ITRM20070327A1 - COLLOIDAL VECTORS WITH POLYAMINOACIDIC STRUCTURE FOR THE ORAL RELEASE OF PEPTIDES AND PROTEINS AND ITS RELATED PRODUCTION METHOD. - Google Patents

Description

DESCRIPTION

accompanying a patent application for an industrial invention entitled:

"Colloidal vectors with polyaminoacidic structure for the oral release of peptides and proteins and related production method"

The present invention relates to colloidal vectors with a polyaminoacidic structure, the oral release of peptides and proteins and the related production method. More specifically, the invention relates to systems for the release of active ingredients, specifically peptides and proteins, through their incorporation into nanoparticles, nanoaggregates or complexes consisting of suitably derivatized synthetic polyamino acids. Such polymeric systems are proposed to effectively release peptide drugs and proteins from oral dosage forms, as well as to increase the chemical-physical stability of proteins in liquid and solid pharmaceutical formulations.

As is known, interest in peptides and proteins with therapeutic activity has grown extremely significantly in recent decades. The reasons for this attention are easily understood: on the one hand, the availability of ever greater quantities of peptides and proteins, both of natural and synthetic origin, and more commonly of products obtained with recombinant DNA technology, has made it possible to overcome, albeit with some reservations of an economic nature, the quantitative limitations of the recent past; on the other hand, the deepening of knowledge in the field of molecular biology and pharmaceutical biotechnology has given continuous inputs in identifying peptides and proteins with therapeutic activity in diseases of various kinds, including tumors and viral infections. Furthermore, biotechnological advances have been, in recent years, well supported by successes in the technological field, in the design and implementation of therapeutic systems capable of delivering the new protein drugs in a safe and effective manner.

The relevance of the technological and preformulative aspect is well known, since protein drugs require particular attention due to the multiple causes of instability, both physical (denaturation, aggregation, adsorption on surfaces) and chemical (deamination, oxidation, racemization, formation of disulfide bridges) which can lead to a decrease or total disappearance of biological activity.

In addition to the limitations due to the instability of these molecules, further problems arise from the choice of alternative routes of administration to the parenteral one, which is still the most widely practiced today. There are numerous studies aiming at the evaluation of unconventional dosage forms that may be useful for the administration of proteins by routes other than parenteral.

The oral route is the route of administration that certainly offers the most significant advantages, including dosing accuracy, ease of use and high patient compliance. Despite this, some drawbacks inherent in the oral administration of peptides and proteins are not easy to solve. In addition to having to take into account, in fact, any phenomena of chemical and physical instability intrinsic to the protein molecule, transit through the gastrointestinal tract places these molecules in contact with media whose pH and enzymatic content are incompatible with their permanence in an intact form in this environment, and with their possible absorption. For these reasons, oral administration could only be carried out by resorting to gastro-resistant forms, and with the possible co-administration of enzymatic inhibitors of peptidases.

Furthermore, it must be taken into account that proteins and peptides are generally characterized by a relatively high molecular weight and by a discrete hydrophilicity and therefore have limited permeability through cell membranes and low absorption.

It is therefore easy to understand that, although extremely interesting, the oral administration of peptides and proteins still represents a goal that is not easy to achieve, despite the presence of extensive and authoritative literature, both in terms of scientific publications and patents [ review in: des Rieux A., Fievez V., Grarinot M., Scheider Y-J., Préat V., Nanoparticles as potential orai delivery systems of proteins and vaccines: A mechanistic approach, J. Control. Release 116 (2006) 1-27],

For example, US patent 5641515 (assignee Elan Corporation) describes biodegradable nanoparticles for the controlled release of insulin, which are also proposed for the oral administration of this protein, conventionally taken by diabetic patients several times a day in the form of subcutaneous injection. In the formulation proposed by the cited patent, the insulin is trapped inside nanoparticles of a polycanoacrylate, carrying out the polymerization of the starting cyanoacrylate monomer in the presence of insulin, at low pH.

However, it is a widely shared assumption that a possible solution to the problem of developing an efficient release system for the oral administration of polypeptide active ingredients can be found in the careful combination of therapeutic systems of different chemical nature and of suitable composition.

An approach of significant interest in the oral delivery of peptides and proteins is represented by the use of suitably derivatized polymeric carriers, in which the derivatization is such as to confer a series of advantageous properties to the carrier system constituted by them, including ability to be mucoadhesive and / or to be capable of acting as protease inhibitors themselves [see, e.g., Valenta C., Marschutz M.K., Egyed C., Bernkop-Schnurch A., Evaluation of the inhibitory effect of thiolated poly (acrylates) on vaginal membrane bound aminopeptidase N, J. Pharm. Pharmacol. 54 (2001) 603-610 .; Bernkop-Schnurch A., Thaler S.C., Polycarbophil-cysteine conjugates as platforms for orai polypeptide delivery systems, J. Pharm. Sci. 89 (2000) 901-909]. Another property that can be conferred on polymers by derivatization is the ability to act as absorption promoters themselves [see, e.g., Clausen A.E., Bernkop-Schnurch A., In vitro evaluation of the permeation-enhancing effect of thiolated poiycarbophil, J. Pharm. Sci. 89 (2000) 1253-1261; Kast C.E., Bernkop-Schnurch A., Influence of the molecular mass on the permeation enhancing effect of different poly (acrylates), STP Pharma Sci. 12 (2002) 351-356]. Such polymeric carriers therefore offer the advantage of a lower degradation of the protein drug conveyed by them in the gastrointestinal tract and of a greater absorption.

