COLLOIDAL VECTORS WITH POLYAMINOACID STRUCTURE FOR ORAL RELEASE OF PEPTIDES AND PROTEINS AND METHOD FOR THEIR

PRODUCTION

SPECIFICATION

The present invention concerns colloidal vectors with polyaminoacid structure for oral release of peptides and proteins and a method for their production. Specifically, the invention concerns systems for the release of active substances, specifically peptides and proteins, by means of their incorporation in nanoparticles, nano-aggregates or complexes based on properly derivat- ized synthetic polyaminoacids. These polymeric systems are proposed to release peptide drugs or proteins from oral dosage forms in an effective manner, besides increasing the physicochemical stability of proteins in liquid or solid pharmaceutical dosage forms.

As it is known, in the last decades there has been a growing interest toward therapeutic peptides or proteins. The reasons for this interest are easily understood: on the one hand, the increased availability of peptides and proteins, both natural and synthetic, and more commonly obtained by DNA recombinant technology, that allowed to overcome, even with some economic reservations, the quantitative limitations of the recent past, on the other hand, the increased knowledge of molecular biology and pharmaceutical biotechnologies have given a continuous input for identification of peptides and proteins with therapeutic activity in various pathologies, such as cancer and viral infec- tions. In addition, the progresses in the biotechnology field are well supported in the latest years by technological advances, in the design and realization of therapeutic systems able to carry peptides and proteins in a safe and effective manner.

Technological points of view as well as aspects in pre-formulation step are known to be very important, since peptides and proteins need a particular attention because of several physical (denaturation, aggregation, surface adsorption) or chemical (deamination, oxidation, racemization, formation of

disulfide bridges) causes that can result in a reduction or in the total disappearance of biological activity.

Besides the restrictions due to the instability of these molecules, further problems derive from the choice of alternative administration routes dif- ferent from the parenteral route, that is to date the most used. There are several studies concerning the evaluation of non-conventional dosage forms that can be useful for the administration of proteins through routes different from the parenteral way.

Certainly, the oral route is the administration route that offers the most significant advantages, including accuracy of dosage, an easy use and a high patients' compliance. In spite of these advantages, some drawbacks due to the administration of peptides and proteins by oral route are difficult to solve. Besides having to consider physical and chemical instability phenomena due to the protein structure, the transit in the gastrointestinal tract causes the con- tact of these molecules with physiological media whose pH value or enzymatic content are not compatible with their active form and their potential absorption. For these reasons the oral administration could be performed only by using gastro-resistant dosage forms and possibly with co-administration of peptidase enzyme inhibitors. In addition, it must be considered that generally proteins and peptides have a high molecular weight and an adequate hydrophilic character. As a consequence they possess a reduced permeability across cell membranes and a low absorption.

Therefore, it is evident that, even if very interesting, the oral admini- stration of peptides and proteins is, to date, a difficult goal to realize, although there are several authoritative scientific papers and also patent literature [see review in: des Rieux A., Fievez V., Grarinot M., Scheider Y-J., Preat V., Nanoparticles as potential oral delivery systems of proteins and vaccines: A mechanistic approach, J. Control. Release 116 (2006) 1-27]. For example, the US patent US 5641515 (assignee Elan Corporation) reports biodegradable nanoparticles for controlled release of insulin, that are also proposed for oral administration of this protein, that is conventionally

taken by diabetics many times a day as subcutaneous injection. In the formulation proposed in the above patent, insulin is entrapped into nanoparticles based on a polycyanoacrylate, prepared by a polymerization of the cyanoacry- late monomer at low pH and in the presence of insulin. However, it is a widely shared the opinion that a possible solution for the realization of an efficient system for the oral administration of peptides and proteins could be found in a suitable combination between therapeutic systems of different nature and suitable composition.

An interesting approach in the oral delivery of peptides and proteins is the use of polymeric carriers, properly chemically derivatized, where the deri- vatization confers to the polymeric carrier several profitable properties, such as the ability to be mucoadhesive and/or to act as protease inhibitors [see for example Valenta C, Marschutz M. K., Egyed C, Bernkop-Schnurch A., Evaluation of the inhibitory effect of thiolated poly(acrylates) on vaginal mem- brane bound aminopeptidase N, J. Pharm. Pharmacol. 54 (2001) 603-610.; Bernkop-Schnurch A., Thaler S. C, Polycarbophil-cysteine conjugates as platforms for oral polypeptide delivery systems, J. Pharm. Sci. 89 (2000) 901- 909]. Another property that can be given to polymers by derivatization is the ability to act as permeation enhancers [see for example Clausen A. E., Bernkop-Schnurch A., In vitro evaluation of the permeation-enhancing effect of thiolated polycarbophil, 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]. Therefore, such polymeric vectors offer the advantage of a reduced degradation of the peptide or protein drug they carry in the gastrointestinal tract, and a greater absorption.

