CZ2003605A3 - pH sensitive polymeric conjugates of anthracycline cancerostatic for targeted therapy - Google Patents

Abstract

In the present invention, there are disclosed conjugates comprising a polymeric carrier consisting of 30 to 3000 monomeric units linked in a polymeric chain and consisting of a) 60 to 98,5 percent of the N-(2-hydroxypropyl)methacrylamide units, b) 1 to 25 percent of oligopeptide methacryloylated hydrazone units terminated by an anthracycline cancerostatic molecule, c) 0.5 to 15 percent of methacryloylated oligopeptide units or sodium salts thereof and optionally d) 0.5 to 10 percent of oligopeptide methacryloylated hydrazide units, intended for targeted therapy of neoplastic diseases in human medicine.

Description

pH sensitive anthracycline cancerostatic polymer conjugates for targeted therapy

Technical field

The invention relates to polymeric cancerostatics enabling targeted transport in the body and directed to the targeted therapy of cancer in human medicine.

State of the art

In recent years, the development of new drugs and dosage forms has increasingly focused on the use of polymeric substances, particularly water-soluble polymers, as drug carriers. A variety of polymeric conjugates of cancerostatics with soluble polymers have been prepared and studied in which a drug having an antitumor effect has been attached to the polymer by a non-cleavable covalent bond, a hydrolytically unstable ionic bond, or a covalent bond prone to enzymatic or simple hydrolysis. The aim was to prepare drugs with improved pharmacokinetic and pharmacodynamic behavior, enabling targeted therapy of cancer. An important group consists of polymer drugs prepared on the basis of ΗΡΜΑ copolymers. In these agents, the cancerostatic is coupled to a polymeric V- (2-hydroxypropyl) methacrylamide carrier by an enzymatically cleavable oligopeptide sequence, prepared as a substrate for lysosomal enzymes (enzymes present in mammalian cells). The structure, synthesis and properties of such conjugates are described in the patent [Duncan 1985]. An overview of the results achieved so far in this area was prepared very well by Kopeček et al. [Kopecek et al. 2000]. Polymeric drugs of the type described have been effective in treating a variety of tumors in mice and rats. Two polymer conjugates are currently even tested clinically [Vasey 1999 et al., Julyan et al. 1999, Thomson et al. 1999]. The results of clinical testing have shown that the polymeric conjugate of doxorubicin is less non-specifically toxic than the free drug. Its maximum tolerated dose (MTD) is 320

C Λ mg / m, which is 4, 5, (x more than the normally used clinical dose of free doxorubicin (60 80 80 mg / m). No significant effect on cardiac function was observed after administration of polymeric drug, although the individual cumulative dose reached up to All the other toxicities observed with the administration of free, non-directed anthracycline antibiotics have been significantly reduced.The disadvantage of the previously clinically tested polymer drug conjugates is the relatively low specificity of the effect, since these conjugates either do not contain a targeting unit at all, such as PK1. and / or relatively little specific (// '/ <á Fá' ί: carbohydrate (galactosamine in PK2 conjugate, which is tested for its ability to target polymeric drug to the liver). Conjugates are also under development, with a specific target effect by attaching a specific targeting molecule (e.g. ad antibody, but also lectin, growth hormone, transferrin, etc.) to the carrier molecule.

Another disadvantage of clinically tested conjugates, including poly (HPMA) conjugates to doxorubicin, is that the drug is released intracellularly in pharmacologically active form only by an enzymatic reaction that occurs in lysosomes. This means that such a drug is only effective in cells with high concentrations of lysosomal enzymes - peptidases. Another disadvantage is the relatively complex structure of the conjugate, which must contain a sequence from which the drug is released by peptidases, mostly the tetrapeptide linker GlyPheLeuGly, which makes the synthesis more expensive and complicated.

There is a great deal of information in the literature about the preparation and study of the properties of polymers bearing attached cancerostatic by a linkage prone to hydrolysis in aqueous media. The results are summarized in a review article by Kratz [Kratz et al., 1999]. Natural macromolecules, such as albumin, dextrans, transferrin, alginates or antibodies, have mostly been used as carriers of cancerostatics. Synthetic polymeric carriers, poly (ethylene glycol) and polyglutamines have also been used in several cases. The drugs were attached to the carriers by linkages allowing hydrolysis-controlled release of the active drug both in the extracellular space and within the cells. In the case of doxorubicin (DOX), it was most often a cis-aconitic acid ester bond or a hydrazone bond. However, in vivo testing of all of the above-mentioned pH-sensitive conjugates in experimental animals did not produce conclusive results and thus none of the polymer conjugates were used or clinically tested in the treatment of cancer.

