WO2018146506A1 - Dérivés thérapeutiques - Google Patents

Dérivés thérapeutiques Download PDF

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Publication number
WO2018146506A1
WO2018146506A1 PCT/IB2017/000222 IB2017000222W WO2018146506A1 WO 2018146506 A1 WO2018146506 A1 WO 2018146506A1 IB 2017000222 W IB2017000222 W IB 2017000222W WO 2018146506 A1 WO2018146506 A1 WO 2018146506A1
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Prior art keywords
therapeutic
derivative according
saccharide
senescent cells
damaged
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PCT/IB2017/000222
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English (en)
Inventor
Ramón MARTÍNEZ MAÑEZ
Manuel SERRANO MARUGÁN
José Ramón MURGUÍA IBÁÑEZ
Andrea BERNARDOS BAU
Borja DÍAZ DE GREÑU PUERTAS
Irene GALIANA GUILLEM
Beatriz LOZANO TORRES
Miguel ROVIRA DEL OLMO
Félix SANCENÓN GALARZA
Original Assignee
Universitat Politècnica De València
Fundación Centro Nacional De Investigaciones Oncologicas Carlos Iii
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Priority to PCT/IB2017/000222 priority Critical patent/WO2018146506A1/fr
Publication of WO2018146506A1 publication Critical patent/WO2018146506A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • 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
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome

Definitions

  • the invention relates to therapeutic derivatives comprising a therapeutic agent and a saccharide.
  • the derivative may also comprise a liposome.
  • the invention extends to the use of these derivatives to eliminate damaged and/or senescent cells and to treat, ameliorate or prevent diseases or conditions characterized by the presence of such cells.
  • Senescent cells show a radically altered phenotype which is believed to harm the function of tissues and to predispose them to the development and progression of diseases. This is in part mediated by the fact that senescent cells release a plethora of pro-inflammatory cytokines, known as the Senescence Associated Secretory Phenotype (SASP). Senescence is an essential process in the deposition of extracellular matrix required for wound healing, and the excess presence of senescent cells can lead to pathological fibrosis. Although the immune system destroys many senescent cells, it becomes much less effective at this task with ageing.
  • SASP Senescence Associated Secretory Phenotype
  • senescent or "death-resistant” cells accumulate in tissues, thus accelerating inflammation, ageing and the development of diseases such as fibrosis. Furthermore, the accumulation of senescent cells alters the behavior of neighboring cells, favors degradation of the extracellular matrix, reduces the source of cells capable of suffering mitosis and stimulates cancer.
  • telomeres In WS or RTS erosion of the telomeres in most tissues can be observed, even if telomerase is not the main cause of the disease.
  • Other diseases are more related to accelerate ageing in specific tissues, such as Dyskeratosis Congenita (DS), Idiopathic Pulmonary Fibrosis (IPF), Liver Fibrosis, Renal Fibrosis or Cancer.
  • DS Dyskeratosis Congenita
  • IPF Idiopathic Pulmonary Fibrosis
  • Liver Fibrosis Liver Fibrosis
  • Renal Fibrosis Cancer.
  • telomeres a defective activity of the telomerase in the stem cell compartment of tissues with high cell regeneration such as the skin, pulmonary epithelium and bone marrow.
  • a frequent secondary effect in these diseases is the induction of cancer, especially of those cancers which imply a shortening of the telomeres.
  • the treatment of tumors with cytotoxic drugs can induce the appearance of senescence in healthy tissue, leading to accelerated ageing and age-related diseases in cancer survivors.
  • such therapies would contribute to the treatment of accelerated cell ageing and would also perpetuate the idea that rejuvenation is possible.
  • these new therapies could be used to target fibrotic diseases such as, for example, idiopathic pulmonary fibrosis, cardiac fibrosis, hepatic fibrosis, or renal fibrosis.
  • Senescent cells are required for wound healing, where it is hypothesized that the pro- inflammatory cytokines released as part of the SASP are required for the deposition of the fibrin and collagen extracellular matrix that causes fibrous or "scar" tissue.
  • Senescent cells may, therefore, play a critical role in pathological fibrosis, for example idiopathic pulmonary fibrosis, cardiac fibrosis, hepatic fibrosis, or renal fibrosis.
  • Senescent cells may also play a role in altered skin appearance, for example, the formation of skin moles, which may contain melanocytic nevi that have undergone cellular senescence. Removing senescent cells may, therefore, result in altered cosmetic appearance.
  • a major step towards achieving the above objective of eliminating senescent cells would be the development of formulations capable of selectively killing senescent cells.
  • Bioactive agents that exhibit limited solubility and stability or possess high toxicity may be chemically modified by conjugation to pro-drugs, or included in liposomes as means to overcome the aforementioned limitations and reduce their toxicity.
  • Traditional methods of doing this are based upon reformulating the bioactive drug in a less toxic form.
