SK278506B6 - THE POLYMERIC DRUG AND PREPARATION METHOD THEREOF m cells. - 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

Technical field

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

BACKGROUND OF THE INVENTION

Many low-molecular-weight drugs used in chemotherapy penetrate rapidly into a wide variety of cell types by random diffusion across the cell membrane. This lack of selectivity reduces their applicability to the desired target tissue and sometimes causes undesirable side effects. The attachment to the cell is rapid, so that the therapeutic effect does not last long. In addition, the drug can be rapidly removed from the bloodstream by glomerular filtration.

The covalent attachment of low molecular weight bioactive molecules to a soluble carrier polymer prevents both glomerular filtration and cell penetration by simple diffusion. The entrapment is restricted to cells capable of absorbing substrates by a selective mechanism known as pinocytosis, whereby a portion of the cell-bounding membrane extends inside the cell, absorbing the macromolecule. Then, a portion of the cell membrane is detached inward to form an intracellular organelle containing the entrapped material.

This difference in the mechanism of capture provides a potential method of allowing drugs to be directed specifically to those cells where a therapeutic effect is desired.

Another difference lies in the different fate of the two types of drugs upon penetration into the mtracellular space. Small molecules that enter cells due to diffusion tend to penetrate into all parts of the cell, while macromolecules after pinocytosis are transported in intracellular organelles (pinosomes) directly to the lysosome compartment where a number of hydrolytic enzymes are located.

Thus, pinocytic capture of a polymeric drug that contains a drug-carrier linkage that is subject to hydrolysis in lysosomes provides a mechanism for the controlled intracellular release of a bioactive molecule that results in it appearing 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 Tailored" Prepared Soluble Polymeric Carriers "in" Recent Advances in Drug Delivery Systems "(Plenum Press, 1984).

Two criteria must be met when designing such a system. Firstly, it is desirable to design a drug-carrier linkage capable of undergoing controlled lysosomal hydrolysis but at the same time resistant to the effects of enzymes in the bloodstream. Second, the polymeric drug must be capable of specifically being captured in those target cells where a therapeutic effect is desired, while at the same time being minimally captured in other cells.

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

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 preferably undergoes lysosome hydrolysis to facilitate secretion of the polymer from the body; (b) it must contain a degradable bond between the drug and the carrier resistant to extracellular hydrolysis but subject to controlled lysosome hydrolysis; determinant.

Such a molecule can be represented schematically as follows:

(D) specific determinant © drug attached by biodegradable bond optional biodegradable crosslink in polymer carrier

Although natural macromolecules have been used as carriers, synthetic polymers provide the advantages that the molecular weight is easier to adapt to achieve optimal cell selectivity and, unlike many natural macromolecules, are not immunogenic. 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 USP 4062831 and 4097470, these polymers are water-soluble and have good biocompatibility.In addition, due to the incorporation of n-methacryidovyligigopeptide p-nitrophenyl esters, Polymer chains can be crosslinked below the gelatinization point to achieve optimal molecular weight and, due to the presence of biodegradable cross-links, to provide a polymer degradation method facilitating 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 amide linkage would provide the possibility of lysosomal hydrolysis. However, this is not the case in practice. Peptide spacers located between the drug and the carrier have been found to be subject to degradation by the action of lysosomal enzymes at 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, e.g. j. the rate of drug release greatly depends on the number and nature of the amino acid residues in the peptide spacer. In general, spacers containing less than three amino acids were not subject to 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.

Modification of glycoproteins leading to the formation of galactose-terminated oligosaccharide side chains has been shown to lead to a steep 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 the targeted action of drugs in the case of hepatoma, i. j. a type of cancer that is particularly difficult to treat. Moreover, galactosamine bound to HPMA copolymers by amide bond provides a similar result and thus suggests that receptors on hepatocyte membranes recognize galactose units not only when bound by glycosidic bond, but also when these units are present as Nacylgalactosamine. Many other "recognition" systems are known, such as the Nacetylglucosamine / mannose "recognition" system of Kupffer cells and macrophages, and the phosphohexose "recognition" system of vibroblastomas.

Another possible targeted 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 bound 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 polymer "intermediate chain" between the drug and the polymer has been shown to greatly simplify these problems.

