WO2013054298A2 - Modified polyethylene glycols and their supramolecular complexes with biologically active macromolecules - Google Patents

Modified polyethylene glycols and their supramolecular complexes with biologically active macromolecules Download PDF

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WO2013054298A2
WO2013054298A2 PCT/IB2012/055542 IB2012055542W WO2013054298A2 WO 2013054298 A2 WO2013054298 A2 WO 2013054298A2 IB 2012055542 W IB2012055542 W IB 2012055542W WO 2013054298 A2 WO2013054298 A2 WO 2013054298A2
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peg
acid
ch
polymer
biologically active
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WO2013054298A3 (en )
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Giancarlo Tonon
Paolo Caliceti
Gaetano Orsini
Rodolfo Schrepfer
Fabio SELIS
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Bio-Ker S.r.l
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    • C08GMACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32Polymers modified by chemical after-treatment
    • C08G65/329Polymers modified by chemical after-treatment with organic compounds
    • C08G65/331Polymers modified by chemical after-treatment with organic compounds containing oxygen
    • C08G65/332Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof
    • C08G65/3322Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof acyclic
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08GMACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32Polymers modified by chemical after-treatment
    • C08G65/329Polymers modified by chemical after-treatment with organic compounds
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08GMACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32Polymers modified by chemical after-treatment
    • C08G65/329Polymers modified by chemical after-treatment with organic compounds
    • C08G65/331Polymers modified by chemical after-treatment with organic compounds containing oxygen
    • C08G65/332Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof
    • C08G65/3324Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof cyclic
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08GMACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2650/00Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G2650/28Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule characterised by the polymer type
    • C08G2650/50Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule characterised by the polymer type containing nitrogen, e.g. polyetheramines or Jeffamines(r)
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08LCOMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2203/00Applications
    • C08L2203/02Applications for biomedical use

Abstract

The present invention in a first aspect relates to a polymer based on modified polyethylene glycol (PEG) of formula CH3-(OCH2CH2)n-O-X-A, wherein (OCH2CH2)n- is a polyoxyethylene chain wherein n varies from 100 to 5000, X is a saturated or unsaturated C1C20 linear alkyl group or a saturated or unsaturated C3-C5 branched alkyl group having at least one end functionalised with a hydroxyl, amino, carboxylic or thiol group, and A is a functional lipophilic group. In another aspect, the invention relates to supramolecular complexes of the polymers of polyethylene glycol (PEG) that is modified with a biologically active macromolecule or a biopharmaceutical and their applications in therapy.

Description

MODIFIED POLYETHYLENE GLYCOLS AND THEIR SUPRAMOLECULAR COMPLEXES WITH BIOLOGICALLY ACTIVE MACROMOLECULES

Field of the invention

The present invention relates to modified polyethylene glycols and their supramolecular complexes with biologically active macromolecules.

The present invention specifically relates to polyethylene glycol polymers functionalised with lipophilic molecules suitable for forming supramolecular complexes with macromolecules provided with biological and/or pharmacological activity.

According to some aspects, the invention further concerns the use of modified PEG-based polymers provided with amphiphilic characteristics for the controlled release of biologically active macromolecules.

Prior art

The present invention takes its origin in general from the pharmaceutical technology sector, and in particular from systems for the controlled release of biologically active principles, of biopharmaceuticals.

Pharmaceutical technology has moved to the design and production of new pharmaceutical forms capable of improving the therapeutic profile of medicinal products.

The design of new pharmaceutical forms is necessary to improve or extend the therapeutic uses of active principles, which are also provided with elevated pharmacological activity and have physicochemical, biopharmaceutical, biological, pharmacodynamic or pharmacokinetic features which limit their potential use in medicine.

In the case of biotechnological drugs or biopharmaceuticals, some of the principal limitations to their therapeutic use can be traced back to their low solubility, elevated chemical, physical, and microbiological instability, their enzyme- denaturing action, their low absorption via biological membranes, their rapid elimination from the systemic circulation, and their inadequate distribution at the sites of action.

The use in medical treatment of biotechnological drugs, in particular those based on peptides, proteins or oligonucleotides [Weng Z. and DeLisi C, Protein therapeutics: Promises and challenges for the 21st century], which have been obtained using the most recent biotechnological techniques, has considerably increased.

Nevertheless, the clinical use of biotechnological drugs is limited by a number of problems of formulation.

Indeed, in contrast with conventional drugs, biotechnological macromolecules are structurally highly complex and are therefore readily subject of a to phenomena of degradation, denaturing and inactivation.

Methods for formulating biotechnological drugs therefore utilise means and systems capable of stabilising the biological macromolecules and protecting them from denaturing [Orive G. et al, Drug delivery in biotechnology: present and future, Curr. Opinion Biotechnol, 14:659-664, 2003; Cleland J. et al., Emerging protein delivery methods, Curr Opin. Biotechnol. 12:212-219, 2001 ; Brayden Controlled release technologies for drug delivery, Drug Discov, Today 8: 976-978, 2003; .Razzacki S.Z., Thwar P.K., Yang M., Ugaz V.M., Burns M.A. Integrated microsystems for controlled drug delivery, Adv. Drug Del. Rev, 56:185-198, 2004; Wang W. Protein aggregation and its inhibition in biopharmaceutics Int. J. Pharm. 289:1-30, 2005].

