ITTO20070415A1 - BIOCOMPATIBLE POLYMERS OF BISACRYLAMIDS AND AMINO ACIDS - Google Patents

Description

DESCRIPTION

of the patent for Industrial Invention

The present invention relates to soluble and biocompatible polymers, to a composition comprising the same, and to their use for the preparation of a medicament.

Biocompatible polymers are known which comprise disulfide bridges in the main chain. These polymers can be considered stimulus-sensitive release systems since drug release is favored by the reductive breakdown of the disulfide bridge with consequent degradation of the polymer chain.

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

Following oral administration, the disulfide bridges are not affected by changes in pH in the digestive tract, and therefore allow the delivery of the active substance to the colon, where the reduction occurs thanks to the action of the bacterial flora.

Among the biocompatible polymers, conjugates of PEG with bioactive molecules are also known, such as for example peptides or enzymes, drugs or liposomes linked to PEG by means of disulfide bridges. These conjugates proved to be stable at neutral pH, but disadvantageously unstable in strongly reducing physiological environments.

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

Poly (amidoamines) (PAA) are a family of synthetic polymers characterized by the presence of tertiary amine groups and amide groups regularly arranged along the polymer structure. They are normally degradable in an aqueous environment and are generally 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 that carry a lateral carboxyl function are also less toxic and exhibit a biocompatibility comparable to that of dextran.

The same PAAs, if injected into animals, are able to concentrate in solid tumors thanks to the EPR effect (Enhanced Permeation and Retention effect). Furthermore, both amphoteric and non-amphoteric PAAs have shown potential as endosomalytic polymers for the delivery 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).

PAAs are obtained by Michael polyaddition of primary monoamines or diamines secondary 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, the preparation of poly (amidoamine) containing disulfide groups along the polymer chain obtained by reaction of 2-methylpiperazoine with N, N'-bis (acryloylcystamine) and with N, N'-bis (acryloylcystine) is reported .

It is known, however, that sterically hindered amines react very slowly with bisacrylamides to give high molecular weight polymers (Ferruti et al., Polymer 1985, 26, 1336; Ferruti et al., "Ion-Chelating Polymers (Medicai Applications"), in : “Polymeric Materials Encyclopedia”, vol. 5, J. C. Salamone, Ed., CRC Press Ine., Boca Raton, Florida 1996, p. 3334 ± 3359).

Polymers of bisacrylamides are known with amino acids which do not carry substituents in position a to the amino group, such as gliein and β-alanine. These polymers are cross-linked and therefore insoluble and not usable for in vivo administration. Generally, natural α-amino acids do not react to form polymers under the normal reaction conditions used for the preparation of PAAs.

Currently we are looking for polymers without the disadvantages of the polymers of the known art.

In particular, the need is felt for soluble and biocompatible synthetic polymers, which are stable in the bloodstream but easily degradable once internalized by the cells.

The object of the present invention is to find polymers which do not have the problems described above and which are therefore soluble, biocompatible, stable in the bloodstream, but easily biodegradable.

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

Hereinafter, the term "biocompatible" means biologically compatible with the tissues, organs and functions of the organism and which does not cause toxic or immunological responses of the same.

In particular, according to a first aspect of the present invention, polymers formed from monomers obtained by 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-withdrawing group are provided. and a group that is not hydrogen.

Advantageously, these amino acids and / or peptides behave as 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 by Michael type polyaddition starting from monomers of Formula I

in which:

Ri and R2 are independently selected from the group consisting of H, alkyl group comprising a number of carbon atoms between 1 and 18, cycloalkyl group comprising a number of carbon atoms between 4 and 7, phenyl group and benzyl group, substituted or unsubstituted with substituents selected from the group consisting of carboxyl group, hydroxyl group, tertiary amino group and sulphonic group; or, the residues Ri, R2 and X can form a cycle, substituted or unsubstituted with selected substituents in the group consisting of H, alkyl group comprising a number of carbon atoms between 1 and 18, cycloalkyl group comprising a number of carbon atoms between 4 and 7, phenyl group and benzyl group.

X is selected from 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 position a with an electron-withdrawing group and a group other than 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 from 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 position a with an electron-withdrawing group belongs to an amino acid group.

Advantageously, it has been noted that the serum bulk and / or a reduction in the nucleophilicity of the substituted amino group in a compared to that of the corresponding non-sterically hindered primary amine allow the rate of the second addition to acrylamide to be much lower than that of the corresponding non-sterically hindered primary amine. of the first addition. Furthermore, the reactivity of the amino groups of the α-amino acids is influenced by the presence on the carbon in a of the carboxylic group.