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

As mentioned earlier, the rate of lysosomal hydrolysis of a peptide spacer depends on the number and nature of the amino acid residues. This is reflected in both spatial and structural factors. 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 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. The part of the enzyme where the substrate interacts 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 catalyzes the reaction, such as cleavage. Investigation of the 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 a single bond in the peptide chain depends not only on the nature of the structure close to the cleaved bond, but also on the nature of the amino acid residues relatively distant from the cleavage bond, but important in maintaining the enzyme at a certain position during hydrolysis. The detailed structure of the lysosome active sites hinders the preparation of peptide spacers that are subject to lysosome hydrolysis at a suitable rate and are intended for use in polymeric drugs.

SUMMARY OF THE INVENTION

The invention is based on the discovery of peptide sequences that can act as spacers capable of lysosomal hydrolysis at a satisfactory rate to allow controlled intracellular drug release but which are further resistant to extracellular hydrolysis and can also be used as binding for specific determinants. Other systems have been developed to allow easy cleavage of the peptide cross-linker. Hydrolysis of these bonds is particularly sensitive to steric hindrance due to polymer chains, more than hydrolysis of terminal units from peptide side chains.

The present invention provides a polymeric drug consisting of an inert synthetic carrier linked via amino acid or peptide spacer to a bioactive molecule and a specific determinant, which may contain cross-links. This polymeric drug comprises:

(a) as polymeric carrier from 55 to 99.7 mol. % of polymerized N- (2-hydroxypropyl) methacrylamide units; (b) as peptide spacers of from 0.2 to 20 mol. % of units consisting of Gly-Leu-Gly, Gly-Phe-Ala, Gly-Phe-Ala, Gly-Leu-Phe, Gly-Leu-Ala, Gly-Phe-Leu-Gly, Gly- Phe-Phe-Leu, Gly-Phe-Tyr-Ala, Gly-Phe-Gly-Phe, Gly-Phe-Gly-Gly, Gly-Phe-Gly, Gly-Phe-Gly, Gly-Phe- Leu-Gly-Phe or Gly-Gly-Phe-Leu-Gly-Phe terminated by a bioactive molecule bound by an ester bond or an amide bond to a peptide spacer.

(c) as an amino acid or peptide spacer of from 0.1 to 20 mol. % units consisting of Leu-Phe, Gly-Gly, Gly-Leu-Gly, Gly-Leu-Phe, Gly-Leu-Phe, Gly-Leu-Phe Val-Ala, Gly-Phe-Leu-Gly, Gly-Phe-Phu-Leu, Gly-Phe-Tyr-Ala, Gly-Phe-Gly-Phe, Gly-Phe-Gly-Phe, Ala-Gly-Val- Phe, Gly-Phe-Phe-Gly, Gly-Phe-Leu-Gly-Phe or Gly-Gly-Phe-Leu-Gly-Phe terminated with a specific determinant capable of reacting with specific cell surface receptors, (d) optionally up to 5 mol. % crosslinks containing peptide spacers, consisting of Gly-Leu-Gly, Gly-Val-Ala, Gly-Phe-Ala, Gly-Leu-Phe, Gly-Leu-Ala, Ala-Val-Ala, Gly-Phe-Leu -Gly, Gly-Phe-Phe-Gly, Gly-Phe-Tyr-Gly, Gly-Phe-Gly-Phe, Gly-Phe-Gly-Phe , Gly-Phe-Leu-Gly-Phe or Gly-Gly-Phe-Leu-Gly-Phe attached to a linker that is attached to a similar peptide spacer attached to another polymer chain and (e) optionally as radioactive labeling units up to 2 mol. % of units containing an amide bonded tyrosinamide.

The bioactive molecule found in the polymeric drug is preferably daunomycin, puromycin, adriamycin, melphalan (sarcolysin), IgG (immunoglobulin), bestatin or tritylcysteine.

The determinant used is preferably a monosaccharide or oligosaccharide, which is preferably bound amido 3 enzymes has not yet been determined, as shown by binding to the polymer chain directly or via an amino acid or peptide spacer or antibody. Particularly preferred determinants are galactosamine glucosamine or mannosamine.

The linker has the preferred composition - (amino acid-NH (CH ₂ x NH- (amino acid)) - where x is an integer from 1 to 12 and is attached to adjacent peptide spacer amide bonds. Particularly preferred linking groups are those in which x is 6 and the amino acid is Phe, Tyr, Ala, Gly or Leu, and those in which x is 2 and the amino acid is Ala.

