WO2007110003A2

WO2007110003A2 - Micellar carriers for drugs with anti-cancer activity

Info

Publication number: WO2007110003A2
Application number: PCT/CZ2007/000020
Authority: WO
Inventors: Petr Chytil; Tomas Etrych; Martin Hruby; Karel Ulbrich; Blanka Rihova
Original assignee: Zentiva, A.S.
Priority date: 2006-03-28
Filing date: 2007-03-28
Publication date: 2007-10-04
Also published as: WO2007110003A3; CZ2006207A3

Abstract

A micellar system destined for controlled release of medical drug is formed by a micellar structure, constituted by a hydrophilic or amphiphilic polymer, to which the drug is bound by a covalent linkage, the molecules of which are arranged on the hydrophilic surface of the micelle, while the nucleus of the micelle is constituted by hydrophobic components of the system, which are linked with the polymer on the surface by a chemical bond.

Description

Micellar Carriers for Drugs with Anti-cancer Activity

Technical Field

The invention concerns micellar polymeric carriers of cancerostatics allowing for targeted transport of anti-cancer medical drugs, especially doxorubicin, into solid tumors and designed for targeted treatment of tumor diseases in human medicine.

Background Art

In recent years, development of new drugs and drug forms has been focused more and more on the use of polymeric substances, especially water-soluble polymers, as drug carriers. This development has been focused very often to the field of new cancerostatics. High molecular weight of polymers prevents fast release of the drug from the organism by glomerular filtration and, in this way, ensures prolonged period for circulation in blood and for retention in the organism and hence better bioavailability of the drug. Furthermore, macromolecular substances and especially synthetic polymers can accumulate in solid tumors thanks to the EPR (enhanced permeability and retention) effect [Maeda 2000, 2001]. It is possible to make use of this property, in case of binding of the cancerostatic to a macromolecular carrier, for its targeted accumulation in the tumor. A number of systems that are based on this principle have been developed. For example, polymeric conjugates of cancerostatics with soluble polymers have been synthesized and studied, in which a drug with anti-cancer activity was bound to the polymer by a non-cleavable covalent linkage, hydrolytically unstable ionic linkage or a covalent linkage susceptible to enzymatic or simple chemical hydrolysis. Such systems are able to release the cancerostatic from the carrier in its active form either in the tumor, or even more specifically, directly in the tumor cell. Polymeric drugs prepared on the basis of copolymers of N-(2-hydroxypropyl) methacrylamide (HPMA) [Duncan 1985, Rihova 2000, Kopecek 2001, 2000] make up one important group. Literature offers a lot of information about preparation and study of properties of polymers carrying a cancerostatic bound to the polymer by a linkage susceptible to hydrolysis in aqueous media [Kratz 1999]. An important place among them belongs to HPMA copolymers carrying the cancerostatic doxorubicin bound to the polymeric chain by a hydrolytically cleavable hydrazone linkage [Etrych 2002, Ulbrich 2004a, Ulbrich 2004b, Ulbrich - patents]. This linkage is relatively stable in the blood vessel environment (during transport in the organism) and it is hydrolytically labile in the mildly acidic environment of the living cell. The rate of hydrolysis of the linkage also regulates the rate of drug release, i.e. it also regulates concentration of the active substance in the location of the desired effect. Such polymeric cancerostatics showed, in in vitro and in vivo tests in mice, significantly higher anti-cancer effectiveness against a number of tumor lines compared to the free drug and in many cases, their use led to complete recovery of the experimental animals even in the case of therapeutic form of administration [Rihova et al., 2001, Etrych et al., 2001].

Polymer micelles are another carrier system developed for tumor specific transport of cytostatics to solid tumors, which takes advantage of the EPR effect of solid tumors for increased accumulation of the macromolecular drug. They are usually prepared by arranging amphiphilic diblock copolymers into macromolecular micellar formations, in which the drug is bound to the hydrophobic nucleus of the micelle by physical (hydrophobic interaction, ionic linkages) or covalent linkages [Kataoka 2001, Yokoyama 1999, Bae 2003, Yoo 2002, Bronich 1999]. The hydrophobic nucleus of the micelle is enveloped with a hydrophilic layer composed of blocks of a hydrophilic polymer, usually poly(oxyethylene), which protects the whole system from aggregation and undesired interactions with the components of the living organism. Although carrier and micellar systems are not yet commonly used in human medicine, they represent, along with liposomes, a new generation of more effective and safer cancerostatics compared to currently used classic chemotherapeutics.

