CZ278551B6 - Polymeric medicament, process for preparing thereof and pharmaceutical compositions based thereon - Google Patents

Abstract

The polymeric medication consists of an inert synthetic carrier connected with a molecule having biological activity and a specific determinate with the use of amino acid or peptide spacers and this connection is allowed to contain the transversal bonds. The nested chains of the specified polymer can be created based on the connection using the transversal bonds. The polymeric units closed to N-(2-hydroxypropyl)- metakrylamide, 55 up to 99,7 molar percent are used as the polymeric carrier. Monosaccharide or oligosaccharide linked based on the amide bond to the peptide spacer is suitable to be used as the determinant. The polymeric medication can be prepared based on this principle, that N-(2-hydroxipropyl)metakrylamide co-polymerisation is done either with N-metakryloyl peptide or with p-nitrophenyl ester of that, while the final co-polymer is undertaken to the reaction with a reactive bio-active molecule, reactive determinant or the diamide nesting facility, while the ester or amide bonds are created. The advantage of this medication is a controlled intracellular unlocking in the ai

Description

The invention relates to polymeric medicaments comprising an inert synthetic polymeric carrier coupled by means of a biodegradable spacer with a low molecular weight bioactive molecule and to a process for their preparation.

Numerous low-molecular-weight drugs used in chemotherapy rapidly penetrate various cell types by accidental diffusion through the cell membrane. This lack of selectivity reduces their applicability to the desired target tissue and sometimes causes undesirable side effects. The capture in the cell is rapid, so the therapeutic effect does not last long. In addition, the drug can be rapidly removed from the bloodstream by glomerular filtration.

Covalent attachment of low molecular weight bioactive molecules to soluble polymeric carriers prevents both glomerular filtration and cell penetration by simple diffusion. The capture is limited to cells capable of absorbing substrates by a selective mechanism known as pinocytosis, wherein the cell-flanking portion of the membrane is entrapped inside the cell, absorbing the macromolecule. Then, a portion of the cell membrane is detached inwardly to form an intracellular organelle containing the entrapped material.

This difference in the capture mechanism provides a potential method to allow intracellular space penetration. Small molecules that enter cells due to diffusion tend to penetrate all parts of the cell, while macromolecules after pinocytosis are transported in intracellular organelles (pinosomes) directly to the lysosomal compartment where a number of hydrolytic enzymes are found.

Thus, the pinocytic capture of a polymeric drug that contains a drug-carrier linkage that is subject to lysosomal hydrolysis provides a mechanism for the controlled intracellular release of a bioactive molecule that results in its appearance within the cytoplasm of the target cell. Theoretical considerations leading to the design of such a drug system have recently been reviewed in J. Kopeček's Synthesis of tailor-made soluble polymeric carriers in Recent Advances in Drug Delivery Systems (Plenum Press, 1984).

Two criteria must be met when designing such a system. First, it is desirable to design a link between a drug and a carrier capable of being subjected to controlled lysosomal hydrolysis, but at the same time stable to the action of enzymes in the bloodstream. Secondly, the polymeric drug must be capable of specifically capturing in these target cells, where a therapeutic effect is desired, with simultaneous minimal capture in other cells.

Although the pinocytic process provides a degree of selectivity towards macromolecules that can be optimized by molecular weight change, a higher target selectivity can be achieved by incorporating a specific determinant into the molecule. Cells contain specific receptors and antigens on their surface that recognize and interact with certain molecules or portions thereof, known as specific determinants. High cellular specificities can be achieved by incorporating into the polymeric drug a determinant that is recognized by the type of cells for which a therapeutic effect is desired.

-1GB 278551 B6

This means that a controlled drug delivery system that allows a specific target effect, followed by intracellular drug release, must have the following features:

(a) it must contain a polymeric carrier which is preferably subjected to lysosomal hydrolysis to facilitate secretion of the polymer from the body; (b) it must contain a degradable drug-carrier linkage resistant to extracellular hydrolysis but subject to controlled lysosomal hydrolysis; specific determinant.

Such a molecule can be represented schematically as follows:

wherein the specific determinant of the drug attached by the biodegradable bond is an optional biodegradable crosslink in the polymeric carrier

Although natural macromolecules have been used as carriers, synthetic polymers provide the advantages that the molecular weight is more easily adapted to achieve optimal cell selectivity and that, unlike many natural macromolecules, they are non-ionic. They are also easier to prepare on a commercial scale.

Synthetic polymers based on N- (2-hydroxypropyl) methacrylamide (HPMA) have been suggested as potential drug carriers, see US Pat. Nos. 4,062,831 and 4,097,470; these polymers are soluble in aqueous media and have good biocompatibility. In addition, due to the incorporation of p-nitrophenyl esters, N-methacryloyloligopeptides, they can be combined with many drugs containing primary

-2C 278551 B6 amino. Polymeric. the chains may be propagated below the gelation point to achieve optimal molecular weight and, due to the presence of biodegradable cross-links, to provide a method of degrading the polymer to facilitate its secretion from the body.

Since lysosomal enzymes contain a number of proteinases capable of hydrolyzing peptide bonds, it would appear that direct attachment of the bioactive molecule to the polymer chain via an amidic linkage would provide the possibility of lysosomal hydrolysis. However, this is not the case in practice. However, peptide spacers located between the drug and the carrier have been found to be subject to degradation by the action of lysosomal enzymes over a wide range of rates. The bond that cleaves is usually the bond between the drug and the adjacent amino acid, although this is not always the case. Hydrolysis, i.e., the rate of drug release, appears to depend greatly on the number and nature of the amino acid residues in the peptide spacer. In general, spacers containing less than three amino acids have not undergone lysosomal hydrolysis. Peptide spacers, designed to correspond to the known substrate specificity of thiol proteinases, which are known to be contained in lysosomes, are cleaved particularly efficiently.

Furthermore, it has been shown that modification of glycoproteins leading to the formation of galactose-terminated oligosaccharide side chains results in a sharp increase in glycoprotein deposition in parenchymal liver cells. The galactose unit acts as a specific determinant that reacts with receptors located on the plasma membrane of liver cells. This circumstance offers a potential mechanism for targeting drugs in the case of hepatoma, a type of cancer that is particularly difficult to treat. In addition, galactosamine, coupled to HPMA copolymers via an amidic bond, provides a similar effect and thus suggests that receptors on hepatocyte membranes recognize galactose units not only when bound by glycosidic linkage, but also when these units are present as N-acylgalactosamine. A number of other recognition systems are known, such as the N-acetylglucosamine / mannose recognition system of Kupfer cells and macrophages, and the phosphohexose fibroplastic recognition system.

Another possible targeting mechanism is to bind the polymeric drug to an antibody that is specifically recognized by cells having the corresponding membrane antigens. Drug molecules have also been linked directly to immunoglobulin, but this can lead to loss of drug activity, loss of antibody activity, or solubility of the conjugate. The introduction of a polymeric intermediate chain between drug and polymer has been shown to greatly simplify these problems.

Another mechanism of targeting agents is based on the binding of a protein or hormone to a polymer, for example, transferrin or a melanocyte stimulating hormone that binds specifically to the target cell type.

While the need for the synthesis of targeted polymer drugs with hydrolyzable peptide spacers has been reported earlier (see Kopecka, cited previously), peptide spacers capable of controlled intracellular drug release at appropriate rates have eluded the attention of researchers.

As mentioned previously, the rate of lysosomal hydrolysis of pep

The amount of acidic spacer depends on the number and nature of the amino acid residues. This reflects both spatial and structural facts

The rate of terminal hydrolysis of a spacer containing 2-4 amino acid residues generally depends on the number of residues present, which is attributed to the spatial interaction between the polymer chain and the enzyme. For a peptide of given length, the rate of hydrolysis depends on the nature (and sequence) of the amino acid residues. This dependence is due to the substrate specificity of the lysosomal enzymes responsible for cleavage of the peptide spacer, and the part of the enzyme where the interaction with the substrate takes place is known as the active site of the enzyme. The active site plays a dual role - it binds the substrate and at the same time catalysis is a reaction, such as cleavage. Investigation of structures of proteolytic enzyme complexes with peptides shows that the active site of these enzymes is relatively large and is bound to several amino acid residues in the peptide.

Thus, the degradability of an individual bond in the peptide chain depends not only on the nature of the structure near the cleaved bond, but also on the nature of the amino acid residues relatively distant from the cleaved bond, but playing an important role in maintaining the enzyme at a certain position during hydrolysis. The detailed structure of lysosomal enzyme active sites has not yet been determined, which has been shown to hinder the preparation of peptide spacers that undergo lysosomal hydrolysis at an appropriate rate and are intended for use in polymeric drugs.

The invention is based on the discovery of such peptide sequences which may act as spacers capable of lysosomal hydrolysis at a satisfactory rate so as to allow controlled intracellular drug release but which remain resistant to extracellular hydrolysis, and may also be used to bind specific determinants. In addition, systems have been developed to facilitate the cleavage of the basic polymeric carrier. Biodegradability is achieved by introducing oligopeptide sequences containing biodegradable linkages into the polymer backbone. Hydrolysis of these bonds is particularly sensitive to steric hindrance caused by polymer chains, rather than hydrolysis of terminal units from peptide side chains.

The present invention provides a polymeric drug consisting of an inert synthetic, polymeric carrier based on repeating units of (N - (2-hydroxypropyl) methacrylamide, which is linked to a biologically active molecule by amino acid or peptide spacer and optionally linked by the same or other spacer with a targeting unit and optionally crosslinked, characterized in that it consists of (a) 5.0 to 99.7 mol% units derived from N- (2-hydroxypropyl) methacrylamide of the formula

OH ch ₃ - C - CO - NH - ch ₂ -CH-ch ₃ ,

(B) 0.2 to 20.0 mol% units derived from N-methacryloylated peptide of the formula

CH ₉

II ch ₃ - C - CO - [NH - R - CO] - [B] where - [NH-R-CO] - is a residue derived from Gly-Gly, Gly-Phe-Gly, GlyPhe-Phe, Gly- Gly-Phe-Ala, Gly-Phe-Ala, Gly-Phe-Ala, Gly-Phe-Tyr, Gly-Phe-Tyr, Gly-Phe-Tyr Gly-Phe-Gly-Phe, Gly-Phe-Gly-Phe, Gly-Phe-Gly-Phe, Gly-Phe-Gly-Phe, Gly-Phe-Gly-Phe Gly-Gly-Phe-LeuGly-Phe, α - [B] is a biocative molecule, and (c) 0.1 to 94.8 mol% units of the formula

ch ₃ ch ₀ II - c - what - NH - [D] or CH ₀ 11 ch ₃ II - c - what - [D] or ch ₉ II ch ₃ - c - what - [NH - R = CO] -D

where - [D] is a determinant and - [NH-R-CO] - is a residue derived from Leu, Phe, Gly-Gly, Gly-Leu-Gly, Gly-Val-Ala, Gly-Phe-Ala, Gly- Gly-Phe-Leu-Gly, Gly-Phe-Leu-Gly, Gly-Phe-Leu-Gly, Gly-Phe-Tyr-Ala, Gly-Phe-Tyr-Ala, Gly-Phe- Gly-Phe, Gly-Phe-Le-Gly-Phe, Gly-Phe-Le-Gly-Phe, Gly-Phe-Le-Gly-Phe.

