WO2014012093A1 - Promédicaments dirigés contre le cancer de la prostate et leurs méthodes d'utilisation - Google Patents

Promédicaments dirigés contre le cancer de la prostate et leurs méthodes d'utilisation Download PDF

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WO2014012093A1
WO2014012093A1 PCT/US2013/050474 US2013050474W WO2014012093A1 WO 2014012093 A1 WO2014012093 A1 WO 2014012093A1 US 2013050474 W US2013050474 W US 2013050474W WO 2014012093 A1 WO2014012093 A1 WO 2014012093A1
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psa
prodrug
cells
prostate cancer
pi3k
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PCT/US2013/050474
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English (en)
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George Kulik
Mark E. Welker
Freddie R. Salsbury, Jr.
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Wake Forest University Health Sciences
Wake Forest Forest University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the fields of oncology and modulation of signal transduction pathways for inducing targeted cell death. More specifically, compositions and methods for inhibiting cancer, particularly prostate cancer, are disclosed.
  • Prostate cancer remains the second leading cause of cancer-related death in men in the United States.
  • Conventional treatments as surgery, radiation and androgen suppression are effective if prostate cancer is confined into the organ.
  • many patients experience recurrence that typically translates into androgen-ablation insensitivity with limited or transient response to systemic chemotherapy (Wang et al. (2011) Mol.Cancer Then, 10:1728-1739; Sarker et al. (2009) Clin.Cancer Res., 15:4799-4805).
  • prodrugs for the treatment of cancer particularly prostate cancer
  • the prodrugs comprise a
  • the PSA cleavable compound comprises the amino acid sequence LEHSSKLQX (SEQ ID NO: 1).
  • the PSA cleavable peptide is Mu-LEHSSKLQL (SEQ ID NO: 2).
  • the PI3K inhibitor may be selected from the group consisting of LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, PX-866, ZSTK474, TGX221, SF 1126, BEZ235, VQD-002, KRX-0401 , GSK690693 , BKM- 120, liphagal, and
  • compositions comprising at least one prodrug of the instant invention and at least one
  • precursor compounds and methods of synthesizing the prodrugs are also provided.
  • methods of inhibiting, treating, and/or preventing cancer, particularly prostate cancer comprise administering at least one prodrug of the instant invention to a subject in need thereof.
  • Figure 1 A provides a ribbon diagram of an X-ray structure of the PI3K ATP- binding site with LY294002 and an active site lysine. Structurally similar docking poises for LY294002 (light grey) and the L-O-CH 2 -LY294002 (dark grey) are shown for comparison.
  • Figure IB provides the Mu-LEHSSKLQL (SEQ ID NO: 2)-0-CH 2 - LY294002 PI3K inhibitor prodrug formula. The predicted prostate-specific antigen (PSA) cleavage site is depicted.
  • PSA prostate-specific antigen
  • Figures 2A-2C provide schematics of the synthesis of LY294002 analogues and prodrug-LY294002.
  • Figure 2A shows the preparation of the H-0-CH 2 - LY294002 analog (8).
  • Figure 2B shows the attachment of leucine to 8, thereby generating the L-LY294002 (10).
  • Figure 2C shows the attachment of the Mu- LEHSSKLQL (SEQ ID NO: l)-peptide to 8, thereby generating the Mu- LEHSSKLQL (SEQ ID NO: 1)-LY294002 PI3K inhibitor prodrug (11).
  • Figure 3 shows that L-LY294002 exerts similar PI3K inhibitory properties as LY294002.
  • L-LY294002 exerts similar PI3K inhibitory properties as LY294002.
  • Fig. 3A prostate cancer C4-2
  • Fig. 3B breast cancer BT-549 cells
  • Figure 4 shows that prodrug-LY294002 selectively inhibits PI3K/Akt signaling in prostate cancer cells that secrete PSA.
  • prostate cancer C4-2 and breast cancer BT-549 cells were treated with prodrug-LY294002 for indicated periods of time.
  • Time-dependent reduction of p-Akt T308 was seen in prostate cancer C4-2 cells (top panel).
  • No significant differences in p-Akt T308 levels were detected in breast cancer BT-549 cells treated with the prodrug- LY294002 in the same time-frame (bottom panel).
  • Control cells treated with PBS.
  • Figure 4C provides a total PSA assay (tPSA) showing significant increase of secreted PSA in prostate cancer C4-2 cells in conditioned media. No PSA was detected in breast cancer BT-549 cells conditioned media.
  • tPSA total PSA assay
  • Figure 5 demonstrates that PSA is required to convert prodrug-LY294002 into an active PI3K inhibitor.
  • Figure 5A shows that the addition of prodrug-LY294002 previously incubated for 6 hours in the media conditioned by prostate cancer C4-2 cells resulted in the significant reduction of p-Akt T308 in breast cancer BT-549 cells.
  • prodrug-LY294002 incubated in the media conditioned by BT-549 cells did not inhibit PDK/Akt pathway.
  • Control cells treated with PBS.
  • Figure 5B shows that the prodrug-LY294002 incubated for 6 hours with an enzymatically active PSA (0-10 4 ng/ml) and then added to breast cancer BT-549 cells media for 30 minutes resulted in significant p-Akt T308 decrease, if compared to prodrug incubated without PSA.
  • Control cells treated with DMSO. As positive control cells were treated with activated prodrug L-LY294002. Representative Western blots, out of three independent experiments, are shown.
  • Figure 6 shows that prodrug-LY294002 dose-dependently inhibits PI3K/Akt pathway in prostate cancer cells that secrete PSA.
  • Figure 6A provides a total PSA (tPSA) assay confirming secretion of PSA in prostate cancer LNCaP and C4-2 cells media at 3 hours. RPMI media was used as control.
  • tPSA total PSA
  • Breast cancer BT-549 and glioblastoma-astrocytoma U-87 MG cells did not express PSA.