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

Furthermore, since the polymerization of amino acids and / or peptides with bisacrylamides is due to the formation of amide groups, already naturally present in the corresponding peptide chains, no reactive groups extraneous to the peptide are added, thus not altering its chemical-physical properties.

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

Preferably the monomers are selected from the group consisting of:

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

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

According to a third aspect of the present invention, the use of the polymer or its composition for the preparation of a medicament is finally provided.

Furthermore, the polymer or its composition can be used for the delivery of an active principle, and / or with peptide-mimetic functions as well as active constituents of biosensors thanks to their ability to selectively bind surface cell structures.

Further characteristics of the present invention will emerge from the following description of some merely illustrative and non-limiting examples.

The following abbreviations will be used in the following examples: BP (1,4-bisacryloylpiperazine), BAC (2,2-bis (acrylamido) 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 (methylenbisacrylamide) NMR (Nuclear Magnetic Resonance), HPLC ( High Performace Liquid Chromatography), MS (Mass Spectroscopy).

Where not expressly indicated, the succession in the polymers of the amino acid or peptide units occurs with random concatenation.

Example 1

Synthesis of BP-L-Cysteine

BP (500 mg, 2.57 mmol) is dissolved in 0.86 ml of degassed distilled water and 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 solution is obtained. clear and then left at room temperature (20 ± 3 ° C), under nitrogen, in the dark and with occasional shaking for 72 hours. The crude solution is then acidified to pH ~ 4 with the dropwise addition of hydrochloric acid and the crude product, a yellowish solid, is recovered by reprecipitation in acetone and drying under vacuum. The raw product is purified following dissolution in distilled water, ultrafiltration through a regenerated cellulose membrane with a nominal "cut-off" of 5000, and freeze-drying of the retained portion. The final product, a white solid, is obtained in the form of hydrochloride: yield 676 mg (75%).

1H NMR (D20) δ = 2.8 (N-CH2-CH2-CO), δ = 2.95 (CH2-CH2-CO), δ = 3.60-3.67, δ = 3.4 (CH2 -CH2S), δ = 3.13-3.22 (S-CH2-CH), δ = 3.98 (S-CH2-CH (COOH) -NH).

<13> C NMR (D20) δ = 35 (N-CH2-CH2-CO), δ = 26 (CH2-CH2-CO), δ = 170 (CH2-CH2-CO), δ = 42 (a), δ = 41 (CH2-CH2S), δ = 31 (S-CH2-CH), δ = 60 (S-CH2-CH (COOH) -NH), δ = 174 (S-CH2-CH (COOH) -NH ).

Elemental analysis: (C13H23.2N304.5C1O.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; C1 = 2.21.

Specific rotary power [α] <2> ° 589 = 1.7 degrees dm <'1> g <' 1> cm <3>.

The characteristics of molecular weight (determined by chromatographic methods) and solubility are reported 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 other lithium hydroxide until the final pH is brought to ~ 9. The mixture is stirred until a clear solution is obtained and then processed exactly as in example 1. The yield final is 1283 mg, (69.7%).

1H NMR (D20, all broad peaks) δ = 2.6 (N-CH2-CH2-CO), δ = 2.82 (C3⁄4-C3⁄4-CO), δ = 5.8 (s, NH -CH (COOH) -NH), δ = 3.4 (CH2-CH2S), δ = 3.2 (S-CH2-CH), δ = 4.1 (S-CH2-CH (COOH) -NH) .

<13> C NMR (D20) δ = 34 (N-CH2-CH2-CO), δ = 27 (CH2-CH2-CO), δ = 170 (CH2-CH2-CO), δ = 57 (NH-CH (COOH) -NH), δ = 174 (NH-CH (COOH) -NH), δ = 42 (CH2-CH2S), δ = 30.6 (S-CH2-CH), δ = 63 (S-CH2 -CH (COOH) -NH) δ = 172 (S-CH2-CH (COOH) -NH).

Elemental analysis: (CnH ^ NaOnCli ^ S); Calculated 0 = 30.36, H = 6.07, 0 = 35.4, N = 11.02, S = 7.36, Cl = 9.77; Found C = 29.83, H = 5.28, 0 = 33.32, N = 9.28, S = 7.44; C = 9.56.

