Classifications

• • 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
  • A61K47/58—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 obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
• • 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
  • A61K47/59—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/595—Polyamides, e.g. nylon
• • 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
  • A61K47/62—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 a protein, peptide or polyamino acid
  • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
  • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
  • C08F220/52—Amides or imides
  • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
  • C08F220/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen

Definitions

• Copolymer with hydrolytic release of cancerostatic agent cytarabine method of its preparation and use thereof
• the invention relates to the structure and properties of water-soluble polymer therapeutics based on cytarabine derivatives intended primarily for the treatment of leukaemias and non-Hodgkin's lymphomas in human medicine.
• polymeric conjugates of anticancer drugs with soluble polymers have been prepared and studied, in which a drug with antitumor effect has been attached to the polymer by a non-cleavable covalent bond, a hydrolytically unstable ionic bond or a covalent bond enabling controlled release of the drug and thus its activation by enzymatic or simple chemical hydrolysis of this bond.
• polymeric carrier systems are designed to be capable of releasing a therapeutically active anticancer drug from a carrier either in a tumour, or more specifically, directly in a tumour cell.
• polymer therapeutics are polymeric drugs prepared on the basis of copolymers of N-( 2- hydroxypropyl)methacrylamide (HPMA), many of which are actively targeted to tumours through a targeting structure (antibody, lectin, hormone) attached to the polymer.
• HPMA N-( 2- hydroxypropyl)methacrylamide
• a targeting structure antibody, lectin, hormone
• polymer cancerostatics it has also been demonstrated that it is not necessary to use an actively targeted carrier for specific drug delivery to the tumour site, but significantly increased accumulation of polymer cytostatics, especially in solid tumours, can be achieved by increasing the molecular weight of the polymer therapeutic (so-called passive targeting to solid tumours).
• This ability of macromolecules to accumulate in solid tumours has been termed the Enhanced Permeability and Retention (EPR) effect, and this effect has been shown to be also significant in HPMA copolymer-based carriers.
• EPR Enhanced Permeability and Retention
• HPMA copolymers as passively targeted high-molecular- weight carriers is their non-cleavable carbon chain. Only polymers not exceeding a molecular weight of 40,000 to 50,000 g/mol can be eliminated from the body. That is, if the polymer is not to accumulate in the body after repeated administration of the drug, and if the molecular weight of the carrier is to be as high as possible to make passive targeting as effective as possible, the polymeric carrier must be designed to be degradable in the body.
• HPMA copolymers carrying the cancerostatic drug doxorubicin bound to the polymer chain by a hydrolytically cleavable hydrazone bond play an important role [Etrych T, Chytil P, Jelinkova M, Rihova B, Ulbrich K, Synthesis of HPMA Copolymers Containing Doxorubicin Bound via a Hydrazone Linkage. Effect of Spacer on Drug Release and in vitro Cytotoxicity. Macromolecular Biosci.
• cytarabine is used both in the first-line treatment and as part of consolidation and rescue regimes. It is cytosine arabinoside, where cytosine is linked to an arabinose sugar. Because cytarabine is sufficiently structurally similar to deoxycytosine, it can be incorporated into DNA instead. However, cytarabine must first enter the tumour cell. At low plasma concentrations, cytarabine is predominantly actively transported into the cell by transporters Solute Carrier Family 29 Members 1 and 2 (SLC29A1, SLC29A2). Upon reaching high plasma concentrations ( ⁇ 10 ⁇ M), cytarabine gets into tumour cells passively according to a concentration gradient. Cytarabine is itself a prodrug without a direct antitumor effect.
• cytarabine Inside tumour cells, cytarabine must first be activated by sequential phosphorylation to cytarabine triphosphate and then incorporated into DNA. A key step in activating cytarabine is thought to be its phosphorylation by the enzyme deoxycytidine kinase (dCK) to cytarabine monophosphate. Repeated studies have shown that the acquired resistance of tumour cells to cytarabine is due to a decrease in dCK expression.
• dCK deoxycytidine kinase
• the metabolism, activation and deactivation of cytarabine and its metabolites is a highly complex process and depends, in addition to the dose and route of administration, on the microenvironment (different concentrations of cytidine deaminase in different parts of the cell) and on congenital genetic factors.
• Cytarabine is one of the cytostatics that specifically affect the S phase of the cell cycle, as it strongly interferes with DNA synthesis and thus damages DNA in the replication phase. Due to their high rate of proliferation, rapidly dividing cells of haematological malignancies are far more affected than healthy cells.
• the main degradation mechanism of cytarabine and its metabolites is deamination, in particular by the enzyme cytidine deaminase to the corresponding inactive uracil derivatives.