The mucoadhesion of a polymer, i.e. its ability to adhere to mucous membranes of the organism (and, in particular, to gastrointestinal mucosa) is an important property for the development of drug delivery systems, especially for peptides and proteins, since thanks to mucoadhesive properties it is possible to obtain a close contact between the polymeric systems and the mucous membrane, thus preventing the alteration of peptides and proteins in the gas-

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trointestinal tract. In addition, it is possible to increase the residence time of peptides and proteins in the adsorption site, where the high concentration obtained will promote their absorption.

Thiolated polymers (thiomers or thiopolymers), i.e. polymers where thiol groups are linked to the backbone, represent an example of mucoadhe- sive polymers useful for the preparation of solid oral dosage forms [Bernkop- Schnurch A., Krauland A.H., Leitner V.M., Palmberger T., Thiomers: potential excipients for non-invasive peptide delivery systems, Eur. J. Pharm. Bio- pharm. 58 (2004), 253-263]. Among these polymers, the above literature re- ports chitosan-cysteine, polycarbophil cysteamine, polyacrylic acid- homocysteine, that show strong mucoadhesive properties thanks to the formation of covalent linkages (S-S bonds) between thiol groups of the polymer and cysteine domains of glycoproteins present in the mucus. The formation of covalent linkages between thiopolymers is very important too. This phenome- non, that causes a decrease in free thiol groups in comparison with total thiol groups, produces an increase in the medium viscosity. Such further ability of thiolated polymers, i.e. the ability to form disulfide bonds, both inter- and intramolecular, can be exploited when these polymers are used as excipients for tablet formulation [Bernkop-Schnurch A.,. Kast CE, Richter M. F., Im- provement in the mucoadhesive properties of alginate by the covalent attachment of cysteine, J. Control. Release 71 (2001) 277-285]. In fact, by using thiolated polymers it is possible to prolong the disintegration time of these tablets in comparison with the use of non-thiolated excipients; this because the inter-chain disulfide bridges confer high stability to the drug-carrier system based on thiopolymers and increase the medium viscosity.

On the other hand, both ionic and neutral polymers can interact with proteins, thus forming macromolecular aggregates: the former polymers act through the formation of hydrogen bonds, van der Waal's and electrostatic interactions with the polar groups of the proteins; the latter polymers act mainly through hydrophobic interactions. In addition, the presence of hydrophobic regions in a polymer can promote the interaction of a hydrophobic nature with the lipophilic portions of biological membranes, thus increasing the

permeability of polymer-protein complex.

With reference to systems proposed for peptide and protein release based on polymers whose backbone is derivatized with hydrophobic and ioni- zable groups, the European patent EP 0734720 (Flamel Technologies), corre- sponding to US patent 5904936, discloses polyaminoacidic microparticles, based on α-aminoacids and obtained by block or statistical polymerization of two different types of α-aminoacids: a hydrophobic or neutral aminoacid (such as Leu, VaI, Ala, Pro, Phe) and an aminoacid with a ionizable side chain (such as aspartic or glutamic acid). Specifically, the preferred polyaminoacids re- ported in the patent are poly(Leu/Glu), with a ratio Leu/(Glu+l_eu) of not less than 6% for block polyaminoacids, and not less than 20% for statistical polyaminoacids, with a molecular weight not less than 4000 Da. Pratically, in the preferred examples here reported, the fraction of leucine must be adequately high to avoid the complete water solubility of the polymer and to give it suit- able hydrophobic properties, in order to cause an association of polymeric chains to form nanoparticulate systems. The obtained nanoparticles form a colloidal dispersion in aqueous medium, that can be loaded with the peptide drug. Alternatively, particles containing the active ingredient can be obtained by dispersion of α-polyaminoacids in the solution containing the peptide drug, before aggregation.

As a further progress of the above indicated system, the European patent EP 1131056 (Flamel Technologies), which corresponds to the US patent US 6630171 , discloses a similar release system, that has been called "Medusa ^® " probably to remind the action mechanism of polymeric vectors in the incorporation of polypeptide substances, i.e. the ability to interact with the active ingredient and to "wrap up" the active ingredient, thus protecting it from degradation phenomena and carrying it as particulate form (nano or micro) until the action site. In this system the polyaminoacidic carriers that form the principal backbone of the particles are homopolymers or copolymers of glu- tamic acid (AcGIu) and/or aspartic acid (AcAsp) and their salts (recurring ami- noacids, both ionizable), and a percentage of them present a hydrophobic

side chain, linked to the carboxyl group of AcGIu or AcAsp by ester (or amide) linkages. The length in the polymeric chain ranges from 200 to 1000 aminoac- ids, i.e. with a molecular weight equal to 26,000-65,000 for an AcGIu ho- mopolymer and 23,000-57,000 for an AcAsp homopolymer. The hydrophobic groups (R) linked to the polymeric backbone can be linear or branched carbon chains or they can contain double bonds, and preferably they are linear groups C ₂ -C ₁₈ . The molar percentage of recurring ami- noacids derivatized with hydrophobic functions ranges from 3 to 70%. Some examples of derivatized polymers reported in the patent are: sodium polyglu- tamate-ib/oc/c-exadecyl glutamate; sodium polyglutamate-co-ethyl glutamate; sodium polyaspatic acid-Jb/oc/c-propyl aspatate; sodium polyaspatic acid-b/oc/c- benzyl aspartate.