The mucoadhesiveness of a polymer, that is its ability to adhere to the mucous membranes of the organism (and, in particular, to the mucous membrane of the gastrointestinal tract) constitutes an important property in the development of drug delivery systems, especially of a peptide nature, from the moment that thanks to the mucoadhesive properties it is possible to establish an intimate contact between the transport system and the mucosa, preventing the presystemic metabolization of the peptides during absorption in the gastrointestinal tract. Consequently, the residence time of the drug in the absorption site will also increase, where a concentration gradient will form such as to allow the drug to diffuse.

An example of these mucoadhesive polymers is represented by thiolated polymers (thiomers, or thiopolymers), ie polymers whose central structure (or polymeric "backbone") are linked to thiol groups, to be used as excipients in the composition of solid oral forms [Bernkop-Schnurch A., Krauland A.H., Leitner V.M., Palmberger T., Thiomers: potential excipients for non-invasive peptide delivery systems, Eur. J. Pharm. Biopharm. 58 (2004), 253-263]. These polymers, among which the last mentioned review mentions, for example, chitosan-cysteine, polycarbophilcysteamine, polyacrylic acid-homocysteine, have strong mucoadhesive properties thanks to the establishment of covalent bonds (S-S bridges) between the thiol groups of the polymer and the domains rich in cysteine of the glycoproteins of which the mucus is made up. No less important are the covalent bonds that are also formed between the same thiomers. This phenomenon, which leads to the reduction of free thiol groups with respect to the total ones, leads to an increase in the viscosity of the medium in which they are dispersed. This further property of thiolated polymers, ie the ability to give rise to S-S, inter- and intramolecular bridges, can be positively exploited if they are used as excipients for the formation of tablets [Bernkop-Schnurch A.,. Kast C.E, Richter M.F., Improvement in the mucoadhesive properties of alginate by the covalent attachment of cysteine, J. Control. Release 71 (2001) 277-285]. In fact, the disintegration time of these tablets can be considerably prolonged with respect to that observed using the same non-thiolated excipient; this is because the inter-chain disulfide bridges that are formed give high stability to the drug-carrier system based on thiomers and increase the viscosity of the medium.

On the other hand, both ionic and neutral polymers can have the property of interacting with protein molecules, forming macromolecular aggregates: the former, through the formation of hydrogen bonds, van der Waal's and electrostatic interactions with the polar functional groups of proteins; the second for the establishment of mainly hydrophobic interactions. The presence of hydrophobic portions in a polymer can also favor the formation of hydrophobic interactions with the lipophilic portions of biological membranes, thus increasing the permeability of the polymer-protein complex. With reference to proposed release systems for the delivery of active ingredients preferably of the peptide and protein type in which the central polymer chain is derivatized with hydrophobic and ionizable groups, the European Patent EP 0734720 (Flamel Technologies), corresponding to the US patent 5904936, describes (micro) release particles of polyamino acid type, based on α-amino acids and obtained by block or statistical polymerization of two different types of α-amino acids: a hydrophobic or neutral type (such as Leu, Val, Ala, Pro, Phe) and a type having an ionizable side chain (such as aspartic acid and glutamic acid). Specifically, the preferred polyaminoacidic polymers described in the document are of the poly (Leu / Glu) type, with a Leu / (Glu + Leu) ratio of not less than 6% in the case of block polyamino acids, and not less than 20% if these are statistical polyaminoacids, and molecular weight not less than 4000 Da. In practice, in the preferred examples referred to here, the leucine fraction must be high enough to prevent the polymer from being completely soluble in water and to provide suitable hydrophobic properties, so that polymer chains can associate to form nanoparticulate systems. The particles formed constitute a colloidal dispersion in an aqueous medium, which can then be loaded with the peptide active principle to be conveyed. Alternatively, the particles containing the active ingredient can be obtained by directly dispersing the α-polyamino acids in the active ingredient solution, before aggregation.

As a further evolution of the aforementioned system, the European Patent EP 1131056 (Flamel Technologies), corresponding to the US patent 6630171, describes a similar release system, which was then called "Medusa <®>" probably to recall the mechanism of action of polymeric vectors in incorporating the polypeptide active ingredients, that is their ability to interact with the active principle and to “wrap” it, protecting it from degradation phenomena and conveying it in the form of a particle (nano- or micro-) to the site of action. In this system, the polyaminoacidic carriers that make up the main polymer chain of the particles consist of homopolymers or copolymers of glutamic acid (AcGlu) and / or aspartic acid (AcAsp) and their salts (recurrent amino acids, both of the ionizable type), and a certain percentage of them carry a lateral hydrophobic chain, linked to the carboxyl function of AcGlu or AcAsp through ester (or amide) bonds. The length of the polymer chain varies between 200 and 1000 amino acids, which translates into a molecular weight of 26000-65000 in the hypothesis of an AcGlu homopolymer and of 23000-57000 in the hypothesis of an AcAsp homopolymer.