• cytarabine Due to the rapid degradation, cytarabine is therefore dosed either in repeated short-term infusions at high doses (so-called high-dose cytarabine, usually 2-3 grams of araC/m 2 of the patient's body surface area given in a short-term 2-3 hour infusion, 4 to 6 applications after 12 hours), or is administered continuously in significantly lower doses (usually 100 - 200 mg araC/m 2 in a continuous infusion usually for 7 days).
• Palliative therapy for acute leukaemias and myelodysplastic syndromes also uses repeated subcutaneous applications of small doses of cytarabine (usually 20 mg araC s.c. twice daily for 10 days).
• cytarabine therefore has a crucial impact on its anti-tumour effect and toxicity (especially haematological).
• Intrathecal applications of small doses of cytarabine 50 mg are used to treat patients with central nervous system disorders with meningeal leukaemia or lymphoma.
• liposomal cytarabine (DepoCyt®) was approved in the United States for intrathecal administration in the indication of lymphomatous or leukaemic cells infiltrating the meninges to achieve longer exposure due to the gradual release of cytarabine from the liposome.
• liposomal cytarabine is as effective as or more effective than cytarabine alone.
• liposomal cytarabine offers an excellent response rate, improved quality of life of patients and prolonged time to neurological progression. If the cause of meningitis is a solid tumour, liposomal cytarabine will prolong the time to neurological progression and improve quality of life.
• Depocyt® was discontinued in 2017 due to unclear (unspecified technical) reasons, was withdrawn from the market and is therefore no longer available. Recently, a paper has been published describing the preparation of a system for the controlled delivery of cytarabine.
• the present invention relates to the structure, synthesis and use of a novel polymeric drug with a narrow molecular weight distribution ( ⁇ ⁇ 1.1 to 1.4) and with a defined content of covalently bound cytarabine.
• the described polymeric drug is characterized by prolonged pharmacokinetics, increased accumulation in the tumour and significant anti-tumour activity. It is a random linear or star polymeric conjugate composed of monomeric units N-(2-hydroxypropyl) methacrylamide) and monomeric units carrying cytarabine attached to the polymer backbone via linkers that allow hydrolytic release of cytarabine in the human body.
• An object of the present invention is a polymeric conjugate comprising from 80 to 95 mol% of monomeric units of HPMA and from 5 to 20 mol% of monomeric units of the general formula I and optionally II (based on the sum of monomeric units of HPMA, (I) and (II)) wherein
• X is a covalent bond or an aminoacyl attached via an amide bond to the carbonyl group of the monomeric unit (I) and, optionally, to the carbonyl of the monomeric unit (II) and selected from the group consisting of
• n is an integer from I to 6; q is selected from 1, 2 and 3; A is selected from the group consisting of methyl, isopropyl, cyclohexyl and pyridyl; L is a covalent bond or 1,4-phenylene; wherein -CH 2 - of group X may be further substituted with one or more of the same or different natural amino acid side chains; and/or the phenylene of group X may be further substituted with one or more of the same or different natural amino acid side chains; preferably the chains of the natural amino acid are methyl, isopropyl, isobutyl, -CH(CH 3 )(CH 2 CH 3 ), -CH 2 OH, -CH(OH)(CH 3 ), -CH 2 -(C 6 H4)OH, -(CH
• amino acids By natural amino acids are meant histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, cysteine, glutamine, glycine, proline, tyrosine, alanine, aspartic acid, asparagine, glutamic acid, serine, selenocysteine.
• the side chains are chains attached to the alpha-carbon of the amino acid.
• X is a covalent bond or X is selected from the group consisting of:
• the polymeric conjugate is a linear statistical copolymer of HPMA and monomeric units of general formula (I) and optionally (II).
• This linear statistical copolymer can be represented by the general formula (III).
• the molar mass of the linear statistical copolymer is from 4,000 to 100,000 g/mol, preferably from 40,000 to 70,000 g/mol.
• Groups X and Z are defined as above.
• the end groups of the linear statistical copolymer contain parts of polymerisation initiator molecules (e.g., azo initiator 2,2'-azobis(2-methylpropionitrile) (AIBN), 4,4'-azobis(4-cyanopentanoic acid)
• polymerisation initiator molecules e.g., azo initiator 2,2'-azobis(2-methylpropionitrile) (AIBN), 4,4'-azobis(4-cyanopentanoic acid
• ACVA 2,2'-azobis(4-methoxy-2,4-dimethylpentanenitrile (V70)
• a transfer agent selected from the group of 2-cyano-2-propylbenzodithioate, 4-cyano-4- (thiobenzoylthio)pentanoic acid, 2- cyano-2-propyldodecyl trithiocarbonate, 2-cyano-2-propylethyl trithiocarbonate and 4-cyano-4 - [(dodecylsulphanylthiocarbonyl)sulphanyl]pentanoic acid.