The polymeric drug of the invention may further comprise (d) up to 5 mol% units of the formula

CH ₃ - C - CO - [NH - R - CO] - [L] - [CO - R - NH] - CO - C - CH ₃ where - [NH-R-CO] is a residue derived from Gly-Leu -Gly, Gly-Phe-Ala, Gly-Phe-Ala, Gly-Phu-Leu, Gly-Phu-Leu, Gly-Phu-Leu, Gly-Phu-Leu, Gly-Phu-Leu -Leu-Gly-Gly-Phe-Tyr-Ala, Gly-Phe-Gly-Phe, Gly-Phe-Phe-Gly, Gly-Phe-Leu-Gly-Phe and Gly-Gly-Phe -Leu-Gly-Phe and - [L] - is a linker, and may further comprise

(E) as radiolabelled units up to 2 mol% of the units of the formula

NH ₉

AND

CH, CO

II ² I

CH ₃ - C - CO - NH - CH - CH ₂ - C ₆ H ₄ - OH

Not all combinations of the oligopeptide sequences mentioned under (b) and biologically active molecules or drug molecules undergo intracellular lysosomal hydrolysis; for example, as shown below, the combination of Gly-Gly with daunomycin is not subject to this hydrolysis and therefore Gly-Gly should not be used as a peptide spacer with daunomycin. However, Gly-Gly is an acceptable spacer for other drugs, for example, for deacetylcolchicine and colcemid.

As shown in connection with (c), the determinant may be attached directly to the polymer chain either by an amide or ester linkage, i.e. without a spacer, or may be linked via an amino acid or peptide spacer. The considerations affecting the choice of binding to be used for a particular determinant are complex and depend, inter alia, on the nature and number of drug-bearing units, or on whether the polymer has a single chain or is branched and of course the determinant. The predominant requirement is that the determinant be available for specific receptors on targeted cells, which is largely a function of the geometry of the polymer drug molecule.

The biologically active molecule present in the polymeric drug is primarily an anticancer agent, an antimicrobial agent, an anti-parasitic agent, an anti-inflammatory agent, a cardiovascular drug or a drug having an effect on the nervous system. Particularly preferred are, for example, daunomycin, puromycin, adriamycin, melphalan isopropyl ester (sarcolysin), bleomycin, desferioxamine, bestatin and tritylcysteine.

In particular, the determinant is a monosaccharide, disaccharide, oligosaccharide or O-methacryloylated carbohydrate unit, which are preferably bound by an amide bond, antibodies such as IgG (rat immunoglobulin) and an anti-O antibody, or a protein such as transferrin or melanocyte stimulating hormone (MSH). . Particularly suitable determinants are galactose, galactosamine, glucosamine, mannosamine and fucosylamine.

The L group forming the linker between the polymer chains preferably has an A-type composition. ΝΗ (Οι. _{Ζ] χ} -NH-A -, wherein A is an amino acid and x is an integer of 1 to 12, and is connected to the adjacent peptidic spacers amide-bonds. Particularly preferred linking groups are those in which x is 6 and the amino acid A is Phe, Tyr, Ala, Gly or Leu, and those in which x is 2 and amino acid A is Ala.

A particularly preferred embodiment of the invention comprises single or cross-linked chains of the specified polymer with daunomycin as

The bioactive molecule, galactose, galactosamine, or fucosylamine as determinant, Gly-Phe-Leu-Gly as the peptide spacer between the polymeric carrier and both daunomycin and galactosamine, and optionally with Gly-Phe-Leu-Gly as the peptide spacer and - (Phe) NH-fCH _{2 -} .gNH (Phe) - as the linking group.

The invention also encompasses the synthesis of polymeric drugs as set forth in the disclosure.

Since the same oligopeptidic spacer can be used for both the binding of a bioactive molecule and the determinant, and the preparation of these compounds is easier than those in which these spacers are of different kinds, we will first describe the procedure for synthesizing a polymeric carrier with the same spacer. In principle, the synthesis takes place in two stages.

The initial step involves the copolymerization of HPMA and the N-methacryloylated oligopeptide p-nitrophenyl ester (incorporation of N-methacryloyltyrosinamide is also possible to obtain a radiolabelled unit) to yield a polymeric precursor containing terminal p-nitrophenoxy end-cleavage groups on the peptide side chains. in the next addition reaction. The second step involves the sequential addition of a reactive drug, a reactive determinant, and optionally a diamine crosslinker. If the polymeric drug carrier is desired in the form of a single chain, the drug and the determinant are gradually added to the polymeric precursor in any order. If a cross-linked product is desired, then a suitable cross-linking agent, drug, and determinant are added sequentially to the polymer precursor, again in any order.

The above procedure can be illustrated as follows:

HPMA + MA-AT-QNp

Copolymerization

ONp = p-nitrophenoxy

MA = methacryloyl (d) = drug determinant

-7EN 278551 B6

Single Chain Product í —νί— / Avv-®

© ΆΙΑ — i cross-linked © -44Λ— / product

In cases where the drug and the determinant do not have the same binding units, two synthetic procedures may be used.

For those cases where the drug and determinant binding groups contain common amino acid residues adjacent to the carrier, the polymer precursor containing these amino acids is sequentially reacted with the amino acid or peptidyl derivative of the determinant and optionally, if a crosslinked product is desired, the diamine networking agent.

1) AA] _ - ©

HPMA + MA ~ ^/ W ^at ONp

2) ΑΑ _χ -.

3) AA ₂ - ©

Single chain product

Cross-linked product

₂

1-AA-1

11A — AA-1 - ©

AA ₂ - ©

AA ^ —ΛΜ— a and ₂

Alternatively, a polymerizable form of a determinant can be prepared by reacting an N-methacryloyl oligopeptide containing the desired amino acid sequence with a determinant. The determinant thus modified is incorporated into the polymer precursor by copolymerization. The polymer precursor is then reacted with the reactive drug and optionally, when a crosslinked product is desired, with a diamine crosslinker.

-8EN 278551 B6

This procedure can be illustrated as follows.

HPMA + MA - ^ - ONp + ΜΑ-37} -Τ 'Copolymerization

IN

1) h ₂ N 1 | —nh ₂

2) ©

Single chain product

C / VH-pRA-®

-W-®

It is also possible to prepare polymeric precursors similar to those described above but containing peptide side chains terminated not by p-nitrophenyl ester groups but by carboxyl groups. In the presence of a suitable reagent, drugs and determinants can be attached to these polymeric precursors via terminal carboxyl groups; in this case, the hydroxyl rather than the p-nitrophenoxy group is released during the addition.

Drugs and determinants containing reactive amino groups provide products identical to those obtained from polymeric precursors with terminal p-nitrophenyl ester groups, i.e., the reactive molecule is bound to the side chain via an amide bond. However, polymeric precursors containing terminal carboxyl groups, unlike precursors containing terminal p-nitrophenyl ester groups, also react with drugs and determinants containing reactive hydroxy groups to produce products in which the reactive molecule is bound to the side chain via an ester bond.

Polymeric side chain precursors terminated with carboxyl groups can be prepared by (a) basic hydrolysis of the corresponding polymeric side chain precursors terminated with p-nitrophenyl ester groups as described above, (b) copolymerizing HPMA by any of the synthetic procedures described above for preparing polymeric precursors with terminal p-nitrophenyl ester groups but using an unesterified N-methacryloylated peptide as a comonomer. The resulting copolymer can then be reacted with a suitable drug and determinant.

. The methods by which polymeric side chain precursors terminated with carboxyl groups can be prepared can be illustrated as follows:

-9EN 278551 B6

ΗΡΜΑ + Ma-AV ^ -OH

Copolymerization (b)

ΜΑ-3ΤΓ-Τ — 4¼ — OH / - TD - 0

Suitable reagents for the addition of a reactive drug or determinant to polymeric precursors containing carboxyl groups include carbodiimides such as dicyclohexylcarbodiimide in organic solvents and N-cyclohexyl-N '- (2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate and aqueous solutions and Woodward's reagent, (N-ethyl-5-phenylisoxazolium-3'-sulfonate).

The order in which some or all of the reactive determinants of the reactive drug and the crosslinking agent are added to the polymer precursors using any of the methods described above is not critical. However, the amount of reactants used must be such as to provide the desired molar content of each component in the polymer within the range indicated above. The total molecular weight is determined by the polymerization conditions, particularly the concentration of the initiator, the presence of transporters, and the concentration of the comonomeric p-nitrophenoxy groups.

The single chain products of the invention preferably have a molecular weight in the range of 5,000 to 60,000, more preferably 20,000 to 40,000; crosslinked products preferably in the range of 10,000 to 500,000, more preferably 30,000 to 400,000.

The starting materials required to carry out the above syntheses are either commercially available or can be prepared by general methods known to those skilled in the art.

Thus, the peptide derivatives required for copolymerization with N- (2-hydroxypropyl) methacrylamide (HPMA) can be prepared and copolymerization can be carried out as follows:

(i) A commercially available dipeptide (containing the first two amino acid residues of the desired peptide spacer) is N-methacryloylated, as described, for example, in Macromol. Chem. 177, 2833 (1976). (N-methacryloylated tyrosinamide for radiolabeling purposes can be obtained in the same manner).

(ii) The N-methacryloylated dipeptide is then esterified with p-nitrophenol as described, for example, in Makromol Chem. 178, 2169

-10GB 278551 B6 (1977). (Methacryloylated p-nitrophenyl esters of Leu and Phe, providing amino acid spacers suitable for use as determinant, may be prepared in the same manner).

(iii) The peptide chain of the N-methacryloylated dipeptidyl-p-nitrophenyl ester is then extended (unless the dipeptide is not Gly-Gly, where the dipeptide itself is one of the spacer that can be used to bind determinants) by attaching an oligopeptide containing the necessary amino acid residues the desired peptide sequences, as described, for example, in Makromol. Chem. 182, 1917 (1981).

(iv) The N-methacrylated oligopeptide obtained from (iii) can be copolymerized with HPMA to form a polymeric side chain precursor containing carboxyl end groups, or it can be re-esterified with p-nitrophenol. In the latter case, the N-methacryloylated peptidyl-p-nitrophenyl ester may be copolymerized with HPMA as described, for example, in J. Polymer Sci., Polymer Symp. 66, 15 (1979), or if the drug and determinant are to have different spacer, it can be reacted with the determinant to form an M-methacryloylated oligopeptide determinant and then copolymerized with the N-methacryloylated ester with HPMA, as described, for example, in Biochem. Biophys. Acta, 757,518 (1983).

The preparation of N- (2-hydroxypropyl) methacrylamide is described in Angew. Makromol. Chem. 70, 109 (1978); Suitable drugs and determinants for binding are all commercially available. Amino acid and peptidyl derivatives of drugs and determinants can be prepared by the methods described in Makromol. Chem. 184, 1997 (1983) and crosslinking agents may be prepared as described, for example, in Makromol. Chem. 182, 1899 (1981).

The invention also includes pharmaceutical compositions comprising at least one polymeric drug of the invention and an inert physiologically acceptable carrier. The polymeric drug may be administered orally or as an injection, e.g., intraperitoneally, intraveneously, or intramuscularly.