Disclosure of Invention

Introducing hydrophobic substituents to the structure of a water-soluble polymer - drug carrier - leads to arrangement of such macromolecules in aqueous medium into polymeric micellar formations with the nucleus composed of hydrophobic substituents and the envelope composed of hydrophilic polymeric chains. These formations can be of an intramolecular or intermolecular character. The micellar formation described in the present patent is composed of a hydrophilic top layer constituted by hydrophilic polymeric chains, e.g. of a HPMA copolymer, vinylpyrrolidone copolymer or a multiblock poly(oxyethylene) [Pechar 2000, 2001, 2003], which carry a cancerostatic, e.g. doxorubicin, linked to the polymer by linkers containing pH-sensitive hydrolytically cleavable hydrazone linkages, or by means of an enzymatically cleavable sequence, e.g. GlyPheLeuGly. Other cancerostatics, such a mitomycin C, cis-Pt derivatives, raltitrexate, methotrexate, taxol and other cancerostatics, can be linked to the polymer by a chemical linkage. The hydrophobic nucleus of the micelle is composed of hydrophobic molecules (saturated as well as unsaturated aliphatic chains and their derivates, cholesterol and its derivates, cholic acid and its derivates), linked to the HPMA copolymer chain by ester or amidic linkages or in similar fashion as drug molecules, i.e. by a hydrolytically labile hydrazone linkage. The nucleus of the micelle is held together thanks to hydrophobic interactions between hydrophobic substituents. Contrary to the classic micelles, where the drag is usually situated in the hydrophobic nucleus of the micelle, in this case the drag is linked by an enzymatically or pH-labile covalent linkage to the hydrophilic envelope of the micelle. The whole micellar system having the size of 8 to 150 nm (depending on the composition and structure of the hydrophobic substituent) is stable during transport through the organism (pH 7.4) and is activated and releases the drag only after prospective accumulation in solid tumors (thanks to EPR effect) and after penetration to target tumor cells (pH 5 to 6). Depending on the detailed structure of the copolymer, the macromolecular micellar system is hydrolyzed; in the first phase the drug is released, optionally in the second phase of the degradation the hydrophobic substituents that can be metabolized are also released. The whole macromolecular micellar system thus breaks up into relatively small water-soluble polymeric fragments based on the HPMA copolymer, which can be secreted from the organism, e.g. by glomerular filtration.

Description of the Micellar Drug

• Micellar systems prepared according to the invention are characterized by the fact that the micellar structure is composed of amphiphilic molecules of the HPMA copolymer, arranged in a supramolecular micellar structure, with the nucleus of the micelle composed of hydrophobic components of the system and with a hydrophilic outer layer of the micelle composed of hydrophilic parts of the polymeric chain carrying the covalently-bound cancerostatic. The micelle is schematically depicted in Figure 1.

• The main polymeric chain is composed of a iV-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, which contains from 80 to 98 mol.% of HPMA units and from 2 to 20 mol.% of units of comonomers - esters of methacrylic acid and/or methacryloylated, the carboxy group-modified α-aminoacids, ε-aminoacids, aromatic aminoacids or oligopeptides, from 0.5 to 10 mol.% of which is terminated with hydrophobic molecules and from 0.5 to 8 mol.% is terminated with the cancerostatic doxorubicin or another cancerostatic, linked to the methacryloylated residue of the α-aminoacid, ε-aminoacid, aromatic aminoacid or oligopeptide by a hydrolytically cleavable hydrazone linkage or a linkage cleavable with lysosomal enzymes.

• The hydrophobic molecule can preferably be the acyl of oleic, cholic and cholanic or deoxycholic acids, the acyls of fatty acids with a C10 - C18 chain or, generally, the acyl of unsaturated acids (oleic acid, linoleic acid, linolenic acid), linked to the residue of the comonomer unit with a hydrazide, amide or ester linkage, or

- a hydrazone of 2-keto olefmes with a C12 - C18 chains, or the hydrazone of cholest-4-en-3-one, or an ester of cyclic or polycyclic hydrocarbons or their derivates, preferably cholesterol, 7-dehydrocholesterol, cholestanol, vitamin D, or

- an ester of an aliphatic alcohol with a C9 - C30 chain, preferably a C9 - Cl 8 chain.

• One can preferably use, as the α-aminoacid: glycine, alanine, valine; as the ε- aminoacid: β-alanine, δ-aminobutanoic acid, ε-aminocaproic acid; as the aromatic aminoacid: 4-aminobenzoic acid; as the oligopeptide: GIyGIy, GlyLeuGly, GlyPheGly, GlyPheLeuGly, GlyLeuPheGly and the like.

• The hydrophobic molecule can be incorporated in the structure of the copolymer by polymeranalogous reaction of the chloride of the respective acid, its reactive ester (4- nitrophenyl, succinimidyl), thiazolidine-2-thione amide and the like, by reaction of the acid itself after activation with carbodiimides, or by copolymeration with the respective methacrylamide or methacryloyl ester monomer.

• The molecular weight of the copolymers ranges from 3,000 to 60,000 g/mol.

The structures of basic types of copolymers used for preparation of the micellar carriers for the cancerostatic doxorubicin are presented in Figures 2 and 3.

Brief Description of Drawings

Figure 1 represents a schematic depiction of a polymeric micelle.

Figure 2 depicts the structure of copolymers with DOX bound by a hydrazone linkage and the hydrophobic substituent bound to the polymer in the form of an ester. X is an aminoacid or an oligopeptidic spacer, RE is a residue of cholesterol, dehydrocholesterol, vitamin D or an aliphatic alcohol.

Figure 3 depicts the structure of copolymers with DOX bound by a hydrazone linkage and the hydrophobic substituent bound to the polymer in the form of the acyl of the respective hydrophobic acid (its acyl is depicted in the scheme as RK) by a hydrazide or amidic linkage, or of a 2-ketoolefine bound to the polymer by a hydrazone linkage. X is an aminoacidic or oligopeptidic spacer.

Figure 4 depicts the structure of a polymeric precursor with cholesterol, prepared according to

Example 2.

Figure 5 depicts the structure of a polymeric precursor containing hydrophobic oleoyl, prepared according to Example 4.

Figure 6 depicts the structure of a polymeric precursor with hydrophobic dodecyl substituent bound by an ester linkage, prepared according to Example 5.

Figure 7 depicts the structure of a polymeric precursor with hydrazide groups and with the hydrazide of deoxycholic acid, prepared according to Example 6.