• the end groups of the copolymer contain at one end a radical leaving group of a transfer agent, preferably 2- cyano-2-propyl, or 5-carboxy-2-cyano-2-pentyl.
• a radical leaving group of a transfer agent preferably 2- cyano-2-propyl, or 5-carboxy-2-cyano-2-pentyl.
• a dithiobenzoate or trithiocarbonate group which can be removed by reaction with excess AIBN or can be converted in situ after its reduction to other reactive groups, for example to dibenzocyclooctyne (DBCO), azide, carboxyl group, amino group, which are further usable for preparation of star copolymers.
• DBCO dibenzocyclooctyne
• the polymeric conjugate is a star copolymer of general formula (IV), wherein the multivalent carrier is a second or third generation poly(amidoamine) (PAMAM) dendrimer (for example with ethylenediamine, 1,4-diaminobutane, 1,6-diaminohexane, 1,12- diaminododecane, cystamine core) or 2,2-bis(hydroxymethyl)propionic acid based second to fourth generation dendrimer or dendron (e.g. with trimethylolpropane nucleus);
• PAMAM poly(amidoamine) dendrimer
• X and Z are as defined above;
• Y is selected from the group consisting of a primary amino group; alkyne group of 3 to 6 carbons, such as propargyl; and a cyclooctyne group which may be optionally further independently substituted with one or more groups selected from an alkyl group of 1 to 6 carbons and aryl of 6 carbons, e.g. DBCO; and W is an amide bond between the primary amino group Y of the multivalent carrier and the carboxyl end group of the polymer of general formula (III); or a triazole linker formed by the reaction of an azide end group of the polymer of general formula (III) and an alkyne or cycloalkyne group Y of a dendrimer or dendron (multivalent carrier).
• the star copolymer consists of a central molecule of PAMAM dendrimer or 2,2-bis (hydroxymethyl)propionic acid-based dendrimer or dendron, to the end groups of which at least one chain of the linear statistical copolymer of general formula (III), as defined above, is attached.
• the end groups of the multivalent carrier are amino groups or alkyne groups of 3 to 6 carbons, e.g. propargyl, or cyclooctyne groups, which may be optionally further independently substituted with one or more groups selected from alkyl group of 1 to 6 carbons and aryl of 6 carbons, e.g. DBCO, and the linear copolymer of formula (III) is attached to them via an amide or triazole linker.
• the star copolymer comprises from 2 to 48 linked chains of the linear statistical copolymer of general formula (III), more preferably 8 to 32 linked chains of the linear statistical copolymer of general formula (III), most preferably 16 to 24 linked chains of the linear statistical copolymer of general formula (III).
• Molar mass M n of the star copolymer of general formula (IV) is in the range of from 60,000 to 1,000,000 g/mol, preferably from 100,000 to 400,000 g/mol.
• the molar mass of each chain of the linear statistical copolymer of general formula (III) bound to the multivalent carrier is from 4,000 to 100,000 g/mol, preferably 40,000 to 70,000 g/mol, while the molar mass of the multivalent carrier alone which is part of the copolymer of general formula (IV) does not exceed 50,000 g/mol.
• Scheme 1 Scheme of the preparation of a star copolymer, wherein n is an integer in the range of from 1 to 48, Y is a primary amino group, a C3-C6 alkyne group or a cyclooctyne group (e.g. DBCO group) of poly(amidoamine) or 2,2-bis(hydroxymethyl)propionic acid based dendrimer or dendron:
• V is an azide or TT end reactive group introduced by a transfer agent to the end of the polymer chain of general formula (III);
• W is an amide bond or a triazole linker formed by the reaction of the general formula (III) polymer azide and an alkyne group or cycloalkynyl group of a dendrimer or dendron.
• a further object of the present invention is a method for the preparation of the linear statistical copolymer of general formula (III) according to the present invention, which comprises the following steps: (i) providing an HPMA monomer and a monomer of general formula (V) and/or (VI) wherein
• Z is a reactive substitution derivative of X as defined above, preferably Z is selected from the group comprising -COOH; thiazoline-2-thione (TT); (4-nitrophenyl)oxy group; (2, 3, 4,5,6- pentafluorophenyl)oxy group; (succinimidyl)oxy group; -OH; Boc-NH-NH- (tert-butyloxycarbonyl hydrazide);
• step (iii) in case the step (ii) is the polymerisation of HPMA with a monomer of general formula (VI), the step of attaching cytarabine to the Z group of the polymer of step (ii), as defined above;
• HPMA N-(2-hydroxypropyl)methacrylamide
• Monomers of general formula (VI) are prepared by acylation of the corresponding amino acids (from which aminoacyls X described above are derived) or derivatives thereof with reactive methacrylic acid derivatives, preferably with methacroyl chloride, and subsequent modification of the end aminoacyl with the reactive group Z defined above in a non-aqueous medium with using carbodiimide -based condensing agents.
