EP2413973A1 - Polymeric conjugates of paclitaxel and docetaxel with ph-controlled releasing of the cancerostatic agent - Google Patents

Polymeric conjugates of paclitaxel and docetaxel with ph-controlled releasing of the cancerostatic agent

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Publication number
EP2413973A1
EP2413973A1 EP10716267A EP10716267A EP2413973A1 EP 2413973 A1 EP2413973 A1 EP 2413973A1 EP 10716267 A EP10716267 A EP 10716267A EP 10716267 A EP10716267 A EP 10716267A EP 2413973 A1 EP2413973 A1 EP 2413973A1
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Prior art keywords
polymeric
ptx
drug
dtx
acid
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German (de)
French (fr)
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Tomas Etrych
Karel Ulbrich
Blanka Rihova
Milada Sirova
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Zentiva KS
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Zentiva KS
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    • 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
    • A61K47/62—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

Definitions

  • the invention deals with the structure and properties of water-soluble polymeric therapeutic agents (pro-drugs) based on derivatives of paclitaxel (PTX), docetaxel (DTX) and larotaxel (LTX), destined mainly for treatment of solid tumours in therapy of tumour diseases in human medicine.
  • PTX paclitaxel
  • DTX docetaxel
  • LTX larotaxel
  • Polymeric conjugates of cancerostatic agents with soluble polymers were prepared and studied where the drug with an anti-cancer effect was attached to the polymer with a non-cleavable covalent bond, hydrolytically instable ionic bond and/or covalent bond allowing controlled release of the drug and thus its activation on the basis of enzymatic or plain chemical hydrolysis of this bond.
  • Polymeric carrier systems are generally constructed in such a way to be able to release the therapeutically active cancerostatic agent from the carrier either in the tumour, or more specifically, directly in a tumour cell.
  • polymeric therapeutic agents An important group of polymeric therapeutic agents is represented by polymeric drugs prepared on the basis of copolymers of N-(2- hydroxypropyl)methacrylamide (HPMA), a number of which are actively directed to tumours by means of a targeting structure attached to the polymer (antibody, lectin, hormone).
  • HPMA N-(2- hydroxypropyl)methacrylamide
  • a targeting structure attached to the polymer antibody, lectin, hormone
  • HPMA copolymers as passively targeted high-molecular earners is due to their non-cleavable carbon chain and only polymers not exceeding the molecular weight of 40 - 50,000 g/mol can be excreted from the organism. This means that if accumulation of the polymer in the organism after repeated administration of the drug is not to occur and if the molecular weight of the carrier is to be as high as possible to make the passive targeting as efficient as possible, the polymeric carrier must be constructed as degradable in the organism.
  • Literature mentions a lot of information about the preparation and studying of properties of polymers carrying a cancerostatic agent, attached to the polymer by a bond susceptible to hydrolysis in an aqueous environment [Kratz 1999].
  • HPMA copolymers carrying the cancerostatic agent doxorubicin, bound to the polymeric chain by a hydrolytically cleavable hydrazone bond take an important position [Etrych 2002, Ulbrich 2004a, Ulbrich 2004b, Ulbrich pat CZ 293787 B6].
  • This bond is relatively stable in the bloodstream environment (in the course of transport in the organism) and hydrolytically instable in the slightly acidic environment of the living cell.
  • Paclitaxel and docetaxel belong to the group of taxanes, the anti-cancer drugs routinely used for the treatment of ovarian tumours and breast, lung, prostate and other tumours [Vanhoefer at al., 1997]. Besides their secondary toxicity, common for cancerostatic agents, they have another disadvantage of very low solubility in aqueous solutions, resulting in the necessity of administration in various excipients, especially in Cremophor EL, which also leads to other side effects of the dosage form. In some administration schemes the administration of taxanes in Cremophor EL may even significantly reduce the efficiency of the therapy [Ng at al., 2006].
  • paclitaxel was covalently bound to poly( ethylene glycol) (PEG) by a non-cleavable ester or 7-carbamate bonds (C2 -OH group), or by means of a hydrolytically instable amino-acid spacer (Ala, GIy) [Greenwald et al., 1995; Greenwald, 2001; Greenwald et al., 2003; Pendri et al., 1998]. It has been shown that the anti-tumour activity of the conjugate depends on the structure of the bond used between the polymer and the drug and that the molecular weight of the carrier is another important factor. The conjugate with PTX was tested in phase I of clinical trials [Satchi-Fainaro et al., 2006], but apparently with little success.
  • conjugate of paclitaxel with poly(glutamic acid) appears to be much more successful in clinical trials [Winter 2005, Kratz et al., 2008].
  • PTX is bound to a polyamino-acid earner by an ester bond via the -OH group in position 2.
  • the drug is released due to degradation of the polymeric chain, formation of the GIu derivative of PTX and its subsequent hydrolysis.
  • this conjugate is in phase III of clinical trials.
  • Paclitaxel has also been bound to HPMA copolymers by an ester bond using a biodegradable oligopeptide GlyPheLeuGly spacer. It has been demonstrated that PTX, after incubation with lysosomal enzymes, is released from the carrier and this release is important for achieving the anti-cancer activity in vivo.
  • PNU166945 is a conjugate of a HPMA copolymer with ester-bound PTX, which was clinically tested in phase I [Terwogt et al., 2000; Terwogt et al., 2001], however, after this phase further testing was stopped.
  • a polymeric drug in accordance with this invention is characterized in that a cancerostatic agent from the group of taxanes, paclitaxel (PTX), docetaxel (DTX), or larotaxel (LTX) (hereinafter drugs) is bound to a water-soluble polymeric carrier, prepared on the basis of a linear or grafted HPMA copolymer.
  • the drug is bound to the polymeric chains via an ester group produced by acylation of the -OH group in position 2 by means of spacers, containing pH-sensitive hydrolytically cleavable hydrazone bonds.
  • spacers may consist of acid residues of various oxo acids, by means of which the carbonyl group is introduced to the drug structure, linked with residues of individual amino acids, oligopeptides, or other structures, allowing termination of the side chains of the polymeric earner with the hydrazone group.
  • the molecular weight of the polymeric chain is selected below the excretion limit of HPMA copolymers from the organism, preferably in the range of 10 - 50,000 g/mol.
  • a grafted copolymer the molecular weight is selected in the range of 50 — 250,000 g/mol.
  • the polymeric drug in accordance with the invention is destined for intravenous (injection or infusion) administration in a solution, but it may also be administered intratumourally or intraperitoneally and is intended for treatment of solid tumours.
  • the polymer with the chemically bound cytostatic agent is constructed in such a way to be stable during circulation in the bloodstream and to prevent hydrolysis of the hydrazone bond between the given taxane and the polymer, or, possibly, to keep the hydrolysis rate in the course of transport through the organism as low as possible (at the pH value 7.4 in the bloodstream), so that no cytotoxic effect of the released drug or its derivative can be manifested.
  • the entire system is constructed as a two-phase system.
  • the polymeric conjugates with a targeted anti-cancer effect in accordance with the invention are characterized in that the cytostatic agent (taxol, docetaxel, larotaxel) is attached, by means of an ester bond and spacer, to the polymeric carrier formed by a linear [Etrych pat CZ 297827 B6, publ. 2008], or grafted [Etrych pat CZ 298945 (B6), publ.
  • cytostatic agent taxol, docetaxel, larotaxel
  • HPMA copolymer through a hydrolytically instable hydrazone group produced by a reaction of the carbonyl group of the molecule of the drug derivative with the hydrazide group of the polymeric carrier.
  • the polymeric carriers are generally prepared by radical solution copolymerization of HPMA with comonomers corresponding to the desired composition.
  • the binding of the corresponding taxane to the polymeric carrier results in considerable reduction of its cytotoxicity, considerable increase of the molecular weight of the drug, and thus extension of the circulation time in the bloodstream; i.e. extension of the total residence time of the drug in the organism and hence increase of its bioavailability.