Figure 8 represents distribution of micelles in the physiological solution measured by means of light dispersion (QELS). The micelles were prepared from copolymers containing the oleyl

( ), dodecyl ( ) and cholesteryl ( ) hydrophobic groups. A conjugate without hydrophobic groups (-^•-—) is attached for comparison.

Figure 9 represents release of doxorubicin from micellar solutions of polymers differing in the structure of the hydrophobic substituent. Phosphate buffer pH 5.0, 37 ⁰C, polymer concentration 5 mg/ml. The micelles were prepared from copolymers containing the oleyl

(α ), dodecyl (o ) and cholesteryl (Δ ) hydrophobic groupa. A conjugate without hydrophobic groups (T -^•-^•-^•) is attached for comparison.

Figure 10 depicts the size of the tumor (current state day 42) in B/6 mice with inoculated mouse EL 4 lymphoma, treated with micellar conjugates DOX or with "classic" soluble conjugate (B-578) after intravenous application of one dose 10 mg/kg or two doses 2x5 mg/kg of DOX equivalent in a therapeutic regime of drug administration. Samples B-651 are HPMA copolymers with the oleyl substituent, samples B-652 with the cholesteryl substituent. Examples

Example 1: Synthesis of monomers

HPMA was prepared according to 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)hexanoylhydrazine (N¹-(6-hydrazino-6-oxohexyl)-2-methylacrylamide) (MA-AH-NHNH₂) was prepared according to the previously described procedure [Ulbrich patents, Etrych patent].

6-(methacryloylamino)hexanoyl-N^Λ-(tert-butyloxycarbonyl)hydrazine (MA-AH-NHNH-Boc) was prepared according to the previously described procedure [Ulbrich 2004]

lH-cyclopenta[a]phenanthren-3-yl ester of 6-(2-methacryloylamino)hexanoic acid (MA-AH- chol) was prepared by reaction of methacrylated 6-aminohexanoic acid (prepared according to the previously described procedure [Ulbrich patents]) (MA-AH-OH) with cholesterol with use of the conjugating agent dicyclohexyl carbodiimide (DCC) in tetrahydrofuran (THF). 250 mg of MA-AH-OH (1.26 mmol) and 456 mg of cholesterol (1.26 mmol) was dissolved in freshly redistilled THF. 1.2 molar excess of DCC (311 mg, 1.51 mmol) was dissolved in 0.75 ml THF; the solution contained several crystals of ΛζN-dimethylaminopyridine (DMAP). Both solutions were cooled down to -18 °C. One hour after cooling, the solutions were poured together and the reaction mixture was left at -18 °C for 1 h and at 4 °C for 16 hours. Unreacted DCC was removed by reaction with 50 μl of concentrated acetic acid while stirred at room temperature, rn 0.5 hour the precipitated dicyclohexylurea (DCU) was removed by filtration, THF was evaporated and the product dissolved in ethyl acetate. Residues of precipitated DCU were again removed by filtration. Unreacted MA-AH-OH was removed by extraction with a 2 w. % NaHCO₃ solution and MA-AH-chol was separated by crystallization from acetone and purified by recrystallization.

Y=46 %, 329 mg. melting point: 93-95 °C, elementary analysis: theor. 78.25 % C, 10.83 % H, 2.47 % N, exp. 78.73 % C, 10.85 % H, 2.34 % N, TLC: ethyl acetate: hexane 1:1, Rf =0.8. Example 2: Synthesis of a polymeric precursor - HPMA copolymer with MA-AH-NHNH₂ and MA-AH-chol (PoIy(HPMA-Co-MA-AH-NHNH₂-CO-MA-AH-ChOl))

The poly(HPMA-co-MA- AH-NHNH₂-Co-MA- AH-chol) copolymer was prepared by solution radical copolymerization of HPMA, MA- AH-ISIHNH₂ and MA-AH-cholesteryl in methanol at 6O ⁰C.

601.3 mg (4.20 mmol) of HPMA, 69.3 mg (0.327 mmol) Of MA-AH-NHNH₂ and 79.46 mg (0.140 mmol) of MA-AH-chol (14 w. % of monomers) and 53.6 mg (0.326 mmol) of ABIN (1 w. %) was dissolved in 5.76 ml of methanol. The solution was transferred into an ampoule and nitrogen was bubbled through the solution for 15 min. The ampoule was sealed and put into a thermostat. The small, at normal temperature undissolved, residue dissolved upon heating of the mixture to the polymerization temperature of 60 °C. After polymerization at 60 °C for 20 hours the reaction mixture was precipitated with 150 ml of ethyl acetate and the precipitate was removed by centrifugation. The polymer was re-precipitated from methanol into ethyl acetate, filtered through S4 sintered glass and dried until the constant weight. For structure see Figure 4.

Generally, all the polymeric precursors were characterized by determination of the content of cholesterol or of the aliphatic component by means of ¹H-NMR, and water-soluble precursors by determination of weight average of molecular weights M_w and of polydispersity by means of a GPC system (AKTA™ Explorer, Pharmacia, Sweden) equipped with RI, UV and a multiangle LS detector (DAWN DSP-F, Wyatt Technology Corp., USA). For characterization, the TSK 3000 column was used and 0.3 M acetate buffer with pH 6.5. (20%) and methanol (80%) as the eluent, or alternatively the Superose™ 12 or Superose™ 6 columns and 0.3 M acetate buffer with pH 6.5. The polymeric precursors forming polymeric micelles in the aqueous media were characterized by means of static or dynamic light dispersion (LS, QELS) and the particle sizes were determined by means of a Zetasizer Nano instrument (Malvern, model ZEN 3600, UK).