• Ad (ii) The radical polymerisation takes place at a temperature in the range of from 30 to 100 °C, preferably from 40 to 80 °C, in a solvent preferably selected from the group consisting of water, aqueous buffers, dime thy lsulphoxide, dimethylacetamide, dimethylformamide, methanol, ethanol, dioxane, tert-butyl alcohol or mixtures thereof, initiated by an initiator, preferably selected from the group comprising, in particular, azo initiators 2,2'-azobis(2-methylpropionitrile) (AIBN), 4,4'- azobis(4-cyanopentanoic acid) (ACVA), 2,2'-azobis(4-methoxy-2,4-dimethylpentanenitrile) (V70), optionally in the presence of a transfer agent, preferably selected from the group comprising 2-cyano- 2-propylbenzodithioate, 4-cyano-4-(thiobenzoylthio)penta
• Ad (iii) The optional reaction of the monomeric unit of general formula (II) with cytarabine takes place at a temperature between 20 and 100 °C, preferably from 30 to 60 °C in a solvent preferably selected from the group comprising pyridine, dimethylsulphoxide, dimethylacetamide, dimethylformamide, methanol, ethanol or mixtures thereof.
• the molar mass M n of the linear conjugates thus prepared is in the range of from 4,000 to 100,000 g/mol, preferably from 40,000 to 70,000 g/mol.
• the reaction with cytarabine is preceded by removal of the Boc protecting group, for example by acid hydrolysis with HC1 or CF 3 COOH, or by thermal hydrolysis in water at 100 °C.
• Ad (iv) The step of removing the end reactive groups of the linear copolymers in the preparation of which the transfer agent was used.
• the end groups of the linear polymer of step (ii) or (iii) are reacted with an excess of azo initiator selected from the group of initiators described in step (ii) at a temperature in the range of from 50 to 100 °C, preferably from 60 to 80 °C, in a solvent preferably selected from the group comprising dimethylsulphoxide, dimethylacetamide and dimethylformamide, to form the polymer of general formula (III).
• Another object of the present invention is a method for the preparation of the star copolymer as defined above, comprising the following steps:
• the transfer agent is selected from the group comprising [4-(3-azidopropylamino)-1- cyano-1-methyl-4-oxo-butyl]benzenecarbodithioate, N-(3-azidopropyl)-4-cyano-4-ethylsulphanyl- carbothioylsulphanyl-pentanamide, [1-cyano-1-methyl-4-oxo-4-(2-thioxothiazolidin-3-yl)butyl] benzenecarbodithioate or 2-ethylsulphanylcarbothioyl-sulphanyl-2-methyl-5-oxo-5-(2-thioxo- thiazolidin-3-yl)pentanenitrile.
• the molar mass M n of the linear copolymers thus prepared is in the range of from 4,000 to 100,000 g/mol, preferably from 25,000 to 50,000 g/mol.
• the resulting semitelechelic linear copolymer of general formula (III) contains end reactive groups (azide and TT) introduced by the transfer agent are further used in step (iii).
• a multivalent carrier for grafting the semitelechelic copolymer of step (ii), wherein the multivalent carrier is selected from the group comprising second or third generation poly (amidoamine) (PAMAM) dendrimer and 2,2-bis(hydroxymethyl) propionic acid (bis-MPA) based second, third or fourth generation dendrimer or dendron, terminated by Y groups selected from primary amino groups, alkyne groups of 3 to 6 carbons, e.g. propargyl, and cyclooctyne groups which may be optionally further independently substituted with one or more groups selected from an alkyl group of 1 to 6 carbons and aryl of 6 carbons, e.g. DBCO.
• PAMAM poly (amidoamine)
• bis-MPA 2,2-bis(hydroxymethyl) propionic acid
• Said multivalent carriers are commercially available, optionally the DBCO end groups can be prepared by reacting primary amino end groups with DBCO-NHS.
• the grafting reaction of the polymers onto the cores takes place in a solvent preferably selected from the group comprising dimethylsulphoxide, dimethylacetamide, dimethylformamide, methanol and ethanol.
• the molar mass M n of the copolymers thus prepared is in the range of from 60,000 to 1,000,000 g/mol, preferably from 100,000 to 400,000 g/mol.
• step (v) optionally, if the resulting star copolymer of step (iv) contains monomeric units of general formula (II), the step of attaching cytarabine to the Z group of the polymer of step (iv) at a temperature between 20 and 100 °C, preferably from 30 to 60 °C, in a solvent preferably selected from the group comprising pyridine, dimethylsulphoxide, dimethylacetamide, dimethylformamide, methanol, ethanol or mixtures thereof.