  • Figure 6B shows the incubation of prostate cancer LNCaP cells and C42 cells, which secrete PSA, with prodrug-LY294002 for 3 hours resulted in a dose-dependent reduction of p-Akt T308 levels.
  • Figure 6C shows that no significant reduction of p- Akt T308 levels was observed in glioblastoma-astrocytoma U 87MG cells or in breast cancer BT-549 cells, which do not secrete PSA.
  • Control cells treated with PBS. Representative Western blots, out of three independent experiments, are shown.
  • Figure 7 shows that the prodrug-LY294002 induces apoptosis in C4-
  • Figure 8A provides the formulas for two ZSTK analogs.
  • Figure 8B provides a schematic of the synthesis of ZSTK prodrugs.
  • Figure 8C provides a schematic of the synthesis of PX-866 prodrugs.
  • Figures 8D and 8E provide schematics of the synthesis of BKM- 120 prodrugs.
  • Figure 9A provides the structures of two LY294002 based prodrugs.
  • the top structure comprises the peptide sequence LEHSSKLQL (SEQ ID NO: 2) and the bottom structure comprises the peptide sequence LELEHSSKLQL (SEQ ID NO: 3).
  • Figure 9B provides the structure of a ZSTK474 based prodrug. DETAILED DESCRIPTION OF THE INVENTION
  • the phosphatidylinositol 3 kinase/ Akt (PI3K/Akt) pathway is one of the mechanisms that allow prostate cancer cells to maintain continued proliferation in a low-androgen environment (Cohen et al. (2009) J. Cell Biochem., 106:363-371).
  • the PI3K pathway is a key signal-transduction pathway initiated by a signaling cascade coming from receptor tyrosine kinases that activate the PI3 , resulting in an accumulation of phosphatidylinositol-3, 4, 5 -triphosphate (PIP3) in plasma membrane.
  • This lipid second messenger recruits Akt and the phosphoinositide dependent protein kinase 1 (PDK1) to the cell membrane, where Akt is phosphorylated by PDK1 at threonine 308.
  • Partially activated Akt recruits the mammalian target of rapamycin (mTOR) that, acting with Rictor protein, forms the mTORC2 complex which completes the activation of Akt by phosphorylation at serine 473.
  • mTOR mammalian target of rapamycin
  • Fully activated Akt translocates to the cytoplasm and nucleus phosphorylating downstream substrates involved in angiogenesis, cell cycle progression, growth, migration, proliferation, and survival (Morgan et al. (2009) Curr. Cancer Drug Targets, 9: 237-249).
  • PI3K/Akt pathway Constitutive activation of the PI3K/Akt pathway in prostate cancer is often caused by functional loss of the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) or mutations in PI3K itself that lead to increased Akt phosphorylation and correlate with higher Gleason grade, advanced stage, and unfavorable prognosis (Mazzoletti et al. (2011) Cancer Res., 71 :4573-4584; Erlich et al. (2012) Br. J. Cancer, 106:107-1 15).
  • the PI3K/Akt pathway is constitutively activated in a significant proportion of advanced prostate tumors, and is considered one of the key mechanisms supporting progression toward an androgen-independent status, for which no effective therapy is available. For these reasons, PI3K inhibitors have been considered as an adjuvant therapy for advanced prostate cancer.
  • the PI3K inhibitor molecule is converted into a prostate-selective prodrug by attaching a specific prostate-specific antigen (PSA) cleavable peptide, thereby increasing the delivery to tumor sites while minimizing the systemic toxicity and thus improving the efficacy of the PI3K inhibitors to treat prostate cancer patients.
  • PSA prostate-specific antigen
  • PSA is a protease with chymotrypsin-like activity, involved in the hydrolytic processing of semenogelins, necessary for seminal fluid liquefaction.
  • PSA concentration is high, but inactive in blood serum, as PSA is complexed with the ai-anti-chymotrypsin (PS A- ACT) or a 2 - macroglobulin (Bjork et al. (1999) BJU. Int., 84:1021-1027; LeBeau et al. (2009) Biochemistry, 48:3490-3496).
  • PSA is free (fPSA) and enzymatically active, able to activate cytotoxic prodrugs based on a PSA- cleavable peptidic sequence (Evans-Axelsson et al. (2012) Cancer Biother.
  • the synthesis and characterization of superior prostate cancer-specific PSA-activated PI3K inhibitor prodrugs are provided.
  • the prodrugs are exemplified with the quercetin analog LY294002.
  • the PI3K inhibitor LY294002 was linked with the Mu-LEHSSKLQL (SEQ ID NO: 2) peptide, containing the HSSKLQ (SEQ ID NO: 8) sequence, which is cleaved by PSA.
  • the resulting PI3K inhibitor prodrug-LY294002 is water soluble and shows superior specificity for the prostate cancer environment over other prodrugs as it requires PSA cleavage for activation.
  • prodrug-LY294002 is specifically activated in the media conditioned by the prostate cancer cells C4-2 that secrete PSA as well as by purified PSA.
  • the prodrugs of the instant invention e.g., those comprising longer PSA cleavable peptides such as LEHSSKLQX (SEQ ID NO: 1), demonstrated surprisingly superior specificity for prostate cancer cells over a prodrug with only HSSKLQL (SEQ ID NO: 4) linked to a PI3K inhibitor, which was capable of penetrating cells and inhibiting PI3K regardless of PSA cleavage and thereby leading to undesirable toxic side effects.
  • the PI3K inhibitor prodrug-LY294002 Upon activation, the PI3K inhibitor prodrug-LY294002 shows consistent time-dependent and concentration-dependent inhibition of the PI3 kinase and induction of apoptosis. Specificity of the PI3K inhibitor prodrug-LY294002 for PSA- secreting prostate cancer cells was confirmed using the BT-549 breast cancer cell line and the glioblastoma-astrocitoma U-87 MG cells, which do not produce PSA.
  • PI3K/Akt pathway is constitutively activated in these cells and is not affected by the prolonged administration of the prodrug-LY294002 unless it was previously incubated in C4-2 conditioned medium or in the presence of enzymatically active purified PSA.