Example 3: Synthesis of a polymeric precursor — reaction of cholesteryl chloroformate with poly(HPMA-co-MA-AH-NHNH₂)

The poly(HPMA-co-MA-AH-NHNH₂-co-MA-AH-NHNH-chol) copolymer was prepared by polymeranalogous reaction of poly(HPMA-co-MA-AH-NHNH2) with cholesterol chloroformate. Example of the reaction: 250 mg Of PoIy(HPMA-Co-MA-AH-NHNH₂) (1.30.10^"4 mol) was dissolved in 1.5 ml of N,7V^Λ-dimethylformamide (DMF). 15.1 mg (7.78.10^"5 mol) of cholesterol chloroformate was dissolved in 0.02 ml of dichloromethane and the solution was added to a stirred solution of the polymer. The reaction proceeded at 4 °C (cooling with the mixture water-ice) for 0.5 hours. The reaction mixture was then precipitated in a twenty-fold amount of ethyl acetate and centrifuged. The polymer was dissolved in methanol, precipitated with ethyl acetate, filtered with S4 sintered glass and dried until the constant weight.

Example 4: Synthesis of a polymeric precursor — reaction of oleic acid N- hydroxysuccinimide ester with poly(HPMA-co-MA-AH-NHNH^)

The PoIy(HPMA-CO-MA-AH-NHNH₂-CO-MA-AH-NHNH-OIeOyI) terpolymer was prepared by polymeranalogous reaction of poly(HPMA-co-MA-AH-NHNH₂) with oleic acid N- hydroxysuccinimide ester.

Example of the reaction: 245.2 mg Of PoIy(HPMA-Co-MA-AH-NHNH₂) (1.17.10^"4 mol) was dissolved in 1 ml of methanol. 29.5 mg of oleic acid N-hydroxysuccinimide ester (7.78.10^"5 mol) was dissolved in 0.3 ml methanol and added to the stirred solution of the polymer. The reaction proceeded at room temperature for 5 hours and then at 4 °C for 16 hours. The reaction mixture was then precipitated with ethyl acetate and the polymer was separated by centrifugation. The polymer was dissolved in methanol, precipitated in a twenty-fold amount of ethyl acetate, filtered with S4 sintered glass and dried until the constant weight. For structure see Figure 5.

Example 5: Synthesis of a polymeric precursor — a terpolymer of HPMA, MA-AH-NHNH2 and dodecylmethacrylate (PoIy(HPMA-CO-MA-AH-NHNH₂-CO-DDM))

The poly(HPMA-co-MA-AH-ΝHΝH₂-co-DDM) terpolymer was prepared in two steps. The first step involved solution radical copolymerization of HPMA with MA-AH-NHNH-Boc and dodecylmethacrylate (DDM) in methanol at 60 °C. hi the second step the protecting groups were removed by means of trifluoroacetic acid (TFA). Example of the reaction:

Step I: 782 mg (5.46 mmol) of HPMA, 155 mg (0.496 mmol) of MA-AH-NHNH-Boc, 72.6 mg (0.285 mmol) of freshly distilled DDM (14 w. % of monomers) and 71.4 mg (0.435 mmol) of ABIN (1 w. %) was dissolved in 7.7 ml of methanol. An ampoule with the solution of the polymerization mixture was bubbled through with nitrogen for 10 minutes, then sealed and put into a thermostat at 60 ⁰C for 20 hours. The reaction mixture was precipitated with 175 ml of a mixture acetone:diethyl ether and the precipitate was separated by centrifugation. The polymer was re-precipitated from methanol again into the mixture acetone : diethyl ether 3:1, the precipitate was filtered with S4 sintered glass and dried until the constant weight. Step II: 288 mg of the polymer was dissolved in 3 ml of the mixture TFA : triisopropylsilane : water 95 : 2.5 : 2.5. 20 minutes later the mixture was concentrated in a vacuum evaporator, dissolved in a five-fold amount of methanol, and evaporated again. Dissolution and evaporation were repeated several times until small crystals fell out after evaporation. The product was dissolved in water and pH of the aqueous solution was increased to pH = 7 - 8 by means of NaOH. The polymer was further purified and isolated by means of dialysis against water (3 days) and subsequent lyophilization. For structure see Figure 6.

Example 6. Preparation of a polymeric precursor containing deoxycholic acid bound by a hydrazide linkage (PoIy(HPMA-Co-MA-AH-NHNH₂-CO-MA-AH-NHNH-ChOlA))

Preparation started from the succinimide ester of deoxycholic acid and the precursor poly(HPMA-co-MA-AH-NHKH₂). 300 mg of poly(HPMA-cø-MA-AH-NHNH₂) (1.56.10^'4 mol of hydrazide groups) were dissolved in 2 ml of DMF. 30 mg (5.7.1O^"5 mol) of deoxycholic acid succinimide ester was dissolved in 0.25 ml of dichloromethane and the solution was added into a stirred solution of the polymer. The reaction mixture was stirred at room temperature for 1.5 hours, then the polymer was precipitated with a twenty-fold amount of ethyl acetate and the precipitate was separated by centrifugation. The polymer was precipitated from methanol into a mixture ethyl acetate-diethyl ether, the precipitate was washed with diethyl ether, filtered with S4 sintered glass, and the final polymeric precursor was dried until the constant weight. For structure see Figure 7.