• a solvent preferably selected from the group comprising pyridine, dimethylsulphoxide, dimethylacetamide, dimethylformamide, methanol, ethanol or mixtures thereof.
• the reaction with cytarabine is preceded by removal of the Boc protecting group, for example by acid hydrolysis with HC1 or CF 3 COOH, or by thermal hydrolysis in water at 100 °C.
• Another object of the present invention is a pharmaceutical composition
• a pharmaceutical composition comprising as active ingredient the linear statistical polymeric conjugate of general formula (III) according to the present invention and/or a star polymeric conjugate of general formula (IV) according to the present invention; wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient selected from the group comprising anti-adhesives, binders, coating agents, colourants, swelling agents, flavouring agents, lubricants, preservatives, sweeteners, sorbents.
• solvents especially water or saline
• ionising additives antioxidants, anti-microbial additives.
• Another object of the present invention is the use of the polymeric conjugate of cytarabine as defined above in human medicine, in particular in the treatment of malignancies, preferably for the treatment of solid tumours and/or haematological tumours, more preferably for the treatment of leukaemias and non-Hodgkin's lymphomas.
• the polymeric conjugate of cytarabine according to the present invention in which cytarabine is bound to the polymeric carrier by an amide bond to the carbonyl group of group X as defined above, does not allow deamination of cytarabine in plasma after drug administration, and thus disables its degradation to uracil derivative. For this reason, the circulation time of bound cytarabine in the body is significantly prolonged, which makes it possible to transport cytarabine on high-molecular polymer carrier to the site of action and to release it there in its original form.
• the polymeric drug according to the present invention is further characterized in that the binding of the drug to the polymeric carrier is relatively stable, so that a maximum of 7 % of the drug is released in 24 h during transport in the bloodstream and body fluids, and the binding is also hydrolytically cleavable in the tumour environment and within target tumour cells in lysosomes.
• This means that the drug is transported through the bloodstream in an inactive, polymer-bound form, and its release and activation occurs mainly after entering the tumour tissue or penetrating the target tumour cells. Activation of the drug only in the target cells leads to the protection of cytarabine against premature degradation in the organism, elimination of side effects and targeting of its effect preferentially on tumour cells.
• the binding of cytarabine to the polymeric carrier significantly increases the molecular weight of the drug and thus prolongs its circulation in the bloodstream in an inactive form, prolongs the total residence time of the drug in the body and thus increases its bioavailability.
• the targeted (passive) transport to the tumour or tumour cells is the responsibility of the polymeric carrier prepared on the basis of HPMA copolymers, the molecular weight and thus the efficiency of accumulation in tumour tissue of which can be controlled by changes in the structure of the polymeric carrier (linear polymer, high- molecular-weight biodegradable star polymer).
• the linear polymeric precursor has a molecular weight below the limit of renal filtration, and is thus easily removed from the body by renal filtration. After cytarabine binding, the molecular weight increases above the limit of renal filtration and thus there is a significant prolongation of the circulation time and accumulation in solid tumours and lymphomas. Upon release of cytarabine, the molecular weight of the polymeric carrier decreases again to a value below the limit of renal filtration.
• the basic monomers are synthesized: HPMA, monomers of general formulas (V) and (VI), which are methacryloylated derivatives of amino acids and oligopeptides terminated by an amino-reactive group, methacryloylated derivatives of amino acids and oligopeptides with amide-linked cytarabine.
• precursors i.e. linear statistical copolymers of HPMA and monomeric units of general formulas (I) and/or (II), bearing functional groups serving as polymeric drug carriers, or cytarabine polymeric conjugates themselves, are synthesized by radical polymerisation.
• the polymeric precursor bearing an amino-reactive functional group along the chain can be prepared either by radical copolymerisation of the above-mentioned functional monomers with HPMA or by their controlled radical (RAFT) polymerisation.
• RAFT controlled radical
• a polymeric precursor is copolymer of HPMA and methacryloylated amino acids or oligopeptides, or hydrazides of amino acids selected from the group consisting of aminomethanoyl (AM), 2- aminoethanoyl (AE), 6-aminohexanoyl (AH), 3-aminopropanoyl (AP), 4-aminobutanoyl (AB), 5- aminopentanoyl (VAL), glycylglycyl (GG), glycyl (Gly), 4-aminobenzoyl (BA) and acyls derived from oligopeptides of 2 to 3 amino acids, characterised in that it contains 80 to 95 mol% of HPMA and 5 to 20 mol% of units with amino-reactive functional groups.
