WO2008152475A2

WO2008152475A2 - Biocompatible polymers of bisacrylamides and amino acids

Info

Publication number: WO2008152475A2
Application number: PCT/IB2008/001495
Authority: WO
Inventors: Paolo Ferruti; Roberta Cavalli; Elisabetta Ranucci; Michele Trotta
Original assignee: Universita' Degli Studi Di Torino
Priority date: 2007-06-12
Filing date: 2008-06-11
Publication date: 2008-12-18
Also published as: ITTO20070415A1; WO2008152475A3

Abstract

Polymer formed from monomers obtained by the reaction of a bisacrylamide with at least one amino acid and/or at least one peptide comprising a thiol group and/or a primary amine substituted in position a with an electron-attracting group and a group that is not hydrogen. The monomers preferably have Formula I. The polymer may be used to prepare a medicament, preferably for the vehiculation of an active ingredient, or with peptide -mimetic functions, or again as an active constituent of biosensors.

Description

"BIOCOMPATIBLE POLYMERS OF BISACRYLAMIDES AND AMINO ACIDS"

TECHNICAL FIELD The present invention concerns soluble and biocompatible polymers, a composition comprising the same, and their use for preparing a medicament .

BACKGROUND ART Biocompatible polymers are known that comprise disulphide bridges in their main chain. These polymers may be considered release systems sensitive to stimuli, since the release of the drug is favoured by the reductive breakage of the disulphide bridge, with consequent degradation of the polymer chain.

These systems allow the intracellular vehiculation of a drug since the marked reducing environment present inside the cells rather than in the extracellular fluids facilitates its release .

After oral administration, the disulphide bridges are not influenced by the variations of the pH in the digestive tract, so they allow the vehiculation of the active substance towards the colon, where reduction takes place thanks to the action of the bacterial flora.

Among the biocompatible polymers, conjugates of PEG with bioactive molecules are also known, for example peptides or enzymes, drugs or liposomes bonded to the PEG by disulphide bridges These conjugates have proved to be stable in a neutral pH, but they are disadvantageously unstable in highly reducing physiological environments.

In the early Seventies, new soluble, and biocompatible polymers were therefore studied which allow a controlled release of drugs in vivo, poly (amidoamines) (Danusso, F.; Ferruti, P. Polymer 1970, 11, 88) .

Poly (amidoamines) (PAA) are a family of synthetic polymers characterised by the presence of tertiary amine groups and amide groups arranged regularly along the structure of the polymer. They are normally degradable in an aqueous environment and generally are not toxic, despite their polycationic nature (Ferruti et al., Biomaterials 1994, 15, 1235; Ferruti et al . , J. Biomater. Sci. Polym. Ed. 1994, 6, 833) .

Amphoteric PAAs which carry a lateral carboxylic function are also less toxic and have a biocompatibility comparable to that of dextran.

If injected into animals, the same PAAs are able to concentrate in solid tumours thanks to the EPR effect (Enhanced Permeation and Retention effect) . Moreover, both amphoteric and non amphoteric PAAs have shown a potential as endosome-lithic polymers for the vehiculation of genes and toxins (Richardson et al . , J. Drug Targeting 1999, 6, 391; Richardson et al., Proceedings of the International Symposium on Controlled Release of Bioactive Materials, Boston, 1999; Vol. 26, pp 165-166) .

The PAAs are obtained by means of Michael polyaddition of primary monoamines or secondary diamines to bisacrylamides . The reaction is specific and generates a great variety of polymeric structures. For example, in Emilitri et al . , J. Polymer Sci.: Part A: Polymer Chem. 2005, 43, 1404, there is a report on the preparation of poly (amidoamines) containing disulphide groups along the polymer chain obtained by means of the reaction of 2 -methyl piperazine with N, N' -bis (acryloil cistamine) and with N, N' -bis (acryloil cisteine) .

However it is known that sterically hindered amines react very slowly with bisacrylamides to give polymers with a high molecular weight (Ferruti et al., Polymer 1985, 26, 1336; Ferruti et al . , "Ion-Chelating Polymers (Medical Applications")/ in: "Polymeric Materials Encyclopedia", vol. 5, J. C. Salamone, Ed., CRC Press Inc., Boca Raton, Florida 1996, p. 3334+3359) .

