofluorescence staining with phospho-RPS6 (CST) for 2 hours at room temperature.
  • CST phospho-RPS6
  • Ultrathin sections (approximately 70 nm thick) were cut on a ultracut UC7 (Leica) ultramicrotome, mounted on 200 mesh copper grids coated with 1:1,000 polylisine, and stabilized for lday at room temperature (RT) and, contrasted with uranyl acetate. Sections were examined with a Jeol 1400JEM (Tokyo, Japan) transmission electron microscope equipped with a Orius 600 camera and Digital Micrograph.
  • Jeol 1400JEM Tokyo, Japan
  • Dual-Luciferase reporter assay for 5’TOP constructs NCL-depleted cells (3000/well) or N6L-treated cells or not (2000/well) were seeded in 96-well plates over 24 hours.
  • 5’TOP activity assay cells were co-transfected with 25 ng of monocistronic reporter containing 5’ TOP motif of Rps3 , Rpl36 or Eif3a genes (GenScript) and 25 ng of pRL construct constitutively expressing the Renilla luciferase gene as an internal control for transfection efficiency. Luciferase assays were performed 2 hours after transfection with the reporter plasmids using X-tremeGENE 9 reagent (Roche).
  • Dual luciferase assays were performed using the Dual-Glo luciferase reagent (Promega) according to the manufacturer’s instructions, and a Tecan Ml 000 plate reader. The relative luciferase activity was calculated by firefly luciferase activity I Renilla luciferase activity.
  • SUnSET for Global protein synthesis Global protein synthesis analysis by puromycylation followed by puromycin detection was performed as previously described (David et al. 2012). Briefly, puromycin (1 pg/mL) was added to the N6L treated or non-treated cell medium for 2 hours at 37°C. Cells were harvested, lysed in Laemmli buffer and loaded onto polyacrylamide gel. Puromycin incorporation was detected by Western blot on whole-cell protein extracts.
  • Cells were homogenized using Precellys® Evolution homogenizer (OZYME) and successively centrifuged at 700g and 1200g to eliminate the nuclei and the mitochondria, respectively.
  • Cytoplasmic lysates (1-2 mg of protein) were loaded onto 15%-47% sucrose density gradients and centrifuged at 217,000 g (SW 40 Ti rotor, Beckman Coulter, Inc.) for 2 hours at 4°C. Gradients were fractionated into 19 fractions and the OD at 254 nm was continuously recorded using an ISCO fractionator (Teledyne ISCO).
  • RNA from cytosolic and polysomal pooled fractions was extracted and purified using TRIzol reagent (Invitrogen).
  • TRIzol reagent Invitrogen
  • cne hundred nanograms of total RNA was reverse-transcribed using hexamer random primers and first-strand cDNA synthesis kit (Fermentas) and the synthesized cDNA was used for RT-QPCR using FastStart Universal SYBR Green Master (ROX) (Roche).
  • the relative quantities of the ingredients may be varied to optimize the desired effects, additional ingredients may be added, and/or similar ingredients may be substituted for one or more of the ingredients described. Additional advantageous features and functionalities associated with the systems, methods, and processes of the present disclosure will be apparent from the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.
  • Nucleolin Antagonist Triggers Autophagic Cell Death in Human Glioblastoma Primary Cells and Decreased in Vivo Tumor Growth in Orthotopic Brain Tumor Model. Oncotarget 6 (39).
  • LARPl Functions as a Molecular Switch for MTORC1 -Mediated Translation of an Essential Class of MRNAs. ELife 6: e25237.

Abstract

A method of treating cancer (e.g., pancreatic cancer) in a subject is disclosed. The method includes administering to a cell or a subject in need thereof an effective amount of a mechanistic target of rapamycin inhibitor and an effective amount of a polyvalent synthetic compound, wherein the mTOR inhibitor and the polyvalent synthetic compound are effective for treating or ameliorating at least one symptom of the cancer, wherein the polyvalent synthetic compound includes a support comprising at least 3 pseudopeptides coupled or grafted thereto, wherein the support is a linear peptide support having the formula (la) or (lb), and each pseudopeptide independently has formula (Ila) or formula (IIb).