The preparation of the release particles based on the above polymers occurs by a decrease in solubility of the hydrophobic fraction, by adding a saline solution that causes the precipitation of the polymer with a consequent formation of nanoparticles. In order to increase the particle size to obtain mi- croparticles, during the formation of the suspension, a further aggregation step is made to occur, by using an aggregation agent, such as an electrolyte, an acid, a base or a ionic polymer (polylisine, polyethyleneimine). As in the previ- ous case, the particles can be loaded with the active protein by performing their formation in a medium containing the active ingredient, or by dispersion and incubation of empty lyophilized particles in a medium where the active ingredient is dissolved or dispersed.

In view of the foregoing, a main object of the present invention is thus to increase the oral bioavailability of peptide and protein active ingredients, e.g. insulin, by exploiting the ability of carrier copolymers to interact with the protein molecules, to reduce their degradation by digestive enzymes and gastric pH and to promote their oral absorption.

Another object of this invention is to employ the proposed copolymers as excipients for the production of oral or liquid dosage forms containing peptides or proteins in order to prolong their stability both in the dosage form and in physiological environment.

As already observed, it is evident that to improve the performance of systems for the release of peptides and proteins based on colloidal polymeric vectors (both to increase the oral bioavailability thereof and to increase the physicochemical stability of said peptides and proteins in liquid or solid dos- age forms), it is necessary to combine in the same polymer different chemical functionalities, each with a specific technological property. In this way, it is possible to optimize and to modulate the advantageous properties of biocompatible polymers for their use in the realization of dosage forms for the release of peptides and proteins, also intended for oral adminisration. To that end there is proposed, according to the present invention, to use for the realization of polymeric vectors useful as carriers for peptides and proteins specific polymeric structures of a polyaspartamide type, containing hydrophobic functionalities, ionizable functionalities and thiol functionalities as side chains, each individually or in combination between them. In particular, the approach employed in the present invention is the synthesis of copolymers based on a poly-hydroxyethyl-aspartamide or PHEA, more exactly α,β- poly(N-2-hydroxyethyl)-D,L-aspartamide, having the following structure:

or its copolymers, that present, in the side chains, hydrophobic, ionizable and thiol functionalities, covalently linked through the hydroxyl groups that are present one in each repeating unit of these polymers. The obtained copolymers are able to form pharmaceutical systems for the release of peptides and

proteins, through their incorporation into nanoparticles or nano-aggregates or complexes, formed by the interaction between these copolymers and the proteins or peptides at issue.

As it known, PHEA is a derivative of a high molecular weight polysuc- cinimide (PSI), obtained by aminolysis of the latter, by reaction with ethanola- mine. As it can be observed in the above structure and is more evident in the following structure - that represents the same polymer in a different graphic form - due to the cyclic structure or the starting polysuccinimide, the coupling of ethanolamine can occur in such a way as to leave a methylene group either in the polymeric backbone or in the pendent functional group. Therefore, the repeating unit can have a structure slightly different in the first or in the second case, but its molecular weight is the same.

It is known that PHEA is a polymer with excellent biopharmaceutical properties, such as biocompatibility, in fact in the past it has been proposed as plasma expander [Neri P., Antoni G., Benvenuti F., Cocola F., Gazzei G., Synthesis of α,β-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 (PHEA) as a plasma substitute, // farmaco 34 (1978) 146-156]. The polymer is also characterized by an optimal reactivity, since it presents hydroxyl groups in side chains.

The technology proposed by the present invention consists in incorporating peptides or proteins into nanoparticles, nano-aggregates or complexes

based on synthetic linear polyaminoacids, specifically polyaspartamide-like polyaminoacids, properly derivatized with thiol, hydrophobic and ionizable groups. The suitable combination of these functionalities in side chains of the copolymer confers to it the property to interact with the protein of interest, through non-covalent interactions, such as hydrophobic, electrostatic and hydrogen bonds, and to form supramolecular systems with colloidal size, having a high kinetic stability and mucoadhesive properties, besides all advantageous properties of thiomers, such as ability to act as protease inhibitors and permeation enhancers. Besides PHEA, other polymers with polyaspartamide structure with nucleophilic pendent groups along the chain can be also used and properly derivatized with hydrophobic, ionizable and thiol functionalities, in such a way to result in compositions and materials according to this invention, each time with specific properties. Therefore, the present invention specifically provides a colloidal polymeric vector for the formulation and the release of peptides and proteins from nanoparticle, aggregate or complex dosage forms, which vector consists of a polymer or copolymer with polyaspartamide structure, carrying hydrophobic functionalities, ionisable functionalities and thiol functionalities in side chains, each individually or in combination with each other.