Specific rotational power [a] 589 = 2.7 degrees dm <'> g <'> cm.

The characteristics of molecular weight (determined by chromatographic methods) and solubility are reported in Table 1.

Example 3

Synthesis of BP-L-Gre

BP (0.3065g, 1.579 mmol) is dissolved in water (0.54 mL) with the precautions given in Example 1. Reduced L-glutathione (0.5002 g, 1.578 mmol) and potassium carbonate (0.2203 g, 1.578 mmol), are added in the order. The resulting mixture, whose pH must be 9.5 ± 0.25, is then processed exactly as in example 1. Yield 0.4822 g (59.76%).

1H NMR (D20) δ = 3.62 (CH2-CH (NH2) -COOH); δ = 3.6 (NH-CH2-COOH); δ = 4.5 (CH2-CH (NHCO) -CONH); δ = 2.75-3 (S-CH2-CH); δ = 2.4 (CONH-CH2-CH2); δ = 3.3 (NH-CH2-CH2); δ = 2.95 (CH2-CH2-CON); δ = 2.10 (CH2-CH2-CH); δ = 3.75; δ = 3.0 (NCO-CH2-CH2); δ = 2.8 (CH2-CH2-S).

<13> C NMR (D20) δ = 62.5 (CH2-CH2-COOH); δ = 45 (NH-CH2-COOH); δ = 53 (CH2-CH (NHCO) -CONH); δ = 33 (S-CH2-CH); δ = 32 (CONH-CH2-CH2); δ = 43 (NH-CH2-CH2); δ = 29 (CH2-CH2-CON); δ = 25 (CH2-CH2-CH); δ = 42; δ = 28 (NCO-CH2-CH2); δ = 34 (CH2-CH2-S); δ = 170-174 (quaternary carbons).

The characteristics of molecular weight (determined by chromatographic methods) and solubility are reported in Table 1.

Example 4

Synthesis of BAC-L-Gre

BAC (0.3270g, 1.578 mmol) and potassium carbonate (0.2245g, l, 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 order. The resulting mixture, whose pH must be 9.5 ± 0.25, is then processed exactly as in example 1. Yield 0.5433 g (65.68%). The NMR spectra and elementary analysis correspond to the hypothesized structure.

The characteristics of molecular weight (determined by chromatographic methods) and solubility are reported in Table 1.

Example 5

Synthesis of BP-L-Gox

The same procedure as in Example 3 is followed, starting with BP (0.1940 g, 0.9988 mmol), distilled water (0.75 mL), oxidized L-glutathione (0.6452 g, 1.000 mmol) and potassium carbonate (0.1527 mg, 1.093 mmol). The mixture, processed in the same way, gives a yield of 0.7136g (85.03%).

1H NMR (D20) δ = 3.65 (CH2-CH (NH2) -COOH); δ = 3.6 (NH-CH2-COOH); δ = 4.75 (CH2-CH (NHCO) -CONH); δ = 2.8-3.2 (S-CH2-CH); δ = 2.5 (CONH-CH2-CH2); δ = 3.9 (piperazine ring); δ = 2.9 (NCO-CH2-CH2); δ = 3.25 (NH-CH2-CH2);

<13> C NMR (D20) δ = 62 (CH2-CH (NH2) -COOH); δ = 53 (NH-CH2-COOH); 5 = 39 (CH2-CH (NHCO) -CONH); δ = 29 (S-CH2-CH); δ = 43 (CONH-CH2-CH2); δ = 29 (piperazine ring); δ = 25 (NCO-CH2-CH2); δ = 44 (NH-CH2-CH2); 170-175 (Quaternary coals).

The characteristics of molecular weight (determined by chromatographic methods) and solubility are reported in Table 1.

Example 6

Synthesis of BP-L-Gox

The same procedure described in Example 4 is followed, starting with BAC (0, .2041 g, 1.030 mmol), distilled water (0.50 mL), oxidized L-glutathione (0.6447 g, 1.024 mmol) and potassium carbonate added in two shots as indicated in Example 2 (first addition 0.1405 g, 1.006 mmol; second addition 0.1397 g, 0.1000 mmol). The mixture, processed in the same way, gives a yield of 0.3106 g (36.62%).

The characteristics of molecular weight (determined by chromatographic methods) and solubility are reported in Table 1.

Example 7

Summary of BP-RGDC

a) Preparation of the modified peptide.