The therapeutic use of biotechnological drugs is also a limited by their immunological and pharmacokinetic characteristics. Biotechnological drugs often exhibit an elevated immunogenic and antigenic characteristic which can lead to their inactivation or even to the development of immune reactions, potentially with anaphylactic shock (Schellekens H., Immunogenicity of therapeutic proteins: clinical implications and future prospects, Clin.Ther. 24:1720-1740, 2002).

These problems have been at least partially overcome by adopting methods for the quantitative production of homologous proteins that are potentially non- immunogenic and non-antigenic. However, these methods do not always allow the problem of the elevated immunogen characteristic to be overcome.

One technological solution to resolve the problem of immunogenicity and antigenicity of biotechnological products provides for the surface modification of the biotechnological drug, forming covalent bonds with polymers which mask the drug from the immune system and from antibodies (Chirino A. J. et aL Minimizing the immunogenicity of protein therapeutics, Drug Discov. Today 82-90, 2004). This technology exposes to the risk of rendering immunogenic proteins which are non-immunogenic prior to the structural modification.

Another problem of proteins and oligonucleotides for their therapeutic use is represented by their low bioavailability, caused by their poor absorption via biological membranes and their rapid elimination from the body by means of phenomena of degradation or denaturing, cellular uptake, or elimination via the renal or hepatic pathways, as well as via the immune system [Blanchette J. et al., Principles of transmucosal delivery of therapeutic agents, Biomed. Pharmacother. 58:142-151 , 2004;].

Moreover, biotechnological drugs based on oligopeptides and oligonucleotides are of this high molecular weight and are highly hydrophilic, limiting their absorption via biological membranes and making parenteral administration necessary.

A number of pharmaceutical techniques provide for the use of polymers for producing pharmaceutical formulations capable of overcoming the problems expounded above in relation to stabilisation and release control according to desired kinetics.

Other techniques for formulating polypeptide-based and oligonucleotide-based biotechnological is drugs provide for the use of polymeric or lipid microspheres and nanospheres, liposomes, monolithic or reservoir polymeric matrices, supramolecular systems obtained by the physical interaction of molecules or macromolecules with proteins to form complexes, colloidal aggregates, or micellar systems [US 5,904,936 (1999) Particles based on polyamino acid(s) and capable of being used as delivery carrier for active principles and method for preparing them; US 6,180,141 (2001 ) Composite gel microparticles as active principle carriers; WO 01/37809 (2000); WO 02/28521 (2001 ) Colloidal suspension of sub micronic particles as vectors for active principles and method for preparing same; US 2001/0051189 A1 (2001 ) Application of nanoparticles based on hydrophilic polymers as pharmaceutical forms; Panyam J., Labhasetwar V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue, Adv. Drug Del. Rev. 55: 329-347, 2003]. Further formulations provide for the use of bioconjugates, that is, macromolecules modified with polymers, lipophilic molecules, sugars or directional agents via the formation of a covalent bond. However, bioconjugates must be considered as new molecular entities rather than as technological formulations proper [Veronese F.M., Harris J.M., Introduction and overview of peptide and protein pegylation, Adv. Drug Del. Rev. 54:453-456, 2002; WO2007/112794 (2007) New activated polyethylene glycols and related polymers and their applications ].

An alternative formulation of biotechnological drugs provides for the use of supramolecular systems obtained by non-covalent interaction between the biopharmaceutical and polymeric macromolecules. These non-covalent interactions may be stabilised by the presence of opposite charges or chelating agents, by the interaction of lipophilic chains, or even by interaction via specific functions.

Electrostatic interactions induced by the presence of opposite charges are typical in the formation of complexes between oligonucleotides negatively charged by the presence of phosphate residues and polycationic carrier macromolecules such as chitosans, po!yamidoamines and polylysines. This type of interaction is widely studied for the delivery of oligonucleotide drugs and for transfection [Jeong J.H., Kim S.W, Park T.G. A new antisense oligonucleotide delivery system based on self-assembled ODN-PEG hybrid conjugate micelles; El-Aneed A. An overview of current delivery systems in cancer gene therapy J. Control. Rel. 94, 1-14, 2004]. However, the level of drug stabilisation obtained with the use of these formulations has not yet been adequately studied. Furthermore, release of the biopharmaceutical from these formulations is not yet adequate.

At the present time there is therefore a perceived need for the availability of new formulations for biotechnological drugs, in particular ones based on peptides, proteins and oligoneucleotides, which enable their physicochemical and biopharmaceutical characteristics and the therapeutic profile to be improved.

Summary of the invention

The inventors of the present patent application have surprisingly found that selected amphilic polymers based on modified polyethylene glycol (PEG) stabilise a hydrophobic interaction with biotechnological drugs, in particular those based on peptides, proteins or oligoneucleotides, forming soluble supramolecular complexes which stabilise the biotechnological drugs and improve their profile and release into the body.