Description

COMBINATION THERAPY OF NUCANT AND mTOR INHIBITORS
FOR TREATING CANCER
CROSS-REFERENCE TO RELATED APPLCIATIONS
[0001] The present disclosure claims priority to and the benefit of U.S. Provisional Patent Application No. 63/176,647, filed 19 April 2021, and U.S. Provisional Patent Application No. 63/211,189, filed 16 June 2021, each titled COMBINATION THERAPY OF NUCANT AND mTOR INHIBITORS FOR TREATING CANCER, and both of which are incorporated herein by reference in their entirety for all purposes.
INCORPORATION BY REFERENCE
[0002] In compliance with 37 C.F.R. § 1.52(e)(5), the sequence information contained in electronic file name: URZ0024PCT_Sequence_Listing_13APR2022_ST25.txt; size 11.9 KB; created on: 13 April 2022; using Patent-In 3.5, and Checker 4.4.0 is hereby incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The description relates to a method or treating cancer, such as pancreatic cancer, with a combination treatment with at least one mechanistic target of rapamycin (mTOR) inhibitor and at least one polyvalent synthetic compound.
BACKGROUND
[0004] Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease, for which mortality closely parallels incidence with 5-year survival rate less than 6% (Kamisawa et al. 2016). It is predicted to be the second leading cause of cancer-related death by 2030 (Soreide et al. 2019). The biology of PDAC contributes to early recurrence and metastasis, resistance to chemotherapy and radiotherapy, because of its complexity at the genomic, epigenetic and metabolic levels, with multiple activated biological pathways and crosstalk. Although there are four major driver genes identified in PDAC including one oncogene, KRAS and three tumor suppressor genes such as CDKN2A (encoding pi 6), TP 53 and SMAD4, none of these genes have currently provided clues to treat the disease.
[0005] Currently, a cure can only be achieved through resection. However, for many patients with advanced disease, surgery is not an option. The FOLFIRNOX (FOLinic acid or leucovorin, Fluorouracil, IRinotecan, and OXaliplatin) and Gemcitabine/NAB-paclitaxel (Nanoparticle Albumin-Bound paclitaxel) are the neoadjuvant treatments for patients who are not surgical candidates, but have a good performance status (Bachet et al. 2017; Gunturu et al. 2013). Indeed, therapeutic options are limited and discovering effective drug therapies for affected patients is of paramount importance.
[0006] Mechanistic target of rapamycin (mTOR), or FK506-binding protein 12- rapamycin-associated protein (FRAP) is a kinase that is a member of the phosphatidylinositol 3 -kinase-related kinase family and is encoded by the MTOR gene in humans. mTOR interacts with other proteins to form the mTOR complex 1 (mTORCl) and mTOR complex 2 (mTORC2), which regulate different cellular processes, including cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy, and transcription. mTOR inhibitors inhibit the mTOR serine/threonine protein kinase, and is comprised of two families of inhibitors. The first generation of mTOR inhibitors are allosteric mTORCl inhibitors and include inhibitors, such as, rapamycin (SIROLIMUS), CCI-779 (TEMSIROLIMUS), RAD001 (EVEROLIMUS), AP-23573 (RID AF OROLIMU S), UMIROLIMUS, and ZOTAROLIMUS. The second generation of MTOR inhibitors are ATP- competitive mTOR kinase inhibitors that inhibit both mTORCl and mTORC2, and includes inhibitors, such as, AZD2014 (VISTUSERTIB), INK 128 (SAPANISERTIB), and AZD8055. [0007] Nucleolin (NCL) is an RNA- and protein-binding multifunctional protein that has become the focus of interest in the cancer biology field in recent years for several reasons. First, NCL expression seems to correlate with the proliferation rate of the cells with an overexpression in several cancer types, including hepatocellular carcinoma (Guo et al. 2014), acute myeloid leukemia (Marcel et al. 2017), non-small-cell lung cancer (Zhao et al. 2013), gastric cancer (Qiu et al. 2013), and PDAC (Gilles et al. 2016). Second, in addition to its nucleolar, nucleoplasmic and cytoplasmic localizations, NCL is present on the surface of many cell types, and this extracellular form of NCL is a hallmark of proliferative and cancer cells (Ugrinova et al. 2018; Hovanessian et al. 2000; Koutsioumpa et Papadimitriou 2014; Farin et al. 2011).
[0008] Indeed, NCL is a key protein for the regulation of several processes required for the proliferation and division of cells. In particular, it plays an important role in the coordination of cell metabolism with cell division and in the biogenesis of ribosomes which are required to sustain the higher level of translation in cells that have a high proliferation rate. The high expression level of NCL in cancer cells is related to high levels of protein synthesis. [0009] Several molecules were developed to target NCL on the cell surface of cancer cells. For example, W02007/125210 discloses polyvalent or multivalent synthetic peptides made of at least three particular pseudopeptide units grafted to a support. This family of compounds (which have been named Nucant compounds), has been shown to interact with surface nucleolin RGG domain and to have both anti -proliferative and anti-angiogenic properties and to be useful for the treatment of cancer or inflammatory diseases. They have a fairly broad spectrum of activity against angiogenic factors, a good solubility in aqueous media, improved resistance to in vivo breakdown processes (due to the presence of a modified bond in pseudopeptide units), showed very few side effects, and have a synthesis process that is easily adaptable to an industrial scale. Specific exemplified compounds include compound HB 19 and compounds Nucant 1, 2, 3, 6 and 7. This document is incorporated herein by reference in its entirety.
[0010] WO2009/141687 discloses improved Nucant compounds, in which lysine residues in the pseudopeptide units are all in the same L or D configuration. This document notably describes compound Nucant 6L (N6L), which corresponds to compound Nucant 6 as disclosed in W02007/125210, in which all lysine residues of the pseudopeptide units are in L configuration. This compound showed improve anti-cancer activity compared to complex compound Nucant 6 in which the lysine residue of each pseudopeptide unit may be in L or D configuration. The compounds were also shown to improve wound healing. This document is incorporated by reference in its entirety. WO2009/141687 also discloses compounds Nucant 4, 8 and 9.
[0011] As discussed above, the pseudo-peptides HB19 and N6L were shown to interact withNCL and to inhibitthe proliferation of cancer cells (Krust et al. 2011; Damien Destouches et al. 2008). In addition, N6L as well as a NCL-blocking antibody impairs both in vitro and in vivo angiogenesis by targeting ECs and tumor vessels (Destouches et al. 2011; Birmpas et al. 2012). Many studies reported the antitumor activities of N6L in several cancer types including, breast cancer (Destouches et al. 2011), glioblastoma (Benedetti et al. 2015)(Dhez et al. 2018), non-small cell lung carcinoma (Ramos et al. 2020), and PDAC (Gilles et al. 2016; Sanhaji et al. 2019). Interestingly in PDAC, NCL targeting by N6L blocks both tumor progression and normalizes tumor vasculature, improving the delivery and efficacy of chemotherapeutic drug (Gilles et al. 2016).
[0012] WO 2012/045750 discloses compositions comprising a mixture of Nucant multivalent synthetic compound and glycosaminoglycan (GAGs) forming microspheres. The GAGs mediated and/or enhances the beneficial therapeutic activity of the Nucant compounds within the composition. This document is incorporate herein by reference in its entirety. [0013] Pancreatic cancer is an aggressive disease characterized by high invasiveness, rapid progression, and resistance to conventional therapy. There is an urgent need to identify new molecules to improve current cancer therapies, including pancreatic cancer therapies, with better efficacy and less toxicity. Thus, there is an urgent need for improved therapies, with better efficacy and decreased toxicity, for the treatment of cancer, such as pancreatic cancer.
SUMMARY
[0014] The present disclosure describes the surprising and unexpected discovery that the coadministration of a mechanistic target of rapamycin (mTOR) inhibitor and a polyvalent synthetic compound of the present disclosure have a synergistic effect in inhibiting cancer cell growth and cancer cell viability.
[0015] As such, an aspect the disclosure provides a method of treating cancer (e.g., pancreatic cancer) in a subject, the method comprising administering or co-administering to a cell or a subject in need thereof an effective amount of at least one (e.g., 1, 2, 3, or 4) mTOR inhibitor and an effective amount of at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound, wherein the mTOR inhibitor and the polyvalent synthetic compound are effective for treating or ameliorating at least one symptom of the cancer (e.g., pancreatic cancer), wherein the polyvalent synthetic compound comprises a support comprising at least 3 pseudopeptides coupled or grafted thereto, wherein the support is a linear peptide support having the formula (la) or (lb):
(Lys-Aib-Gly)z or (SEQ ID No. l)z (la), or (Aib-Lys-Aib-Gly)z or (SEQ ID No. 2)z (lb), wherein: z is an integer from 3 to 20; and each pseudopeptide independently has formula (Ila) or formula (lib):
[(X)n — Y1 — ¾ — Y2 — (X)m] (Ha or SEQ ID No. 3 ), or
Figure imgf000006_0001
(lib or SEQ ID No. 4), wherein: each X is independently any amino acid (e.g. any proteinogenic amino acid);
Z is proline or derivative thereof; each Y1 and Y2 is independently a basic amino acid (e.g., arginine, lysine, lysine derivative, or lysine analog); n is 0 or 1; m is 0, 1, 2, or 3; and
Y represents a reduced bond (-CH2NH-), a retro-inverso bond (-NHCO-), a methyleneoxy bond (-CH2O-), a thiomethylene bond (-CH2-S-), a carba bond (-CH2CH2-), a ketomethylene bond (-CO-CH2-), a hydroxyethylene bond (-CHOH-CH2-), an E-alkylene bond , or a C=C bond, or a pharmaceutically acceptable salt or pharmaceutically acceptable derivative thereof. [0016] In any aspect or embodiment described herein, the polyvalent synthetic compound is present/administered in a composition that includes a pharmaceutically acceptable carrier, excipient, or diluent.
[0017] In any aspect or embodiment described herein, the support has the formula (la). [0018] In any aspect or embodiment described herein, n and m are each 0.
[0019] In any aspect or embodiment described herein, z is an integer from 3 to 10.
[0020] In any aspect or embodiment described herein, z is 6.
[0021] In any aspect or embodiment described herein, the polyvalent synthetic compound comprises 3 to 15 pseudopeptides.
[0022] In any aspect or embodiment described herein, one or more of the pseudopeptides (e.g., each/all of the pseudopeptides) is coupled or grafted directly on the support.
[0023] In any aspect or embodiment described herein, the pseudopeptides are grafted or coupled to the support via a lysine of the support.
[0024] In any aspect or embodiment described herein, the pseudopeptides are optically pure pseudopeptides.
[0025] In any aspect or embodiment described herein, the Y1 and the Y2 have an L configuration or D configuration.
[0026] In any aspect or embodiment described herein, the Y1 and the Y2 have an L configuration.
[0027] In any aspect or embodiment described herein, each Y1 and Y2 is independently lysine or arginine.
[0028] In any aspect or embodiment described herein, each Y1 and Y2 is a lysine having an L configuration.
[0029] In any aspect or embodiment described herein, each Y1 and Y2 is independently selected from ornithine, homolysine, and diaminoheptanoic acid (e.g., 2,7-diaminoheptanoic acid).
[0030] In any aspect or embodiment described herein, the Y represents a reduced bond. [0031] In any aspect or embodiment described herein, the m is 0 or 1.
[0032] In any aspect or embodiment described herein, one or more X is a proteinogenic amino acid.
[0033] In any aspect or embodiment described herein, the polyvalent synthetic compound has the structure:
Figure imgf000008_0001
[0034] In any aspect or embodiment described herein, the mTOR inhibitor is an allosteric mTOR inhibitor (such as, one or more (e.g., 1, 2, 3, or 4) of rapamycin (SIROLIMUS), CCI- 779 (TEMSIROLIMUS), RAD001 (EVEROLIMUS), AP-23573 (RID AF OROLIMU S), UMIROLIMUS, and ZOTAROLIMUS).
[0035] In any aspect or embodiment described herein, the mTOR inhibitor is an ATP- competitive mTOR inhibitor (such as, one or more (e.g., 1, 2, 3, or 4) of AZD2014 (VISTUSERTIB), INK128 (SAPANISERTIB), and AZD8055).
[0036] In any aspect or embodiment described herein, the mTOR inhibitor is an mTORCl inhibitor, such as an allosteric mTORCl inhibitor or an ATP -competitive mTOR kinase inhibitor.
[0037] In any aspect or embodiment described herein, the mTOR inhibitor is an mTORC2 inhibitor, such as an ATP-competitive mTOR kinase inhibitor.
[0038] In any aspect or embodiment described herein, the mTOR inhibitor is selected from Rapamycin, AZD2014, and INK 128.
[0039] In any aspect or embodiment described herein, the cancer is pancreatic cancer.
[0040] In any aspect or embodiment described herein, the cancer or the pancreatic cancer is pancreatic ductile adenocarcinoma.
[0041] In any aspect or embodiment described herein, the cancer or the pancreatic cancer is pancreatic ductile adenocarcinoma, pancreatic adenocarcinoma, pancreatic squamous cell carcinoma, pancreatic adenosquamous carcinoma, pancreatic colloid carcinoma, preferably pancreatic ductile adenocarcinoma.
[0042] In any aspect or embodiment described herein, the polyvalent synthetic compound and the mTOR inhibitor have a synergistic inhibitory effect on the pancreatic cancer cell growth and/or cell viability.
[0043] The preceding general areas of utility are given by way of example only and are not intended to be limiting on the scope of the present disclosure and appended claims. Additional objects and advantages associated with the compositions, methods, and processes of the present disclosure will be appreciated by one of ordinary skill in the art in light of the instant claims, description, and examples. For example, the various aspects and embodiments of the disclosure may be utilized in numerous combinations, all of which are expressly contemplated by the present description. These additional aspects and embodiments are expressly included within the scope of the present disclosure. The publications and other materials used herein to illuminate the background of the disclosure, and in particular cases, to provide additional details respecting the practice, are incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] The accompanying drawings, which are incorporated into and form a part of the specification, illustrate several embodiments of the present disclosure and, together with the description, serve to explain the principles of the disclosure. The drawings are only for the purpose of illustrating an embodiment of the disclosure and are not to be construed as limiting the disclosure. Further objects, features and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure, in which:
[0045] Figure 1. Structure of nucleolin protein. Human nucleolin consists of 707 amino acids. Nucleolin can be broken down into two main parts (Ginisty, H., Sicard, H., Roger, B., and Bouvet, P. Structure and functions of nucleolin. (1999) J. Cell Science 112, 761-772; Srivastava, M., and Pollard, H. B. Molecular dissection of nucleolin's role in growth and cell proliferation: new insights. (1999) FASEB J. 13, 1911-1922): N-terminal (aa 1-308), and C- terminal (aa 309-707). The N-terminal domain consists of 4 long acidic domains, consisting of an uninterrupted repetition of glutamic acid and aspartic acid (Al, A2, A3, A4). The C-terminal domain, consists of alternating hydrophobic and hydrophilic regions forming 4 RNA Binding Domains (RBD I, II, III, and IV), and its extremity (aa 644-707 or SEQ ID No. 5) carries the highly basic RGG domain comprised of Arg-Gly-Gly repetitions. [0046] Figures 2A and 2B. (A) Amino acid sequence of exemplary compound of the present disclosure Nucant 6L (N6L or SEQ ID No. 6). (B) Structure of exemplary compound of the present disclosure N6L.
[0047] Figures 3A, 3B, 3C, and 3D. NCL targeting by N6L decreases mPDAC cell proliferation by inducing apoptosis. mPDAC cells were seeded in 96-well plates and allowed to adhere overnight. Cells were treated with increasing concentrations of N6L. (A) mPDAC cell growth was assessed by time-lapse Incucyte® over 120 hours. (B) In parallel, mPDAC cell viability was determined by MTS assay periodically over 72 hours. (C) and (D) Cells were seeded in 96-well places, allowed to adhere overnight. Apoptosis induction assay upon increasing doses of N6L on mPDAC cells. Curves (C) and representative images (D) from the InCucyte ZOOM® live-imaging. Software counted the number of caspase-3/7-positive cells (green color) per surface (mm2).
[0048] Figures 4A, 4B, 4C, 4D, and 4E. NCL targeting by N6L impacts/ mPDAC cell translation. (A) Experimental design of translatome analysis procedure. mPDAC cells were treated (T) or not (NT) with 50 mM of N6L for 48 hours. Cytosolic lysates from cells were sedimented on sucrose gradient (15-47%). Following ultracentrifugation, 40S and 60S ribosome subunits, the 80S monosomes, and polysomes are separated. RNAs were extracted from polysome pooled fractions (mRNA-associated polysomes) and cytoplasmic fractions (cytoplasmic mRNA). The RNA quality and integrity were verified using a Bioanalyser 2100 before sequencing and bioinformatics analysis. (B) and (C) Polysome profiles of mPDAC cells non-treated (mPDAC NT in (B)) or treated with N6L (mPDAC T in (C)). Curves show absorbance at 254 nm as a function of sedimentation. (D) and (E) mPDAC cells, treated or not treated with N6L, were incubated with puromycin for the measurement of overall protein synthesis by SUnSET method and then subjected to western-blotting (D). Protein synthesis rates were quantified by measuring the signal intensity in each lane and normalizing the values to that of the control lane (E). w/o Puro, without puromycin. Data represented are the means ± SD of at least three experiments. *P<0.05; **P<0.01; ***P<0.001 and n.s. for not significant, as determined by Student t-test on final data point.
[0049] Figures 5A, 5B, 5C, 5D, 5E, and 5F.: Bioinformatics analysis of mPDAC cell translatome upon N6L treatment. (A) Percentage of translationally deregulated mRNAs in response to N6L treatment. Among the 14,384 blasted genes, 39% (5,610) were significantly expressed in both NT and T conditions (A). About 6% (886) of these 5,610 genes were significantly deregulated at translational levels (TI cut-off = 1.5, p < 0.05) (B). Among these 886 translationally deregulated genes, 24.04% were up-regulated and 75.96% were down- regulated (C). Distribution of the Translational Index (TI) calculated using the following formula TI=X1/X2, with XI and X2 expressing the ratio between polysomal-to-cytoplasmic fraction in mPDAC treated and non-treated cells, respectively (D) (complete gene list in Table 1, below). (D) and (E) Ontology analysis was determined using the Functional annotation clustering tools from EnrichR software. Translationally down-regulated genes are involved in cell cycle, Fanconi Anemia, and RNA transport (E). While translationally up-regulated genes are mainly involved in Ribosome/translation and metabolism (F).
[0050] Figures 6A, 6B, 6C, 6D, 6E, and 6F. NCL targeting by N6L dysregulates mPDAC cell translation. Bioinformatics analysis. (A) and (B) Venn diagram showing the number of transcriptionally up-regulated, down-regulated or stable genes per Translation Index (TI) DOWN (C) or TI UP (D) category obtained in FIG. 5 A- FIG. 5F. (C) and (D) GO terms for genes which are translationally down-regulated and transcriptionally up-regulated (n=340, (C)) or unchanged (n= 315, (D)). (E) and (F) GO terms for genes which are translationally up- regulated and transcriptionally down -regulated (n=131, (E)) or unchanged (n= 73, (F)).
[0051] Figures 7A, 7B, 7C, 7D, 7E, and 7F.: NCL targeting by N6L decreases the translation of mRNAs encoding the canonical subunits of EiF3 factor, Eif3a and Eif3c. (A) Translation Index (TI) for eukaryote Initiator Factors (elF) mRNA under N6L treatment. (B)- (E) mPDAC cells were treated (T) or not treated (NT) with N6L for 48 hours following which EiF3 subunits protein expression was analyzed using western-blotting ((B) and (D)) and quantified in relation to Actin ((C) and (E)). (F) Validation of relative eIF3a and eIF3c expression using RT-QPCR in mPDAC treated or not treated with N6L after normalization to actin housekeeping gene. (P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 and n.s. for not statistically significant, as determined by Student t-test on final data point.
[0052] Figures 8A, 8B, 8C, 8D, 8E, and 8F. NCL targeting by N6L increases 5’ TOP and 5’TISU mRNA translation by activating mTOR pathways. (A) TI for 5’ TOP-containing mRNA under N6L treatment (right). (B) Venn diagram comparing 5’ TOP-gene overlap between our TI UP mRNAs and 5’TOP gene lists from Gentilella et al. 2017. (C) Table 2 summarizes the distribution of 5’ TOP and non-5’TOP mRNAs according to their transcriptional expression (up, down or stable). (D) mPDAC cells were treated (T) or not treated (NT) with N6L for 48 hours following which RPL36 and RPS3 expression at protein level was analyzed using western-blotting (upper panel) and quantified in relation to H3 (lower panel). (E) Illustrates the design of 5’ TOP Firefly-luciferase reporter constructs, with the first C of the TOP motif as the +1 Transcription Start Site (TSS). (F) Fold change of Firefly luciferase activities under 5’TOP motifs of Rpl36 and Rps3 mRNAs upon 48 hours of increasing concentrations of N6L treatment in mPDAC, after normalization to mPDAC NT. *P<0.05; **P<0.01; ***P<0.001; ****p<0.0001 and n.s. for not statistically significant, as determined by Student t-test on final data point.
[0053] Figures 9A, 9B, 9C, 9D, 9E, and 9F. The impact of mTOR inhibitors treatment on mPDAC and PDX cell growth. (A)-(C) mPDAC cell lines were seeded in 96-well plates and allowed to adhere overnight. Cells were treated with 10 or 25nM of Rapamycin (RAPA, (A)), AZD2014 (AZD, (B)) or INK128 (INK, (C)) over 72 hours. (D)-(F), PDAC PDX (PAN014) cell lines were seeded in 96-well plates and allowed to adhere 2 days. Cells were treated with 1, 10 or lOOnM of RAPA (D), AZD (E) or INK (F) over 96 hours. Cell growth was assessed by time-lapse InCucyte ZOOM®. The AUC (Area Under Curves) was calculated by InCucyte ZOOM® software. Growth for treated cells was normalized to untreated cells on the same plate.
[0054] Figures 10A, 10B, IOC, and 10D. NCL targeting by N6L increases 5’ TOP mRNA translation by activating mTOR pathways. (A) mTOR pathway activation was analyzed using western-blotting in mPDAC cells, non-treated (NT) or treated (T) with N6L at 50mM for 48 hours (lanes 1,2) or after NCL siRNA depletion (lanes 3,4). g and b represent hyper- phosphorylated and a hypo-phosphorylated forms of 4EBP1. The phosphorylated-to-total protein ratio was quantified for RPS6, AKT and 4EBP1. (B) and (C) mTOR pathway activation was analyzed in mPDAC, non-treated (NT) or treated (T) with N6L at increasing doses using western-blotting (B) and Immunofluorescence staining (C) by anti-phosphorylated RPS6 (red) and DAPI for nuclei (blue). (D) Western-blotting analysis of LARPl, RPS6, 4EBP1 and EiF4E expression in mPDAC treated or not treated with N6L at 30 and 50mM for 48 hours.
[0055] Figures 11A, 11B, 11C, 11D, HE, 11F, 11G, 11H, 111, 11J, 11K, 11L, 11M, and 11N. N6L and mTORi combination acts synergistically to decrease PDAC cell and spheroid growth and organoid viability. (A)-(C) mPDAC cell lines were seeded in 96-well plates and allowed to adhere overnight. Cells were treated with 10 or 25nM of Rapamycin (RAPA, (A)), AZD2014 (AZD, (B)) or INK128 (INK, (C)) in combinations with N6L at 10mM over 72 hours. D-F, PDAC PDX (PAN014) cell lines were seeded in 96-well plates and allowed to adhere 2 days. Cells were treated with 1, 10 or lOOnM of RAPA (D), AZD (E) or INK (F) in combinations with N6L at 10mM over 96 hours. Cell growth was assessed by IncuCyte ZOOM® live-imaging. The AUC (Area Under Curves) was calculated by IncuCyte ZOOM® live-imaging software. Growth for treated cells was normalized to untreated cells on the same plate. (G) Synergy/antagonism calculation. The Contour views from LOEWE model were selected as graphical outputs for the synergy distribution. (I) and (J) Representative images of mPDAC (I) and PDAC PDX (J) spheroids non-treated (NT) or treated with N6L alone or combined with INK128 over 96 hours. (K) and (L), Spheroid area was calculated using ImageJ software and data were normalized to control for combinations of N6L and INK 128. (M) mPDAC organoid viability assessed with MTS assay upon treatment with INK 128 alone or in combination with N6L at 1 OmM and 30mM. (N) Contour view showing the synergy distribution from MTS assay on mPDAC organoid. Scale bar, 500 pm (#) if comparing each treatment to NT, (*) if comparing combination to single treatment. *P<0.05; **P<0.01; ***P<0.001; ****p<0 0001 and n.s. for not statistically significant, as determined by Student t-test on final data point.
[0056] Figures 12A, 12B, 12C, 12D, and 12E. Synergistic effects of N6L/mTORi combination on Pane cells. (A) Pane cell lines were seeded in 96-well plates and allowed to adhere overnight. Cells were treated with concentrations of N6L alone at 10, 30 or 50mM (A, upper left), Rapamycin (RAPA) alone at 25 or 50nM (A, upper middle), INK128 (INK) alone at 10 or 25nM (A, upper right) or in combinations (lower) over 72 hours. Cell growth was assessed by time-lapse Incucyte®. The AUC (Area Under Curves) was calculated by Incucyte® software. Growth for treated cells was normalized to untreated cells on the same plate. (B) Synergy/antagonism calculation. Additive and synergistic effects when using combinations of N6L with RAPA (left) or INK 128 (right) were determined using Combenefit software. The software calculates a synergy score for each combination, where a positive score indicates synergy, a score of 0 is additive, and a negative score indicates antagonism. The Contour (upper), Surface (middle) and Matrix (lower) views from LOEWE model were selected as graphical outputs for the synergy distribution. (C) Representative images of Pane untreated (NT) or treated with N6L alone or combined with RAPA (upper panels) or with INK128 (lower panels) at 48 hours after treatment. Scale bar, 50pm. (D) Representative images of Pane spheroids untreated (NT) or treated with N6L alone (upper panels) or combined with RAPA (middle panels) or with INK128 (lower panels) over 96 hours. € Pane spheroid area was calculated using ImageJ software and data were normalized to control for combinations of N6L and RAPA (upper graphs) or INK128 (lower graphs). (#) if comparing each treatment to NT, (*) if comparing combination to single treatment. *P<0.05; **P<0.01; ***P<0.001; ****p<0 0001 and n.s. for not statistically significant, as determined by Student t-test on final data point.
[0057] Figures 13A and 13B. N6L combined to mTORi treatment inactivate mTOR pathway by decreasing RPS6 and 4EBP1 phosphorylation. mTOR pathway inhibition was analyzed using western-blotting in mPDAC (A) and PDX PDAC (B) cells non-treated (NT) or treated with N6L, RAPA, INK 128 and AZD2014 alone or in combination (N6L+RAPA, N6L+INK, or N6L+AZD).
[0058] Figures 14A, 14B, 14C, 14D, and 14E. Synergistic effects of N6L/mTORi combination on MiaPaca cells. (A) MiaPaca cell lines were seeded in 96-well plates and allowed to adhere overnight. Cells were treated with concentrations of N6L alone at 10, 30 or 50mM (upper left), Rapamycin (RAPA) alone at 25 or 50nM (upper middle), INK128 (INK) alone at 10 or 25nM (upper right) or in combinations (lower) over 72 hours. Cell growth was assessed by time-lapse Incucyte®. The AUC (Area Under Curves) was calculated by Incucyte® software. Growth for treated cells was normalized to untreated cells on the same plate. (B) Synergy/antagonism calculation. Additive and synergistic effects when using combinations of N6L with RAPA (left) or INK 128 (right) were determined using Combenefit software. The software calculates a synergy score for each combination, where a positive score indicates synergy, a score of 0 is additive, and a negative score indicates antagonism. The Contour (upper), Surface (middle) and Matrix (B, lower) views from LOEWE model were selected as graphical outputs for the synergy distribution. (C) Representative images of MiaPaca untreated (NT) or treated with N6L alone or combined with RAPA (upper panels) or with INK128 (lower panels) at 48 hours after treatment. Scale bar, 50pm. (D) Representative images of MiaPaca spheroids untreated (NT) or treated with N6L alone (upper panels) or combined with RAPA (middle panels) or with INK128 (lower panels) over 96 hours. (E) MiaPaca spheroid area was calculated using ImageJ software and data were normalized to control for combinations of N6L and RAPA (upper graphs) or INK128 (lower graphs). (#) if comparing each treatment to NT, (*) if comparing combination to single treatment. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 and n.s. for not statistically significant, as determined by Student t-test on final data point.
DETAILED DESCRIPTION
[0059] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety. For example, U.S. Patent Application Publication No. 2011/0065649 Al, published 17 March 2011, U.S. Patent Application Publication No. 2011/0201559 Al, published 18 August 2011, and U.S. Patent Application Publication No. 2014/0080759 Al, published 20 March 2014, are each incorporated herein by reference in their entirety for all purposes. Furthermore, all references cited herein are incorporated by reference herein in their entirety for all purposes.
[0060] Presently described are methods that relate to the surprising and unexpected discovery that the use of a mechanistic target of rapamycin (mTOR) inhibitor and a polyvalent synthetic compound of the present disclosure in combination has a synergistic inhibitory effect on cancer cell growth and cancer cell viability.
[0061] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise (such as in the case of a group containing a number of carbon number of amino acids in which case each carbon number or amino acid number falling within the range is provided), between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the disclosure.
[0062] The following terms are used to describe the present disclosure. In instances where a term is not specifically defined herein, that term is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present disclosure. [0063] The articles "a" and "an" as used herein and in the appended claims are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article unless the context clearly indicates otherwise. By way of example, "an element" means one element or more than one element.
[0064] The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
[0065] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." [0066] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
[0067] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. [0068] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.