Any of the conventional ingredients and additives of the dosage forms can in principle also be used for the polymeric drug of the invention. In the case of injections, it is recommended to use sterile aqueous media as carriers.

The following non-limiting examples illustrate the invention.

Examples 22 to 35 relate to the biological testing of polymeric drugs of the invention and analogs thereof, and the remaining examples relate to the synthesis of these polymeric drugs.

As noted, Examples 22-35 relate to biological assays of polymer drug analogs of the present invention, wherein the analogs are compounds containing feature (s) and in some cases one or both of optional features (d) and (e) of the compounds of the invention. , but contain only one of characters (b) and (c), and not both. In these examples, analogs were used rather than the compounds of the invention to show more clearly the effect of changing features (b) and (c) individually.

-11EN 278551 B6

Example 1

Preparation of a single chain polymer containing daunomycin bound to a tetrapeptide spacer, galactosamine bound to a dipeptide spacer, and tyrosinamide bound directly to the backbone.

HPMA

Ma-Tyr-NH ₂

Ma-Gly-Glygaactact from amine (2)

MA-Gly-Phe-Leu-Gly-ONβ (3) daunomycin

- ^- copolymerization

--------------: ----------- y • Tyr-NH ₂

Gly-Gly-gal

Gly-Phe-LeuGly-ONp

Preparation of MA-Tyr-NH ₂ (1).

MA-Tyr-NH ₂ was prepared by reacting 3.4 g (0.017 mol) of p-nitrophenylmethacrylate and 3.0 g (0.017 mol) of Tyr-NH ₂ in dimethylformamide with stirring the reaction mixture at 25 ° C for 16 hours. After evaporation of the solvent, the viscous oil was mixed with dry ether and a small amount of hydroquinone. The yield was 3 g of solid (mp 194-196 ° C; crystallized from ethanol). Molar extinction coefficient e 280 nm ^{= 1.7} * ^1q3 (ethanol).

Preparation of MA-Gly-Gly-Galactosamine (2).

To a mixture of 3.5 g (1.08. 10 ^"2 mol) of MA-Gly-ONp (p-nitrophenyl methakryloylglycylglycinu) and 2.8 g (1.3 .10 ² mol) of galactosamine hydrochloride in 18 ml of dimethylformamide was slowly added 1.8 mL (1.3. 10 ^"2 mole) of triethylamine and a mixture was stirred at 25 ° C for 16 hours. The triethylamine hydrochloride was then filtered off and the remainder of the reaction mixture was evaporated to a viscous oil. Addition of ethanol and cooling gave the crude product. After recrystallization 1.5 g of product were obtained; mp 112 ° C (ethanol).

Preparation of MA-Gly-Phe-Leu-Gly-ONp (3).

A mixture containing 4.1 g (1.0.10 ^-2 mol) of MA-Gly-Phe-ONp (prepared according to J. Polym. Sci. 66, 15 (1979) in 50 ml of dioxane, 2.1 g (1). 1.10 ^"2 mol) of H-Leu-Gly-OH in 50 ml of water and 1.8 g (2.2. 10" ² mol) of NaHCO ₃ was stirred at room temperature for 48 hours. the solvent was evaporated under reduced pressure until the volume of the mixture was

The reaction mixture was acidified with dilute hydrochloric acid to pH 2 after addition of a small amount of inhibitor (hydroquinone). The crude product was extracted into ethyl acetate and precipitated with diethyl ether. The yield was 74% MA-Gly-Phe-Leu-Gly-OH; m.p. Mp 145-150 ° C. TLC analysis showed the product to be free of impurities. (Note: it is recommended to use a crude product for the next reaction step as the pure material readily polymerizes during purification).

MA-Gly-Phe-Leu-Gly-OH was 4.6 g (1.0. 10 ^"2 mol) esterified with p-nitrophenol (1.1. ² 10 mmol) in 55 mL of THF using dicyclohexylcarbodiimide (DCC) (1.1-10 " ² mol). After 3 hours at 15 ° C and 12 hours at 25 ° C, dicyclohexylurea was filtered off and the remaining solution was evaporated under reduced pressure. The residue upon addition of dry ether solidified and recrystallized from acetone-ether (3: 1) to give MA-Gly-Phe-Leu-Gly-ONp in 76% yield and analytical purity; mp 124-128 ° C. Amino acid analysis gave a ratio of Gly: Leu: Phe = 2.0: 1.0: 0.99.

Preparation of a reactive polymer precursor (4).

HPMA copolymerization + MA-Tyr-NH ₂ (1) + MA-Gly-Gly-galactosamine (2) + MA-Gly-Phe-Leu-Gly-OMp (3)

To a solution of 2.5 g (1.75. 10 ^"2 mole) of HPMA (92 mol.%), 0.047 g (1.90. 10" ⁴ mol) of MA-Tyr-NH ₂ (1 mol.%) And 0.555 g (9.54. 10 ^"4 mol) of MA-Gly-Phe-Leu-Gly-ONp (5 mol.% in 25 ml of acetone was added a solution of 0.138 g (3.82. 10 ^-4 mol) of MA-Gly-Gly -galactosamine (2 mol%) in 1 ml DMSO was then added a solution of 0.153 g azobisisobutyronitrile in 3 ml acetone, poured into ampoules, purged with nitrogen and sealed, and polymerized at 50 ° C for 24 hours. The precipitated polymer was separated and purified by dissolution in methanol and reprecipitation in acetone to yield 1.9 g (60%) of the product containing 4.7 mol% of side chains -Gly-Phe-Leu-ONp (determined from 274 274 nm). 10 ³ in DMSO),

1.8 mol. % of side chains -Gly-Gly-galactosamine (determined by GPC after acid hydrolysis) and 1.0 mol.% of side chains -Tyr-NH ₂ . The weight-average molar mass M M of the aminolysed polymer was 22,000; molar mass polydispersity M _w / M _n = 1.4. (M _n is the number-average molar mass).

Preparation of a polymer containing daunomycin (5). Binding of daunomycin to a polymer precursor.

A solution of 0.16 g (2.8. 10 ^"4 mol) of daunomycin hydrochloride in 1 M DMSO was added to a solution of 1.0 g of copolymer (4), which contained 2.8. 10 ^"4 mol of reactive p-nitrophenoxy with ^the rpin in 4 ml of dimethylsulfoxide (DMSO). Thereto was added 0.028 g (2.8. 10 ^"4 mole) of triethylamine. The mixture was stirred at room temperature in the dark for 16 hours, after which 20 μΐ aminopropanol was added after removal of any residual ONp groups. The polymer was precipitated by pouring the reaction mixture dropwise into 400 mL of acetone / diethyl / ether (9: 1), the precipitate was filtered off, washed thoroughly with acetone and ether and dried. The polymer was purified by double gel

-13GB 278551 B6 by filtration on Sephadex LH-20 (2 x 100 cm) using methanol as eluent. The methanol was evaporated under reduced pressure and the pure polymer was isolated by lyophilization from an aqueous solution. Yield 0.75 g. The polymer contained 3.5 mol. % of side chains terminated by daunomycin (10.2 wt.% daunomycin) 1.8 mol. % of galactosamine-terminated side chains (1.8 wt.% galactosamine) and 1 mol. % Of the side chains of Tyr-NH _second The polymer contained less than 0.1% unbound daunomycin based on the amount of bound daunomycin.

Determination of unbound daunomycin: Free DNM was determined by extraction with ethyl acetate; 1.5 mg of the polymer was dissolved in 1 ml of water and shaken with a mixture of 1 ml of buffer (0.2 M Na ₂ CO ₃ / NaHCO ₃ , pH 9.8) and 2 ml of ethyl acetate. The organic layer was separated, dried with a small amount of dry MgSO _4, and the DNM concentration was determined spectrophotometrically at 485 nm (e = 1.0, 10 ⁴ in ethyl acetate).

Example 2

Preparation of a single chain polymer containing daunomycin and galactosamine bound to a tetrapeptide spacer and tyrosinamide bound directly to the main polymer chain.

f-tyrosinamide (-Gly-Phe-Leu-Gly-ONp

1) Daunomycin

........ 2) Galactosamine ___

T-tyrosinamide

-Gly-Phe-Leu-Gly-Daunomycin

-Gly-Phe-Leu-Gly-galactosamine

A copolymer of HPMA, MA-tyrosinamide and MA-Gly-Phe-Leu-Gly-ONp (0.1 g, 2.85, 10 ^-5 mol -ONp groups) was dissolved in dimethylsulfoxide (0.4 mL) and a solution was added. daunomycin hydrochloride (8.5 mg,

1.5. 10 ^"5 M in dimethyl sulfoxide (0.1 mL) followed by triethylamine (1.5 mg, 1.5. 10 ^-5 mol). The mixture was stirred at room temperature for 90 minutes and then a solution of galactosamine hydrochloride (6.5 mg, 3.0, 10 ^-5 mol) in dimethylsulfoxide-R (0.1 mL) was added followed by the addition of triethylamine (3.0 mg, 3). (0.10 mol). The mixture was stirred at room temperature for 16 hours and then poured into acetone / diethyl ester (9: 1). The precipitated polymer was filtered off, washed thoroughly with acetone and diethyl ether and dried in vacuo. The polymer was purified by dialysis in a dialysis tube against dilute acid and then water. The pure polymer was isolated by lyophilization and contained 2.17 mol. % of side chains terminated by daunomycin, i.e. 7.5 wt.% of active product, and 1.8 mol.

B6% of galactosamine-terminated side chains, i.e. 2.0 wt. % galactosamine.

Example 3

Preparation of a single chain polymer containing daunomycin bound to a dipeptide. spacer, and tyrosinamide, bound directly to the polymer backbone.

HPMA

MA-Tyr-NH ₂

MA-Tyr-Gly-ONp (6) copolymerization

--- Tyr-NH ₂ -Gly-Gly-ONp (7) daunomycin —------— s>

-Tyr-NH ₂ -Gly-Gly-DNM (8) p-nitrophenyl methakryloylglycylglycinu (6) was prepared according to Makromol. Chem. 177, 2833 (1976). The preparation of the polymer precursor was carried out as in Example 1. M _w / M _n = 1.4 (aminolysed precursor).

Preparation of a polymer containing daunomycin (8).

To a solution of 0.50 g of a polymer precursor (7) containing

5.5 mol. % Gly-Gly-ONp side chains (1.8. 10 ^"4 mol of ONp groups) in 2 mL of DMSO was added 0.038 g (1.35. 10 ^-4 mol) of daunomycin hydrochloride in 0.3 ml DMSO and then 0.014 g (1.35. 10 ^"4 mole) of triethylamine. The further procedure was the same as in Example 1. Yield: 0.38 g of polymer which contained 3.1 mol. % side chains -Gly-Gly-daunomycin (10.2 wt% daunomycin);

e ₄₈₅ nm = 9,800 (H ₂ O).

Example 4

Preparation of a single chain polymer comprising daunomycin and anti-O antibodies bound to the tetrapeptide side chain and tyrosinamide bound directly to the main polymer chain.