• AM aminomethanoyl
• AE 2- aminoethanoyl
• AH 6-aminohexanoyl
• AP 3-aminopropanoyl
• a polymeric conjugate is a compound of the polymeric precursor with cytarabine or its derivative with an oxo acid on the amino group, where cytarabine is attached to the polymeric precursor by an amide bond prepared by reacting the amino group of the drug with amino-reactive groups of the polymer, or prepared by reacting the amino group of the drug with amino-reactive groups of the monomer and subsequent copolymerization with HPMA, or cytarabine is first modified with an oxo acid to form an amide bond between cytarabine and oxo acid, levulinic acid (LEV), 4-pyridyl-4-oxo- butanoic acid (PYR), 4-2-oxopropyl-benzoic acid (OPB), 5 -cyclohexyl-5 -oxo-pentanoic acid (COP) and 5-methyl-4-oxo-hexanoic acid (ILE), and subsequently the oxo group of the derivative is used to bind via
• Figure 1 Graph showing the rate of cytarabine release from linear polymeric conjugates in pH 7.4 buffer (bloodstream model).
• Figure 2 Graph showing the rate of cytarabine release from linear polymeric conjugates in pH 7.4 buffer (bloodstream model) and pH 5 buffer (cell model).
• Figure 3 Demonstration of in vivo biological activity.
• Polymeric araC conjugates are more effective in treating MCE compared to araC alone.
• the following dosages were used: araC 4 mg/mouse (rhomb), polymeric conjugate p(HPMA-co-Ma-GFLG-araC) 4 mg/mouse (cross), polymeric conjugate p(HPMA-co-MA-AH-araC) 4 mg/mouse (circle).
• HPMA was prepared according to the procedure previously described (Ulbrich K, ⁇ ubr V, Strohalm J, Plocová D, Jel ⁇ nková M, ⁇ hová B, Polymeric Drugs Based on Conjugates of Synthetic and Natural Macromolecules I. Synthesis and Physico-chemical Characterisation. J. Controlled Rel. 64, 63-79 (2000)). Elemental analysis: calculated 58.8 % C, 9.16 % H, 9.79 % N; found 58.98 % C, 9.18 % H, 9.82 % N. The product was chromatographically pure.
• MA-AH-TT 3-(6-Methacrylamidohexanoyl)thiazolidine-2-thione
• MA-VAL-TT 3-(5-methacrylamidopentaoyl)thiazolidine-2-thione
• MA-AH-NHNH-Boc 1-(tert-butoxycarbonyl)-2-(6-methacrylamidohexanoyl)hydrazine
• MA-VAL-NHNH-Boc 1-(tert-butoxycarbonyl)-2-(6-methacrylamidopentanoyl)hydrazine
• Monomer with MA-AH-araC was prepared by reacting said reactive monomer Ma-AH-TT according to the following procedure.
• MA-AH-TT 100 mg was dissolved together with 81 mg of cytarabine in 1 mL of pyridine under Ar atmosphere at 50 °C and stirred for 3 days. The solution was then precipitated into diethyl ether. The product was precipitated several times from methanol into diethyl ether and characterized by HPLC and NMR.
• the value of the coefficient thus determined corresponded to the value of the coefficient obtained from the cytarabine content determined by 1H NMR analysis of the polymeric conjugates.
• the purity of all monomers was determined using the HPLC system [Shimadzu HPLC system equipped with the Chromolith Performance RP-18e reverse phase column (100 ⁇ 4.6 mm) and Shimadzu SPD-lOAVvp UV-VIS detector (230 nm); eluent water - acetonitrile with a gradient of 0- 100 vol.% of acetonitrile, flow rate 5 mL min -1 .
• Example 2 Synthesis of cytarabine polymeric conjugate by direct copolymerisation
• the polymeric conjugate poly(HPMA-co-MA-AH-araC) was prepared by controlled solution radical copolymerisation of HPMA (95 mol%, 100 mg) and MA-AH-araC (5 mol%, 15.6 mg) in methanol at 60 °C in the presence of AIBN initiator 4-cyano-4-thiobenzoylsulphanylpentanoic acid as transfer agent.
• the polymerisation mixture was dissolved in tert-butyl alcohol and transferred to a glass ampoule to be bubbled with Ar and sealed. After 16 h at 70 °C, the polymer was isolated by precipitation into acetone.
• the precipitate was then washed with diethyl ether and dried in vacuum.
• the end dithiobenzoate groups were removed from the copolymer by reaction with AIBN (10-fold molar excess) in DMSO (15% polymer solution) under argon atmosphere for 3 h at 70 °C in a sealed vial.
• the polymeric conjugate was isolated by precipitation into acetone.
• the precipitate was washed with diethyl ether and dried in vacuum to dryness.