Consequently, the class of PEG-based amphiphilic polymers identified by the Applicant finds application in the bio-pharmaceutical sector as a system for the vehiculation of biotechnological drugs suitable for overcoming the problems of stability, solubility and pharmacokinetics of the biopharmaceutical in the free form. In accordance with a first aspect of the present invention, polymers based on modified polyethylene glycol (PEG) of formula (I) are described

Y- (OCH2CH2)n-0-CH2-CH2-X-A (I)

wherein

(OCH2CH2)n- is a polyoxyethylene chain wherein n varies from 100 to 9000, preferably from 500 to 5000,

Y is -CH3 or -H,

X, if present, is

a) a saturated or unsaturated C1-C20 linear alkyl group, having each end functionalised with a group selected from hydroxyl, amino or thiol,

b) a saturated or unsaturated C3-C5 branched alkyl group, having each end functionalised with a group selected from hydroxyl, amino or thiol,

c) a heteroatom selected from N, S, O or an NH group,

A is a lipophilic group selected from

a) a saturated or unsaturated aliphatic carboxylic acid comprising 8 to 36 carbon atoms, or

b) a polycyclic group selected from colanic acid, cholesterol, tocopherol and their analogues or derivatives.

In one aspect of the present invention supramolecular systems of the previously described PEG-based polymers with a biologically active macromolecule are provided.

According to a second aspect of the invention, a supramolecular complex or system is therefore provided comprising a modified polyethylene glycol polymer (PEG) of formula (I) and a biologically active macromolecule. In certain embodiments, the biologically active macromolecule is a peptide, protein or oligoneucleotide.

According to a third aspect of the invention, a method is provided for producing a supramolecular complex or system comprising a modified polyethylene glycol polymer (PEG) of formula (I) and a biologically active macromolecule.

According to a further aspect of the invention, use is provided of a supramolecular complex or system comprising a modified polyethylene glycol (PEG) polymer of formula (I) and a biologically active macromolecule for the controlled release of the biologically active macromolecule.

The advantages achievable with the modified PEG polymers will be more obvious to the person skilled in the field from the following detailed description of particular embodiments of the method and of the compounds obtainable therewith, which are provided by way of non-limiting examples.

Brief description of the drawings

The present invention will be described in detail in what follows with reference to the drawings, wherein:

Fig. 1 shows the 1 H NMR spectrum of mPEG 5 Kda-stearic acid.

Fig. 2 shows the mass spectrum of tocopherol functionalised with dodecyldiamine. Fig. 3 illustrates the 1 H NMR spectrum of the derivative mPEG 5kDa-colanic acid. Fig. 4 shows the mass spectrum of the derivative colanic acid-NH-(CH2)12NH2 of Example 1.5.

Fig. 5 illustrates the viscosity profile of the derivative mPEG5000-colanic acid as a function of the polymer concentration and the correlation function.

Fig. 6 illustrates the micellar association profile obtained by fluorimetry techniques with the mPEG2000-colanic acid series.

Fig. 7 illustrates the size distribution profile of mPEG-5000-colanic acid.

Fig. 8 A-E illustrates the elution profiles obtained by incubating r-h-GH with different quantities of mPEG 2kDa-stearic acid.

Figs. 9 and 10 show the calibration curves for r-G-CSF and r-h-GH.

Figs. 1 1-18 display the association profiles obtained with a number of modified

PEG polymers and the proteins r-h-GH and r-h-GCSF. Fig. 19 illustrates the results of graph analysis obtained with r-h-G-CSF and mPEG-5000-colanic acid.

Fig. 20 illustrates the CMC/association profiles obtained with modified PEG polymers and r-h-GH.

Fig. 21 illustrates the profile for serum content of G-CSF over time.

Fig. 22 illustrates the pharmacodynamic profile of the association r-G-CSF- mPEG 5kDa- colanic acid.

Fig. 23 illustrates the in-vitro stability curves of native GLP- and GLP-1 associated with mPEG 5KDa-colanic acid with the use of DPP IV in aqueous solution.

Detailed description of the invention

In particular the Applicant has surprisingly identified that a polymers of PEG advantageously functionalised with lipophilic molecules interact with macromolecules having biological activity, forming supramolecular structures usable within the field of pharmaceutics.

In particular, said PEG polymers functionalised with lipophilic molecules are suitable for modifying the physicochemical, biopharmaceutical and biological characteristics of biologically active macromolecules such as peptides and therapeutic proteins, improving their stability, administration, pharmacokinetic profile and bioavailability, and thus the therapeutic profile.

According to a first aspect of the present invention, a polymer based on modified polyethylene glycol (PEG) of formula (I) is therefore provided

Y- (OCH2CH2)n-0-CH2-CH2-X-A (I)

wherein

(OCH2CH2)n- is a polyoxyethylene chain wherein n varies from 100 to 9000, preferably from 500 to 5000,

Y is -CH3 or -H,

X, if present, is

a) a saturated or unsaturated C1-C20 linear alkyl group, having each end functionalised with a group selected from hydroxyl, amino or thiol,

b) a saturated or unsaturated C3-C5 branched alkyl group, having each end functionalised with a group selected from hydroxyl, amino or thiol, c) a heteroatom selected from N, S, or an NH group;

A is a lipophilic group selected from

i) a saturated or unsaturated aliphatic carboxylic acid comprising from 8 to 36 atoms of carbon, preferably from 10 to 20C, or

ii) a polycyclic group selected from colanic acid, cholesterol, tocopherol and/or their analogues and derivatives.