We developed polymeric cancerostatics based on HPMA copolymers with hydrazone pH sensitive binding cancerostatics, showed in vitro and in vivo tests in mice significantly higher antitumor activity against a number of tumor lines than conjugates in which the drug was attached to a polymeric carrier by enzymatically cleavable binding via The release of the drug is due to a change in the pH of the environment and therefore the presence of lysosomal enzymes is not required for activation. The release rate (and thus the instantaneous cytostatic concentration) is much higher than that of conjugates containing enzymatically cleavable sequences only [Říhová et al., 2001, Etrych et al., 2001].

SUMMARY OF THE INVENTION

The present invention provides linear and branched polymer conjugates of doxorubicin, daunomycin, farmo rubicin and other carbonyl-containing anthracycline carcinostatics with copolymers prepared based on V- (2-hydroxypropyl) methacrylamide (HPMA) and optionally with antibodies, fragments thereof or non-specific immunoglobulin. 3). These conjugates are characterized in that the polymeric carrier together with the antibody (immunoglobulin) ensures prolonged circulation of the polymeric drug in the bloodstream and its subsequent preferential passive or active accumulation in the tumor. The cancerostatic is attached to the carrier by a blood-stable linkage (at pH 7.4). Upon binding of the drug to the polymer, the cancerostatic loses its biological effect, is transported through the bloodstream in an inactive form, and activation of the cytotoxic drug occurs only intracellularly in the organelles of the target cells due to pH drop and hydrolysis of the drug to drug attachment.

The conjugates consist of a polymeric carrier consisting of 30 to 3000 monomeric units linked to the polymer chain, of which 60 to 98.5% are N- (2-hydroxypropyl) methacrylamide units, 1 to 25% are methacryloylated hydrazones of oligopeptides terminated with an anthracycline carcinostatic molecule ( preferably doxorubicin) and 0.5 to 15% are methacryloylated oligopeptide units or their sodium salts, optionally 0.5 to 10% methacryloylated oligopeptide hydrazide units are present and the conjugate may optionally have 0.5 to 5% methacryloylated amino acids, ε- amino acids or oligopeptides terminated by an immunoglobulin molecule or a specific monoclonal or polyclonal antibody.

The branched high molecular weight structures contain, in addition to the above units, 0.1 to 5% of the linker units linking the individual polymer chains to the high molecular weight branched structure, consisting of enzymatically degradable methacryloylated oligopeptides, preferably GlyPheGly, GlyLeuGly or GlyPheLeuGlyendi , hexamethylenediamine).

The essence of the polymeric conjugates with a targeted antitumor effect according to the invention is the binding of the active ingredient - a cytostatic agent - to the polymeric carrier via a hydrazone group formed by the reaction of the carbonyl group in the drug molecule with the hydrazide group of the polymer. Binding of the drug to the polymeric carrier will significantly increase the molecular weight

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X the weight of the drug, thereby increasing the circulation time in the bloodstream and the total residence time of the active substance in the body. The drug-polymer binding is relatively stable during drug transport in the bloodstream and hydrolytically cleavable in a slightly acidic environment within cells, namely, in cell organelles characterized by an acidic pH. That is, the drug is transported through the bloodstream in an inactive, polymer-bound form, and is released and activated only after penetration into the target tumor cells. Activation of the drug only in the target cells leads to the elimination of side effects of otherwise toxic cytostatics and to the targeting of their effect preferably on the tumor cells. The polymer carrier based on HPMA copolymers is responsible for targeted delivery to the tumor or tumor cells. In the case of non-directed or branched high molecular weight poly (HPMA) carrier, polymer drug is deposited in solid tumor tissue due to passive targeting and EPR effect (Enhanced

Permeability and Retention effect). The polymer may be released from the body after enzymatic degradation of the oligopeptide linkers in the form of shorter polymer chains, e.g., by glomerular filtration. In the case of antibody-containing carriers (immunoglobulin), the antibody is attached to the carrier via a hydrazone linkage resulting from the reaction of aldehyde groups introduced into the Fc fragment of the antibody by oxidation of sodium periodate with the hydrazone groups of the carrier.

The antibody (immunoglobulin) can also be attached to the polymeric carrier chain using bifunctional reagents. In this case, the antibody molecule is modified by reaction with 2iminothiolane (introduction of -SH groups), or the -SH groups can be introduced into the antibody by reducing some -S-S- groups (bridges) by reduction, eg, glutathione. The polymer are ib. . . . Maleimide groups are introduced by reacting the hydrazide groups of the polymer with the succinimide groups

A 3-maleimidopropionic acid ester and conjugation occurs by addition of the -SH group of the antibody to the double bond of the maleimide group of the polymer. Similarly, other bifunctional reagents, such as SPDP (3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester), can be conjugated to conjugate -SH groups containing the antibody or fragments thereof (immunoglobulin) directly to the hydrazide groups of the polymer. carriers.