  • One such example is the polyene antibiotic Amphotericin B, which is presently available in a less toxic micellar form as a liposomal formulation, as a colloidal dispersion and as a lipid complex.
  • the development of water-soluble polymer-drug conjugates has, thus, been pursued as a means for achieving targeted drug delivery and lower drug toxicity.
  • cellular damage and cellular senescence are closely associated concepts; damaged cells often become senescent cells.
  • cellular senescence (initiated by means that do not necessarily imply cellular damage) is characterized by aberrant mitochondrial function that results in high levels of reactive oxygen species (ROS) that self-inflict damage to the cell, thereby ensuring that senescent cells are indeed themselves damaged cells.
  • ROS reactive oxygen species
  • a therapeutic derivative comprising a therapeutic agent and a saccharide.
  • the saccharide is attached to the therapeutic agent.
  • the therapeutic derivative as well as comprising a therapeutic agent and a saccharide may also comprise a liposome.
  • the saccharide may be attached to the liposome, preferably the outside of the liposome, and the therapeutic agent may be present inside the internal cavity of the liposome.
  • the saccharide comprises at least one unit of monosaccharide. In another embodiment, the saccharide comprises between one and 20 units, or between one and 50 units, or between one and 100 units, or between one and 15 units, or between five and 10 units, of monosaccharide.
  • the monosaccharide may be galactose and/or fucose. Preferably, at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the unit/s of monosaccharide maybe galactose and/or fucose.
  • the therapeutic derivative comprises galactose and/or fucose oligomers. In another embodiment the therapeutic derivative comprises between one and 5 oligomers, or between one and 10 oligomers, or between one and 20 oligomers, or between one and 50 oligomers, or between 2 and 20 oligomers.
  • the therapeutic agent may comprise a protein, peptide, antibody, chemical compound, genetic material, small molecule or imaging agent.
  • the derivative may also comprises a further therapeutic agent, wherein the further therapeutic agent comprises a protein, peptide, antibody, chemical compound, genetic material, small molecule or imaging agent.
  • Cellular senescence is characterized by three properties: (1) an epigenetic reorganization that prevents further cell proliferation; (2) an intense secretory program or Senescent Associated Secretory Phenotype (SASP) that includes cytokines that recruit inflammatory cells; and (3) up-regulation of lysosomal-associated ⁇ - galactosidase activity (SA- ⁇ gal) and lysosomal-associated a-fucosidase (SA-a-fuc) activity. These properties are not necessarily linked.
  • SASP Senescent Associated Secretory Phenotype
  • the activation of the therapeutic agent is triggered by the presence of senescence associated ⁇ -galactosidase (SA- -gal) activity, or senescence associated a-fucosidase (SA-a-fuc) activity in damaged and/or senescent cells, or cells causing fibrosis.
  • SA- -gal senescence associated ⁇ -galactosidase
  • SA-a-fuc senescence associated a-fucosidase
  • the present invention is able to advantageously utilize a more specific therapeutic agent, which is able to act selectively in cells which overexpress ⁇ -galactosidase or ⁇ -fucosidase, such as damaged and/or senescent cells, or cells involved in causing fibrosis.
  • the present invention thus, provides modified therapeutic agents having reduced toxicity relative to the unmodified parent therapeutic agent, while retaining substantially the same degree of therapeutic activity compared to the unmodified parent.
  • a therapeutic derivative according to the first aspect for use in eliminating damaged and/or senescent cells.
  • a therapeutic derivative also according to the first aspect of the invention, for use in the treatment, amelioration or prevention of a disease or condition characterized by the presence of damaged and/or senescent cells.
  • a method of eliminating damaged and/or senescent cells comprising administering to a subject a therapeutically effective amount of a therapeutic derivative according to a first aspect of the invention.
  • a method of treating, ameliorating or preventing a disease or condition characterized by the presence of damaged and/or senescent cells comprising administering to a subject in need of such treatment, a therapeutically effective amount of a therapeutic derivative according to the first aspect.
  • the present invention relates to therapeutic derivatives which may advantageously comprise (i) a liposome loaded with drugs and coated with saccharides; (ii) drugs linked to saccharides, wherein said saccharides comprise at least ⁇ unit of monosaccharides, and wherein at least 30% of the monosaccharides are galactose and/or fucose.
  • the conjugate comprises 1 to 100 units of monosaccharides and even more preferably 3 to 6 units of monosaccharides.
  • therapeutic derivatives is understood in the context of the invention to mean a complex capable of acting as a therapeutic agent in a selective manner.
  • cytotoxic compound means a compound which is toxic for cells. In contact with cytotoxic compounds, the cells may suffer necrosis, apoptosis or may cease to grow and divide.