In certain embodiments, X is a) as previously described, where a) is

- NH - (CH2)m -NH -, wherein m is within the range from 1 to 20, preferably from

8 to 14,

- R - CO - (CH2)p - CO - R -, wherein p is within the range from 1 to 20, preferably from 8 to 14, wherein R, if present, is O or S or another residue deriving from the carboxyl activating group.

Typical examples of carboxyl activating groups include R= CI; O-CO-O-R1.

In some embodiments X is a) a saturated or unsaturated Ci-C20 linear alkyl group, preferably C3-C10, having two ends functionalised with a hydroxyl, amino, carboxyl or thiol group.

In some embodiments, the group X is absent.

In particular, the present invention relates to a polymer based on modified polyethylene glycol (PEG) of formula (I)

Y- (OCH2CH2)n-O-CH2-CH2-X-A (I)

wherein

Y is -CH3,

X is NH

n is within the range from 100 to 9000,

A is a polycyclic group selected from colanic acid, cholic acid, chenodeoxycholic acid, deoxycholic acid or their analogues, and is preferably colanic acid.

According to the present invention, the preferable polymer based on polyethylene glycol (PEG) has the following structure: NH-CH2-CH2-0-(CH2-CH2-0)n-Y

Figure imgf000011_0001
wherein Y is -CH3 and preferably

n is within the range from 100 to 9000.

In the modified PEGs described in the present invention and with reference to formula (1 ), the lipophilic functional group A functions as a modifying group. In certain embodiments, group A comprises organic molecules having aliphatic or alicyclic residues.

In certain embodiments, group A is a lipophilic group selected from

a) a saturated or unsaturated aliphatic carboxylic acid comprising from 10 to 20 carbon atoms, or

b) a polycyclic group selected from colanic acid, cholesterol, tocopherol and their derivatives.

In certain embodiments, A is an aliphatic carboxylic acid having a chain provided with a number of carbon atoms within the range from 10 to 20.

In certain embodiments, said group A is stearic acid or palmitic acid.

Typical examples of PEG polymer modified with fatty acids or derivatives or esters, for example with glycerol, include mPEG-stearic acid, mPEG-arachidic acid, or distearyl-glycerol and their mixtures.

In other embodiments, the group A contains a polycyclic molecule for example cholic acid, colanic acid, chenodeoxycholic acid, deoxycholic acid and their analogues or derivatives.

According to the preferred embodiment, A is colanic acid. Typical examples of PEG polymer modified with polycyclic molecules include methoxyPEG-tocopherol and methoxyPEG-colanic acid and their mixtures. Typically, the polymers of modified PEG described in the invention form micelles and are capable of solubilising hydrophobic molecules.

In particular, these modified PEGs are capable of solubilising hydrophobic molecules and exhibit their own characteristic critical micellar concentration (CMC) which depends on the nature of the moleceule bound to the PEG and on the molecular weight of the PEG itself.

On the modified PEG polymers, the critical micelle concentration (CMC) values were determined by means of fluorimetric techniques in which the solubilisation of fenantrene was evaluated as the polymer concentration increased [Lavasanifar A, Samuel J., Know G.S. The effect of alkyl-core structure on micellar properties of poly(ethyleneoxyde)-block-poly(L-aspartamide) derivatives, Coll. Surf. B Biointerf.

22:115-126, 2001]. The data obtained according to the described protocol are reproduced in Example 3 and elaborated as shown in Fig. 6.

Table I below presents the critical micelle concentrations (CMC) obtained with modified PEGs described in the invention.

Table I

Figure imgf000013_0001

. The dimensions of the polymeric micelles were measured by dynamic light scattering analysis (PCS, photon correlation spectroscopy).

By way of example, Table II summarises the size values of some of the modified PEG polymers described in the invention.

Table II. Mean micellar dimensions obtained with the various polymers.

Figure imgf000014_0001

The modified PEG polymers of the invention are typically substantially devoid of acute and chronic, local or systemic, toxicity and biological activity, and are easily eliminated from the body, essentially via the renal or hepatic routes, avoiding undesirable accumulation in certain regions, organs, tissues and cells. Furthermore, modified PEGs are not toxic, immunogenic or antigenic.

The PEG polymers of the invention are typically obtained by polymerisation of the oxide of ethylene of molecular weight variable for example from 200 to 50,000 dalton. The polymers of the invention are obtainable by means of conventional modification and/or activation methods of low cost and high degree of purity. Due to the repetitive nature of their oxyethylene monomeric units, the polyethylene glycols of the invention are amphiphilic polymers, that is, they dissolve freely in aqueous solutions and in suitable organic solvents, as a function of the molecular weight. High molecular-weight polyethylene glycols are typically soluble in polar organic solvents, whereas at low molecular weight they are also soluble in non- polar organic solvents. Moreover, the ability to coordinate 3 molecules of water for each monomeric unit induces in them a large increase in the hydrodynamic volume.