The starting modified peptide, L-Arginyl-glycyl-L-glutamyl-L-cysteine (RGDC), is prepared according to a standard peptide synthesis using an "ABI 433 Peptide Synthetiser (Applied Biosystems)" peptide synthesizer loaded with protected cysteine (FMOC -Cys-Trityl) conjugated with Wang resin (0.7 mmol Cis / g). The amino acid derivatives used are, in order, FMOC acid-L-aspartic 4-tert-butyl ester, FMOC-Glycine 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 loaded with the resin (0.143 g, 0.1 mmol of cysteine) and with six containers (two for amino acid residue, because each step is repeated twice) each containing 1 mmol of protected amino acid, i.e. 0.411 g, 0.297 g and 0.648 g, respectively. Coupling reactions are carried out in NMP with hexafluorophosphate of N, N, N ', N'-tetramethyl-0- (1H-benzotriazoll-yl) uronium (HBTU) (1.896 g, 5xl0 <"3> mol) and 1- hydroxybenzotriazole (HOBt) (675.5 mg, 5x10 <'> mol) dissolved in 10 ml of dimethylformamide as coupling agents. Deprotection of amino groups is performed with piperidine and 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 lyophilized. Final purification is carried out by preparative HPLC Yield 42 mg (93.5%).

1H NMR (D20) 6 = 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 (NH2) -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 2.178xl0 <'3> mol / g, in agreement with a molecular weight of 459.

b) Preparation of the polymer.

The same procedure used in Example 3 followed, starting with 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 titer of the peptide is determined iodometrically and the quantity to be used is calculated based on the titer. The pH of the solution is determined potentiometrically and if appropriate adjusted to the value of 9.5 ± 0.51. The reaction mixture is then processed as indicated in Example 3. Yield 25 mg (38%).

1H NMR (D20, broad peaks) δ = 1.06 (S-CH2-CH2-CO) 1.60 (CH (NH2) -CH2-CH2-NH), 1.90 (CH (NH2) -CH2-CH2 -NH), 3.60-3.67 (piperazine ring), 4.59 (S-CH2-CH (COOH) -NH), 4.26 (CO-CH2-CH (COOH) -NH).

The characteristics of molecular weight (determined by chromatographic methods) and solubility are reported in Table 1.

Example 8

Summary of BAC-RGDC

We operate exactly as indicated 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 carbonate of potassium (13.8 mg and 10 mg in the next two additions). The reaction mixture is then processed in the same way. Yield 36 mg (55.6%).

The NMR spectra and elementary analysis correspond to the hypothesized structure.

The characteristics of molecular weight (determined by chromatographic methods) and solubility are reported in Table 1.

Example 9

Synthesis of BP-CRGDC

First, a peptide analogous to RGDC, L-Cysteinyl-L-Arginyl-glycyl-L-glutamyl-L-cysteine, is prepared with a similar technique, containing a cysteine residue at both ends. It should be noted that the cysteine residue in a is bound through its carboxyl, while the one in w is bound through its amino group. Therefore, the CRGDC starting peptide contains two sulfhydryl groups and one terminal amino group. The former are able to add to the double bonds of bisacrylamides even at pH <7, while the latter is added only as a free base, i.e. at pH> 8. It follows that the two groups can be made to react in a differentiated way, that is, in the specific case, to avoid interference of the amino group on the polymerization by working at pH 6.8.

Once the peptide has been prepared, proceed exactly 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 careful addition of a 10% lithium hydroxide solution. remaining in a nitrogen atmosphere. The reaction mixture is then worked as indicated in Example 7. Yield 37 mg (47.9%).

The NMR spectra and elementary analysis correspond to the hypothesized structure. It should be noted that the obtained polymer contains free amino groups which lend themselves to labeling with fluorescein isothiocyanate or other similar fluorescent markers to follow their biodistribution, intracellular trafficking and metabolic fate.

The molecular weight characteristics (determined by chromatographic methods) are reported in Table 1.

Example 10

Synthesis of a BAC-f2-methylniperazine CRGDC1 conolymer

One operates starting from 0.1 mmol of BAC, 0.05 mol CRGDC and 0.05 mol 2-methylpiperazine (MeP). It operates in two stages. First, 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 careful addition of dilute hydrochloric acid, while maintaining an inert gas atmosphere, the peptide is added, the solution is homogenized after adjusting the pH afterwards. addition if necessary, and finally left at room temperature under nitrogen with occasional stirring for 8 days. This procedure is adopted to avoid the interference of the free amino group of the peptide. The reaction mixture is then worked as indicated in the preceding examples. Yield 39 mg (72.8%).