  • cytotoxic compounds valid in the context of the invention would be wherein the core of the cytotoxic is a mitotic inhibitor, such as vinca alkaloids, including vincristine, vinblastine, vinorelbine, vindesine, vinflunine; podophyllotoxin; taxanes, including docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel; epothilones, such as ixabepilone; topoisomerase I inhibitors, such as topotecan, irinotecan, camptothecin, rubitecan, and belotecan; topoisomerase type II inhibitors, including amsacrine, etoposide, etoposide phosphate, and teniposide; anthra mitotic
  • Senolytic compound refers to a small molecule that can selectively induce death of senescent cells. Senolytic comprise at least one of the listed compounds: quercetin, dasatinib, navitoclax, obatoclax, veliparib, ABT-737, ABT-199 and all the anti-cancer drugs acting as a PARP, Bcl-2, Bcl-XL and Bcl-w proteins inhibitor and thereof.
  • the therapeutic derivatives of the present invention can selectively deliver drugs to damaged and/or senescent cells, to alter their activity, and/or cause their removal, and also may detect and/or mark senescent cells.
  • Damaged and/or senescent cells present in a specific manner an overexpression of endogenous liposomal ⁇ -galactosidase and/or a-fucosidase.
  • This ⁇ -galactosidase and/or a-fucosidase is capable of hydrolyzing the saccharides galactose and fucose respectively, thus releasing the parent drug.
  • saccharide is understood to mean a carbohydrate which consists of 1 to 100 units of monosaccharides (basic units of sugars), with the general formula CnH 2n 0n. Also, in a preferred embodiment, at least 30% of the monosaccharides are galactose and/or fucose.
  • At least 30% of the monosaccharides being galactose and/or fucose means that if there are 2 monosaccharides, 1 or 2 of them will be galactose and/or fucose; if there are 3 monosaccharides, 2 or 3 of them will be galactose and/or fucose; if there are 4 monosaccharides, 2, 3 or 4 of them will be galactose and/or fucose; if there are 5 monosaccharides, 3, 4 or 5 will be galactose and/or fucose and if there are 6 monosaccharides, 3, 4, 5 or 6 of them will be galactose and/or fucose.
  • liposome is understood to mean a closed vesicle which membrane is formed by the aggregation of lipids.
  • a schematic diagram is shown in Figure 1; a liposome of the present invention includes a saccharide covalently bonded to its membrane surface, for example through a linker. The saccharide can also be bonded to the liposome through non covalent bonds.
  • cytotoxic and/or senolytic compounds, and any combinations thereof may be encapsulated by the lipid bilayer of the liposome.
  • the lipid constituting the liposomes of the present invention may include phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids, gangliosides, glycolipids, phosphatidylglycerols, and cholesterol.
  • the phosphatidylcholines preferably include dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.
  • the phosphatidylethanolamines preferably include dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, and distearoylphosphatidylethanolamine.
  • the phosphatidic acids preferably include dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, distearoylphosphatidic acid, and dicetylphosphoric acid.
  • the gangliosides preferably include ganglioside GMi, ganglioside GDia, and ganglioside GTib.
  • the glycolipids preferably include galactosylceramide, glucosylceramide, lactosylceramide, phosphatide, and globoside.
  • the phosphatidylglycerols preferably include dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, and distearoylphosphatidylglycerol.
  • the liposome itself can be produced through any conventional method including a thin film method, a reverse phase evaporation method, an ethanol injection method, and a dehydration-rehydration method.
  • the particle size of the liposome can be controlled through an ultrasonic radiation method, an extrusion method, a French press method, a homogenization method or any other suitable conventional method.
  • the size of the liposomal drug delivery system described here is within the range of 50 to 350 nm, and more preferably 60 to 100 nm.
  • the nano size of the liposomes enables passive targeting of the particles in disease sites.
  • Surface grafted saccharide chains bring about the necessary characteristics for increasing the circulation time of the nano liposomes and benefits from its active targeting mechanisms.
  • therapeutic derivative refers to a compound comprising an oligomer, preferably a therapeutic agent, chemically bonded to monomers or oligomers of saccharides, including galactose and/or fucose.
  • the chemical bond is preferably covalent bonding, most preferably via a N or O atom of the parent drug molecule and a C atom of the saccharide, said N or O atoms being an inherent part of the structure of said drug or appended thereto following chemical modifications.
  • the therapeutic agent linked or otherwise bound to the saccharide can be selected from the list which comprises senolytic compounds, cytotoxic compounds and any combinations thereof.
  • the therapeutic derivatives of the invention based on therapeutic agents linked to saccharide may be characterized as follows:
  • the therapeutically active drug may be conjugated to the saccharide backbone via a
  • the conjugates may have reduced toxicity in non-senescent cells in comparison with the unmodified conjugates
  • the conjugates may have decreased biological and/or therapeutic activity compared to the unmodified conjugates, until such time as they are cleaved by a senescent cell or cell with elevated ⁇ -galactosidase activity or a-fucosidase activity;
  • the conjugates may retain the structure of the drug conjugated to the saccharide
  • the conjugates may retain most of the physical and chemical characteristics which allow the use thereof in a fashion similar to the use of the unmodified conjugates.