  • the polymeric drug in accordance with the invention is further characterized in that the bond of the drug to the polymeric carrier is relatively stable in the course of the transport in the bloodstream and in body liquids and is hydrolytically cleavable in the slightly acidic environment of the tumour and especially inside the target tumour cells in endosomes characterized by slightly acidic pH.
  • the bond of the drug to the polymeric carrier is relatively stable in the course of the transport in the bloodstream and in body liquids and is hydrolytically cleavable in the slightly acidic environment of the tumour and especially inside the target tumour cells in endosomes characterized by slightly acidic pH.
  • the drug is transported via the bloodstream in an inactive form bound to the polymer and it is released and activated predominantly after the penetration into the target tumour cells.
  • the fact that the drug is only activated in the target cells eliminates the side effects of otherwise toxic cytostatic agents and directs their effect preferentially to the tumour cells.
  • the efficiency of accumulation in the tumour tissue may be controlled by the changes in the structure of the skeleton of the polymeric carrier (non- degradable linear polymer, high-molecular biodegradable grafted polymer) that is responsible for the targeted (passive) transport to the tumour or tumour cells.
  • the polymeric carrier non- degradable linear polymer, high-molecular biodegradable grafted polymer
  • the sphere of application of the presented invention includes the use of polymeric drugs of the invention for the treatment of solid tumours in malignant diseases in the human medicine.
  • the basic monomers are synthesized: HPMA, methacryloylated derivatives of amino acids and of oligopeptides, terminated with the hydrazide (CONHNH 2 ) group, or possibly terminated with the hydrazide group protected with the t-butyloxycarbonyl group (Boc).
  • polymeric precursors are synthesized, i.e. HPMA copolymers carrying the functional groups (random copolymers), serving as the polymeric carriers for the drugs.
  • a polymeric precursor, carrying functional hydrazide groups along the chain can be prepared either by radical copolymerization of the above-mentioned functional monomers with HPMA, or by polymer-analogous transformation of the basic copolymer carrying the functional groups.
  • Grafted copolymers are prepared from multivalent and semitelechelic HPMA copolymers in accordance with the procedure described in [Etrych 2008 pat, publ.].
  • the basic copolymer is a copolymer of HPMA and methacryloylated hydrazides of amino acids or of oligopeptides selected from the group of glycyl, glycylglycyl, ⁇ -alanyl, 6-aminohexanoyl (AH), 4-aminobenzoyl, or a mixed acyl derived from the oligopeptides GlyPheGly, GlyLeuGly, GlyLeuPheGly and GlyPheLeuGly), characterized in that it contains 70 - 98 mole % of HPMA and 2 - 30 mole % of units with hydrazide functional groups (see Scheme 2 with the spacer consisting of 6-aminohexanoyl).
  • a drug derivative is a compound of the drug (PTX, DTX or LTX) produced by acylation of the hydroxyl group in position 2 of the drug with the corresponding oxo acid.
  • the following oxo acids have been preferably used: levulic acid, 4-(2-oxopropyl) benzoic acid, 4-oxo-pent-2-enoic acid and 5-oxo-hex-2-enoic and 6-oxo-hept-2-enoic acids (see Scheme 1).
  • a polymeric conjugate is a compound of a polymeric precursor with a drug derivative, wherein the drug derivative is bound to the polymeric precursor by a hydrazone bond prepared by reaction of the carbonyl group of the drug derivative with the hydrazide groups of the polymer, characterized in that it contains 70 - 98 mole % of HPMA, 1.5 - 29.5 mole % of units with hydrazide functional groups and 0.5 - 10 mole % of units with the hydrazone-bound drug derivative (see Scheme 3; with the spacer consisting of 6-aminohexanoyl and levulic acid).
  • FIGURES Fig. 1 Diagram of the release rate of PTX and its derivatives from linear polymeric conjugates in a buffer of pH 5 (an intracellular environment model).
  • Fig. 2 Diagram of the release rate of PTX and its derivatives from linear polymeric conjugates in a buffer with pH 7.4 (a bloodstream model).
  • the drug doses were 2x20 mg of the DTX equivalent/kg
  • PHPMA- AH-NH-N DTX-LEV linear polymeric conjugates with the DTX content of 8.2 % and 16.3 % in the dose of 2x30 mg of the DTX equivalent DTX i.v., on days 9 and 13.
  • HPMA was prepared in accordance with the previously described procedure [Ulbrich et al., 2000].
  • the product was chromatographically pure.
  • the ester of levulic acid and paclitaxel (in the -OH 2 position) was prepared by reaction of levulic acid with paclitaxel by means of the carbodiimide method (dicyclohexylcarbodiimide, DCC) in A ⁇ iV'-dimethylformamide (DMF).
  • Levulic acid (19.4 mg, 0.167 mmol) and DCC (37.5 mg, 0.182 mmol) were dissolved each in 0.15 mL of DMF at the laboratory temperature. Both solutions were cooled to -18 0 C and mixed.
  • the product was purified of low-molecular admixtures by means of chromatography on a column (60cm x 4cm) filled with silica gel in ethyl acetate.
  • the fraction containing the PTX-LEV product was collected and concentrated to 0.4 mL and the product was precipitated with 20 mL diethyl ether.
  • the product was aspirated, washed with a small amount of diethyl ether and dried in vacuum until the constant weight.
  • the yield was 98 mg of the product (84 %) with the melting point of 136 to 138 0 C.
  • TLC ethyl acetate : hexane 1:1
  • one spot at Rf 0.15.
  • MALDI-TOF MS 970 (M+Na).
  • Levulic acid 38 mg, 0.327 mmol
  • DCC 100 mg, 0.487 mmol
  • Both solutions were cooled to -18 0 C and mixed.
  • a solution of docetaxel 200 mg, 0.247 mmol
  • iV,N-dimethylaminopyridine 28 mg, 0.229 mmol
  • the reaction went on at -18 0 C for 30 minutes and at 4 0 C for 16 h.
  • ester of 4-(2-oxopropyl)benzoic acid and paclitaxel (in the -OH 2 position) (OPB-PTX) was prepared by the same above-described method for the preparation of LEV-PTX, namely by reaction of 4-(2-oxopropyl) benzoic acid with paclitaxel using the conjugation reagent DCC in
  • the ester of 4-oxo-pentenoic [3-acetylacrylic] acid and paclitaxel (in the -OH 2 position) was prepared by the same above-described method for the preparation of LEV-PTX, namely by reaction of 4-oxo-pentenoic acid with paclitaxel using DCC in DMF.
  • the ester of 5-oxo-hexenoic acid and paclitaxel (in the -OH 2 position) was prepared by the same above-described method for the preparation of PTX-LEV, namely by reaction of 5- oxo-hexenoic acid with paclitaxel using the conjugation reagent DCC in DMF.
  • the ester of 5-oxo-hexenoic acid and docetaxel (in the -OH 2 position) was prepared by the same above-described method for the preparation of DTX-LEV, namely by reaction of 5-oxo-hexenoic acid with docetaxel using the conjugation reagent DCC in DMF.
  • Example 2 Synthesis of a polymeric precursor - a copolymer of HPMA with MA-AH-NHNH 2
  • the PoIy(HPMA-Co-MA-AH-NHNH 2 ) copolymer was prepared by solution radical copolymerization of HPMA and MA-AH-NHNH 2 in methanol at 60 0 C in accordance with a previously described procedure [Etrych patent].
  • Copolymers with derivatives of PTX, DTX and LTX linked to the PHPMA carrier by a hydrolytically cleavable hydrazone bond were prepared by reaction of polymeric precursors containing hydrazine groups with the corresponding drug derivative in methanol under catalysis with acetic acid.
  • the polymeric fraction was isolated, concentrated in a vacuum evaporator and the product was precipitated with 50 niL of ethyl acetate, isolated by filtration on frit S4, washed with 150 mL of ethyl acetate and dried until the constant weight.
  • the content of total PTX or its derivative in the polymeric conjugate was determined by the HPLC method (HPLC Shimadzu system) after complete hydrolysis of the polymeric conjugate in an HCl solution (pH 2) at 37 °C for 1 hour and extraction of the PTX derivative with chloroform.