  • Prostate tumor PSA-dependent inhibition of PI3K provides at least two benefits to patients: 1) by completely inhibiting the PI3K pathway at the tumor site and 2) by reducing side effects due to inhibition of PI3K in normal tissues, as systemic exposure will be minimal, resulting only from the active drug redistributed out from the cancerous tissues (Holmes, D. (201 1) Nat. Rev. Drug Discov., 10:563- 564; Garlich et al. (2008) Cancer Res., 68:206-215). Indeed, increasing the specificity of the PI3K prodrug for pancreatic cancer environment allows for increased drug dosage, accomplishing complete inhibition of PI3K activity in prostate cancer cells and reducing the systemic cytotoxic effects.
  • a PSA-activated PI3K inhibitor prodrug will be effective against hormone- refractory prostate tumors with constitutively activated PI3K pathway that become “addicted” or dependent to a PI3K signaling (Rudner et al. (2010) Radiat. Oncol., 5:108; Ellwood-Yen et al. (201 1) Cancer Res., 71 :3052-3065). Normal tissues are not "addicted” to a constitutive PI3K activity and the "leakage" of the activated PI3K inhibitor outside of tumor sites will not produce substantial toxic side effects, as evident from phase I clinical trials with several non-targeted PI3K inhibitors (Yuan et al. (201 1) Mol.
  • PI3K inhibitor prodrugs cannot enter into cells (and be metabolized) until "released” from the inhibiting peptide, they will show an improved pharmacokinetics comparing to unmodified PI3K inhibitors, as reported for SF1126, a LY294002 derivative coupled with a RGDS peptide (Garlich et al. (2008) Cancer Res., 68:206- 215). It is also noteworthy that, unlike the original LY294002 which showed poor water solubility and needed to be dissolved in dimethyl sulfoxide, the peptide-coupled prodrug-LY294002 is water soluble and, therefore, is easily formulated as an orally or intravenous-delivered therapeutic.
  • the prodrug of the instant invention comprises a prostate specific antigen (PSA) cleavable peptide attached to a phosphatidylinositol-3 -kinase (PI3K) inhibitor, i.e. an agent which is effective to inhibit PI3K activity.
  • PSA prostate specific antigen
  • PI3K phosphatidylinositol-3 -kinase
  • the inhibitor reduces the activity of the ⁇ isoform of PI3K.
  • PI3K inhibitors include, without limitation, LY294002, LY292223, LY292696, LY293684, LY293646 (see, e.g., Vlahos et al. (1994) J. Biol. Chem. 269:5241-5248),
  • wortmannin PX-866 (a wortmannin derivative; Oncothyreon), ZSTK474 (Zenyaku Kogyo Co.; optionally wherein the CHF 2 group has been replaced with CF3), TGX221 (WO 2004/016607), SF1 126 (Semaphore Pharmaceuticals), BEZ235 (Novartis), VQD-002 (VioQuest Pharmaceuticals), KRX-0401 (Keryx Biopharmaceuticals) GSK690693 (GlaxoSmithKine), BKM-120 (Novartis), XL147 (Exelixis), liphagal (Mehta et al. (Tetrahedron Lett.
  • the PI3K inhibitor is a LY294002, ZSTK474, PX-866, or BKM-120.
  • the PI3K inhibitor is LY294002.
  • the PI3K inhibitor is wortmannin.
  • the PSA cleavable peptide comprises an amino acid sequence that can be cleaved by PSA.
  • the PSA cleavable peptide may comprise, for example, about 9 to about 15 amino acids, particularly about 9 to about 11 amino acids.
  • the PSA cleavable peptide comprises the sequence LEHSSKLQX (SEQ ID NO: 1) or LELEHSSKLQX (SEQ ID NO: 7), wherein X is any amino acid, particularly glycine, alanine, valine, isoleucine, or leucine.
  • LEHSSKLQX SEQ ID NO: 1
  • LELEHSSKLQX SEQ ID NO: 7
  • X is leucine.
  • the PSA cleavable peptide comprises about 9 to about 15 amino acids, particularly about 9 to about 11 amino acids, wherein the C-terminal amino acids are HSSKLQX (SEQ ID NO: 5),
  • EHSSKLQX (SEQ ID NO: 6), LEHSSKLQX (SEQ ID NO: 1), or LELEHSSKLQX (SEQ ID NO: 7), where X is defined as above.
  • the peptide may further comprise a cap or amine protecting group (e.g., Boc) at the amino terminus.
  • the peptide may comprise a morpholino (e.g., acetyl morpholino, morpholino urea (Mu)), N-methyl piperazine urea, or benzyloxycarbonyl at the amino terminus, particularly Mu.
  • the PSA cleavable peptide is Mu-LEHSSKLQL (SEQ ID NO: 2). While the instant invention exemplifies the linkage of the PSA cleavable peptide to a PI3K inhibitor, the PSA cleavable peptide may be linked to any compound or agent (e.g., a toxin or a chemotherapeutic) to be targeted to a PSA expressing cell (e.g., prostate cancer cell).
  • a PSA expressing cell e.g., prostate cancer cell
  • Figure 9A provides structures of two prodrugs of the instant invention. While the provided structures show LY294002, any PI3K inhibitor can be used in place of LY294002, such as ZSTK474, PX-866, or BKM-120. Indeed, Figure 9B provides an example of a prodrug comprising ZSTK474, wherein the CHF 2 group within
  • ZSTK474 has been replaced with CF 3 .
  • the instant invention also encompasses compositions comprising at least one prodrug of the instant invention and at least one pharmaceutically acceptable carrier.
  • the compositions may also further comprise at least one toxin, at least one chemotherapeutic agent, and/or at least one other agent conventionally used to treat prostate cancer.
  • toxins include, without limitation: Pseudomonas exotoxin (PE) A, PE40, ricin, ricin A-chain, diphtheria toxin, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, and immunotoxins and prodrugs thereof.