The class of polyethylene glycol-based polymers derivatised with lipophilic functions of the invention interacts with biologically active macromolecules in particular peptides and proteins to form stable supramolecular systems of micellar, aggregate or complex type.

The modified PEG polymers of the invention typically form supramolecular systems with peptides, proteins and oligoneucleotides by means of non-covalent or electrostatic interactions.

In certain embodiments, the mean molecular weight of polyethylene glycol is within the range from 200 to 40,000 Da, preferably between 1000 to 20,000 Da, and yet more preferably from 2000 to 5000 Da.

In a second aspect of the invention, a method is provided for the production of PEG polymers of the invention comprising the binding of a lipophilic compound to one end of a monomethoxypolyethylene glycol (mPEG) of the type previously described.

As described in the present invention, the lipophilic molecules modifying the mPEG polymer are bound to the polyethylene glycol directly or via suitable spacers X, while mPEG-stearic acid and mPEG-arachidic acid polymers can be obtained by reacting the chloride of the acid and a polyethylene glycol functionalised with an amino group at the end of the chain (mPEG-NH2) with formation of an amide bond between the PEG and the acyl residue.

The mPEG-stearic acid and mPEG-arachidic acid polymers can be obtained by reacting the acid chloride and a polyethylene glycol containing a free hydroxyl group at the end of the chain (mPEG-OH) with formation of an ester bond between the PEG and the acyl residue, a bond which is unstable in vivo on account of the esterases.

In one embodiment of the present invention, the mPEG-tocopherol and mPEG- colanic acid polymers can be obtained by reacting the chloride of the acid of the polycyclic molecule with a polyethylene glycol functionalised with an amine group at the end of the chain (1T1PEG-NH2) with formation of an amide bond between the PEG and the polycyclic residue.

The mPEG-tocopherol and mPEG-colanic acid polymers, containing a spacer X between the lipophilic molecule and the PEG, can be obtained by reacting a single amine group of an α,ω-alkyl-diamine with a polyethylene glycol functionalised with an activated carboxyl group at the end of the chain (e.g. mPEG-OSu) and then reacting the product obtained with tocopherol chloride or with colanyl chloride. Use of the modified PEG polymers of the invention comprises a number of advantages. In particular, the modified PEGs can be used successfully to protect vehicled pharmaceuticals from degradation by endogenous enzymes, as well as to optimise the profiles of release of the active principles, thus improving the therapeutic performance thereof.

According to a further aspect, the invention relates to a supramolecular complex or system comprising at least one modified polyethylene glycol (PEG) polymer formula (I) and at least one biologically active macromolecule.

In the supramolecular complex according to this aspect of the invention, the modified PEG polymer is non-covalently bound to the biologically active macromolecule.

The term "biologically active macromolecule" used within the scope of the present invention refers to molecules which exert any type of biological activity and in particular of the therapeutic type when administered to the body.

Suitable biologically active macromolecules comprise proteins, polypeptides or oligoneucleotides, for example produced using recombinant techniques of molecular genetics, which exert an activity, typically therapeutic, when administered to the human body.

According to some embodiments, the biologically active macromolecule of the supramolecular complex of the invention is the biologically active protein, typically obtained using recombinant techniques of genetic engineering.

In some embodiments, the biologically active macromolecule is the growth hormone (h-GH) or a homologue or derivative thereof having a degree of homology equal to at least 90%. Within the scope of the invention, the term h-GH hormone and its homologues or derivatives is used to mean a protein having an amino-acid sequence which is at least 90% identical to the amino-acid sequence of human growth hormone of 191 amino acids and possibly modified by conjugation to biocompatible polymers which partially or totally maintain the biological activity of interacting with the specific receptor of h-GH and/or of stimulating in the liver or other tissues the production of the insulin-like growth factor of type I (IGF-I), which mediates the effects of h.GH on tissue and body growth. The variations in the amino-acid sequence of the homologues and derivatives of h-GH encompassed by the present invention may derive from addition, subtraction, substitution or chemical modification of one or more amino acids of the sequence of natural h-GH of 91 amino acids.

In some embodiments, the biologically active macromolecule is the granulocyte colony-stimulating factor (G-CSF) or a homologue or derivative thereof having a degree of homology equal to at least 90%.

Within the scope of the invention, the term G-CSF or homologues or derivatives is used to mean a protein having an amino-acid sequence which is at least 90% identical to the amino-acid sequence of the principal variant, both glycosylated and non-glycosylated, of natural human G-CSF of 174 amino-acids, which partially or totally maintain the activity of native human G-CSF of stimulating proliferation and the differentiation of progenitor cells to mature neutrophils.

The variations in the amino-acid sequence of the homologues and derivatives of G-CSF included in the present invention may derive from addition, subtraction, substitution or chemical modification of one or more amino acids of the sequence ofnatural human G-CSF, both glycosylated and non-glycosylated, of 174 amino acids.

In certain embodiments, the biologically active macromolecule is the peptide hormone GLP-1 (Glucagon-Like Peptide-1 ) or a homologue or derivative thereof having a degree of homology equal to at least 90%.