  • ⁇ M W > and the molecular weight distribution were determined by means of liquid chromatography (TSKGeI 4000 column (300x10 mm), 20% 0.3 M acetate buffer (CH 3 COONa/CH 3 COOH; pH 6.5; 0.5 g/L Of NaN 3 ) and 80% methanol, flow rate 0.5 mL/min, detection with a differential refractometer, a light dispersion detector (DAWN-DSP-F, Wyatt Technology, USA) and a UV detector (250 nm).
  • TSKGeI 4000 column 300x10 mm
  • 20% 0.3 M acetate buffer CH 3 COONa/CH 3 COOH; pH 6.5; 0.5 g/L Of NaN 3
  • flow rate 0.5 mL/min
  • Amounts of PTX, DTX or their derivatives released from the polymeric conjugates were measured after their incubation in a phosphate buffer with pH 5.0 (0.1M phosphate buffer containing 0.05M of NaCl), modelling the intracellular environment, and a phosphate buffer with pH 7.4, modelling the bloodstream environment.
  • the amounts of the released drugs or their derivatives in the incubation solutions were determined by means of HPLC (Shimadzu).
  • the spacer consisting of levulic acid or 4-(2-oxopropyl)benzoic acid allows very fast release of the drug derivative at pH 5; however, hydrolysis of the ester bond between the drug and the acid occurs only very slowly.
  • the spacer consisting of 4-oxo-pentenoic acid stabilizes both the hydrazone and the ester bond, so there is no significant release of the drug or its derivative.
  • partial hydrolysis of the hydrazone bond occurs in the case of polymeric conjugates with a spacer consisting of levulic acid or 4-(2-oxopropyl)benzoic acid, with a significantly lower rate as compared to the environment with pH 5.
  • Example 5 Demonstration of in vitro biological activity of linear polymeric conjugates of docetaxel and palitaxel during incubation with cells of tumour lines; EL-4 T cell lymphoma and 4Tl mammary gland carcinoma.
  • Example 6 Demonstration of in vivo biological activity of linear polymeric conjugates of docetaxel and palitaxel in mice inoculated with EL4 T cell lymphoma
  • mice of the C57BL/6 strain females
  • mice of the C57BL/6 strain were subcutaneously implanted with 1x10 5 EL-4 tumour cells on day 0.
  • the drugs were administered intravenously (i.v.), in two doses administered on day 8 and day 12 after the transplantation of the tumour cells.
  • the first dose was administered at the time when the tumours were well developed, palpable, with the size of approx. 300 mm 3 .
  • the tumour size, body weight of the mice, overall health condition and survival rate were observed in the experiment.
  • the effect of the conjugate was always compared to the effect of the free drug (DTX, PTX) and its derivatives (DTX-LEV, PTX-LEV).
  • the average time of survival of untreated control mice with EL-4 lymphoma was 31.3 days (SD (SD 3.66, mean survival time 30.5 days).
  • DTX, PTX and the DTX-LEV, PTX-LEV derivatives were dissolved for the i.v. application in a mixture of Cremophor EL (Sigma, USA) and ethanol (1 :1); after dissolution of the drug the volume was complemented with 4 volume parts of PBS (phosphate-buffered physiological solution).
  • A. Anti-cancer activity of the PHPMA-AH-NH-N DTX-LEV conjugate (Fig. 3) Free DTX induced complete regression of EL-4 tumours in 4 mice out of 7 tested mice. The LEV- DTX derivative had lower effect and induced complete regression of the EL-4 tumour in 1 out of 8 tested mice.
  • mice that experienced complete regression of the tumour remained without any symptoms of tumour growth or toxicity until day 94, when they were transplanted EL-4 cells again in the same (i.e. lethal) dose and the mice were left without treatment. This second transplantation was carried out to prove immunologically mediated resistance against the tumour.
  • the PHPMA-AH-NH-N DTX-LEV conjugate had a significant anti-cancer effect in C57BL/6 mice with the EL-4 lymphoma, its administration was not accompanied by toxic side effects and enabled establishing of resistance against the tumour in 71% of the cured individuals.
  • B. Anti-cancer activity of the PHPMA-AH-NH-N PTX-LEV conjugate (Fig. 4)
  • the PTX-LEV derivative (2x30 mg of PTX eq./kg) or the conjugate containing the PTX derivative (2x60 mg of PTX eq./kg) did not have any therapeutic effect in the treatment of syngenic EL-4 lymphoma in mice.
  • Example 7 Demonstration of in vivo biological activity of a linear polymeric conjugate of paclitaxel in mice inoculated with the 4Tl mammary gland carcinoma (Fig. 5)
  • a model of murine syngenic 4Tl mammary gland carcinoma was used.
  • Mice of the BALB/c strain females
  • the drugs were administered intravenously (i.v.) in two doses administered on day 8 and day 12 after the transplantation of the tumour cells.
  • the first dose was administered at the time when the tumours were well developed, palpable, with the size of approx. 300 mm 3 .
  • the tumour size, body weight of the mice, the overall health condition and survival rate were observed in the experiment.
  • the effect of the conjugate was always compared to the effect of the free drug (PTX) and its derivative (PTX-LEV).
  • the PHPMA-AH-NH-N PTX-LEV conjugate completely cured 3 out of 8 tested mice.
  • PTX-LEV derivative After administration of the PTX-LEV derivative, regression of the 4Tl tumour occurred in 1 out of 8 mice.
  • Administration of the free drug (PTX) was accompanied by significant side effects: the i.v. administration of the second dose induced a severe reaction (spasms, later poor overall condition - bristled hair, slackness, which took at least 24 hours) in the first of the mice.
  • the second dose was reduced by 10% and administered intraperitoneally instead of i.v. Only one mouse was cured that received the dose by i.v. injection, in the other mice no therapeutic effect of PTX was manifested. In no test group any weight loss was recorded as a drug toxicity indicator.
  • mice were transplanted again 129 days after the first transplantation of tumour cells.
  • IxIO 5 4Tl cells were injected s.c. and the mice were left without treatment.
  • the tumours did not grow, which means that these mice were resistant to the given tumour.
  • Example 8 Demonstration of in vivo biological activity of linear polymeric conjugates of docetaxel with a variable content of the drug in mice inoculated with the EL4 T cell lymphoma (Fig. 6)
  • the tumour model of the EL-4 lymphoma was used.
  • the conjugate with the lower content of the drug (8.2 % of DTX) cured 1 out of 8 tested mice and in the other ones it extended the survival time in a statistically significant way (untreated controls: average survival time 27.25 days, SD 1.64, median 28 days; treated mice: average 41.75 days, SD 6.54, median 45 days; p ⁇ .01).
  • the conjugate with the higher drug content (16.3 % DTX) also cured 1 out of 8 tested mice but the survival of the other mice in the group was not significantly extended.

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Abstract

The present invention describes a polymeric conjugate consisting of a derivative of a cytostatic agent selected from the group of taxanes, in particular paclitaxel (PTX), docetaxel (DTX) or larotaxel (LTX), and a polymeric carrier prepared on the basis of a linear or biodegradable grafted copolymer consisting of units of the basic copolymer N-(2-hydroxypropyl)methacroylamide (HPMA) and units containing methacryloylated hydrazones of amino acids or oligopeptides. The invention also deals with a method of preparation of the above mentioned polymeric conjugate, in which the polymeric carrier is subjected to polymer-analogous transformation of the carrier by reaction with an ester of the oxo acid and the cytostatic agent. The polymeric conjugate is used for the preparation of a medicament for treatment of tumour diseases.

Description

Polymeric conjugates of paclitaxel and docetaxel with pH-controlled releasing of the cancerostatic agent
TECHNICAL FIELD
The invention deals with the structure and properties of water-soluble polymeric therapeutic agents (pro-drugs) based on derivatives of paclitaxel (PTX), docetaxel (DTX) and larotaxel (LTX), destined mainly for treatment of solid tumours in therapy of tumour diseases in human medicine.