The hydrophobic groupings (R) bonded to the polymeric backbone can be linear or branched carbon chains or contain double bonds, and preferably are linear C2-Ci8 groups. The percentage in moles of recurrent amino acids derivatized with hydrophobic functions is variable between 3 and 70%. Some examples of derivatized polymers described in the patent are: sodium-bloc-methyl glutamate polyglutamate; sodium-bloc-glutamate hexadecyl polyglutamate; sodium polyglutamate-ethyl co-glutamate; sodium polyaspartate-propyl blocaspartate; sodium polyaspartate-benzyl bloc-aspartate. The preparation of the release particles starting from the polymers described above substantially decreases the solubility of the hydrophobic fraction, by adding saline solution, so that this induces the precipitation of the polymer forming particles (nano). To increase the size of the particles and obtain microparticles, an additional aggregation step is provided in the preparation of the suspension, with the help of an aggregating agent, such as a salt, acid, base or an ionic polymer (polysine, polyethyleneimine). As in the previous case, the particles can be loaded with the protein active principle by forming them directly in a medium containing the active principle or by dispersion and incubation of the empty particles already formed, freeze-dried, in a medium containing the dissolved active principle or missing.

In light of the foregoing, the main objective of the invention is therefore to increase the oral bioavailability of peptide drugs and proteins, such as insulin, by exploiting the ability of carrier copolymers to interact with protein molecules, to reduce their degradation to work of digestive enzymes and gastric pH and favor their absorption.

A further object of the present invention is to use the copolymers proposed as excipients in the production of both liquid and solid pharmaceutical formulations, containing peptide molecules or proteins in order to prolong their stability over time both in the formulation itself and in a physiological environment.

As already noted, it is evident that to improve the performance of the release systems of peptide or protein active ingredients based on colloidal polymeric vectors (both in the sense of increasing the bioavailability of proteins after oral administration, and in the sense of increasing chemical stability -physics of proteins in liquid and solid pharmaceutical formulations) it appears necessary to combine different chemical functions on the same polymer, each of which is endowed with specific technological properties. In this way it is possible to try to optimize and modulate the favorable properties of biocompatible polymers for their use in the realization of pharmaceutical systems for the release of peptides and proteins, also for the oral route.

For this purpose, the present invention proposes to use, for the realization of polymeric vectors useful as carriers for active principles of the polypeptide type, particular polymeric structures of the polyaspartamide type, to which both hydrophobic functions and ionizable and thiol functions are connected as side chains, each individually or in combination with each other. In particular, the approach used in the present invention was to synthesize copolymers based on polyhydroxyethyl-aspartamide or PHEA, more properly α, β poly (N-2-hydroxyethyl) -D, L-aspartamide having the following structural formula

or its copolymers, which present in the side chains, covalently linked through the hydroxyl function which is present in these polymers at the rate of one for each starting monomer unit, hydrophobic, ionizable and thiol functions. The copolymers thus formed are capable of forming pharmaceutical systems for the release of peptides and proteins through their incorporation in nanoparticles or nanoaggregates or complexes, constituted precisely by the interaction between the copolymers themselves and the proteins of interest.

As is known, PHEA is a derivative of a high molecular weight polysuccinimide (PSI) obtained by aminolysis of the latter, by reaction with ethanolamine. As can be seen in the structural formula above and it is more evident in the one that follows, which represents the same polymer in a different graphic form, due to the cyclic structure of the starting polysuccinimide, ethanolamine etching can occur in a different way. to leave indifferently a methylene group in the body of the main chain or in the pendant functional group. Therefore, the repetitive unit can have a strut

slightly different in one or the other case, while always having the same molecular weight.

It is known that PHEA is a polymer that has excellent biopharmaceutical properties, in terms of biocompatibility, so much so that it has been proposed in the past as a plasma expander [Neri P., Antoni G., Benvenuti F., Cocola F., Gazzei G., Synthesis of a, p-poly [(2-hydroxyethyl) -DL-aspartamide], a new plasma expander, J. Med. Chem., 16 (1973) 893-897; Antoni G., Arezzini G., Cocola F., Gazzei G., Neri P., Pharmacological and toxicological evaluation of poly-hydroxyethylaspartamide (PFIEA) as a plasma substitute, The drug 34 (1978) 146-156]. The polymer is also characterized by an excellent reactivity, since it is endowed with hydroxyl groups in the side chain.

The technology proposed with the present invention consists in the incorporation of protein molecules in nanoparticles, nanoaggregates or complexes consisting of synthetic linear polyamino acids, specifically of the type of polyaspartamides, suitably derivatized with thiol, hydrophobic and ionizable groups. The appropriate combination of these side chain functions to the copolymer gives it the property of interacting with the protein of interest, through non-covalent interactions of hydrophobic, electrostatic and hydrogen bonds, and of forming supramolecular systems of colloidal dimensions, with of high kinetic stability and mu coadhesion, as well as of all the favorable properties of thiomers, such as the ability to act as inhibitors of proteases and as absorption promoters.

In addition to PHEA, other polymers with a polyaspartamide structure with nucleophilic groups hanging along the chain can be equally used and suitably derivatized with hydrophobic, ionizable and thiol functions, in such a way as to give compositions and materials according to the invention, each time provided with specific requirements.

Therefore, the specific object of the present invention is a colloidal polymeric vector for the delivery and release of peptides and proteins, in the form of nanoparticles, aggregates or complexes, consisting of a polymer or copolymer with a polyaspartamide structure carrying in the side chain, each individually or in combination among them, hydrophobic functions, ionizable functions and thiol functions.

The proposed polymeric vectors are obtained starting from macromolecules with a polypeptide bearing structure (specifically, polyaspartamide) having nucleophilic groups in the ratio of one for each repeating unit of the chain, and their copolymers. The above mentioned functions are covalently linked to these nucleophilic groups, which within the same macromolecule are both hydrophobic, ionizable and thiol type, where each of said functions can be connected to the polymeric backbone individually or in combination with several other such functions.