The conjugate with the antibody (immunoglobulin) or other targeting moiety (antibody fragment, lectin, biologically active oligopeptide) accumulates passively and actively in the target organs / tissues. Passive accumulation is facilitated by the higher molecular weight of the conjugate and the so-called EPR effect is exerted; active accumulation is ensured by a process whereby the binding site of the targeting structure (e.g., antibody) interacts with the corresponding receptors on the surface of the target cells. In this case, the HPMA copolymer plays a role not only of the carrier but also of the protective sheath, which substantially reduces the immunogenicity of the targeting glycoprotein in the conjugate.

with.,

The polymeric drug according to the invention can be used in three forms differing in the detailed structure. The first structure represents a linear polymer conjugate with a drug without a targeting antibody (immunoglobulin) (Fig. 1), the second structure is high molecular weight and represents a branched polymer structure with biodegradable oligopeptide linkages (Qbr. 2) and the third structure also includes a targeting structure - antibody (immunoglobulin) , protein /

or lectin (Fig. 3).

The preparation of non-directed linear polymeric drugs takes place in several reaction steps. In the first step, polymer precursor I (see Fig. 4) is prepared as a copolymer of HPMA with methacryloylated reactive esters of (4-nitrophenyl, N-hydroxysuccinimide, etc.) oligopeptides, in the second step leading to the preparation of polymeric precursors II are terminal reactive ester groups converted to hydrazides by hydrazinolysis and in a third step, a cancerostatic containing a keto group in the molecule is attached to the hydrazide groups by chemical hydrazone linkage. The second way of synthesis of polymeric drug or polymer precursor II is based on preparation of protected (eg Z-butyloxycarbonyl, Boc) hydrazide of methacryloylated oligopeptides, its copolymerization with HPMA and after removal of the protecting group (trifluoroacetic acid). the previous case.

For the preparation of the branched polymeric drug, it is necessary to start from polymeric precursors I containing an enzymatically degradable oligopeptide in the side chains, preferably GlyPheGly, GlyLeuGly, GlyPheLeuGly or GlyLeuPheGly. The second step is initiated by gently branching the polymer by reacting the polymeric precursors I with alkylenediamine (ethyl, butyl, hexyl) and completed by subsequent hydrazinolysis of the remaining hydrazine reactive groups with the hydrate as in the previous case.

Antibody (immunoglobulin) -druged drugs can be prepared from both previous types of drugs by subsequent reaction with an antibody (immunoglobulin) into whose structure they have been introduced by mild oxidation of the KIO ₄ or NaIO ₄ aldehyde group or by conjugating them to the antibody (immunoglobulin) using bifunctional agents. as described above. In this case, it is necessary to use a polymer precursor with a higher content of reactive ONp and hence hydrazide groups than in the preparation of a specifically non-directional polymeric drug.

The invention is applicable to the preparation of polymeric drugs useful for the treatment of various types of cancer in human medicine.

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Overview of pictures

Giant. 1 represents the structure of a linear polymeric drug conjugate. It is a non-directed polymer drug. For biological experiments, a conjugate with x = 94 mol%, a == 3 mol%, b) = 2 mol%, c = 1 mol% and X- = GlyPheLeuGly or GlyGly was preferably used.

Giant. 2 is a schematic of the preparation and structure of branched high molecular weight conjugates with biodegradable oligopeptide linkages. A suitable molecular weight of the conjugate is 120,000 g / mol. Crosslinking content 0.5 mol%, DOX content 9 wt%, hydrazone group content 2 mol%. and carboxyls 2 mol%.

Giant. 3 depicts the general structure of a conjugate comprising a targeting antibody. For biological experiments, a conjugate with x = * / 2 mol%, a = 3 mol%, b-d, 7 mol%, c = 2 mol%, d = 2.3 mol% was preferably used. and X = GlyPheLeuGly or GlyGly.

A A A š

Giant. 4 shows the structure of a polymer precursor.

Giant. 5 shows the results of measuring the release rate of doxorubicin from the conjugates of the invention differing in spacer composition; A) at the pH modeling environment of endosomes (pH 5); B) at pH modeling blood pH (7.4). The drug content of the conjugates was 10% by weight.

Giant. 6 depicts the results of the in vivo anti-tumor activity of the conjugates of the invention containing hydrolytically released doxorubicin (protective regimen). EL4 T cell lymphoma: 10 ⁵ cells / mouse, sc

DETAILED DESCRIPTION OF THE INVENTION

Example 1.