Polymers of bisacrylamides with amino acids are known which do not carry substituents in a position in the amine group, such as glycin and β-alanin. These polymers are cross-linked and are therefore not soluble and cannot be used for administration in vivo. However, natural a-amino acids generally do not react to form polymers in the normal conditions of reaction used for the preparation of PAAs .

So at present polymers are being sought which do not have the disadvantages of the polymers of the prior art.

In particular, the need was felt for synthetic soluble and biocompatible polymers that would be stable in the blood circulation but easily degradable once internalised by the cells.

DISCLOSURE OF INVENTION The aim of the present invention is to find polymers that do not have the problems described above and that are therefore soluble, biocompatible, stable in the blood circulation, but easily biodegradable.

According to the present invention, this aim is achieved by means of the polymers according to claim 1.

Below, the term "biocompatible" means biologically compatible with the tissues, organs and functions of the organism and which does not provoke toxic or immunological responses of the same . In particular, according to a first aspect of the present invention, polymers are supplied that are formed of monomers obtained from the reaction of a bisacrylamide with at least one amino acid and/or at least one peptide comprising a thiol group and/or a primary amine substituted in position a with an electron-attracting group and a group that is not hydrogen.

Advantageously these amino acids and/or peptides behave like difunctional monomers in the reaction with bisacrylamides, allowing the synthesis of the polymers according to the present invention.

Preferably the polymers of the invention are obtained from Michael type polyaddition starting from monomers with Formula

I

in which:

Ri and R₂ are selected independently in the group consisting of H, an alkyl group comprising a number of carbon atoms between 1 and 18, a cycloalkyl group comprising a number of carbon atoms between 4 and 7, a phenyl group and a benzyl group, substituted or not substituted with substituents selected in the group consisting of a carboxyl group, hydroxyl group, tertiary amine group and sulphonic group; or the residues R₁, R₂ and X may form a cycle, substituted or not substituted with substituents selected in the group consisting of H, an alkyl group comprising a number of carbon atoms between 1 and 18, a cycloalkyl group comprising a number of carbon atoms between 4 and 7, a phenyl group and a benzyl group. X is selected in the group consisting of an alkylene group comprising a number of carbon atoms between 1 and 18, a cycloalkyl group comprising a number of carbon atoms between 4 and 7;

Y is a residue deriving from amino acids or peptides comprising a thiol group and/or a primary amine substituted in α position with an electron-attracting group and a group that is not hydrogen.

Preferably the alkyl group comprises a number of carbon atoms between 1 and 4, and the cycloalkyl group comprising 6 carbon atoms .

Preferably, X is selected in the group consisting of -NH-CH₂- NH-, -NH-CH(COOH)-NH- and

Preferably the thiol group belongs to a cysteine and the primary amine substituted in a position with an electron- attracting group belongs to an amino acid group.

Advantageously, it has been noted how the steric hindrance and/or a reduction of the nucleophilicity of the amine group substituted in a, compared with that of the corresponding non sterically hindered primary amine, allow the speed of the second addition to the acrylamide to be much lower than that of the first addition. Moreover the reactivity of the amine groups of the a-amino acids is influenced by the presence on the carbon in a of the carboxyl group.

Therefore the amine group substituted in a, advantageously, behaves like a monofunctional group and reacts only once with the bisacrylamide, leading to the formation of linear and consequently soluble polymers .

Moreover, since the polymerisation of amino acids and/or of peptides with bisacrylamides is due to the formation of amide groups, already naturally present in the corresponding peptide chains, no reactive groups are added that are extraneous to the peptide itself, so its chemical and physical properties are not altered.

In particular, in a preferred embodiment of the invention Y is chosen in the group consisting of cysteine, cystine, reduced glutathione, oxidized glutathione, lysine, RGDC, CRGDC, YLRGDC .

Preferably the monomers are selected in the group consisting

Advantageously the polymers of the present invention have proved stable in plasma, but sensitive to reductive breakage which allows their rapid degradation after internalisation in the cells .