Preferably, according to the invention, said polymer or copolymer with a polyaspartamide structure is alpha, beta-poly (N-2-hydroxyethyl) aspartamide (PHEA, and its copolymers, carrying in the side chain hydrophobic, ionizable and thiol functions, linked to hydroxyl groups of the starting polymer through amide, enamine, urethane or ester bonds), depending on the type of conjugate group.

The length of the polyamine acid chain is preferably such that the molecular weight is between 10,000 and 60,000 daltons. In the preferred case of PHEA, this is equivalent to a number of repetitive units of hydroxyethyl-aspartamide, comprised between 63 and 315 units.

The hydrophobic groupings bonded to the polymeric backbone can be preferably selected from the group consisting of: linear, branched or cyclic alkyl with up to 18 carbon atoms, linear, branched or cyclic alkenyl with up to 18 carbon atoms, linear, branched alkynyl or cyclic with up to 18 carbon atoms, an isocyclic aromatic or aralkyl group with up to 18 carbon atoms. According to some specific embodiments of the invention, said hydrophobic groups are selected from ethyl (C2), propyl (C3), butyl (C4), dodecyl (Ci2) or hexadecyl (0Ί6) linear groups, propyl (C3), butyl groups (C4), dodecyl (C12) 0 hexadecyl (Ci6) branched 0 containing double bonds and aromatic groups. The molar ratio between the moles of molecules carrying the hydrophobic group and the moles of repeating units of polymer is preferably comprised between 0.01 and 0.6.

The ionizable functions that are preferably related to the polyaspartamide structure of the invention consist of linear or branched alkyl, alkenyl or alkynyclic groups with a number of carbon atoms between 2 and 20, carrying carboxylic functions (-COOH); groups carrying phosphoric functions (-P03H2), such as o-phosphocolamine; sulfuric (-S04H) or sulphonic (-S03H) functions such as taurine; primary amino functions (-NH2), secondary amino (-NHR) or tertiary amino functions (-NR'R ") or quaternary ammonium salts [- <+> N (R) 3], The molar ratio between the moles of carrier molecules the ionizable group and the moles of repeating units of polymer is comprised between 0.01 and 0.6.

The thiol functions linked to the polymeric backbone can be derived from thiolated molecules such as cysteine, homocysteine, cysteamine and their derivatives (preferably cysteine). The molar ratio between the moles of molecules carrying the thiol group and the moles of repeating units of polymer is between 0.01 and 0.5.

According to a preferred embodiment of the invention, the colloidal polymeric vector, bearing on the polyaspartamide chain hydrophobic functions of the linear alkyl type, ionizing functions of the carboxylic type and thiol functions derived from cysteine, is constituted by the polymer (a, p-poly (N -2-hydroxyethyl) -co- [N-2- (butylcarbamate) ethylene] -co- [N-2 (2'carboxysuccinyl) ethylene] -co- [N-2- (2'-L-cysteine-succinylamide) ethylene] aspartamide). This polymer has been synthetically named PHEA-C4-CS-Cist.

The preparation of some of the aforesaid copolymers can be carried out through a series of successive reactions that allow the following molecules to be linked in sequence to the hydroxyl groups of the starting polyaspartamide polymer:

• hydrophobic groups

• groups with ionizable functions;

• groups carrying thiol functions.

According to a further aspect, therefore, the present invention relates to a process for the production of colloidal polymeric vectors as defined above, comprising the following operations in succession:

a) activation of the reactive groups of a starting polyaspartamide structure polymer with activating agents;

b) reaction of the polymer thus activated with amines corresponding to said desired hydrophobic functions in the side chains, using a molar ratio between moles of amine and moles of repeating units of polymer comprised between 0.01 and 0.6;

c) conjugation of the polymer obtained from the previous operation with molecules carrying said ionizable functions, using a ratio between moles of molecules carrying the ionizable functions and moles of repetitive units of polymer between 0.01 and 0.6;

d) conjugation of the polymer obtained from the previous operation with molecules carrying said thiol functions, in the presence of activating agents, using a ratio between moles of molecules carrying thiol functions and moles of repeating units of polymer between 0.1 and 0.5 .

Preferably, as noted, the starting polyaspartamide structure polymer is alpha, beta-poly (N-2-hydroxyethyl) aspartamide or PHEA.

According to some preferred solutions for the synthetic method of the invention, the activating agents of operation a) consist of bis (4-nitrophenyl) carbonate (4-NPBC) in a solution of dimethylformamide (DMF), and said operation is carried out at temperature constant, between 25 and 60 ° C. Furthermore, the activating agents of step d) preferably consist of N-hydroxysuccinimide (NHS) and N-3 (3-dimethylaminopropyl) -N-ethyl-carbodiimide hydrochloride (EDC-HCI) in aqueous solution, and said operation is conducted at a temperature between 15 and 40 ° C.

In a specific case reported for illustrative purposes, the hydroxyl groups of the starting polyaspartamide polymer were linked, in succession:

• an aliphatic chain with four carbon atoms (C4) (obtaining the copolymer which has been called PHEA-C4);

• succinic groups (obtaining the copolymer which has been called PHEA-C4-CS);

• molecules carrying thiol groups such as cysteine (obtaining the copolymer called PHEA-C4-CS-Cist).