Preparation of non-directed linear polymer drug based on doxorubicin

Polymer drug synthesis was performed in four synthetic steps. In the first step, the monomers N- (2-hydroxypropyl) methacrylamide (HPMA) and 4-nitrophenyl esters of methacryloylated oligopeptide (MA-X-ONp) were prepared. The second step was the preparation of polymer precursor I by radical copolymerization of HPMA with MA-X-ONp. In the third step, polymeric precursor II was prepared by hydrazinolysis of precursor I, and in the last step DOX was coupled to the polymeric precursor.

Preparation of monomers:

t ';

AND

HPMA was prepared by the previously described method [Ulbrich 2000]. Methacryloylated reactive esters were prepared by methacryloying the appropriate oligopeptide (X = diGly, GlyLeuGly, GlyPheGly, GlyPheLeuGly, etc.) by a Schotten-Bauman reaction followed by reaction of the resulting methacrylic acid derivative with 4-nitrophenol hexane in the presence of d-nitro-cyclohexyl).

The methacryloylated derivatives of GlyLeuGly, GlyPheGly, GlyPheLeuGly and GlyLeuPheGly were prepared by methacryloying the oligopeptide as described in [Ulbrich 2000, Subr 1992]. The synthesis is similar for derivatives of all said oligopeptides, therefore only an example for the synthesis of Gly-Gly-Gly.

MA-GlyLeuGly-ONp: Dissolve 0.024 mol (5.88 g) of the tripeptide HGlyLeuGly-OH in a 15 ml solution of 0.024 mol (0.97 g) of sodium hydroxide in a three-necked flask. To the aqueous solution of sodium amino acid salt was added dropwise, with stirring and cooling (5 ° C), 0.024 mol (2.55 g) of methacryloyl chloride and a solution of 0.024 mol (0.97 g) of sodium hydroxide dissolved in 10 ml of water. During the reaction, the pH of the reaction mixture must not exceed pH 9. The reaction mixture was stirred for 1 hour and after acidifying the reaction mixture to pH 2, the methacryloylated tripeptide Ma = GlyLeuGly-OH was extracted into ethyl acetate and isolated by crystallization. The product was purified by crystallization from ethanol-water.

The 4-nitrophenyl ester was prepared by reacting 0.014 mol (4.40 g) of N-methacryloyl glycylleucylglycine with 0.014 mol (1.93 g) of 4-nitrophenol in tetrahydrofuran at -10 ° C in the presence of 0.0153 mol (3.16 g) of dicyclohexylcarbodiimide. The reaction was allowed to proceed for 10 hours, then the precipitated dicyclohexylurea was filtered off and the product purified by crystallization from acetone-water.

Preparation of polymer precursors I: 1 g of a mixture of HPMA (95 mol%, --0.89 g) and MA-GlyLeuGlyONp (5% hiol, 0.157 g) and 0.048 g of azo-bis-isobutyronitrile were dissolved in 6.95 g of acetone and

Λ The solution is put into a polymerization ampoule. After purging the polymerization mixture with nitrogen, the vial was sealed and polymerization was carried out at 50 ° C for 24 hours. The precipitated polymer was filtered off, washed three times with acetone, diethyl ether and dried under vacuum. The content of the side chains terminated by reactive -ONp groups (copolymer composition) can be controlled by the composition of the polymerization feed (monomer ratio). In the same way, all polymer precursors differing in side chain composition were prepared.

Preparation of Polymer Precursors II: Poly (HPMA-co-MA-GlyLeuGly-NHNH2) copolymer was prepared by reacting polymer precursors I with shydrazine hydrate. 300 mg of polymer precursor I (1.3 x 10 ^-4 mol ONp) was dissolved in 2 ml methanol and a solution of 69 mg NH 2 NH 2 H 2 O in 1 ml methanol (1.3V 10 ^-3 mol, 10 µl, was added with vigorous stirring). fold over ONp). The reaction mixture was allowed to react for 3 hours, then the methanol solution was diluted to 30 ml with distilled water and the product was freed of low molecular weight components by dialysis against distilled water (2 days). The final product was isolated from the aqueous solution by lyophilization. The molecular weight (Mw) and the polydispersity of the polymer were determined by gel chromatography equipped with a light scattering detector. The amount of hydrazide groups was determined spectrally after reaction of the hydrazide groups with TNBS (trinitrobenzenesulfonic acid). Precursors containing other amino acids and oligopeptides were prepared in the same manner.