The polymeric drug of the invention may further comprise as radiolabelled units up to 2 mol. % of units containing tyrosinamide bonded to the polymer chain via an amide bond,

A particularly preferred embodiment of the invention comprises single or cross-linked chains of a specified polymer with daunomycin as a bioactive molecule, galactosamine as determinant, Gly-Phe-Leu-Gly as a peptide spacer between polymer carrier and daunomycin as well as galactosamine and optionally with Gly-Phe-Leu-Gly as the peptide spacer contained in the crosslink and - (Phe) -NH (CH ₂ ) ₆ NH- (Phe) - as the linker.

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

Since the same peptide-spacer can be used for both the bioactive molecule and the determinant, and the preparation of these compounds is easier than those in which the spacer is of different species, we will first describe the synthesis procedure 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 peptide p-nitrophenyl ester (incorporation of N-methacryloyltyrosinamide is also possible to obtain a radiolabelled unit) to yield a "polymer precursor" containing terminal p-nitrophenoxy end groups on the peptide side chains. in the next coupling reaction. The second step involves the sequential addition of a reactive drug, a reactive determinant, and optionally a diamine crosslinker. When the polymeric drug carrier is desired in the form of a single chain, the drug and the determinant are sequentially added to the polymeric precursor in any order. If a crosslinked product is desired, then a suitable crosslinking agent, drug and determinant are added sequentially to the polymer precursor, again in any order.

This can be illustrated as follows:

HPMn <MA "W ^Cr, p

For example, two synthetic procedures can be used.

For those cases where the drug and determinant binding moieties contain common amino acid residues 5 adjacent to the carrier, the polymer precursor containing these amino acids is sequentially reacted with the amino acid or peptidyl derivative of the drug, the amino acid or peptidyl derivative of the determinant, and optionally, if desired. crosslinked product, with diamine crosslinker.

HPKA + MA - W ~ OHp

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 polymeric precursor by copolymerization. The polymer precursor is then reacted with the reactive drug and optionally when a crosslinked product with a diamine crosslinker is desired.

This procedure is plural as follows:

It is also possible to prepare polymeric precursors similar to those described but containing peptide side chains terminated not by p-nitrophenyl ester groups but by carboxyl groups. In the presence of a suitable catalyst, it is possible to attach reactive drugs and determinants 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 having terminal p-nitrophenyl ester groups, i. j. the reactive molecule is bound to the side chain via an amide bond. However, polymeric precursors containing terminal carboxyl groups as opposed to precursors containing terminal p-nitrophenyl ester groups 4

SK 278506-6

In cases where the drug and the determinant do not have reactive hydroxy groups to form products in which the reactive molecule is bound to the side chain by an ester bond.

Polymeric side chain precursors terminated with carboxyl groups can be prepared by (a) basic hydrolysis of the corresponding polymeric side chain precursor terminated with the p-nitrophenyl ester groups described above, (b) copolymerization of HPMA by any of the synthetic processes described above terminal p-nitrophenyl ester groups, but using unesterified N-methacryloylated peptide as a comonomer. The resulting copolymer is then capable of reacting with a reactive drug and a reactive determinant.

Methods by which polymorphic precursors with side-chain terminated carboxyl groups can be prepared can be illustrated as follows:

Suitable catalysts for the addition of a reactive drug or determinant to carboxyl-containing polymeric precursors include carbodiimides such as dicyclohexylcarbodiimide in organic solvents and N-cycohexyl-N '- (2-morpholinoethyl) carbodiimide metho-p-toluenesulfonic acid and wood-solvent solutions. , (N-ethyl-5-phenylisoxyzolium-3'-sulfonate).

The order in which some or all of the reactive determinants of the reactive drug and the crosslinking agent is 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, in the range already mentioned. The total molecular weight is determined by the polymerization conditions, particularly the initiator concentration, the presence of transporters, and the concentration of 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 in the range of 10,000 to 500,000, more preferably 30,000 to 400,000.

The starting materials required for carrying out the described syntheses are either commercially available or can be prepared by general methods known to those of skill in the art.

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, also reacting with drugs and determinants the contained tyrosinamide for radioactive purposes can be obtained in the same manner).

(ii) The N-methacryloylated dipeptide is then esterified with p-nitrophenol as described, for example, in Macromol. Chem. 178, 2169 (1977). (Methacryloylated p-nitrophenyl esters of Leu and Phe, providing amino acid spacers suitable for use with a determinant, may be prepared in the same manner).