Within the scope of the present invention, the term GLP-1 hormone or analogues or derivatives thereof are intended to mean the compound GLP-1 of 30 amino acids amidated at the C-terminal position and the compound GLP-1 of 31 amino acids with the C-terminal extension of a glycine residue and their biologically active forms obtained by deletion, addition or substitution of one or more amino- acid residues by conjugation to biocompatible polymeric molecules as well as the natural or synthetic insulinotropic peptides which have at least 50% identity of sequence relative to human GLP-1 of 31 amino acids with the C-terminal extension of a glycine residue.

More specifically, with reference to the peptide analogues of GLP-1 and derivatives, the term biologically active forms used in the present invention indicates the products with incretin mimetic effects in that they are capable, following administration in vivo, of potentiating the secretion of insulin induced by glucose.

The growth hormone and recombinant preparations of h-GH are commercially available and are used as substitution therapy by means of subcutaneous injection, principally for the treatment of dwarfism in babies having a GH deficit. G-CSF and recombinant preparations of G-CSF, both in the glycosylated and the non-glycosylated form, are used clinically for the treatment of febrile neutropenias and to prevent infective complications thereof which frequently arise in cancer patients undergoing chemotherapy and which frequently limit the use of effective dosages of cytotoxic anti-cancer drugs.

According to another aspect of the invention, a method is provided for producing supramolecular complexes comprising a modified polyethylene glycol (PEG) of formula (I) and a biologically active macromolecule comprising the mixture of a solution containing the modified polyethylene glycol (PEG) of formula (I) with a solution containing [the] biologically active molecule for a time necessary for formation of the complex, typically within the range from 2 to 24 hours. In some embodiments, both solutions comprise a buffering agent.

The interaction between derivatised PEG polymer and biologically active macromolecule is of a non-covalent nature because in the polymers that are the subject of the present invention there are no reactive functional groups capable of forming covalent bonds with the macromolecules.

The macromolecule / PEG polymer complex is formed by spontaneous interaction of the lipophilic function covalently bound to PEG with particular areas of the molecule for example a protein, in that the PEG per se is not capable of forming combination products with biologically active macromolecules such as proteins and peptides.

The results reported in the present invention demonstrate that the biologically active macromolecules or biopharmaceuticals, typically proteins or polypeptides, combine with modified PEG polymers according to defined association constants which depend on the physicochemical characteristics of the macromolecule and on the physicochemical characteristics of the polymer.

For example, with the two biopharmaceuticals r-h-GH and r-h-GCSF, various degrees of charging with the polymers are obtained.

The association studies were conducted by incubating defined quantities of one of the two proteins with increasing quantities of polymer. The formation of the product of association and the disappearance of free protein not associated with the polymers were determined by means of gel-filtration HPLC.

In particular Fig. 8, relating to Example 5, presents the gel-filtration elution profiles obtained with the association r-h-GH and mPEG-2000-stearic acid. By addition of the modified PEG polymer, a peak appears that has a lesser elution volume relative to that of the protein in the absence of polymer. This peak corresponds to the association product and its area increases with the increase in the polymer content, while the area of the peak corresponding to the non-associated protein progressively diminishes. The areas relating to the peak corresponding to the polymer/protein complex and the peak corresponding to the non-associated protein were measured, and the concentration of protein present in the two forms were determined by means of suiTable calibration curves obtained with protein in the absence of polymer (Figs. 9 and 10).

The values thus obtained were represented graphically as a function of the quantity of polymer. By way of example, Figs. 1 1-18 present the association profiles obtained with a number of test polymers, and r-h-GH and r-h-G-CSF. The data obtained were processed so as to calculate the degree of charging of the macromolecular drug on the polymer expressed as quantity of protein in milligrams completely associated with 100 mg of polymer (% of association). The experimental data used to determine the degree of loading were processed according to the following three methods:

1 . Calculation of the quantity of polymer necessary for complete association of the protein incubated on the basis of achieving the association plateau;

2. Determination of the quantity of polymer which associates all the protein on the basis of the initial gradient of the curve for appearance of associated protein;

3. Determination of the quantity of polymer which associates all the protein on the basis of the initial gradient of the curve for appearance of non- associated protein.

In a number of cases, rapid formation of associated product is achieved even with small quantities of polymer, indicating an elevated drug/polymer affinity. In other cases, no association plateau is reached, indicating that the association is not complete but corresponds to an equilibrium between associated protein and non- associated protein. In this case, the degree of charging was calculated using methods 2 and 3.

The charging results obtained are presented in Table III, which shows the mean charging values, expressed as milligrams of r-h-GH or r-h-G-CSF associated with 100 milligrams of modified PEG polymer.

Table III. Mean values for charging of r-h-GH and r-G-CSF on functionalised PEG

Figure imgf000021_0001

The data obtained demonstrate that the optimum charging occurs with PEG of molecular weight between 2000-5000. By increasing or lowering the molecular weights and keeping the lipophilic modifier equal, the degree of association diminishes. This result may be due to the high degree of hydration of the PEG, to its great flexibility which impedes an adequate approach to the protein, and to the stability of the drug/protein system.