In the first step of the synthesis, the hydroxyl groups of PHEA were activated by reaction with bis (4-nitrophenyl) carbonate (4-NPBC) in a solution of dimethylformamide (DMF), at a constant temperature (preferably between 25 and 60 ° C). Following the reaction with butylamine, using a molar ratio between moles of butylamine and moles of repeating units of PFIEA between 0.01 and 0.6, allowed to derivatize the polymer with butyl groups in the side chain, obtaining the PHEA copolymer- C4 (a, p-poly (N-2-hydroxyethyl) -co- [N-2- (butylcarbamate) ethylene] -aspartamide)

The introduction of butylamine chains into the PHEA polymeric backbone was confirmed by <1> H-NMR and FT-IR analysis of the PHEA-C4 polymer after purification with exhaustive dialysis against distilled water and lyophilization. The percentage of alkylamine functions bound in the PHEA-C4 copolymer (expressed as the number of moles of bound butylamine / number of moles of repeating units of the polymer x 100), determined by <1> H-NMR, is variable and can be between N % and 60% by moles (preferably 15-20%).

The average molecular weight of the PHEA-C4 derivative, determined by molecular exclusion chromatography (SEC), is between 30 and 50 kDa, depending on the degree of derivatization, compared to a calibration carried out with molecular standards of PEO / PEG.

In the second step of the synthesis, the PHEA-C4 copolymer, obtained as previously described, was placed to react in a DMF solution with succinic anhydride (AS) in the presence of triethylamine (TEA) for a time between 4 and 24 hours, to depending on the ratio between moles of AS and moles of repeating units of PHEA, and at a constant temperature, preferably between 15 and 40 ° C. The ratio of moles of AS to moles of repeating units of PHEA can be between 0.01 and 0.6. In such conditions, the still free hydroxyl groups of PHEA-C4 easily react with the anhydride, according to the scheme below.

PHEA-C,

After purification, by exhaustive dialysis against distilled water, the copolymer PHEA-C4-CS (a, p-poly (N-2-hydroxyethyl) -co- [N-2- (butylcarbamate) ethylene] -co- [N-2 (2'carboxysuccinyl) ethylene] -aspartamide) obtained was characterized by FT-IR and <1> H-NMR analysis. The spectroscopic data confirm the introduction of the succinic group in the copolymer.

The percentage of bound succinic functions in the PHEA-C4-CS copolymer (expressed as the number of moles of bound succinic acid / number of moles of repeating units of the polymer x 100), determined by <1> H-NMR, is variable and can be between Γ1 and 60% by moles (preferably 30-50%).

The average molecular weight of the PHEA-C4-CS derivative, determined by molecular exclusion chromatography (SEC), is between 30 and 50 kDa, depending on the degree of derivatization, compared to a calibration performed with PEO / PEG molecular standards.

In the third step of the synthesis, the PHEA-C4-CS copolymer obtained as previously described was reacted in the presence of the activators N-hydroxysuccinimide (NHS) and N-3 (3-dimethylaminopropyl) -N-ethyl-carbodiimide hydrochloride (EDC- HCl) in aqueous solution, for a time ranging from 1 to 18 hours, depending on the ratio between moles of cysteine and moles of succinic acid bound to PHEA, at a constant temperature, preferably between 15 and 40 ° C. The ratio of moles of cysteine to moles of succinic acid bound to PHEA can be between 0.5 and 2, corresponding to a molar ratio between the moles of molecules carrying the thiol group and the moles of polymer repeating units of 0, 1 -0.8. Under these conditions, the amino groups of cysteine react with the carboxyl groups of the succinic chains of PHEA-C4-CS giving rise to the copolymer PHEA-C4-CS-Cist (a, p-poly (N-2-hydroxyethyl) -co- [ N-2- (butylcarbamate) ethylene] -co- [N-2 (2'carboxysuccinyl) ethylene] -co- [N-2- (2'-L-cysteine-succinylamide) ethylene] aspartamide).

PHEA-C4-CS

r

After purification, by exhaustive dialysis against water, the obtained PHEA-C4-CS-Cist copolymer was characterized by FT-IR and 1H-NMR analysis.

The percentage of bound thiol functions in the PHEA-C4-CS-Cist copolymer (expressed as the number of moles of bound cysteine / number of moles of repetitive units of the polymer x 100), was determined by Ellman's assay, which allows to determine the number of thiol functions (SH) and therefore the percentage of cysteine molecules bound to the polymer. This percentage can be between Γ1 and 50% by moles (preferably 10% by moles with respect to the repeating units of the PHEA or approximately 30% with respect to the succinic groups).

The average molecular weight of the PHEA-C4-CS-Cist derivative, determined by molecular exclusion chromatography (SEC) is between 30 and 50 kDa, depending on the degree of derivatization, compared to a calibration carried out with molecular standards of PEO / PEG .

For all the synthesized copolymers the yields were in the range 94-100% with respect to the starting copolymer.

According to a further aspect, the invention relates to pharmaceutical preparations which are obtained by loading the proposed polymeric carriers with the active ingredients, preferably but not exclusively of peptide nature. The invention therefore concerns a pharmaceutical composition for the release of one or more active ingredients, specifically of a peptide or protein nature, in the form of nanoparticles, aggregates or complexes, in which said one or more active ingredients are conveyed in a polymeric vector colloidal as defined above.

Advantageously, the proposed pharmaceutical composition can be formulated for oral administration. Said one or more active ingredients included in the colloidal polymeric carrier according to the invention can also be accompanied by one or more pharmaceutical excipients acceptable for oral administration.

The invention further concerns pharmaceutical compositions based on either one or more active ingredients of a peptide or protein nature as defined above, however formulated in a liquid or solid release form.