(iii) The peptide chain of the N-methacryloylated dipeptidyl-p-nitrophenyl ester is then elongated (unless the dipeptide is Gly-Gly, where the dipeptide itself is one of the spacer that can be used for the determinant) by attaching a peptide containing the necessary amino acid residues of the desired peptide sequence, as described, for example, in Makromol. Chem. 182, 1917 (1981).

(iv) The N-methacryloylated pcptid obtained from (iii) can be copolymerized with HPMA to form a polymeric side chain precursor containing terminal carboxyl 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 the determinant are to have different spacer, it may be reacted with a reactive determinant to form an N-methacryloylated oligopeptide determinant and then copolymerized with the N-methacryloylated ester and HPMA as described, for example, in Biochem. Biophys. Acta, 755,518 (1983).

The preparation of N- (2-hydroxypropyl) methacrylamide is described in Angew. Dep. Chem. 70, 109 (1978); reactive drugs and determinants are mostly 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 can be prepared as described, for example, in Makromol. Chem. 182, 1899 (1981).

DETAILED DESCRIPTION OF THE INVENTION

The following are non-limiting examples of embodiments of the invention.

Example 1

Preparation of single-chain polymers containing daunomycin, tayrosinamide and galactosamine!

-C, 1y-Fhe-Leti-Sly-Cl »c

T.ilaktč’as: -

A copolymer of HPMA, Methyrosinamide and Ma-Gly-Phe-Leu-Gly-ONp (0.1 g, 2.85 x 10 ^-5 moles -ONp groups) was dissolved in dimethylsulfoxide (0.4 mL) and a solution was added. daunomycin hydrochloride (8.5 mg, 1.5 x 10 ^-5 moles in dimethylsulfoxide (0.1 ml) followed by addition of triethylamine (1.5 mg, 1.5 x 10 ^-5 moles)) was stirred at room temperature 90 minutes and after

SK 278506-6

Dep. Chem. 177, 2833 (1976). (N-methacrylic (6.5 mg, 3 x 10 ^-5 moles) in dimethylsulfoxide (0.1 ml) followed by addition of triethylamine (3.0 mg, 3.0 x 10 ^-5 moles)) The mixture was stirred for 16 hours at room temperature and then poured into acetone / diethyl ether (9: 1) The precipitated polymer was filtered off, washed thoroughly with acetone and diethyl ether and dried under vacuum The polymer was purified by dialysis in a dialysis tube against established acid and then against water. isolated by lyophilization and contained 2.17 mol% of daunomycin-terminated side chains, ie 7.5 wt% of active product and 1.8 mol% of galactosamine-terminated side chains, ie 2.0 wt% of galactosamine.

Example 2

Preparation of a cross-linked polymer containing daunomycin and galactosamine.

Gly-Phe-Leu-Gly-ONp

1) Daunoaycía

2) H (Phe) HH (CH 2) eNB (Phe) H, 3) Galtosanine

I-Gly-Phe-Leu-Gly-daunoinycin (

-Gly-Pie-Leu-Gly-galactosamine /

Gly-Phe-Leu-Gly-Phe-KH (CH2) e-NH-Phe-Gly-Leu-Phe-Gly-j

HPMA and MA-Gly-Phe-Leu-Gly-ONp copolymer (0.2 g, 5.9 x 10 ^-5 moles of ONp groups) were dissolved in dimethylsulfoxide (0.8 mL) and daunomycin hydrochloride solution (16.2 mL) was added. mg, 2.85 x 10 ^-5 moles) in dimethylsulfoxide (0.2 ml) followed by the addition of triethylamines (2.85 x 10 ^-5 moles). The mixture was stirred at room temperature for 60 minutes and then a solution of N, N'-bis (H-Phe) -hexamethylenediamine (5.74 mg, 1.4 x 10 ^-5 moles) in dimethylsulfoxide (0.05 mL) was added. The mixture was stirred for an additional 10 minutes and then a solution of galactosamine hydrochloride (25.44 mg, 1.18 x 10 ^-4 moles) in dimethylsulfoxide (0.4 mL) was added followed by the addition of triethylamine (11.94 mg, 1.18 x 10 ⁴ moles). The mixture was stirred at room temperature for 18 hours and then poured into acetone / diethyl ether (9: 1). The precipitated polymer was filtered off, washed thoroughly with acetone and diethyl ether, and dried under vacuum. The polymer was purified by dialysis in a dialysis tube against dilute acid and then water. Pure polymer was isolated by lyophilization.