[0069] The terms "co-administration" and "co-administering" or “combination therapy” refer to both concurrent administration (administration of two or more therapeutic agents at the same time) and time varied administration (administration of one or more therapeutic agents at a time different from that of the administration of an additional therapeutic agent or agents), as long as the therapeutic agents are present in the patient to some extent, preferably at effective amounts, at the same time. In certain preferred aspects, one or more of the present compounds described herein, are co-administered at least one (e.g., 1, 2, 3, or 4) mTOR inhibitor and at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound. In particularly preferred aspects, the co-administration of at least one (e.g., 1, 2, 3, or 4) mTOR inhibitor and at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound results in synergistic activity and/or therapy, including anticancer activity, such as inhibiting cell growth and/or decreasing cell viability, or anti-angiogenic activity.
[0070] As used herein, the term "Nucant" can mean but is in no way limited to a nucleolin- binding compound or entity, e.g., nucleolin antagonist, including, for example, a nucleolin binding peptide or pseudopeptide, or a derivative or analog thereof (collectively "Nucant peptide") or polyvalent synthetic compound as described herein.
[0071] The term “compound”, as used herein, unless otherwise indicated, refers to any specific chemical compound disclosed herein, including the polyvalent synthetic compound described herein, and includes tautomers, regioisomers, geometric isomers, and where applicable, stereoisomers, including optical isomers (enantiomers) and other stereoisomers (diastereomers) thereof, as well as pharmaceutically acceptable salts and derivatives, including prodrug and/or deuterated forms thereof where applicable, in context. Deuterated compounds contemplated herein are those in which one or more of the hydrogen atoms contained in the drug molecule have been replaced by deuterium.
[0072] Within its use in context, the term compound generally refers to a single compound, but also may include other compounds such as stereoisomers, regioisomers and/or optical isomers (including racemic mixtures) as well as specific enantiomers or enantiomerically enriched mixtures of disclosed compounds. The term also refers, in context to prodrug forms of compounds which have been modified to facilitate the administration and delivery of compounds to a site of activity. It is noted that in describing the present compounds, numerous substituents and variables associated with same, among others, are described. It is understood by those of ordinary skill that molecules which are described herein are stable compounds as generally described hereunder. When the bond is shown, both a double bond and single bond are represented or understood within the context of the compound shown and well-known rules for valence interactions.
[0073] The term "derivatives", as used herein, are compositions formed from the native compounds either directly, by modification, or by partial substitution.
[0074] The term "analogs", as used herein, are compositions that have a structure similar to, but not identical to, the native compound.
[0075] The term "peptides" can mean, but is in no way limited to, recombinant polypeptide having at least 4 amino acids connected by peptide bonds. Furthermore, peptides of the present disclosure may include amino acid mimentics, and analogs. Recombinant forms of the peptides can be produced according to standard methods and protocols which are well known to those of skill in the art, including for example, expression of recombinant proteins in prokaryotic and/or eukaryotic cells followed by one or more isolation and purification steps, and/or chemically synthesizing peptides or portions thereof using a peptide synthesizer.
[0076] The term, "biologically active" or "bioactive", as used herein, can mean, but is in no way limited to, the ability of an agent, such as the compounds provided by the present disclosure, to effectuate a physiological change or response. The response may be detected, for example, at the cellular level, for example, as a change in growth and/or viability, gene expression, protein quantity, protein modification, protein activity, or combination thereof; at the tissue level; at the systemic level; or at the organism level. Techniques used to monitor these phenotypic changes include, for example, measuring: the binding of a ligand to its receptor in or on a cell, activation of cell signaling pathways, stimulation or activation of a cellular response, secretion or release of bioactive molecules from the cell, cellular proliferation and/or differentiation, or a combination thereof. In one example, the biological activity of a peptide provided by the present disclosure can be determined by detecting its ability to inhibit the growth and/or proliferation of a cell.
[0077] The term “patient” or “subject” is used throughout the specification to describe an animal, preferably a human or a domesticated animal, to whom treatment, including prophylactic treatment, with the compositions according to the present disclosure is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal, including a domesticated animal such as a dog or cat, or a farm animal such as a horse, cow, sheep, etc. In general, in the present disclosure, the term patient refers to a human patient unless otherwise stated or implied from the context of the use of the term.
[0078] The term “effective” is used to describe an amount of a compound, composition, or component which, when used within the context of its intended use, effects an intended result. The term effective subsumes all other effective amount or effective concentration terms, which are otherwise described or used in the present application.
[0079] The term "effective amount/dose," "pharmaceutically effective amount/dose," "pharmaceutically effective amount/dose" or "therapeutically effective amount/dose" can mean, but is in no way limited to, that amount/dose of the active pharmaceutical ingredient sufficient to prevent, inhibit the occurrence, ameliorate, delay or treat (alleviate a symptom to some extent, preferably all) the symptoms of a condition, disorder or disease state. The effective amount depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 1000 mg/kg body weight/day of active ingredients is administered dependent upon potency of the agent. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
[0080] The term “independently” is used herein to indicate that the variable, which is independently applied, varies independently from application to application.
[0081] The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the present disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
[0082] The term "pharmacological composition," "therapeutic composition," "therapeutic formulation" or "pharmaceutically acceptable formulation" can mean, but is in no way limited to, a composition or formulation that allows for the effective distribution of an agent provided by the present disclosure, which is in a form suitable for administration to the physical location most suitable for their desired activity, e.g., systemic administration.
[0083] Non-limiting examples of agents suitable for formulation with the, e.g., compounds of the present disclosure include: cinnamoyl, PEG, phospholipids or lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into various tissues, for example the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers, such as poly(DL-lactide-coglycolide) microspheres for sustained release delivery after implantation (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).
[0084] The term "pharmaceutically acceptable" or "pharmacologically acceptable" can mean, but is in no way limited to, entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. [0085] The term "pharmaceutically acceptable carrier" or "pharmacologically acceptable carrier" can mean, but is in no way limited to, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. [0086] The term "systemic administration" refers to a route of administration that is, e.g., enteral or parenteral, and results in the systemic distribution of an agent leading to systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect. Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compositions of the present disclosure can potentially localize the drug, for example, in certain tissue types, such as the tissues of the pancreas. A liposome formulation which can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful.
[0087] The term "any amino acid", as used herein, means any natural or synthetic amino acid, possibly modified by the presence of one or more substituents.
[0088] The term "conservative mutations" refers to the substitution, deletion or addition of nucleic acids that alter, add or delete a single amino acid or a small number of amino acids in a coding sequence where the nucleic acid alterations result in the substitution of a chemically similar amino acid. Amino acids that may serve as conservative substitutions for each other include the following: Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q); hydrophilic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Hydrophobic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C). In addition, sequences that differ by conservative variations are generally homologous. The term “non-conservative multations” refers to substitutions, deletions or addition of nucleic acids that alter, add or delete a single amino acid or a small number of amino acids in a coding sequence where the nucleic acid alterations results in the substitution of an amino acid that is not chemical similar — i.e., an alteration that does not result in a conservative mutation.
[0089] The term "binding", as used herein, can mean, but is in no way limited to, the physical or chemical interaction, direct or indirect, between two molecules (e.g., compounds, amino acids, nucleotides, polypeptides, or nucleic acids). Binding includes covalent, hydrogen bond, ionic, non-ionic, van der Waals, hydrophobic interactions, and the like.
[0090] The term "cell", as used herein, can mean, but is in no way limited to, its usual biological sense, and does not refer to an entire multicellular organism. The cell can, for example, be in vivo , in vitro or ex vivo , e.g., in cell culture, or present in a multicellular organism, including, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
[0091] The term "support", as used herein, refers to any pharmaceutically acceptable molecule, in other words without intrinsic toxicity, on which at least 3 pseudopeptide units can be grafted or coupled. An acceptable support therefore has to be of sufficient size to allow at least 3 pseudopeptide units to be grafted on it. For example, in any aspect or embodiment described herein, there are 3 to 20 pseudopeptides grafted or coupled to the support (preferably 3 to 8 pseudopeptide). Such an acceptable support should also preferably be large enough to allow at least 3, preferably 3 to 8, pseudopeptide units can come together to interact in the RGG domain of one or more nucleolin molecules. In any aspect or embodiment described herein, the support is not immunogenic.
[0092] The term “amino acid with a basic side chain” means any natural or non-natural amino acid whose side chain R has a pKa value greater than 7 (pKa(R)>7). Thus, any amino acid can be used for Yi and Y2, as long as its side chain has a pKa value greater than 7, preferably greater than 7.5, greater than 8, greater than 8.5 or greater than 9. In particular, among the natural amino acids those whose side chain has a pKa value greater than 7 include lysine (K, pKa(R)~10.5), arginine (R, pKa(R)~12.5), ornithine (inferior homologue of lysine, pKa(R)~10.8), generally considered to be natural basic amino acids. Thus, in an advantageous embodiment, Yi and Y2 are independently selected from arginine (R), lysine (K) and ornithine. Even more advantageously, Yi is a lysine (K) and Y2 is an arginine (R). However, other non natural amino acids can be used instead as long as the pKa value of their side chain R is greater than 7, preferably greater than 7.5, greater than 8, greater than 8.5, or greater than 9.
[0093] The term "grafted" or "coupled" as used herein for the pseudopeptide or pseudopeptide units described herein means being bound to the support by means of a covalent bond, either directly or through the intermediate of a spacer compound between the pseudopeptide and support. As a result of this, in any aspect or embodiment described herein, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, or even all) of the pseudopeptides are grafted or coupled directly on the support without a spacer compound between them and the support. In any aspect or embodiment described herein, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, or even all) of the pseudopeptides are grafted or coupled on the support through an intermediate of a spacer. Examples of acceptable spacers include an alkyl group, ethylene glycol, piperazine, or an amino acid of the type aminohexanoic acid or beta-alanine.
[0094] The term "pharmaceutically acceptable salt" is used throughout the specification to describe, where applicable, a salt form of one or more of the compounds described herein which are presented to increase the solubility of the compound in the gastic juices of the patient's gastrointestinal tract in order to promote dissolution and the bioavailability of the compounds. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids, where applicable. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium, magnesium and ammonium salts, among numerous other acids and bases well known in the pharmaceutical art. Sodium and potassium salts are particularly preferred as neutralization salts of the phosphates according to the present disclosure.
[0095] The term "pharmaceutically acceptable derivative" is used throughout the specification to describe any pharmaceutically acceptable prodrug form (such as an ester, amide other prodrug group), which, upon administration to a patient, provides directly or indirectly the present compound or an active metabolite of the present compound.
Methods of the Present Disclosure
[0096] In another aspect, the present disclosure provides methods for treating and/or preventing a disease or disorder related to the detrimental growth and/or proliferation of a cell, e.g., a cancer cell. In any aspect or embodiment described herein, the method comprises administering or co-administering a composition comprising an effective amount of at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound of the present disclosure and at least one (e.g., 1, 2, 3, or 4) mTOR inhibitor to a subject or a cell, in vivo , ex vivo , or in vitro , wherein the composition is effective in inhibiting or preventing the growth and/or proliferation and/or viability of a cancer cell.
[0097] As discussed above, the present disclosure describes the surprising and unexpected discovery that the coadministration of a mTOR inhibitor and a polyvalent synthetic compound of the present disclosure have a synergistic effect in inhibiting cancer cell growth and cancer cell viability. As such, an aspect the disclosure provides a method of treating cancer (such as pancreatic cancer) in a subject, the method comprising administering or co-administering to a cell or a subject in need thereof an effective amount of at least one (e.g., 1, 2, 3, or 4) mTOR inhibitor and an effective amount of at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound described herein, wherein the mTOR inhibitor and the polyvalent synthetic compound are effective for treating or ameliorating at least one symptom of the cancer (such as pancreatic cancer).
[0098] In any aspect or embodiment described herein, the polyvalent synthetic compound is present/administered in a composition, such as a therapeutic composition or a pharmaceutical composition, that includes a pharmaceutically acceptable carrier, excipient, or diluent.
[0099] In any aspect or embodiment described herein, the cancer is pancreatic cancer. [00100] In any aspect or embodiment described herein, the cancer or the pancreatic cancer is pancreatic ductile adenocarcinoma.
[00101] In any aspect or embodiment described herein, the pancreatic cancer is pancreatic ductile adenocarcinoma, pancreatic adenocarcinoma, pancreatic squamous cell carcinoma, pancreatic adenosquamous carcinoma, pancreatic colloid carcinoma, preferably pancreatic ductile adenocarcinoma. In any aspect or embodiment described herein, the polyvalent synthetic compound and the mTOR inhibitor have a synergistic inhibitory effect on the pancreatic cancer cell growth and/or cell viability.
[00102] As described in WO 2007/125210, the Nucant or polyvalent synthetic compounds described therein are known to be useful for the treatment of a disease involving deregulation of cell proliferation and/or angiogenesis. Therefore, in an additional aspect, the present disclosure provides methods of treating a disease or disorder involving deregulation of cell proliferation and/or angiogenesis comprising the step of administering or co-administering to a cell, a subject or an individual at least one (e.g., 1, 2, 3, or 4) mTOR inhibitor and an effective amount of a therapeutic composition comprising at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound of the present disclosure.
[00103] The term "disease involving deregulation of cell proliferation and/or angiogenesis" means, in the context of the present disclosure, any human or animal disease affecting one or more organs in which one or more abnormal cell proliferation phenomena are observed, as well as groups of cells or tissues and/or abnormal neovascularisation. Such diseases include all types of cancer, such as adenoma, sarcoma, carcinoma, lymphoma, and especially cancer of the ovary, breast, pancreas, lymphatic ganglion, skin, blood, lung, brain, kidney, liver, nasopharyngeal cavity, thyroid, central nervous system, prostate, colon, rectum, uterine neck, testicles or bladder. Any composition of the present disclosure as described herein may thus be for use with an mTOR inhibitor in the treatment of disease involving deregulation of cell proliferation and/or angiogenesis. [00104] A further aspect of the present disclosure provides methods for treating and/or preventing a disease or disorder related to the growth and/or proliferation of a cancer cell in an individual. In any aspect or embodiment described herein, the composition of the present disclosure is for use with an mTOR inhibitor in the treatment and/or prevention of a disease or disorder related to the growth and/or proliferation of a cancer cell in an individual, in particular for the treatment and/or prevention of cancer.
[00105] In any aspect or embodiment described herein, the methods described herein comprise administering or co-administering a mTOR inhibitor and (1) a composition comprising an effective amount of at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound of the present disclosure or (2) a composition comprising microspheres of at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound of the present disclosure, to a cell, a subject, or an individual, wherein the compound/composition and mTOR inhibitor are effective in inhibiting or preventing the growth and/or proliferation and/or viability of the cancer cell. [00106] As indicated in Background section, Nucant compounds have been shown to bind surface nucleolin RGG domain. Moreover, it has also been shown that surface nucleolin is expressed at the surface of tumor cells, such as tumor cells derived from hepatic carcinoma (Semenkovich, C. F., Ostlund, R. E. T, Olson, M. O., and Yang, J. W. A protein partially expressed on the surface of HepG2 cells that binds lipoproteins specifically is nucleolin. (1990) Biochemistry 29, 9708-9713), T-lymphocyte leukemia (Callebaut, C., Jacotot, E., Krust, B., Guichar, G., Blanco, J., Svab, J., Muller, S., Briand, J. P., and Hovanessian, A. G. Compose TASP inhibitors of HIV entry bind specifically to a 95-kDa cell surface protein. (1997) J. Biol. Chem. 272, 7159-7166; and Callebaut, C., Blanco, J., Benkirane, N., Krust, B., Jacotot, E., Guichard, G., Seddiki, N., Svab, J., Dam, E., Muller, S., Briand, J. P., and Hovanessian, A. G. Identification of V3 loop-binding proteins as potential receptors implicated in the binding of HIV particles to CD4(+) cells. (1998) J. Biol. Chem. 273, 21988-2199) and uterine cancer cells (Callebaut, C., Jacotot, E., Krust, B., Guichar, G., Blanco, J., Svab, J., Muller, S., Briand, J. P., and Hovanessian, A. G. Compose TASP inhibitors of HIV entry bind specifically to a 95-kDa cell surface protein. (1997) J. Biol. Chem. 272, 7159-7166), as well as at the surface of activated endothelial cells (Christian, S., Pilch, J., Akerman, M. E., Porkka, K., Laakkonen, P., and Ruoslahti, E. Nucleolin expressed at the cell surface is a marker of endothelial cells in angiogenic blood vessels. (2003) J. Cell Biol. 163, 871-878), cells which are involved in the angiogenesis process. Because the binding of the polyvalent synthetic compounds described herein to nucleolin is a generalized effect, the combination therapy provided by the present disclosure would be suitable for treating any known cancer. For example, types of cancer suitable for treatment with the compositions and methods provided by the present disclosure include, Acute Lymphoblastic Leukemia; Acute Myeloid Leukemia; Adrenocortical Carcinoma; Adrenocortical Carcinoma; AIDS-Related Cancers; AIDS-Related Lymphoma; Anal Cancer; Appendix Cancer; Astrocytomas; Atypical Teratoid/Rhabdoid Tumor; Basal Cell Carcinoma; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bone Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma; Brain Stem Glioma; Brain Tumor; Embryonal Tumors; Astrocytomas; Craniopharyngioma; Ependymoblastoma; Medulloepithelioma; Pineal Parenchymal Tumors of Intermediate Differentiation; Supratentorial Primitive Neuroectodermal Tumors and Pineoblastoma; Brain and Spinal Cord Tumors; Breast Cancer; Bronchial Tumors; Burkitt Lymphoma; Carcinoid Tumors; Cervical Cancer; Childhood Cancers; Chordoma; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Colon Cancer; Colorectal Cancer; Craniopharyngioma; Cutaneous T-Cell Lymphoma; Endometrial Cancer Ependymoblastoma; Ependymoma; Esophageal Cancer; Esthesioneuroblastoma; Ewing Sarcoma Family of Tumors; Extracranial Germ Cell Tumor; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer; Intraocular Melanoma; Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastrointestinal Carcinoid Tumor; Gastrointestinal Stromal Tumor (GIST); Germ Cell Tumor, Extracranial; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma; Hairy Cell Leukemia; Head and Neck Cancer; Heart Cancer; Hepatocellular (Liver) Cancer; Histiocytosis, Langerhans Cell; Hodgkin Lymphoma; Hodgkin Lymphoma, Childhood; Hypopharyngeal Cancer; Intraocular Melanoma; Islet Cell Tumors (Endocrine Pancreas); Kaposi Sarcoma; Kidney (Renal Cell) Cancer; Langerhans Cell Histiocytosis; Laryngeal Cancer; Laryngeal Cancer, Childhood; Lip and Oral Cavity Cancer; Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Non-Hodgkin Lymphoma; Macroglobulinemia, Waldenstrom; Malignant Fibrous Histiocytoma of Bone and Osteosarcoma; Medulloblastoma; Medulloepithelioma; Melanoma; Merkel Cell Carcinoma; Mesothelioma; Metastatic Squamous Neck Cancer; Mouth Cancer; Multiple Endocrine Neoplasia Syndromes; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Neoplasms; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Neuroblastoma; Non-Small Cell Lung Cancer; Oral Cancer; Oropharyngeal Cancer; Osteosarcoma and Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Papillomatosis; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pharyngeal Cancer; Pineal Parenchymal Tumors of Intermediate Differentiation; Pineoblastoma and Supratentorial Primitive Neuroectodermal Tumors; Pituitary Tumor Plasma Cell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Pelvis and Ureter, Transitional Cell Cancer; Respiratory Tract Cancer with Chromosome 15 Changes; Retinoblastoma; Rhabdomyosarcoma; Salivary Gland Cancer; Sarcoma, Ewing Sarcoma Family of Tumors; Sarcoma, Kaposi; Sarcoma, Soft Tissue; Sezary Syndrome Skin Cancer (Nonmelanoma or squamous cell); Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Stomach (Gastric) Cancer; Supratentorial Primitive Neuroectodermal Tumors; T-Cell Lymphoma, Cutaneous, see Mycosis Fungoides and Sezary Syndrome; Testicular Cancer; Throat Cancer; Thymoma and Thymic Carcinoma; Thyroid Cancer; Transitional Cell Cancer of the Renal Pelvis and Ureter; Urethral Cancer; Uterine Cancer, Endometrial; Uterine Sarcoma; Vaginal Cancer; Vulvar Cancer; Waldenstrom Macroglobulinemia; and Wilms Tumor.
[00107] As described in WO 2012/045750, the polyvalent synthetic compounds described therein form microspheres when admixed with a GAG. Therefore, in an additional aspect, the present disclosure provides methods of treating a disease or disorder, e.g., cancer, comprising the step of administering or co-administering to a cell, a subject, or an individual an effective amount of at least one (e.g., 1, 2, 3, or 4) mTOR inhibitor and an effective amount of a therapeutic composition comprising microspheres of at least one (e.g., 1, 2, 3, or 4) of the polyvalent synthetic compounds described herein and a GAG. In certain embodiments, the microspheres are formed prior to administration. In an additional embodiment, the microspheres are formed after administration to the subject or patient, e.g., in vivo.
[00108] In any aspect or embodiment described herein, the therapeutic composition of the polyvalent synthetic compounds of the present disclosure is any pharmaceutically acceptable form and administered by any pharmaceutically acceptable route, for example, the therapeutic composition can be administered as an oral dosage, either single daily dose or unitary dosage form, for the treatment of a muscle disorder or conditions, e.g., diabetes. Such pharmaceutically acceptable carriers and excipients and methods of administration will be readily apparent to those of skill in the art, and include compositions and methods as described in the USP-NF 2008 (United States Pharmacopeia/National Formulary), which is incorporated herein by reference in its entirety. Therefore, another aspect of the present disclosure provides pharmaceutically acceptable formulations of the polyvalent synthetic compounds of the present disclosure. In any aspect or embodiment described herein, pharmaceutically acceptable formulations include salts of the polyvalent synthetic compounds described herein (e.g., acid addition salts, such as salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid).
[00109] In any aspect or embodiment described herein the polyvalent synthetic compounds described herein are formulated for parenteral administration, e.g., formulated for injection via the intravenous, intraarthricular, intrathecal, intramuscular, sub -cutaneous, intra-lesional, or even intraperitoneal routes. In addition, the preparation of an aqueous composition that contains a cancer marker antibody, conjugate, inhibitor or other agent as an active component or ingredient will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
[00110] A “pharmacological composition”, “pharmacological formulation”, or the like, as used herein, refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or subject, preferably a human.
[00111] By "systemic administration", as used herein, is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (e.g., a cell to which the negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations, such as toxicity and forms which prevent the composition or formulation from exerting its effect. [00112] Preparations for administration of the therapeutic of the present disclosure include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non- aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Exemplary aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles including fluid and nutrient replenishers, electrolyte replenishers, and the like. Preservatives and other additives may be added such as, for example, antimicrobial agents, anti-oxidants, chelating agents and inert gases and the like.
[00113] A pharmaceutical composition of the polyvalent synthetic compound of the present disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral — e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, intraperitoneal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In any aspect or embodiment described herein, the parenteral preparation is enclosed in ampoules, disposable syringes or multiple dose vials, each of which may be made of glass or plastic.
[00114] Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the polyvalent synthetic compounds of the present disclosure can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). In any aspect or embodiment described herein, the composition of the present disclosure includes or is a liposome formulation with the polyvalent synthetic compounds described herein, wherein the liposome formulation facilitates the association of the polyvalent synthetic compounds described herein with the surface of cells, such as, lymphocytes and macrophages. [00115] The formulations and the polyvalent synthetic compounds of the present disclosure can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising polyvalent synthetic compounds of the present disclosure and a pharmaceutically acceptable carrier. One or more polyvalent synthetic compound of the present disclosure can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions comprising polyvalent synthetic compounds of the present disclosure can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
[00116] Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.
[00117] Excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
[00118] Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
[00119] Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti -oxidant such as ascorbic acid.
[00120] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present. Pharmaceutical compositions having polyvalent synthetic compounds of the present disclosure can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.
[00121] Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non -toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. [00122] For administration by inhalation, the polyvalent synthetic compounds for use according to the present disclosure are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethan-e, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The polyvalent synthetic compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
[00123] The polyvalent synthetic compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the polyvalent synthetic compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the polyvalent synthetic compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. [00124] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor.TM.. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
[00125] In any aspect or embodiment described herein, the polyvalent synthetic compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
[00126] It is especially advantageous to formulate oral or parenteral compositions in dosage unit faun for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
[00127] Sustained-release preparations can be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT. TM. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
[00128] For administration to non-human animals, the therapeutic compositions having the polyvalent synthetic compounds of the present disclosure can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects. mTOR Inhibitors
[00129] In any aspect or embodiment described herein, the mTOR inhibitor is an mTORCl inhibitor, such as an allosteric mTORCl inhibitor or an ATP -competitive mTOR kinase inhibitor. [00130] In any aspect or embodiment described herein, the mTOR inhibitor is an mTORC2 inhibitor, such as an ATP-competitive mTOR kinase inhibitor.