According to a second aspect of the present invention, a composition is also supplied comprising the polymer of the invention as described above .

According to a third aspect of the present invention, the use of the polymer or of its composition for preparing a medicament is lastly supplied.

Moreover, the polymer or its composition may be used for the vehiculation of an active ingredient, and/or with peptide- mimetic functions and as an active constituent of biosensors thanks to their capacity of selectively bonding superficial cell structures.

Further characteristics of the present invention will be clear from the following description of some examples, supplied purely for illustrative purposes without limitation.

The following abbreviations will be used in the following examples: BP (1, 4-bisacryloil piperazine) , BAC (2,2- bis (acrylamide) acetic acid (obtained as indicated in "Ferruti, P.; Ranucci, E.; Trotta, F.; Gianasi, E.; Evagorou, G. E.; Wasil, M.;. Wilson, G.; Duncan, R Macromol . Chem. Phys . 1999, 200, 1644"), MBA (methylenebisacrylamide) NMR (Nuclear Magnetic Resonance) , HPLC (High Performance Liquid Chromatography) , MS (Mass Spectroscopy) .

Where not expressly indicated, the succession in the polymers of amino acid or peptide units takes place with a random chain effect.

Example 1

Synthesis of BP-L-Cysteine

BP (500 mg, 2.57 mmol) is dissolved in 0.86 ml of degassed distilled water under a nitrogen atmosphere. L-Cysteine (312 mg, 2.57 mmol) and lithium hydroxide (107.8 mg, 2.57 mmol) are then added: the final pH is thus brought to ~ 9. The mixture is stirred until a limpid solution is obtained and then left at ambient temperature (20+3⁰C), under nitrogen, in the dark, stirring occasionally, for 72 hours. The crude solution is then acidified to pH~ 4 with drop by drop addition of hydrochloric acid and the crude product, a yellowish solid, is recovered by reprecipitation into acetone and drying in a vacuum. The crude product is purified after dissolving it in distilled water, ultrafiltration through a regenerated cellulose membrane with nominal cut-off 5000, and freeze- drying of the captured portion. The final product, a white solid, is obtained in the form of hydrochloride: yield 676 mg

(75%) .

¹H NMR (D₂O) d =2.8 (N-CH₂-CH₂-CO), d =2.95 (CH₂-CH₂-CO), d = 3.60-3.67, d= 3.4 (CH₂-CH₂S), d=3.13-3.22 (S-CH₂-CH), d=3.98 (S-CH₂-CH (COOH) -NH) .

¹³C NMR (D₂O) d=35 (N-CH₂-CH₂-CO) , d =26 (CH₂-CH₂-CO) , d=170 (CH₂-CH₂-CO) , d= 42 (a) , d=41 (CH₂-CH₂S) , d=31 (S-CH₂-CH) , d=60 (S-CH₂ -CH (COOH) -NH) , d =174 (S-CH₂ -CH (COOH) -NH ) . Elementary analysis: (C₁₃H_23-2N₃O_4-5Cl_0-2S); Calculated C=46.93, H= 7.02, 0=21.64, N=12.63, S=9.60, Cl=2.13 ; Found C=46.81, H =6.77, 0=21.34, N =12.43, S =9.11; Cl =2.21. Specific rotary power [a] ²⁰ ₅₈₉=l .7 degrees dm^"1 g^"1 cm³.

The characteristics of molecular weight (determined with chromatography methods) and solubility are shown in Table 1.