BACKGROUND ART At present, trends of drug development very often focus on the development of medicament forms enabling the specific effect of the drug in the place of the desired therapeutic effect only. Biologically active substances with such a targeted effect are mainly applied in the fields where undesirable side effects of the drug may damage healthy parts of the organism. This danger is especially associated with cytostatic substance treatment in chemotherapy of tumour diseases. It is known that attachment of a cytostatic agent to a water-soluble polymeric carrier by a chemical bond makes it possible to increase solubility of otherwise insoluble or poorly soluble drugs and to considerably reduce their direct toxicity. The high molecular weight of polymers prevents fast excretion of the drug from the organism by glomerular filtration, ensuring an extended time of circulation in the blood and presence in the organism, and thus also longer time of bioavailability of the drug.
In the past, many polymeric conjugates of cancerostatic agents with soluble polymers were prepared and studied where the drug with an anti-cancer effect was attached to the polymer with a non-cleavable covalent bond, hydrolytically instable ionic bond and/or covalent bond allowing controlled release of the drug and thus its activation on the basis of enzymatic or plain chemical hydrolysis of this bond. Polymeric carrier systems are generally constructed in such a way to be able to release the therapeutically active cancerostatic agent from the carrier either in the tumour, or more specifically, directly in a tumour cell. An important group of polymeric therapeutic agents is represented by polymeric drugs prepared on the basis of copolymers of N-(2- hydroxypropyl)methacrylamide (HPMA), a number of which are actively directed to tumours by means of a targeting structure attached to the polymer (antibody, lectin, hormone). [Duncan 1985, Rihova 2000, Kopecek 2001, 2000, Duncan, 2005; Satchi-Fainaro et al., 2006], However, their synthesis is quite complex. What has also been proved during the development of polymeric cancerostatic agents is that for specific transport of the drug to the place of occurrence of the tumour there is no need to use an actively targeted carrier, but considerably increased accumulation of polymeric cytostatic agents, especially in solid tumours, can be achieved by increasing of the molecular weight of the polymeric therapeutic agent above the excretion limit of the carrier by kidneys (i.e. passive targeting to solid tumours). This ability of macromolecules to accumulate in solid tumours has been called the EPR effect (Enhanced Permeability and Retention) and this effect has proved to be significantly manifested in carriers on the basis of HPMA copolymers as well [Noguchi et al., 1998; Seymour et al., 1995].
One of the main problems of using HPMA copolymers as passively targeted high-molecular earners is due to their non-cleavable carbon chain and only polymers not exceeding the molecular weight of 40 - 50,000 g/mol can be excreted from the organism. This means that if accumulation of the polymer in the organism after repeated administration of the drug is not to occur and if the molecular weight of the carrier is to be as high as possible to make the passive targeting as efficient as possible, the polymeric carrier must be constructed as degradable in the organism. Such polymeric earners based on HPMA copolymers have been recently developed and their structure has been patented [Chytil et al., 2008; Etrych et al., 2008, Chytil PV 2006-207, Etrych PV 2006 - 592].
Literature mentions a lot of information about the preparation and studying of properties of polymers carrying a cancerostatic agent, attached to the polymer by a bond susceptible to hydrolysis in an aqueous environment [Kratz 1999]. Among them, HPMA copolymers carrying the cancerostatic agent doxorubicin, bound to the polymeric chain by a hydrolytically cleavable hydrazone bond, take an important position [Etrych 2002, Ulbrich 2004a, Ulbrich 2004b, Ulbrich pat CZ 293787 B6]. This bond is relatively stable in the bloodstream environment (in the course of transport in the organism) and hydrolytically instable in the slightly acidic environment of the living cell. The speed of hydrolysis of this bond also governs the speed of releasing of the drug and consequently the concentration of the active substance in the place of the desired effect. During in vitro as well as in vivo tests in mice such polymeric cancerostatic agents exhibited a considerably higher anti-tumour effect against a number of tumour lines than the free drug and in a number of cases their use resulted in complete recovery of the test animals even in case of a therapeutic method of administration. [Rihova 2001, Etrych 2001]. Increasing the molecular weight of the carrier (biodegradable grafted polymers, micellar and nanogel systems) always led to an increase of efficiency of the anti-tumour activity of the drug, verified in in vivo systems. Paclitaxel and docetaxel belong to the group of taxanes, the anti-cancer drugs routinely used for the treatment of ovarian tumours and breast, lung, prostate and other tumours [Vanhoefer at al., 1997]. Besides their secondary toxicity, common for cancerostatic agents, they have another disadvantage of very low solubility in aqueous solutions, resulting in the necessity of administration in various excipients, especially in Cremophor EL, which also leads to other side effects of the dosage form. In some administration schemes the administration of taxanes in Cremophor EL may even significantly reduce the efficiency of the therapy [Ng at al., 2006]. By binding these drugs to polymeric carriers (PEG, HPMA copolymer, poly(glutamic acid)), very well water-soluble pro-drugs have been obtained, having an extended circulation time in the organism and increased accumulation in solid tumours. Their anti-cancer activity has been verified in vivo in animal models and in some cases clinically as well. Thus, paclitaxel (PTX) was covalently bound to poly( ethylene glycol) (PEG) by a non-cleavable ester or 7-carbamate bonds (C2 -OH group), or by means of a hydrolytically instable amino-acid spacer (Ala, GIy) [Greenwald et al., 1995; Greenwald, 2001; Greenwald et al., 2003; Pendri et al., 1998]. It has been shown that the anti-tumour activity of the conjugate depends on the structure of the bond used between the polymer and the drug and that the molecular weight of the carrier is another important factor. The conjugate with PTX was tested in phase I of clinical trials [Satchi-Fainaro et al., 2006], but apparently with little success.
The conjugate of paclitaxel with poly(glutamic acid) (Xyotax) appears to be much more successful in clinical trials [Winter 2005, Kratz et al., 2008]. In this conjugate PTX is bound to a polyamino-acid earner by an ester bond via the -OH group in position 2. The drug is released due to degradation of the polymeric chain, formation of the GIu derivative of PTX and its subsequent hydrolysis. At present, this conjugate is in phase III of clinical trials.
Paclitaxel has also been bound to HPMA copolymers by an ester bond using a biodegradable oligopeptide GlyPheLeuGly spacer. It has been demonstrated that PTX, after incubation with lysosomal enzymes, is released from the carrier and this release is important for achieving the anti-cancer activity in vivo. PNU166945 is a conjugate of a HPMA copolymer with ester-bound PTX, which was clinically tested in phase I [Terwogt et al., 2000; Terwogt et al., 2001], however, after this phase further testing was stopped. The above mentioned results show that conjugation of paclitaxel with a polymeric carrier results in a considerable improvement of properties of the drug (solubility, improved bioavailability). However, if the drug is to find its use in the human medicine, the structure and molecular weight of the carrier as well as the structure of the spacer between the polymer and the drug, controlling the rate and thus the concentration of the drug in the place of the desired effect, has to be carefully selected and tailor-made prepared. In the development of optimum structures it is necessary to verify the function of these structures in suitable in vivo models.
DISCLOSURE OF INVENTION
A polymeric drug in accordance with this invention is characterized in that a cancerostatic agent from the group of taxanes, paclitaxel (PTX), docetaxel (DTX), or larotaxel (LTX) (hereinafter drugs) is bound to a water-soluble polymeric carrier, prepared on the basis of a linear or grafted HPMA copolymer. The drug is bound to the polymeric chains via an ester group produced by acylation of the -OH group in position 2 by means of spacers, containing pH-sensitive hydrolytically cleavable hydrazone bonds. These spacers may consist of acid residues of various oxo acids, by means of which the carbonyl group is introduced to the drug structure, linked with residues of individual amino acids, oligopeptides, or other structures, allowing termination of the side chains of the polymeric earner with the hydrazone group. In the case of a linear polymer the molecular weight of the polymeric chain is selected below the excretion limit of HPMA copolymers from the organism, preferably in the range of 10 - 50,000 g/mol. In the case of a grafted copolymer the molecular weight is selected in the range of 50 — 250,000 g/mol.