• poly(HPMA-co-MA-BA-araC), poly(HPMA-co-MA-G-araC), poly(HPMA-co-MA-GG-araC) were prepared analogously to this procedure.
• Example 3 Synthesis of polymeric precursor - copolymer of HPMA with MA-AH-TT
• copolymer poly (HPMA-co-MA-AH-TT) was prepared by controlled RAFT solution radical copolymerization of HPMA and MA-AH-TT in methanol at 60 °C according to the same procedure as described in Example 2.
• Other polymeric precursors, poly(HPMA-co-MA-BA-TT), poly(HPMA- co-MA-G-TT), poly(HPMA-co-MA-VAL-TT) and poly(HPMA-co-MA-GG-TT) containing other amino acid or oligopeptide linkers were prepared analogously to this procedure.
• the polymeric precursors had molecular weights below the renal filtration limit for pHPMA copolymers, determined to be 50,000 g/mol, which subsequently allows the carrier itself to be removed after drug delivery without the risk of its accumulation in the body.
• Lev-Cyt derivative was prepared by modification of cytarabine with activated levulinic acid. Cytarabine (18.0 mg, 74 ⁇ mol) and Lev-TT (19.8 mg, 74 ⁇ mol) were dissolved in 0.35 mL of pyridine. The reaction mixture was bubbled with argon and the reaction was run at 50 °C. The course of reaction was monitored by HPLC. After 2 days the reaction was complete, the solution was precipitated into ethyl ether and the product was dried, characterised by ESI MS (364 g/mol M + Na + ) and HPLC.
• the copolymer poly(HPMA-co-Ma-AH-NH-NH 2 ) was prepared by a two-step synthesis.
• the copolymer poly(HPMA-co-MA-AH-NH-NH-Boc) was prepared by controlled RAFT solution radical copolymerisation of HPMA and MA-AH-NHNH-Boc in methanol at 60 °C according to the same procedure as described in Example 2.
• the polymeric precursor poly(HPMA-co-MA-AH- NH-NH 2 ) was prepared from HPMA-co-MA-AH- NH-NH-Boc by removing Boc protecting groups by incubation in water at 100 °C for 30 min. The final product was obtained by lyophilisation.
• poly(HPMA-co-Ma-AM-NH-NH 2 ), poly(HPMA-co-Ma-AE-NH-NH 2 ), poly (HPMA-co-Ma- AP-NH-NH 2 ) , poly(HPMA-co-Ma-AB-NH-NH 2 ) and poly(HPMA-co-Ma- VAL-NH-NH 2 ) were prepared analogously to this procedure.
• the polymeric precursors had molecular weights below the renal filtration limit for pHPMA copolymers, determined to be 50,000 g/mol, which subsequently allows the carrier itself to be removed after drug delivery without the risk of its accumulation in the body.
• Polymeric conjugates of cytarabine with various linkers attached to a PHPMA carrier by a hydrolytically cleavable bond were prepared by reacting polymeric precursors containing amino- reactive groups with cytarabine in pyridine at 50 °C for 3 days.
• the polymeric conjugate poly(HPMA- co-MA-AH-araC) was prepared according to the following procedure: 850 mg of copolymer poly(HPMA-co-MA-AH-TT) and 150 mg of cytarabine was dissolved in 10 mL of pyridine and the solution was bubbled with argon and stirred at 50 °C for 3 days.
• the characterisation of the polymeric drug is given in Table 2. Total yield of drug binding reaction: 860 mg (86 %). The procedure for binding cytarabine to polymeric precursors with other linkers was similar. The molecular weights of the polymeric conjugates are just above the limit of renal filtration, which should allow a prolonged circulation of these polymeric cytarabine conjugates and thus a greater accumulation within the lymphomatous tissue. Upon drug release, these polymeric carriers with molecular weights below the limit of renal filtration should be well excretable from the body.
• Example 5 To 100 mg of the polymeric precursor poly(HPMA-co-MA-AH-NH-NH 2 ) prepared in Example 5, 15 mg of Lev-Cyt, the oxo acid-modified cytarabine of Example 4, was bound in DMA in the presence of acetic acid (100 mg of polymer / 40 ⁇ l CPECOOH/l mL of DMA). The course of reaction was monitored by TLC (methanol : CHCl 3 (3: 1)). After binding of all cytarabine, the polymerisation mixture was precipitated into diethyl ether and the precipitate was centrifuged.
• the product, p(HPMA- co-MA-AH-hydr-Lev-araC), was purified by gel chromatography on a column packed with Sephadex LH-20 in water. The polymeric fraction was isolated and lyophilised. The content of total cytarabine in the polymeric conjugate was determined by UV/Vis spectrophotometry as described in Example 1. Mw and molecular weight distribution and hydrodynamic radius Rh were determined by liquid chromatography as described in Example 3.