The interaction between the copolymers object of the invention and the protein molecules conveyed by them allows to protect the latter from both enzymatic and chemical degradation phenomena and to release them intact at the administration site, thus offering the advantage of a lower degradation of the protein drug and greater absorption.

The polymeric delivery systems according to the invention have been shown to increase the oral bioavailability of insulin, used as model protein, and to induce a lowering of plasma glucose levels in rats equal to 30% of the hypoglycemic effect observed after subcutaneous administration of insulin. In addition, these vectors did not show any cellular cytotoxicity in vitro and excellent tolerability in vivo on the models used. Furthermore, the proposed systems have been shown to be capable of increasing the chemical stability of insulin in the presence of proteolytic enzymes such as Γα-chymotrypsin and in means mimicking gastrointestinal fluids.

The use of the vectors according to the invention, therefore, allows to increase the oral bioavailability of peptide drugs and proteins, such as insulin, and to prolong the stability of peptide molecules and proteins when they are used as excipients in both liquid formulations that solid.

The specific characteristics of the invention, as well as the advantages of the same and the relative operating methods, will be more evident with reference to the detailed description presented by way of example below, together with the results of the experiments carried out on it and the comparison data with the prior art. Some experimental results are also illustrated in the attached drawings, in which:

Figure 1 shows the trend of cell viability, assessed by MTS test, of human intestinal epithelium cells after 24 hours of incubation with increasing concentrations (0.1, 0.5 and 1 mg / ml) of the PHEA-C4- copolymers CS and PHEA-C4-CS-Cist;

Figure 2 shows the trend of transmittance as a function of time of the insulin samples, PHEA-C4 and of the insulin / PHEA-C4 complexes in different weight ratios (1/1, 1/3, 1/6);

Figure 3 shows the trend of transmittance as a function of time of the insulin samples, PHEA-C4-CS and of the insulin / PHEA-C4-CS complexes in the different weight ratios (1/1, 1/3, 1/6) ;

Figure 4 shows the trend of transmittance as a function of time of the insulin samples, PHEA-C4-CS-Cist and of the insulin / PHEA-C4-CS-Cist complexes in the different weight ratios (1/1, 1/3, 1/6);

Figure 5 represents the electrophoresis on agarose gel of insulin, PHEA-C4 / insulin, PHEA-C4-CS / insulin, PHEA-C4-CSCist / insulin to confirm the integrity of the complexed protein structure;

Figure 6 shows the percentage of intact insulin released over time from the insulin / copolymer complexes after incubation in the presence of a-chymotrypsin;

Figure 7 shows the percentage of insulin released from the tablets containing the different insulin / copolymer systems in the fluid mimicking medium g.i. (pH 1, up to 120 min, pH 6.8 from 121 to 360 min); And

Figure 8 shows the trend of serum glucose levels in rats after oral administration of capsules containing the insulin / PHEA-C4-CS (P1) complex; non-complexed insulin (os insulin); the copolymer (P1 os) and subcutaneous insulin (insulin se) as a positive control.

The copolymers object of the present invention were subjected to in vitro biocompatibility studies on human intestinal epithelial cells in order to evaluate any cytotoxic effects by means of an MTS assay. As shown in the attached Figure 1, cell viability data after 24 hours of incubation with copolymer concentrations of 0.1, 0.5 and 1 mg / ml did not show any cytotoxic effect on the cell model employed.

By way of example only, the results relating to a series of copolymers, used as model protein vectors for interaction and oral release studies, are reported below. In order to evaluate the possibility of using the synthesized copolymers as agents for the delivery of proteins, and among these insulin, chosen as a model protein, studies were carried out on the interaction between the copolymers according to the invention and insulin:

• turbidimetric studies; electrophoresis on agarose gel, measurements of light scattering and zeta potential.

Some experimental results are illustrated in the attached figures.

The turbidimetric studies were performed by recording the transmittances, at 500 nm, of insulin, individual copolymers and insulin / copolymer complexes at different insulin / copolymer weight ratios, in phosphate buffer pH 7.4.

The insulin / PHEA-C4 or insulin / PHEA-C4-CS systems described by way of example above show a decrease in transmittance as a function of time. This phenomenon is much more marked in the case of the insulin / PHEA-C4-CS-Cist complex, with a drastic decrease in transmittance as a function of time (up to 24 hours) and as a function of the insulin / copolymer ratio. The data are reported as transmittance versus time in Figures 2, 3 and 4.

In all cases, this behavior can be explained by assuming the formation of colloidal aggregates between the protein and the copolymers which, due to their size, are "opaque" to the incident light beam with respect to the single copolymers and the naked protein, even if invisible to naked eye. In the case of the PHEA-C4-CS-Cist copolymer in particular, in addition to physical bonds, such as Van der Waals and hydrophobic interactions, also chemical bonds, such as S-S bridges between the copolymer chains themselves, are involved in the formation of the aggregates.

In order to verify the structural integrity of the complexed protein, the electrophoretic analysis of the insulin / copolymer complexes was carried out on agarose gel. In Figure 5 attached it is reported that the electrophoretic run, conducted for 30 min at 150 V, did not show changes in the electrophoretic mobility of the insulin complexed with the copolymers compared to the native insulin (control) and no further bands were evident, index of possible degradation of the protein.

It is therefore deduced that following the interaction of the protein with all the copolymers, the chemical structure of the same, as well as the secondary and tertiary ones, remain unaltered.