  • the toxins may be immunotoxins which comprise the toxin linked to an antibody which is
  • Prostate antigens include, without limitation, prostate specific membrane antigen (PSMA), prostate cancer antigen (PC A), mucin 1 (MUC1), epidermal growth factor receptor, platelet-derived growth factor, platelet-derived growth factor receptor, urokinase plasminogen activator, and urokinase plasminogen activator receptor.
  • PSMA prostate specific membrane antigen
  • PC A prostate cancer antigen
  • MUC1 mucin 1
  • epidermal growth factor receptor epidermal growth factor receptor
  • platelet-derived growth factor platelet-derived growth factor
  • platelet-derived growth factor receptor urokinase plasminogen activator
  • urokinase plasminogen activator receptor urokinase plasminogen activator receptor
  • the prostate antigen is PSMA.
  • An example of a PSMA antibody is J591 (Ross et al. (2005) Cancer Metastasis Rev., 24:521-537).
  • the prodrug may also be
  • Suitable chemotherapeutic agents include, but are not limited to: alkylating agents (e.g., nitrogen mustards such as chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, and uracil mustard; aziridines such as thiotepa;
  • alkylating agents e.g., nitrogen mustards such as chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, and uracil mustard
  • aziridines such as thiotepa
  • methanesulphonate esters such as busulfan; nitroso ureas such as carmustine, lomustine, and streptozocin; platinum complexes such as cisplatin and carboplatin; bioreductive alkylators such as mitomycin, procarbazine, dacarbazine and
  • altretamine inhibitors of protein synthesis (e.g., omacetaxine mepesuccinate); DNA strand-breakage agents (e.g., bleomycin); topoisomerase II inhibitors (e.g., amsacrine, dactinomycin, daunorubicin, idarubicin, mitoxantrone, doxorubicin, etoposide, and teniposide); DNA minor groove binding agents (e.g., plicamydin); antimetabolites (e.g., folate antagonists such as methotrexate and trimetrexate; pyrimidine antagonists such as fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, and floxuridine; purine antagonists such as mercaptopurine, 6-thioguanine, fludarabine, pentostatin; asparginase; and ribonucleotide reductase inhibitor
  • tubulin interactive agents e.g., vincristine, vinblastine, and paclitaxel (Taxol®)
  • hormonal agents e.g., estrogens; conjugated estrogens; ethinyl estradiol; diethylstilbesterol; chlortrianisen; idenestrol; progestins such as hydroxyprogesterone caproate, medroxyprogesterone, and megestrol; and androgens such as testosterone, testosterone propionate, fluoxymesterone, and methyltestosterone
  • adrenal corticosteroids e.g., prednisone, dexamethasone, methylprednisolone, and
  • leutinizing hormone releasing agents or gonadotropin-releasing hormone antagonists e.g., leuprolide acetate and goserelin acetate
  • gonadotropin-releasing hormone antagonists e.g., leuprolide acetate and goserelin acetate
  • bicalutamide e.g., leuprolide acetate and goserelin acetate
  • leuprorelin or leuprolide acetate bicalutamide
  • hormone ablating agents e.g., tamoxifen, antiandrogen agents such as flutamide; and antiadrenal agents such as mitotane and aminoglutethimide.
  • the chemotherapeutic agent is selected from the group consisting of: omacetaxine mepesuccinate, placitaxel (Taxol®), cisplatin, docetaxol, carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide, CPT-11, 5-fluorouracil (5- FU), gemcitabine, estramustine, carmustine, adriamycin (doxorubicin), etoposide, arsenic trioxide, irinotecan, and epothilone derivatives.
  • omacetaxine mepesuccinate placitaxel (Taxol®)
  • cisplatin docetaxol
  • carboplatin vincristine
  • vinblastine methotrexate
  • CPT-11 5-fluorouracil
  • gemcitabine gemcitabine
  • estramustine carmustine
  • adriamycin doxorubicin
  • the instant invention also encompasses prodrug precursor compounds, such as the PI3K inhibitors modified with a linker or with an amino acid (e.g., leucine) and, optionally a linker.
  • the PI3K inhibitors may be modified to be attached to an amino acid (e.g., leucine), optionally via a linker, and then the remainder of the PSA cleavable peptide may be added to the amino acid to produce the prodrug.
  • the full PSA peptide may linked to the PI3K inhibitor, optionally via a linker.
  • the linker may be attached to the PI3K inhibitor at any chemically feasible position, particularly where the presence of the linker does not reduce the activity of the PI3K inhibitor.
  • the linker may be attached to the phenyl group (e.g., 4 position) of LY294002, to the benzimidazole group (e.g., ortho or meta position) of ZSTK474, in place of the methyl ether of PX-866 (or wortmannin), to open the furan ring of wortmannin, or to the aniline NH 2 of BKM-120.
  • the linker may be a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches the PI3K inhibitor to the PSA peptide.
  • the linker may contain from 0 (i.e., a bond) to about 500 atoms, about 1 to about 100 atoms, or about 1 to about 50 atoms.
  • Linkers may comprise at least one optionally substituted; saturated or unsaturated; linear, branched or cyclic alkyl, alkenyl, or aryl group.
  • the linker comprises an alkyl, particularly a lower alkyl, which comprises a functional group (e.g., -OH, -N3 ⁇ 4, C0 2 H, CONH 2 ) to attach an amino acid and/or peptide.
  • the precursor compounds may also have an amino acid, particularly leucine, linked to the PI3K inhibitor via the linker.
  • the precursor compounds may also have a blocking moiety (e.g., N-FMOC, N-Boc, Boc, etc.) attached to the amino acid or amine group.
  • precursor compounds include, without limitation: N-FMOC- Leu-O-CH 2 -LY294002, N-BOC-Leu-O-CH 2 -LY294002, Leu-(CH 2 ) 11 -CO-0-CH 2 - LY294002, N-BOC-Leu-(CH 2 ) !
  • the instant invention also encompasses methods of synthesizing the prodrugs of the instant invention.