The proposed polymeric vectors are obtained starting from macro- molecules with polypeptide structure (specifically, polyaspartamide) having nucleophilic groups in each repeating unit and from their copolymers. The moieties or functionalities reported above are covalently linked to these nu- oleophilic groups. Within the same macromolecule these moieties or functionalities are either of a hydrophobic, or ionizable or thiol type, and each of such functionalities can be linked to the polymeric backbone individually or in combination with other different functionalities.

Preferably, according to the present invention, said polymer or co- polymer with polyaspartamide structure is α,β-poly(N-2-hydroxyethyl)-D,L- aspartamide (PHEA) and its copolymers, carrying in side chains hydrophobic, ionizable and thiol functionalities, linked to the hydroxyl groups of the starting

polymer through amide, enamine, urethane or ester bonds, as a function of the conjugated group.

Preferably, the length of the polyaminoacid chain is such that the molecular weight ranges from 10,000 to 60,000 dalton. In the preferred case of PHEA, this corresponds to a number of repeating units of hydroxyethyl- aspartamide ranging from 63 to 315.

The hydrophobic groups linked to the polymer backbone are preferably chosen in 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 at- oms; linear, branched or cyclic alkynyl with up to 18 carbon atoms; an aromatic or isocyclic arylalkyl group with up to 18 carbon atoms. According to some specific embodiments of this invention, these hydrophobic groups are chosen among: linear ethyl (C ₂ ), propyl (C ₃ ), butyl (C ₄ ), dodecyl (C1 ₂ ) or hexa- decyl (Ci ₆ ) groups; branched propyl (C ₃ ), butyl (C ₄ ), dodecyl (Ci ₂ ) or hexade- cyl (Ciβ) groups; propyl (C ₃ ), butyl (C ₄ ), dodecyl (C12) or hexadecyl groups containing double bonds and aromatic groups Preferably, the molar ratio of molecules carrying the hydrophobic groups to moles of repeating units of the polymer ranges from 0.01 to 0.6.

The ionizable functionalities that preferably are linked to the polyas- partamide structure of this invention are made of: linear or branched alkyl, alkenyl or alkynyl groups, with a number of carbon atoms ranging from 2 to 20, carrying carboxyl functionalities (-COOH); groups carrying phosphoric functionalities (-PO ₃ Ha), such as O-phosphocolamine; groups carryng sulfuric (-SO ₄ H) or sulfonic (-SO ₃ H) functionalities, such as taurine; groups carrying primary amines (-NH ₂ ), groups carrying secondary amines (-NHR) or groups carrying tertiary amines (-NR ¹ R") or groups carrying ammonium quaternary salts [- ⁺ N(R) ₃ ]. The molar ratio between moles of molecules carrying the ionizable group and the moles of repeating units of the polymer ranges from 0,01 to 0,6. The thiol functionalities 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 moles of molecules carrying the thiol group and moles of repeating units of the polymer ranges from 0.01 to 0.5.

According to a preferred embodiment of this invention, the colloidal polymeric vector carrying, in the polyaspartamide chain, hydrophobic functionalities such as linear alkyl, ionizable functionalities such as carboxyl groups and thiol functionalities deriving from cysteine, is made of the polymer α,β-poly(N-2-hydroxyethyl)-co-[N-2-(butylcarbamate)ethylen ]-co-[N-2(2'car- boxysuccinyl)ethylen]-co-[N-2-(2'-L-cysteine-succinylamide)e thylen]aspart- amide). This polymer has been called PHEA-C ₄ -CS-CySt.

The preparation of some of the copolymers referred to above can be performed by means of a sequence of reactions that allow to link the following molecules to the hydroxyl groups of the starting polyaspartamide:

^■ hydrophobic groups ^■ ionizable groups

^■ thiol groups.

According to a further aspect thereof, the present invention concerns a procedure for the production of the above-defined polymeric colloidal vectors, including the following sequence of steps: a) activation of reactive groups of a starting polymer with polyaspartamide structure by means of activating agents; b) reaction of the polymer thus activated with amines corresponding to desired hydrophobic functionalities in the side chains, by using a molar ratio between moles of amine and moles of repeating units of polymer ranging from 0.01 to 0.6; c) conjugation of the polymer obtained from the previous reaction with molecules carrying ionizable functionalities, by using a molar ratio between moles of ionizable functionalities and moles of repeating units of polymer ranging from 0.01 to 0.6; d) conjugation of the polymer obtained from the previous reaction with molecules carrying thiol groups, in the presence of activating agents, by using a molar ratio between moles of thiol groups and moles of repeat-

ing units of polymer ranging from 0.1 to 0.5.

Preferably, as evidenced, the starting polyaspartamide is α,β-poly(N- 2-hydroxyethyl)-D,L-aspartamide or PHEA

According to some preferred embodiments of the synthesis method of the invention, the activating agents of step a) consist of bis (4- nitrophenyl)carbonate (4-NPBC) in dimethylformamide (DMF) solution, and this reaction is carried out at constant temperature, ranging from 25 to 60 ⁰ C.