>: r-NH ₂ '-Gly-Phe-Leu-Gly-ONp

I (9) daunomycin _'-Tyr-NH2

-Gly-Phe-Leu-Gly-DNM -Gly-Phe-Leu-Gly-ONp '(10)

Anti-O antibodies aqueous buffer, pH 8.0

-Tyr-NH ₂

-Gly-Phe-Leu-Gly-DNM

-Gly-Phe-Leu-Gly-anti-0

-15GB 278551 B6

Ma-Gly-Phe-Leu-Gly-ONp (3) was prepared as described in Example 1, and the polymer precursor (9) was prepared by copolymerization of HPMA and (3) by the procedure described in Example 1. Polymer precursor (9) ) contained 8.0 mol. % __ -Gly-Phe-Leu-Gly-ONp side chains and 1 mol. % Of Tyr-NH _second M _{w of the} aminolysed precursor (9) = 17,000; = 1.3.

Preparation of a copolymer with daunomycin and free reactive groups.

To a solution of 0.31 g of polymer precursor (9) (the content of ONp groups of 1.35. 10 ^-4 mol) in 1.2 of DMSO was added 0.028 g (5.1. 10 ^-5 mol) of daunomycin hydrochloride in 0.1 ml DMSO and 0.0052 g (5.10 ^-5 mol) of triethylamine. The reaction mixture was stirred at room temperature for 60 minutes and then dropped into 400 mL of acetone ether (3: 1) to precipitate the copolymer. The polymer contained 2.5 mol. % of side chains terminated with daunomycin and 4.3 mol% of side chains terminated with an unreacted ONp group.

Binding of anti-O antibodies to a daunomycin-containing polymer.

mg of the copolymer described in the previous paragraph was dissolved at 5 ° C in 0.5 ml of dilute hydrochloric acid (pH 3.0). 0.6 ml of Sorensen buffer (pH 8.0) containing 0.15 M NaCl was then added, followed by dropwise addition of 0.67 ml of anti-O antibody solution in the same buffer (the solution contained 39.5 mg of protein). The reaction was allowed to proceed for 30 minutes at 5 Ό. The temperature was gradually raised to 20 ° C over the next 30 minutes. (At the start of the reaction, the molar ratio of reactive groups ONp: NH ₂ = 1: 1 and the concentration of macromolecules was 6.6 wt%). After one hour of reaction, a solution of 20 μΐ-1-aminopropan-2-ol in 0.2 ml Sorensen buffer (pH 8.0) containing 0.15 M NaCl was added. After 10 minutes, the reaction mixture was transferred to a Visking dialysis tube and dialyzed against phosphate buffered saline to pH 7.2.

Example 5

Preparation of a single-chain polymer containing puromycin and galactosamine bound to a tetrapeptide spacer.

Tyr-NH ₂ -Gly-Phe-Phe-Leu · puromycin

-Gly-Phe-Phe-Leugalactosamine (14)

Preparation of Ma-Gly-Phe-Phe-Leu-ONp.

A mixture containing 4.1 g (1.0.10 ^-2 mol) MA-Gly-Phe-ONp (see Example 1) in 50 ml dioxane and 3.06 g (1.1.10 ² mol. Sigma) H -Phe-Leu-OH in 50 mL dioxane and 1.85 g NaHCO ₃ (2.2.10 10 ² mol) in 50 mL water was stirred at room temperature for 45 h. 3) in Example 1.

-16GB 278551 B6

The crude product MA-Gly-Phe-Leu-OH (51% yield) was esterified with p-nitrophenol by the method described in Example 1. The pure product was obtained in 50% yield after recrystallization from acetone / ether 3: 9). Amino acid analysis gave a ratio of Gly: Phe: Leu =

1.0: 1.98: 1.0.

Preparation of the polymer precursor (13).

Copolymerization of HPMA (95 mol%) with MA-Tyr-NH ₂ (1 mol%) and MA-Gly-Phe-Phe-Leu-ONp (12) (5.0 mol%) according to the procedure described in the example 1, provided a polymeric precursor containing 3.0 mol. % of side chains -Gly-Phe-Phe-Leu-ONp with 1.0 mol. % Of the side chains of Tyr-NH _second = 23,000; ΐ ^ / ϊ ^ = 1.3.

Binding of puromycin and galactosamine.

To a solution of 0.36 g of a polymer precursor (13) containing

6.5. 10 ^_5 mole of ONp groups in 1.4 ml DMSO was added a solution of 0.026 g (4.4. 10 ^-5 mol) of puromycin dihydrochloride in 0.1 ml DMSO and 0.0088 g (8.8. 10 @ ^-5 mol) triethylamine. After stirring at room temperature for 30 minutes, 0.094 g (4.4.10 mol) of galactosamine and 0.044 g (4.4.10 ^-5 mol) of triethylamine were added to the mixture. The reaction mixture was stirred for 16 hours and then 10 μΐ aminopropanol was added.

The polymer was immediately, precipitated in 400 ml of acetone, washed and dried. The polymer was precipitated from solution in methanol with acetone and purified by dialysis of the aqueous solution in a Visking dialysis tube. After isolation of the product by lyophilization, 0.265 g of a polymer containing 1.3 moles was obtained. % puromycin-terminated side chains (3.7 wt.% puromycin; determined by a molar extinction coefficient e ₂₇₂ nm = 2.0, 10 ⁴ , ethanol), and 1.0 mol%. % of galactosamine-terminated side chains. (1.0 wt% galactosamine). The purity of the product, ie the presence of low molecular puromycin, was controlled by extraction of the polymer into ethylene acetate as described in Example 1 and confirmed by GPC on Sephadex G-15.

Example 6

Preparation of a single chain polymer containing deacetylcolchicine bound to a tripeptide spacer and a small amount of Tyr-NH ₂ .

-Tyr-NH ₂

-Ala-Val-Ala-ONp (16) deacetylcolchicine

------------>

-Tyr-NH ₂

-Ala-Val-Ala-Deacetylcolchicine (17)

-17GB 278551 B6

Preparation of MA-Ala-Val-Ala-ONp (15).

g (0.17 mol) of alanine was dissolved in 40 ml of methanol and 20 g of thionyl chloride was slowly added to the solution at -15 ° C. After 1 hour at room temperature and 2 hours of boiling, methanol was removed and 12 g of Ala-OMe.HCl was isolated with diethyl ether.

To a solution of 10 g (0.086 mol) of valine and 3.4 g (0.086 mol) of NaOH in dioxane-water (2: 1) was slowly added at. ° C with vigorous stirring 20.5 g (0.094 mol) of di-tert-butyl dicarbonate. The reaction mixture was stirred for another hour at room temperature, dioxane was removed under reduced pressure, and acidified to pH 2-3 to obtain BOC-Val-OH. The crude product, consisting of 18 g of a viscous oil, was obtained after evaporation of the solvent from the ethyl acetate extract.

The condensation of BOC-Val-OH with Ala-OMe.HCl was carried out by the mixed anhydride method; 12 g (0.055 mol) of BOC-Val-OH was dissolved in 40 ml of dry THF and 7.7 g (0.055 mol) of Ala-OMe.HCl and 5.6 g (0.055 mol) were added to the solution cooled to -15 ° C. mol) of triethylamine. The reaction mixture was then stirred at room temperature for an additional 16 hours. The THF was evaporated and the product was extracted with ethyl acetate. Recrystallization from ether gave 8.1 g of analytically pure BOC-Val-Ala-OMe, m.p. 138-148 ° C.

Removal of the BOC group was accomplished by dissolving 8.1 g of BOC-Val-Ala-OMe in 60 mL of methanol and adding 20 mL of 30% methanolic HCl. After stirring for 2 hours, the solvent was evaporated and crude HCl-Val-Ala-OMe was isolated.

N-methacryloylalanine was prepared by Schotten-Bauman acylation. Condensation of MA-Ala-OH (5.35 g; 0.034 mol) with HCl. Val-Ala-OMe (8.1 g) was carried out in dimethylformamide with dicyclohexylcarbodiimide (7.0 g) in the presence of triethylamine (3.4 g) ). Recrystallization from CH ₂ C1 ₂ (with addition of isopentane) afforded 9.5 g of MA-Ala-Val-Ala-OMe.

g MA-Ala-Val-Ala-OMe was hydrolyzed in methanol with a small excess of 2N BaOH and acidified with citric acid to pH 3 after removal of methanol; 4 g of crude product was used in the next reaction.

From 3.6 g of MA-Ala-Val-Ala-OH (0.012 mol), 1.5 g (0.012 mol) of p-nitrophenol and 2.5 g (0.0125 mol) of dicyclohexylcarbodiimide in 50 ml of tetrahydrofuran were prepared 2, 0 g (40% yield) MA-Ala-Val-Ala-ONp; m.p. 174-176 ° C, which was recrystallized from ethyl acetate / hexane. Molar extinction coefficient? 74 nm = 9 300 (DMSO). ·

Preparation of polymer precursor.

Copolymerization of HPMA (95 mol%), MA-Tyr-NH ₂ (1 mol%) and MA-Ala-Val-Ala-ONp (4 mol%) yielded a polymer precursor containing 2.9 mol. % of side chains with ONp groups. M ^. = 27,000; M + / Mn = 1.4 (determined for aminolyzed precursor).

A mixture of deacetylcolchicine and isodeacetylcolchicine was prepared by reaction of trimethylcolchicine acid with diazomethane,

-18GB 278551 B6 by my procedure (Biochemistry 25, 2463 (1966)).

Deacetylcolchicine binding.

To a solution of 0.41 g polymer precursor (16) containing 4.0 x 10 ^-5 mol ONp groups in 1.6 ml DMSO was added 0.021 g (6.0 x 10 ^-5 mol) deacetylcolchicine in DMSO. The mixture was stirred for 20 hours at room temperature in the dark and then the polymer was precipitated in acetone. Purification was performed by ultrafiltration (membrane UM-02) in water with 20% methanol. The polymer product was isolated by lyophilization and contained 2.7 mol. % of the side chains terminated by deacetylcolchicine. (Calculated from the molar extinction coefficient ε ₃₅ θ _nm = 1.6. 10 ⁴ (ethanol)).

Example 7

Preparation of a branched polymer containing daunomycin and galactosamine, prepared by subsequent aminolysis.

-Tyr-NH ₂ , -Gly-Phe-Leu-Gly-ONp (18)

1. H (Phe) -NH (CH ₂ ) ₆ NH- (Phe) H

2. daunomycin

3. galactosamine ---------------------------------->

-Tyr-NH ₂

-Gly-Phe-Leu-Gly-Daunomycin) -Gly-Phe-Leu-Gly-Gal

-Gly-Phe-Leu-Gly-Phe-NH (CH ₂ ) ₆ NH-Phe-Gly-Leu-Phe-Gly '(19)

Polymer precursor (18) was prepared by copolymerization of HPMA (90 mol%), MA-Tyr-NH ₂ (1 mol%), and MA-Gly-Phe-Leu-Gly-ONp (9.0 mol%). The polymer precursor (18) contained 8.0 mol. % of side chains terminated by ONp M groups _w = 17,000; M _w / M _n = 1.3 (determined for aminolysed precursor).