  • Analogs of PI3K inhibitors may be synthesized which contain a functional group (e.g., an alcohol or amine functional group) which can be used to link amino acids (e.g., leucine) and/or peptides such as peptides comprising the PSA cleavable peptides. Examples of synthesis methods for LY294002 prodrugs are provided in the Example.
  • two analogs may be synthesized.
  • One analog comprises a primary alcohol in the meta position on the benzene ring (i) and one with the primary alcohol functional group in the ortho position of the benzene ring (ii) ( Figure 8A).
  • the primary alcohols serve as the linkage point to leucine and/or the peptide.
  • the prodrug syntheses in each case start from commercially available 3,4-diamino- benzoic acid (iii) or 2,3-diamino-benzoic acid.
  • Figure 8B shows the preparation of the meta isomer, but the ortho follows an analogous path.
  • cyanuric chloride (vii) was then treated sequentially with morpholine and compound (vi) to produce (ix).
  • Compound (ix) was then treated with morpholine and the Boc protecting group removed to prepare PI3K inhibitor (xi).
  • PX-866 analogs with a primary alcohol linker attachment point for the PSA peptide sequence may be made by starting with wortmannin (xii) (Fig. 8C).
  • wortmannin xii
  • Fig. 8C The opening of the tetrahydrofuran ring of wortmannin with diallylamine has been reported previously (Wipf et al. (2004) Org. Biomol. Chem., 2:1920).
  • Selective cleavage of methyl ethers to yield primary alcohols in the presence of ester and ketone functional groups has also been reported (Schafer et al. (1966) Chem. Ber., 99:160- 164).
  • These two steps may be performed (in either order) to yield the preparation of the desired material (xiii).
  • Boc protected leucine may then be added to (xiii) and deprotected as described above for the ZSTK analogs.
  • BKM-120 (xiv; Fig. 8D) may be prepared in 4 steps as described previously
  • the leucine and/or the PSA peptide may be attached to the aniline NH 2 .
  • the N3 ⁇ 4 may be acetylated with methoxy acetyl chloride (Burke et al. (1983) J. Org. Chem., 48:5221- 5228) and then the methyl ether can be cleaved to expose the primary alcohol linker (xv) as described above under the PX-866 analog syntheses (Fig. 8D).
  • BKM-120 analogs may be synthesized with a primary alcohol linker.
  • a CF 3 and CH 2 OH substituted pyridine may be used in the synthesis method described in Burger et. al. (ACS Med. Chem. Lett. (201 1) 2:774-779) to make the BKM-120 analog with the CH 2 OH group for the attachment point to the peptide (Fig. 8E).
  • click chemistry may be used to add the leucine and/or the PSA peptide.
  • wortmannin may be reacted with
  • leucine propargyl amide or peptide propargyl amide can be reacted with the azidopropylamine-wortmannin in a click reaction.
  • Propargyl amide constructs may be synthesized by reacting leucine or peptide (e.g., Boc protected) with propargyl amine using diisopropylcarbodiimide (DICD) and
  • the resultant product comprises the leucine or peptide linked to wortmannin via a triazole containing linker.
  • the PI3K inhibitor may be liphagal or derivatives or analogs thereof.
  • Methods of synthesis are provided in Mehta et al. (Tetrahedron Lett. (2009) 50:5260-5262) and Deore et al. (Syn. Commun. (2011) 41 :177-183). In a the formula:
  • X is a halogen (e.g., Br) or C(0)H and Y is a functional group which allows for linking of an amino acid and/or peptide (e.g., OH or N3 ⁇ 4).
  • the prostate cancer may be an androgen- independent or androgen-dependent prostate carcinoma.
  • the prostate cancer is advanced.
  • the method comprises administering at least one prodrug of the instant invention (inclusive of pharmaceutically acceptable salts thereof) to a subject in need thereof.
  • the prodrug is administered as a composition with at least one pharmaceutically acceptable carrier.
  • the methods may further comprise the administration of at least one toxin, at least one chemotherapeutic agent, and/or at least one other agent conventionally used to treat prostate cancer.
  • the other agents may be contained within the same composition as the prodrug of the instant invention of may be contained in separate compositions with at least one pharmaceutically acceptable carrier.
  • the other agents may also be administered simultaneously and/or sequentially with the prodrug of the instant invention.
  • the methods of the instant invention may also comprise the administration of radiation therapy and/or include surgical removal of the tumor and/or prostate.
  • Radiation therapy includes the use of high-energy radiation from x-rays, gamma rays, neutrons, protons and other sources to target cancer cells. Radiation may be administered externally or it may be administered using radioactive material given internally (e.g., radioactive seed implantation).
  • the compositions of the instant invention may be administered at the same time and/or at different times (e.g., sequentially) with the radiation therapy and/or surgical excision.
  • compositions of the instant invention can be administered to an animal, in particular a mammal, more particularly a human, in order to treat/inhibit/prevent prostate cancer.
  • the compositions comprising the prodrugs of the instant invention may be conveniently formulated for administration with any pharmaceutically acceptable carrier(s).
  • the prodrugs may be formulated with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof.
  • concentration of the prodrug in the chosen medium may be varied and the medium may be chosen based on the desired route of administration of the pharmaceutical preparation. Except insofar as any conventional media or agent is incompatible with the prodrug to be administered, its use in the pharmaceutical preparation is contemplated.
  • a suitable pharmaceutical preparation will also depend upon the mode of administration chosen.
  • the prodrug of the invention may be administered by direct injection (e.g., intratumor or to the surrounding area), orally, or intravenously.
  • a pharmaceutical preparation comprises the prodrug dispersed in a medium that is compatible with the site of injection.
  • Prodrugs of the instant invention may be administered by any method.
  • the prodrugs of the instant invention can be administered, without limitation parenterally,
  • Dosage forms for oral administration include, without limitation, tablets (e.g., coated and uncoated, chewable), gelatin capsules (e.g., soft or hard), lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders/granules (e.g.,
  • Dosage forms for parenteral administration include, without limitation, solutions, emulsions, suspensions, dispersions and powders/granules for reconstitution.