Example 7. Preparation of polymeric conjugates containing doxorubicin and a hydrophobic substituent (PHPMA-AH-NH-N^DOX-co-MA-AH-choVoleyVdodecyl)

Copolymers with DOX bound to a PHPMA carrier by a hydrolytically cleavable hydrazone linkage containing a hydrophobic substituent were prepared by reaction of polymeric precursors containing hydrazide and hydrophobic groups (an aliphatic substituent, an ester and hydrazide of cholesterol or cholic acid) with DOX.HC1 in methanol under catalysis of acetic acid.

A solution of 15.384 g of the poly(HPMA-co-MA-AH-NHNH₂-co-MA-AH-chol) copolymer in 92.1 ml of methanol (167 mg polymer/ml) was put into a thermostated cell, in which 2.5 g of D0X.HC1 (4.3 mmol) had been placed. The non-homogeneous suspension was stirred in dark at 25 ⁰C and 1 minute later, 4.9 ml of acetic acid was added (total volume 116 ml). In the course of the reaction, dissolution of the suspension slowly occurred; after reacting for 22 hours, the polymeric product was isolated from the homogenous solution by precipitation with 1 1 of ethyl acetate, the precipitate of the polymeric drug was isolated by filtration with S4 sintered glass, washed with 150 ml of ethyl acetate and dried until the constant weight. The content of total DOX was determined spectrally. M_w and M_n were determined using liquid chromatography (LC AKTA) with light dispersion detection (Multiangel detector DAWN DSP, Wyatt). Characterization of the polymeric drug: Total yield of the drug-binding reaction: 17.2 g (96 %), content of the total DOX 11.3 w. %, content of free DOX 1.52 % of the total content of DOX. The procedure for binding doxorubicin to polymeric precursors through the hydrazone linkage was the same for all types of precursors.

Example 8. Preparation of a polymeric conjugate with the drug and a hydrophobic substituent bound by a hydrolytically labile hydrazone linkage.

(A) A HPMA (92.5 mol.%) with MA-AH-NHNH₂ (7.5 mol.%) copolymer, (poly(HPMA-co- MA-AH-NHNH₂) with M_w = 22,000, 100 mg; (0.052 mmol hydrazide groups) was reacted with 2-dodecanone (37 mg, 0.20 mmol) in methanol (2.0 ml) with addition of acetic acid (50 μl) at room temperature under stirring for 3 days. The polymer was then isolated by precipitation with a mixture acetone (20 ml) - diethyl ether (40 ml) and purified by re- precipitation from methanol into the mixture acetone-diethyl ether. The yield was 98 mg. The resulting polymer contains 6.1 mol.% of monomeric units of residual hydrazide groups (determined with TNBSA, Etrych et al. 2001), i.e. 81 % conversion of the hydrazide groups to hydrazone. DOX was bound to the polymer in the subsequent step using the procedure described in Example 7.

(B) The ρoly(HPMA-co-MA-AH-NHNH₂) (7.5 mol.% of hydrazides) copolymer with M_w = 22,000, 100 mg; 0.052 mmol of hydrazide groups) was reacted with 2-octadecanone (54 mg, 0.20 mmol) in methanol (2.0 ml) with addition of acetic acid (50 μl) at room temperature under stirring for 3 days. The polymer was then isolated by precipitation with a mixture acetone (20 ml) - diethyl ether (40 ml) and purified by re-precipitation from methanol into the mixture acetone-diethyl ether. The yield was 91 mg. The resulting polymer contains 5.9 mol.% of monomelic units of residual hydrazide groups (determined with TNBSA), i.e. 79 % conversion of the hydrazide groups to hydrazone. DOX was bound to the polymer in the subsequent step using the procedure described in Example 7.

(C) A conjugate prepared by copolymerization of monomers

Synthesis of the MA-AH-NHN=dodecane monomer

2.00 g Of MA-AH-NHNH₂ (9.38 mmol), 1.73 g of dodecanone (9.38 mmol), 100 microliters of acetic acid, ca 5 mg of an inhibitor and 25 ml of 96 % ethanol was refluxed for 5 hours.

Subsequently, triethylamine (600 μl) was added to the still hot solution and the total was evaporated until dryness. The monomer was purified by liquid chromatography in a chromatographic column filled with a fifty-fold weight excess of silica gel 60 using ethyl acetate as the mobile phase, R_F hydrazone = 0.25, Rp hydrazide = 0.00, R_F dodecanone =

1.00. The theoretical yield was 3.56 g (9.38 mmol), the isolated yield was 2.26 g (63 %; 5.95 mmol).

The resulting polymer was prepared by radical copolymerization of 92.5 mol.% HPMA,

4.5 mol.% MA-AH-NHNH₂ and 3 mol.% MA-Acap-NHN=dodecane and subsequent binding of DOX to the free hydrazide groups. The polymer was precipitated with ethyl acetate, washed with diethyl ether, and dried under vacuum. DOX was bound using the method described in

Example 7. The copolymer contained 9.5 w.% of DOX and 2.8 mol.% of dodecane groups.

Example 9. Preparation of micelles in aqueous media

General procedure: 20 mg of the polymeric conjugates the preparation of which was described in Examples 6 through 8 (containing DOX bound by a hydrazone linkage and a hydrophobic substituent) were dissolved in 200 ml of saline (dissolving can be also carried out in distilled water). In order to achieve perfect dissolution to a micellar solution, a K 5 ultrasound bath (Kraintek s.r.o.) was used; sonication was performed for 1 - 5 min, depending on the composition of the sample being dissolved. The samples - polymeric micelles - were filtered on a Whatman membrane, pore size 0.22 μm, before characterization with light dispersion and in vitro and in vivo testing. The polymeric precursors forming polymeric micelles in aqueous media were characterized by means of static or dynamic light dispersion (LS, QELS) and particle sizes were determined in saline by means of a Zetasizer Nano instrument (Malvern, model ZEN 3600, UK.) using polymer concentration of 2 mg/ml. An example of distribution curve is presented in Figure 8.