Example 3

The preparation of a polymer comprising a single chain of IgG (immunoglobulin) s ^β s> Gly-Gly-QH ----------------- * 5 E-Gly-Gly-IgG

HPMA and MA-Gly-Gla-OH copolymer (15.0 mg, 5.0 x 10 ^-6 moles of COOH groups) was dissolved in Sorensen buffer pH 5 (75 μ () and N-cycohexyl-N '- (2- morpholinoethyl) carbodiimide (0.002 mg) in the same buffer (40 μΐ) was added at 1 ° C. The mixture was stirred for 15 minutes and then an IgG solution (6.0 mg) in the same buffer (235 μΐ) was slowly added. The mixture was stirred for 5 hours and then allowed to stand overnight at 4 ° C. The crude reaction mixture was dialyzed against phosphate buffered, and galactosamine hydrochloride solution was added. Example 4

Release of daunomycin and puromycin from HPMA-based polymeric drugs by lysosome enzymes in vitro.

Three polymeric drug analogs of the compounds of the invention were prepared. Analogon 1 was a single chain HPMA polymer carrying -Gly-Phe-Leu-Gly-daunomycin and tyrosinamide substituents but containing no specific determinants, analogon 2 was a single chain HPMA polymer carrying a -Gly-Phe-Leu-Gly-puromacin and tyrosinamide substituents but do not contain any specific determinants; analogon 3 was a single-chain HPMA polymer carrying Gly-Phe-Phe-Leu-daunomycin and tyrosinamide substituents but containing no specific determinants. The peptide spacer of analogue 3 is not in accordance with the invention.

The analog (25 mg in 1.5 ml) was incubated with 1 ml of enzyme solution at 37 ° C. The enzyme solution contained either a mixture of isolated lysosomal enzymes from rat liver prepared according to the method of Trouet et al. (1974) in buffer pH 5.5 containing 1 mM EDTA, 5 mM reduced glutathione and 0.2% Triton X-100 ^(R) (a surfactant) or a purified lysosomal enzyme, cathepsin L (3, 8 x 10 ^-7 mol / L) in phosphate buffer pH 6.0 containing 5 mM reduced glutathione and 1 mM EDTA.

The concentration of free drug was determined at various time intervals by extracting it and spectrophotometrically determining the absorbance at a suitable wavelength. Incubation samples (0.15 mL) were taken and added to 1.5 mL of buffer and 1.5 mL of ethyl acetate layer, and extinction was measured at 485 nm for daunomycin or 272 nm for puromycin.

The results are graphically depicted in Figure 1, where three curves (for analogs 1, 2, 3 in top-down order) were obtained by plotting the amount of drug released (in percent) originally present in the analogon versus time (in hours). The results obtained for the mixture of lysosomal enzymes are shown by open circles, the results obtained with purified enzyme Cepsin L represent solid circles.

The results show:

(a) differences in the release rate of puromycin and daunomycin from the same peptide spacer;

(b) the effect of altering the peptide spacer on the release rate of daunomycin (comparison of the upper and lower curves); and (c) differences in the ability of the purified lysosomal enzyme and the mixture of lysosomal enzymes (derived from different sources) to degrade the same peptide spacer.

(Trouet, A (1974)) Methods in Enzymology, (Flieisher, S &amp; Packer, L., Eds), Vol. XXXI, Hs. 323-329, Academic Press, New York.

EDTA = ethylandiaminetetraacetic acid

Triton-100 = surfactant containing quaternary ammonium groups

Example 5

In vitro cytotoxicity of N- (2-hydroxypropyl) methacrylamide polymeric drugs and analogues thereof against mouse leukemia (L 1210) These experiments were carried out using the polymeric drug of the invention

2786 saline solution (pH 7.2).

do not contain specific determinants; all of these compounds were of the single chain type. The detailed composition was as follows:

(A) HPMA copolymer containing -Gly-Phe-Leu-Gly-daunomycin, -Gly-Phe-Leu-Gly-galactosamine and tyrosinamide in the side chains;