The polymeric drug in accordance with the invention is destined for intravenous (injection or infusion) administration in a solution, but it may also be administered intratumourally or intraperitoneally and is intended for treatment of solid tumours. The polymer with the chemically bound cytostatic agent is constructed in such a way to be stable during circulation in the bloodstream and to prevent hydrolysis of the hydrazone bond between the given taxane and the polymer, or, possibly, to keep the hydrolysis rate in the course of transport through the organism as low as possible (at the pH value 7.4 in the bloodstream), so that no cytotoxic effect of the released drug or its derivative can be manifested. The entire system is constructed as a two-phase system. Due to suitable selection of the molecular weight of the earner, allowing extravasation as well as efficient accumulation in the tumour tissue, an interaction with the cell membrane should occur after the initial accumulation of the drug in the tumour tissue and the molecularly dissolved polymeric drug should penetrate into individual tumour cells by pinocytosis. Inside the target cells, fast hydrolysis of the hydrazone bond and release of the drug or its derivative from the carrier should occur due to the drop of pH from the external value (7.4) to the intracellular value (5 - 6). In the subsequent step hydrolysis of the ester bond of the drug derivative, already released from the polymer, should occur by chemical hydrolysis, or, more specifically, by the effect of intracellular enzymes, e.g. carboxyesterases. Feasibility of the above-suggested mechanism of the action of polymeric drugs of the invention is evidenced by experiments of model releasing of drugs from the polymeric carrier. The results of these tests, including the tests of anti-cancer activity, are presented in the experimental part of the application. The polymeric conjugates with a targeted anti-cancer effect in accordance with the invention are characterized in that the cytostatic agent (taxol, docetaxel, larotaxel) is attached, by means of an ester bond and spacer, to the polymeric carrier formed by a linear [Etrych pat CZ 297827 B6, publ. 2008], or grafted [Etrych pat CZ 298945 (B6), publ. 2008] HPMA copolymer through a hydrolytically instable hydrazone group produced by a reaction of the carbonyl group of the molecule of the drug derivative with the hydrazide group of the polymeric carrier. The polymeric carriers are generally prepared by radical solution copolymerization of HPMA with comonomers corresponding to the desired composition. The binding of the corresponding taxane to the polymeric carrier results in considerable reduction of its cytotoxicity, considerable increase of the molecular weight of the drug, and thus extension of the circulation time in the bloodstream; i.e. extension of the total residence time of the drug in the organism and hence increase of its bioavailability. The polymeric drug in accordance with the invention is further characterized in that the bond of the drug to the polymeric carrier is relatively stable in the course of the transport in the bloodstream and in body liquids and is hydrolytically cleavable in the slightly acidic environment of the tumour and especially inside the target tumour cells in endosomes characterized by slightly acidic pH. This means that the drug is transported via the bloodstream in an inactive form bound to the polymer and it is released and activated predominantly after the penetration into the target tumour cells. The fact that the drug is only activated in the target cells eliminates the side effects of otherwise toxic cytostatic agents and directs their effect preferentially to the tumour cells. It is the polymeric carrier prepared in the basis of HPMA copolymers whose molecular weight, i.e. the efficiency of accumulation in the tumour tissue may be controlled by the changes in the structure of the skeleton of the polymeric carrier (non- degradable linear polymer, high-molecular biodegradable grafted polymer) that is responsible for the targeted (passive) transport to the tumour or tumour cells.
The sphere of application of the presented invention includes the use of polymeric drugs of the invention for the treatment of solid tumours in malignant diseases in the human medicine.
Synthesis and structures of polymeric conjugates
Synthesis of polymeric conjugates in accordance with the invention is carried out in several steps; the detailed final structure of the conjugate considerably depends on the selected synthetic way.
In the first step of the synthesis the basic monomers are synthesized: HPMA, methacryloylated derivatives of amino acids and of oligopeptides, terminated with the hydrazide (CONHNH2) group, or possibly terminated with the hydrazide group protected with the t-butyloxycarbonyl group (Boc).
In the second step polymeric precursors are synthesized, i.e. HPMA copolymers carrying the functional groups (random copolymers), serving as the polymeric carriers for the drugs. A polymeric precursor, carrying functional hydrazide groups along the chain, can be prepared either by radical copolymerization of the above-mentioned functional monomers with HPMA, or by polymer-analogous transformation of the basic copolymer carrying the functional groups.
Grafted copolymers are prepared from multivalent and semitelechelic HPMA copolymers in accordance with the procedure described in [Etrych 2008 pat, publ.].
The basic copolymer (precursor) is a copolymer of HPMA and methacryloylated hydrazides of amino acids or of oligopeptides selected from the group of glycyl, glycylglycyl, β-alanyl, 6-aminohexanoyl (AH), 4-aminobenzoyl, or a mixed acyl derived from the oligopeptides GlyPheGly, GlyLeuGly, GlyLeuPheGly and GlyPheLeuGly), characterized in that it contains 70 - 98 mole % of HPMA and 2 - 30 mole % of units with hydrazide functional groups (see Scheme 2 with the spacer consisting of 6-aminohexanoyl). A drug derivative is a compound of the drug (PTX, DTX or LTX) produced by acylation of the hydroxyl group in position 2 of the drug with the corresponding oxo acid. The following oxo acids have been preferably used: levulic acid, 4-(2-oxopropyl) benzoic acid, 4-oxo-pent-2-enoic acid and 5-oxo-hex-2-enoic and 6-oxo-hept-2-enoic acids (see Scheme 1).
A polymeric conjugate is a compound of a polymeric precursor with a drug derivative, wherein the drug derivative is bound to the polymeric precursor by a hydrazone bond prepared by reaction of the carbonyl group of the drug derivative with the hydrazide groups of the polymer, characterized in that it contains 70 - 98 mole % of HPMA, 1.5 - 29.5 mole % of units with hydrazide functional groups and 0.5 - 10 mole % of units with the hydrazone-bound drug derivative (see Scheme 3; with the spacer consisting of 6-aminohexanoyl and levulic acid).
DESCRIPTION OF FIGURES Fig. 1: Diagram of the release rate of PTX and its derivatives from linear polymeric conjugates in a buffer of pH 5 (an intracellular environment model).
Fig. 2: Diagram of the release rate of PTX and its derivatives from linear polymeric conjugates in a buffer with pH 7.4 (a bloodstream model).
Fig. 3: Survival rate of C57BL/6 mice with EL-4 lymphoma, which were administered DTX, a LEV-DTX derivative and a PHPMA-AH-NH-N=DTX-LEV linear polymeric conjugate. The drug doses were 2x20 mg of the DTX equivalent/kg, the PHPMA-AH-NH-N=DTX-LEV conjugate was administered in doses of 2x20 and 2x40 mg of the DTX equivalent/kg.
Fig. 4: Survival rate of C57BL/6 mice with EL-4 lymphoma, which were administered a LEV- PTX paclitaxel derivative and a PHPMA-AH-NH-N=PTX-LEV linear polymeric conjugate. Due to limited solubility PTX could not be administered in the free form. Fig. 5: Survival rate of BALB/c mice with 4Tl mammary gland carcinoma, which were administered paclitaxel, a LEV-PTX paclitaxel derivative and a PHPMA-AH-NH-N=PTX-LEV linear polymeric conjugate. PTX and PTX-LEV were injected in the dose of 2x30 mg of the PTX equivalent/kg on days 8 and 12 (see the text) and the PHPMA-AH-NH-N=PTX-LEV conjugate in the dose of 2x60 mg of the PTX equivalent/kg on days 8 and 12.
Fig. 6: Survival rate of C57BL/6 mice with EL-4 lymphoma, which were administered PHPMA- AH-NH-N=DTX-LEV linear polymeric conjugates with the DTX content of 8.2 % and 16.3 % in the dose of 2x30 mg of the DTX equivalent DTX i.v., on days 9 and 13. EXAMPLES
Examples of carrying out the synthesis of the intermediates and conjugates of the invention
Example 1: Synthesis of monomers and drug derivatives
HPMA was prepared in accordance with the previously described procedure [Ulbrich et al., 2000]. Elementary 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.