Alternatively, polymeric precursors II were also prepared by radical precipitation copolymerization of the corresponding N-tert. methacryloylated oligopeptide butyloxycarbonylhydrazide (poly (HPMA-co-MA-X-NHNH-BOC)) with HPMA under the conditions described above. MA-X-NHNH-BOC monomers (X-GlyLeuGly, GlyPheLeuGly and GlyPheGly) were prepared from the corresponding methacryloylated oligopeptide (acid) by reaction with hydrazine hydrate using dicyclohexylcarbodiimide as usual in peptide synthesis. BOC groups of poly (HPMA-co-MA-X-NHNH-BOC precipitated into acetone-diethyl ether and the BOC protecting group was removed from the polymer with trifluoroacetic acid. The resulting polymer precursor II was dissolved in water, dialysed against water and lyophilized.

Binding of doxorubicin to polymeric precursor: 250 mg of polymeric precursor II (poly (HPMA-co-MA-GlyLeuGly-NHNH ₂ ) (0.1 mmol NHNH ₂ ) was dissolved in 3 ml methanol. The thus prepared polymer solution was added to 23 mg DOX HCl (40 pmol) 2 drops of acetic acid were added to the reaction mixture, and this inhomogeneous solution was stirred at room temperature (in the dark) After 48 hours of reaction, the homogeneous solution was purified twice by gel filtration from free DOX on a column filled with Sephadex LH- The polymer fraction was isolated, concentrated on a vacuum evaporator, and the product precipitated into diethyl ether.The product was precipitated from methanol into diethyl ether and dried to constant weight The DOX content was determined spectrally (λ 480 nm, ε 11,500 L mol ^-1 cm) ^1, water) and the content of hydrazide groups with TNBS method. _<Mw> and molecular weight distribution were determined by liquid chromatography (Superose ™ 6 column (3Ο (| χίθ mm), 0.3 ^ Μ acetates buffer (CH ₃ COONa / CH ₃ COOH; pH 6.5; 0.5 g / L NaN _3), flow rate 0.5 ml / min, detection by differential refractometer II, light dispersion detector (DAWN-DSP-F, Wyatt Technology , USA) and a UV detector (280 and 488 nm).

This procedure was used to prepare all other polymeric conjugates of doxorubicin.

Example 2.

Preparation of branched high molecular weight conjugate.

400 mg of polymeric precursors I containing the oligopeptide sequences GlyPheGly, GlyLeuGly or GlyPheLeuGly (0.19 mmol ONp) were dissolved in 1.1 ml DMSO. With vigorous stirring of the polymer solution, 105 μΐ of a 0.2 M solution of 1,2-ethylenediamine in DMSO (21 μιηοΐ EDA) was added and the reaction mixture was allowed to react at room temperature for 1 hour. While vigorously stirring the polymer solution, a solution of 100 mg hydrazine hydrate (2 mmol) in 0.3 mL DMSO was added and the reaction mixture was stirred vigorously for a further 3 hours. Then the reaction mixture was diluted to 30 ml with distilled water and the product was purified by dialysis against distilled water (2 days). The high molecular weight precursor was lyophilized. <M _W > and molecular weight distribution was determined by liquid chromatography, the hydrazide group content was determined spectrally by the TNBS method. Doxorubicin was bound to the polymer and the conjugate characterized under the conditions described in Example 1.

Example 3.

Preparation of antibody (immunoglobulin) -conjugated conjugate I

By partially modifying the hydrazide groups of polymeric precursors II containing 10 to 15 mol% hydrazide groups with an X-hydroxysuccinimidyl ester of maleimido-propionic acid (SMP), a copolymer bearing in the side chains of the hydrazide and maleimide (MI) (poly (HPMA-co-MA-X) -NHNH 2 -co-MA-XNHNH-MI)). 250 mg of poly (HPMA-co-MA-diGly-NHNH ₂ (125 pmol hydrazide groups) precursors II) was dissolved in 1.2 ml DMSO and a solution of 16.6 mg SMP (62.5 pmol) in 0.6 ml DMSO was added. The reaction mixture was stirred at room temperature for 8 hours After purification on a PD-10 column (eluent: distilled water), the <M _W > and the molecular weight distribution of the conjugate were determined by liquid chromatography,

Maleimide groups were determined by a modified Ellman assay and the content of the remaining hydrazide groups by the TNBS method. Doxorubicin was bound to this polymer in the same manner as described in Example 1.

Introduction of -SH groups into antibody (immunoglobulin) molecule: 15 mg of antibodies (polyclonal anti-thymocyte IgG) (0.1 pmol) were transferred to a 1 ml phosphate buffer (0.05 µM NaH ₂ PO ₄ / Na ₂ HPO ₄ ; 0.1 M / NaCl; 0.01 EDTA; pH 7.4). 0.67 mg of 2iminothiolane (4 pmol) was dissolved in 50 µL of DMF and added to the vortex antibody solution. After 2.5 hours of stirring at room temperature, the product was isolated by gel filtration on a PD-10 column (phosphate buffer, pH 7.4). The degree of antibody (immunoglobulin) modification was determined by a method using a reaction of sulfhydryl groups with an Ellman reagent.