  • Dosage forms for topical administration include, without limitation, creams, gels, ointments, salves, patches and transdermal delivery systems.
  • compositions containing a prodrug of the present invention as the active ingredient in intimate admixture with a pharmaceutically acceptable carrier can be prepared according to conventional pharmaceutical compounding techniques.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral, and direct injection.
  • a pharmaceutical preparation of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art.
  • the dose and dosage regimen of prodrug according to the invention that are suitable for administration to a particular patient may be determined by a physician considering the patient's age, sex, weight, general medical condition, and the specific condition for which the prodrugs are being administered and the severity thereof. The physician may also take into account the route of administration, the pharmaceutical carrier, and the biological activity of the prodrug.
  • the pharmaceutical preparation comprising the prodrug may be administered at appropriate intervals, for example, at least twice a day or more until the
  • pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level.
  • the appropriate interval in a particular case would normally depend on the condition of the patient.
  • a pharmaceutical composition of the present invention can be delivered in a controlled release system, such as using an intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
  • a pump may be used (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press (1985); Handbook of Pharmaceutical Controlled Release Technology, Wise (ed.), CRC Press (2000); Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201 ; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med.
  • polymeric materials can be used (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press (1985); Handbook of Pharmaceutical Controlled Release Technology, Wise (ed.), CRC Press (2000);
  • a controlled release system can be placed in proximity of the target tissues of the animal, thus requiring only a fraction of the systemic dose (see, e.g., Medical Applications of Controlled Release).
  • a controlled release device can be introduced into an animal in proximity of the site of inappropriate immune activation or a tumor. Other controlled release systems are discussed in the review by Langer (Science (1990) 249:1527-1533). Definitions:
  • Prostate cancer refers to the presence of malignant cells in the prostate.
  • the terms "advanced prostate cancer”, “locally advanced prostate cancer”, “advanced disease” and “locally advanced disease” mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage C 1 -C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system.
  • AUA American Urological Association
  • TNM tumor, node, metastasis
  • Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base.
  • Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.
  • composition refers to an amount sufficient to modulate tumor growth or metastasis in an animal, especially a human, including without limitation decreasing tumor growth or size or preventing formation of tumor growth in an animal lacking any tumor formation prior to administration, i.e., prophylactic administration.
  • subject refers to both animals and humans.
  • an “antibody” or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen.
  • the term includes polyclonal, monoclonal, chimeric, single domain (Dab) and bispecific antibodies.
  • antibody or antibody molecule contemplates recombinantly generated intact immunoglobulin molecules and immunologically active portions of an immunoglobulin molecule such as, without limitation: Fab, Fab', F(ab') 2 , F(v), scFv, scFv 2 , scFv-Fc, minibody, diabody, tetrabody, single variable domain (e.g., variable heavy domain, variable light domain), bispecific, Affibody® molecules (Affibody, Bromma, Sweden), and peptabodies (Terskikh et al. (1997) PNAS 94:1663-1668).
  • an immunoglobulin molecule such as, without limitation: Fab, Fab', F(ab') 2 , F(v), scFv, scFv 2 , scFv-Fc, minibody, diabody, tetrabody, single variable domain (e.g., variable heavy domain, variable light domain), bi
  • Antibodies immunospecific for antigens present on prostate cells are particularly preferred for use in the present invention.
  • antigens include, without limitation, PMSA, PSCA, MUC1, Epidermal growth factor receptor, platelet-derived growth factor, platelet-derived growth factor receptor, urokinase plasminogen activator, and urokinase plasminogen activator receptor.
  • proteins/polypeptides particularly antibodies, that bind to one or more epitopes of a protein or compound of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
  • a "toxin” refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells.
  • the term includes small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof which are effective to inhibit protein synthesis in a target cell.
  • toxins include, but are not limited to: Pseudomonas exotoxin (PE) A, PE40, ricin, ricin A-chain, diphtheria toxin, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, and calicheamicin.
  • Antibodies may also be conjugated to such toxins, thereby forming an "immunotoxin" to facilitate targeting to the prostate cancer cell.
  • Such immunotoxins may also be generated as prodrugs which comprise an operably linked PSA cleavable peptide which masks the antigen binding site until cleaved by PSA elaborated by prostate cancer cells.
  • prodrug refers to a precursor form of the drug which is metabolically converted in vivo to produce the active drug.
  • PI3K inhibitors in the form of prodrugs are administered to a subject in accordance with the present invention which undergo subsequent metabolic activation and regenerate active forms of the PI3K inhibitor (optionally comprising a portion of the prodrug which does not interfere with the activity of the inhibitor) at the site of interest (e.g., at the prostate) in vivo, e.g., following exposure to endogenous PSA protease in the body.
  • “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • a “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., Tween 80, Polysorbate 80), emulsifier, buffer (e.g., Tris HCl, acetate, phosphate), bulking substance (e.g., lactose, mannitol), excipient, auxilliary agent or vehicle with which an active agent of the present invention is administered.
  • preservative e.g., Thimersol, benzyl alcohol
  • anti-oxidant e.g., ascorbic acid, sodium metabisulfite
  • solubilizer e.g., Tween 80, Polysorbate 80
  • emulsifier e.g., Tris HCl, acetate, phosphate
  • bulking substance e.g
  • Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
  • the compositions can be incorporated into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes or micelles. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of components of a pharmaceutical composition of the present invention.
  • the pharmaceutical composition of the present invention can be prepared, for example, in liquid form, or can be in dried powder form (e.g., lyophilized). Suitable
  • the term "prevent” refers to the prophylactic treatment of a subject who is at risk of developing a condition (e.g., cancer) resulting in a decrease in the probability that the subject will develop the condition.
  • a condition e.g., cancer
  • treat refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.
  • lower alkyl refers to an alkyl which contains 1 to 3 carbons in the hydrocarbon chain.