  • Example of ABT-737 compounds covalently bonded to saccharides and their possible combinations are listed in Table 2.
  • Example of ABT-199 compounds covalently bonded to saccharides and their possible combinations are listed in Table 2.
  • the invention has the potential for treatment of various malignancies or conditions including but not limited to fibrosis and diseases of aging.
  • the present invention relates to a composition which comprises therapeutic derivatives such as those described above.
  • the composition defined in a general manner, is a set of components which is made up of at least the product as it is described in the invention.
  • the composition is a pharmaceutical.
  • This pharmaceutical composition preferably comprises pharmaceutically acceptable excipients.
  • the pharmaceutical composition is a set of components which may be made up of at least the product of the invention (the liposomes or the prodrug) in any concentration, and which has at least one application in improving the physical or physiological or psychological wellbeing of a subject, which implies an improvement in their general state of health, for example, in the treatment of pathological fibrosis.
  • the therapeutic derivatives of the invention may be provided, preferably, in a pharmaceutically acceptable or substantially pure form, in other words, which has a pharmaceutically acceptable level of purity, excluding normal pharmaceutical additives such as diluents and carriers, and not including material considered toxic at normal dosage levels.
  • the levels of purity for the active principle are preferably higher than 50%, more preferably higher than 70%, and even more preferably higher than 90%. In a preferred embodiment, they are higher than 95% of the compounds described above, or of their salts, solvates or pro-pharmaceuticals.
  • the term "therapeutically effective quantity” refers to that quantity of the component of the pharmaceutical composition which when administered to a mammal, preferably a human, is sufficient to produce the prevention and/or treatment, as defined below, of a disease or pathological condition of interest in the mammal, preferably human.
  • the therapeutically effective quantity will vary, for example, depending on the patient's age, body weight, general state of health, gender and diet; the mode and moment of administration; the speed of excretion, the combination of drugs; the severity of the specific disorder or pathological condition; and the subject undergoing therapy, but can be determined by a specialist in the technique based on their own knowledge.
  • medicament has a more limited meaning than the meaning of "pharmaceutical composition", as defined in the present invention, as the medicament necessarily implies a preventive or therapeutic effect, in other words, a physiological effect in the subject.
  • the pharmaceutical composition of the invention can comprise another active substance.
  • active principle is any matter, irrespective of its origin, human, animal, vegetable, chemical or other type, which is attributed activity appropriate to constitute a medicament.
  • composition of the present invention may be presented in the form of solutions or any other clinically allowed forms of administration and in a therapeutically effective quantity.
  • the pharmaceutical composition of the invention can be formulated in solid forms, semi-solid, liquid or gaseous, such as tablet, capsule, powder, granule, ointment, solution, suppository, injectable, inhalant, gel, syrup, nebulizer, microsphere or aerosol, preferably in the form of a tablet, capsule, powder, granule, solution, suppository or syrup.
  • compositions mentioned above can be prepared using conventional methods, such as those described in the Pharmacopoeias of different countries and in other reference texts.
  • the compounds and compositions of the present invention can be used together with other medicaments in combined therapies.
  • the other drugs can form part of the same composition or of another different composition, for administration at the same time or at different times.
  • a third aspect of the present invention relates to the use of the therapeutic derivatives as described above, for the preparation of a medicament.
  • the medicament referred to in the present invention may be for human or veterinary use.
  • a "medicament for human use” is any substance or combination of substances which is presented as having properties for the treatment or prevention of diseases in human beings or which can be used in human beings or administered to human beings for the purpose of restoring, correcting or modifying physiological functions, by exerting a pharmacological, immunological, or metabolic action or establishing a medical diagnosis.
  • the "medicament for veterinary use” is any substance or combination of substances which is presented as having healing or preventive properties in relation to animal diseases or which can be administered to the animal for the purpose of restoring, correcting or modifying its physiological functions by exerting a pharmacological, immunological, or metabolic response, or establishing a veterinary diagnosis.
  • "Pre-mixtures for animal feed” prepared for adding to a feed may also be considered “veterinary medicaments”.
  • the present invention relates to the use of the therapeutic derivatives as described above, liposome derivatives and prodrug conjugates, for the preparation of a medicament for the treatment and/or prevention of diseases which result from overexpression of ⁇ -galactosidase and/or a-fucosidase, and which preferably takes place in senescent cells.
  • the disease is selected from the list selected from the Wiedemann-Rautenstrauch syndrome of neonatal progeria, the Werner syndrome of adult progeria, Hutchinson-Gilford syndrome, Rothmund Thompson syndrome, Mulvill-Smith syndrome, Cockayne syndrome, Dyskeratosis Congenita, idiopathic pulmonary fibrosis, liver fibrosis, renal fibrosis, oral mucous fibrosis, cardiac fibrosis, pancreatic fibrosis, cancer, aplastic anaemia, emphysema, type 2 diabetes, degeneration of cartilage, obesity, Alzheimer's disease, Parkinson's disease, sarcopenia, osteoarthritis, intervertebral disc degeneration, macular degeneration, glaucoma, cataracts, pulmonary hypertension, chronic obstructive pulmonary disease, renal transplantation, oral mucositis, and intestinal bowel disease.