Conjugation of 60 mg of poly (HPMA-co-MA-diGly-NHN = DOX-co-MA-X-NHNH2-coΜΑ-Χ-ΝΉΝΗ-ΜΙ containing doxorubicin, hydrazide and maleimide groups with 20 mg of modified antibodies was performed in phosphate buffer pH 7.4 (see above) at room temperature The final product was purified by gel filtration and characterized by UV spectroscopy (DOX content), amino acid analysis (antibody content, immunoglobulin) and Phastsystem - Pharmacia LKB electrophoresis (presence of free protein or drug).

Example 4.

Preparation of antibody (immunoglobulin)-directed conjugate II.

Polymeric precursor II was modified in the first step of SPDP synthesis under similar conditions to the polymer modification by SMP described in Example 3

To 14 mg -SH group of modified immunoglobulin antibodies (see above) containing 0.68 pmol SH groups dissolved in 1.7 ml phosphate buffer (0.05 M NaH ₂ PO 4 / Na ₂ HPO 4; 0.1 µM NaCl; 0.01 EDTA; pH 7.4) a solution of 42 mg of poly (HPMA-co-MA-diGly-NHN = DOX-co-MA-diGly-NHNH2-co-MA-diGlyNHNH-PD (containing 3.3 pmol) was added with stirring The reaction mixture was stirred for 7 hours at room temperature, after which time the low molecular weight constituents and unbound polymer were removed from the conjugate by gel filtration, then the conjugate was concentrated by ultrafiltration and characterized similarly as described in of Example 3.

Example 5.

Preparation of antibody (immunoglobulin)-directed conjugate III.

These conjugates were prepared by the reaction of antibodies in which the aldehyde groups were introduced by mild oxidation of sodium periodate with the hydrazide groups remaining after the reaction of polymer precursors II with doxorubicin.

Preparation of oxidized antibodies): 40 mg of antibodies (polyclonal anti-thymocyte IgG) were transferred to a PD-10 column in acetate buffer (0.02 M CH ₃ COONa / CH ₃ COOH; 0.15 M NaCl; pH 5). A 0.1 M NaICL solution was prepared in the same buffer in the dark. Antibody and periodate solutions were mixed in a 4: 1 ratio so that the final NaICL concentration was 0.02 mol / l and the antibody concentration was 9 mg / ml. The reaction mixture was stirred at room temperature in the dark for two hours and then 25 μΐ of ethylene glycol was added for each ml of the reaction mixture. After 20 minutes, the oxidized antibodies (immunoglobulin) were purified and isolated by liquid chromatography on a PD-10 column. The concentration of antibodies (immunoglobulin) in the solution was determined spectrophotometrically. The number of aldehyde groups was determined by a method employing the reaction of aldehyde groups with the dye Lucifer Yellow CH.

Example of preparation of conjugate sample: 36.5 mg of oxidized antibodies in 5 ml of acetate buffer (0.02 M CH ₃ COONa / CH ₃ COOH; 0.15 M NaCl; pH 5) was cooled to 15 ° C with cooling and mixed with the solution 73 mg of polymer precursor II poly (HPMA-co-MAdiGly-NHN = DOX-co-MA-diGly-NHNH ₂ with DOX bound dissolved in 1 ml of buffer. The reaction mixture was stirred in the dark for 16 hours at 15 ° C. The pH of the reaction mixture was adjusted to 0.1 (M NaOH to pH 7.2). Low molecular weight fractions and unbound polymer were removed from the conjugate by gel filtration, then the conjugate was concentrated and characterized similarly to other antibody conjugates (immunoglobulin).

Human and rabbit non-specific immunoglobulin (IgG), polyclonal anti-thymocyte antibodies (ATG), anti-Thy 1.2 monoclonal antibodies, anti-CD4, anti-CD 71, anti-BCL1, anti-CD4, anti- CD8, anti-CD14, and other antibodies against tumor-associated antigens (anti-TAA).

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Example 6.