  • 2-Morpholin-4-yl-4-oxo-4H-chromen-8-yl trifluoromethanesulfonate 7 (100 mg, 0.263 mmol) was dissolved in 5:1 MeCN/EtOH (lOmL). K 2 C0 3 (2.5 eq., 0.091g, 0.658 mmol) was added and the solution was degassed with N 2 . 4- (Hydroxymethyl)phenylboronic acid (1.21 eq., 0.048 g, 0.318 mmol) and Pd(OAc) 2 (0.1 eq., 0.006 g, 0.0263 mmol) were added and the solution refluxed overnight.
  • the hormone-responsive prostate cancer LNCaP and breast cancer BT-549 cell lines were purchased from ATCC (Manassas, VA).
  • the androgen-independent prostate cancer LNCaP subline C4-2 and the glioblastoma-astrocitoma U-87 MG cells were obtained from Dr. Leland Chung (Cedars-Sinai Medical Center, Los Angeles, CA) and Dr. Frank Furnari (University of California, San Diego, CA) respectively.
  • tissue culture reagents were purchased from Invitrogen (Carlsbad, CA).
  • cells were seeded at 4xl0 5 /6 cm petri dish and kept in culture for 48 hours to reach about 80% confluence.
  • Cells were kept in a serum- free media condition (LNCaP cells were kept in medium supplemented with 2.5% FBS).
  • C4-2LucBAD cells were kept in serum-free medium for 18 hours to increase PSA concentrations.
  • ELIA Total PSA electrochemiluminescence immunoassay
  • Rabbit polyclonal anti-p-Akt Thr308 and anti-Akt were purchased from Cell Signaling Technology (Beverly, MA).
  • Mouse monoclonal anti-P-actin was obtained from Sigma-Aldrich (St. Louis, MO).
  • Secondary goat anti-mouse IRDye 680 and goat-anti rabbit IRDye 800 were both purchased from Li-Cor Biosciences (Lincoln, NE). Protein bands were quantified using Image J software (National Institutes of Health).
  • Apoptosis in whole cell populations was assessed by monitoring caspase 3 activity with the specific fluorogenic substrate Ac-DEVD-7-amido-4- trifluoromethilcoumarin (Bachem, Torrance, CA), as reported (Sastry et al. (2007) J. Biol. Chem., 282: 14094-14100).
  • adherent and floating cells were collected and lysed using a cold lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM HEPES, 1 mM EDTA, 1 mM DTT, and 5 ⁇ g/ml aprotinin, leupeptin, and pepstatin, respectively). Fluorescence was recorded each 15 minutes for 1 hour using a VersaFluorTM (BioRad, Hercules, CA). Caspase 3 activity was expressed in arbitrary units and calculated using Excel 2010 (Microsoft Corporation, Redmond,
  • LC-ESI-MS analysis was performed using an Accela Open 1200 UHPLC coupled to a LTQTM XL Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Separation was accomplished using a ZORBAX Eclipse XDB-C18 (1.8 ⁇ ⁇ ⁇ , 2.1 x 50 mm) analytical column with a ZORBAX® extend C18 Narrow Bore Guard column (5 ⁇ , 2.1 x 12.5 mm) both from Agilent Technologies, Santa Clara, CA. The following mobile phases were used for the separation: Solvent A, water/0.1% formic acid, and solvent B, methanol/0.1% formic acid.
  • the gradient used for separation was 95% to 5% solvent A over 10 minutes at a flow rate of 250 ⁇ / ⁇ .
  • optimized positive mode ESI conditions were found as follows; sheath and aux gas ware 60 and 4 arbitrary units, respectively, spray voltage 4 kV, capillary temperature and voltage were 325 °C and 49 V, tube lens was found to be 115 V.
  • Full scan high resolution mass spectra were collect at 60,000 Hz from 150 to 2000 m/z. The full scan data for standard curve samples and unknowns were processed with Xcalibur software to determine concentrations (Thermo Fisher Scientific, Waltham, MA).
  • C4-2 and BT-549 cells were cultured as described above.
  • Prodrug-LY294002 was added to the medium with C4-2 cells and incubated for 6 hours at 37°C in a humidified atmosphere (5% C0 2 ) in serum-free medium. Media was then collected and added to BT-549 cells for 3 hours. Inhibition of PI3 kinase was assayed by monitoring the p-Akt T308 levels by Western blotting.
  • Prodrug-LY294002 cleavage with purified PSA
  • the prodrug-L Y294002 was incubated in 50 mM Tris-HCl, 0.1 M NaCl, pH
  • LY294002 was redocked into the active site with 256 runs of AutoDock' s Lamarckian genetic algorithm. Default parameters were used except in the number of runs (256) and the size of the initial pool (30000 initial dockings). Subsequent analysis showed that the LY294002 was docked with a similar poise as in the crystal structure with 4.0 ⁇ predicted affinity, using Autodock's estimator of binding affinity (Fig. 1A) (Morris et al. (1998) J. Computational Chem., 19:1639- 1662).
  • a phenyl ring in LY294002 structure has been identified to attach a CH 2 OH moiety, which subsequently was used to couple the Mu- LEHSSKLQL (SEQ ID NO: 2) sequence, containing the PSA substrate peptide (Fig. IB). Since PSA is predicted to cleave Mu-LEHSSKLQL (SEQ ID NO: 2) peptide after glutamine (Q), it leaves leucine (L) attached to the phenyl ring in LY294002 via CH 2 OH moiety.
  • the AutoDock modeling of interaction between this activated prodrug L-O-CH 2 -LY294002 and PI3K has shown that it can still fit into ATP binding site and, thus, predicted to inhibit PI3K activity (Fig. 1 A).
  • the enolate of N-acetyl morpholine (4) was then generated with lithium diisopropyl amide and condensed with the ester in 3 to produce the salicylacetamide (5).
  • the salicylacetamide (5) underwent cyclodehydration after being treated with triflic anhydride to give the triflate (6).