  • the Wiedemann-Rautenstrauch syndrome of neonatal progeria the Wer
  • the present invention also relates to the use of the therapeutic derivatives as described above, for the preparation of a medicament for the treatment of cancer, and in particular the selective elimination of residual cancer cells which have survived prior chemotherapy treatment.
  • Residual cancer cells that have survived prior chemotherapy treatment may experience elevated beta-galactosidase and/or alpha-fucosidase activity, as a result of DNA damage that triggers the cellular senescence program, and these chemotherapy resistant cells may be targeted by the medicament disclosed here.
  • the present invention relates to the use of the therapeutic derivatives as described above, for the alleviation of the side effects and/or accelerated ageing caused by treatment with chemotherapy, which inflicts damage on non-cancerous, healthy tissue and drives cellular senescence in these tissues, which play a pathological role in these side effects.
  • the present invention also relates to the use of the therapeutic derivatives as described above, for the removal of senescent cells following treatment with anti-retroviral drugs, which can cause subcutaneous lipolysis, metabolic dysfunction, and accelerated ageing.
  • anti-retroviral drugs may include abacavir, azacytidine, azidothymidine ( ⁇ ), dideoxycytidine, efavirenz, invirase, ixabepilone, lamivudine (3TC), lopinavir, nevirapine, ritonavir, stavudine, tenofovir, tipranavir, or thiotepa.
  • the present invention also relates to the use of the therapeutic derivatives as described above, for the removal of senescent cells following treatment with corticoids including Cortisol, prednisone, prednisolone, dexamethasone, triamcinolone, beclometasone, fludrocortisone, deoxycorticosterone, and aldosterone.
  • corticoids including Cortisol, prednisone, prednisolone, dexamethasone, triamcinolone, beclometasone, fludrocortisone, deoxycorticosterone, and aldosterone.
  • Chrmoic treatment with these corticoids can lead to cellular senescence and tissue degeneration, for example in tendons following dexamethasone administration, metabolic dysfunction such as obesity and insulin resistance.
  • the present invention also relates to the use of the therapeutic derivatives as described above, for the removal of senescent cells following long term use of PPAR gamma agonists, including thiazolidinediones such as rosiglitazone, pioglitazone, and troglitazone.
  • the removal of senescent cells will prevent the induction of the SASP, which may be associated with increased adverse cardiac events, and increased risk of cancer.
  • the present invention relates to the use of the therapeutic derivatives as described above, for the treatment of cellular hypertrophy, including carciac hypetrtrophy. Senescent cells retain growth factor signaling, but being unable to undergo division, continue to enlarge and undergo hypertrophy.
  • Senescent cells frequently experience constitutive mammalian target of rapamycin (mTOR) complex activation. Removal of senescent cells with the present therapy will treat diseases caused by cellular senescence, for example, cardiac hypertrophy.
  • An eleventh aspect of the present invention relates to the use of the therapeutic derivatives as described above, for the treatment of chronic inflammation, caused by cytokines released as part of the SASP from senescent cells.
  • the present invention relates to reducing the side effects of and accelerated ageing caused by exposure to radiation, which causes DNA damage and cellular senescence, with subsequent release of SASP cytokines.
  • Senescence may be caused by various forms of radiation including alpha radiation, beta radiation, gamma radiation.
  • the present invention also relates to the topical use of the therapeutic derivatives as described above for cosmetic improvements in skin, for example, the removal of moles, which contain melanocytic nevi that have undergone senescence.
  • a dermatologically acceptable carrier is a carrier which is suitable for topical application to the skin.
  • Suitable dermatologically acceptable carriers include: emulsions such as oil-in-water, water-in-oil, oil- in-water-in silicone and water-in-oil- in water emulsions; anhydrous liquid solvents such as oils (e.g. mineral oil), alcohols (e.g. ethanol, isopropanol), silicones (e.g. dimethicone, cyclomethicone); aqueous- based single phase liquid solvents.
  • the cosmetic formulation may comprise skin actives such as vitamins (e.g. vitamin B3, vitamin E, etc), hydroxy acids (e.g. salicylic acid, glycolic acid); exfoliation agents such as zwitterionic surfactants; sunscreen (e.g.
  • the cosmetic formulations may be in the form of ointments, pastes, creams, lotions, gels, powders, solutions or patches.
  • the formulations and compositions are creams, which may further contain saturated or unsaturated fatty acids such as steaeric acid, palmitic acid, oleic acid, palmato-oleic acid, acetyle, or aryl oleyl alcohols, stearic acid. Creams may also contain a non-ionic surfactant, for example, polyoxy-40-stearate.