Example 2

Synthesis of BAC-L-Cysteine BAC (873.9 mg, 4.126 mmol, purity 93.6%) is dissolved in 1.38 ml of degassed distilled water, under a nitrogen atmosphere and in the presence of lithium hydroxide (173.2 mg, 4.127 mmol). L-cysteine (500 mg, 4.127 mmol) is then added with more lithium hydroxide to bring the final pH to 9. The mixture is stirred until a limpid solution is obtained and then worked exactly as in example 1. The final yield is 1283 mg, (69.7%) . ¹H NMR (D₂O, peaks all enlarged) d=2.6 (N-CH₂-CH₂-CO), d=2.82 (CH₂-CH₂-CO), d = 5.8 (s, NH-CH (COOH)-NH) , d= 3.4 (CH₂-CH₂S), d=3.2 (S-CH₂ -CH), d= 4.1 (S-CH₂ -CH (COOH)-NH). 1³C NMR (D₂O) d= 34 (N-CH₂-CH₂-CO) , d=27 (CH₂-CH₂-CO) , d=170 (CH₂-CH₂-CO) , d= 57 (NH-CH (COOH) -NH) , d=174 (NH-CH (COOH) -NH) , d= 42 ( CH₂ -CH₂S ) , d=30 . 6 ( S-CH₂-CH) , d= 63 ( S - CH₂-CH ( COOH) - NH) d = 172 ( S -CH₂-CH (COOH) -NH) .

Elementary analysis: (C₁₁H₂₆₇₂N₃O₁₁CIi₁₂S); Calculated C=30.36, H= 6.07, 0=35.4, N=Il.02, S=7.36, Cl=9.77 ; Found C=29.83, 5 H=5.28, 0=33.32, N =9.28, S =7.44; C =9.56.

Specific rotary power [a] ²⁰ ₅₈₉=2.7 degrees dm^"1 g^"1 cm³.

The characteristics of molecular weight (determined with chromatography methods) and solubility are shown in Table 1.0

Example 3

Synthesis of BP-L-Gre

BP (0.3065g, 1.579 mmol) is dissolved in water (0.54 mL) with the precautions listed in example 1. Reduced L-glutathione5 (0.5002 g, 1.578 mmol) and potassium carbonate (0.2203 g,

1.578 mmol), are added in that order. The resulting mixture, the pH of which must be 9.5 ± 0.25, is then worked exactly as in example 1. Yield 0.4822 g ( 59.76%).

¹H NMR (D₂O) d = 3.62 (CH₂-CH (NH₂) -COOH) ; d = 3.6 (NH-CH₂-COOH);O d = 4.5 (CH₂-CH(NHCO)-CONH) ; d = 2.75-3 (S-CH₂-CH); d = 2.4

(CONH-CH₂-CH₂); d = 3.3 (NH-CH₂-CH₂); d = 2.95 (CH₂-CH₂-CON); d

= 2.10 (CH₂-CH₂-CH); d = 3.75; d = 3.0 (NCO-CH₂-CH₂); d = 2.8 (CH₂-CH₂-S) .

¹³C NMR (D₂O) d = 62.5 (CH₂-CH₂-COOH); d = 45 (NH-CH₂-COOH); d =5 53 (CH₂-CH(NHCO)-CONH) ; d = 33 (S-CH₂-CH); d = 32 (CONH-CH₂-

CH₂) ; d = 43 (NH-CH₂-CH₂) ; d = 29 (CH₂-CH₂-CON) ; d = 25 (CH₂-

CH₂-CH); d = 42; d = 28 (NCO-CH₂-CH₂); d = 34 (CH₂-CH₂-S); d =

170-174 (quaternary carbons) . 0 The characteristics of molecular weight (determined with chromatography methods) and solubility are shown in Table 1.

Example 4

Synthesis of BAC-L-Gre 5 BAC (0.327Og, 1.578 mmol) and potassium carbonate (0.2245g, 1.610mmol) are dissolved in water (0.54 mL) . Reduced L- glutathione (0.5001 g, 1.578 mmol) and potassium carbonate (0.2203 g, 1.578 mmol) are added in that order. The resulting mixture, the pH of which must be 9.5 + 0.25, is then worked exactly as in example 1. Yield 0.5433 g ( 65.68%). The NMR spectrums and the elementary analysis correspond to the hypothesised structure.