Finally, tight scattering analyzes and measurements of the zeta potential in phosphate buffer at pH 7.4 as a function of time were performed, in order to evaluate the hydrodynamic diameter of the polymeric complexes and obtain information on the surface charge of the same complexes.

The complexes with PHEA-C4 and PHEA-C4-CS show smaller dimensions than insulin as it is, suggesting the occurrence of protein compaction phenomena thanks to the interaction with the copolymers themselves. This behavior appears to be time dependent for the PHEA-C4-CS-Cist copolymer. In fact, the dimensions of the complexes decrease over time very markedly, probably due to the establishment of S-S bridges between the chains of the same copolymers. Moreover, these results are in agreement with those obtained from turbidimetric studies.

The zeta potential measurements also show a decrease in the surface potential in the complexes, compared to native insulin. This confirms a shielding effect of the surface charge present on the insulin molecule following complexation with the copolymer.

Stability of protein / copolymer complexes in the presence of achymotrypsin

The ability of the copolymers object of the present invention to improve the stability of protein molecules against degradation in the intestinal environment was evaluated by incubating the previously prepared and lyophilized protein / polymer complexes in the presence of α-chymotrypsin and determining, at intervals of regular time, the percentage of intact protein in the reaction medium.

As shown in Figure 6, the copolymers object of the invention have a protective effect by reducing the enzymatic degradation of insulin in the presence of α-chymotrypsin. This protective effect was found to be increasing in the order PHEA-C4 <PHEA-C4-CS <PHEA-C4-CS-Cist. In the latter case, in fact, the presence of intact insulin is observed up to 140 minutes from the start of incubation with the enzyme. On the contrary, native insulin undergoes complete degradation in the presence of α-chymotrypsin within 40 minutes.

The different extent of the protective effect of the copolymers towards insulin can be explained by considering a different nature and strength of the interactions between insulin and the three copolymers, i.e. mainly hydrophobic interactions with PHEA-C4, hydrophobic interactions and bonds to hydrogen with PHEA-C4-CS; hydrophobic interactions, hydrogen bonds and disulfide bonds with PHEA-C4-CS-Cist.

In vitro release studies in simulated gastrointestinal fluids

The possibility of using the protein / copolymer complexes as systems for the release of proteins from solid dosage forms (e.g. tablets), was evaluated by determining the release of model proteins as a function of time in a medium mimicking the gastrointestinal environment. .

By way of example, the data relating to a formulation of tablets containing the insulin / copolymer complexes with insulin / copolymer weight ratios of 1/6 are reported, using rice starch and microcrystalline silica as excipients. The results show a slow release of intact insulin from tablets containing the insulin / copolymer complexes. As shown in Figure 7, this delay is particularly evident in the case of the PHEA-C4-CS / insulin and PHEA-C4-CS-Cist / insulin complexes.

From these data it can be seen that the synthesized copolymers are able to control the release rate of insulin from the tablets, as well as their disintegration rate in simulated gastrointestinal fluids.

In addition, there is an effect of pH on the rate of insulin release relative to the insulin complexes with the PHEA-C4-CS and PHEA-C4-CS-Cist copolymers. These copolymers, in fact, possessing a certain amount of free carboxyl groups, have a solubility in an aqueous medium that increases with increasing pH. In complexes, this behavior leads to easier release of complexed insulin at intestinal pH (pH 6.8); on the contrary, negligible release and degradation at gastric pH (pH 1).

In vivo release studies in rats

The possibility of using the protein / copolymer complexes as systems for the delivery of proteins from oral solid dosage forms was evaluated by determining the release of insulin, used as a model protein, after oral administration to rats of capsules containing insulin complexed with the copolymers. object of the present invention.

By way of example, the data relating to the oral administration of gelatin capsules containing the insulin / copolymer complexes at insulin / copolymer weight ratios of 1/6 are reported, using microcrystalline silica as excipient.

In vivo experiments were conducted on healthy male rats weighing an average of 250 g, Wistar type. The animals were fasted overnight (approximately 12 hours) in such a way that they all had constant blood glucose levels. Before administration to each animal, 5 μΙ of blood was taken from the tail vein and the glucose levels were immediately measured using a blood glucose reader type Comelab 20 Thermo system (GODPOD).

The animals were divided into 4 groups of 3, each group treated separately with different dosage forms, after sedation with a2Agonists, Medetomidine (Domitor) at the dosage suggested by the pharmaceutical company. The different formulations were administered in the form of a 0.13ml capsule, placed in the inner part of the throat to stimulate the swallowing reflex.

The first group (called P1 os) was administered the dosage form consisting only of the inert excipients and free of in sulin, while the second group (called SC insulin) was administered a sterile physiological solution containing 2 mg (56 USP units) (~ 8 mg / kg) of bovine insulin (pancreas, Sigma) subcutaneously (positive control). The third group (named insulin / P1 os) was given the formulation prepared with the insulin / PHEA-C4-CS complex, containing 2 mg (56 USP units) (~ 8 mg / kg) of bovine insulin (pancreas, Sigma) . Finally, the last group (called os insulin) was administered 200 μΙ of a control aqueous insulin solution containing 2 mg (56 USP units) (~ 8 mg / kg) of bovine insulin (pancreas, Sigma), again by route oral (negative control).

All rats were fasted for 12 hours, leaving free access to water, during which 5 μΙ of blood from the tail were taken at 1 hour intervals and for 8 hours under local anesthesia with Lidocaine at 2. % gel (LUAN) and immediately monitored blood glucose levels. After 12 hours of administration, all the rats were fed; therefore, after a total of 24 hours, a final blood glucose sampling and dosage was carried out.