(B) HPMA copolymer containing -Gly-Phc-Leu-Gly-daunomycin and tyrosinamide in the side chains;

(C) HPMA copolymer containing -Gly-Phe-Phe-Leu-daunomycin and tyrosinamide in the side chains;

(D) HPMA copolymer containing -Gly-Phe-Leu-Gly-puromycin and tyrosinamide in the side chains;

(E) HPMA copolymer containing -Gly-Leu-Phe-sarcolysin in the side chains;

(F) HPMA copolymer containing -Gly-Phe-Leu-tritylcysteine in the side chains;

Mouse leukemia, L 1210, was maintained in suspension culture in RPMI 40 medium containing 10% heat inactivated horse serum. Under these conditions, the number of cells doubled in 15 to 20 hours to a cell density of approximately 1 x 10 ⁵ cells / ml. Cell densities were determined using a Coulter counter.

To assess the cytotoxicity of the polymeric drugs, the cells were transferred to tubes (10 ml) and their initial density was measured. Different concentrations of different polymeric drugs were added and the cells were maintained in a thermostatic growth cabinet for an additional 72 hours. Their density was then measured.

The number of cells found in tubes containing polymeric drug was expressed as the percentage of cells found in tubes incubated at the same time (72 hours) without any additives; the results obtained are shown in FIG. 2 in the accompanying drawings, the upper group of curves referring to compounds (A), (B), (C), and the lower group to compounds (D), (E), (F). In each group of curves, the percentage of cells surviving for 72 hours is plotted depending on the concentration of polymer drug in mg / ml.

From these results it can be concluded that all daunomycin-containing polymeric drugs (A), (B), (C) were cytotoxic to murine L 1210 in vitro. Polymer drug (A) containing both daunomycin and galactosamine was able to inhibit cell growth. It can also be seen that amino acid sequence variations in the peptide spacer alter the degree of cytotoxicity of the preparations.

The results obtained with (D), (E) and (F) show that confusion of daunomycin with puromycin, sarcolysin (melphalan) or tritylcysteine provides polymeric drugs that are almost completely ineffective using the peptide spacer.

Example 6

Targeted HPMA copolymer containing daunomycin to rat liver hepatocytes.

Two HPMA-based polymeric drugs having the structures shown below were prepared. Polymer 2 is prepared according to the invention, polymer 1 is not.

j-TyrNH 2 (1.7 nol *) 1-Tyr 1 TH 2 (1.8

J-Gly-Phe-Leu-Gly-Daunonycin> Gly-Phe-Leu-Gly-Daunomycin (3.5 mol *) \ (2.2 mol *)

J-Gly-Phe-Leu-GLy-phthalactosamine (1.9 moles *) polymer 1 polymer 2 (A) and with five analogs (B), (C), (D), (E), and (F), which ones

Both HPMA copolymers were radiolabelled with the ¹²⁵ I chloramine method. This radiolabel is stable in the biological environment and therefore allows the fate of the polymer to be traced in vivo.

The polymer (about 100 µg in 0.1 ml buffer) was anesthetized by syringe injection into the femoral vein of a male Wistar rat (250-300 g); after 30 minutes the animal was sacrificed and the distribution of radioactivity in the body was measured.

The results are shown below.

Distribution in the body (30 min) % radioactivity Polymer 1 Polymer 2 spleen 0.51 0.19 kidney 4.39 4.83 lungs 1.34 1.37 liver 4.97 23.20 blood 88,77 70.38

From these results, it is evident that the introduction of even a small amount of galactosamine (1.9 mol%) into the HPMA copolymer will result in its targeted transport to the rat liver within 30 minutes after intravenous administration.

Claims (12)