It has also been found, and constitutes a further unanticipated aspect of the present invention, that the conjugation of mPEG to the colanic function gives rise to a polymer having a greater capacity for interaction with peptides and proteins as compared with both cholesterol and tocopherol and, to a greater degree, to the long-chain fatty acid functions.

The experimental association data have been processed to obtain an evaluation of the type of complex between the proteins and the polymers.

Example 6 presents the details of the Scatchard and Klotz analyses used for characterisation of the protein/polymer complex. These methods have been applied successfully for the study of protein/polymer complexes (Salmaso, S. Supramolecular association of recombinant human growth hormone with hydrophobized polyhydroxyethylaspartamides. Eur. J. Pharm. Biopharm, 68: 656- 666, 2008).

Two parameters have been obtained from the Scatchard analysis: Bmax, which expresses the binding sites in the protein for the polymer, and kD, which expresses the association constant. Figure 19 shows, by way of example, the results of the graph analysis obtained with r-h-G-CSF and mPEG-5000-colanic acid. In Table IV below are summarised all the Bmax and kD values calculated from processing of the association analyses.

Table IV Results of the Scatchard analyses obtained with the supramolecular complexes of r-h-GH and r-G-CSF with various PEG derivatives

Figure imgf000023_0001
The Bmax and kD data presented in Table IV show how the maximum association and the association constant depend both on the type of protein and the type of polymer. In particular, it can be seen that the Bmax and kD values obtained with r-h-GH are in some cases very different from those obtained with r-h-G-CSF, indicating a different degree of association. In the case of mPEG5kDa-colanic acid, r-h-G-CSF shows greater association than r-h-GH. However, with regard to the polymer significant variations are observed based on the type of lipophilic function and the length of the PEG.

By observing the homologous series of mPEG-colanic acid (PEG 5, 10 and 20 kDa), it is seen that the maximum association is always of the same order of magnitude, while the disassociation constant increases with increase in the molecular weight of the polymer.

With mPEG 5 kDa and r-h-G-CSF, a disassociation constant is obtained which is around 5-10 times lower as compared with mPEG 10 and 20 kDa, indicating that the affinity of the polymer for the protein diminishes with increase in the molecular weight of the polymer.

With r-h-GH, the polymer modified with an alkyl chain of 12 atoms of carbon and colanic acid, the dissociation constant typically diminishes dramatically, indicating that this polymer has a high affinity for the protein. The Klotz analysis performed for reprocessing of the experimental association data have confirmed the Scatchard results discussed above.

The results obtained therefore indicate that, by appropriately selecting the structure and composition of the polymer, it is possible to obtain products having different degrees of biopharmaceutical/polymer association and to modulate the stability of the complex to give different biopharmaceutical characteristics.

Correlation studies, like the one in Example 7, have been performed between the physicochemical characteristics of the PEG polymers and the protein charging capacity, in particular between the capacity of modified PEG polymers to form micelles, which is evaluated as CMC, and the degree of association with proteins expressed as the charging capacity (mg of protein associated with 100 mg of polymer).

Fig. 20 presents, by way of example, the CMC/association correlation profiles obtained with polymers and r-h-GH which show differing courses according to the type of function bound to the polymeric chain.

In the case wherein the function bound to the polymer is a linear aliphatic chain such as, for example, stearyl or arachidyl, the degree of protein charging is observed to increase with increase in the CMC of the polymer. A similar behaviour is also observed with the polymer derivatised with polycyclic functions, although in this case there is no linear correlation, indicating that the mechanisms of interaction are different for the aliphatic chains and the polycyclic functions. The absence of correlation between the degree of charging and structural properties indicates that there is no unique structural characteristic which determines the degree of association but that they can contribute in part to the interaction with the protein.

It has been observed that the way in which the lipid molecule is bound to the PEG polymer may also have a role in the interaction with the biologically active macromolecule. For example, the difference in behaviour between the colanic derivative as compared with the cholesterol derivative which presents a very similar molecular structure can be ascribed to the different site of conjugation of the modifying agent to the PEG.

In the case of cholesterol, the PEG is typically bound in proximity to the polycyclic functions whereas, in the case of the colanic derivative, PEG is bound on the opposite side. Therefore, the polycyclic functions are efficient in forming an interaction with the biopharmaceutical and are conveniently left available for the interaction. For this, the presence of the highly hydrated PEG in proximity to the function which interacts with the drug can interfere with the formation of the associate. For this reason, derivatives have been prepared wherein the lipophilic modifying molecule has been bound to the PEG via the group X.

A number of preferred embodiments of the invention relate to the derivatives methoxy-PEG-NH-(CH2)12NH-colanic acid and methoxy-PEG-NH(CH2)12NH- tocopherol.

(CH2)12NH-tocopherols give rise to an elevated association which corresponds to a high degree of charging, as shown in Table III above.

In accordance with another aspect of the invention, use is provided of the supramolecular complex or system comprising a modified polyethylene glycol (PEG) of formula (I) and the biologically active macromolecule, as previously described, for the prolonged release of said biologically active macromolecule.