0.2 g of polymer precursor (18) (8.0 mol% of side chains containing ONp groups) in 0.8 ml DMSO (9.8, 10 ^-5 mol ONp) was added to a solution of 0.0045 g (1). 1, 10 ^-5 mol) of N, N'-bis- (H-Phe) hexamethylenediamine in 0.2 ml of DMSO. The mixture was stirred at room temperature for 5 minutes and then 0.036 g (6.3, 10 ^-5 mol) of daunomycin hydrochloride dissolved in 0.3 ml DMSO and 0.0064 g (6.3 g) were added sequentially. 10 ^-5 mol) of triethylamine. The mixture was stirred for 60 minutes and then a solution of galactosamine hydrochloride (0.018 g; 8.5, 10 ^-5 mol) and triethylamine (0.0086 g; 8.5, 10 ^-5 mol) were added. The mixture was stirred for an additional 16 hours

And the polymer was precipitated in acetone. The polymer was purified by first gel filtration using Sephadex LH-20 and methanol and then dialysis in a Visking dialysis tube against water. Pure polymer was isolated by lyophilization. The polymer product contained 2.8 mol. % of side chains terminated with daunomycin (7.8 wt.% daunomycin - determined from _{485 nm} = 9.8. 10 ³ in DMSO) and 3.9 mol.

% of side chains terminated by gaactosamine. Polymer yield 0.15 g; = 110,000; It ^ / Mn of 4.0 (M _w aminolyzed precursor was 17,000; ί ^ / ^ ΐ = 1.3).

Example 8

Preparation of a single chain polymer containing bleomycin and Tyr-NH ₂ tyrosinamide.

-Tyr-NH ₂

-Gly-Leu-piy-ONp (20) bleomycin ------------>

-Tyr-NH ₂

-Gly-Leu-Gly-Bleomycin (21)

The methacryloylglycylleucylglycine p-nitrophenyl ester was prepared as described in Example 6 for compound (15).

The polymer precursor was obtained by copolymerization of HPMA (94 mol%), MA-Tyr-NH ₂ (1 mol%) and MA-Gly-Leu-Gly-ONp (5 mol%). The polymer precursor (20) contained 4.3 mol. % of side chains terminated with ONp groups.

To a solution of 0.137 g of precursor (20) (3.8. 10 ^-5 _mo j ONp groups) in 0.55 ml of dimethylsulfoxide was added a solution of 0.047 g (3,8.10 ^-5 mol), bleomycin sulfate, and 0.0038 g (3, 8 (10 ^-5 mol) triethylamine in 0.3 ml DMSO. The reaction mixture was stirred for 12 hours and the polymer was then isolated by addition of 20 μΐ aminopropanol and precipitation in acetone. After two days of dialysis using a Visking tube, the polymer was lyophilized. Yield 0.102 g. The polymer product contained 3.1 mol. bleomycin-terminated side chains (determined by kinetic measurement of the concentration of ONp groups).

Example 9

Preparation of a single chain polymer containing melanocyte stimulating hormone (MSH) coupled to a dipeptide spacer.

-Gly-Gly-ONp

MSH ------------>

-Gly-Gly-MSH)

-20GB 278551 B6

To a solution of 0.20 g of polymer precursor (22) (5.0 mol% Gly-Gly-ONp) containing 6.6. 10 ^-5 moles of ONp groups, in 0.8 ml DMSO a solution of 0.005 g (3.0, 10 ^-5 mol) of melanocyte stimulating hormone in 0.3 ml DMSO was added. After stirring for 16 hours, 10 μΐ of aminopropanol was added and the polymer product precipitated in acetone. After purification by dialysis and lyophilization, the product was analyzed for bound MSH content. The polymer product (23) contained 1.3 wt. MSH% (determined spectroscopically: for 0.347 mg / ml in water MSH is A ¹ = 1.49).

^J 278.mii '

Example 10

Preparation of a single chain polymer containing desferioxamine bound to a tetrapeptide spacer.

₁ -Tyr-NH ₂ -Gly-Phe-Leu-Gly-ONp (24) desferioxamine -------------->

1 -Tyr-NH ₂

-Gly-Phe-Leu-Gly-> desferioxamine) (25)

Polymer precursor (24) containing 4.3 mol. % of side chains with ONp groups (0.20 g; 5.3. 10 ^-5 mol ONp groups) was dissolved in 0-8 ml DMSO and 0.070 g (1.06. 10 ^-4 mol) was added. of desferioxamine and 0.011 g (1.06, 10 ^-4 mol) of triethylamine in 0.2 ml of DMSO. After stirring at room temperature for 16 hours, the polymer product was precipitated in 200 ml of acetone. Purification was performed by dialysis in water containing 20% methanol using a Visking dialysis tube. ......

The desferioxamine content was calculated from kinetic measurements to determine the content of ONp groups in the reaction mixture. The polymer product contained 3.9 mol. % desferioxamine-bound side chains (12.4 wt.% desferioxamine).

Example 11

Preparation of a single chain polymer containing bestatin and galactosamine bound to a tetrapeptide spacer.

-Tyr-NH ₂

-Gly-Phe-Leu-Gly-ONp

1. bestatin

2. Galactosamine ---------------- _>

-Tyr-NH ₂

-Gly-Phe-LeuGly-bestati.i

-Gly-Phe-LeuGly-gal

0.150 g of polymer precursor (18) containing 6.8. 10 ~ ⁵ mol of ONp groups was dissolved in 0.6 ml DMSO and a solution of 0.030 g (1.3. 10 ^"4 mol) of galactosamine and 0.014 g (1.3.

(21 mol of triethylamine in 0.2 ml of DMSO). The reaction mixture was stirred at room temperature for 16 hours, the product was precipitated in 200 ml of acetone and purified by dialysis for 3 days. The lyophilized product (0.111 g) was analyzed for galactosamine content (see Example 1). The amount of bound bestatin was calculated from kinetic measurements by monitoring the amount of ONp groups in the reaction mixture. The polymer product (27) contained 1.4 mol. % of side chains terminated by bestatin (2.4 wt.% bestatin) and 5.4 mol. % of galactosamine-terminated side chains (5.5 wt.% galactosamine).

Example 12

Preparation of a single chain polymer containing adriamycin and mannosamine bound to a tetrapeptide spacer.

-Tyr-NH ₂

-Gly-Leu-Leu-Gly-ONp

1. adriamycin

2. Mannosamine -------------->

-Tyr-NH ₂ '-Gly-Leu-Leu-Glyl adriamycin

-Gly-Leu-Leu-GlymannOsamine (28)

The methacryloylglycyl leucylglycine p-nitrophenyl ester was prepared in a similar manner to compound (3) in Example 1.

Polymer precursor (28) was prepared by copolymerization of HPMA (89.5 mol%), Ma-Tyr-NH ₂ (1 mol%) and MA-Gly-Leu-Leu-Gly-ONp (9.5 mol%) . The polymer contained 8.1 moles. % of tetrapeptide side chains terminated with ONp groups. = 17,000;

= 1.3 (determined for aminolyzed precursor).

The polymer product (29) was prepared from 0.4 g of polymer precursor (28) containing 1.8. 10 ^-4 mol of ONp groups and dissolved in 1.6 ml DMSO. To the solution was added 0.06 g (1.1, 10 ^-4 mol) of triethylamine. The following procedure was the same as in Example 1. The polymer product contained 3.2 mol. % of side chains containing adriamycin (9.6 wt.% adriamycin) and 4.1 mol. % of side chains containing mannosamine. The yield was 0.31 g. The content of carbohydrate residues was determined after acid hydrolysis by GPC using a known method (Anal. Biochem. 73, 532 (1976)).

Example 13

Preparation of a single chain polymer containing bleomycin coupled to a tripeptide spacer.

rGly-Phe-Tyr-ONp> (30) bleomycin ---------------------> ·

-Gly-Phe-Tyr-Bleomycin (31)

-22GB 278551 B6

The reactive monomer MA-Gly-Phe-Tyr-ONp and the polymeric precursor (30) were prepared according to Makromol. Chem. 182, 799 (1981). The polymer precursor contained 8.5 mol. % of side chains with ONp groups. M / z = 1.3 (determined for aminolysed precursor).

Binding of bleomycin to the polymer precursor was performed according to the procedure described in Example 8. The polymer product (31) contained 5.7 moles. % of side chains with bleomycin.

Example 14

Preparation of a single chain polymer containing puromycin and fucosylamine bound to a tripeptide spacer.

i-Gly-Phe-Phe-ONp

1.puromycin

l.fucosylamine

-Gly-Phe-Phe-puromycin

-Gly-Phe-Phe-Fucosylamine (33)

MA-Gly-Phe-ONp reactive monomer and polymer precursor (32) were prepared according to Makromol. Chem. 182, 799 (1981). The polymer precursor contained 12.9 moles. % of side chains with ONp groups. M _w = aminolysed precursor was 14,000; M / z = 1.28. To a solution of 0.25 g of polymer precursor (32) 1.64. 10 ~ ⁴ mol of ONp groups) in 1 mL of DMSO was added a solution of 0.029 g (4.9. 10 @ ^-5 mol) of puromycin dihydrochloride and 0.010 g (9.8. 10 ^"5 mol) of triethylamine in 0.25 ml DMSO. The reaction was allowed to proceed for 1 hour and then 0.015 g (9.0, 10 ^-5 mol) of fucosylamine in 0.1 ml DMSO was added to the mixture. The following procedure was similar to that described in Example 5. The polymer product (33) contained 4.1 mol. % puromycin-terminated side chains and 6.7 mol. % fucosylamine-terminated side chains.

Example 15

Preparation of a single chain polymer containing transferrin bound to a dipeptide spacer.

-Gly-Gly-ONp (34) transferrin ------------->

-Gly-Gly-Transferrin (35)

27.4 g of polymer precursor (34) containing 11.4 mol. % of the Gly-Gly-ONp side chains were dissolved in 0.25 ml of dilute hydrochloric acid (pH 3.0). 1.25 ml of Sorensen buffer (pH 8) containing 0.1 M NaCl was added, followed by a solution of 14.2 mg of transferrin in 0.5 ml of the same buffer. After 45 minutes, 10 µL of 1-amino-2-propanol dissolved in 0.2 ml Sorensen buffer was added to the mixture. After 30 minutes, the reaction was stopped and the mixture transferred to a Visking dialysis tube and dialyzed against phosphate buffered saline (pH 7.2).

-23GB 278551 B6

Example 16

Preparation of a single chain polymer containing IgG bound to a dipeptide spacer.

-Gly-Gly-OH

IgG ------>

-Gly-Gly-IgG

The copolymer HPMA and MA-Gly-Gly-OH (15.0 mg; 5.0.10 ^-6 mol COOH groups) was dissolved in Sorensen buffer pH 5 (75 μΐ) and a solution of 0,002 was added at 1 ° C. mg of N-cyclohexyl-N '- (2-morpholinoethyl-D-carbodiimide in the same buffer (40 μΐ) was stirred for 15 minutes and then 60.0 mg of IgG in 235 μΐ of the same buffer was slowly added. The crude reaction mixture was dialyzed against phosphate buffered saline (pH 7.2).

Example 17

Preparation of a branched polymer comprising puromycin and galactosamine bound to a tripeptide spacer.