The NMR spectra and elementary analysis correspond to the hypothesized structure. It should be noted that the obtained polymer contains free amino groups which lend themselves to labeling with fluorescein isothiocyanate or other similar fluorescent markers to follow their biodistribution, intracellular trafficking and metabolic fate.

The molecular weight characteristics (determined by chromatographic methods) are reported in Table 1.

Examples 11 and 12

Synthesis of BP-vLRGDC and BAC-YLRGDC

Using standard peptide synthesis methods, the γ-L-Lysil-L-Arginyl-glycyl-L-glutamyl-L-cysteine (yLRGDC) peptide is prepared. This modified RGD is a pentapeptide containing a sulfhydryl group deriving from cysteine and a terminal amino group deriving from the ira-amino group of lysine and is therefore in this respect similar to reduced glutathione. The polymers of this example are then prepared in a completely similar way to BP-L-Gre and BAC-L-Gre (Examples 3 and 4) by replacing the reduced L-glutathione with an equimolecular quantity of yLRGDC and using the same quantities of the other reagents . The yields are 41 and 43 mg, respectively (52.0% and 54.5%, respectively).

The NMR spectra and elementary analysis correspond to the hypothesized structure.

The molecular weight characteristics (determined by chromatographic methods) are reported in Table 1.

Examples 13-18

Summary of MBA-L-Cis. MBA-L-Gre. MBA-L-Gox. MBA-RGDC. MBA-CRGDC. MBA-LRGDC

We operate exactly as indicated in examples 1, 3, 5, 7, 9, 11 by 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

It is the BP-L-Gre polymer neatly concatenated head-head-tail-tail. It operates basically as in Example 3, but operating in two stages. In the first time only one half of the bisacrylamide is added and the pH of the solution is checked, keeping it in the range 6, 5-6, 8. After 36 hours, a sample shows by NMR that there are no longer free double bonds, and an Ellman reaction that there are no longer any appreciable quantities of free sulfhydryl groups. At this point the other half of the BP is added, the pEl is brought to 9.5 with lithium hydroxide, and the reaction is allowed to continue as indicated in example 3, then working the mixture as indicated. Similar yield.

Table 1

Ex. N ° Polymer Molecular weight Solubility at room temperature (* 5 **)

1 BP-Cys = 19600 = 62300 S, a; S, b; I, c; I, d; I, and 2 BAC-Cys <M> n = 10600 <Mw> = 21500 S, a ***; I, b; I, c; I, d;

I, and

3 BP-GluRed Mn- 22700 <Mw> = 36200 S a; I b; I c; I d; I and; 4 BAC-GluRed <M> «= 4200 M <W> = 6200 S a; I b; I c; I d; I and; 5 BP-GluOx Mn- 5200 <Mw> = 13800 S a; I b; I c; I d; I and; 6 BAC-GluOx Mn = 4950 Mw = 7200 S a; I b; I c; I d; I and; 7 BP-RGDC Mn = 6800 = 10300 S, a; S, b; Sw, c; I, d;

I, and

8 BAC-RGDC Mn = 7600 <Mw> = 62300 Not determined 9 BP-CRGDC 66

Mn = 9000 Mw = 22300

10 BAC- (MeP 66

Mn = 7600 <Mw> = 16300

CRGDC)

11 BP-gLRGDC 66

Mn = 8600 Mw = \ i500

12 BAC- 66

<Mn> = 9600 <Mw> = 16200

gLRGDC

13 MBA-L-Cis, u

Mn = 16600 Mw = 34300

14 MBA-L-Gre, 66

Mn = 12600 ^ = 17800

15 MBA-L-Gox, 66

Mn = 13200 = 25900

16 MBA-RGDC, 66

Mn = 10300 Mw = 21200

17 MBA- 66

<Mn> = 16700 Mw = 33700

CRGDC,

19 MBA- 66

<Mn> = 17800 Mw = 34000

LRGDC

19 BP-L-Gre- 66

Mn = 13400 <Mw> = 25200

Ord

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

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

*** Soluble in water for pH values <2 and> 6, insoluble in the range.

Example 20

Synthesis of BP-cystine fBP-CY1

In a flask, equipped with a magnetic stirrer and a nitrogen inlet comes

dissolved L-cystine (1.00 g, 4.12 mmol) in bidistilled water (1.4 ml) in atmosphere

inert, 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. Then the nitrogen inlet is closed and the reaction solution is

placed in a thermostated bath at 25 ° C and away from direct light.