  • therapeutic derivatives according to the invention may be used in a medicament, which may be a monotherapy (i.e. the sole use of that antagonist or medicament), for eliminating damaged and/or senescent cells or for treating, ameliorating or preventing a disease or condition characterized by the presence of damaged and/or senescent cells.
  • therapeutic derivatives according to the invention may be used as an adjunct to, or in combination with, known therapies for eliminating damaged and/ or senescent cells or for treating, ameliorating or preventing a disease characterized by the presence of damaged and/or senescent cells.
  • the therapeutic derivatives may be used in combination with known agents for treating pulmonary fibrosis, such as corticosteroids.
  • the antagonists of the invention may be used in combination with known techniques for treating cancer, such as chemotherapy and/ or radiotherapy.
  • compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used.
  • the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment.
  • vehicle of medicaments according to the invention should be one which is well -tolerated by the subject to whom it is given.
  • Medicaments comprising therapeutic derivatives according to the invention may be used in a number of ways.
  • compositions comprising therapeutic derivatives of the invention may be administered by inhalation (for example, intranasally).
  • Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin, for example, adjacent the treatment site.
  • Therapeutic derivatives according to the invention may also be incorporated within a slow- or delayed-release device.
  • Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months.
  • the device may be located at least adjacent the treatment site.
  • Such devices may be particularly advantageous when long-term treatment with antagonists used according to the invention is required and which would normally require frequent administration (for example, at least daily injection).
  • therapeutic derivatives and compositions according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment.
  • Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion).
  • amount of therapeutic derivative that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of therapeutic derivatives, and whether it is being used as a monotherapy or in a combined therapy.
  • the frequency of administration will also be influenced by the half-life of the therapeutic derivatives within the subject being treated.
  • Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular therapeutic derivatives in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the disease or condition being treated. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet and time of administration.
  • a daily dose of between o.ooi g/kg of body weight and 10 mg/kg of body weight of the therapeutic derivatives according to the invention may be used for eliminating damaged and/or senescent cells or for treating, ameliorating or preventing a disease or condition characterized by the presence of damaged and/or senescent cells, depending upon which therapeutic derivatives is used. More preferably, the daily dose is between o.oi ⁇ g/kg of body weight and 1 mg/kg of body weight, more preferably between o.i ⁇ g/kg and 100 ⁇ g/kg body weight, and most preferably between approximately ⁇ ⁇ g/kg and 10 ⁇ g/kg body weight.
  • the therapeutic derivatives may be administered before, during or after onset of the condition or disease characterized by the presence of damaged and/or senescent cells.
  • Daily doses may be given as a single administration (for example, a single daily injection).
  • the therapeutic derivatives may require administration twice or more times during a day.
  • therapeutic derivatives may be administered as two (or more, depending upon the severity of the disease or condition being treated) daily doses of between 0.07 ⁇ g and 700 mg (i.e. assuming a body weight of 70 kg).
  • a patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two-dose regime) or at 3- or 4-hourly intervals thereafter.
  • a slow release device may be used to provide optimal doses of therapeutic derivatives according to the invention to a patient without the need to administer repeated doses.
  • a damaged and/or senescent cells-treatment composition comprising a therapeutic derivative according to a first aspect of the invention and a pharmaceutically acceptable vehicle.
  • damaged and/or senescent cells-treatment composition can mean a pharmaceutical formulation used in the therapeutic amelioration, prevention or treatment of any disease condition characterised by the presence of damaged and/ or senescent cells in a subject.
  • the invention also provides in a seventh aspect, a process for making the damaged and/or senescent cells-treatment composition according to the fifth aspect, the process comprising contacting a therapeutically effective amount of a therapeutic derivative according to a first aspect of the invention and a pharmaceutically acceptable vehicle.
  • a "therapeutically effective amount" of a therapeutic derivative of the invention is any amount which, when administered to a subject, is the amount of antibody that is needed to eliminate damaged and/or senescent cells, or produce the desired effect.
  • the therapeutically effective amount of a therapeutic derivative according to a first aspect of the invention used may be from about o.ooi mg to about ⁇ mg, and preferably from about o.oi mg to about 500 mg. It is preferred that the amount of agent is an amount from about 0.1 mg to about 100 mg, and most preferably from about 0.5 mg to about 50 mg.
  • a "pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.
  • compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (for example, cattle), pets, or may be used in other veterinary applications. Most preferably, though, the subject is a human being.
  • the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet.
  • a solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavoring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings or tablet-disintegrating agents.
  • the vehicle may also be an encapsulating material.
  • the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention.
  • the active agent may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain up to 99% of the active agents.
  • Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
  • the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.
  • the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution.
  • Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
  • the active agent according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
  • the liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilisers or osmo-regulators.