6-(Methacryloylamino)hexanoyl hydrazine (N1 -(6-hydrazino-6-oxohexyl)-2-methylacrylamide) (MA-AH-NHNH2) was prepared in accordance with the previously described procedure [Ulbrich patents, Etrych patent].
The ester of levulic acid and paclitaxel (in the -OH 2 position) (LEV-PTX) was prepared by reaction of levulic acid with paclitaxel by means of the carbodiimide method (dicyclohexylcarbodiimide, DCC) in AζiV'-dimethylformamide (DMF). Levulic acid (19.4 mg, 0.167 mmol) and DCC (37.5 mg, 0.182 mmol) were dissolved each in 0.15 mL of DMF at the laboratory temperature. Both solutions were cooled to -18 0C and mixed. After 20 minutes a solution of paclitaxel (100 mg, 0.117 mmol) and ΛζN-dimethylaminopyridine (DMAP) (14 mg, 0.117 mmol) in 0.3 mL of DMF was added to this solution. The reaction went on at -18 0C for 30 minutes and at 4 0C for 16 h. The course of the reaction was monitored with TLC - 60 F254 silica gel plates (ethyl acetate: hexane 1: 1, Rf(PTX)= 0.25, Rf(LEV-PTX)= 0.15, Rf(Levulic acid)= 0.45). The product was purified of low-molecular admixtures by means of chromatography on a column (60cm x 4cm) filled with silica gel in ethyl acetate. The fraction containing the PTX-LEV product was collected and concentrated to 0.4 mL and the product was precipitated with 20 mL diethyl ether. The product was aspirated, washed with a small amount of diethyl ether and dried in vacuum until the constant weight. The yield was 98 mg of the product (84 %) with the melting point of 136 to 138 0C. TLC (ethyl acetate : hexane 1:1): one spot at Rf = 0.15. MALDI-TOF MS: 970 (M+Na).
Purity of all the monomers and drug derivatives was determined using an HPLC system [Shimadzu HPLC system equipped with a Chromolith Performance RP-ISe (10Ox 4,6 mm) reverse phase column and an UV-VIS detector - Shimadzu SPD- lOAVvp (230 nm); eluent: water-acetonitrile with the gradient of 50-100 vol.% of acetonitrile, flow rate 0.5 mL-min"1]. The ester of levulic acid and docetaxel (in the -OH 2 position) (LEV-DTX) was prepared similarly to LEV-PTX by reaction of levulic acid with docetaxel by the carbodiimide method (DCC) in DMF.
Levulic acid (38 mg, 0.327 mmol) and DCC (100 mg, 0.487 mmol) were dissolved each in 0.25 mL of DMF at the laboratory temperature. Both solutions were cooled to -18 0C and mixed. After 20 minutes a solution of docetaxel (200 mg, 0.247 mmol) and iV,N-dimethylaminopyridine (DMAP) (28 mg, 0.229 mmol) in 0.6 mL of DMF was added to this solution. The reaction went on at -18 0C for 30 minutes and at 4 0C for 16 h. The course of the reaction was monitored with TLC 60 F254 silica gel plates (ethyl acetate : hexane 1:1, Rf(DTX) = 0.3, Rf(LEV-DTX) = 0.2, Rf(Levulic acid) = 0.45). The product was purified of low-molecular admixtures twice using chromatography on a column (60cm x 4cm) filled with silica gel in ethyl acetate. The fraction containing the DTX-LEV product was collected each time and concentrated to 0.6 mL and the product was precipitated with 20 mL of diethyl ether. The product was aspirated, washed with a small amount of diethyl ether and dried in vacuum until the constant weight. The yield was 179 mg of the product (80 %) with the melting point of 86 to 89 0C. TLC (ethyl acetate : hexane 1:1): one spot at Rf= 0.20. MALDI-TOF MS: 929 (M+Na).
Scheme 1. Structures of the esters of levulic acid and 4-(2-oxopropyl)benzoic acid with paclitaxel and docetaxel: A) Ester of levulic acid and paclitaxel, LEV-PTX; B) Ester of 4-(2-oxopropyl)benzoic acid and paclitaxel, OPB-PTX; C) Ester of levulic acid and docetaxel, LEV-DTX.
The ester of 4-(2-oxopropyl)benzoic acid and paclitaxel (in the -OH 2 position) (OPB-PTX) was prepared by the same above-described method for the preparation of LEV-PTX, namely by reaction of 4-(2-oxopropyl) benzoic acid with paclitaxel using the conjugation reagent DCC in
DMF.
The yield was 85 %. Melting point 143 to 145 0C. TLC (ethyl acetate : hexane 1:1): one spot at
Rf= 0.25. MALDI-TOF MS: 1032 (M+Na).
The ester of 4-(2-oxopropyl)benzoic acid and docetaxel (in the -OH 2 position) (OPB-PTX) was prepared by the same above-described method for the preparation of DTX-LEV, namely by reaction of 4-(2-oxoproρyl) benzoic acid with docetaxel using DCC in DMF. The yield was 81 %. The melting point 94 to 96 0C. TLC (ethyl acetate ; hexane 1:1): one spot at Rf= 0.28. MALDI-TOF MS: 990 (M+Na).
The ester of 4-oxo-pentenoic [3-acetylacrylic] acid and paclitaxel (in the -OH 2 position) (AAK- PTX) was prepared by the same above-described method for the preparation of LEV-PTX, namely by reaction of 4-oxo-pentenoic acid with paclitaxel using DCC in DMF.
The yield was 86 %. Melting point 138 to 139 0C. TLC (ethyl acetate : hexane 3:1): one spot at Rf= 0.6. MALDI-TOF MS: 968 (M+Na).
The ester of 5-oxo-hexenoic acid and paclitaxel (in the -OH 2 position) (OHE-PTX) was prepared by the same above-described method for the preparation of PTX-LEV, namely by reaction of 5- oxo-hexenoic acid with paclitaxel using the conjugation reagent DCC in DMF.
The yield was 85 %. Melting point 132 to 134 0C, TLC (ethyl acetate : hexane 3:1): one spot at Rf = 0.65. MALDI-TOF MS: 982 (M+Na).
The ester of 5-oxo-hexenoic acid and docetaxel (in the -OH 2 position) (OHE-DTX) was prepared by the same above-described method for the preparation of DTX-LEV, namely by reaction of 5-oxo-hexenoic acid with docetaxel using the conjugation reagent DCC in DMF.
The yield was 86 %. Melting point 84 to 86 0C. TLC (ethyl acetate : hexane 3:1): one spot at Rf = 0.7. MALDI-TOF MS: 940 (M+Na).
Example 2: Synthesis of a polymeric precursor - a copolymer of HPMA with MA-AH-NHNH2
The PoIy(HPMA-Co-MA-AH-NHNH2) copolymer was prepared by solution radical copolymerization of HPMA and MA-AH-NHNH2 in methanol at 60 0C in accordance with a previously described procedure [Etrych patent].
Scheme 2. Structures of a polymeric precursor, the poly(HPMA-co-MA-AH-NHNH2) copolymer.
Example 3 Preparation of polymeric conjugates containing drug derivatives (PHPMA-AH-NH-N=LEV-PTX, PHPMA-AH-NH-N=LEV-DTX, PHPMA-AH-NH-N^OPB-PTX, PHPMA-AH-NH-N=OPB-DTX, PHPMA-AH-NH-N=OHE-PTX, PHPMA-AH-NH-N=OHE-DTX, PHPMA-AH-NH-N=AKK-PTX and PHPMAΑH-NH-N=AKK- DTX)
Copolymers with derivatives of PTX, DTX and LTX linked to the PHPMA carrier by a hydrolytically cleavable hydrazone bond were prepared by reaction of polymeric precursors containing hydrazine groups with the corresponding drug derivative in methanol under catalysis with acetic acid.