In addition, preferably, the activators of step b) consist of N-hydroxy- succinimide (NHS) and N-3(3-dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride (EDC-HCI) in aqueous solution, and this reaction is carried out at a temperature ranging from 15 and 40 ⁰ C.

In a specific case, reported for illustrative purposes, the following groups are linked in sequence to hydroxyl groups of the starting polyaspartamide: ^» an aliphatic chain with four carbon atoms (C ₄ ) (thus obtaining the copolymer referred to as PHEA-C ₄ );

^■ succinic groups (thus obtaining the copolymers referred to as PHEA-C ₄ - CS);

^■ molecules carrying thiol groups such as cysteine (thus obtaining the co- polymer referred to as PHEA-C ₄ -CS-CySt).

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

The introduction of butylamine chains in the PHEA polymeric back- bone has been confirmed by ¹ H-NMR and FT-IR analysis of the resulting

PHEA-C ₄ copolymer after exhaustive dialysis against distilled water and Iy- ophilization. The percentage of alkylamine functionalities linked to PHEA in the PHEA-C ₄ copolymer (expressed as number of moles of linked bu- tylamine/number of polymer repeating units x100), determined by ¹ H-NMR, is in the range between 1 and 60 mol % (by preference between 15 and 20 mol%).

The weight average molecular weight of the PHEA-C ₄ derivative, as

determined by size exclusion chromatography (SEC), is comprised between 30 and 50 kDa, depending on the degree of derivatization, as determined by using a calibration curve obtained with PEO/PEG standards.

In the second step, the PHEA-C ₄ copolymer, obtained as previously reported, reacted with succinic anhydride (SA) in DMF solution, in the presence of triethylamine (TEA) for a time period ranging from 4 to 24 hours, depending on the molar ratio between SA and PHEA repeating units, and at constant temperature, preferably between 15 and 40 ⁰ C.

The molar ratio between SA and PHEA repeating units can be comprised in the range between 0.01 and 0.6. In such a condition the still free hydroxyl groups of the PHEA-C ₄ copolymer react easily with succinic anhydride, according to the scheme reported scheme below.

After purification by exaustive dialysis against distilled water, the resulting PHEA-C ₄ -CS copolymer (α,β-poly(N-2-hydroxyethyl)-co-[N-2- (butylcarbamate)ethylen]-co-[N-2(2'carboxysuccinyl)ethylen]- aspar- tamide) has been characterized by FT-IR e ¹ H-NMR. Analytical and spectral data of copolymer confirmed the introduction of succinic group into the copolymer.

The percentage of succinic functionalities linked to PHEA-C ₄ -CS copolymer (expressed as number of linked succinic groups/number of polymer repeating units x 100), determined by ¹ H-NMR, is in the range between 1 and 60 mol% (preferably between 30 and 50 mol%).

The weight average molecular weight of PHEA-C ₄ -CS copolymer, determined by size exclusion chromatography (SEC) is included between 30 and 50 kDa, depending on the degree of derivatization, by using a calibration curve obtained with PEO/PEG standards. In the third step of the synthesis process, the obtained PHEA-C ₄ -CS copolymer reacted with cysteine, in the presence of N-hydroxysuccinimide (NHS) and N-3(3-dimethylaminopropyl)-N-ethyl-carbodiimmide hydrochloride (EDC-HCI) in aqueous solution, for a period time ranging from 1 to 18 hours, depending on the molar ratio between cysteine and succinic groups linked to PHEA, at constant temperature, preferably between 15 and 40 ⁰ C. The molar ratio between cysteine and succinic acid groups linked to PHEA can be in the range between 0.5 and 2, corresponding to a molar ratio between thiol- bearing molecules and copolymer repeating units in the range between 0.1 and 0.8. In such a condition the amine groups of cysteine react with carboxylic groups of succinic chains of PHEA-C ₄ -CS-Cyst (α,β-poly(N-2-hydroxyethyl)- co-[N-2-(butylcarbamate)ethylen]-co-[N-2(2'carboxysuccinyl)e thylen]-co- [N-2-(2'-L-cysteine-succinylamide)ethylen]aspartamide).

PHEA-C ₄ -CS

After purification by extaustive dialysis against distilled water, the PHEA-C ₄ -CS-CySt copolymer has been characterized by FT-IR and ¹ H-NMR.

The percentage of thiol groups linked in the PHEA-C ₄ -CS-CySt copolymer (expressed as moles of linked cysteine/moles of copolymer repeating units x 100), has been determined by Ellman assay, allowing the evaluation of thiol group amount (SH) and then the percentage of cysteine linked to copolymer. This percentage is from 1 to 50 mol % (the most preferred being 10 % of repeating units of PHEA i.e. 30 % of succinic groups).

The weight average molecular weight of PHEA-C ₄ -CS-CySt copolymer, determined by size exclusion chromatography (SEC) is between 30 and 50 kDa,

depending on the degree of derivatization, by using a calibration curve obtained with PEO/PEG molecular standards.