  • liquid vehicles for oral and parenteral administration include water (partially containing additives as above, for example, cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, for example, glycols) and their derivatives, and oils (for example, fractionated coconut oil and arachis oil).
  • the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate.
  • Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration.
  • the liquid vehicle for pressurised compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
  • Liquid pharmaceutical compositions which are sterile solutions or suspensions, can be utilised by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection.
  • the a therapeutic derivative according to a first aspect of the invention may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline or other appropriate sterile injectable medium.
  • the therapeutic derivative according to a first aspect of the invention and compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 8o (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
  • the antagonists used according to the invention can also be administered orally either in liquid or solid composition form.
  • compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets and powders, and liquid forms, such as solutions, syrups, elixirs and suspensions.
  • forms useful for parenteral administration include sterile solutions, emulsions and suspensions.
  • nucleic acid or peptide or variant, derivative or analogue thereof which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof.
  • substantially the amino acid/nucleotide/peptide sequence can be a sequence that has at least 40% sequence identity with the amino acid/nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequences referred to herein.
  • amino acid/nucleotide/peptide sequences with a sequence identity which is greater than 50%, more preferably greater than 65%, 70%, 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to herein are also envisaged.
  • the amino acid/nucleotide/peptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90%, 92%, 95%, 97%, 98%, and most preferably at least 99% identity with any of the sequences referred to herein.
  • the percentage identity for two sequences may take different values depending on: (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local versus global alignment, the pair-score matrix used (for example, BLOSUM62, PAM250, Gonnet etc.) and gap-penalty, for example, functional form and constants.
  • the method used to align the sequences for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison
  • the parameters used by the alignment method for example, local versus global alignment, the pair-score matrix used (for example, BLOSUM62, PAM250, Gonnet etc.) and gap-penalty, for example, functional form and constants.
  • percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length-dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.
  • calculation of percentage identities between two amino acid/nucleotide/peptide sequences may then be calculated from such an alignment as (N/T)*ioo, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs.
  • a substantially similar nucleotide sequence will be a sequence which hybridises to a nucleotide sequence encoding a peptide according to the first aspect, or a functional fragment or functional variant thereof, or their complements, under stringent conditions.
  • stringent conditions is meant that the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45 °C followed by at least one wash in 0.2.x SSC/0.1% SDS at approximately 20-65 °C.
  • a substantially similar peptide may differ by at least 1, but less than 3, 4, 5, 6, 7, 8, 9 or 10 amino acids from the sequences disclosed.
  • nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the peptide, polypeptide or protein encoded thereby, to provide a functional variant thereof.
  • Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
  • Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
  • small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine.
  • Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
  • the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
  • the positively charged (basic) amino acids include lysine, arginine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids maybe replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.
  • Table ⁇ lists the different combinations for covalently bonding doxorubicin or epirubicin compounds with saccharides
  • Table 2 lists the different combinations for covalently bonding Navitoclax or ABT-263, ABT-737 and ABT-199 compounds with saccharides;
  • Figure 1 illustrates a schematic presentation of one embodiment of a liposome coated with saccharide and loaded with a drug.
  • Figure 2 is an illustration of the structures of navitoclax and navitoclax linked to acetyl-galactose (compound 13 or NGia).
  • Figure 3 is an illustration of the results of proton nuclear magnetic resonance ( ⁇ - NMR) of navitoclax linked to acetyl-galactose (compound 13 or NGia).
  • Figure 4 is an illustration of the results of high resolution mass spectroscopy (HRMS) of navitoclax linked to acetyl-galactose (compound 13 or NGia).
  • HRMS high resolution mass spectroscopy
  • Figure 5 is an illustration of the results of a cell viability test after 24 hours of navitoclax and navitoclax linked to acetyl-galactose (compound 13 or NGia).
  • Figure 6 is an illustration of the results of a cell viability test after 48 hours of navitoclax and navitoclax linked to acetyl-galactose (compound 13 or NGia).
  • Example 1 Synthesis of the liposomes coated with saccharide and loaded with a drug
  • sugars are covalently bounded to phospholipids. Details of the preparation steps of the invention are given in the following example for a hexagalactooligosaccharide (galactan).
  • Hydrogenated Soy phosphatidylcholine (HSPC), cholesterol, N-(Carbonyl- methoxypolyethylene glycol 2000 ⁇ -i,2-distearyl-sn-glycero-3- phosphoethanolamine (mPEG-2000-DSPE) and dipalmitoylphosphatidylethanolamine at a 30:10:10:2 HSPC:Chol:mPEG-2000-DSPE:DPPEA (mol ratio) were solved in ethanol under stirring and the temperature was maintained at 65°C. Aqueous ammonium sulfate solution was prepared in a glass container with water at 65°C. The lipid mixture was slowly injected into the ammonium sulfate solution through an orifice of the nozzle with stirring.