A solution of 100 mg of the poly(HPMA-cσ-MA- AH-NHNH2) copolymer in 1.1 mL of methanol was mixed with a solution of LEV-PTX in 0.2 mL of methanol. After 1 minute 40 μL of acetic acid were added to the stirred reaction mixture at 25 °C. The course of the reaction (loss of LEV- PTX) was monitored by means of TLC (60 F254 silica gel plates, ethyl acetate, Rf(LEV-PTX)= 0.8). After 2 h the reaction mixture was purified from the free derivative of the drug by gel filtration in a column filled with Sephadex LH-20 in methanol. The polymeric fraction was isolated, concentrated in a vacuum evaporator and the product was precipitated with 50 niL of ethyl acetate, isolated by filtration on frit S4, washed with 150 mL of ethyl acetate and dried until the constant weight. The content of total PTX or its derivative in the polymeric conjugate was determined by the HPLC method (HPLC Shimadzu system) after complete hydrolysis of the polymeric conjugate in an HCl solution (pH 2) at 37 °C for 1 hour and extraction of the PTX derivative with chloroform. <MW> and the molecular weight distribution were determined by means of liquid chromatography (TSKGeI 4000 column (300x10 mm), 20% 0.3 M acetate buffer (CH3COONa/CH3COOH; pH 6.5; 0.5 g/L Of NaN3) and 80% methanol, flow rate 0.5 mL/min, detection with a differential refractometer, a light dispersion detector (DAWN-DSP-F, Wyatt Technology, USA) and a UV detector (250 nm). Characterization of the polymeric drug: Total yield of the drug binding reaction: 96 mg (88 %), content of the total LEV-PTX 9.2 % by weight, content of free PTX <0.2 % out of the total content of PTX. The procedure of binding the PTX derivative to polymeric precursors by the hydrazone bond was the same for all the types of precursors and drug derivatives.
Scheme 3. Structures of the PHPMA-AH-NH-N=DTX-LEV polymeric conjugate with the bound derivative, ester of levulic acid and docetaxel
Example 4: Releasing of PTX, DTX or their derivatives from polymeric conjugates
Amounts of PTX, DTX or their derivatives released from the polymeric conjugates were measured after their incubation in a phosphate buffer with pH 5.0 (0.1M phosphate buffer containing 0.05M of NaCl), modelling the intracellular environment, and a phosphate buffer with pH 7.4, modelling the bloodstream environment. The amounts of the released drugs or their derivatives in the incubation solutions were determined by means of HPLC (Shimadzu). In predetermined time intervals 200 μL doses of the incubation solution were sampled and after the extraction of released drugs and their derivatives with chloroform their amounts were determined using an HPLC system [Shimadzu HPLC system equipped with a Chromolith Performance RP- ISe (100* 4.6 mm) reverse phase column and a UV-VIS detector - Shimadzu SPD- lOAVvp (230 nra); eluent: water-acetonitrile with the gradient of 50- 100 vol.% of acetonitrile, flow rate 0.5 mL-rnin"1.
After incubation of the conjugates (concentration 5 mg/mL) in the physiological environment at 37 0C (phosphate buffer, pH 7.4) the drugs (PTX and DTX) (or their derivatives) are released considerably more slowly (Fig. 2) than in a slightly acidic environment with pH 5.0 modelling the environment of the endosomes and lysosomes of tumour cells (Fig. 1). In both the drugs the rate of releasing from the polymeric conjugate and the proportion of released constituents (drug and its derivative) are principally influenced by the structure of the oxo acid spacer. The spacer consisting of levulic acid or 4-(2-oxopropyl)benzoic acid allows very fast release of the drug derivative at pH 5; however, hydrolysis of the ester bond between the drug and the acid occurs only very slowly. On the other hand, the spacer consisting of 4-oxo-pentenoic acid stabilizes both the hydrazone and the ester bond, so there is no significant release of the drug or its derivative. During incubation in the physiological environment with pH 7.4 partial hydrolysis of the hydrazone bond occurs in the case of polymeric conjugates with a spacer consisting of levulic acid or 4-(2-oxopropyl)benzoic acid, with a significantly lower rate as compared to the environment with pH 5. Stability of the ester bond between levulic acid and the drug at pH 7.4 is provably lower and the free drug is released to the extent of one third. On the other hand, in the case of a spacer consisting of 4-(2-oxopropyl)benzoic acid only the drug derivative is released; the ester bond is stable. Decomposition of the drug derivative to the free drug or direct cleaving of the drug from the polymeric conjugate is presumably due to the effect of enzymes, predominantly carboxy esterases.
Example 5: Demonstration of in vitro biological activity of linear polymeric conjugates of docetaxel and palitaxel during incubation with cells of tumour lines; EL-4 T cell lymphoma and 4Tl mammary gland carcinoma.
Table 1. IC50 values (in ng/ml)
Example 6: Demonstration of in vivo biological activity of linear polymeric conjugates of docetaxel and palitaxel in mice inoculated with EL4 T cell lymphoma
For in vivo demonstration of the activity of docetaxel and palitaxel conjugates a model of murine syngenic EL-4 T cell lymphoma was used. Mice of the C57BL/6 strain (females) were subcutaneously implanted with 1x105 EL-4 tumour cells on day 0. The drugs were administered intravenously (i.v.), in two doses administered on day 8 and day 12 after the transplantation of the tumour cells. The first dose was administered at the time when the tumours were well developed, palpable, with the size of approx. 300 mm3. The tumour size, body weight of the mice, overall health condition and survival rate were observed in the experiment. The effect of the conjugate was always compared to the effect of the free drug (DTX, PTX) and its derivatives (DTX-LEV, PTX-LEV). The average time of survival of untreated control mice with EL-4 lymphoma was 31.3 days (SD (SD 3.66, mean survival time 30.5 days). DTX, PTX and the DTX-LEV, PTX-LEV derivatives were dissolved for the i.v. application in a mixture of Cremophor EL (Sigma, USA) and ethanol (1 :1); after dissolution of the drug the volume was complemented with 4 volume parts of PBS (phosphate-buffered physiological solution). The PHPMA-AH-NH-N=DTX-LEV; PHPMA-AH-NH-N=PTX-LEV conjugates were dissolved in PBS. The volume of each individual dose of the drug was 0.2 ml. The following drug doses were applied: DTX and the DTX-LEV derivative 2x20 mg of the DTX equivalent/kg, PHPMA-AH-NH-N=DTX-LEV conjugate 2x20 and 2x40 mg of the DTX equivalent/leg, PTX and PTX-LEV derivative 2x30 mg of the PTX equivalent/kg, PHPMA-AH-NH-N=PTX-LEV conjugate 2x60 mg of the PTX equivalent/kg.
A. Anti-cancer activity of the PHPMA-AH-NH-N=DTX-LEV conjugate (Fig. 3) Free DTX induced complete regression of EL-4 tumours in 4 mice out of 7 tested mice. The LEV- DTX derivative had lower effect and induced complete regression of the EL-4 tumour in 1 out of 8 tested mice. The PHPMA-AH-NH-N=DTX-LEV conjugate in a dose equivalent to free DTX (2x20 mg of the DTX equivalent/kg) also cured 1 mouse out of a group of 8 mice. However, doubling the conjugate dose (2x40 mg of the DTX equivalent/kg) resulted in curing of 7 mice from the group (n=8). In none of the tested groups a weight loss was observed (as an indicator of toxicity of the drug). The mice that experienced complete regression of the tumour remained without any symptoms of tumour growth or toxicity until day 94, when they were transplanted EL-4 cells again in the same (i.e. lethal) dose and the mice were left without treatment. This second transplantation was carried out to prove immunologically mediated resistance against the tumour. A statistically significant proportion of mice cured with the PHPMA-AH-NH-N=DTX- LEV conjugate (2x40 mg of the DTX eq./kg) were resistant against the EL-4 tumour (5 resistant mice out of 7, i.e. 71%). Mice cured with free DTX also manifested a similar proportion of resistant individuals (3 mice out of 4 cured were resistant).