1. -Gly-Phe-Tyr-ONp 1. H-Ala-Gly-HN (CH ₂ ) g NH-Gly-Ala-H

2. puromycin (36); 3. galactosamine

------------------------------------------------>

/ -Gly-Phe-Tyr-puromycin)

S-Gly-Phe-Tyr-gal

) -Gly-Phe-Tyr-Ala-Gly-HN _{(CH2) 6} -NH-Gly-Ala-Tyr-Phe-Gly- (<(37) 5

Polymer precursor (36) containing 4.8 mol. % of side chains with ONp groups were prepared according to Makromol. Chem. 182, 1899 (1981).

2.0 g of polymer precursor (36) containing 5.9. 10 ^-4 mol of ONp groups were dissolved in 8 ml of DMSO and a solution of 0.038 g (9.8, 10 ^-5 mol) of 1,2-bis-alanylglycylamino) ethane dihydrochloride in 0.2 ml of DMSO and 0.020 g ( 2.0 (10 ^-4 mol) of triethylamine. After stirring for 20 minutes? the reaction mixture was added a solution of 0.120 g (2.10 ^"4 mol) of puromycin dihydrochloride in ml of DMSO and 0.040 g (4.10" ⁴⁾ of triethylamine. After stirring the reaction mixture was added a solution of 0.129 g (6.0. 10 ~ ⁴ mol) of galactosamine and 0.060 g (6.0. 10 ^"4 mol) of triethylamine in 1 mL of DMSO. The mixture was stirred for an additional 16 hours at room temperature. The product was isolated and purified as described in Example 5. Product

-24GB 278551 B6 contained 1.5 mol% galactosamine-terminated side chains.

Example 18

Preparation of a branched polymer containing puromycin and fucosylamine bound to a tripeptide spacer.

-Gly-Phe-Phe-ONp

H-Gly-HN (CH ₂ ) ₆ NH-Gly-H

2. puromycin

3. Fucosylamine

-Gly-Phe-Phe-puromycin

-Gly-Phe-Phe-fucosylamine

-Gly-Phe-Phe-Gly-HN _(CH2) θΝΗ-Gly-Phe-Gly (36)

Polymer precursor (38) containing 4.8 mol. % of side chains with ONp groups were prepared according to Makromol. Chem. 182, 1899 (1981).

2.0 g of polymer precursor (38) containing 5.9. 10 ^-4 mol groups were dissolved in 8 ml DMSO and a solution of 0.029 g (9.8. 10 ^-5 mol) 1,2-bis (glycylamino) ethane diacetate in 0.2 ml DMSO, prepared according to Makromol, was gradually added. . Chem. 182, 1899 (1981), and 0.020 g (2.0. 10 ^-4 mol) of triethylamine. Crosslinking was performed for 5 minutes. 0.120 g ( ^2.0.10-4 mol) of puromycin dihydrochloride in 1 ml DMSO and 0.040 g (4.10 ^-4 mol) of triethylamine were then added to the mixture. The reaction mixture was stirred at room temperature for 16 hours. The product was isolated and purified according to the procedure described in Example 5. The product contained 1.4 mol. % puromycin-terminated side chains (4.2 wt.% puromycin) and 3.0 mol. % fucosylamine-terminated side chains.

Example 19

Preparation of a single chain polymer containing daunomycin bound to a tetrapeptide spacer and galactose bound directly to the polymer chain.

—Gal

-Gly-Phe-Leu-Gly-ONp (40) daunomycin

------------->

-Gal

-Gly-Phe-Leu-Glydaunomycin

-25GB 278551 B6

Preparation of the polymer precursor (40).

In 13.0 ml of dimethyl sulfoxide was dissolved 0.71 g (5 · 10 ^-3 mol) of 1,2,3,4-di-0-isopropylidene-6-0-methacryloyl-D-galacto-pyranose (prepared according to J. Chem. Soc (London) C, 1913 (1986)). To this solution was added 3 mg of azobisisobutyronitrile (1.8. 10 ^"4 mole) in 1.8 ml of dimethylsulfoxide and thereafter 291 mg of MA-Gly-Phe-Leu-Gly-ONp (5.10" ⁴ mole) in 1 ml DMSO prepared according to the method described in Example 1. The mixture was purged with nitrogen and then polymerized in a sealed vial at 60 ° C for 30 hours. The resulting polymer was precipitated with ether and deacetonated by dissolving in 80% aqueous formic acid for 20 hours at room temperature. The yield of polymer precursor after formic acid evaporation was 1.2 g; the content of ONp groups was 9.5 mol. % and the galactose content was 52 mol. %.

Binding of daunomycin to the polymer precursor (40).

In 1.6 ml of DMSO, 374 mg (1.6. 10 ^-4 mol) of polymer precursor (40) was dissolved, and 50 mg (8.10 ^-5 mol) of daunomycin hydrochloride was added in 0.3 ml of DMSO, and 0111 ml (8.

10 ^-5 mol) of room and the product dissolved in triethylamine. The reaction mixture was stirred at min. 20 μΐ of aminopropanol was then added and immediately precipitated in 200 ml of acetone. The precipitate was in methanol and the polymer fraction was separated on a 100 x 2.5 cm Sephadex LH-20 column, washed with methanol. The yield was 0.28 g; the content of tetrapeptide side chains terminated with daunomycin was 3.5 mol. %.

Example 20

Preparation of a single chain polymer containing a tripeptide spacer bound isopropyl ester of melphalan and galactose bound directly to the backbone k-AcGalactose / -Gly-Phe-ONp Glycyl melphalan (-AcGalactose isopropyl ester ( (Gly-Me-ipe) (-Gly-Phe-Gly-Me-ipe / (42) ...........> \ (43) formic acid / -Galactose ) -Gly-Phe-Gly-Me-Ipe

/ (44)

Preparation of melphalan isopropyl ester (Meipe).

A solution of 5 g melphalan in 20% HCl in isopropyl alcohol was refluxed for 5 hours. After cooling in a refrigerator, white crystals of analytically pure product were isolated and dried in vacuo; mp 200-201 ° C. A solution of Meipe in 50 mL of diethyl ether was bubbled through NH _{3 for} 3 hours; the solvent was then evaporated and the oily product used in the next step of the synthesis.

• Preparation of glycyl melphalan isopropyl ester (Gly-Meipe).

Gly-Meipe was prepared by reacting 3.03 g of BOC-Gly-OH with 6.0 g Meipe in 50 ml of tetrahydrofuran using the DCC method. The BOC protecting group was removed in 15% methanolic HCl and the free phase was prepared by bubbling a slurry of Gly-Meipipe hydrochloride in diethyl ether with ammonia. The ammonium chloride was filtered off and the oily product was isolated after evaporation of the solvent.

Preparation of polymeric precursors (42).

Polymer precursor (42) was prepared by radical precipitation copolymerization of DCM (1,2,3,4-di-O-isopropylidene-6-O-methacryloyl-α-D-galactopyranose) and methacryloylglycylphenylalanine p-nitrophenyl ester (MA-Gly-Phe- ONp) (prepared according to Makromol. Chem. 178, 2169 (1977)) according to the procedure described in Example 1.

Polymerization mixture: HPMA 3.43 (85 mol%) MA-Gly-Phe-ONp 0.926 g (8 mol%) DGM 0.65 g (7 mol 5%) azobisisobutyronitrile 0.24 g. hours at 50 ° C in 44 ml of acetone.

Binding of Gly-Meipe to the polymer precursor (42).

3 g of polymeric precursors (42) were dissolved in 20 ml of dimethyl sulfoxide and 0.816 g of Gly-Meipe was added. The mixture was stirred at room temperature for 3 days and the polymer was then precipitated in acetone and precipitated twice from methanol into a 1: 1 mixture of acetone and diethyl ether. The protected galactose, bound to the polymeric support, was deacetonylated in 80% formic acid (see Example 19) and the polymeric drug was isolated by lyophilization and reprecipitation from methanol into acetone-diethyl ether (1: 1). Polymer yield 2.9 g. Content of drug bound to polymeric carrier (isopropyl ester of melphalan): 106 mg / g polymer (calculated from elemental analysis minus chlorine content). Molecular weight of polymer carrier: = 35,000. The absence of low molecular weight substances (free drug) was demonstrated by GPC using Sephadex G-25 as a column fill.

Example 21

Preparation of a single chain polymer comprising a tetrapeptide spacer coupled with isopropyl ester of melphalan and a backbone galactose.

(-AcGalactose Leucylglycyl melphalan isopropyl ester (Leu-gly-me-ipe) 1-Gly-Phe-ONp ------------------------------------------ _> ) -AcGalactose Formic acid / -Galactose -----------------: -------------> 2

-Gly-Phe-Leu-Gly-Me-ipe / -Gly-Phe-Leu-Gly-Me / ipe (43)) (46)

-27GB 278551 B6

Preparation of leucylglycyl melphalan isopropyl ester.

In 35 mL of THF, 3.1 g of BOC-Leu-Gly-OH was reacted with 3.7 g of Meipe using the DCC method. BOC-Leu-Gly-Me-ipe was crystallized from acetone. The BOC protecting group was removed by bubbling ammonia through a suspension of hydrochloride in diethyl ether. The pure oily product was isolated after separation of ammonium chloride and evaporation of the solvent. The polymer precursor (42) was prepared by the method described in Example 20.

Binding of leucylglycyl melphalan isopropyl ester to a polymer precursor.

The reaction was carried out as in Example 18. To a solution of 1.5 g of polymer precursor (42) in 10 ml of DMSO was added 0.5 g of Leu-Gly-Meipe and the mixture was stirred for 3 days. Polymer (45) was precipitated in acetone-diethyl ether (1: 1) and precipitated twice from methanol into the same mixture. Galactose deacetonylation and polymer isolation and characterization (46) were performed as described in Example 18, a 35,000 polymer carrier; drug content (polymer bound isopropyl ester of melphalan): 118 mg / g polymer. The absence of free drug was controlled by GPC (Sephadex G-25).

Examples 22 to 25

Effect of different peptide spacers on the efficacy of the same drug.

Example 22

Effect on L1210 activity of mouse leukemia in vitro.

These experiments were performed with three HPMS analogs that did not contain specific determinants. All three analogs contained daunomycin as the biologically active moiety and peptide spacer to the drug of (b). In detail, the analogues looked like this:

(a) HPMA with -Gly-Gly-Daunomycin (b) HPMA with -Gly-Phe-Leu-Gly-Daunomycin (c) HPMA with -Gly-Phe-Phe-Leu-Daunomycin.

L1210 mouse leukemia was maintained as a suspension culture in RPMI 40 medium containing 10% heat inactivated horse serum. Under these conditions, doubling the number of cells takes 15 to 20 hours at a cell density of up to about 1. 10 ⁵ cells / ml. Cell densities were determined using a Coulter counter.

When evaluating the cytotoxicity of the polymer drug, the cells were seeded in 10 ml tubes and the initial cell density was measured. Various polymeric drugs were added at various concentrations and cells were stored in the culture chamber for a further 72 hours before further cell density measurement.