For all synthesized copolymers, yields were in the range between 94 and 100 % starting from the parent polymer. According to a further aspect thereof, the present invention concerns pharmaceutical products obtained by loading the proposed polymeric vectors with active ingredients preferably, but not exclusively, of protein or peptide nature. Therefore, the invention concerns a pharmaceutical composition for the delivery of one or more active ingredients, specifically with protein or pep- tide structure, in the form of nanoparticles, aggregates or complexes, wherein said one or more active ingredients are carried by a polymeric colloidal vectors as defined above.

The proposed pharmaceutical formulation can advantageously be used for oral administration. The said one or more active ingredients incorpo- rated into polymeric vectors according to the present invention can be formulated together with one or more pharmaceutical excipients acceptable for oral administration.

The present invention further concerns pharmaceutical formulations containing one or more active agents with peptide or protein structure as above defined, administered as liquid or solid dosage forms.

The interaction between the copolymers object of the present invention and protein or peptide molecules allows to protect them from both chemical and enzymatic degradation and to release them in an intact form in the administration site, thus offering the advantages of a minor degradation of the protein drug and of a greater absorption.

Polymeric delivery systems according to this invention have shown to be able to increase oral bioavailability of insulin, used as model protein, and to induce a decrease of the plasmatic glucose levels in the rats equal to 30 % of the ipoglicemic effect obtained after subcutaneous administration of insulin. Moreover, these vectors have not evidenced any in vitro citotoxicity and showed a very good in vivo tolerability on the animal models used. In addition, the proposed systems have been able to increase chemical stability of insulin

in the presence of proteolytic enzymes such as α-chimitripsin, and in media mimicking gastrointestinal fluids.

The use of the vectors according to the present invention, therefore, allows to increase the oral bioavailability of peptide and protein drugs, such as for example insulin, and to prolong the stability of peptide and protein molecules when administered in the presence of these copolymers as excipients, used in either liquid or solid formulations.

The specific features of the invention, as well as its advantages and the corresponding operating conditions, will be more evident with reference to the detailed description presented by way of an example below, together with some results of the experiments performed on the invention and data for comparison with the prior art. Some experimental results are also reported in the attached figures, wherein:

Figure 1 shows the cell availability profile, evaluated by MTS test, on intestinal epithelial cells after 24 hours of incubation with increasing concentration (0,1 , 0,5 e 1 mg/ml) of PHEA-C ₄ -CS and PHEA-C ₄ -CS-CySt copolymers;

Figure 2 shows the transmittance profile as a function of time of insulin, PHEA-C ₄ and insulin/PHEA-C ₄ complex sample solutions at different weight ratios (1/1 , 1/3, 1/6);

Figure 3 shows the transmittance profile as a function of time of insulin, PHEA-C ₄ -CS and insulin/PHEA-C ₄ -CS complex sample solutions at different weight ratios (1/1, 1/3, 1/6);

Figure 4 shows the transmittance profile as a function of time of insu- lin, PHEA-C ₄ -CS-CySt and insulin/PHEA-C ₄ -CS-Cyst complex sample solutions at different weight ratios (1/1 , 1/3, 1/6);

Figure 5 shows the agarose gel electrophoresis of insulin and PHEA- C ₄ /insulin, PHEA-C ₄ -CS/insulin, PHEA-C ₄ -CS-Cyst/insulin complexes to confirm the structural integrity of complexed protein; Figure 6 shows the percentage of intact insulin released as a function of time from insulin/copolymer complexes after incubation in the presence of

α-chymotrypsin;

Figure 7 shows the percentage of insulin released from tablets containing different insulin/copolymer complexes in media mimicking gastrointestinal fluids (pH 1 , up to 120 min, pH 6.8 from 121 to 360 min); Figure 8 shows the glycemic profile of rats after administration of: insulin/PHEA-C ₄ -CS complex (insulin/P1 os), not complexed insulin (free oral, insulin os); free copolymer (P1 os) and subcutaneous insulin (insulin sc) as positive control;

Figures 9 a) and b) show the observation under UV transilluminator of rat intestin segments after oral administration of FITC-insulin/PH EA-C ₄ - CS complexes; and

Figures 10 a), b) and c) show confocal laser scanning microscope images of rat intestinal tissues after oral administration of FITC- insulin/PHEA-C ₄ -CS complexes. The copolymers object of the present invention have been subjected to in vitro biocompatibility studies on human intestinal epithelial cells to evaluate the possible toxic effects by MTS test. As shown in the enclosed Figure 1 , cell availability data after 24 hours of incubation with copolymer concentration of 0,1 , 0,5 and 1 mg/ml have not shown any cytotoxic effects on the cellular model used.