Example 10: Release of doxorubicin from polymeric micelles constituted by polymers with different hydrophobic substituents

Amounts of doxorubicin released from the polymeric micelles after their incubation in a phosphate buffer with pH 5.0 (0.1 M phosphate buffer containing 0.15 M NaCl), modeling the intracellular environment, and in a phosphate buffer with pH 7.4, modeling the blood vessel, were measured. The amount of released DOX in the incubation solution was measured using HPLC (Shimadzu). hi predetermined time intervals amounts of 50 μl of the incubation solution were collected and analyzed in the TSKGeI G 3000 column, isocratic flow 0.5 ml/min of the mobile phase composed of the mixture methanol : acetate buffer with pH 6.5 (80 : 20 v.%). The amount of DOX was determined from peak areas of free and bound DOX (UV-VIS detection at 488 nrn). After incubation of the micelles (concentration 5 mg/ml) in the physiological environment at 37 °C (phosphate buffer, pH 7.4), release of DOX does not take place or only a small amount of the drug is released (up to 8 %/24 hours); release rates of DOX from micelles at pH 5.0 are presented in Figure 9. It is apparent that the release rate of DOX, and hence the rate of activation of a cytotoxic drug, is remarkable in mildly acidic environment; while the hydrophobic substituents decrease the release rate of the drug in the micellar system, this effect is minimal.

Example 11: Hydrolytic disintegration of a micelle with DOX and a hydrophobic substituent bound by a hydrolytically cleavable hydrazone linkage

Release of doxorubicin from micellar solutions of the polymers differing in the structure of the hydrophobic substituent. Phosphate buffer pH 5.0, 37 °C, polymer concentration 5 mg/ml. The copolymer prepared according to Example 8(A) (containing the dodecanone hydrophobic substituent) was dissolved in PBS buffer (0.15 mol.l^"1, pH 5.0 and 7.4, resp.) into a micellar solution with concentration 2 mg/ml and disintegration of the micelles was observed using the dynamic light dispersion method (QELS). Relatively large micelles with the hydrodynamic diameter of 274 nm disintegrate at 37 ⁰C significantly faster at pH 5.0, the disintegration proceedeing up to a soluble polymeric product which can be secreted from the organism (a tumor tissue model; intensity of dispersed light 6.9 % of the original value after 24 hours) than at pH 7.4 (a blood plasma model; intensity of dispersed light 87 % of the original value after 24 hours). This function is similar to that of the release of the drug.

The polymer prepared according to Example 7(B) (an octadecanone substituent) forms micelles with the hydrodynamic diameter close to 200 nm and the rate of their disintegration is comparable to disintegration of the polymer prepared according to Example 8(A). The final product of micelle disintegration after 4 day incubation at both pH (7.4 and 5.0) is a hydrazide polymer with the hydrodynamic diameter of 7 nm, i.e. a polymer which can be secreted from the organism. Simultaneously, low-molecular-weight hydrophobic ketones precipitate from the solution as a macroscopic precipitate.

Example 12. An example of in vitro biological activity ofmicellar conjugates of doxorubicin during incubation with cells of different tumor lines.

During in vitro experiments, anti-proliferation cytostatic activity was observed using the method of incorporation of ³H-thymidine in five tumor lines of human and mouse origin and in T-mitogen (concanavaline A (Con A))-stimulated mouse splenocytes. The tumor lines of mouse origin were: T-cell lymphome EL4, B-cell lymphome 38C13, fibroblast line 3T3 and B-cell leukemia. The tumor line of human origin was a metastasing line of colorectal carcinoma SW 620. The tumor lines were, inter alia, selected on the basis of sensitivity to the used cytostatic. The results of measurement are presented in Table 1.

Table 1 : Cytotoxicity of micellar polymeric conjugates of doxorubicin for various cell lines measured using the method of incorporation of ³H-thymidine. The table presents values of IC₅₀, i.e. the concentration in μg/ml at which proliferation is inhibited in half of the tested cells.

The mouse line 38C13 was the most sensitive to the original drug with IC₅₀ = 0.0005 μg/ml. The other lines and also stimulated normal T splenocytes (Con A) are one to two orders less sensitive. In in vitro tests, there is no substantial difference between samples with the oleoyl and cholesteryl substituents, they behave similarly. After binding DOX to the polymeric carrier, IC₅₀ decreases by one to two orders. The smallest difference was recorded for normal splenocytes.

Example 13. An example of in vivo biological activity of micellar conjugates of doxorubicin in mice inoculated with T-cell lymphome EL4