Example 5

Synthesis of BP-L-Gox

The procedure followed is the same one described in Example 3, starting from BP (0.1940 g, 0.9988 mmol), distilled water (0.75 mL) , oxidised L-glutathione (0.6452 g, 1.000 mmol) and potassium carbonate (0.1527 mg, 1.093 mmol). The mixture, worked in the same way, gives a yield of 0.7136g (85.03%) . 1H NMR (D₂O) d = 3.65 (CH₂-CH (NH₂) -COOH) ; d = 3.6 (NH-CH₂-COOH); d = 4.75 (CH₂-CH(NHCO)-CONH) ; d = 2.8-3.2 (S-CH₂-CH); d = 2.5 (CONH-CH₂-CH₂); d = 3.9 (piperazine ring); d = 2.9 (NCO-CH₂- CH₂); d = 3.25 (NH-CH₂-CH₂);

¹³C NMR (D₂O) d = 62 (CH₂-CH (NH₂) -COOH) ; d = 53 (NH-CH₂-COOH); d = 39 (CH₂-CH(NHCO)-CONH); d = 29 (S-CH₂-CH); d = 43 (CONH-CH₂- CH₂) ; d = 29 (piperazine ring) ; d = 25 (NCO-CH₂-CH₂) ; d = 44 (NH-CH₂-CH₂) ; 170-175 (quaternary carbons) .

Example 6

Synthesis of BP-L-Gox

The procedure followed is the same one described in Example 4 , starting from BAC (0.2041 g, 1.030 mmol), distilled water (0.50 mL) , oxidised L-glutathione (0.6447 g, 1.024 mmol) and potassium carbonate added on two occasions as indicated in example 2 (first addition 0.1405 g, 1.006 mmol; second addition 0.1397 g, 0.1000 mmol) . The mixture, worked in the same way, gives a yield of 0.3106 g (36.62%).

Example 7

Synthesis of BP-RGDC a) Preparation of modified peptide.

The starting modified peptide, L-Arginil-glycil-L-glutamyl-L- cisteine (RGDC) , is prepared according to a standard peptide synthesis using a peptide synthesizer "ABI 433 Peptide Synthesizer (Applied Biosystems) " filled with deprotected cysteine (FMOC-Cys-Tritile) combined with Wang resin (0.7 mmoles Cys/g) . The amino acid derivatives used are, in order, FMOC L-aspartic acid 4-tert-butyl ester, FMOC-Glycin and Na- FMOC-N' - (2,2,4,6, 7-pentamethyl-dihydrobenzofuran-5-sulfonyl) - L-arginine. The reagents, N,N-diisopropylethylamine (DIPEA) and piperidine, and the solvents 1-methyl-pyrrolidinone (NMP) and dichloromethane (DCM) are pure commercial products suitable for peptide synthesis. The reactor is filled with the resin (0.143 g, 0.1 mmol of cysteine) and with six containers (two for each amino acid residue, because each passage is repeated twice) each containing 1 mmol of protected amino acid, that is 0.411 g, 0.297 g and 0.648 g, respectively. The coupling reactions are carried out in NMP with hexafluorophosphate of N, N, N¹ ,N' -tetramethyl-O- (IH- benzotriazol-l-il)uronium (HBTU) (1.896 g, 5xlO^"3 mol) and 1- hydroxybenzotriazol (HOBt) (675.5 mg, 5xlO^'3 mol) dissolved in 10 ml of dimethylformamide as coupling agents. The deprotection of the amine groups is accomplished with piperidine and the detachment of the peptide from the resin with trifluoroacetic acid (20 mL) in the presence of water (0.5 mL) , 4-thioanisole (1 mL) and 1, 2-ethanedithiol (1 mL) . The final product is precipitated with ether, dissolved in water, filtered and freeze-dried. The final purification is carried out with preparatory HPLC. Yield 42 mg (93.5 %) . 1H NMR (D₂O) d=2.85 (dd, HS-CH₂-CH), 4.76-4.78 (m, HS-CH₂- CH(COOH)-NH), 2.91-2.97 (m, HN-CO-CH₂-CH), 4.56 (t, CH₂- CH(COOH)-NH), 4.06 (dd, HN-CO-CH₂-CO), 3.95 (t, CO-CH(NH₂)- CH₂), 1.68-1.71 (m, CH(NH₂J-CH₂-CH₂), 1.91-1.93 (m, CH₂-CH₂-CH₂- NH), 3.22 (t, CH₂-CH₂-NH-C (NH₂) =NH) .