After 48 hours, the viscous solution is diluted with an aqueous solution of

1M hydrochloric acid up to pH 3 and then precipitated with acetone 3: 1 v / v. The

precipitate is extracted with further two volumes of acetone and optionally dried under vacuum.

Yield 1.98 g (73.8%, calculated after the composition of the product by elemental analysis). Mn <,> = 5 000; = 13 500.

Elemental analysis: Calculated for (Ci4H22N406S223⁄40, 0.2 HC1): 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 insoluble in most organic solvents.

Example 21

Synthesis of BAC-cystine (3⁄4AC-CY1

The same procedure used in example 9 was followed, but using 1.5 times the amount of potassium carbonate. The final product is collected by acidification to pH 3. The precipitate is extracted by adding a further 2 volumes of acetone and dried to constant weight under vacuum. Yield: 1.576 g (74.7%, calculated after the composition of the product by elemental analysis). <M> «= 11 900; goo.

Elemental analysis: Calculated for (C12H18N408S2 2H20 HC1): 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 it is soluble at all other pH values. It is insoluble in organic solvents.

Example 22

Toxicological analyzes on BAC-CY and BP-CY

BP-CY and BAC-CY are dissolved in DMEM at different concentrations between 10mg / ml and 2mg / ml. Murine embryofibroblasts of the cell line 3T3 / BALB-C Clone A31 are incubated for 24 hours with the polymer solutions and their viability is analyzed with WST-1 tetrazolinium salt, which allows the quantitative evaluation of metabolically active cells. In viable cells, mitochondrial dehydrogenase enzymatically converts the WST-1 tetrazolinium salt into soluble formazan, which is quantified spectrophotometrically at 450 nm and correlates directly with the number of viable cells present in culture.

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

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

In the case of the BAC-CY polymer no appreciable cytotoxicity was shown even at the highest tested concentrations (10 mg / ml), therefore it was not possible to establish an ICso value and the material can be considered completely cytocompatible (Figure 2).

Claims (14)

CLAIMS 1. Polymer formed from monomers obtained by 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-withdrawing group and a group other than hydrogen. 2. Polymer according to claim 1, characterized in that said monomers have Formula I: in which: Ri and R2 are independently selected from the group consisting of H, alkyl group comprising a number of carbon atoms between 1 and 18, cycloalkyl group comprising a number of carbon atoms between 4 and 7, phenyl group and benzyl group, substituted or unsubstituted with substituents selected from the group consisting of carboxyl group, hydroxyl group, tertiary amino group and sulphonic group; or, the residues Ri R2 and X can form a cycle, substituted or unsubstituted 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, phenyl group and benzyl group. X is selected from 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 position a with an electron-withdrawing group and a group other than hydrogen. 3. Polymer according to claim 2, characterized in that said alkyl group comprises a number of carbon atoms between 1 and 4. 4. Polymer according to claim 2, characterized in that said cycloalkyl group comprising 6 carbon atoms. 5. Polymer according to any one of the preceding claims, characterized in that X is selected from the group consisting of -NH-CH2-NH-, -NH-CH (COOH) -NH- and 6. Polymer according to any one of the preceding claims, characterized in that said thiol group belongs to a cysteine. 7. Polymer according to any one of the preceding claims, characterized in that said primary amine substituted in position a with an electron-withdrawing group belongs to an amino acid group. 8. Polymer according to any one of the preceding claims, characterized in that said group Y is selected from the group consisting of cysteine, cystine, reduced glutathione, oxidized glutathione, lysine, RGDC, CRGDC, yLRGDC. 9. Polymer according to any one of the preceding claims, characterized in that said monomers are selected from the group consisting of: 10. Composition comprising a polymer according to any one of claims 1 to 9. Use of a polymer according to any one of claims 1 to 9 or a composition according to claim 10, for the preparation of a medicament. Use of a polymer according to any one of claims 1 to 9 or of a composition according to claim 10, for the delivery of an active principle. 13. Use of a polymer according to any one of claims 1 to 9 or a composition according to claim 10, as a peptide mimetic. Use of a polymer according to any one of claims 1 to 9 or a composition according to claim 10, as a biosensor.