For in vivo experiments a model of mouse T-cell lymphome EL 4 was used. The conjugates were administered intravenously either 1 x 10 mg/kg, or 2 x 5 mg/kg. One-shot administration was effected on the eight day after transplantation of the tumor cells; in the case of two-shot administration, the first dose was administered also on the eight day after transplantation of the tumor cells and the second dose was administered on the twelfth day. All the experimental groups exhibited significantly slower cancer growth already on the third day after the first administration compared with the controls. The sample with bound cholesterol has markedly better anti-cancer effect; a higher one-shot dose (10 mg/kg) is the most effective. On the fourteenth day after transplantation of the tumor cells, there is apparent certain progression in the group of animals treated with the sample with oleoyl, which is, of course, much smaller than in the controls. In experimental groups treated with the sample with cholesteryl, there is apparent marked reduction of the tumor at this time; the one-larger-dose treatment is the most effective. On the eighteenth day after transplantation and on the tenth day after the first administration of the drug, first cured mice occur, in high percentage (seven animals out of eight) for the higher dose, four out of eight for the lower dose. In the control treatment with a non-micellar soluble conjugate, three mice out of eight were cured. On the fourteenth day after the first administration of the drug, the mice treated with the conjugates with cholesterol administered in either of the scheme are 100 % cured and this situation still continues until the 42^nd day. The conjugates with oleoyl are less effective, even though the anti-cancer effect is evident compared with controls. The LTS (long-term survivors) in the group treated with the oleoyl sample is 1/8 in the case of the higher dose and 2/8 in the case of two lower doses 8/8 in the group treated with the cholesteryl sample in both schemes of administration. For "classical" non-micellar hydrazone it is (with the selected dosage) 4/8. The controls are all dead; the shortest interval of survival is 20 days and the longest one 30 days. The average was 22 days. Results of in vivo test are presented in Figure 10 and Table 2.

Table 2. Survival of B/6 mice with inoculated mouse lymphome EL 4 treated with micellar conjugates of DOX after i.v. administration of one dose of 10 mg/kg or two doses 2x5 mg/kg of DOX equivalent in a drug-administration therapeutic regime. Samples B-651 are HPMA copolymers with the oleyl substituent, samples B-652 are with the cholesteryl substituent. Sample B-578 is a control, "classical", soluble non-micellar conjugate. The days mean individual days of death of the animals, LTS stands for surviving animals without a palpable tumor, LTS? stands for surviving animals with a palpable tumor.

Literature.

Bae, Y. S.; Fukushima, S.; Harada, A.; Kataoka, K. pH responsive drug-loaded polymeric micelles: Intracellular drug release correlated with in vitro cytotoxicity on human small cell lung cancer SBC-3. 2003. Salt Lake City, Utah, U.S.A. Winter Symposium and 11th International Symposium on Recent Advances in Drug Delivery Systems. 2003.

Bronich, T. K.; Nehls, A.; Eisenberg, A.; Kabanov, V. A.; Kabanov, A. V. Colloids Surf. B 1999, 76, 243-251.

R. Duncan, J.B. Lloyd, J. Kopecek, P. Rejmanova, J. Strohalm, K. Ulbrich, B. Rihova, V. Chytry: Synthetic Polymeric Drugs (1985). Czech. PV 0095/85, Australia 589587, Canada 130053, Denmark 164485, Europe 0187547, US 5,037,883, Japan 000137/86

Etrych, T., Jelinkova, M., Rihova, B. and Ulbrich K., New HPMA copolymers containing doxorubicin bound by pH sensitive linkage. Synthesis, in vitro and in vivo biological properties. J. Controlled Release 73, 89-102 (2001).

T. Etrych, P. Chytil, M. Jelinkova, B. Rihova, K. Ulbrich, Synthesis of HPMA Copolymers Containing doxorubicin Bound by a Hydrazone Linkage. Effect of Spacer on Drug Release and in vitro Cytotoxicity. Macromolecular Biosci. 2, 43-52 (2002)

Etrych T., Chytil P., Pechar M., Studenovsky M., Rihova B., Ulbrich K.: A method of preparation of polymeric doxorubicin conjugates with pH-regulated drug release, CZ PV 2005-558

Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Delivery Rev. 2001, 47, 113-131.

J. Kopecek, P. Kopeckova, T. Minko, Z. Lu, HPMA Copolymer- Anticancer Drug Conjugates: Design, Activity, and Mechanism of Action. Europ. J. Pharm. Biopharm. 50, 61 - 81 (2000) Kopecek, J., Kopeckova, P., Minko, T., Lu, Z.R., Peterson, CM. (2001) "Water soluble polymers in tumor targeted delivery". J. Controlled Release IA, 165-173.

F. Kratz, U. Beyer, M.T. Schutte, Drug-polymer conjugates containing acid-cleavable bonds, Crit. Rev. Ther. Drug Carrier Syst. 16 (1999) 245-288.

Maeda, H., J. Wu, T. Sawa, Y. Matsumura, and K. Hori. 2000. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release 65:271-284.

Maeda, H. 2001. The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Adv Enzyme Regul 41:189-207.

M. Pechar, J. Strohalm, K. Ulbrich, E. Schacht, Biodegradable Drug Carriers Based on Poly(ethyleneglycol) Block Copolymers. Macromol. Chem.Phys. 198, 1009-1020 (1997)

M. Pechar, K. Ulbrich, V. Subr, L. W. Seymour, E. Schacht, Poly(Ethylene Glycol) Multiblock Copolymers as a Carier of Anti-Cancer Drug doxorubicin. Bioconjugate Chem. 11, 131-139, 2000

M. Pechar, K. Ulbrich, M. Jelinkova, B. Rihova: Conjugates of Antibody-Targeted PEG Multiblock Polymers with doxorubicin in Cancer Therapy. Macromol. Biosci. 3, 364-372 (2003)

B. Rihova, M. Jelinkova, J. Strohalm, V. Subr, D. Plocova, O. Hovorka, M. Novak, D. Plundrova, Y. Germano, K. Ulbrich, Polymeric Drugs Based on Conjugates of Synthetic and Natural Macromolecules II. Anti-cancer Activity of antibody or (Fab')₂-targeted Conjugates and Combined Therapy with Immunomodulators. J. Controlled ReI. 64, 241-261 (2000)