  • HSPC Hydrogenated Soy phosphatidylcholine
  • mPEG-2000-DSPE N-(Carbonyl- methoxypolyethylene glycol 2000 ⁇ -i,2-distearyl-sn-glycer
  • Multilamellar vesicles (MLVs) produced were extruded through a thermobarrel extruder while maintaining the temperature of suspension at 65°C.
  • the extrusion process comprised of passing the suspension through a 200 nm (1 time) and 8onm (3 times) pore size polycarbonate membrane filter sequentially in order to obtain a particle size in the range 80 nm to 100 nm (determined by using dynamic light scattering technique).
  • the liposomal suspension was subjected to dialysis with 10% w/v sucrose solution as the exchange buffer and to remove external phase ammonium sulfate ions. The process of dialysis was continued until the external ion concentration was less than 100 ppm.
  • the doxorubicin liposomes were sterile filtered using a 200 nm syringe filter and the suspension transferred to glass vials under nitrogen purging for their storage.
  • acetylated- saccharide-bromide mono, di, tri, tetra, penta, hexa-saccharide or galacto-oligosaccharide (GOS) a round bottom flask was charged with the desired saccharide (1 eq) and acetic anhydride (100 eq). The mixture was stirred and perchloric acid was added dropwise. Then the reaction was treated with hydrobromic acid in acetic acid (33% w/w, 0.6 eq) for 90 minutes. The acetylated and brominated product was extracted with dichloromethane, dried over magnesium sulfate and the solvent was eliminated under vacuum.
  • N-protecting group e.g Fmoc or Boc
  • a round bottom flask was charged with N-protected doxorubicin or epirubicin (1 eq) and 2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl- bromide or any acetylated- saccharide-bromide derivative (1.5 eq).
  • the mixture was stirred with an excess of DIPEA at 6o°C for 4 h under argon atmosphere.
  • the isolation of the product involved the purification by flash chromatography on silica gel or precipitation by converting doxorubicin derivative in HC1 salt. See Table 1.
  • doxorubicin or epirubicin must be protected with any iV-protecting group (e.g Fmoc or Boc) and hydroxyl group in amino-sugar moiety with any O-protecting group (e.g THP).
  • a round bottom flask was charged with N- and O-protected doxorubicin or epirubicin (1 eq) and 2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl- bromide or any acetylated- saccharide-bromide derivative (1.5 eq).
  • the mixture was stirred with an excess of DIPEA at 6o°C for 7 h under argon atmosphere.
  • the isolation of the product involved the purification by flash chromatography on silica gel or precipitation by converting doxorubicin derivative in HC1 salt. See Table 1.
  • a round bottom flask was charged with ABT-263 or ABT-737 (1 eq), 2,3,4,6-tetra-O- acetyl-a-D-galactopyranosyl-bromide or any acetylated- saccharide-bromide derivative (1.5 eq) and K 2 C0 3 (1.9 eq).
  • Anhydrous acetonitrile was added (3 ml/eq). The mixture was stirred at 70°C for 3 h under argon atmosphere. The solvent was eliminated under vacuum.
  • the product was purified by flash chromatography on silica gel and ethyl acetate-hexane (7:3, v/v). See Table 2 and Figure 2 for compound 13.
  • Example 3 Characterization of the navitoclax linked to acetyl-galactose (compound 13 or NGia).
  • the navitoclax and the new compound 13 or NGia were synthesized as it was described above in Example 2 and their structures are shown in Figure 2.
  • the new compound 13 or NGia was characterized by ⁇ -NMR and HRMS.
  • the chemical shift of the anomeric proton observed by ⁇ -NMR clearly indicated the formation of the desired product compound 13 or NGia, see Figure 3.
  • Formation of the compound 13 or NGia was also confirmed by HRMS. MS spectrum showed the exact mass of the desired product, thus indicating its formation. See Figure 4.
  • Example 4 Cell viability test after 24 or 48 hours of navitoclax and navitoclax linked to acetyl-galactose (compound 13 or NGia).
  • SK-MEL-103 cells human melanoma acquired from the American Type Culture Collection were used for cell viability assays.
  • Cells were maintained in DMEM and supplemented with 10% FBS and penicillin-streptomycin (all from Gibco), and incubated in 20% O2 and 5% CO2 at 37 °C.
  • DMEM media containing palbociclib (Pfizer, 5 ⁇ ) for 2 weeks.
  • Control and senescent cells were plated in flat-bottom-clear 96 well plates (Greiner Bio- One, 655087) at a density of 6000 and 4000 cells per well respectively.

Abstract

La présente invention concerne de nouvelles formulations de dérivés thérapeutiques pour une thérapie de la sénescence. Les molécules comprennent des conjugués modifiés de liposomes ou de médicaments liés aux saccharides qui conservent le même degré d'activité thérapeutique que le composé parent non modifié. Par conséquent, l'invention se situe dans les domaines thérapeutiques cliniques.
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