MS (70 eV) : m/z = 450 (M+) . The concentration of SH groups, titrated according to Ellman was 2.178xlO^~3 mol/g, agreeing with a molecular weight of 459.

b) Preparation of the polymer.

The procedure followed is the same one used in Example 3, starting from BP (19.4 mg, 0.1 mmol) , degassed distilled water

(0.3 mL) , RGDC (44.9 mg, 0.1 mmol) and potassium carbonate (10 mg, 0.072 mmol). Immediately before use the title of the peptide is iodometrically determined and the quantity to be used is calculated according to the title. The pH of the solution is potentiometrically determined and, if necessary, adjusted to the value of 9.5 ± 0.51. The reaction mixture is then worked as indicated in Example 3. Yield 25 mg (38%) .

¹H NMR (D₂O, enlarged peaks) d= 1.06 (S-CH₂-CH₂-CO) 1.60

(CH(NH₂) -CH₂-CH₂-NH) , 1.90 (CH(NH₂J-CH₂-CH₂-NH), 3.60-3.67 (piperazine ring), 4.59 (S-CH₂-CH(COOH)-NH), 4.26 (CO-CH₂-

CH(COOH) -NH) .

Example 8

Synthesis of BAC-RGDC

The procedure is exactly the same one as in Example 4 , starting from BAC (19.8 mg, 0.1 mmol), degassed distilled water (0.3 mL) , RGDC (44.9 mg, 0.1 mmol) and potassium carbonate (13.8 mg and 10 mg in the two adding operations) . The reaction mixture is then worked in the same way. Yield 36 mg (55.6%) .

The NMR spectrums and the elementary analysis correspond to the hypothesised structure.

Example 9

Synthesis of BP-CRGDC

Using a similar technique, a peptide similar to RGDC is first prepared, L-Cysteinyl-L-Arginyl-glycyl-L-glutamyl-L-cysteine, having a cysteine residue at both ends . It must be noted that the cysteine residue in a is bonded through its carboxyl, while the residue in w is bonded through its amine group. The starting peptide therefore contains two sulfhydryl groups and one terminal amine group. The first are able to be added to the double bonds of the bisacrylamides even with pH<7, while the second is added only as a free base, that is at pH>8. Consequently the two groups can be made to react in a different way, that is, in the specific case, to avoid interferences of the amine group on polymerisation, working at pH 6.8.

After preparing the peptide, the procedure followed is exactly the same as in Example 7, using 0.1 mmol of CRGDC and the same quantities of BP and water, but bringing the pH to 6.8 with the cautious addition of a 10% solution of lithium hydroxide and remaining in a nitrogen atmosphere. The reaction mixture is then worked as indicated in Example 7. Yield 37 mg (47.9%) .

The NMR spectrums and the elementary analysis correspond to the hypothesised structure. It must be observed that the polymer obtained contains free amine groups which are suitable for marking with fluorescein isothiocyanate, or other similar fluorescent markers to follow its biodistribution, the intracellular traffic and the metabolic destiny.

The characteristics of molecular weight (determined with chromatography methods) are shown in Table 1.

Example 10

Synthesis of a copolymer BAC- (2-methylpiperazine + CRGDC)

The procedure starts from 0. lmmol of BAC, 0.05 mol CRGDC and 0.05 mol 2-methylpiperazine (MeP). There are two stages. In the fist stage, all the BAC is added to the 2-methylpiperazine and 0.1 mol of lithium hydroxide monohydrate is added. It is left to react for 36 hours, then the pH of the reaction mixture is lowered to 6.8 with the cautious addition of diluted hydrochloric acid; remaining in an inert gas atmosphere the peptide is added, the solution is homogenised after having added the pH, if necessary after the addition, and then it is left at ambient temperature under nitrogen, stirring occasionally for 8 days. This procedure is adopted to avoid the interference of the free amine group of the peptide . The reaction mixture is then worked as indicated in the previous examples. Yield 39 mg (72.8%) .