K. Ulbrich, V. Subr, J. Strohalm, D. Plocova, M. Jelinkova, B. Rihova, Polymeric Drugs Based on Conjugates of Synthetic and Natural Macromolecules I. Synthesis and Physico- chemical Characterisation. J. Controlled ReI. 64, 63-79 (2000) K. Ulbrich, T. Etrych, P. Chytil, M. Jelinkova, B. Rihova, Antibody-Targeted Polymer- doxorubicin Conjugates with pH-Controlled Activation, J. Drug Targeting 12(8) (2004) 477- 489]. (C)

K. Ulbrich, V. Subr, Polymeric Anticancer Drugs with pH-Controlled Activation, Adv. Drug Delivery Rev. 56/7, 1025-1052 (2004)

K. Ulbrich, T. Etrych, P. Chytil, M. Pechar, M. Jelinkova, B. Rihova, Polymeric Anticancer Drugs with pH-Controlled Activation. Int. J. Pharm. 277/1-2 67-72 (2004)

K. Ulbrich, T. Etrych, B. Rihova, M. Jelinkova, M. Kovar. pH-Sensitive polymeric conjugates of an anthracycline cancerostatic for targeted therapy. CZ 293787 (WO 03/053473), CZ 293886

Yokoyama, M.; Okano, T.; Sakurai, Y.; Fukushima, S.; Okamoto, K.; Kataoka, K. J. Drug Targeting 1999, 7, 171-186.

Yoo, H. S.; Lee, E. A.; Park, T. G. J. Controlled Release 2002, 82, 17-27.

Claims

C L A I M S

1. A micellar system destined for controlled release of a drug, characterized by a micellar structure constituted by a hydrophilic or amphiphilic polymer, to which the drug is bound by a covalent linkage, the molecules of which are arranged on the hydrophilic surface of the micelle, while the nucleus of the micelle is constituted by hydrophobic components of the system, which are linked with the polymer on the surface by a chemical bond.

2. The micellar system according to claim 1, characterized in that the hydrophilic polymer is selected from the group including copolymers of N-(2- hydroxypropyl)methacrylarnide, N-vinylpyrrolidone and multiblock polymers based on poly(oxyethylene) .

3. The micellar system according to claim 2, characterized in that the micellar structure is constituted by amphiphilic molecules of a copolymer of N-Q-- hydroxypropyl)methacrylamide with methacryloylated derivatives of cytostatics and hydrophobic molecules.

4. The system according to any of the preceding claims, characterized in that the drug contained therein is a cancerostatic, preferably doxorubicin, methotrexate, taxol or derivatives of cis-platin.

5. The system according to any of the preceding claims, characterized in that the drug (cancerostatic) is bound to the copolymer by hydrolytically degradable hydrazone linkages or by enzymatically degradable oligopeptidic sequences.

6. The system according to any of the preceding claims, characterized in that the size of the micelle ranges from 8 to 150 nm.

7. The system according to any of claims 3 to 6, characterized in that it contains from 80 to 98 mol.% of units of N-(2-hydroxypropyl)methacrylamide and from 2 to 20 mol.% of units of comonomers, i.e. of esters of methacrylic acid and/or of methacryloylated, carboxy-group modified α-aminoacids, ε-aminoacids, aromatic aminoacids or oligopeptides, from 0.5 to 10 mol.% of which is terminated with hydrophobic molecules and from 0.5 to 8 mol.% of which is terminated with a cancerostatic.

8. The system according to any of the preceding claims, characterized in that its hydrophobic part is constituted by aliphatic hydrocarbons with a C9 to C30 chain or their derivatives or by cyclic or polycyclic hydrocarbons or their derivatives, preferably by cholesterol, cholestanol or their derivatives, or by vitamin D.

9. The system according to claim 8, characterized in that the hydrophobic molecule is selected from the group of acyls of ClO to Cl 8 fatty acids, or unsaturated fatty acids, or cholic acid, cholanic acid (5β-cholan.24-oic acid) or 7-dehydrocholic acid.

10. The system according to claim 8, characterized in that the hydrophobic molecule linked to the polymer by a hydrolytically labile hydrazone linkage is selected from the group of 2- keto olefins with a C12 to C18 chain or cholest-4-en-3-on has been used for the linkage.

11. The system according to claim 8, characterized in that the hydrophobic molecule is selected from the group of esters of cholesterol or 7-dehydrocholesterol, cholestanol, vitamin D or aliphatic alcohols with C9 to Cl 8 chains.

12. The system according to claims 7 through 11, characterized in that it contains an α- aminoacid selected from the group of glycine, alanine, valine, and/or an ε-aminoacid selected from the group of β-alanine, δ-aminobutanoic acid, ε-aminocaproic acid, and/or 4-aminobenzoic acid as an aromatic aminoacid, and/or an oligopeptide selected from the group of GIyGIy, GlyLeuGly, GlyPheGly, GlyPheLeuGly, GlyLeuPheGly.

13. The system according to any of the preceding claims, characterized in that its molecular weight ranges from 3,000 to 70,000 g/mol.

14. A method for the manufacture of the systems according to any of the preceding claims, characterized in that the hydrophobic molecule is incorporated into the polymeric chain by polymeranalogous reaction, the reactive groups being contained in the polymeric precursor, or the hydrophobic molecule is pre-activated by introducing an active substituent.

15. A method for the manufacture of the systems according to any of claims 1 through 11 , characterized in that the hydrophobic molecule, after its optional activation by introducing an active substituent, reacts first with the respective monomelic unit, which is subsequently polymerized.