In vitro release of doxorubicin from polymer conjugates

The hydrolytic stability of the conjugates and the rate of release of the active drug from the polymer were measured in model systems at various pH and temperatures. Fig. 5 shows the results of measuring the release rates of doxorubicin from polymers differing in the composition of the spacer. at a pH modeling the blood pH (7.4) and pH modeling the endosome or lysosomes (pH _5? 6). In experiments, the concentration of bound DOX was constant (0.5 mmol.l ^-1 ) in 0.1 µM acetate buffer (CH ₃ COONa / CH ₃ COOH, 0.05 M NaCl). Samples were incubated in the dark at 37 ° C and at a predetermined time interval, 0.1 ml aliquots of samples were taken and analyzed by HPLC after extracting DOX from the solution. From the results, it is apparent that the structure of the link between the drug and the polymeric carrier affects the rate of drug release from the carrier, the rate of release being relatively low at blood pH and more than an order of magnitude faster at pH modeling cell environment. Considering that the residence time of the drug in the bloodstream is relatively short, the experiment confirms the relative stability of the polymer conjugate during transport and its ability to release the active drug only after penetration into the cells.

Example 7.

Cytotoxic activity of in vitro polymer conjugates containing hydrolytically released doxorubicin in cells with normal lysosome content (Table 1)

The ability of polymer conjugates to reduce cell division or proliferation of target cells is referred to as cytotoxic or cytostatic activity. This cytotoxic activity was detected in vitro by inhibiting the incorporation of ³ H-thymidine into target cell nuclei [Říhová et al., 2000], Thymidine incorporation and conjugate activity are inversely proportional. Low thymidine incorporation means high cytotoxic activity of test substances. Cells of selected tumor lines (murine T cell leukemia EL4, murine B cell leukemia BCL1, murine B cell lymphoma 38C13, human colorectal carcinoma primary SW 480, human colorectal carcinoma metastatic SW 620, human colorectal carcinoma SW620 genetically modified with the mouse Thy 1.2 gene (SW620 / T) and T cell mitogen, ie concanavalin A stimulated mouse splenocytes and human peripheral lymphocytes were at doses of 1 x 10 ⁴ - 5 x 10 ⁵ (depending on test cell systems used) in growth RPMI 1640 medium was pipetted into 96-well FB-tissue plates (NUNC, Denmark). Further cultivation was carried out either without further stimulation (all tumor lines and unaffected mice and human lymphocytes) or in the presence of concanavalin A T cell mitogen as far as the effect on normal intensively proliferating T cells was investigated. The concanavalin A concentration at which the cells were cultured was 1.25 pg / well. Test samples or free doxorubicin at final concentrations of 0.0016 / 80 pg / ml were added to each experimental well of the tissue plate containing test cells in medium with or without concanavalin A. The total well volume was 250 µl. Each concentration was tested in parallel on one sample and one line three to five times. Microtiter tissue plates were incubated at 37 ° C in 5% CO 2 for 24-72 hours. After cultivation, 1 µCi of ³ H-thymidine was added to each well. After another five to six hours (depending on the test system), cells were harvested (Tomtec cell collector) on a glass fiber filter (Filtermat, Wallac), dried, and evaluated for incorporated radioactivity (MicroBeta Trilux, Wallac). The IC 50 for non-directed conjugates containing hydrolytically released doxorubicin ranged from 0.01 µg doxorubicin / ml to 1.41 µg doxorubicin / ml depending on the resistance (sensitivity of the target line used to therapy) and the type of conjugate. Cytotoxicity has been confirmed to be an exclusive property of conjugates containing hydrolytically released doxorubicin since the drug-free conjugate is not cytotoxic.

In vitro assays have shown that a) the activity of the hydrolytically releasable drug conjugates does not depend on the enzymatic degradability of the covalent bond between the drug and the oligopeptide linker.

Table 1. Comparison of In vitro Inhibitory Activities of Samples Containing Drug-Bound Different Couplers; EL4 mouse leukemia test line (EL4 T cell lymphoma).

Sample IC50 (pgDOX / ml) P-GFLG-DOX 19.1 P-GG-DOX (Hydrazone) 0.40 P-GFLG-DOX (hydrazone) 0.26 Doxorubicin (DOX) 0.02

IChe drug concentration causing 50% inhibition of proliferation.

Example 8.

Cytotoxic activity of in vitro polymer conjugates containing hydrolytically released doxorubicin in low lysosome-containing cells

The in vitro cytotoxic activity assay was performed as described in Example 7. Low lysosome erythroleukemic lineage K 562 cells were selected as target cells. Conjugates containing hydrolytically released doxorubicin have been shown to be significantly cytotoxic even in this line where cytotoxic conjugates with enzymatically released doxorubicin are very limited (Table 2). This result demonstrates that conjugates with hydrolytically released doxorubicin are also effective in lysosome-poor cell systems.

Table 2. Comparison of In vitro Inhibitory Activities of Samples Containing Drug-Bound Different Couplers; erythroleukemic line K 562 tested.