The results of this study are shown in Figure 8.

The studies have shown that insulin complexed with the copolymers described in the present invention induces a hypoglycemic effect that lasts up to 5 hours following the oral administration of the complex in rats. The data is significant, taking into account that insulin degrades in the gastric environment within two hours of administration at the same dose. In addition, the lowering of the glycemic level is equal to at least 30% of the effect observed after administration of insulin by the traditional route (SC) for the same amount of time; these results make the system feasible also for the oral administration of insulin in diabetic patients.

The present invention has been described with reference to some specific embodiments thereof, but it is to be understood that variations or modifications may be made to it by those skilled in the art without thereby departing from the relative scope of protection.

Claims (17)

CLAIMS 1. Colloidal polymeric vector for the delivery and release of peptides and proteins, in the form of nanoparticles, aggregates or complexes, consisting of a polymer or copolymer with a polyaspartamide structure bearing in the side chain, each individually or in combination with each other, hydrophobic and ionizable functions and thiol functions. 2. Polymeric vector according to claim 1, wherein said polyaspartamide structure polymer or copolymer is alpha, beta-poly (N-2-hydroxyethyl) aspartamide (PHEA). 3. Polymeric carrier according to claims 1 or 2, wherein said polymer or copolymer with polyaspartamide structure has a molecular weight comprised between 10,000 and 60,000 daltons. 4. Polymeric vector according to any one of claims 1-3, wherein said hydrophobic functions are selected from the group consisting of: linear, branched or cyclic alkyl with up to 18 carbon atoms, linear, branched or cyclic alkenyl with up to 18 carbon atoms carbon, linear, branched or cyclic alchemies with up to 18 carbon atoms, an isocyclic aromatic or aralkyl group with up to 18 carbon atoms. 5. Polymeric vector according to claim 4, wherein said hydrophobic functions are selected from the group consisting of: ethyl (C2), propyl (C3), butyl (C4), dodecyl (C12) or hexadecyl (Ci6) linear groups, propyl groups (C3), butyl (C4), dodecyl (Ci2) or hexadecyl (Ci6) branched or containing double bonds and aromatic groups. 6. Polymeric vector according to any one of claims 1-5, wherein said ionizable functions are selected from the group consisting of: linear or branched alkyl, alkenyl or alkynyclic groups with 2-20 carbon atoms carrying carboxy functions (-COOH); groups carrying phosphoric functions (-P03H2); groups carrying sulfuric (-S04H) or sulphonic (-SO3H) functions; carrier groups primary amino functions (-NH2), secondary amino (-NHR) 0 tertiary amino (-NR'R ") 0 quaternary ammonium salts [- <+> N (R) 3], 7. Polymeric vector according to any one of claims 1-6, wherein said thiol functions are derived from cysteine, homocysteine or cysteamine and their derivatives. 8. Polymeric carrier according to claim 7, consisting of the polymer (a, p-poly (N-2-hydroxyethyl) -co- [N-2- (butylcarbamate) ethylene] -co- [N-2 (2'carboxysuccinyl) ethylene] -co- [N-2- (2'-L-cysteine-succinylamide) ethylene] aspartamide) (PHEA-C4-CS-Cist). 9. Process for the production of colloidal polymeric vectors as defined in claims 1-8, comprising the following operations in succession: a) activation of the reactive groups of a starting polyaspartamide structure polymer with activating agents; b) reaction of the polymer thus activated with amines corresponding to said desired hydrophobic functions in the side chains, using a molar ratio between moles of amine and moles of repeating units of polymer comprised between 0.01 and 0.6; c) conjugation of the polymer obtained from the previous operation with molecules carrying said ionizable functions, using a ratio between moles of molecules carrying the ionizable functions and moles of repeating units of polymer between 0.01 and 0.6; d) conjugation of the polymer obtained from the previous operation with molecules carrying said thiol functions, in the presence of activating agents, using a ratio between moles of molecules carrying thiol functions and moles of repeating units of polymer between 0.1 and 0.5 . 10. Process according to claim 9, wherein said starting polyaspartamide structure polymer is alpha, beta-poly (N-2-hydroxyethyl) aspartamide (PHEA). 11. Process according to claims 9 or 10, wherein said activating agents of step a) consist of bis (4-nitrophenyl) carbonate (4-NPBC) in dimethylformamide (DMF) solution, and said operation is carried out at temperature constant, between 25 and 60 ° C. 12. Process according to any one of claims 9-11, wherein said activating agents of step d) consist of N-hydroxysuccinimide (NHS) and N-3 (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride (EDC-HCI) in aqueous solution and said operation is carried out at a temperature comprised between 15 and 40 ° C. 13. Pharmaceutical composition for the release of one or more active principles, in the form of nanoparticles, aggregates or complexes, in which said one or more active principles are conveyed in a colloidal polymeric vector as defined in claims 1-8. 14. Pharmaceutical composition according to claim 13, wherein said active ingredients are of peptide or protein nature. 15. Pharmaceutical composition according to claims 13 or 14, formulated for oral administration. 16. Composition according to claim 15, wherein said one or more active ingredients are included in said colloidal polymeric carrier, further comprising one or more pharmaceutical excipients acceptable for oral administration. 17. Pharmaceutical composition for the release of one or more active principles of peptide or protein nature according to claims 13 or 14, formulated in a liquid or solid release form.