Examples 11 and 12 Synthesis of BP-γLRGDC and BAC-γLRGDC

Proceeding with standard methods of peptide synthesis, the peptide y-L-Lysyl-L-Arginyl-glycyl-L-glutamyl-L-cysteine

(γLRGDC) is prepared. This modified RGD is a pentapeptide containing a sulfhydril group derived from cysteine and a terminal amine group derived from the a-amine group of lysine and from this point of view it is therefore similar to reduced glutathione. The polymers of this example are therefore prepared in the same way as BP-L-Gre and BAC-L-Gre (Examples 3 and 4), replacing the reduced L-glutathione with an equimolecular quantity of γLRGD0 and using the same quantities of the other reagents. The yields are, respectively, 41 and 43 mg (52.0% and 54.5%, respectively).

Examples 13-18 Synthesis of MBA-L-Cis, MBA-L-Gre, MBA-L-Gox, MBA-RGDC, MBA- CRGDC, MBA-LRGDC

The procedure is exactly as indicated in the examples 1, 3, 5, 7, 9, 11, replacing the BP with an equimolecular quantity of MBA. The reaction yields and the characteristics of the products are completely similar.

Example 19

Synthesis of BP-L-Gre-Ord

This is the polymer BP-L-Gre linked in a head-head-tail-tail structure . The procedure is fundamentally as in Example 3 , but is performed in two stages. In the first stage only half of the bisacrylamide is added and the pH of the solution is checked, keeping it in the interval 6.5-6.8. After 36 hours a sample shows for NMR that there are no longer any double free bonds, and an Ellman reaction shows that there are no longer appreciable quantities of free sulfhydryl groups. At this point the other half of the BP is added, the pH is brought to 9.5 with lithium hydroxide, and the reaction is left to continue as indicated in example 3 , then working the mixture as indicated. Similar yield.

Table 1

* S = soluble, Sw = swellable, I = insoluble.

** a = water, b = methanol, c = acetone, d = chloroform, e = dimethylformamide .

*** Soluble in water for values of pH =2 and =6, insoluble in the interval .

Example 20

Synthesis of BP-cysteine (BP-CY)

In a conical flask, equipped with a magnetic stirrer and an inlet for nitrogen, L-cysteine (1.00 g, 4.12 mmol) is dissolved in bidistilled water (1.4 ml) in an inert atmosphere, together with potassium carbonate (1.115g, 8.24 mmol) .

BP (0.818g, 4.12 mmol) is added and the reaction solution is stirred for 30 minutes. The nitrogen inlet is then closed and the reaction solution is placed in a thermostatic bath at 25⁰C and out of direct sunlight.

After 48 hours, the viscous solution is diluted with an aqueous solution of hydrochloric acid IM to pH 3 and then it is precipitated with acetone 3:1 v/v. The precipitate is extracted with two further volumes of acetone and possibly dried in a vacuum.

Yield 1.98 g (73.8%, calculated after the composition of the product by elementary analysis. ^Mn = 5 000; ^Mw = 13 500. Elementary analysis: Calculated for (0₁4!!₂₂N₄O₆S₂ 2H₂O, 0.2 HCl): C% 40.17, H% 6.32, N% 11.71, S% 13.40; Cl% 1.60. Measured: C% 39.73, H% 5.90, N% 11.57, S% 13.76; Cl% 1.63.

Solubility: the polymer is soluble in an aqueous environment at any pH and in DMSO, but is insoluble in most organic solvents .

Example 21

Synthesis of BAC-cysteine (BAC-CY)

The same procedure used in example 9 was followed, but using 1.5 times the quantity of potassium carbonate. The final product was collected by acidification at pH 3. The precipitate was extracted by adding further 2 volumes of acetone and dried to a constant weight under a vacuum. Yield: 1.576 g (74.7%, calculated after the composition of the product by elementary analysis. ^Mn= 11 900; Mw= _{51 8O}o. Elementary analysis: Calculated for (C12H18N4O8S2 2H2O HCl) : C% 30.19, H% 4.89, N% 10.06, S% 11.51, Cl% 6.90. Measured: C% 33.59, H% 5.35, N% 9.36, S% 10.82, Cl% 7.60.