The number of cells found in polymer drug drug tubes was expressed as the percentage of cells found in the test tubes incubated for the same time (72 h) without drug addition. The results are shown graphically in Figure 1 of the accompanying drawings, in which the percent growth curves of cells versus daunomycin concentration in µg / ml are plotted. The results indicate that all three analogs were cytotoxic to murine L1210 leukemia in vitro to some extent, but analogs (b) and (c) showed a better inhibitory effect than analog (a).

Example 23

Effect on L1210 mouse leukemia activity in vitro.

The experiments were performed with three HPMA analogs not containing specific determinants. All three analogs contained sarcolysin (melphalan-ipe) as the biologically active moiety and peptide spacers to the drug of (b). The following analogs were tested in a detailed composition:

(a) HPMA with the substituents -Gly-Leu-Gly-sarcolysin;

(b) HPMA with the substituents -Gly-Phe-Leu-Gly-sarcolysin;

(c) HPMA with -Gly-Phe-Gly-sarcolysin substituents.

The experiment procedure was the same as in Example 22.

The results are shown graphically in Figure 2 of the accompanying drawings, in which the curves represent the dependence of the percentage cell growth on the sarcolysin concentration in µg / ml. The results show that all three analogs were somewhat cytotoxic to murine L1210 leukemia in vitro, but analog (c) had a significantly better inhibitory effect than analogs (a) and (b), which stimulated growth at low concentrations.

Example 24

Effect on L1210 mouse leukemia activity in vivo.

The following experiments were performed with two HPMA analogs that did not contain specific determinants; both compounds had a single chain and contained daunomycin as a biologically active component and the peptide spacers according to (b). The detailed structure of the analogs was as follows:

(a) HPMA with -Gly-Phe-Leu-Gly-daunomycin substituents (b) HPMA with -Gly-Gly-daunomycin substituents.

L1210 mouse leukemia was inoculated (10 ⁵ viable cells) intraperitoneally with DBA ₂ mice at day zero. HPMA copolymers were administered on days 1, 2 and 3. Survival of all animals was noted. Control (untreated) animals always died between day 15 and day 20. The results are summarized in the table.

-29GB 278551 B6

Table 1

Therapy Dose Mean survival days Long-term survival (-50 days) Analog (a) 5 mg / kg 32 3/5 Analog (a) 2 mg / kg 31 2/5 Analog (b) 5 mg / kg 18 1/5 - Analog (b) 8 mg / kg (x1) 16 1/5 + 5 mg / kg (x1) None 17 2/10

It can be seen from Table 1 that the survival of the animals when administering analogue (b). In contrast, animals (a) survived noticeably longer and much of their time.

did not prolong treatment with the analog survived long. Example 25

Effect on drug release rate in vitro.

Two single-chain HPMA analogs were prepared that did not contain specific determinants. Both analogs contained daunomycin as a biologically active moiety bound at the end of the peptide spacer. The detailed composition of the analogs was:

(a) HPMA with -Gly-Phe-Leu-Gly-daunomycin (b) HPMA with -Gly-Phe-Phe-Leu-daunomycin.

Each analog (25 mg in 1.5 ml) was incubated with 1 ml enzyme solution at 37 ° C. The enzyme solution contained purified lysosomal enzyme cathepsin L (3.8, 10 ^-7 mol / l) in phosphate buffer (pH 6.0) containing 5 mM glutathione and 1 mM EDTA.

Drug release was measured at various time intervals by extraction at the corresponding wavelength. Samples of the incubation mixture (0.15 mL) were collected and added to 1.5 mL of buffer (pH 9.8) and 1.5 mL of ethyl acetate. After five minutes of vigorous shaking, a sample (1 mL) of the ethyl acetate layer was taken and the extinction was measured at 485 nm.

The results are graphically shown in Figure 3 of the accompanying drawings, in which the curves of the amount of analog released in percent of the initially present drug versus time in hours are plotted. The results show that the release rate of daunomycin is significantly higher when -Gly-Phe-Leu-Gly- was used as a spacer than -Gly-Phe-Phe-Leu.

Examples 26 to 28

Effect of different drugs having the same peptide spacers.

-30GB 278551 B6

Example 26

Effect on L1210 mouse leukemia activity in vitro.

The experiments were performed with four HPMA analogs that did not contain determinants; all substances had a single chain and contained different drugs. The spacers of all four drugs were in accordance with the invention: two had -Gly-Phe-Leu-Gly- and two -Gly-Leu-Phe- spacers. The detailed composition of the analogs was:

(a) HPMA with -Gly-Phe-Leu-Gly-Daunomycin and Tyrosinamide (b) HPMA with -Gly-Phe-Leu-Gly-Puromycin and Tyrosinamide (c) HPMA with -Gly-Leu-Phe-Sarkolysin and (d) HPMA with -Gly-Leu-Phe-tritylcysteine substituents.

The experimental procedure was the same as in Example 22.

The results are graphically shown in Figure 4 of the accompanying drawings in which the curves obtained by plotting the percentage of cells that survived after 72 hours versus the concentration of polymer drug in mg / ml. The results show that to some extent all four polymeric drugs are cytotoxic to L1210 leukemia in vitro, but that peptide-linked daunomycin was significantly more potent than puromycin, and tritylcysteine was slightly more potent than sarcolysin.

Example 27

Effect on L1210 mouse leukemia activity in vitro.

Two HPMA analogs were prepared that did not contain determinants. The analogs contained different drugs, but the spacer drugs were the same in all cases in accordance with the invention. The detailed structure of the analogs was:

(a) HPMA with the substituents -Gly-Gly-deacetylcolchicine and (b) HPMA with the substituents -Gly-Gly-colcemid.

The experimental procedure is described in Example 22.

The results are graphically shown in Figure 5 of the accompanying drawings, in which the curves were obtained by plotting the percentage growth of cells versus polymer drug concentration in mg / ml. The results show that both analogs were cytotoxic to murine L1210 leukemia in vitro, but that analog (b) had a higher inhibitory effect than analog (a).

Example 28

Effect on drug release rate in vitro in the presence of enzymes.

Two single-chain HPMA analogs were prepared that did not contain determinants. The analogs contained different drugs, but the spacer of the drug was the same in both cases and was in accordance with the invention. The detailed structure of the analogs was:

(A) HPMA with the substituents -Gly-Phe-Leu-Gly-daunomycin and tyrosinamide.

(b) HPMA with the substituents -Gly-Phe-Leu-Gly-puromycin and tyrosinamide.

Both compounds were investigated using two different enzyme solutions, namely (1) a mixture of lysosomal enzymes isolated from rat liver according to the method of Trouet et al. (1974) in a buffer (pH 5.5) containing 1 mM EDTA, 5 mM reduced glutathione and 0.2% Triton X-100, and (2) purified lysosomal cathepsin L enzyme (3.8.10 ⁷ mol / l). ) in phosphate buffer (pH 6.0) containing 5 mM glutathione and 1 mM EDTA.

Each analog (25 mg in 1.5 ml) was incubated with 1 ml enzyme solution. The drug released was measured at various time intervals by extracting the free drug and measuring the absorbance spectrophotometrically at the corresponding wavelength. Samples of the incubation mixture (0.15 mL) were collected and added to 1.5 mL of buffer (pH 9.8) and 1.5 mL of ethyl acetate. After five minutes of vigorous shaking, a sample (1 mL) of the ethyl acetate layer was taken and the extinction was measured at 485 nm for daunomycin or at 272 nm for puromycin.

The results are shown graphically in Figure 6 of the accompanying drawings in which the curves were obtained by plotting the amount of drug released as a percentage of the drug present initially present in analogue versus time in hours. The results obtained with the mixture of lysosomal enzymes (1) are plotted as open circles and the results with purified cathepsin L (2) as filled circles.

These results show:

(a) differences in puromycin and daunomycin release rates having the same peptide spacer; and (b) differences in the ability of purified lysosomal enzyme and a mixture of lysosomal enzymes obtained from different sources to degrade the same peptide spacer bearing the same terminal drug.

(Trouet, A. (1974) Methods in Enzymology, (Fleisher, S. and Packer, L. Eds.), Vol. XXXI, pp. 323-329, Academic Press, New York. EDTA = ethylenediaminotetraacetic acid, Triton X- 100 = quaternary ammonium surfactant).

Examples 29 to 34

Influence of specific determinants.

Example 29

Influence on polymer capture rate by melanoma cells in vitro.

an analogue containing a hormone-stimulating determinant from a spacer-stimulating hormone was -Gly-Gly-HPMA melanocytes (MSH) were prepared as α-malanocytes; peptide in accordance with the invention. The analog was of the branched type (via MSH) and the non-32EN 278551 B6 contained a biologically active component. The analog preparation was labeled with ¹²⁵ I radionuclide and its ability to bind mouse melanoma cells. M3 591 screen was compared to that of a control HPMA polymer containing tyrosinamide substituents and no determinate substituents. Three culture samples were inoculated 2. 10 ⁶ cells and incubated for three days before the experiment. 100 μám of each polymer was added to the cultures and incubated for up to 60 minutes. At given intervals, the cultures were terminated and a sample was taken from the medium; the total number of cells and the number of cells associated with radioactivity were determined.

The results are graphically depicted in Figure 7 of the accompanying drawings, in which curves were plotted by capture in μΐ medium per mg cell protein versus time in minutes. The results show that the analog containing MSH showed higher melanoma cell binding compared to the control polymer.

Example 30

Influence on the rate of polymer consumption by human fibroser in vitro.

HPMA analogue containing apotransferin (75 μΐ, 580 μg / ml) was radiochemically labeled with the iodogenic method; -Gly-Gly- was used as a spacer to the determinant in accordance with the invention. The substance did not contain a biologically active component. Chromatography showed that the analog had a molecular weight of about 260,000, corresponding to the formula polymer (apotransferin) ₃ .

After four days of cultivation of the subculture (cell confluent), fibroblast cells (human skin) were washed and incubated for a predetermined time in Hank's balanced salt solution (3 ml) containing a radionuclide-labeled analog (200 μΐ, ^ g / ml jr). Cell counts were determined in environmental samples (1 mL) in two separate groups to differentiate between binding and internalization, the group was digested with trypsin to remove ligand binding to the transferrin receptor, and Group 2 was not treated in this way. The capture rate was compared to a control HPMA polymer containing -Gly-PhePhe-Leu-aminopropanol substituents and no determinant substituents.

The results are shown graphically in Figure 8 of the accompanying drawings, in which the curves were obtained by plotting capture in μΐ medium per mg cell protein versus time in hours. The results show that the apotransferin-containing polymer exhibited increased internal and binding internalization to human fibroblasts compared to control polymers.

Example 31

Effect on polymer capture by human hepatoma in vitro

A series of HPMA analogs containing galactose as a specific determinant in which galactose was bound directly were prepared

-33GB 278551 B6 to the polymer chain via an ester bond. The analogues contained increasing amounts of galactose in the range of 0 to 99 moles. %; biologically active components were not present. Analogs were labeled with ¹²⁵ L with chloramine T and tested for their ability to penetrate human hepatoma (Hep G2) cells in vitro. Cells were grown in monolayer cultures in EMEM containing 10% fetal bovine serum. Three cultures were inoculated with 2. 10 ⁶ cells and incubated 3 days before use for the experiment. Cells were incubated with each analog (100 μΐ) for 15 seconds. A sample of the medium was then taken and the monolayer cells were washed extensively. 2.5 ml of M NaOH was added to dissolve the cells and samples were taken to determine the radiolabel and total protein content.