[00131] In any aspect or embodiment described herein, the mTOR inhibitor is one or more (e.g., 1, 2, 3, or 4) inhibitors selected from allosteric mTOR inhibitors and ATP-competitive mTOR inhibitors. In any aspect or embodiment described herein, the mTOR inhibitor is one or more (e.g., 1, 2, 3, or 4) allosteric mTOR inhibitors. In any aspect or embodiment described herein, the mTOR inhibitor is one or more (e.g., 1, 2, 3, or 4) ATP-competitive mTOR inhibitors.
[00132] In any aspect or embodiment described herein, the allosteric mTOR inhibitors is one or more (e.g., 1, 2, 3, or 4) inhibitors selected from rapamycin (SIROLIMUS), CCI-779 (TEMSIROLIMUS), RAD001 (EVEROLIMUS), AP-23573 (RID AF OROLIMU S), UMIROLIMUS, and ZOTAROLIMUS.
[00133] In any aspect or embodiment described herein, the ATP-competitive mTOR inhibitor is one or more (e.g., 1, 2, 3, or 4) inhibitors selected from AZD2014 (VISTUSERTIB), INK 128 (SAPANISERTIB), and AZD8055.
[00134] In any aspect or embodiment described herein, the mTOR inhibitor is one or more (e.g., 1, 2, 3, or 4) inhibitors selected from Rapamycin, AZD2014, and INK 128.
Polyvalent Synthetic Compounds of the Present Disclosure
[00135] Located essentially in the nucleus of normal cells where it is protected, nucleolin is, however, abundant at the surface of the cells that are proliferating and cells that of active endothelial cells where it can be a target for the polyvalent synthetic compounds described herein and their derivatives, which binds with specificity to surface nucleolin and/or glycoaminoglycans (GAGs).
[00136] In any aspect or embodiment described herein, the polyvalent synthetic compound comprises a support comprising at least 3 pseudopeptides coupled or grafted thereto, wherein the support is a linear peptide support having the formula (la) or (lb):
(Lys-Aib-Gly)z or (SEQ ID No. l)z (la), or (Aib-Lys-Aib-Gly)z or (SEQ ID No. 2)z (lb), wherein: z is an integer from 3 to 20; and each pseudopeptide independently has formula (Ila) or formula (lib):
[(X)n — Y1— ¾ — Y2 — (X)m] (Ila or SEQ ID No. 3), or
Figure imgf000036_0001
(lib or SEQ ID No. 4), wherein: each X is independently any amino acid (e.g. any proteinogenic amino acid); each Y1 and Y2 is independently a basic amino acid (e.g., arginine, lysine, lysine derivative, or lysine analog); n is 0 or 1; m is 0, 1, 2, or 3; and
Y represents a reduced bond (-CH2NH-), a retro-inverso bond (-NHCO-), a methyleneoxy bond (-CH2O-), a thiomethylene bond (-CH2-S-), a carba bond (- CH2CH2-), a ketomethylene bond (-CO-CH2-), a hydroxyethylene bond (-CHOH- CH2-), an E-alkylene bond , or a C=C bond, or a pharmaceutically acceptable salt or pharmaceutically acceptable derivative thereof. [00137] An advantage of them polyvalent synthetic compounds described herein demonstrate dual activity; the polyvalent synthetic compounds are capable of blocking independently both tumor proliferation and angiogenesis. Another major advantage of the polyvalent synthetic compounds of the present disclosure is that they demonstrate an excellent safety profile. Additionally, because of its small size of the polyvalent synthetic compounds, they are not immunogenic and are easy to manufacture, thereby making the cost of production reasonable.
[00138] The polyvalent synthetic compounds described herein bind surface nucleolin, which is present in active endothelial cells responsible for angiogenesis as well as in cancer cells, in a quasi irreversible manner. The polyvalent synthetic compounds of the present disclosure have shown a binary effect: direct blockade of the growth of tumor cells and inhibition of the angiogenesis which led to the complete eradication of implanted tumor in animal models. The specific binding occurs with the RGG domain located in the C-terminal region of the protein. After interaction the complex is internalized rapidly through a temperature dependent mechanism. The polyvalent synthetic compounds of the present disclosure can be considered as a stable and irreversible ligand of the surface nucleolin. After internalization, the polyvalent synthetic compounds of the present disclosure remains in the cytoplasm and does not cross the nuclear membrane.
[00139] As used herein, the term "Nucant" and polyvalent synthetic compounds of the present disclosure also encompasses peptides having minor modifications, for example, conservative amino acid modifications, chemical modification to mimic valence properties, and modifications that serve to increase its stability, solubility, biouptake and/or bioavailability; for example, absorption from the gut or penetration through the blood-brain barrier (BBB). For a review of strategies for increasing bioavailability of peptides and peptide drugs in the brain, and of methods for determining the permeability of peptides through the BBB using in vitro and in vivo assays, see Engleton et al., Peptides 9:1431-1439 (1997), the teachings of which are incorporated herein by reference. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that include carbohydrates such as glucose, sucrose or dextrans; antioxidants, such as ascorbic acid or glutathione; chelating agents; and low molecular weight proteins. Additional modifications to a peptide or polyvalent synthetic compounds described herein that can increase its bioavailability include conjugating the peptide to a lipophilic moiety, such as a lipophilic amino acid or compound.
[00140] The polyvalent synthetic compounds of the present disclosure is intended to encompass pseudopeptides as described herein having one or several minor modifications to the sequence. Contemplated modifications include chemical or enzymatic modifications (e.g. acylation, phosphorylation, glycosylation, etc.), and substitutions of one or several amino acids to the sequence. Those skilled in the art recognize that such modifications can be desirable in order to enhance the bioactivity, bioavailability or stability of the peptide, or to facilitate its synthesis or purification.
[00141] In any aspect or embodiment described herein, the polyvalent synthetic compounds of the present disclosure can be conjugated to one or more of a carrier or a cytotoxic agent, either directly or indirectly (e.g., via a linker moiety). For example, in any aspect or embodiment described herein, the cytotoxic agent may be covalently bound to a linker moiety, which is in turn covalently bound to the carrier. In any aspect or embodiment described herein, a linker moiety can be, for example, an amino acid (including mimetics, analogs, and derivatives), a peptide or polypeptide (including mimetics, analogs, and derivatives), a sterically labile compound, lipid, aliphatic group, carbohydrate, glyceride, a nucleotide or nucleic acid, peptide nucleic acid, nucleic acid derivative, and the like. For example, a conjugate in which the compounds of the present disclosure is conjugated to is cytotoxic drug 5-fluoro-uracyl ("5-FU").
[00142] Contemplated amino acid substitutions to the compounds of the present disclosure and provided for by the polyvalent synthetic compounds of the present disclosure, include conservative changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of an apolar amino acid with another apolar amino acid; replacement of a charged amino acid with a similarly charged amino acid, etc.). Those skilled in the art also recognize that nonconservative changes (e.g., replacement of an uncharged polar amino acid with an apolar amino acid; replacement of a charged amino acid with an uncharged polar amino acid, etc.) can be made without affecting the function of the compounds of the present disclosure. Furthermore, non-linear variants of the sequence of the polyvalent synthetic compounds described herein, including branched sequences and cyclic sequences, and variants that contain one or more D-amino acid residues in place of their L-amino acid counterparts, may be made.
[00143] In any aspect or embodiment described herein, the polyvalent synthetic compound(s) of the present disclosure is incorporated into liposomes (Gregoriadis, Liposome Technology, Vols. I to III, 2nd ed. (CRC Press, Boca Raton Fla. (1993)). Liposomes, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. In any aspect or embodiment described herein, the polyvalent synthetic compounds of the present disclosure can be prepared as nanoparticles. For example, adsorbing polyvalent synthetic compounds onto the surface of nanoparticles has proven effective in delivering peptide drugs to the brain (see Kreuter et al., Brain Res. 674:171-174 (1995)). Exemplary nanoparticles are colloidal polymer particles of poly-butylcyanoacrylate with the polyvalent synthetic compounds of the present disclosure adsorbed onto the surface and then coated with polysorbate 80.
[00144] Those skilled in the art can determine which residues and which regions of the compounds of the present disclosure are likely to be tolerant of modification and still retain the ability to bind nucleolin with clinically relevant affinity. For example, amino acid substitutions, or chemical or enzymatic modifications, at residues that are less well conserved between species are more likely to be tolerated than substitutions at highly conserved residues. Therefore, in certain embodiments the carrier may be modified so that the modified version of the carrier may be more easily conjugated to a diagnostic agent.
[00145] The polyvalent synthetic compounds described herein may or may not be optically pure, which means that the residues, such as lysine residues, in the pseudopeptide units may either be in random L or D configuration (not optically pure), or be all in D configuration (optically pure) or all in L configuration (optically pure). In any aspect or embodiment described herein, the polyvalent synthetic compounds described herein are optically pure, e.g. the amino acids residues (such as lysine residues) in the pseudopeptide units are all in D configuration or all in L configuration, preferably all in L configuration. Such optically pure polyvalent synthetic compounds can be obtained by the method described in WO2009/141687. [00146] In any aspect or embodiment described herein, the polyvalent synthetic compound comprises 3 to 15 pseudopeptides.
[00147] In any aspect or embodiment described herein, one or more of the pseudopeptides (e.g., each/all of the pseudopeptides) is coupled or grafted directly on the support.
[00148] In any aspect or embodiment described herein, the pseudopeptides are grafted or coupled to the support via a lysine of the support.
[00149] In any aspect or embodiment described herein, the pseudopeptides are optically pure pseudopeptides.
[00150] In any aspect or embodiment described herein, the polyvalent synthetic compound has the structure:
Figure imgf000039_0001
[00151] PSEUDOPEPTIDE
[00152] In any aspect or embodiment described herein, each pseudopeptide independently has formula (Ha):
[(X)n — Y1 — ¾ — Y2 — (X)m] (Ila or SEQ ID No. 3 ), or wherein: each X is independently any amino acid (e.g. any proteinogenic amino acid);
Z is proline or derivative thereof; each Y1 and Y2 is independently a basic amino acid (e.g., arginine, lysine, lysine derivative, or lysine analog); n is 0 or 1; m is 0, 1, 2, or 3; and
Y represents a reduced bond (-CH2NH-), a retro-inverso bond (-NHCO-), a methyleneoxy bond (-CH2O-), a thiomethylene bond (-CH2-S-), a carba bond (-CH2CH2-), a ketomethylene bond (-CO-CH2-), a hydroxyethylene bond (-CHOH-CH2-), an E- alkylene bond , or a C=C bond.
[00153] In any aspect or embodiment described herein, each pseudopeptide independently has formula (lib):
[(X)n — Y1 — ¾O — Y2 — (X)m] (lib or SEQ ID No. 4), wherein: each X is independently any amino acid (e.g. any proteinogenic amino acid); each Y1 and Y2 is independently a basic amino acid (e.g., arginine, lysine, lysine derivative, or lysine analog); n is 0 or 1; m is 0, 1, 2, or 3; and
Y represents a reduced bond (-CH2NH-), a retro-inverso bond (-NHCO-), a methyleneoxy bond (-CH2O-), a thiomethylene bond (-CH2-S-), a carba bond (-CH2CH2-), a ketomethylene bond (-CO-CH2-), a hydroxyethylene bond (-CHOH-CH2-), an E-alkylene bond , or a C=C bond.
[00154] In any aspect or embodiment described herein, n and m are each 0.
[00155] In any aspect or embodiment described herein, the Y1 and the Y2 have an L configuration or D configuration.
[00156] In any aspect or embodiment described herein, the Y1 and the Y2 have an L configuration.
[00157] In any aspect or embodiment described herein, each Y1 and Y2 is independently lysine or arginine.
[00158] In any aspect or embodiment described herein, each Y1 and Y2 is a lysine having an L configuration.
[00159] In any aspect or embodiment described herein, each Y1 and Y2 is independently selected from ornithine, homolysine, and diaminoheptanoic acid (e.g., 2,7-diaminoheptanoic acid).
[00160] In any aspect or embodiment described herein, the Y represents a reduced bond.
[00161] In any aspect or embodiment described herein, the m is 0 or 1.
[00162] In any aspect or embodiment described herein, one or more X is a proteinogenic amino acid. [00163] SUPPORT
[00164] In any aspect or embodiment described herein, the support is a linear peptide support having the formula (la):
(Lys-Aib-Gly)z or (SEQ ID No. l)z (la), wherein z is an integer from 3 to 20.
[00165] In any aspect or embodiment described herein, the support is a linear peptide support having the formula (lb):
(Aib-Lys-Aib-Gly)z or (SEQ ID No. 2)z (lb), wherein z is an integer from 3 to 20.
[00166] In any aspect or embodiment described herein, z is an integer from 3 to 10.
[00167] In any aspect or embodiment described herein, z is 6.
[00168] In any aspect or embodiment described herein, the support is selected from a linear peptide, a cyclic peptide, a peptoid (N-substituted glycine oligomer) that is linear or cyclic, a foldamer (oligomer or polymer with a strong tendency to adopt a compact, well-defined and predictable conformation in solution), a linear polymer or a spherical dendrimer (macromolecule consisting or polymers which combine according to a tree like process around a multifunctional central core), a sugar or a nanoparticle. In any aspect or embodiment described herein, the support is selected from a linear peptide, a cyclic peptide, a linear peptoid, or a cyclic peptoid.
[00169] In any aspect or embodiment described herein, the support is a linear peptide having lysine present in an amount of greater than 25%. In any aspect or embodiment described herein, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more, or even all) of the pseudopeptides are grafted or coupled to a lysine present on the support.
[00170] For example, in any aspect or embodiment described herein, the support linear peptide has a sequence selected from KKKGPKEKGC (SEQ ID No. 7), KKKKGC (SEQ ID No. 8), KKKKGPKKKKGA (SEQ ID No. 9) or KKKGPKEKAhxCONEE (SEQ ID NO: 10), wherein Ahx represents hexanoic amino acid and CONEE represents the fact that the acid group is replaced by an amide group, AhxCONEE, representing (2S)-2-aminohexanamide, or a linear sequence consisting of 2-4 units (KAKPG, SEQ ID No. 11), namely sequence AcKAKPGKAKPGKAKPGCONEE (SEQ ID No. 12) where Ac represents an acetyl group CEE— CO-, and CONEE means that the acid group COOH of glycine is replaced by an amide group CONEE. In any aspect or embodiment described herein, the support linear peptide is peptide KKKGPKEKAhxCONEE (SEQ ID No. 10), wherein Ahx represents hexanoic amino acid and CONEE represents the fact that the acid group is replaced by an amide group, or peptide AcKAKPGKAKPGKAKPGCOME (SEQ ID No. 12) where Ac represents an acetyl group CH3-CO- and CONH2 means that the acid group COOH of glycine is replaced by an amide group CONH2.
[00171] Among the linear peptides, some are known to adopt a helicoidal structure. These linear peptides can also be used as supports in the compounds of the present disclosure. In any aspect or embodiment described herein, the linear peptide support that forms a helicoidal structure comprises at least 3 (e.g., 3 to 20, 3 to 10 or 3 to 8) repetitions of peptide units having the sequence Aib-Lys-Aib-Gly (SEQ ID No. 2) or Lys-Aib-Gly (SEQ ID No. 1), where Aib represents 2-amino-isobutyric acid. In any aspect or embodiment described herein, at least half of the SEQ ID No. 2 or SEQ ID No. 1 peptide units (e.g., each of the peptide units) have a pseudopeptide of the present disclosure coupled or grafted thereupon (e.g., grafter to the lysine of each peptide unit).
[00172] For example, in any aspect or embodiment described herein, a quadrivalent compound with 4 pseudopeptide units of the present disclosure includes a support that is a linear peptide forming a helicoidal structure having the formula Ac-Lys-Aib-Gly-Lys-Aib- Gly-Lys-Aib-Gly-Lys-Aib-Gly-COME (SEQ ID No. 13), where Ac represents a CH3-CO- group and CONH2 means that the acid group COOH of glycine is replaced by an amid group COME.
[00173] Alternatively, in any aspect or embodiment described herein, a pentavalent compound with 5 pseudopeptide units of the present disclosure includes a support that is a linear peptide forming a helicoidal structure having the formula Aib-Lys-Aib-Gly-Aib-Lys- Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly (SEQ ID No. 14) or Lys-Aib- Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly (SEQ ID No. 15). In any aspect or embodiment described herein, the support in a linear peptide forming a helicoidal structure having a formula selected from Ac-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly- Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-COME (SEQ ID No. 16) where Ac represents an acetyl group CH3-CO- and COME means that the COOH acid group of glycine is replaced by an amide group COME, or Ac-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib- Gly-COME (SEQ ID No. 17) where the Ac group represents an acetyl group CH3-CO- and COME means that the COOH acid group of glycine is replaced by an amide group COME. [00174] Alternatively, in any aspect or embodiment described herein, a hexavalent compound with 6 pseudopeptide units of the present disclosure includes a support that is a linear peptide forming a helicoidal structure having the formula Ac-Aib-Lys-Aib-Gly-Aib- Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly- CONH2 (SEQ ID No. 18) where Ac represents a CH3-CO- group and CONH2 means that the acid group COOH of glycine is replaced by an amide group CONH2, or Ac-Lys-Aib-Gly-Lys- Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-COME (SEQ ID No.19) where Ac represents a CH3-CO- group and CONH2 means that the acid group COOH of glycine is replaced by an amid group COME.
[00175] Alternatively, in any aspect or embodiment described herein, an octavalent compound with 8 pseudopeptide units of the present disclosure includes a support that is a linear peptide forming a helicoidal structure having the formula Ac-Lys-Aib-Gly-Lys-Aib- Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly- CONH2 (SEQ ID No. 20), where Ac represents a CH3-CO- group and COME means that the acid group COOH of glycine is replaced by an amid group COME.
[00176] In any aspect or embodiment described herein, the support is a cyclic peptide or a cyclic peptoid. This allows the flexibility of the structure of the support to be restricted. In any aspect or embodiment described herein, the cyclic peptide or a cyclic peptoid support is selected from hexa-cyclic peptide, octa-cyclic peptide, deca-cyclic peptide, dodeca-cyclic peptide, or a chain of N-alkyl Glycine residue (an exemplary cyclic peptoid). In any aspect or embodiment described herein, the support has amino acid residues in the L (levorotatory) and D (dextrorotatory) configuration in alternation (D,L-cyclopeptide). For example, in any aspect or embodiment described herein, the support is a cyclic hexapeptide consisting of alternate alanine (A) residues of configuration D and lysine residues (K) of configuration L with 3 KPR units with a Y (-CH2N-) bond between K and P.
[00177] In any aspect or embodiment described herein, the support includes or has 5 lysine residues linked by amide bonds at the e amino group of each Lysine residue may also be used. [00178] In any aspect or embodiment described herein, the support is selected from: (i) a cyclic hexapeptide consisting of alternating alkaline (A) residues of configuration D and Lysine (K) residues of configuration L; (ii) 5 lysine residues linked by amide bonds at the e amino group of each Lysine residue; and (iii) a linear peptide of sequence SEQ ID No. 7 through SEQ ID No. 20.
[00179] In any aspect or embodiment described herein, the support is a linear peptide or cyclic peptide having the pseudopeptide units directly coupled or grafted to the peptide to lysine residues of the peptide support, at the amino group in the a or e position (e.g., at the amino group in the position (on the side chain) of lysine). In any aspect or embodiment described herein, the direct grafting or coupling of pseudopeptide units on the peptide support is carried out by means of an amide bond between the acid group COOH of the amino acid in the C-terminal position of the pseudopeptide unit and an amino group of a lysine residue (e.g., the amino group in the e position (on the side chain) of lysine).
[00180] In any aspect or embodiment described herein, at least 3 pseudopeptide units (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) are grafted or coupled on or to the support. The inventors have demonstrated previously the importance of binding to the RGG domain of nucleolin for exceptional anti -tumor efficacy of the Nucant peptides, derivatives, and analogues thereof. The binding to the RGG domain of nucleolin is obtained by means of multivalent presentation of several pseudopeptide units, such as those described herein. In any aspect or embodiment described herein, the compound includes at least 3 pseudopeptide units grafted on or coupled to the support. For example, in any aspect or embodiment described herein, the compounds has a support is a linear peptide of sequence KKKGPKEKGC, KKKKGC, KKKKGPKKKKGA or KKKGPKEKAhxCOME, and has at least three (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) pseudopeptides directly or indirectly grafted or coupled thereupon. By way of further example, in any aspect or embodiment described herein, the compound described herein includes 3 to 8 pseudopeptide units (e.g., 4-7, 4-6, 4 or 5, or 5 or 6 pseudopeptide units) grafted on or coupled to the support.
[00181] As used herein, the term "any amino acid" means any natural or synthetic amino acid, possibly modified by the presence of one or more substituents. More precisely the term amino acid means an alpha aminated amino acid with the following general structure:
Figure imgf000044_0001
wherein R represents the side chain of the amino acid. In any aspect or embodiment described herein, R represents the side chain of a side or non-side amino acid. The term "natural amino acid" means any amino acid which is found naturally in vivo in a living being. Natural amino acids therefore include amino acids coded by mRNA incorporated into proteins during translation but also other amino acids found naturally in vivo which are a product or by-product of a metabolic process, such as for example ornithine, which is generated by the urea production process by arginase from L-arginine. Thus, in any aspect or embodiment described herein, the amino acids used can therefore be a natural amino acid or not a natural amino acid. Natural amino acids generally have an L configuration but also, an amino acid of the present disclosure can have the L or D configuration. Moreover, R is of course not limited to the side chains of natural amino acid but can be freely chosen. For example, in any aspect or embodiment described herein, the any amino acid can be a non-proteinogenic amino acid. [00182] Various chemical bonds are likely to significantly increase resistance to at least one protease are known. In any aspect or embodiment described herein, the compounds of the present disclosure include a modified peptide bond Y, which provides significantly more resistant to at least one protease than a standard peptide. The inventors have also discovered that the presence of the modified bond Y makes it possible to significantly increase the efficacy of binding to nucleolin. This phenomenon may be due to the fact that this allows the compounds of the present disclosure to form an irreversible complex with nucleolin.
[00183] The term "standard peptide bond", as used herein, means an amide bond of formula (-CONH-) which is normally present between 2 amino acids in a natural protein. Such a bond is sensitive to the action of proteases. The term "modified peptide bonds Y" means a chemical bond between 2 amino acids of chemical formula distinct from the standard peptide bond of formula (-CONH-). This modified bond Y is such that it is significantly more resistant to at least one protease than a standard peptide bond of formula (-CONH-). The term "protease", also known as "peptidase" or "proteolytic enzyme", means any enzyme which cleaves the standard peptide bonds in proteins. This process is known as proteolytic cleavage. This involves the use of a water molecule, which is what leads to proteases being classified as hydrolases. The proteases namely include proteases known as N-peptidases which carry out the cleavage of the N-terminal end of proteins. These proteases are particularly inconvenient in terms of the in vivo stability of peptides without modified peptide bonds. This is why pseudopeptide units of the compounds of the present disclosure include a modified bond Y between Y1 and proline such that the resistance of the sub-unit of formula (I) is significantly increased which is essential for binding to nucleolin, namely to these N-peptidases. For example, in any aspect or embodiment described herein, Y represents a reduced bond (- CH2NH-) or (-CH2N-) in the case where bonding takes place at the level of a secondary amine group as is the case with the bond with proline, a retro-inverso bond (-NHCO-), a methyleneoxy bond (-CH2-O-), a thiomethylene bond (-CH2-S-), a carba bond (-CH2-CH2-), a ketomethylene bond (-CO-CH2-), a hydroxyethylene bond (-CHOH-CH2-), , an E-alkene bond or a (- CH=CH-) bond. In any aspect or embodiment described herein, Y represents a reduced bond (-CH2-NH-), which significantly increase the compounds resistance to at least one protease. [00184] The Y bond should therefore make it possible to significantly increase resistance to at least one N-peptidase. This makes it possible to significantly increase the half-life of compounds of the present disclosure in vivo and in vitro. For example, HB19, which has a modified bond Y, has a half-life of more than 24 hours in human serum or fetal calf serum at 37°C, whereas the same compound with a standard peptide bond instead of the Y bond only has a half-life of one hour under these same conditions.
[00185] The presence of additional Y bonds is expected to further increase resistance of the compounds of the present disclosure to proteases. However, additional Y bonds does further complicate the process of synthesizing compounds of the present disclosure and is thus optional. Therefore, in any aspect or embodiment described herein, further Y may be present on the pseudopeptide, the support, or both.
[00186] Immunochemical assays useful for practicing methods of the disclosure are well known to those skilled in the art, as described, for example, in Klug, T. L. et al ., Cancer Res., 44:1048 (1984), Herlyn, M. et al, J. Clin. Immunol., 2:135 (1982), Metzgar, R. S. et al., Proc. Natl. Acad. Sci., USA. 81:5242 (1984), Papsidero, L. D. et al. , Cancer Res., 44:4653 (1984), Hayes, D. F. et al, J. Clin. Invest., 75:1671 (1985), Killian, C. S. et al, J. Natl. Cancer Inst., 76:179 (1986), Killian, C. S. et al, Cancer Res., 45:886 (1985), Hedin, A. et al, Proc. Natl. Acad. Sci., USA. 80:3470 (1983), Pekary, A. E. etal, Clin. Chem., 30:1213-1215 (1984), Bast, R. C. et al, New England J. Med., 309:883-887 (1983) and Bellet, D. H. et al, Proc. Natl. Acad. Sci., USA, 81:3869-3873 (1984), all of which are specifically incorporated herein by reference.
[00187] The compounds described herein can be produced using well known recombinant methods or via well-known synthetic methods. There are several well-known methods for performing peptide synthesis including liquid-phase and solid-phase synthesis. Detailed discussions of various methods can be found at, for example, Atherton, E.; Sheppard, R. C. (1989). Solid Phase peptide synthesis: a practical approach. Oxford, England: IRL Press; Stewart, J. M.; Young, J. D. (1984). Solid phase peptide synthesis, 2nd edition, Rockford: Pierce Chemical Company, 91; R. B. Merrifield (1963). "Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide". J. Am. Chem. Soc. 85 (14): 2149-2154; L. A. Carpino (1993). "1- Hydroxy-7-azabenzotriazole. An efficient peptide coupling additive". J. Am. Chem. Soc. 115 (10): 4397-4398; which are hereby incorporated by reference in their entirety for all purposes.
EXAMPLES [00188] Pancreatic cancer (PDAC) is an aggressive disease characterized by high invasiveness, rapid progression, and resistance to conventional therapy. There is an urgent need to identify new molecules to improve current therapies, with better efficacy and less toxicity. It was previously shown that N6L, a synthetic pseudo-peptide that targets nucleolin (NCL) impairs PDAC growth and normalizes tumor vessels in animal models.
[00189] Here, the translatome of PDAC cells treated with N6L was analyzed to identify the pathways that were either repressed or activated during treatment with N6L. A strong decrease of global protein synthesis was observed. However, it was found that about 6% of the mRNAs were enriched in the polysomal fractions from N6L-treated cells. Interestingly, a 5'-terminal oligopyrimidine (5’ TOP) motif was identified in many of these mRNAs, and it was observed that a chimeric RNA bearing a 5 ‘TOP motif was translationally up-regulated by N6L. It was further demonstrated that N6L activates the mTOR signaling pathway which is required for the translation of these mRNAs. Interestingly, when N6L was combined with several mTOR inhibitors, a synergistically inhibition of cell growth and viability in PDAC cells lined, including patient derived-xenografts and tumor derived-organoids.
[00190] Thus, N6L treatment strongly reduces pancreatic cells proliferation, which then undergoes a translational reprogramming through the activation of the mTOR pathway. The inventors made the surprising and unexpected discovery that N6L and mTOR inhibitors act synergistically to inhibit the proliferation of several PDAC and human PDX cell lines. This new combination therapy of N6L and mTOR inhibitors constitutes a new alternative to treat pancreatic cancer.
[00191] In order to better understand how the NCL antagonist, N6L, inhibits cancer cell growth, a translatome analysis in mPDAC cell lines was carried out. Using this approach, this approach demonstrated that N6L induces a translational reprogramming through the activation of the mTOR pathway during N6L treatment. The inventors surprisingly discovered that N6L and mTOR inhibitors act synergistically to inhibit the proliferation of several PDAC and human PDX cell lines.
[00192] Results.