• Polymeric conjugates p(HPMA-co-MA-AH-hydr-OPB- araC), p(HPMA-co-MA-AH-hydr-PYR-araC) and p(HPMA-co-MA-AH-hydr-ILE-araC) were prepared analogously. The characteristics of all prepared polymeric conjugates are given in Table 3.
• a reactive copolymer p(HPMA- co-MA-AH-TT)-N 3 was prepared using azide-CTA, N-(3-azidopropyl)-4- ethylsulphanylcarbothioylsulphanyl-4-methyl-pentanamide as transfer agent containing an azide group, in a manner similar to the reactive copolymer in Example 3.
• the copolymer of cytarabine was prepared from this reactive polymeric precursor as in Example 4.
• the second generation PAMAM dendrimer was modified by reacting its end amino groups with dibenzocyclooctyne N-hydroxysuccinimidyl ester (DBCO-NHS) as follows: 20 mg of PAMAM dendrimer G2 with amino groups (6.7 ⁇ mol of dendrimer, 0.1 mmol of amino groups) was dissolved in 1 mL of methanol and a solution of 5 mg of DBCO-NHS in 0.2 mL of methanol was added. After 1 h, unreacted DBCO was removed from the reaction mixture by gel chromatography (Sephadex LH20, methanol). The result was a PAMAM dendrimer containing end DBCO groups.
• DBCO-NHS dibenzocyclooctyne N-hydroxysuccinimidyl ester
• the modified dendrimer PAMAM-DBCO in the last step reacted with p(HPM A-co-Ma-AH-araC)-N 3 in methanol for 2 h.
• this star conjugate proceeded in two steps.
• the reactive copolymer p(HPMA- co-Ma-AH-araC)-TT was prepared using the transfer agent TT-CTA, [1-cyano-1-methyl-4-oxo-4- (2- thioxothiazolidin-3-yl)butyl]benzenecarbodithioate containing a TT group in a manner similar to the copolymer in Example 2.
• the amino group-containing bis-MPA dendrimer was reacted with p(HPMA-co-Ma-AH-araC)-TT in methanol for 2 h.
• the resulting star polymeric conjugate was precipitated into acetone and dried to constant weight.
• Example 10 Release of cytarabine from polymeric conjugates The amount of cytarabine released from polymeric conjugates with cytarabine linked via an amide bond to the polymer side chain after their incubation in phosphate buffer pH 7.4 (0.1 M phosphate buffer containing 0.05 M NaCl) modelling the bloodstream environment and phosphate buffer pH 5.0 modelling the intracellular environment was determined using the HPLC system (Shimadzu) equipped with the Chromolith Performance RP-18e reverse phase column (100 ⁇ 4.6 mm) and Shimadzu SPD- 10AVvp UV-VIS detector (230 nm); eluent water-acetonitrile with a gradient of 0 to 100 vol.% of acetonitrile, flow rate 0.5 mL ⁇ min -1 .
• HPLC system Shimadzu SPD- 10AVvp UV-VIS detector
• the polymeric conjugates of cytarabine described herein allow for significantly longer circulation of cytarabine in an inactive form and at the same time significantly protect the transported cytarabine against its degradation in the body.
• the polymeric conjugates have clearly shown high hydrolytic stability in the bloodstream environment, which prevents the release and subsequent deamination of transported cytarabine in the circulation, but the drug can accumulate in the form of polymeric conjugate in lymphoma, where the gradually released araC exhibits its anti-tumour activity.
• the release of cytarabine from polymeric conjugates with cytarabine bound by a combination of amide and hydrazone bonds was similarly performed (Fig. 2).
• Example 11 Demonstration of in vivo biological activity of linear polymeric conjugates of cytarabine in mice inoculated with mantle cell lymphoma (MCL)
• VFN-M1 model A mantle cell lymphoma model derived from a patient with relapsed disease (VFN-M1 model) was used to demonstrate the activity of cytarabine conjugates in vivo.
• the immunodeficient mice NOD.Cg-Prkdcscid I12rgtm1Wj1/SzJ (females) were xenotransplanted with 1x10 6 VFN-M1 cells subcutaneously on day 0.
• the drug was administered once intravenously (i.v.) at a time when the tumours were well developed, palpable, and approximately 300 mm 3 in size.
• the tumour sizes in three perpendicular dimensions, the body weight of the mouse, overall health of the mouse and survival were monitored throughout the experiment.
• conjugate p(HPMA-co-MA-AH-araC) was compared with the effect of free cytarabine, the polymeric conjugate p(HPMA-co-MA-GFLG-araC) described in the literature and with the untreated control (Fig. 3).
• the polymeric drug conjugates were dissolved in PBS. The volume of each single dose of the drug was 0.2 mL.

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