Sample IC ₅₀ (pg DOX / ml) P-GFLG-DOX 9.71 P-GG-DOX (Hydrazone) 0.06 Doxorubicin (DOX) 0.28

ICs of the drug concentration causing 50% inhibition of proliferation.

Example 9.

Antitumor activity in vivo of polymer conjugates containing hydrolytically released doxorubicin (Fig. 6)

C57BL / 10 inbred mice were transplanted subcutaneously with T cell lymphoma (ELA). Tumor cells were injected into the lower back of experimental animals at a dose of 1 x 10 ⁵ cells / mouse. The day of cell transplantation was designated as day 0. Then they were '7 ··' ..:

polymer conjugates with hydrolytically released doxorubicin administered intraperitoneally on either days 1, 3, 5, 7, and 9 (protective regimen), on days 10, 12, 14, 16, and 18, or on days 12, 14, 16, 18, and 20 (therapeutic regime). The daily dose of doxorubicin was 50 pg / mouse. The conjugates significantly suppressed tumor growth and increased the number of long-term surviving experimental animals. While the drug-free polymer conjugate was completely ineffective and the classical ie unmodified doxorubicin had very limited effect (the maximum survival time of the experimental animals was 40 days versus 35 days in the control group), the administration of polymer conjugates with hydrolytically released doxorubicin allowed than 80 days in the protective regimen in 40% of the mice, in the therapeutic regimen in 20% of the mice.

/ 'Λ Λ 4

Balb / c inbred mice were ip transplanted with BCL1 mouse B cell leukemia. Tumor cells were injected at a dose of 5 x 10 ⁵ cells / mouse. The transplant day was designated as day 0. Thereafter, the polymer conjugates with hydrolytically released doxorubicin were administered intravenously on days 11, 13 and 17 (therapeutic regimen). The daily dose of doxorubicin was 100 pg / mouse. The conjugates tested had significant anti-tumor activity. While control mice survived for a maximum of 40 days, 20% of the mice treated with the test conjugates survived for more than 100 days and 10% for more than 140 days.

Literature

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Claims (11)

PATENT CLAIMS 1. Conjugates consisting of a polymeric carrier consisting of 30 to 3,000 monomer units linked to a polymer chain, consisting of (a) 60 to 98.5% units of N- (2-hydroxypropyl) methacrylamide b) 1 to 25% units of methacryloylated hydrazones of oligopeptides terminated by anthracycline cancerostatic molecule (c) 0.5 to 15% units of methacryloylated oligopeptides or their sodium salts, and optionally d) 0.5 to 10% units of methacryloylated hydrazide oligopeptides. The conjugates of claim 1, further comprising 0.5 to 5% of methacryloylated oligopeptides terminated with an immunoglobulin molecule or a specific monoclonal or polyclonal antibody. Conjugates according to claim 1 or 2, further comprising 0.1 to 5% units of enzymatically degradable methacryloylated oligopeptides linked by alkylenediamines, which form a bivalent linker between the individual polymer chains, thereby forming a branched structure of the conjugate. The conjugates of claim 1, 2 or 3, wherein the anthracycline cancerostatic is doxorubicin. The conjugate of claim 1, wherein the individual structural units are randomly arranged in the chain and wherein X is a suitable linker formed by an oligopeptide residue and x is from 20 to 3000, and is from 1 to 750, b and c are from 1 to 450. A conjugate according to claim 2 having the formula CH ₃ CH _{2 -} c · c = o X CH, CH, C = 0 AND X COOH nh ₂ AND NH AND Al 1 -.-. 0-Λ.,. fkcMX, x, a, b and c are as defined above and have a value of 1 to 150, S is either an antibody selected from the following series: γ-globulin for iv, administration, autologous antibodies, anti-17-ΙA antibodies, anti- CA 15-3 et al., Attached to a polymer chain (indicated by the structure in Mrc, a, _t or S means a biodegradable oligopeptide linker by which other similar chains are attached). Conjugates according to any one of the preceding claims, wherein the oligopetides used are selected from the series > GlyGly, GlyLeuGly, GlyPheGly, GlyPheLeuGly and GlyLeuPheGly. Conjugates according to claim 2 or 6, wherein antibodies selected from the γ-globulin series for i.v. applications, autologous antibodies, anti-17-ΙΑ antibodies, anti-CA 15-3, and other tumor-specific and tumor-associated antibodies. The conjugates of claim 3, wherein the bivalent linear chain linking units are selected from the series GlyPheLeuGly, GlyLeuGly, GlyPheGly, GlyLeuPheGly. Pharmaceutical composition for the treatment of cancer, characterized in that it comprises as an active ingredient a conjugate according to any one of the preceding claims. Pharmaceutical composition according to claim 10, for the treatment of tumors, such as a breast tumor or a colorectal tumor.