By way of example only, results relevant to a series of copolymers are reported below, where these copolymers are used as vectors of a model protein for interaction studies and oral delivery studies. In order to evaluate the possibility to use the synthesized copolymers as agents for the delivery of proteins, and among them insulin, chosen as model protein, studies of interaction between insulin and copolymer according to the invention have been performed: • turbidimetric studies; gel agarose electrophoresis; light scattering and zeta potential measurements. Some experimental results are reported in enclosed figures.

Turbidimetric studies have been performed by recording the transmit-

tance at 500 nm of insulin, single copolymers and insulin/copolymer complexes at different weight ratios in buffer solution at pH 7.4.

The insulin/ PHEA-C ₄ or insulin/PHEA-C ₄ -CS complexes, described in the foregoing as an example, show a transmittance decrease as a function of time. This result is much more pronounced in the case of the insulin/ PHEA- C ₄ -CS-Cyst complex, with a strong trasmittance decrease as a function of time (up to 24 hours) and as a function of the insulin/copolymer ratio. Transmittance results as a function of time are reported in Figures 2, 3 and 4.

In all cases this behaviour can be explained hypothesizing the forma- tion of colloidal aggregates between protein and copolymer that, for their dimensions, turn out to be "opaque" to incident light rays in comparison with single copolymers and free protein, also if they are invisible to the naked eye. In the case of the PHEA-C ₄ -CS-CySt copolymer particularly, not only physical bonds are involved in the aggregation formation, such as Van der Waals and hydrophobic interactions, but also chemical linkages, such as S-S bridges among the same copolymers chains.

In order to verify the structural integrity of the complexed protein, electrophoresis analyses on agarose gel have been performed on insulin/copolymer complexes. In the enclosed Figure 5 the electrophoretic run of insulin/copolymer complexes, carried out for 30 min at 150 V, is reported; this figure does not show any modifications in the electrophoretic mobility of insulin complexed by copolymers in comparison with naked insulin (control), and no further bands have been evidenced showing the possible degradation of protein. Therefore, it is possible to conclude that the chemical structure of proteins, as well the protein secondary and tertiary structures, remain unchanged after interacting with any of one the copolymers.

Finally, light scattering analyses and zeta potential measurements have been performed in buffer solutions at pH 7.4, as a function of time, in order to evaluate the hydrodynamic diameter of the polymeric complexes and to obtain information on surface charges of the same complexes.

Insulin/PHEA-C ₄ and insulin/ PHEA-C ₄ -CS complexes showed a

smaller size than free insulin, thus suggesting the occurrence of protein condensation processes due to the interaction with copolymers. Such a behaviour seems to be depending on the time for PHEA-C ₄ -CS-CySt copolymer. In fact the complex size decreases strongly as a function of time, probably due to the occurrence of S-S bridges between copolymer chains. These results are, on the other hand, in agreement with that obtained from turbidimetric studies.

Moreover zeta potential measurements show a decrease of the complex surface potential in comparison with naked insulin; that confirms a screening effect on the surface charge of the insulin molecule after the com- plexation with the copolymer.

Stability of protein/copoiymer complexes in the presence of α- chymotrypsin

The ability of copolymers according with the present invention, to improve the stability of protein molecules to intestinal degradation, has been evaluated after incubation of the protein/copoiymer complexes, previously prepared and lyophilised, in the presence of α-chymotrypsin and evaluating, at regular time intervals the percentage of intact protein in the reaction medium.

As shown in Figure 6, the copolymers object of the present invention show a protection effect reducing the enzymatic degradation of insulin in the presence of α-chymotrypsin. This effect of protection appears to increase in the order PHEA-C4 < PHEA-C ₄ -CS < PHEA-C ₄ -CS-CySt. In the latter case, in fact the presence of insulin in the intact form is recorded until 140 min from the beginning of the enzymatic incubation. On the contrary, naked insulin under- goes complete degradation in the presence of α-chymotrypsin within 40 min.

The different impact of the protecting effect towards insulin can be explained considering the different nature and strength of the interactions between insulin and the three copolymers considered, i.e. mainly hydrophobic interaction with PHEA-C ₄ , hydrophobic interactions and hydrogen bonds with PHEA-C ₄ -CS and hydrophobic interactions, hydrogen bonds and S-S bridges with PHEA-C ₄ -CS-CySt.

Release studies in artificial gastrointestinal fluids

The possibility to use protein/copolymer complexes as systems for the release of proteins in oral dosage form (for example tablets) has been evalu- ated by measuring the amount of intact model protein released as a function of time in a medium mimicking gastro-intestinal fluids.

By way of example, data related to a tablet formulation containing protein/copolymer complexes at a weight ratio 1/6, by using rice starch and microcrystalline cellulose as excipients are reported. Results show a slow release of intact insulin from the tablets containing the insulin/copolymer complexes. As shown in Figure 7, a delayed release is particularly evident in the cases of lnsulin/PHEA-C ₄ -CS and insulin/PHEA-C-rCS-Cyst.

From these data there can be concluded that the synthesized copolymers are able to control the release rate of insulin from tablets, as well tablet disgregation rate in mimicking gastrointestinal fluids.