[00193] N6L decreases ribosome biogenesis and protein synthesis in PDAC cells. NCL is a master regulator protein required for rRNA transcription and ribosome biogenesis. Whether the N6L pseudo-peptide had any effect on the production of ribosomes in PDAC cells was first examined. Time course analysis of mPDAC treated with different concentrations of N6L shows that at 50 mM a strong reduction of cell proliferation was observed (FIG. 3A) while metabolic activity which reflected cell viability, assessed by MTS test, was reduced by 56% (FIG. 3B). With N6L treatment impairing cell viability of mPDAC cells, whether the death of the cells was due to caspase dependent apoptosis was examined. Apoptosis induction assay was performed using the IncuCyte ZOOM®. A fluorescent reagent, activated when effector caspases 3/7 were activated too, allowed the device to count the number of apoptotic cells per mm2 every 2 hours until 72 hours. A strong and significantly increase of caspase dependent apoptosis in mPDAC treated cells in a dose dependent manner was observed, as compared to non-treated cells (FIG. 3C). The morphology of the N6L-treated cells appeared different to non-treated cells, more “thorny”.
[00194] The impact of N6L on ribosome production was evaluated by treating mPDAC cells at 50 mM for 48h (FIG. 4A, FIG. 4B, and FIG. 4C). Total RNA extraction was performed from the same number of cells treated or not treated with N6L, and the rRNA were analyzed (FIG. 4A). The electrophoretic profiles of 18S and 28S rRNA showed that the amount of rRNA was significantly decreased upon N6L treatment. When compared to non-treated cells, the rRNA quantity (28S+18S) in treated mPDAC was reduced by 61% (FIG. 4A).
[00195] Ribosome biogenesis requires the initial transcription of rDNA genes to a single pre-rRNA (47S) that is subsequently cleaved in several pre-rRNA to ultimately provide 5.8S, 18S and 28 S rRNAs. The level of rDNA transcription was measured by RT-qPCR using 5’ETS probe (before the early cleavage), which is able to detect short-lived rRNA sequence of 47S. It was found that the 47S ribosomal RNA synthesis was repressed by 58% after N6L treatment (FIG. 4B).
[00196] This suggests that N6L could affect the synthesis of rRNA. This result agrees with an inhibition of NCL function by N6L in the nucleolus as it was previously shown thatNCL inhibition impacts rRNA transcription (Cong et al. 2012). The consequences are the disorganization of the nucleolar structure as shown in (FIG. 4C) by electronic microscopy analysis, which was very similar to what was observed upon nucleolin knock-down by siRNA (Ugrinova et al. 2007).
[00197] This decrease of rRNA synthesis could be the consequence of a decrease of NCL protein accumulation upon the N6L treatment of PDAC (FIG. 4D) while the level of transcription of the NCL gene seems to be unaffected (FIG. 4E).
[00198] In order to evaluate the ability of mPDAC cells to synthesize proteins following N6L treatment, we used the SUrface SEnsing of Translation (SUnSET) technique (David et al. 2012). This technique is based on the incorporation of puromycin to newly synthesized proteins and its detection with anti -puromycin antibodies (FIG. 4F and FIG. 4G). As shown in FIG. 4F, mPDAC treated-cells displayed a significant decrease in the amount of puromycin- labeled peptides compared to non-treated mPDAC as soon as 24 hours of N6L treatment (by 26%; lane 4) with a maximum decrease after 48 hours (by 52%; lane 6) (FIG. 4G).
[00199] Altogether, the data reveals that N6L impaired ribosome biogenesis and protein synthesis in PD AC cells.
[00200] NCL targeting by N6L impairs protein synthesis and induces a translational reprogramming in mPDAC cells. mRNA translation is a central cellular process that regulates growth and metabolism (Sonenberg et Hinnebusch 2009). To evaluate the impact of N6L treatment on mPDAC translatome, we performed polysome profiling analysis on cells treated or not treated with N6L for 48 hours (FIG. 4A-FIG. 4F).
[00201] Time course analysis of mPDAC treated with different concentrations of N6L shows that at 30-50 mM a strong reduction of cell proliferation was observed (FIG. 3A) while metabolic activity which reflected cell viability, assessed by MTS test, was reduced by 56%
(FIG. 3B).
[00202] To evaluate the impact of N6L treatment on mPDAC translatome, treated and non- treated mPDAC cells were harvested and RNAs were extracted from an aliquot of the cytoplasmic fractions, while the remaining fraction was analyzed through sucrose gradient to perform polysome profiling. RNAs were then extracted from the polysomal pooled fractions. The extracted RNAs (from cytoplasmic and polysomal pooled fractions) were submitted to deep sequencing (workflow shown in FIG 4A).
[00203] The polysome profile of treated mPDAC cells (mPDAC T) and non-treated mPDAC cells (mPDAC NT) (FIG 4B and FIG. 4C). In contrast, a strong decrease of polysome peaks was observed in cells treated with N6L (FIG. 4C) with a reduced level of 40S and 60S subunits and an important increase of the 80S peak as comparised to non-treated cells, as compared to non-treated cells (FIG. 4B).
[00204] In order to evaluate the ability of mPDAC cells to synthesize proteins following N6L treatment, the SUrface SEnsing of Translation (SUnSET) technique (David et al. 2012) was utilized. This technique is based on the incorporation of puromycin to newly synthesized proteins and its detection with anti -puromycin antibodies (FIG. 4F and FIG. 4G). As shown in FIG. 4F, mPDAC treated-cells displayed a significant decrease (26%) in the amount of puromycin-labeled peptides compared to non-treated mPDAC as soon as after 24 hours of N6L treatment (lane 4) with a maximum decrease of 52% after 48 hours (by 52%; lane 6) (FIG. 4G).
[00205] The results indicate a decrease of protein synthesis in N6L-treated cells and agree with the decrease in the mPDAC translation capacity upon N6L treatment. [00206] To determine how the translatome was affected by N6L treatment, results from the deep sequencing of cytoplasmic and polysomal fractions were analyzed (FIG. 5A-FIG. 5F). The RNA-sequencing analysis using the package DeSeq2 showed that among the 14,384 mRNAs present in mPDAC, 39% (5,610) were significantly expressed on both non-treated cells and N6L-treated cells (p- adj<0.05) (FIG. 5A and FIG. 5B).
[00207] These significantly expressed genes in non-treated and treated mPDAC cells were used to identify bona fide alterations in translational efficiency in response to N6L by calculating the translational index (TI) (FIG. 5C and FIG. 5D). TI reflects the translation efficiency for each expressed mRNA, by measuring the percentage of mRNA engage in the polysomal (poly) fractions to a total amount of this mRNA found that cytoplasmic (cyto) fractions and comparing these percentages between treated (T) and non-treated (NT) mPDAC cells using the following formula: TI = X1/X2, with XI and X2 representing the ratio between polysomal -to-cytoplasmic fraction in mPDAC treated and non-treated cells, respectively. [00208] By applying a TI cut-off = 1.5 and p- adj < 0.05, 886 genes were identified whose TI was significantly changed in response to N6L over 48 hours (FIG. 5C), indicating that N6L treatment altered translation efficiency of 886 mRNAs out of the 14,384 analyzed. The range of TI varied from -2 to +2.5 (FIG. 5D). The translation of 673 (75.96%) of these mRNAs were down-regulated, whereas 213 (24.04%) of these mRNAs were upregulated (FIG. 5C). Therefore, it was observed that down-regulation of translation efficiency was more frequent than up-regulation (genes list in Table 1).
Table 1. Translational Index (TI) for the completed gene list
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
[00209] To determine the main functions of the genes whose mRNAs recruitment within polysomes was altered in response to N6L treatment, Gene Ontology analysis was performed using the functional annotation clustering analytic modules of EnrichR bioinformatics resources that provides a rank classification of enriched functions based on determination of P-v alues and enrichment scores (Kuleshov et al. 2016; Kuleshov et al. 2019). Gene Ontology analysis revealed functional pathways corresponding to cell cycle, Fanconi anemia pathway and RNA transport for the 673 mRNAs translationally repressed (FIG. 5E) and functional pathways corresponding to Ribosome/translation, Alzeimer’s disease, and oxidative phosphorylation for the 213 mRNAs translationally up-regulated (FIG. 5F).
[00210] Interestingly, among these 673 translationally down-regulated mRNAs, 315 were transcriptionally stable (46.8%) (FIG. 6 A), encoding proteins implicated in phosphatidylinositol biosynthesis (FIG. 6D) and 340 were transcriptionally up-regulated (51%) encoding proteins implicated in mitotic division (FIG. 6C). In addition, among the 213 translationally up-regulated genes shown in FIG. 6B, 73 were transcriptionally stable (34.3%) encoding proteins implicated in translation/ribosomal proteins (FIG. 6F) and 131 were transcriptionally down-regulated 61.5% encoding proteins implicated in the regulation of cholesterol storage (FIG. 6E). Altogether, the data shows that the recruitment of these mRNAs into polysomes was controlled upon N6L treatment.
[00211] N6L induces a global decrease of mRNA translation correlated with a decrease of EiF3 mRNAs translation. The expression of the mRNAs coding for essential proteins involved in translation regulation that were subjected themselves to a translational regulation as revealed by our Functional Gene Ontology analysis was examined (FIG. 5E). Among the 673 mRNA that are down regulated, several mRNA encode for protein involved in translation initiation (FIG.2E).
[00212] EiF3a mRNA and EiF3c mRNA, coding for the essential components of the translation initiation EiF3 complex, were strongly translationally repressed under N6L treatment (TI = - 0.99 and - 0.54, respectively) while mRNA encoding the other members ( EiF3e-j ) were mostly unchanged (FIG. 7A). Using Western blotting analysis, EiF3a and EiF3c protein levels were significantly reduced (FIG. 7B and FIG. 7C) while the protein level of the other EiF3 members (EiF3e, EiF3h, and EiF3k) did not change (FIG. 7D and FIG. 7E) upon N6L treatment. Using quantitative PCR, no change was observed in the expression of the EiF3a and EiF3c mRNAs (FIG. 7F) in agreement with the transcriptomic analysis (data not shown). Altogether, these results revealed that N6L decreases the EiF3a and EiF3c expression at the translational level that contribute to the down-regulation of global mRNA translation. [00213] NCL targeting by N6L increases 5’TOP mRNA translation by activating mTOR pathway. Although global translation was overall reduced upon N6L treatment, the translatome analysis identified a subset of mRNAs (24.04%) that were enriched in the polysomal fractions (FIG. 5C-FIG. 5F). Functional Gene Ontology analysis of these 213 mRNAs identified genes encoding proteins involved in ribosome biogenesis, translation or energy metabolism pathways (FIG. 2F). In particular, it was very striking to surprisingly discover many mRNAs coding for ribosomal proteins (FIG. 8A).
[00214] Ribosomal protein transcripts are characterized by the presence of a specific motif on their 5’UTR called 5 terminal oligopyrimidine (TOP) sequence immediately adjacent to the cap structure (Meyuhas et Kahan 2015). This family of TOP mRNAs encode proteins involved in translation initiation, elongation, and termination.
[00215] By comparing the mRNAs enriched in polysomes after N6L treatment mRNA to those classified as 5’TOP mRNAs (Gentilella et al. 2017), out of the 213 genes enriched in polysome fractions, 53 mRNAs (24.2%) were identified (FIG. 8B, Table 1). Gene Ontology analysis of these 53 mRNAs revealed that they are all involved in pathways related to translation, cytoplasmic ribosomal protein, or Cap-dependent translation initiation (FIG. 9A and FIG. 9B). Whereas, the set of non-5’TOP were exclusively involved in pathways related to mitochondrial metabolism, including mitochondrial translation, ATP synthesis, or oxidative phosphorylation (FIG. 9C and FIG. 9D).
[00216] Most of the 53 5’ TOP translationally up-regulated mRNAs (81.1%) were transcriptionally down-regulated (FIG. 8C). This means that, upon N6L treatment, enrichment in the polysome fraction is not a consequences of a higher accumulation of these mRNAs, but due to an increase of the recruitment of these mRNAs in the polysomes.
[00217] Collectively, the data shows that NCL targeting by N6L promotes a selective recruitment to polysomes of some mRNAs bearing 5’ TOP motif in their 5’UTR. This recruitment maintains translation as seen for RPL36 and RPS3 using wester-blotting analysis (FIG. 8D) despite the decreased accumulation of these mRNAs upon N6L treatment.
[00218] To determine if the 5’TOP motif plays an active role in this recruitment, the 5’TOP motif of Rpl36 and Rps3 mRNAs were inserted upstream of firefly gene reporters and downstream a CMV promoter (FIG. 8E). These plasmids, pFL-5’TOP, were co-transfected with pRL (for normalization) in mPDAC cells treated with increasing doses of N6L. After 24 hours, the luciferase activity was quantified to determine if the 5’UTR was required for the increased translation of the chimeric mRNA. Indeed, in the presence of increasing amount of N6L, the presence of these 5’UTR sequences provided an increasing amount of luciferase activity suggesting that the presence of this motif increased the recruitment of these mRNAs in the polysomes upon N6L treatment (FIG. 8F).
[00219] As the 5’TOP motifs are mainly regulated by the mTOR pathway (Thoreen et al. 2012), the data suggested that the N6L treatment could activate the mTOR pathway in mPDAC. Therefore, to determine if the mTOR pathway was activated in mPDAC cells upon N6L treatment, the expression/activation of the three primary downstream effectors of mTOR 4E-BP1, RPS6 (a downstream target of S6K), and ART were analyzed by performing western blotting and quantification of the phosphorylated-to-total form ratios of RPS6, 4EBP1, and ART accumulations (FIG. 10A-FIG. 10D).
[00220] The N6L treatment induced a strong phosphorylation of RPS6, 4EBP1 (y and b forms), and ART, as compared to non-treated mPDAC cells (FIG. 10A; lanes 1-2) while expression of NCL protein is decreased. Whether NCL expression modulates the mTOR pathway was examined. Indeed, the activation of mTOR, via the hyper-phosphorylation of RPS6, ART and 4EBP1, was also observed when NCL was down regulated by siRNA (FIG. 10A, lanes 3-4). In addition, the phosphorylation of RPS6 and ART occurred in a N6L dose- dependent manner as revealed by Western blotting (FIG. 10B) and immunofluorescence analysis of phosphorylated RPS6 (FIG. IOC).
[00221] Collectively, the results demonstrate that NCL inhibition, either through siRNA or with N6L treatment, causes an activation of the mTOR pathway in mPDAC cells.
[00222] The translation of 5’ TOP mRNAs is regulated by binding of La-related protein 1 (LARPl) to the cap and TOP sequence (Aoki et al. 2013; Gentilella et al. 2017; Fonseca et al. 2018). Indeed, while no change was observed on EiF4E protein level upon N6L treatment, western-blotting results showed an accumulation of LARPl in a N6L dose-dependent manner (FIG. 10D).
[00223] Thus, the data demonstrated that the N6L treatment modified the translatome of treated PDAC cells through the activation of the mTOR pathway, thereby allowing the translation of a specific set of mRNAs involved in the translational and metabolic pathways. [00224] Combinations of N6L and mTORi are synergistic on PDAC cell growth and viability inhibition. The activation of the mTOR pathway by N6L tinduces an increase in translation of 5’ TOP mRNAs, which may contribute to a decrease of efficiency of the N6L treatment on cell viability and proliferation, the effects of the combination of mTOR inhibitors with N6L in human and murine PDAC models was examined. Two classes of mTORi were examined, allosteric mTORCl inhibitor Rapamycin (RAPA, Sirolimus) and ATP-competitive mTOR kinase inhibitors AZD2014 (Vistusertib) and INK128 (Sapanisertib), which inhibit both mTORCl and mTORC2 (Pike et al. 2013)(Hsieh et al. 2012).
[00225] Two concentrations of RAPA, AZD2014, or INK 128 were tested alone or in combination with N6L for mPDAC confluence/growth assay over 72 hours using IncuCyte ZOOM® live-imaging (FIG. 9A-FIG.9F and FIG. 11A-FIG. 11N). As previously described in FIG. 3A-Fig.3D, N6L induced a decrease of cell growth in a dose-dependent manner, whereas mTORi alone had no or modest effects on mPDAC cell growth at the tested concentrations (10 and 25 nM) (FIG. 9A-9C). However, the combination of both mTOR inhibitors andN6L had strong inhibitory effects on mPDAC cell growth (FIG. 11A-FIG.11C), demonstrating an additive or synergistic effect between the two molecules. Using the Combenefit® software (Di Veroli et al. 2016) based on the LOEWE method, the combination of N6L (5 or 1 OmM) and mTORi was demonstrated to have a strong synergetic effect on the mPDAC growth inhibition (FIG. 11G; upper panels). The software calculates a synergy score for each combination, where a positive score (blue color) indicates synergy, a score of 0 is additive, and a negative score (yellow-red colors) indicates antagonism.
[00226] These combinations were further validated in patient-derived xenograft (PDX) models and other preclinical cell lines. PDX models faithfully reproduce the molecular features of PDAC human tumors, display a high predictive value for clinical efficacy in patients, and are critically needed for the development of new treatments. Whereas, mTORi alone have no or modest effects on PDX cell growth at the tested concentrations (FIG. 9D-9F), except for INK128 at high concentration of 100 nM, the combination of RAPA, AZD2014 or INK128 with N6L was superior to single agent treatment (FIG. 11D-FIG. 11F) and this effect on PDX growth inhibition was synergistic (FIG. 11G, lower panels).
[00227] The same results were obtained in human PDAC cell lines MiaPaca-2 (FIG. HA- FIG. 14C) and Panc-1 (FIG. 12A-FIG. 12C) upon N6L/mTORi combination. These combinations induce a synergetic anti-proliferative effect in cancerous cells, assessed by IncuCyte ZOOM® live-cell imaging (FIG. 12C and FIG. 14C).
[00228] As the N6L treatment impaired the cell viability of mPDAC cells, it was examined if the death of the cells was due to caspase dependent apoptosis as previously described. Apoptosis induction assay was performed using the IncuCyte ZOOM® live-imaging. A fluorescent reagent, activated when effector caspases 3/7 were activated too, allowed the device to count the number of apoptotic cells per mm2 every 2 hours until 72 hours. A strong and significantly increase of caspase dependent apoptosis was observed in mPDAC treated cells in a dose dependent manner as compared to non-treated cells (FIG. 3C). The morphology of the N6L-treated cells appears different to non-treated cells.
[00229] Cell death was also assessed by Green caspase3/7 apoptosis assay using the IncuCyte ZOOM®. A strong increase of caspase dependent apoptosis was observed in mPDAC cells treated with N6L combined with the dual mTORi INK128 (FIG. 11H).
[00230] To accurately predict the efficacy of these new drug combinations for PD AC therapy, the drug combinations were tested on three-dimensional culture models by establishing spheroids and PDAC tumor-derived organoid cultures. To this end, PDAC and PDX cells were allowed to form a spheroid, as previously described (Ware et al. 2016). Then, the spheroids were treated with different drugs concentrations of N6L or INK 128, alone or in combinations over 96 hours, using IncuCyte ZOOM® live- imaging (FIG. 111-FIG. 11L). To evaluate the impact of the drug combination on spheroid growth, their area was calculated using ImageJ software. The results showed that the spheroid area decreased by 22% and 21%, upon N6L or INK 128 alone respectively (FIG. 111-FIG. UK), while the mPDAC spheroid area decreased more than 41% (p= 0.006) when ING128 is combined with N6L treatment. The same results were obtained for PDX spheroids (FIG. llJ).In PDX models, the combination of N6L and the INK128 significantly decreased the PDX spheroid area more than 60% (p 0 003) compared to non-treated conditions (FIG. 11L).
[00231] The combination of N6L and INK128 on mPDAC tumor-derived organoid viability (FIG. 11M and FIG. 11N). The organoid cultures were established from mPDAC tumors generated in mice as previously described (Boj, S.F., et al. 2014 and Tiriac, FL, et al., 2018). An analysis of MTS data by the LOEWE method in the PDAC derived-organoids models demonstrated that the combination of N6L and INK128 had a synergistic effect on cell viability, as shown in the contour plot of synergy/antagonism with the LOEWE model in FIG. 11M and FIG. 11N.
[00232] Mechanistically, it was confirmed by western-blot analysis that the mTOR pathway was inactivated upon the N6L and mTORi dual treatment (FIG. 13A and FIG. 13B), where a strong decrease of RPS6 and 4EBP1 phosphorylation in mPDAC and PDX cells was observed. Of note, the dual mTORCl/mTORC2 INK128 seemed to have a strong effect in comparison to the other mTOR inhibitors when combined with N6L.
[00233] Collectively, the results demonstrate that NCL targeting by N6L treatment and inhibitor of the TOR pathway synergistically to inhibit PDAC cell growth. [00234] Discussion and Conclusions. Nucleolin (NCL) overexpression is associated with a poor prognostic for many cancers, pancreatic cancer. PDAC remains one of the most aggressive and lethal disease because of the limited therapeutic options. The nucleolin aptamer N6L shows antitumor effects on breast cancer_(Destouches et al. 2011), glioblastoma (Benedetti et al. 2015)(Dhez et al. 2018), non-small cell lung carcinoma (Ramos et al. 2020), and PDAC (Gilles et al. 2016; Sanhaji et al. 2019).
[00235] It was surprisingly discovered that, although N6L strongly inhibits global translation, some mRNAa escape this global translational shutdown (FIG. 5A-FIG. 5F). The expression of these mRNAs that continue to be recruited in polysomes in N6L-treated cells was either stable or lower (FIG. 6A-FIG. 6F) indicating that this increased recruitment in polysomes was not the consequence of an increase of their transcription but rather a consequence of a regulation at the translational level. These translationally up regulated mRNAs are mostly implicated in cell metabolism and translation machinery.
[00236] Many of these mRNAs recruited in the polysomes under N6L treatment contains a 5’ Terminal oligopyrimidine (5’ TOP) in their 5’UTR and encodes for ribosomal proteins and for factors regulating translation.
[00237] The 5’TOP motif begins with a C nucleotide directly adjacent to the cap structure followed by a series of approximately 4-515 pyrimidines often followed by a G-rich region (Meyuhas, O. & Kahan, T 2015). The 5’ TOP motif is highly conserved and is found in all human ribosomal proteins as well as non-ribosomal proteins involved in translation (Yamashita, R. et al. 2008 and Levy, S., et al. 1991). The shared TOP motif allows cells to quickly modulated the expression of proteins involved in ribosome production and protein synthesis in response to changes in cellular homeostasis. It was hypothesized that the cells activate the translation of these 5’ TOP mRNAs to counteract the negative effect of N6L on cell survival. Mechanistically, the translation of these 5’UTR motifs is regulated by the mTOR pathway.
[00238] The mTOR pathway is crucial to control cell growth and survival, in physiological as well as in pathological conditions. It acts in two distinct mTOR complexes, mTORCl and mTORC2, which differ in associated proteins and by sensitivity to rapamycin and its derivatives (Sabatini 2006). The major function of mTORCl is the promotion of cellular growth and proliferation via increasing protein synthesis and inhibition of autophagy. Functions of mTORC2 are less well studied and include organization of the actin cytoskeleton, control of cellular metabolism, and anti-apoptotic properties via stimulation of the AKT- FOXO pathway. [00239] The inhibition of mTORCl pathway leads to a strong decrease in translation of transcripts containing the 5’TOP motif (Thoreen, C.C. et al. 2012 and Philippe, L., et al., 2020). How mTOR regulates 5’ TOP mRNA translation, however, is not completely understood. [00240] Many factors have been proposed as trans-acting factors that could regulate (positively or negatively) the translation of 5’TOP mRNA, including the T-cell intracellular antigen-1 (TIA1) and TIA-l-related (TIAR) proteins, miR-lOA, 4EBP1 andLARPl (Patursky- Polischuk, I. et al.2014). mTORCl enhance 5 'TOP mRNA translation by two mechanisms: phosphorylation of 4EBP1 and/or LARPl (Thoreen, C. C. et al. 2012, Fonseca, B. D., et al. 2018, and Hsieh, A. C. et al. 2012).
[00241] kinder mTOR activation, 4EBP1 is phosphorylated, which prevents 4EBP1 from disrupting the interaction of eIF4E and the mRNA cap, and has been shown to selectively enhance 5'TOP mRNA translation (Thoreen, C. C. et al. 2012 and Hsieh, A. C. et al. 2012). The role of LARPl in the regulation of TOP mRNAs is, however, controversial. While LARPl was shown to regulate TOP mRNA stability, it has also been described as a positive or negative regulator of TOP mRNA translation, depending on the context and the energy status of the cells (Al-Ashtal, H. A., et al. 2021). LARPl interacts with raptor and is phosphorylated by mTORCl, which is thought to modulate its mRNA-binding activity (Philippe, L., et al. 2020 and Hong, S. et al. 2017). Here, the phosphorylation of 4EBP1, RPS6 and ART, and the accumulation of LARPl upon N6L treatment enhanced the 5’TOP mRNA translation, thereby demonstrating that the mTOR pathway is activated and that LARPl could act as a positive regulator.
[00242] The mTOR pathway is reported to be aberrantly active in several cancers including PDAC (Tian, T., et al. 2019 and Bellizzi, A. M., 2010) in part due to mutations in upstream regulatory molecules, including PTEN, ART and TSCl/2. Most PDAC cancers have RAS mutations leading to activation of the MEK/ERK pathway, which can inactivate TSCl/2, thereby activating TORC1. Therefore, mTOR is a relevant target for the treatment of PDAC (Tian, T., et al. 2019). Several types of mTOR inhibitors — such as rapamycin, its rapalogs and dual mTORCl/mTORC2 inhibitors — have been examined in various cancer models, including PDAC (Tian, T., et al. 2019 and Hassan, Z. et al. 2018). The effects of mTOR inhibitors utilized as monotherapy in clinical trials, however, have not shown significant effects and sometimes are dampened by several resistance mechanisms (Guri, Y. & Hall, M. N. 2016). Combined therapies with mTOR inhibitors and other pathway inhibitors or conventional therapies are under investigation in preclinical and clinical trials in different tumor types (Clinical trials. clinical trials. gov/ct2/results?cond=mTOR+inhibitors&term=&cntry=&state=&city=&dist=). Hence, novel therapeutic strategies based on mTOR inhibition still need to be developed. In PDAC, in particular, there is an increased need for new therapeutic strategies with better efficacy and less toxicity.
[00243] Here, NCL targeting by N6L enhanced the mTOR pathway inducing a reprogramming of the translatome by an increase of specific mRNAs (including 5’TOP mRNAs) in the polysomes, while the translation of most of the mRNAs is strongly down regulated. This specific recruitment of mRNAs coding for ribosomal proteins and for factors required for the regulation of translation could be involved in mechanisms put in place by the cell in reaction to N6L to try to better resist the inhibitory effects of the drugs.
[00244] Indeed, when N6L was combined with mTOR inhibitors a synergistic inhibitory effect was observed in different 2D and 3D preclinical models, including PDX and organoid- derived tumors that faithfully reproduce the molecular features of PDAC human tumors, display a high predictive value for clinical efficacy in patients and are critically needed for the development of new treatments. Two classes of mTORi were utilized, allosteric mTORCl inhibitor Rapamycin (RAPA, Sirolimus) and dual mTORCl/mTORC2 inhibitors AZD2014 (Vistusertib) and INK128 (Sapanisertib) (Pike, K. G. etal. 2013 and Hsieh, A. C. etal. 2012). Furthermore, it was demonstrated that the synergistic effect to inhibit cell proliferation and viability is maintained even when the PDAC cells are co-treated with low doses of N6L and mTORi (data not shown), thereby helping to prevent mTORi toxicity side effects observed in the clinic through the reduced mTORi doses. Also, it was confirmed by western-blotting analysis that the inactivation of the mTOR pathway upon the N6L and mTORi dual treatment. [00245] Here, it has been demonstrated that NCL targeting by N6L treatment sensitizes PDAC cells to mTOR inhibitors. Furthermore, a novel therapeutic strategy based on mTOR inhibition combined to NCL targeting by N6L to treat cancers, such as PDAC, has been surprisingly discovered.
[00246] The data described above demonstrates for the first time that Nucleolin targeting by N6L induces a decrease of ribosome biogenesis, protein synthesis, and global translation. This translational reprogramming is orchestrated by the activation of mTOR (mechanistic target of rapamycin) pathway. Then the combination of N6L treatment with mTORi drugs that are clinically validated were examined. It was demonstrated that the mTORi base-combined therapy in association with N6L acts synergistically to inhibit PDAC cell growth and viability. [00247] It was also shown that the level of Ncl mRNA does not change upon N6L treatment as previously reported in mice PDAC tumor treated with N6L (Gilles, Maione, Cossutta, Carpentier, Caruana, Di Maria, et al. 2016). 2016). Unlike here where RNA was extracted from one cell type (mPDAC), RNA was extracted from mice PDAC treated tumors, which consisted of multiple cell types. However, the level of NCL protein decreases upon N6L treatment. NCL may self-regulate its own transcription and/or translation.
[00248] N6L affects the synthesis of NCL protein. The consequences of the NCL down- regulation are the disorganization of the nucleolar structure as shown by electronic microscopy images of the mPDAC nucleolus ultrastructure and a decrease of rDNA transcription by decreasing the expression of the 47S pre-ribosomal RNA. NCL is bound to pre-ribosomal RNA, but it is not found in mature cytoplasmic ribosomes suggesting that NCL is only involved in early steps of ribosome biogenesis. Indeed, NCL plays many functions in ribosome biogenesis, from rDNA chromatin structure, RNA polymerase I transcription regulation to the maturation of pre-rRNA and assembly of pre-ribosomes (for review see (Ugrinova et al. 2018)). The data here demonstrates that N6L affects the nucleolar main function of NCL probably very important to explain vital function of NCL for cell proliferation (Ugrinova et al. 2007).
[00249] Further, it was demonstrated that NCL down-regulation by N6L induces a decrease of protein synthesis and global translation rate in PDAC cells. This result highlights another important cytoplasmic function of nucleolin, although the cytoplasmic fraction of NCL probably does not represent more than a few % of the total protein. It has been reported that NCL binds and regulates mRNA involved in cell proliferation and apoptosis, such as BCL-2 (Sengupta et al. 2004), AKT (Abdelmohsen et al. 2011), or TP53 mRNA (Chen, Guo, et Kastan 2012) thanks to its RNA binding properties. By regulating their stability and translation rate, NCL possesses both anti-apoptotic and proto-oncogenic properties. Indeed, it was found that down-regulation ofNCL by N6L induces a decrease of PD AC cell proliferation and an increase of apoptosis, as previously demonstrated.