Conclusion: The PHPMA-AH-NH-N=DTX-LEV conjugate had a significant anti-cancer effect in C57BL/6 mice with the EL-4 lymphoma, its administration was not accompanied by toxic side effects and enabled establishing of resistance against the tumour in 71% of the cured individuals. B. Anti-cancer activity of the PHPMA-AH-NH-N=PTX-LEV conjugate (Fig. 4)
It was impossible to administer free PTX to mice due to its very limited solubility (the mice died immediately after the injection). The PTX-LEV derivative did not have any anti-cancer effect for the EL-4 lymphoma (it did not extend the average survival time, no mouse was cured; n=8). The PHPMA-AH-NH-N=PTX-LEV conjugate in the dose of 2x60 mg of the PTX equivalent/kg did not cure any mouse or extend the average survival time (n=8). No weight loss was recorded in any group as a drug toxicity indicator.
Conclusion: The PTX-LEV derivative (2x30 mg of PTX eq./kg) or the conjugate containing the PTX derivative (2x60 mg of PTX eq./kg) did not have any therapeutic effect in the treatment of syngenic EL-4 lymphoma in mice.
Example 7: Demonstration of in vivo biological activity of a linear polymeric conjugate of paclitaxel in mice inoculated with the 4Tl mammary gland carcinoma (Fig. 5)
A model of murine syngenic 4Tl mammary gland carcinoma was used. Mice of the BALB/c strain (females) were subcutaneously implanted IxIO6 4Tl carcinoma cells on day 0. The drugs were administered intravenously (i.v.) in two doses administered on day 8 and day 12 after the transplantation of the tumour cells. The first dose was administered at the time when the tumours were well developed, palpable, with the size of approx. 300 mm3. The tumour size, body weight of the mice, the overall health condition and survival rate were observed in the experiment. The effect of the conjugate was always compared to the effect of the free drug (PTX) and its derivative (PTX-LEV). The average time of survival of untreated control mice was 34.9 days (SD 2.59; mean survival time 35 days). PTX, PTX-LEV and the PHPMA-AH-NH-N=PTX-LEV conjugate were prepared for the i.v. administration in the same way as mentioned above (see Example 6 A, B). The following doses were applied: PTX and PTX-LEV in the dose 2x30 mg of the PTX equivalent/kg, the PHPMA- AH-NH-N=PTX-LEV conjugate in the dose of 2x60 mg of the PTX equivalent/kg. The PHPMA-AH-NH-N=PTX-LEV conjugate completely cured 3 out of 8 tested mice. After administration of the PTX-LEV derivative, regression of the 4Tl tumour occurred in 1 out of 8 mice. Administration of the free drug (PTX) was accompanied by significant side effects: the i.v. administration of the second dose induced a severe reaction (spasms, later poor overall condition - bristled hair, slackness, which took at least 24 hours) in the first of the mice. For the other mice in the group the second dose was reduced by 10% and administered intraperitoneally instead of i.v. Only one mouse was cured that received the dose by i.v. injection, in the other mice no therapeutic effect of PTX was manifested. In no test group any weight loss was recorded as a drug toxicity indicator.
To check resistance of the cured mice against the 4Tl tumour the cured mice were transplanted again 129 days after the first transplantation of tumour cells. For the second time IxIO5 4Tl cells were injected s.c. and the mice were left without treatment. In all these mice (3 cured with the PHPMA-AH-NH-N=PTX-LEV conjugate, 1 cured with PTX-LEV) the tumours did not grow, which means that these mice were resistant to the given tumour.
Conclusion: The PHPMA-AH-NH-N=PTX-LEV conjugate had a significant anti-cancer effect in BALB/c mice with the 4Tl mammary gland carcinoma, its administration was not accompanied by any toxic side effect and enabled establishing of resistance to the tumour in 38% of the cured individuals.
Example 8: Demonstration of in vivo biological activity of linear polymeric conjugates of docetaxel with a variable content of the drug in mice inoculated with the EL4 T cell lymphoma (Fig. 6)
The tumour model of the EL-4 lymphoma, described above (Example 6), was used. The PHPMA-AH-NH-N=DTX-LEV conjugates with the drug content of 8.2 weight % of DTX (9.1 weight % of DTX-LEV) and 16.3 weight % of DTX (18.0 weight % of DTX-LEV) were dissolved in PBS (0.2 ml) for the administration and the suboptimal dose of 2x30 mg of the DTX equivalent/kg was applied intravenously on days 9 and 13 after the transplantation of the tumour cells.
The conjugate with the lower content of the drug (8.2 % of DTX) cured 1 out of 8 tested mice and in the other ones it extended the survival time in a statistically significant way (untreated controls: average survival time 27.25 days, SD 1.64, median 28 days; treated mice: average 41.75 days, SD 6.54, median 45 days; pθ.01). The conjugate with the higher drug content (16.3 % DTX) also cured 1 out of 8 tested mice but the survival of the other mice in the group was not significantly extended.
Conclusion: The content of the drug derivative in the PHPMA-AH-NH-N=DTX-LEV conjugate had a significant influence on the anti-cancer effect in C57BL/6 mice with the EL- 4 lymphoma when administered in the suboptimal dose of 2x30 nig of the DTX equivalent/kg. The polymeric conjugate with the lower content proved higher anti-cancer activity.
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Claims

1. A polymeric conjugate, consisting of a derivative of a cytostatic agent selected from the group of taxanes, in particular paclitaxel (PTX), docetaxel (DTX) or larotaxel (LTX), and a polymeric earner prepared on the basis of a linear or biodegradable grafted copolymer consisting of units of the basic copolymer N-(2-hydroxypropyl)methacroylamide (HPMA) and units containing methacryloylated hydrazones of amino acids or oligopeptides.
2. The polymeric conjugate according to claim 1, characterized in that the derivative of the cytostatic agent has been prepared by acylation of the secondary hydroxyl groups of the cytostatic agent with an oxo acid and is linked to the carrier by means of a hydrolytically instable hydrazone bond.
3. The polymeric conjugate according to claims 1 and 2, characterized in that the oxo acid is selected from the group consisting of levulic acid, 4-(2-oxopropyl) benzoic acid, 4-oxo-pent- 2-enoic acid and 5-oxo-hex-2-enoic acid and 6-oxo-hept-2-enoic acids.
4. The polymeric conjugate according to claims 1 and 2, characterized in that the respective amino acids, or oligopeptides, are selected from the group consisting of glycyl, glycylglycyl, β-alanyl, 6-aminohexanoyl (AH), 4-aminobenzoyl and mixed acyls derived from the oligopeptides GlyPheGly, GlyLeuGly, GlyLeuPheGly and GlyPheLeuGly.
5. The polymeric conjugate according to any one of the preceding claims, characterized in that it contains 70 to 98 mole % of the basic copolymer HPMA, 1.5 to 29.5 mole % of units with hydrazide functional groups and 0.5 to 10 mole % of units with the hydrazone-bound derivative of the cytostatic agent.
6. The polymeric conjugate according to any one of the preceding claims, characterized by the molar weight of 10 to 50,000 g/mol in the case of a linear polymer and 50 to 250,000 g/mol in the case of a grafted copolymer.
7. A method for the preparation of the polymeric conjugate according to claims 1-6, characterized in that a polymer obtained by radical copolymerization of HPMA with comonomers containing the cytostatic agent content bound by means of the oxo acid in a proportion which corresponds to the desired composition is used.
8. A method for the preparation of the polymeric conjugate according to claims 1-6, characterized in that the polymeric carrier is subjected to polymer-analogous transformation of the carrier by reaction with an ester of the oxo acid and the cytostatic agent.
9. Use of the polymeric conjugate according to claims 1-6 for the preparation of a medicament for the treatment of tumour diseases such as ovarian tumours, breast, lung and prostate tumours, especially for the treatment of lymphomas and the 4Tl mammary gland carcinoma.
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