A polymeric drug, comprising an inert synthetic polymeric carrier, linked by amino acid or peptide spacer to a bioactive molecule, a specific determinant, and an optional cross-link, comprising (a) as polymeric carrier 55 to 99.7 moles. % polymerized N- (2-hydroxypropyl) methacrylamide units. (b) as a peptide spacer of 0.2 to 20 mol. % units consisting of Gly-Leu-Gly, Gly-Val-Ala, Gly-Phe-Ala, Gly-Leu-Phc, Gly-Leu-Ala, Gly-Phe-Leu-Gly, Gly -Phe-Phe-Leu, Gly-Phe-Tyr-Ala, Gly-Phe-Gly-Phe, Gly-Phe-Gly-Gly, Gly-Phe-Gly-Gly, Gly-Phe-Gly, Gly-Phe -Lcu-Gly-Phe terminated by a bioactive molecule, bound by an ester or amide bond to a peptide spacer, (c) as an amino acid or peptide spacer of 0.1 to 20 mol. % units consisting of Leu, Phe, Gly-Gly-Gly-Gly-Leu-Gly, Gly-Phe-Ala, Gly-Leu-Phe, Gly-Leu-Ala-, Ala-Val-Ala, Gly-Phe-Glu-Gly, Gly-Phe-Glu-Gly, Gly-Phe-Glu-Gly, Gly-Phe-Glu-Phe, Gly-Phe-Glu-Phe, Gly-Phe-Glu-Phe Phe-Phe-Gly, Gly-Phe-Leu-Gly-Phe or Gly-Gly-Phe-Leu-Gly-Phe terminated by a specific determinant capable of reacting with specific cell surface receptors, (d) optionally, as a cross-linking peptide spacer, up to 5 mol. % units, consisting of Gly-Leu-Gly, Gly-Phe-Ala, Gly-Phe-Ala, Gly-Leu-Phe, Gly-Leu-Ala, Gly-Phe-Leu-Gly, Gly -Phe-Phe-Leu, Gly-Phe-Tyr-Ala, Gly-Phe-Gly-Phe, Gly-Phe-Gly-Ge, Gly-Phe-Gly-Gly, Gly-Phe-Gly, Gly-Phc -Leu-Gly-Phe, or Gly-Gly-Phe-Leu-Gly-Phe attached to the connecting 7 And (e) optionally, as a radiolabelled unit, up to 2 mol% of a cell peptide attached to a similar peptide spacer attached to another polymer chain. % units containing 5 tyrosinamide amide bonded. The polymeric drug of claim 1, wherein the determinant is a monosaccharide or oligosaccharide. The polymeric drug of claim 2, wherein the saccharide determinant is galactosamine, glucosamine, or 10 manequins in. The polymeric drug of claim 1, wherein the determinant is an antibody. The polymeric drug of any one of claims 1 to 4, wherein the bioactive molecule is daunomycin, puromycin, adriamycin, melphalan (sarcolysin), IgG, bestatin or tritylcysteine. The polymeric drug of claims 1 to 5, wherein the linker is of the type - (amino acid) -NH (CH ₂ ) x x NH- (amino acid) - wherein x is an integer from 1 to 12 and which is connected to adjacent oligopeptide spacer by amide bonds. The polymeric drug of claim 6, wherein x is 6 and the amino acid is Phe, Tyr, Ala, Gly or Leu, or x is 2 and the amino acid is Ala. 25 The polymeric drug of claims 1 to 7, wherein the bioactive molecule is daunomycin, the determinant is galactosamine, and when the compound contains cross-links, the cross-linking peptide spacer is Gly-Phe-Leu-Gly and the linker is - (Phe) -NH ( CH ₂ ) ₆ NH- (Phe) -. 30 A process for the preparation of a polymeric drug according to claims 1 to 7, characterized in that N- (2-hydroxypropyl) methacrylamide is copolymerized with either the N-methacryloylated peptide or its p-nitrophenyl ester, wherein the resulting copolymer is reacted with a reactive bioactive molecule. , a reactive determinant, and optionally, a diamide crosslinker to form ester or amide bonds. A process for the preparation of a polymeric drug according to claims 1-7, characterized in that 40 is copolymerized with N- (2-hydroxypropyl) methacrylamide with an N-methacryloyl amino acid or peptide or their p-nitrophenyl esters, the resulting copolymer being reacted with a reactive bioactive molecule, a reactive determinant, and optionally, a diamine crosslinker to form ester or amide bonds. Process for the preparation of a polymeric drug according to claims 1 to 7, characterized in that N- (2-hydroxypropyl) methacrylamide is copolymerized with a polymerizable form of a determinant comprising a 50 N-methacryloyl amino acid or peptide and an N-methacryloylated peptide or p-nitrophenyl ester whereupon the resulting copolymer is reacted with a reactive bioactive molecule and optionally a diamine crosslinker to form ester or amide bonds. A process for the preparation of a polymeric drug according to claims 8 to 10, wherein N-methacryloylated tyrosinamide is added to the copolymerization mixture as a radiolabelled substance. 60