[00250] NCL targeting by N6L induces a translational reprogramming revealing that a subset of genes remains unexpectedly translationally active when global translation is inhibited. These genes are implicated in translation machinery and cell metabolism. It is believed that the cells activate this subset of genes to counteract the negative effect of N6L on cell survival, establishing a cell resistant mechanism to this N6L treatment. The strategy adopted by a subset of these genes to counteract the energy-stress-mediated inhibition involves their translational control by the 5’ TOP. The majority of these are well-expressed, supporting a hypothesis whereby TOP motifs permit rapid and reversible regulation of these mRNAs without the metabolic cost of degrading and resynthesizing the transcripts.
[00251] The 5’TOP motif is defined as a +1 C directly adjacent to the 5' cap structure and followed by a series of 4 to 14 pyrimidine nucleotides, which renders translation of the mRNA hypersensitive to a variety of growth signals, including those transmitted by the mTORCl pathway (Meyuhas et Kahan 2015). How mTOR regulates TOP mRNA translation has been a persistent mystery.
[00252] Many potential regulators have been proposed as trans-acting factors that could regulate the translation of 5’TOP mRNA (positively or negatively), including the T-cell intracellular antigen- 1 (TIA1) and TIA-1 -related (TIAR) proteins, miR-lOA, 4EBP1 and LARPl (Patursky -Polischuk et al. 2014). However, the exact role of LARPl in the regulation of TOP mRNAs remains controversial. While LARPl was shown to regulate TOP mRNA stability, it has also been described as a positive or negative regulator of TOP mRNA translation, depending on the context. LARPl interacts with raptor and is phosphorylated by mTORCl, which is thought to modulate its mRNA-binding activity (Hong et al. 2017; Philippe et al. 2020). Here, the accumulation of LARPl upon N6L treatment and the maintain (increase) of 5’ TOP mRNA translation suggested that LARPl could act as positive regulator (interaction of LARPl with other protein/complex in PD AC cells). Mechanistically, the translation of these 5’UTR motifs is under the mTOR pathway.
[00253] The mTOR pathway is crucial to control cell growth and survival in physiological and pathological conditions. It acts in two distinct mTOR complexes, mTORCl and mTORC2, which differ in associated proteins and by sensitivity to rapamycin and its derivatives (Sabatini 2006). The major function of mTORCl is promotion of cellular growth and proliferation via increasing protein synthesis and inhibiting autophagy. Functions of mTORC2 are less well studied and include organization of the actin cytoskeleton, control of cellular metabolism, and anti-apoptotic properties via stimulation of the AKT-FOXO pathway.
[00254] The mTOR pathway is reported to be significantly active in several cancers including PDAC (Tian, Li, et Zhang 2019; Bellizzi et al. 2010). In addition, mTOR is implicated in drug resistance (Guri et Hall 2016). In fact, NCL targeting by N6L induced an activation of the mTOR pathway in the data above, allowing PDAC cells to escape N6L treatment. In order to bypass this drug resistance, mTOR inhibitors were examined in combination with N6L in different preclinical models of human and murine PDAC cells. Two classes of mTORi were examined, allosteric mTORCl inhibitor Rapamycin (RAPA, Sirolimus) and dual mTORCl/mTORC2 inhibitors AZD2014 (Vistusertib) and INK128 (Sapanisertib) (Pike et al. 2013)(Hsieh et al. 2012).
[00255] It was surprisingly discovered that the combo-therapy of N6L and mTORi induces a synergetic effect on PD AC cell growth and viability. In light of these encouraging results, it was demonstrated that NCL targeting by N6L treatment sensitizes PDAC cells to mTOR inhibitors. Furthermore, a novel therapeutic strategy based on mTOR inhibition combined to NCL targeting by N6L to treat PDAC was surprisingly discovered.
[00256] In PDAC, there is an increased need for new therapeutic strategies with better efficacy and less toxicity. mTOR inhibitors should be considered as a valuable addition to chemotherapy or targeted cancer therapy, either as an option for relapsed patients or as a frontline combination therapy to prevent or delay the development of resistance due to sustained mTOR signaling (Rodrik-Outmezguine et al. 2016). However, the effects of mTOR inhibitors utilized as monotherapy in cancer are sometimes dampened by several resistance mechanisms. On the other hand, combined therapies with mTOR inhibitors and other pathway inhibitors or conventional therapies are under investigation in preclinical and clinical trials in different tumor types. Hence, novel therapeutic strategies based on mTOR inhibition still need to be developed to treat PDAC cancers.
[00257] Materials and Methods.
[00258] Material. The nucleolin antagonist N6L (NUCANT®) was obtained from UREKA Pharma-ImmuPharma compagny. The mTOR inhibitors, Rapamycin (Sirolimus from Pfizer®) and INK128 (Sapanisertib, TAK128 or MLN128 from Takeda oncology®) were purchased and stock solutions were made in DMSO.
[00259] Cell lines and RNAi transfection. PDAC cell lines, MiaPaca-2, Panc-1, mPDAC were maintained in DMEM medium and HeLa cells were maintained in aMEM medium containing Glutamax (PAA) 1% non-essential amino acids, supplemented with 10% FBS and 1% Penicillin/Streptomycin. PDAC PDX cell lines were purchased from CTIBIOTECH and maintained in CTIM.Cancer2 medium. All cells were incubated at 37°C in a humidified incubator with 5% C02. Routine Mycoplasma testing was performed by MycoAlert Mycoplasma Detection Kit (catalog no. LT07-118).
[00260] A mixture of functional siRNAs (Eurogentec) specific for nucleolin was used as previously described (Kumar et al. 2017). siRNAs, siRNA #4 (UU CUUU GAC AGGCU CUUCCUU or SEQ ID No. 21) and siRNA #2 (UCCAAGGUAACUUUAUUUCUU or SEQ ID No. 22), were reconstituted at a concentration of 100 nM and stored at -20 °C. As a siRNA control, we used stealth high GC siRNA (Invitrogen). Cells were transfected in a 6-well dish using siRNA at 2 nM final concentration. siRNAs were diluted in 200 mΐ of OptiMEM (Gibco) and plated in a well. 80 mΐ of INTERFERE (Polyplus) diluted 1:10 in RNase-free water were added. After 10 minutes incubation, 2 ml of medium containing 3 x 105 cells were added. RNA extraction and protein analysis were performed 2 days after the initial transfection.
[00261] Live-cell imaging analysis, Spheroid and Organoid formation. Cells were seeded in 96-well plates and allowed to adhere overnight or 2 days. Cells were treated with indicated concentrations of N6L and/or mTORi, for 48 or 72 hours. Cell growth was assessed by IncuCyte ZOOM® live-cell imaging to measure cell confluence after N6L and/or mTORi treatments. IncuCyte cell recognition software calculated values based on cellular size over time, cellular count per field of view or percentage of cell confluence over time (Essen Biosciences, Ann Arbor, MI).
[00262] For spheroid assay, PD AC cell lines were cultured in 96-well ultra -low attachment (ULA) plate and allowed to form spheroid overnight. Next day, PD AC spheroids were treated or not treated with different concentration of N6L alone or in combination with mTORi, RAPA and INK128 in 4 replicates. Spheroid growth was monitored by IncuCyte ZOOM® live-cell imaging. Over 96h, the spheroid area was calculated using ImageJ software and data were normalized to control.
[00263] For Oragnoid formation, mPDAC cells (lxlO3 cells/mouse in 50 pL PBS) were orthotopically injected into the pancrease of FVB/n syngeneic mice, as previously described (Gilles, M.-E., et al. 2016). Mice were sacrificed after 4 weeks and tumor-derived organoids were established by following Tuveson’s laboratory protocol (Boj, S. F. et al. 2015). Briefly, tumors were digested with 0.012% (w/v) collagenase XI (Sigma) and 0.012% (w/v) dispase (Gibco) in DMEM media containing 1% FBS. The resulting cell suspension was incorporated into growth factor-reduced Matrigel (Coming) to obtain a 3D culture.
[00264] Viability assay (MTS) and apoptosis analysis. Cell viability was assessed by MTS assay (CellTiter 96 AQueous MTS Reagent, Promega). Briefly, cell medium was supplemented with MTS reagent 20 mΐ/well, incubated for 2 h, and then the absorbance at 450nm was recorded on TECAN microplate Reader (Sunrise®). Viability for treated cells was normalized to non-treated cells on the same plate.
[00265] Apoptosis analysis was performed on the IncuCyte ZOOM® with Caspase-3/7 Green Reagent for Apoptosis (Essen BioScience). Cells were treated or not treated with increasing concentrations of N6L in 96-well plates in triplicate and Caspase-3/7 reagent reagent was added to the cells. Phase-contrast and fluorescent images were acquired 72 hours later. IncuCyte ZOOM® live-imaging software was used to count the fluorescent object number per mm3 in each well which reflects the caspase 3/7 activation. Staurosporine (ImM) was used as a positive control for inducing apoptosis.
[00266] Immunofluorescence and electronic microscopy. For immunofluorescence staining, mPDAC cells were seeded onto cover slips in 24-well plate (BD Falcon) to adhere overnight. The next day, cells were treated or not treated with the increasing concentrations of N6L. After 48 hours of treatment, cells were rinsed with cold PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature followed by permeabilization with 0.1% Triton X-100. The cells were subjected to immunofluorescence staining with phospho-RPS6 (CST) for 2 hours at room temperature. Then, washed with cold PBS three times for 5 minutes each, cells were incubated with Alexa 555-labeled anti-rabbit secondary antibody (1:500) (BD Bioscience) at room temperature for 1 hour and then washed with cold PBS three times for 5 minutes each. Cell nuclei were counterstained with DAPI and cover slips were rinsed with distilled water and mounted using fluorescent mounting medium (Invitrogen). Cells were visualized under a confocal microscope (Zeiss LSM510).
[00267] For electronic microscopy micrograph, cells were fixed in glutaraldehyde 2%. The sample were washed three times in saccharose 0.4M and Na C-HCl-Cacodylate 0,2M pH7.4 for lhr at 4°C, and post-fixed with 2% Os04 and Na C-HC1 Cacodylate 0.3M pH7.4 30 minutes at RT. Then cells were dehydrated with a increasing ethanol gradient (5minutes in 30%, 50%, 70%, 95%, and 3 times for 10 minutes in absolute ethanol). Impregnation was performed with Epon A (50%) plus Epon B (50%) plus DMP30 (1,7%). Inclusion was obtained by polymerization at 60°C for 72hrs. Ultrathin sections (approximately 70 nm thick) were cut on a ultracut UC7 (Leica) ultramicrotome, mounted on 200 mesh copper grids coated with 1:1,000 polylisine, and stabilized for lday at room temperature (RT) and, contrasted with uranyl acetate. Sections were examined with a Jeol 1400JEM (Tokyo, Japan) transmission electron microscope equipped with a Orius 600 camera and Digital Micrograph.
[00268] Dual-Luciferase reporter assay for 5’TOP constructs. NCL-depleted cells (3000/well) or N6L-treated cells or not (2000/well) were seeded in 96-well plates over 24 hours. For 5’TOP activity assay, cells were co-transfected with 25 ng of monocistronic reporter containing 5’ TOP motif of Rps3 , Rpl36 or Eif3a genes (GenScript) and 25 ng of pRL construct constitutively expressing the Renilla luciferase gene as an internal control for transfection efficiency. Luciferase assays were performed 2 hours after transfection with the reporter plasmids using X-tremeGENE 9 reagent (Roche). Dual luciferase assays were performed using the Dual-Glo luciferase reagent (Promega) according to the manufacturer’s instructions, and a Tecan Ml 000 plate reader. The relative luciferase activity was calculated by firefly luciferase activity I Renilla luciferase activity.
[00269] SUnSET for Global protein synthesis. Global protein synthesis analysis by puromycylation followed by puromycin detection was performed as previously described (David et al. 2012). Briefly, puromycin (1 pg/mL) was added to the N6L treated or non-treated cell medium for 2 hours at 37°C. Cells were harvested, lysed in Laemmli buffer and loaded onto polyacrylamide gel. Puromycin incorporation was detected by Western blot on whole-cell protein extracts.
[00270] Western blot analysis. Twenty micrograms of total protein lysates were run on a 10-15% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane. The membrane was blocked with 3% nonfat milk in TBST. The antibodies used are shown in Table 3 below. Antibodies were incubated for 90 minutes in 3% milk-TBST. Proteins were detected by chemiluminescence using an anti-rabbit or anti-mouse peroxidase-conjugated antibody (Cell Signaling) diluted 1 : 10,000, and Clarity ECL substrate (Bio- Rad). Images were collected on a ChemiDoc XRS+ (Bio-Rad) and the signal analyzed using the Bio-Rad ImageLab software.
Table 3. Antibodies utilized in Western blot analysis
Figure imgf000083_0001
Figure imgf000084_0001
[00271] Polysome profiling and RNA extraction. Cells were cultured in 15 cm-dishes and were allowed to adhere overnight. Cells were then treated or not for48 hours with 50 mM N6L. Before cell lysis, cells were incubated with emetine (25 pg/mL) for 15 minutes then washed with cold PBS. For cell fractionation, lOxlO6 cells were suspended in lysis hypotonic buffer (10 mM KC1, 0.5mM MgC12, 10 mM Tris-HCl pH 7.4) for 10 minutes at 4°C. Cells were homogenized using Precellys® Evolution homogenizer (OZYME) and successively centrifuged at 700g and 1200g to eliminate the nuclei and the mitochondria, respectively. Cytoplasmic lysates (1-2 mg of protein) were loaded onto 15%-47% sucrose density gradients and centrifuged at 217,000 g (SW 40 Ti rotor, Beckman Coulter, Inc.) for 2 hours at 4°C. Gradients were fractionated into 19 fractions and the OD at 254 nm was continuously recorded using an ISCO fractionator (Teledyne ISCO).
[00272] RNA from cytosolic and polysomal pooled fractions was extracted and purified using TRIzol reagent (Invitrogen). For RT-QPCR, cne hundred nanograms of total RNA was reverse-transcribed using hexamer random primers and first-strand cDNA synthesis kit (Fermentas) and the synthesized cDNA was used for RT-QPCR using FastStart Universal SYBR Green Master (ROX) (Roche).
[00273] Library preparation and Illumina sequencing were performed at the Ecole Normale Superieure genomic core facility (Paris, France). Messenger (polyA+) RNAs were purified from 1 pg of total RNA using oligo(dT). Libraries were prepared using the strand specific RNA-Seq library preparation TruSeq Stranded mRNA kit (Illumina). Libraries were multiplexed by 12 on 1 high-output flow cells. A 75 bp single read sequencing was performed on a NextSeq 500 (Illumina). A mean of 39,7 ± 10 millions passing Illumina quality filter reads was obtained for each of the 12 samples. The analyses were performed using the Eoulsan pipeline (Jourdren et al. 2012), including read filtering, mapping, alignment filtering, read quantification, normalization and differential analysis. Before mapping, poly N read tails were trimmed, reads <40 bases were removed, and reads with quality mean <30 were discarded. Reads were then aligned against the Mus musculus genome from Ensembl version 88 using STAR (version 2.5.2b) (Dobin et al. 2013). Alignments from reads matching more than once on the reference genome were removed using Java version of samtools (Li et al. 2009). To compute gene expression, Mus musculus GTF genome annotation version 88 from Ensembl database was used. All overlapping regions between alignments and referenced genes were counted using HTSeq-count 0.5.3 (Anders, Pyl, et Huber 2015). The RNASeq gene expression data and raw fastq files are available on the GEO repository (www.ncbi.nlm.nih.gov/geo/) under accession number: GSEXXX. The sample counts were normalized using DESeq2 1.8.1 (Love, Huber, et Anders 2014). Statistical treatments and differential analyses were also performed using DESeq2 1.8.1.
[00274] Analysis of synergy/antagonism from combination studies. To determine possible additive and synergistic effects when using combinations of nucleolin antagonist (N6L) with mTOR inhibitors (mTORi), the data from cell growth assays (IncuCyte ZOOM® live-imaging software) were analyzed using the freely available software, Combenefit (Di Veroli et al. 2016) from CRUCK Cambridge Institut), which simultaneously assesses synergy/antagonism using three published models [Highest single agent (HSA), Bliss, and Loewe] The software calculates a synergy score for each combination, where a positive score indicates synergy, a score of 0 is additive, and a negative score indicates antagonism. The "3D Surface", “contour” and "Matrix" views were selected as graphical outputs for the synergy distribution and are represented in the Results/Figures section.
[00275] Statistics. Statistical analyses were performed by using GraphPad Prism software (version 6). Bars represent mean ± SEM (n>3). For two-group comparisons, we analyzed the data using two-tailed Student t test. For multiple group comparisons, 1-way ANOV A Rank with Dunn method was used (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001) and n.s. for not statistically significant.
[00276] The contents of all references, patents, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. [00277] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims. It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only, and are in no way considered to be limiting to the disclosure. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application and are considered within the scope of the appended claims. For example, the relative quantities of the ingredients may be varied to optimize the desired effects, additional ingredients may be added, and/or similar ingredients may be substituted for one or more of the ingredients described. Additional advantageous features and functionalities associated with the systems, methods, and processes of the present disclosure will be apparent from the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.
References:
Abdelmohsen, Kotb, Kumiko Tominaga, Eun Kyung Lee, Subramanya Srikantan, Min-Ju Kang, Mihee M. Kim, Roza Selimyan, et al. 2011. Enhanced Translation by Nucleolin via G-Rich Elements in Coding and Non-Coding Regions of Target MRNAs. Nucleic Acids Research 39 (19): 8513-30.
Al-Ashtal, H. A., Rubottom, C. M., Leeper, T. C. & Berman, A. J. The LARPl La-Module recognizes both ends of TOP mRNAs. RNA Biol. 18, 248-258 (2021).
Anders, S., Pyl, P. T. & Huber, W. HTSeq— a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166-169 (2015).
Aoki, Kazuma, Shungo Adachi, Masae Homoto, Hideo Kusano, Katsuyuki Koike, et Tohru Natsume. 2013. LARPl Specifically Recognizes the 3' Terminus of Poly(A) MRNA. FEBS Letters 587 (14): 2173-78.
Bachet, Jean-Baptiste, Pascal Hammel, Jerome Desrame, Aurelia Meurisse, Benoist Chibaudel, Thierry Andre, Philippe Debourdeau, et al. 2017. Nab-Paclitaxel plus Either Gemcitabine or Simplified Leucovorin and Fluorouracil as First-Line Therapy for Metastatic Pancreatic Adenocarcinoma (AFUGEM GERCOR): A Non-Comparative, Multicentre, Open-Label, Randomised Phase 2 Trial. The Lancet Gastroenterology & Hepatology 2 (5): 337-46.
Bellizzi, Andrew M., Mark Bloomston, Xiao-Ping Zhou, Obiajulu Hans Iwenofu, et Wendy L. Frankel. 2010. The MTOR Pathway Is Frequently Activated in Pancreatic Ductal Adenocarcinoma and Chronic Pancreatitis. Applied Immunohistochemistry & Molecular Morphology, juillet, 1.
Benedetti, Elisabetta, Andrea Antonosante, Michele d’ Angelo, Loredana Cristiano, Renato Galzio, Damien Destouches, Tiziana Marilena Florio, et al. 2015. Nucleolin Antagonist Triggers Autophagic Cell Death in Human Glioblastoma Primary Cells and Decreased in Vivo Tumor Growth in Orthotopic Brain Tumor Model. Oncotarget 6 (39).
Boj, S. F. et al. Organoid Models of Human and Mouse Ductal Pancreatic Cancer. Cell 160, 324-338 (2015).
Birmpas, Charalampos, Jean Paul Briand, Jose Courty, etPanagiotis Katsoris. 2012. Nucleolin Mediates the Anti angiogenesis Effect of the Pseudopeptide N6L. BMC Cell Biology 13 (1): 32.
Chen, Jing, Kexiao Guo, et Michael B. Kastan. 2012. Interactions of Nucleolin and Ribosomal Protein L26 (RPL26) in Translational Control of Human P53 MRNA. Journal of Biological Chemistry 287 (20): 16467-76. Clinical trials clinical trials. gov/ct2/results?cond=mTOR+inhibitors&term=&cntry=&state=&city=&dist
Cong, Rong, Sadhan Das, Iva Ugrinova, Sanjeev Kumar, Fabien Mongelard, Jiemin Wong, et Philippe Bouvet. 2012. Interaction of Nucleolin with Ribosomal RNA Genes and Its Role in RNA Polymerase I Transcription. Nucleic Acids Research 40 (19): 9441-54.
David, Alexandre, Brian P. Dolan, Heather D. Hickman, Jonathan J. Knowlton, Giovanna Clavarino, Philippe Pierre, Jack R. Bennink, et Jonathan W. Yewdell. 2012. Nuclear Translation Visualized by Ribosome-Bound Nascent Chain Puromycylation. The Journal of Cell Biology 197 (1): 45-57.
Destouches, Damien, Diala El Khoury, Yamina Hamma-Kourbali, Bernard Krust, Patricia Albanese, Panagiotis Katsoris, Gilles Guichard, Jean Paul Briand, Jose Courty, et Ara G. Hovanessian. 2008. Suppression of Tumor Growth and Angiogenesis by a Specific Antagonist of the Cell-Surface Expressed Nucleolin. PloS One 3 (6): e2518.
Destouches, Damien, Nicolas Page, Yamina Hamma-Kourbali, Valerie Machi, Olivier Chaloin, Sophie Frechault, Charalampos Birmpas, et al. 2011. A Simple Approach to Cancer Therapy Afforded by Multivalent Pseudopeptides That Target Cell -Surface Nucleoproteins. Cancer Research 71 (9): 3296-3305.
Dhez, Anne-Chloe, ElisabettaBenedetti, Andrea Antonosante, Gloria Panella, Brigida Ranieri, Tiziana M. Florio, Loredana Cristiano, et al. 2018. Targeted Therapy of Human Glioblastoma via Delivery of a Toxin through a Peptide Directed to Cell Surface Nucleolin. Journal of Cellular Physiology 233 (5): 4091-4105.
Di Veroli, Giovanni Y., Chiara Fomari, Dennis Wang, Severine Mollard, Jo L. Bramhall, Frances M. Richards, et Duncan I. Jodrell. 2016. Combenefit: An Interactive Platform for the Analysis and Visualization of Drug Combinations. Bioinformatics 32 (18): 2866-68.
Dobin, A. etal. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21 (2013).
Farin, Keren, Sari Schokoroy, Roni Haklai, Ifat Cohen-Or, Galit Elad-Sfadia, Merit E. Reyes- Reyes, Paula J. Bates, Adrienne D. Cox, Yoel Kloog, et Ronit Pinkas-Kramarski. 2011. Oncogenic Synergism between ErbBl, Nucleolin, and Mutant Ras. Cancer Research 71 (6): 2140-51.
Fonseca, Bruno D., Roni M. Lahr, Christian K. Damgaard, Tommy Alain, et Andrea J. Berman. 2018. LARPl on TOP of Ribosome Production. Wiley Interdisciplinary Reviews: RNA 9 (5): el480.
Gentilella, Antonio, Francisco D. Moron-Duran, Pedro Fuentes, Guilherme Zweig-Rocha, Ferran Riano-Canalias, Joffrey Pelletier, Marta Ruiz, et al. 2017. Autogenous Control of 5'TOP MRNA Stability by 40S Ribosomes. Molecular Cell 6Ί (1): 55-70. e4.
Gilles, Maud-Emmanuelle, Federica Maione, Melissande Cossutta, Gilles Carpentier, Laure Caruana, Silvia Di Maria, Claire Houppe, et al. 2016. Nucleolin Targeting Impairs the Progression of Pancreatic Cancer and Promotes the Normalization of Tumor Vasculature. Cancer Research 76 (24): 7181-93.
Gunturu, Krishna S., Xiaopan Yao, Xiangyu Cong, Jaykumar R. Thumar, Howard S. Hochster, Stacey M. Stein, et Jill Lacy. 2013. FOLFIRINOX for Locally Advanced and Metastatic Pancreatic Cancer: Single Institution Retrospective Review of Efficacy and Toxicity. Medical Oncology 30 (1): 361.
Guo, XiaoDong, Lu Xiong, Lingxiang Yu, Ruisheng Li, ZhaoHai Wang, Bo Ren, JingHui Dong, Boan Li, et Dadong Wang. 2014. Increased Level of Nucleolin Confers to Aggressive Tumor Progression and Poor Prognosis in Patients with Hepatocellular Carcinoma after Hepatectomy. Diagnostic Pathology 9 (1): 175.
Guri, Yakir, et Michael N. Hall. 2016. MTOR Signaling Confers Resistance to Targeted Cancer Drugs. Trends in Cancer 2 (11): 688-97.
Hassan, Z. et al. MTOR inhibitor-based combination therapies for pancreatic cancer. Br. .!. Cancer 118, 366-377 (2018).
Hong, Sungki, Mallory A Freeberg, Ting Han, Avani Kamath, Yao Yao, Tomoko Fukuda, Tsukasa Suzuki, John K Kim, et Ken Inoki. 2017. LARPl Functions as a Molecular Switch for MTORC1 -Mediated Translation of an Essential Class of MRNAs. ELife 6: e25237.
Hovanessian, Ara G., Francine Puvion-Dutilleul, Sebastien Nisole, Josette Svab, Emmanuelle Perret, Jau-Shyong Deng, et Bernard Krust. 2000. The Cell-Surface-Expressed Nucleolin Is Associated with the Actin Cytoskeleton. Experimental Cell Research 261 (2): 312-28.
Hsieh, Andrew C., Yi Liu, Merritt P. Edlind, Nicholas T. Ingolia, Matthew R. Janes, Annie Sher, Evan Y. Shi, et al. 2012. The Translational Landscape of MTOR Signalling Steers Cancer Initiation and Metastasis. Nature 485 (7396): 55-61.
Jourdren, L., Bernard, M., Dillies, M.-A. & Le Crom, S. Eoulsan: a cloud computing-based framework facilitating high throughput sequencing analyses. Bioinformatics 28, 1542-1543 (2012).
Kamisawa, Terumi, Laura D Wood, Takao Itoi, et Kyoichi Takaori. 2016. Pancreatic Cancer. The Lancet 388 (10039): 73-85.
Koutsioumpa, Marina, et Evangelia Papadimitriou. 2014. Cell Surface Nucleolin as a Target for Anti-Cancer Therapies. Recent Patents on Anti-Cancer Drug Discovery 9 (2): 137-52. Krust, Bernard, Diala El Khoury, Isabelle Nondier, Calaiselvy Soundaramourty, et Ara G. Hovanessian. 2011. Targeting Surface Nucleolin with Multivalent HB-19 and Related Nucant Pseudopeptides Results in Distinct Inhibitory Mechanisms Depending on the Malignant Tumor Cell Type. BMC Cancer 11 : 333.
Kuleshov, Maxim V, Jennifer E L Diaz, Zachary N Flamholz, Alexandra B Keenan, Alexander Lachmann, Megan L Wojciechowicz, Ross L Cagan, et Avi Ma’ayan. 2019. ModEnrichr: A Suite of Gene Set Enrichment Analysis Tools for Model Organisms. Nucleic Acids Research 47 (Wl): W183-90.
Kuleshov, Maxim V., Matthew R. Jones, Andrew D. Rouillard, Nicolas F. Fernandez, Qiaonan Duan, Ziehen Wang, Simon Koplev, et al. 2016. Enrichr: A Comprehensive Gene Set Enrichment Analysis Web Server 2016 Update. Nucleic Acids Research 44 (Wl): W90-97.
Kumar, Sanjeev, Elizabhet Cruz Gomez, Mounira Chalabi-Dchar, Cong Rong, Sadhan Das, Iva Ugrinova, Xavier Gaume, Karine Monier, Fabien Mongelard, et Philippe Bouvet. 2017. Integrated Analysis of MRNA and MiRNA Expression in HeLa Cells Expressing Low Levels of Nucleolin. Scientific Reports 7 (1): 9017.
Levy, S., Avni, D., Hariharan, N., Perry, R. P. & Meyuhas, O. Oligopyrimidine tract at the 5’ end of mammalian ribosomal protein mRNAs is required for their translational control. Proc. Natl. Acad Sci. 88, 3319-3323 (1991).
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078- 2079 (2009).
Love, M. T, Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Marcel, Virginie, Frederic Catez, Caroline M. Berger, Emeline Perrial, Adriana Plesa, Xavier Thomas, Eve Mattel, et al. 2017. Expression Profiling of Ribosome Biogenesis Factors Reveals Nucleolin as a Novel Potential Marker to Predict Outcome in AML Patients. Edite par Arun Rishi. PLOS ONE 12 (1): e0170160.
Meyuhas, Oded, et Tamar Kahan. 2015. The Race to Decipher the Top Secrets of TOP MRNAs. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1849 (7): 801-11.
Patursky -Polischuk, Ilona, Judith Kasir, Rachel Miloslavski, Zvi Hayouka, Mirit Hausner- Hanochi, Miri Stolovich-Rain, Pinchas Tsukerman, et al. 2014. Reassessment of the Role of TSC, MTORC1 and MicroRNAs in Amino Acids-Meditated Translational Control of TOP MRNAs. Edite par Thomas Preiss. PLoS ONE 9 (10): el09410.
Philippe, Lucas, Antonia M. G. van den Elzen, Maegan J. Watson, et Carson C. Thoreen. 2020. Global Analysis of LARP1 Translation Targets Reveals Tunable and Dynamic Features of 5' TOP Motifs. Proceedings of the National Academy of Sciences 117 (10): 5319-28.
Pike, Kurt G., Karine Malagu, Marc G. Hummersone, Keith A. Menear, Heather M.E. Duggan, Sylvie Gomez, Niall M.B. Martin, Linette Ruston, Sarah L. Pass, et Martin Pass. 2013. Optimization of Potent and Selective Dual MTORC1 and MTORC2 Inhibitors: The Discovery of AZD8055 and AZD2014. Bioorganic & Medicinal Chemistry Letters 23 (5): 1212-16.
Qiu, Wensheng, Fang Zhou, Qian Zhang, Xiaoxiao Sun, Xiaoli Shi, Ye Liang, Xiumei Wang, et Lu Yue. 2013. Overexpression of Nucleolin and Different Expression Sites Both Related to the Prognosis of Gastric Cancer. APMIS 121 (10): 919-25.
Ramos, Kenneth S., Sara Moore, Isabel Runge, Marco A. Tavera-Garcia, Ilaria Cascone, Jose Courty, et Elsa M. Reyes-Reyes. 2020. The Nucleolin Antagonist N6L Inhibits LINE1 Retrotransposon Activity in Non-Small Cell Lung Carcinoma Cells. Journal of Cancer 11 (3): 733-40.
Rodrik-Outmezguine, Vanessa S., Masanori Okaniwa, Zhan Yao, Chris J. Novotny, Claire McWhirter, Arpitha Banaji, Helen Won, et al. 2016. Overcoming MTOR Resistance Mutations with a New-Generation MTOR Inhibitor. Nature 534 (7606): 272-76.
Sabatini, David M. 2006. MTOR and Cancer: Insights into a Complex Relationship. Nature Reviews Cancer 6 (9): 729-34.
Sanhaji, Mourad, Julia Goring, Pierre Couleaud, Antonio Aires, Aitziber L. Cortajarena, Jose Courty, Adriele Prina-Mello, et al. 2019. The Phenotype of Target Pancreatic Cancer Cells Influences Cell Death by Magnetic Hyperthermia with Nanoparticles Carrying Gemicitabine and the Pseudo-Peptide NucAnt. Nanomedicine: Nanotechnology, Biology and Medicine 20: 101983.
Sengupta, Tapas K., Sumita Bandy opadhyay, Daniel J. Fernandes, et Eleanor K. Spicer. 2004. Identification of Nucleolin as an AU-Rich Element Binding Protein Involved in Bcl-2 MRNA Stabilization. Journal of Biological Chemistry 279 (12): 10855-63.
Sinvani, Hadar, Ora Haimov, Yuri Svitkin, Nahum Sonenberg, Ana Tamarkin-Ben-Harush, Benoit Viollet, et Rivka Dikstein. 2015. Translational Tolerance of Mitochondrial Genes to Metabolic Energy Stress Involves TISU and EIF1-EIF4GI Cooperation in Start Codon Selection. Cell Metabolism 21 (3): 479-92.
Sonenberg, Nahum, et Alan G. Hinnebusch. 2009. Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets. Cell 136 (4): 731-45.
Soreide, Kjetil, Florian Primavesi, Knut J. Labori, Martin M. Watson, et Stefan Stattner. 2019. Molecular Biology in Pancreatic Ductal Adenocarcinoma: Implications for Future Diagnostics and Therapy. European Surgery 51 (3): 126-34.
Thoreen, Carson C., Lynne Chantranupong, Heather R. Keys, Tim Wang, Nathanael S. Gray, et David M. Sabatini. 2012. A Unifying Model for MTORC1 -Mediated Regulation of MRNA Translation. Nature 485 (7396): 109-13.
Tian, Tian, Xiaoyi Li, etJinhua Zhang. 2019. MTOR Signaling in Cancer and MT OR Inhibitors in Solid Tumor Targeting Therapy. International Journal of Molecular Sciences 20 (3): 755.
Tiriac, H. et al. Organoid Profiling Identifies Common Responders to Chemotherapy in Pancreatic Cancer. Cancer Discov. 8, 1112-1129 (2018).
Ugrinova, Iva, Karine Monier, Corinne Ivaldi, Marc Thiry, Sebastien Storck, Fabien Mongelard, et Philippe Bouvet. 2007. Inactivation of nucleolin leads to nucleolar disruption, cell cycle arrest and defects in centrosome duplication. BMC Molecular Biology 8 (1): 66.
Ugrinova, Iva, Maria Petrova, Mounira Chalabi-Dchar, et Philippe Bouvet. 2018. Multifaceted Nucleolin Protein and Its Molecular Partners in Oncogenesis. In Advances in Protein Chemistry and Structural Biology , 111 : 133-64. Elsevier.
Ware, Matthew L, Kevin Colbert, Vazrik Keshishian, Jason Ho, Stuart J. Corr, Steven A. Curley, et Biana Godin. 2016. Generation of Homogenous Three-Dimensional Pancreatic Cancer Cell Spheroids Using an Improved Hanging Drop Technique. Tissue Engineering Part C: Methods 22 (4): 312-21.
Yamashita, R. et al. Comprehensive detection of human terminal oligo-pyrimidine (TOP) genes and analysis of their characteristics. Nucleic Acids Res. 36, 3707-3715 (2008).
Zhao, Hongyun, Yan Huang, Cong Xue, Yang Chen, Xue Hou, Ying Guo, Liping Zhao, et al. 2013. Prognostic Significance of the Combined Score of Endothelial Expression of Nucleolin and CD31 in Surgically Resected Non-Small Cell Lung Cancer. Edite par Mitsunobu R. Kano. PLoS ONE 8 (1): e54674.