The results are graphically depicted in Figure 9, in which the curves were plotted by capture in% counts x 10 ³ mg cell protein versus mol. % galactose in each analog. The results show that capture of HPMA galactose copolymers with human hepatoma is proportional to moles. % galactose present.

Example 32

Effect on L1210 mouse leukemia activity in vitro.

The experiments were performed with two HPMA copolymers, one of which is the subject of the present invention. Both compounds contained daunomycin as a biologically active component with -Gly-Phe-Leu, Gly as a spacer drug. The compound of the invention further contained fucosylamine as a determinant and -Gly-Phe-Leu-Gly- as a spacer determinant.

The experimental procedure was the same as in Example 22.

The results are graphically shown in Figure 10, in which the curves were obtained by plotting the percentage cell growth versus daunomycin concentration in µg / ml. The results indicate that the cytotoxicity of the polymeric drug to murine L1210 leukemia in vitro will increase significantly when fucosylamine is present in the molecule as a determinant.

Example 33

Effect on polymer targeting in vivo.

Two HPMA copolymers were prepared having the following structure and composition:

-Tyr-NH ₂ (1.7 mol%)

-Gly-Phe-Leu-Gly-Daunomycin, (3.5 mol%)

Polymer 1

-Tyr-Nri ₂ (1.8 mol%)

-Gly-Phe-Leu-Gly-Daunomycin (2.2 mol%)

-Gly-Phe-Leu-Gly-galactosamine (1.9 mol%)

Polymer 2

-34GB 278551 B6

Polymer 2 is an object of the present invention, polymer 1 is not.

Both HPMA polymers were labeled with ¹²⁵ I radionuclide with chloramine T. This radiolabel is stable in the biological environment and therefore allows the fate of the polymer to be traced in vivo.

The polymer (approximately 100 µg in 0.1 ml buffer) was injected into the femoral vein of a male Wistar rat (250-350 g), anesthetized, and after 30 minutes the animal was sacrificed and the radioactivity distribution measured in its body.

The results obtained are shown in Table 2

Table 2

Division in the body % of measured radioactivity Polymer 1 Polymer 2 Spleen 0.51 0.19 Kidneys 4.39 4.83 Lung 1.34 1.37 Liver 4.97 23.20 Blood 88.77 70.38

The results indicate that attachment of even a small amount of galactosamine (1.9 mol%) effectively directs a significant portion of the HPMA copolymer to rat liver within 30 minutes of intravenous administration.

Example 34

Effect on L1210 mouse leukemia activity in vivo.

The experiments were carried out with two HPMA analogues, one of which is the subject of the present invention. Both analogs contained daunomycin as a biologically active component, -Gly-Phe-Leu-Gly- as spacer drugs and tyrosinamide residues. The polymer of the invention still contained fucose as a determinant; The determinant spacer was -Gly-PheLeu-Gly-. The detailed structure of the polymers was:

S-Gly-Phe-Leu-Gly-Daunomycin-Gly-Phe-Leu-Gly-Daunomycin

-Tyr-NH ₂ (-Gly-Phe-Leu-Gly-Fucose <-Tyr-NH ₂

Polymer 1 ‘Polymer 2

L1210 mouse leukemia was inoculated (10 ⁵ viable cells) intraperitoneally with DBA ₂ mice on day zero. The animals were then divided into two groups, which were treated separately. Animals of the first group were treated with polymer 1, the animals of the second group were treated with polymer 2; a dose equivalent to 7.5 g of daunomycin / kg was administered in both groups. The survival of the animals in both groups was recorded.

-35GB 278551 B6

The results showed that animals in Group 2 treated with the polymer of the invention survived at least twice as long as animals in Group 1 treated with the polymer without a determinant; Thus, in one series of experiments, 57% of animals survived long term in Group 2, compared to Group 1, where only 25% of animals survived long term.

Example 35

Effect of different peptide spacers on the same drug and specific determinant.

The experiments were carried out with two HPMA copolymers, both in accordance with the present invention. Both copolymers contained daunomycin as a biologically active component and anti-O antibody as determinant. For each of the polymers, the same peptide spacer was used for both drug and determinant, but spacers differed in both polymers. The detailed structure of the copolymers was:

-Gly-Phe-Leu-Gly-Daunomycin

-Gly-Phe-Leu-Gly anti-O antibody

Polymer 1

-Gly-daunomycin

-Gly-Gly-anti-O antibody

Polymer 2

The activity of the copolymers was tested against T cells in vivo by measuring the decrease in anti-sheep red blood cell response in mice. The results obtained are shown in Table 3.

Table 3

Polymer 1 Polymer 2 Total dose of anti-0 antibody per mouse (mg) 3.0 3.0 Total dose of daunomycin per mouse (mg) 0.4 0.6 % immune response suppression 98 0

As can be seen from the results, polymer 1 containing -Gly-PheLeu-Gly-spacers to the drug and the determinant is considerably more effective at killing T cells than the corresponding polymer 2 containing -Gly-Gly-spacer.

Claims (7)

A polymeric drug consisting of an inert synthetic polymeric carrier based on repeating units of N- (2-hydroxypropyl) -methacrylamide, which is linked to a biologically active molecule by means of amino acid or peptide spacer and optionally linked to a targeting unit by the same or other spacer; which is optionally crosslinked, characterized in that it consists of (a) 5.0 to 99.7 mol. % of units derived from N- (2-hydroxypropyl) methacrylamide of formula ch ₃ CH ₉ II ^z OH II C - CO - NH - CH ₂ - CH - CH ₃ (b) 0.2 to 20.0 mol. % units derived from the N-methacryloylated peptide of formula CH ₉ II CH ₃ -C-CO- [NH-R-CO] - [B] wherein - [NH-R-CO] is a residue derived from Gly-Gly, Gly-Phe-Gly, Gly-Phe-Phe, Gly-Leu -Gly, Gly-Val-Ala, Gly-Phe-Ala, Gly-Phe-Ala, Gly-Phe-Tyr, Gly-Phe-Tyr, Gly-Phe-Tyr , Gly-Phu-Le-Gly, Gly-Phe-Tyr-Ala, Gly-Phe-Gly-Phe, Gly-Phe-Gly-Phe, Gly-PheLeu-Gly-Phe, and Gly -Gly-Phe-Leu-Gly-Phe, and - [B] is a bioactive molecule and (c) 0.1 to 94.8 mol% units of the formula CH ₃ - C - CO - NH - [D] or CH _O II CH ₃ - C - CO - [D] or CH ₃ - C - CO - [NH - R - CO] - D -37GB 278551 B6 where - [D] is a determinant and - [NH-R-CO] - is a residue derived from Leu, Phe, Gly-Gly, Gly-Leu-Gly, Gly-Val-Ala, Gly-PheAla, Gly-Leu-Phe, Gly-Phe-Leu-Gly, Gly-Phe-Leu-Gly, Gly-Phe-Leu-Gly, Gly-Phe-Tyr-Ala Phe-Gly-Phe, Ala-Glyr-Val-Phe, Gly-Phe-Phe-Gly, Gly-Phe-Leu-Gly-Phe, Gly-Gly-Phe-Leu-Gly-Phe. Polymeric drug according to claim 1, characterized in that it comprises (d) up to 5 mol. % units of the formula where - [NH-R-CO] is a residue derived from Gly-Leu-Gly, Gly-ValAla, Gly-Phe-Ala, Gly-Leu-Phe, Gly-Leu-Ala, Ala-Val-Ala Gly-Phe-Leu-Gly, Gly-Phe-Phu-Leu, Gly-Phe-Gly-Ala, Gly-Phe-Gly-Phe, Gly-Phe-Gly-Phe, Gly-Phe -Phe-Gly, Gly-Phe-Leu-Gly-Phe and Gly-Gly-Phe-Leu-Gly-Phe, and - [L] - is a linking group. Polymeric drug according to claims 1 and 2, characterized in that it comprises (e) as units capable of radiolabeling up to 2 mol% of the units of the formula CH, CO II I CH ₃ - C - CO - NH - CH - CH ₂ - C ₆ H ₄ - OH Polymeric drug according to claims 1 to 3, characterized in that the determinant is selected from the group consisting of a monosaccharide, a disaccharide or an oligosaccharide, bound by an amide or ester bond, an antibody, a hormone, or another protein. The polymeric drug of claim 4, wherein the carbohydrate determinant is selected from the group consisting of galactose, galactosamine, glucosamine, mannosamine or fucosylamine. Polymeric drug according to claims 1 to 3, characterized in that the determinant is an IgG or an anti-O-antibody. The polymeric drug of claims 1 to 6, wherein the biologically active molecule is selected from the group consisting of antibiotics, cytostatics, immunosuppressants, antiparasitics, bacteriostats, anti-inflammatory agents, cardiovascular agents and psychopharmaceuticals. -38GB 278551 B6 The polymeric drug of claim 7, wherein the biologically active molecule is selected from the group consisting of daunomycin, puromycin, adriamycin, melphalan isopropyl ester, bleomycin, desferoxamine. bestatin and tritylcysteine. Polymeric drug according to claims 2 to 8, characterized in that the linking group L is of the type -A -NH (CH ₂ ) _X NH -A-, wherein x is an integer from 1 to 12 and A is an amino acid, and is attached to adjacent peptide groups by amide bonds. A polymeric drug according to claim 9, wherein x is 6 and amino acid A is Phe, Tyr, Ala, Gly or Leu. A polymeric drug according to claim 9, wherein x is 2 and amino acid A is Ala. Polymeric drug according to claims 1 to 11, characterized in that it contains daunomycin as the biologically active molecule, the determinant is selected from the group consisting of galactose, galactosamine and fucosylamine, and wherein, when the compound is crosslinked, the crosslinking spacer is Gly- Phe-Leu-Gly and - (Phe) NH (CH ₂ ) g NH (Phe) - linker. Process for the preparation of a polymeric drug according to claims 1 to 12, characterized in that the monomers selected from the group consisting of N- (2-hydroxypropyl) methacrylamide, p-nitrophenyl ester of N-methacryloylated peptide are subjected to radical solution copolymerization or radical precipitation polymerization. , and the resulting copolymer is sequentially coupled with a biologically active molecule and, optionally, a polymer-analogous reaction with a reactive determinant, and optionally reacting with the diamine crosslinker by aminolysis in solution. according to a claim selected from the group of methacrylamide, 13. A process for the preparation of a medicament by polymerizing N-2 (hydroxypropyl) N-methacryloylated peptide monomers with an N-methacryloylated determinant and reacting the resulting copolymer with a biologically active molecule and optionally a diamine crosslinker. to form ester or amide bonds. Method according to claims 13 and 14, characterized in that the copolymerization mixture contains N-methacryloylated tyrosinamide as a radiolabelled substance. A pharmaceutical composition comprising at least one polymeric drug according to items 1 to 12 and an inert physiologically acceptable diluent. 14. The pharmaceutical composition of claim 16 wherein the carrier is a sterile aqueous medium and the composition is formulated for injection.