Solubility: the polymer is insoluble in an aqueous environment at pH between 1.0 and 4.0 while is is insoluble at all other pH values . It is insoluble in organic solvents .

Example 22

Toxicological analyses on BAC-CY and BP-CY

BP-CY and BAC-CY are dissolved in DMEM in different concentrations between 10mg/ml and 2 mg/ml. Mouse embryo fibroblasts of the cell line 3T3/BALB-C Clone A31 are incubated for 24 hours with the polymer solutions and their vitality is analysed with tetrazoline salt WST-I, which allows the quantitative assessment of metabolically active cells . In the vital cells, mitochondrial dehydrogenase enzymatically converts the tetrazoline salt WST-I into soluble formazan, which is spectrophotometrically quantified at 450 nm and directly correlated with the number of vital cells present in the culture .

Both the polymers studied were found to be highly cytocompatible, even though some differences were noted between the two .

In the case of the polymer BP-CY the high value of IC₅₀ obtained (6.90 g/1) , certainly confirms a good cytocompatibility of the compound tested (Figure 1) .

In the case of the polymer BAC-CY an appreciable cytotoxicity was not found even at higher concentrations tested (10 mg/ml) , so it was not possible to establish a value of IC₅₀ and the material may be considered completely cytocompatible (Figure 2) .

Claims

1. Polymer formed from monomers obtained by the reaction of a bisacrylamide with at least one amino acid and/or at least one peptide,- said amino acid and/or said peptide comprising a thiol group and/or a primary amine substituted in position a with an electron-attracting group and a group that is not hydrogen.

2. Polymer according to claim 1, characterised in that said monomers have the Formula I :

in which:

R₁ and R₂ are selected independently in the group consisting of H, an alkyl group comprising a number of carbon atoms between 1 and 18, a cycloalkyl group comprising a number of carbon atoms between 4 and 7, a phenyl group and a benzyl group, substituted or not substituted with substituents selected in the group consisting of a carboxyl group, hydroxyl group, tertiary amine group and sulphonic group; or the residues Ri, R₂ and X may form a cycle, substituted or not substituted with substituents selected in the group consisting of H, an alkyl group comprising a number of carbon atoms between 1 and 18, a cycloalkyl group comprising a number of carbon atoms between 4 and 7, a phenyl group and a benzyl group.

X is selected in the group consisting of an alkylene group comprising a number of carbon atoms between 1 and 18, a cycloalkyl group comprising a number of carbon atoms between 4 and 7; Y is a residue deriving from amino acids or peptides comprising a thiol group and/or a primary amine substituted in α position with an electron-attracting group and a group that is not hydrogen.

3. Polymer according to claim 2, characterised in that said alkyl group comprises a number of carbon atoms between 1 and 4.

4. Polymer according to claim 2, characterised in that said cycloalkyl group comprises 6 carbon atoms.

5. Polymer according to any one of the preceding claims, characterised in that X is selected in the group consisting of -NH-CH₂-NH-, -NH-CH(COOH)-NH- and

-N N-

6. Polymer according to any one of the preceding claims, characterised in that said thiol group belongs to a cysteine.

7. Polymer according to any one of the preceding claims, characterised in that said primary amine substituted in position a with an electron-attracting group belongs to an amino acid group.

8. Polymer according to any one of the preceding claims, characterised in that said group Y is chosen in the group consisting of cysteine, cystine, reduced glutathione, oxidized glutathione, lysine, RGDC, CRGDC₇- yLRGDC .

9. Polymer according to any one of the preceding claims, characterised in that said monomers are selected in the group consisting of :

10. Composition comprising a polymer according to any one of the claims from 1 to 9.

11. Use of a polymer according to any one of the claims from 1 to 9 or of a composition according to claim 10, for preparing a medicament .

12. Use of a polymer according to any one of the claims from 1 to 9 or of a composition according to claim 10, for the vehiculation of an active ingredient.

13. Use of a polymer according to any one of the claims from 1 to 9 or of a composition according to claim 10, as a peptide-mimetic .

14. Use of a polymer according to any one of the claims from 1 to 9 or of a composition according to claim 10, as a biosensor.