Claims

CLAIMS What Is Claimed Is:
1. A method of treating cancer (e.g., pancreatic cancer) in a subject, the method comprising administering to a cell or a subject in need thereof an effective amount of at least one (e.g., 1, 2, 3, or 4) mechanistic target of rapamycin (mTOR) inhibitor and an effective amount of at least one (e.g., 1, 2, 3, or 4) polyvalent synthetic compound, wherein the mTOR inhibitor and the polyvalent synthetic compound are effective for treating or ameliorating at least one symptom of the cancer, wherein the polyvalent synthetic compound comprises a support comprising at least 3 pseudopeptides coupled or grafted thereto, wherein the support is a linear peptide support having the formula (la) or (lb):
(Lys-Aib-Gly)z (la), or
(Aib-Lys-Aib-Gly)z (lb), wherein: z is an integer from 3 to 20; and each pseudopeptide independently has formula (Ila) or formula (lib):
Figure imgf000093_0001
wherein: each X is independently any amino acid (e.g. any proteinogenic amino acid);
Z is proline or derivative thereof; each Y1 and Y2 is independently a basic amino acid (e.g., arginine, lysine, lysine derivative, or lysine analog); n is 0 or 1; m is 0, 1, 2, or 3; and
Y represents a reduced bond (-CH2NH-), a retro-inverso bond (-NHCO-), a methyleneoxy bond (-CH2O-), a thiomethylene bond (-CH2-S-), a carba bond (-CH2CH2-), a ketomethylene bond (-CO-CH2-), a hydroxyethylene bond (-CHOH-CH2-), an E-alkylene bond , or a C=C bond, or a pharmaceutically acceptable salt or pharmaceutically acceptable derivative thereof.
2. The method of claim 1, wherein the polyvalent synthetic compound is administered in a composition that includes a pharmaceutically acceptable carrier, excipient, or diluent.
3. The method of claim 1 or 2, wherein the support has the formula (la).
4. The method of any one of claims 1-3, wherein n and m are each 0.
5. The method of any one of claims 1-4, wherein z is an integer from 3 to 10.
6. The method of any one of claims 1-5, wherein z is 6.
7. The method of any one of claims 1-6, wherein the polyvalent synthetic compound comprises 3 to 15 pseudopeptides.
8. The method of any of claims 1-7, wherein one or more of the pseudopeptides is coupled or grafted directly on the support.
9. The method of any one of claims 1-8, wherein the pseudopeptides are grafted or coupled to the support via a lysine of the support.
10. The method of any one of claims 1-9, wherein the pseudopeptides are optically pure pseudopeptides.
11. The method of any one of claims 1-10, wherein the Y1 and the Y2 have an L configuration or D configuration.
12. The method of any one of claims 1-11, wherein the Y1 and the Y2 have an L configuration.
13. The method of any one of claims 1-12, wherein each Y1 and Y2 is independently lysine or arginine.
14. The method of any one of claims 1-13, wherein each Y1 and Y2 is a lysine having an L configuration.
15. The method of any one of claims 1-11, wherein each Y1 and Y2 is independently selected from ornithine, homolysine, and diaminoheptanoic acid (e.g., 2,7-diaminoheptanoic acid).
16. The method of any one of claims 1-15, wherein the Y represents a reduced bond.
17. The method of any one of claims 1-16, wherein the m is 0 or 1.
18. The method of any one of claims 1-17, wherein one or more Xis a proteinogenic amino acid.
19. The method of claim 1, wherein the polyvalent synthetic compound has the structure:
Figure imgf000095_0001
20. The method of any one of 1-19, wherein the mTOR inhibitor is an allosteric mTOR inhibitor.
21. The method of any one of 1-20, wherein the mTOR inhibitor is an ATP- competitive mTOR inhibitor.
22. The method of any one of claims 1-21, wherein the mTOR inhibitor is an mTORCl inhibitor, such as an allosteric mTORCl inhibitor or an ATP-competitive mTOR kinase inhibitor.
23. The method of any one of claims 1-22, wherein the mTOR inhibitor is an mTORC2 inhibitor, such as an ATP-competitive mTOR kinase inhibitor.
24. The method of any one of claims 1-23, wherein the mTOR inhibitor is selected from Rapamycin, AZD2014, and INK 128.
25. The method of any one of claims 1-24, wherein the cancer is pancreatic cancer.
26. The method of any one of claims 1-25, wherein the cancer or the pancreatic cancer is pancreatic ductile adenocarcinoma, pancreatic adenocarcinoma, pancreatic squamous cell carcinoma, pancreatic adenosquamous carcinoma, pancreatic colloid carcinoma, preferably pancreatic ductile adenocarcinoma.
27. The method of any one of claims 1-26, wherein the polyvalent synthetic compound and the mTOR inhibitor have a synergistic inhibitory effect on the pancreatic cancer cell growth and/or cell viability.
PCT/EP2022/060288 2021-04-19 2022-04-19 COMBINATION THERAPY OF NUCANT AND mTOR INHIBITORS FOR TREATING CANCER WO2022223544A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202163176647P 2021-04-19 2021-04-19
US63/176,647 2021-04-19
US202163211189P 2021-06-16 2021-06-16
US63/211,189 2021-06-16

Publications (1)

Publication Number Publication Date
WO2022223544A1 true WO2022223544A1 (en) 2022-10-27

Family

ID=81595849

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2022/060288 WO2022223544A1 (en) 2021-04-19 2022-04-19 COMBINATION THERAPY OF NUCANT AND mTOR INHIBITORS FOR TREATING CANCER

Country Status (1)

Country Link
WO (1) WO2022223544A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
US4522811A (en) 1982-07-08 1985-06-11 Syntex (U.S.A.) Inc. Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides
WO2007125210A2 (en) 2006-04-27 2007-11-08 Centre National De La Recherche Scientifique (Cnrs) Use of multivalent synthetic ligands of surface nucleolin for treating cancer or inflammation
WO2009141687A1 (en) 2008-05-22 2009-11-26 Centre National De La Recherche Scientifique (Cnrs) New optically pure compounds for improved therapeutic efficiency
WO2012045750A2 (en) 2010-10-04 2012-04-12 Centre National De La Recherche Scientifique (Cnrs) Compositions comprising multivalent synthetic ligands of surface nucleolin and glycosaminoglycans

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
US4522811A (en) 1982-07-08 1985-06-11 Syntex (U.S.A.) Inc. Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides
WO2007125210A2 (en) 2006-04-27 2007-11-08 Centre National De La Recherche Scientifique (Cnrs) Use of multivalent synthetic ligands of surface nucleolin for treating cancer or inflammation
US20110065649A1 (en) 2006-04-27 2011-03-17 Centre National De La Recherche Scientifique (Cnrs) Use of multivalent synthetic ligands of surface nucleolin for treating cancer or inflammation
WO2009141687A1 (en) 2008-05-22 2009-11-26 Centre National De La Recherche Scientifique (Cnrs) New optically pure compounds for improved therapeutic efficiency
US20110201559A1 (en) 2008-05-22 2011-08-18 Centre National De La Recherche Scientifique (Cnrs) New Optically Pure Compounds for Improved Therapeutic Efficiency
WO2012045750A2 (en) 2010-10-04 2012-04-12 Centre National De La Recherche Scientifique (Cnrs) Compositions comprising multivalent synthetic ligands of surface nucleolin and glycosaminoglycans
US20140080759A1 (en) 2010-10-04 2014-03-20 Centre National De La Recherche Scientifique (Cnrs) Compositions comprising multivalent synthetic ligands of surface nucleolin and glycosaminoglycans

Non-Patent Citations (86)