Moreover, a pH-depending effect can be observed on the insulin release rate in the systems containing insulin/PHEA-C ₄ -CS and insulin/PHEA- C ₄ -CS-CySt complexes. These copolymers, in fact, bearing a certain amount of carboxylic groups, show a water solubility increasing as pH increases. In the complexes this behaviour results in an easier release of the complexed insulin at intestinal pH (pH 6.8) and, on the contrary, a negligible release and degradation at gastric pH (pH 1.1).

In vivo release studies on rats The possibility to use protein/copolymer complexes as systems for the release of proteins in oral dosage forms has been evaluated by measuring the amount of released insulin, used ad model protein, after oral administration of capsules containing insulin complexed by the copolymers of the present invention to rats. By way of example, data concerning the oral administration of gelatin capsules containing protein/copolymer complexes at weight ratio 1/6, by using microcrystalline cellulose as excipients are reported below.

In vivo experiments have been performed on healthymale Wistar rats weighting about 250 g. The animals have been kept fasting for a night (about 12 hours) in a such way to have constant blood glucose levels for all animals. Before each administration, 5 μl of blood have been taken from the tail vein and glucose levels were immediately measured by using a detector Comelab 20 SystemThermo (GODPOD).

The animals were divided in four groups of 3 animals each; each group was treated separately with different dosage forms, upon sedative administration of medetomidine (Domitor) at the dose suggested by the pharmaceutical company.

Different formulations have been administrered as capsules of 0.13 ml, placed deeply into the throat in order to initiate the swallow-reflex.

To the first group (called P1 os) the dosage form containing just inert excipients without insulin has been administered, whereas to the second group (called insulin sc), a sterile physiological solution containing 2 mg (56 USP units) (~ 8 mg/kg) of bovine insulin (spleen, Sigma) has been given by subcutaneous administration (positive control). To the third group (called insulin/Pi os) the formulation insuline/PHEA-C ₄ -CS complex, having 2 mg (56 USP units) (~ 8 mg/kg) of bovine insulin (pancreas, Sigma) has been given. Finally to the last group (called insulin os) 200 μl of an aqueous solution of insulin containing 2 mg (56 USP units) (~ 8 mg/kg) of bovine insulin (pancreas, Sigma), has been administered by the oral route (negative control).

All animals have been kept fasting for 12 hours, with free access to water drinking and during this time, at 1 hour time intervals for 8 hours, sam- pies of 5 μl of blood have been taken from the tail vein, upon local anaesthesia by gel Lidocaine 2 % (LUAN) and blood glucose levels have been immediately evaluated. 12 hours after the administration all rats have been fed and then after further 12 hours (24 hours totally) the last blood sample taking and glycemic analysis have been performed. Results are reported in Figure 8.

The studies have evidenced that the insulin complexed with the co-

polymers described according to the present invention causes an ipoglycemic effect during until 5 hours after the rat oral administration of the complexes.

This result is significant, considering that insulin at the same dose is degraded in the gastric environment within 2 hours. Moreover, the decrease of the glycemic level is equal to almost 30% of the effect observed after the administration of insulin by conventional subcutaneous administration (S. C.) in the same time period. The obtained results make these systems proposable for the oral administartion of insulin in diabetic patients.

Ex vivo evaluation of FITC- insulin intestinal tracking

Fluorescein(FITC)-labeled insulin complexed with PHEA-C ₄ -CS was filled into capsules to yield 2.0 mg of FITC-insulin per capsule and administered by placing the capsules deeply into the throat, in order to initiate the swallow-reflex to Wistar rats fasted overnight (3 rats). The rats were sacrificed 3 h later and intestinal segments localized after laparotomy by observation under UV transilluminator. After washing with isotonic saline, intestinal epithelial cell membranes were stained with formalin (10% paraformaldehyde in PBS pH 7.4) for 24 h. Tissues were rinsed and vertical sections were prepared, mounted on glass slides and observed using a confocal laser scanning micro- scope. Tissue samples were scanned in the x,y plane with λ _e χ _C = 486 nm and λ _em - 529 nm. Control rats (3 rats) were dosed with FITC-insulin or PHEA-C ₄ - CS containing capsules.

The results are reported in Figure 9 a) and b) as well in Figure 10 a), b) and c). After about 3 hours from the administration of the complexes the luminescence of the fluoresceinated insulin confirmed the abundant presence of this protein in the rat intestin (Figure 9 a) and b)).

The microscopy observation (Figure 10 a), b) and c)) of the cross sections of intestin evidences the presence of fluoresceinated insulin also into the cells in deeper layers, showing the effect of the copolymers of the invention as absorption promoters; no fluorescence was detected in the cross section of in-

testin of animals treated with non-complexed fluoresceinated insulin (Figure 10 c).

The present invention has been disclosed with particular reference to some specific embodiments thereof, but it should be understood that modifica- tions and changes may be made by the persons skilled in the art without departing from the scope of the invention as defined in the appended claims.