  • the triflate (6) was then subjected to Suzuki coupling with commercially available 4- (hydroxymethyl)phenyl boronic acid (7) to produce the LY294002 analog with the CH 2 OH functional group (8).
  • the LY294002 analog with the primary alcohol functional group was prepared, it was used to attach leucine or the Mu-LEHSSKLQL (SEQ ID NO: 2)-peptide (Figs. 2B and 2C).
  • the compound (9) was first synthesized where 8 is directly attached to BOC protected leucine.
  • the BOC protecting group can be removed by p-TsOH when desired to produce the compound (10).
  • Compound (10) represents what would remain after PSA has cleaved off substrate peptide from the prodrug.
  • the peptide Mu-LEHSSKLQL (SEQ ID NO: 2) was attached to 8 by AnaSpec, Inc. (Fremont, CA) to give the prodrug (11) as a white powder. Mass calculated as 1487.7 was found to be 1488.0 by LC-MS analysis, conducted at AnaSpec, Inc.
  • L-LY294002 inhibits PI3 Kinase
  • the androgen-independent prostate cancer C4-2 cells were chosen for this study as a model of advanced and PSA secreting prostate cancer.
  • the breast cancer BT-549 cell line that does not secrete PSA was used as a negative control. Both cell lines are PTEN-deficient and show constitutive activation of the PI3K/Akt signaling pathway.
  • PSA is predicted to cleave the Mu-LEHSSKLQL (SEQ ID NO: 2)-LY294002 prodrug after the glutamine, leaving a leucine attached to the phenyl ring of the
  • L-LY294002 inhibits the PI3K/Akt signaling pathway with potency comparable to the LY294002, resulting in a dose-dependent reduction of p- Akt T308 levels in both cell lines 30 minutes after administration (Fig. 3).
  • L- LY294002 induced complete inhibition of pAkt at 30 ⁇ and used this concentration in subsequent experiments.
  • Prodrug-LY294002 inhibits PI3 Kinase in PSA-secreting prostate cancer cells
  • the Mu-LEHSSKLQL (SEQ ID NO: 2)-O-CH 2 -LY294002 prodrug (prodrug- LY294002) effects were monitored in prostate cancer C4-2 and breast cancer BT-549 cells by a time-course, from 30 minutes to 6 hours.
  • a time-dependent reduction of p- Akt T308 levels was observed in prostate cancer C4-2 cells, with almost complete inhibition observed in 3 hours.
  • no significant differences in p-Akt T308 levels were detected in breast cancer BT-549 cells treated with the prodrug- LY294002 during this time-frame (Fig. 4A).
  • PSA concentration is significantly increased in prostate cancer C4-2 conditioned media, while no PSA was detected in breast cancer BT-549 conditioned media (Fig. 4C).
  • PSA is required for Mu-LEHSSKLQL (SEQ ID NO: 2) peptide cleavage and prodrug- LY294002 activation.
  • prodrug-LY294002 was incubated in the medium conditioned by prostate cancer C4-2 cells. After an incubation of 6 hours, this medium with prodrug-LY294002 was transferred into dishes containing breast cancer BT-549 cells.
  • prodrug-LY294002 previously incubated in the media conditioned by prostate cancer C4-2 cells resulted in a significant reduction of p-Akt T308 levels in breast cancer BT-549 cells, which do not secrete PSA, by prodrug-LY294002.
  • prodrug-LY294002 incubated in the media conditioned by BT-549 cells did not inhibit p-Akt T308 levels.
  • prodrug-LY294002 was incubated for 6 hours at 37°C with an enzymatically active PSA purified from human semen.
  • prodrug-LY294002 pre-incubated with PSA
  • BT 549 cells for 30 minutes
  • prodrug-LY294002 incubated for 6 hours at 37°C without enzymatically active PSA did not reduce p-Akt T308 levels in BT-549 cells.
  • Prodrug-LY294002 Promotes Apoptosis in C4-2LucBAD Prostate Cancer Cells
  • LY294002 induces apoptosis in C42LucBAD cells that ectopically express BAD, a BH3-only pro-apoptotic protein of Bcl-2 family (Zha et al. (1996) Cell, 87:619-628; Datta et al. (1997) Cell, 91 :231-241 ; del Peso et al. (1997) Science, 278:687-689; Sastry et al. (2006) J. Biol. Chem., 281 :27367-27377).
  • prodrug-LY294002 inhibited PI3K/Akt pathway and induced apoptosis in C42LucBAD cells, as evident from increased caspase 3 activity and cleavage of caspase substrate PARP (Fig. 7). Somewhat lower levels of caspase activity induced by prodrug-LY294002 are likely due to extra time needed for conversion of prodrug- LY294002 into an active PI3K inhibitor.
  • LY294002 is converted by PSA cleavage into an active PI3K inhibitor that selectively blocks the PI3K/Akt pathway and induces apoptosis in PSA-secreting prostate cancer cells.

Abstract

La présente invention concerne des compositions et des méthodes utilisées pour inhiber la croissance de cellules cancéreuses, en particulier des cellules du cancer de la prostate. Cette invention se rapporte aux domaines de l'oncologie et de la modulation des chemins de transduction du signal pour induire la mort cellulaire ciblée. Des promédicaments pour le traitement du cancer, particulièrement le cancer de la prostate sont présentés. Les promédicaments comprennent un inhibiteur phosphatidylinositol-3-kinase (PI3K) lié à un peptide clivable d'antigène prostatique spécifique.
PCT/US2013/050474 2012-07-13 2013-07-15 Promédicaments dirigés contre le cancer de la prostate et leurs méthodes d'utilisation WO2014012093A1 (fr)

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WO2024036358A1 (fr) * 2022-08-17 2024-02-22 The University Of Sydney Pro-fraction pour former un promédicament sélectivement clivé par un antigène spécifique de la prostate (psa)

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WO2024036358A1 (fr) * 2022-08-17 2024-02-22 The University Of Sydney Pro-fraction pour former un promédicament sélectivement clivé par un antigène spécifique de la prostate (psa)

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