* Cited by examiner, † Cited by third party
Title
"Global Analysis of LARP1 Translation Targets Reveals Tunable and Dynamic Features of 5' TOP Motifs", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 117, no. 10, 2020, pages 5319 - 28
ABDELMOHSENKOTB, KUMIKO TOMINAGAEUN KYUNG LEESUBRAMANYA SRIKANTANMIN-JU KANGMIHEE M. KIMROZA SELIMYAN ET AL.: "Enhanced Translation by Nucleolin via G-Rich Elements in Coding and Non-Coding Regions of Target MRNAs", NUCLEIC ACIDS, vol. 39, no. 19, 2011, pages 8513 - 30
AL-ASHTAL, H. A.RUBOTTOM, C. M.LEEPER, T. C.BERMAN, A. J.: "The LARP1 La-Module recognizes both ends of TOP mRNAs", RNA BIOL, vol. 18, 2021, pages 248 - 258
ANDERS, S.PYL, P. T.HUBER, W: "HTSeq--a Python framework to work with high-throughput sequencing data", BIOINFORMATICS, vol. 31, 2015, pages 166 - 169
AOKI, KAZUMASHUNGO ADACHIMASAE HOMOTOHIDEO KUSANOKATSUYUKI KOIKETOHRU NATSUME.: "LARP1 Specifically Recognizes the 3' Terminus of Poly(A) MRNA", FEBS LETTERS, vol. 587, no. 14, 2013, pages 2173 - 78, XP071252962, DOI: 10.1016/j.febslet.2013.05.035
ATHERTON, E.; SHEPPARD, R. C.: "Solid Phase peptide synthesis: a practical approach", 1989, IRL PRESS
BACHET, JEAN-BAPTISTEPASCAL HAMMELJEROME DESRAMEAURELIA MEURISSEBENOIST CHIBAUDELTHIERRY ANDREPHILIPPE DEBOURDEAU ET AL.: "Nab-Paclitaxel plus Either Gemcitabine or Simplified Leucovorin and Fluorouracil as First-Line Therapy for Metastatic Pancreatic Adenocarcinoma (AFUGEM GERCOR): A Non-Comparative, Multicentre, Open-Label, Randomised Phase 2 Trial", THE LANCET GASTROENTEROLOGY & HEPATOLOGY, vol. 2, no. 5, 2017, pages 337 - 46
BAST, R. C. ET AL., NEW ENGLAND J. MED., vol. 309, 1983, pages 883 - 887
BELLIZZI, ANDREW M., MARK BLOOMSTON, XIAO-PING ZHOU, OBIAJULU HANS IWENOFU, ET WENDY L. FRANKEL: "The MTOR Pathway Is Frequently Activated in Pancreatic Ductal Adenocarcinoma and Chronic Pancreatitis", APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY, vol. 1, 2010
BENEDETTI, ELISABETTAANDREA ANTONOSANTEMICHELE D'ANGELOLOREDANA CRISTIANORENATO GALZIODAMIEN DESTOUCHESTIZIANA MARILENA FLORIO ET : "Nucleolin Antagonist Triggers Autophagic Cell Death in Human Glioblastoma Primary Cells and Decreased in Vivo Tumor Growth in Orthotopic Brain Tumor Model", ONCOTARGET, vol. 6, no. 39, 2015
BIRMPAS, CHARALAMPOSJEAN PAUL BRIANDJOSE COURTYPANAGIOTIS KATSORIS: "Nucleolin Mediates the Antiangiogenesis Effect of the Pseudopeptide N6L", BMC CELL BIOLOGY, vol. 13, no. 1, 2012, pages 32, XP021134598, DOI: 10.1186/1471-2121-13-32
BOJ, S. F. ET AL.: "Organoid Models of Human and Mouse Ductal Pancreatic Cancer", CELL, vol. 160, 2015, pages 324 - 338, XP055484794, DOI: 10.1016/j.cell.2014.12.021
CALLEBAUT, C.BLANCO, J.BENKIRANE, N.KRUST, B.JACOTOT, E.GUICHARD, G.SEDDIKI, N.SVAB, J.DAM, E.MULLER, S.: "Identification of V3 loop-binding proteins as potential receptors implicated in the binding of HIV particles to CD4(+) cells", J. BIOL. CHEM., vol. 273, 1998, pages 21988 - 2199
CALLEBAUT, C.JACOTOT, E.KRUST, B.GUICHAR, G.BLANCO, J.SVAB, J.MULLER, S.BRIAND, J. P.HOVANESSIAN, A. G: "Compose TASP inhibitors of HIV entry bind specifically to a 95-kDa cell surface protein", J. BIOL. CHEM., vol. 272, 1997, pages 7159 - 7166, XP002074337, DOI: 10.1074/jbc.272.11.7159
CHEN, JINGKEXIAO GUOMICHAEL B. KASTAN: "Interactions of Nucleolin and Ribosomal Protein L26 (RPL26) in Translational Control of Human P53 MRNA", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 287, no. 20, 2012, pages 16467 - 76
CHRISTIAN, S.PILCH, J.AKERMAN, M. E.PORKKA, KLAAKKONEN, P.RUOSLAHTI, E: "Nucleolin expressed at the cell surface is a marker of endothelial cells in angiogenic blood vessels", J. CELL BIOL., vol. 163, 2003, pages 871 - 878, XP002533863, DOI: 10.1083/JCB.200304132
CONG, RONGSADHAN DASIVA UGRINOVASANJEEV KUMARFABIEN MONGELARDJIEMIN WONGPHILIPPE BOUVET: "Interaction of Nucleolin with Ribosomal RNA Genes and Its Role in RNA Polymerase I Transcription", NUCLEIC ACIDS RESEARCH, vol. 40, no. 19, 2012, pages 9441 - 54
DAVID, ALEXANDREBRIAN P. DOLANHEATHER D. HICKMANJONATHAN J. KNOWLTONGIOVANNA CLAVARINOPHILIPPE PIERREJACK R. BENNINKJONATHAN W. YE: "Nuclear Translation Visualized by Ribosome-Bound Nascent Chain Puromycylation", THE JOURNAL OF CELL BIOLOGY, vol. 197, no. 1, 2012, pages 45 - 57
DE COLA A ET AL: "N6L pseudopeptide interferes with nucleophosmin protein-protein interactions and sensitizes leukemic cells to chemotherapy", CANCER LETTERS, vol. 412, 1 January 2018 (2018-01-01), pages 272 - 282, XP085268832, ISSN: 0304-3835, DOI: 10.1016/J.CANLET.2017.10.038 *
DESTOUCHES, DAMIENDIALA EL KHOURYYAMINA HAMMA-KOURBALIBERNARD KRUSTPATRICIA ALBANESEPANAGIOTIS KATSORISGILLES GUICHARDJEAN PAUL BR: "Suppression of Tumor Growth and Angiogenesis by a Specific Antagonist of the Cell-Surface Expressed Nucleolin", PLOS ONE, vol. 3, no. 6, 2008, pages e2518, XP055058929, DOI: 10.1371/journal.pone.0002518
DESTOUCHES, DAMIENNICOLAS PAGEYAMINA HAMMA-KOURBALIVALERIE MACHIOLIVIER CHALOINSOPHIE FRECHAULTCHARALAMPOS BIRMPAS ET AL.: "A Simple Approach to Cancer Therapy Afforded by Multivalent Pseudopeptides That Target Cell-Surface Nucleoproteins", CANCER RESEARCH, vol. 71, no. 9, 2011, pages 3296 - 3305
DHEZ, ANNE-CHLOE, ELISABETTA BENEDETTI, ANDREA ANTONOSANTE, GLORIA PANELLA, BRIGIDA RANIERI, TIZIANA M. FLORIO, LOREDANA CRISTIANO: "Targeted Therapy of Human Glioblastoma via Delivery of a Toxin through a Peptide Directed to Cell Surface Nucleolin. ", JOURNAL OF CELLULAR PHYSIOLOGY, vol. 233, no. 5, 2018, pages 4091 - 4105, XP071323184, DOI: 10.1002/jcp.26205
DI VEROLI, GIOVANNI Y.CHIARA FORNARIDENNIS WANGSEVERINE MOLLARDJO L. BRAMHALLFRANCES M. RICHARDSDUNCAN I. JODRELL: "Combenefit: An Interactive Platform for the Analysis and Visualization of Drug Combinations", BIOINFORMATICS, vol. 32, no. 18, 2016, pages 2866 - 68
DOBIN, A. ET AL.: "STAR: ultrafast universal RNA-seq aligner", BIOINFORMATICS, vol. 29, 2013, pages 15 - 21, XP055500895, DOI: 10.1093/bioinformatics/bts635
EMERICH, D F ET AL., CELL TRANSPLANT, vol. 8, 1999, pages 47 - 58
ENGLETON ET AL., PEPTIDES, vol. 9, 1997, pages 1431 - 1439
FARIN, KERENSARI SCHOKOROYRONI HAKLAIIFAT COHEN-ORGALIT ELAD-SFADIAMERIT E. REYES-REYESPAULA J. BATESADRIENNE D. COXYOEL KLOOGRONI: "Oncogenic Synergism between ErbB 1, Nucleolin, and Mutant Ras", CANCER RESEARCH, vol. 71, no. 6, 2011, pages 2140 - 51
FONSECA, BRUNO D., RONI M. LAHR, CHRISTIAN K. DAMGAARD, TOMMY ALAIN, ET ANDREA J. BERMAN: "LARP1 on TOP of Ribosome Production", WILEY INTERDISCIPLINARY REVIEWS: RNA, vol. 9, no. 5, 2018, pages e1480
GENTILELLA, ANTONIOFRANCISCO D. MORON-DURANPEDRO FUENTESGUILHERME ZWEIG-ROCHAFERRAN RIAFIO-CANALIASJOFFREY PELLETIERMARTA RUIZ ET : "Autogenous Control of 5'TOP MRNA Stability by 40S Ribosomes", MOLECULAR CELL, vol. 67, no. 1, 2017, pages 55 - 70, XP085122097, DOI: 10.1016/j.molcel.2017.06.005
GILLES, MAUD-EMMANUELLE, FEDERICA MAIONE, MELISSANDE COSSUTTA, GILLES CARPENTIER, LAURE CARUANA, SILVIA DI MARIA, CLAIRE HOUPPE: " Nucleolin Targeting Impairs the Progression of Pancreatic Cancer and Promotes the Normalization of Tumor Vasculature", CANCER RESEARCH, vol. 76, no. 24, 2016, pages 7181 - 93
GINISTY, H.SICARD, H.ROGER, B.BOUVET, P.: "Structure and functions of nucleolin", J. CELL SCIENCE, vol. 112, 1999, pages 761 - 772
GUNTURU, KRISHNA S., XIAOPAN YAO, XIANGYU CONG, JAYKUMAR R. THUMAR, HOWARD S. HOCHSTER, STACEY M. STEIN, ET JILL LACY: "FOLFIRINOX for Locally Advanced and Metastatic Pancreatic Cancer: Single Institution Retrospective Review of Efficacy and Toxicity", MEDICAL ONCOLOGY, vol. 30, no. 1, 2013, pages 361
GUO, XIAODONGLU XIONGLINGXIANG YURUISHENG LIZHAOHAI WANGBO RENJINGHUI DONGBOAN LIDADONG WANG: "Increased Level of Nucleolin Confers to Aggressive Tumor Progression and Poor Prognosis in Patients with Hepatocellular Carcinoma after Hepatectomy", DIAGNOSTIC PATHOLOGY, vol. 9, no. 1, 2014, pages 175, XP021198667, DOI: 10.1186/s13000-014-0175-y
GURI, YAKIRMICHAEL N. HALL: "MTOR Signaling Confers Resistance to Targeted Cancer Drugs", TRENDS IN CANCER, vol. 2, no. 11, 2016, pages 688 - 97
HASSAN ZONERA ET AL: "MTOR inhibitor-based combination therapies for pancreatic cancer", BRITISH JOURNAL OF CANCER, NATURE PUBLISHING GROUP UK, LONDON, vol. 118, no. 3, 2 January 2018 (2018-01-02), pages 366 - 377, XP037434834, ISSN: 0007-0920, [retrieved on 20180102], DOI: 10.1038/BJC.2017.421 *
HASSAN, Z: "MTOR inhibitor-based combination therapies for pancreatic cancer", CANCER, vol. 118, 2018, pages 366 - 377, XP037434834, DOI: 10.1038/bjc.2017.421
HAYES, D. F. ET AL., J. CLIN. INVEST., vol. 75, 1985, pages 1671
HEDIN, A. ET AL., PROC. NATL. ACAD. SCI., USA, vol. 80, 1983, pages 3470
HERLYN, M. ET AL., J. CLIN. IMMUNOL., vol. 2, 1982, pages 135
HONG, SUNGKIMALLORY A FREEBERGTING HANAVANI KAMATHYAO YAOTOMOKO FUKUDATSUKASA SUZUKIJOHN K KIMKEN INOKI: "LARP1 Functions as a Molecular Switch for MTORC1-Mediated Translation of an Essential Class of MRNAs", ELIFE, vol. 6, 2017, pages e25237
HOVANESSIAN, ARA G.FRANCINE PUVION-DUTILLEULSEBASTIEN NISOLEJOSETTE SVABEMMANUELLE PERRETJAU-SHYONG DENGBERNARD KRUST: "The Cell-Surface-Expressed Nucleolin Is Associated with the Actin Cytoskeleton", EXPERIMENTAL CELL RESEARCH, vol. 261, no. 2, 2000, pages 312 - 28, XP002978746, DOI: 10.1006/excr.2000.5071
HSIEH, ANDREW C.YI LIUMERRITT P. EDLINDNICHOLAS T. INGOLIAMATTHEW R. JANESANNIE SHEREVAN Y. SHI ET AL.: "The Translational Landscape of MTOR Signalling Steers Cancer Initiation and Metastasis", NATURE, vol. 485, no. 7396, 2012, pages 55 - 61, XP055120586, DOI: 10.1038/nature10912
JOLLIET-RIANTTILLEMENT, FUNDAM. CLIN. PHARMACOL., vol. 13, 1999, pages 16 - 26
JOURDREN, L.BERNARD, M.DILLIES, M.-A.LE CROM, S.: "Eoulsan: a cloud computing-based framework facilitating high throughput sequencing analyses", BIOINFORMATICS, vol. 28, 2012, pages 1542 - 1543
KAMISAWA, TERUMI, LAURA D WOOD, TAKAO ITOI, ET KYOICHI TAKAORI: "Pancreatic Cancer", THE LANCET, vol. 388, no. 10039, 2016, pages 73 - 85, XP029627521, DOI: 10.1016/S0140-6736(16)00141-0
KILLIAN, C. S. ET AL., CANCER RES., vol. 45, 1985, pages 886
KILLIAN, C. S. ET AL., J. NATL. CANCER INST., vol. 76, 1986, pages 179
KOUTSIOUMPA, MARINAEVANGELIA PAPADIMITRIOU: "Cell Surface Nucleolin as a Target for Anti-Cancer Therapies", RECENT PATENTS ON ANTI-CANCER DRUG DISCOVERY, vol. 9, no. 2, 2014, pages 137 - 52, XP009511363, DOI: 10.2174/1574892808666131119095953
KREUTER ET AL., BRAIN RES, vol. 674, 1995, pages 171 - 174
KRUST, BERNARD, DIALA EL KHOURY, ISABELLE NONDIER, CALAISELVY SOUNDARAMOURTY, ET ARA G. HOVANESSIAN: "Targeting Surface Nucleolin with Multivalent HB-19 and Related Nucant Pseudopeptides Results in Distinct Inhibitory Mechanisms Depending on the Malignant Tumor Cell Type", BMC CANCER, vol. 11, 2011, pages 333, XP021105119, DOI: 10.1186/1471-2407-11-333
KULESHOV, MAXIM V, JENNIFER E L DIAZ, ZACHARY N FLAMHOLZ, ALEXANDRA B KEENAN, ALEXANDER LACHMANN, MEGAN L WOJCIECHOWICZ, ROSS L CA: "ModEnrichr: A Suite of Gene Set Enrichment Analysis Tools for Model Organisms", NUCLEIC ACIDS RESEARCH, vol. 47, no. Wl, 2019, pages W183 - 90
KULESHOV, MAXIM V.MATTHEW R. JONESANDREW D. ROUILLARDNICOLAS F. FERNANDEZQIAONAN DUANZICHEN WANGSIMON KOPLEV ET AL.: "Enrichr: A Comprehensive Gene Set Enrichment Analysis Web Server 2016 Update", NUCLEIC ACIDS RESEARCH, vol. 44, no. Wl, 2016, pages W90 - 97
KUMAR, SANJEEVELIZABHET CRUZ GOMEZMOUNIRA CHALABI-DCHARCONG RONGSADHAN DASIVA UGRINOVAXAVIER GAUMEKARINE MONIERFABIEN MONGELARDPHI: "Integrated Analysis of MRNA and MiRNA Expression in HeLa Cells Expressing Low Levels of Nucleolin", SCIENTIFIC REPORTS, vol. 7, no. 1, 2017, pages 9017
L. A. CARPINO: "1-Hydroxy-7-azabenzotriazole. An efficient peptide coupling additive", J. AM. CHEM. SOC., vol. 1, 3, no. 10, 1993, pages 4397 - 4398, XP002010111, DOI: 10.1021/ja00063a082
LEVY, S., AVNI, D., HARIHARAN, N., PERRY, R. P. & MEYUHAS, O: " Oligopyrimidine tract at the 5' end of mammalian ribosomal protein mRNAs is required for their translational control", PROC. NATL. ACAD. SCI., vol. 88, 1991, pages 3319 - 3323, XP055044877, DOI: 10.1073/pnas.88.8.3319
LI, H. ET AL.: "The Sequence Alignment/Map format and SAMtools", BIOINFORMATICS, vol. 25, 2009, pages 2078 - 2079, XP055229864, DOI: 10.1093/bioinformatics/btp352
LOVE, M. I.HUBER, W.ANDERS, S: "Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2", GENOME BIOL., vol. 15, 2014, pages 550, XP021210395, DOI: 10.1186/s13059-014-0550-8
MARCEL, VIRGINIEFREDERIC CATEZCAROLINE M. BERGEREMELINE PERRIALADRIANA PLESAXAVIER THOMASEVE MATTEI ET AL.: "Expression Profiling of Ribosome Biogenesis Factors Reveals Nucleolin as a Novel Potential Marker to Predict Outcome in AML Patients. Edite par Arun Rishi", PLOS ONE, vol. 12, no. 1, 2017, pages e0170160
METZGAR, R. S. ET AL., PROC. NATL. ACAD. SCI., USA, vol. 81, 1984, pages 3869 - 3873
MEYUHAS, ODEDTAMAR KAHAN: "The Race to Decipher the Top Secrets of TOP MRNAs", BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - GENE REGULATORY MECHANISMS, vol. 1849, no. 7, 2015, pages 801 - 11
PAPSIDERO, L. D ET AL., CANCER RES., vol. 44, 1984, pages 4653
PATURSKY-POLISCHUKILONA, JUDITH KASIRRACHEL MILOSLAVSKIZVI HAYOUKAMIRIT HAUSNER-HANOCHIMIRI STOLOVICH-RAINPINCHAS TSUKERMAN ET AL.: "Reassessment of the Role of TSC, MTORC1 and MicroRNAs in Amino Acids-Meditated Translational Control of TOP MRNAs. Edite par Thomas Preiss", PLOS ONE, vol. 9, no. 10, 2014, pages e109410
PEKARY, A. E. ET AL., CLIN. CHEM., vol. 30, 1984, pages 1213 - 1215
PIKE, KURT G.KARINE MALAGUMARC G. HUMMERSONEKEITH A. MENEARHEATHER M.E. DUGGANSYLVIE GOMEZNIALL M.B. MARTINLINETTE RUSTONSARAH L. : "Optimization of Potent and Selective Dual MTORC1 and MTORC2 Inhibitors: The Discovery of AZD8055 and AZD2014", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 23, no. 5, 2013, pages 1212 - 16, XP028976469, DOI: 10.1016/j.bmcl.2013.01.019
PROG NEUROPSYCHOPHARMACOL BIOL PSYCHIATRY, vol. 23, 1999, pages 941 - 949
QIU, WENSHENGFANG ZHOUQIAN ZHANGXIAOXIAO SUNXIAOLI SHIYE LIANGXIUMEI WANGLU YUE: "Overexpression of Nucleolin and Different Expression Sites Both Related to the Prognosis of Gastric Cancer", APMIS, vol. 121, no. 10, 2013, pages 919 - 25, XP055429945, DOI: 10.1111/apm.12131
R. B. MERRIFIELD: "Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide", J. AM. CHEM. SOC., vol. 85, no. 14, 1963, pages 2149 - 2154, XP002257754, DOI: 10.1021/ja00897a025
RAMOS, KENNETH SSARA MOOREISABEL RUNGEMARCO A. TAVERA-GARCIAILARIA CASCONEJOSE COURTYELSA M. REYES-REYES: "The Nucleolin Antagonist N6L Inhibits LINE1 Retrotransposon Activity in Non-Small Cell Lung Carcinoma Cells", JOURNAL OF CANCER, vol. 11, no. 3, 2020, pages 733 - 40
RODRIK-OUTMEZGUINEVANESSA S.MASANORI OKANIWAZHAN YAOCHRIS J. NOVOTNYCLAIRE MCWHIRTERARPITHA BANAJIHELEN WON ET AL.: "Overcoming MTOR Resistance Mutations with a New-Generation MTOR Inhibitor", NATURE, vol. 534, no. 7606, 2016, pages 272 - 76, XP055499733, DOI: 10.1038/nature17963
SABATINI, DAVID M.: "MTOR and Cancer: Insights into a Complex Relationship", NATURE REVIEWS CANCER, vol. 6, no. 9, 2006, pages 729 - 34
SANHAJI, MOURADJULIA GORINGPIERRE COULEAUDANTONIO AIRESAITZIBER L. CORTAJARENAJOSE COURTYADRIELE PRINA-MELLO ET AL.: "The Phenotype of Target Pancreatic Cancer Cells Influences Cell Death by Magnetic Hyperthermia with Nanoparticles Carrying Gemicitabine and the Pseudo-Peptide NucAnt", NANOMEDICINE: NANOTECHNOLOGY, BIOLOGY AND MEDICINE, vol. 20, 2019, pages 101983, XP085783846, DOI: 10.1016/j.nano.2018.12.019
SEMENKOVICH, C. F.OSTLUND, R. E. J.OLSON, M. O.YANG, J. W: "A protein partially expressed on the surface of HepG2 cells that binds lipoproteins specifically is nucleolin", BIOCHEMISTRY, vol. 29, 1990, pages 9708 - 9713, XP002978747, DOI: 10.1021/bi00493a028
SENGUPTA, TAPAS K., SUMITA BANDYOPADHYAY, DANIEL J. FERNANDES, ET ELEANOR K. SPICER: "Identification of Nucleolin as an AU-Rich Element Binding Protein Involved in Bcl-2 MRNA Stabilization", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 279, no. 12, 2004, pages 10855 - 63
SINVANI, HADARORA HAIMOVYURI SVITKINNAHUM SONENBERGANA TAMARKIN-BEN-HARUSHBENOIT VIOLLETRIVKA DIKSTEIN: "Translational Tolerance of Mitochondrial Genes to Metabolic Energy Stress Involves TISU and EIF1-EIF4GI Cooperation in Start Codon Selection", CELL METABOLISM, vol. 21, no. 3, 2015, pages 479 - 92
SONENBERG, NAHUMALAN G. HINNEBUSCH: "Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets", CELL, vol. 136, no. 4, 2009, pages 731 - 45
SOREIDE, KJETIL, FLORIAN PRIMAVESI, KNUT J. LABORI, MARTIN M. WATSON, ET STEFAN STATTNER: "Molecular Biology in Pancreatic Ductal Adenocarcinoma: Implications for Future Diagnostics and Therapy", EUROPEAN SURGERY, vol. 51, no. 3, 2020, pages 126 - 34, XP036793924, DOI: 10.1007/s10353-019-0575-z
SRIVASTAVA, M.POLLARD, H. B.: "Molecular dissection of nucleolin's role in growth and cell proliferation: new insights", FASEB J, vol. 13, 1999, pages 1911 - 1922
STEWART, J. M.YOUNG, J. D.: "Solid phase peptide synthesis", vol. 91, 1984, PIERCE CHEMICAL COMPANY
THOREEN, CARSON C.LYNNE CHANTRANUPONGHEATHER R. KEYSTIM WANGNATHANAEL S. GRAYDAVID M. SABATINI: "A Unifying Model for MTORC1-Mediated Regulation of MRNA Translation", NATURE, vol. 485, no. 7396, 2012, pages 109 - 13
TIAN, TIANXIAOYI LIJINHUA ZHANG: "MTOR Signaling in Cancer and MTOR Inhibitors in Solid Tumor Targeting Therapy", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 20, no. 3, 2019, pages 755
TIRIAC, H. ET AL.: "Organoid Profiling Identifies Common Responders to Chemotherapy in Pancreatic Cancer", CANCER DISCOV, vol. 8, 2018, pages 1112 - 1129
UGRINOVA, IVAKARINE MONIERCORINNE IVALDIMARC THIRYSEBASTIEN STORCKFABIEN MONGELARDPHILIPPE BOUVET: "Inactivation of nucleolin leads to nucleolar disruption, cell cycle arrest and defects in centrosome duplication", BMC MOLECULAR BIOLOGY, vol. 8, no. 1, 2007, pages 66, XP021028236, DOI: 10.1186/1471-2199-8-66
UGRINOVA, IVAMARIA PETROVAMOUNIRA CHALABI-DCHARPHILIPPE BOUVET: "Advances in Protein Chemistry and Structural Biology", vol. 111, 2018, ELSEVIER, article "Multifaceted Nucleolin Protein and Its Molecular Partners in Oncogenesis", pages: 133 - 64
WARE, MATTHEW J., KEVIN COLBERT, VAZRIK KESHISHIAN, JASON HO, STUART J. CORR, STEVEN A. CURLEY, ET BIANA GODIN: "Generation of Homogenous Three-Dimensional Pancreatic Cancer Cell Spheroids Using an Improved Hanging Drop Technique", TISSUE ENGINEERING PART C: METHODS, vol. 22, no. 4, 2016, pages 312 - 21
YAMASHITA, R. ET AL.: "Comprehensive detection of human terminal oligo-pyrimidine (TOP) genes and analysis of their characteristics", NUCLEIC ACIDS RES., vol. 36, 2008, pages 3707 - 3715, XP055044886, DOI: 10.1093/nar/gkn248
ZHAO, HONGYUN, YAN HUANG, CONG XUE, YANG CHEN, XUE HOU, YING GUO, LIPING ZHAO: "Prognostic Significance of the Combined Score of Endothelial Expression of Nucleolin and CD31 in Surgically Resected Non-Small Cell Lung Cancer. Edite par Mitsunobu R. Kano", PLOS ONE, vol. 8, no. 1, 2013, pages e54674

Similar Documents

Publication Publication Date Title
Teicher et al. Proteasome inhibitors
US10940151B2 (en) Methods for treating renal disease
US11654149B2 (en) Calmodulin inhibitors for the treatment of ribosomal disorders and ribosomapathies
ES2784234T3 (en) Methods for treating fibrotic cancers
KR20150095780A (en) Apoptosis-inducing agent
CN109789158B (en) RUNX inhibitors
WO2019035866A1 (en) Compositions and methods for treating tuberous sclerosis complex
JP2019532049A (en) Modulation method of PCSK9 and LDLR through DRP1 inhibition
US9610331B2 (en) Methods for hematopoietic precursor mobilization
WO2018193393A1 (en) Tumor suppression by modulation of non-canonical autophagy (lap) in myeloid cells
US20230158153A1 (en) Pi3 kinase inhibitors and uses thereof
US9487559B2 (en) Compositions comprising multivalent synthetic ligands of surface nucleolin and glycosaminoglycans
WO2022223544A1 (en) COMBINATION THERAPY OF NUCANT AND mTOR INHIBITORS FOR TREATING CANCER
JP2020128365A (en) Method of liver cancer treatment with safranal formulations
WO2019113155A1 (en) Oxabicycloheptanes for treatment of secondary acute myeloid leukemia
AU2014247979B2 (en) Inhibitors of metastasis
EP3969027A1 (en) Polypeptides for treatment of cancer
US20150231203A1 (en) Treatment of pain by inhibition of usp5 de-ubiquitinase
WO2020014652A1 (en) Peptoid-peptide macrocycles, pharmaceutical compositions and methods of using the same
Tang et al. Mitochondrial outer membrane protein MTUS1/ATIP1 exerts antitumor effects through ROS-induced mitochondrial pyroptosis in head and neck squamous cell carcinoma.
Tang et al. Mitochondrial outer membrane protein MTUS1/ATIP1 exerts antitumor effects through
WO2014144858A1 (en) Therapeutic methods for treating solid tumors and related diagnostic methods
Class et al. Patent application title: THERAPEUTIC METHODS FOR TREATING SOLID TUMORS AND RELATED DIAGNOSTIC METHODS Inventors: Erdem Bangi (New York, NY, US) Ross Cagan (New York, NY, US) Assignees: Icahn School of Medicine at Mount Sinai
WO2020087126A1 (en) Methods of treatment, prevention and diagnosis
WO2020016855A1 (en) Peptides for specific inhibition of jag1-notch1 pathway

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22722285

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22722285

Country of ref document: EP

Kind code of ref document: A1