The prolonged release of the complexes of the invention is documented in the pharmacokinetic study of Example 8, which is conducted by comparing the association r-h-G-CSF/mPEG5kDa-colanic acid with native G-CSF. The profile of the serum content of G-CSF over time (Fig. 21 ) and the pharmacokinetic parameters tMAX, CMAX, t ½ and AUC, presented in Table V, demonstrate that the formulation with mPEG5kDa-colanic acid confers on the active principle a long release/duration effect. Table V. Pharmacokinetic parameters relating to the study in rats of r-h-G- CSF and rh-G-CSF/mPEG5kDa- colanic acid (1 :21 p/p)

Figure imgf000026_0001

It is also observed that the in-vivo activity of r-G-CSF associated with mPEG5kDa- colanic acid maintains the pharmacological activity thereof (Example 9). The pharmacodynamic profile (Fig. 22) of the association r-G-CSF/mPEG5kDa- colanic acid (administered subcutaneously at a dose of 3 mg/Kg) is comparable with that of native r-G-CSF administered daily for four consecutive days (4 x 0.25 mg/kg). The parameters AUCANC and ANCMAX (where ANC corresponds to the total neutrophil count) presented in Table VI also confirm the conservation of the biological activity and the pharmacological action of the drug as compared with r-G-CSF administered in a non-associated form.

Table VI. Pharmacodynamic parameters relating to the in-vivo activity study in mice of r-h-G-CSF/mPEG5kDa-colanic acid and administered as a single dose by comparison with r-h-G-CSF administered as repeated daily doses for four days.

Figure imgf000026_0002

(*) Corrected values of the baseline value The supramolecular complexes of the invention also have the advantage of reducing or eliminating the instability of the biologically active macromolecule to the hydrolysing action of the enzymes. This phenomenon is observed for example in the case of the endogenous peptide GLP-1 (Glucagon-like peptide I). In the event of exogenous administration of GLP-1 and analogues, the pharmacological activity is expressed only if the peptide is unaltered in its N-terminal portion, on account of which GLP-1 , and derivatives, cannot be used as drugs to the extent that they are rapidly degraded in vivo by means of hydrolytic enzymes, in particular by dipeptidyl peptidase IV (DPP IV).

The Example presents, in particular, the stability of the supramolecular complex mPEG5kDa-colanic acid /GLP-1 and the study of its stability in aqueous solution in the presence of dipeptidyl peptidase IV (DPP IV), an endogenous enzyme which hydrolyses the chain of GLP-1 at the N terminal between the second and third amino acid.

The results presented in Table VII and the stability curves presented in Fig. 23 are evidence of a notable increase in the stability of the peptide when it is associated with mPEG5KDa-colanic acid.

Table VII ln-vitro stability of native GLP-1 and of GLP-1 associated with mPEG5KDa-colanic acid by means of DPP IV in aqueous solution

Figure imgf000028_0001

It is observed that the interaction with the modified PEG polymer can considerably change the pharmacokinetic profile of the biopharmaceutical in that the elevated hydrodynamic volume of the drug/polymer complex can significantly retard the processes of elimination by renal ultrafiltration and absorption by the administration site (for example, subcutaneous). Moreover, interaction with the modified PEG polymer can mask the protein from recognition by proteolytic enzymes, thus providing for stabilisation of the drug.

Another advantage of the invention consists in improving physical stability due to the presence of the modified PEG polymer associated with the protein, which enables aggregation phenomena to be avoided. All these events allow prolonged maintenance of the biopharmaceutical in the body. Consequently, the active principle of biotechnological origin is released slowly in native and active form from the complex by mechanisms of displacement, diffusion or dilution, thus guaranteeing a prolonged action over time.

It is furthermore found that the biopharmaceutical-PEG polymer complexes conjugated to a lipophilic compound according to the invention, in particular those wherein the lipophilic compound is colanic acid, improve the biopharmaceutical profile of the biopharmaceuticals. These formulations have an action of prolonged release of the biopharmaceutical, enabling the use of dosage regimens based on administrations at protracted intervals, and achieving better compliance.

The present invention will now be described with reference to the following examples, which are provided purely for illustrative purposes and must not be understood in a sense limiting the present invention.

EXAMPLE 1

Preparation of the modified PEG polymers

1. Preparation of PEG-lonq-chain fatty acid (from 16 to 36 C).

A) Activation of the lipophilic component.

10 mmol thionyl chloride is added to a solution of 1 mmol of an aliphatic-chain fatty acid (palmitic acid, stearic aciad, arachidic acid) in 1 ml methylene chloride. The organic solution is maintained under reflux for 3 hours in a nitrogen atmosphere and the product is then distilled to separate the chloride from the aliphatic acid. An aliquot part of the product obtained is dissolved in methylene chloride, ethyl amine is added and maintained for one hour at ambient temperature. The derivative is then analysed by mass spectrometry to confirm formation of the aliphatic acid chloride.

B) Formation of the hydrophobised polymer

To a solution of 2 ml of methylene chloride containing 0.08 mmol methoxy-PEG- NH2 ( MW 5, 0 or 20 kDa), 0.24 mmol of the aliphatic acid chloride prepared as described in A) and 0.16 mmol triethylamine are added with agitation. The reaction mixture is maintained under agitation at ambient temperature for 12 hours. At the end of the reaction, the organic solutsion is added dropwise to 50 ml ethyl ether. The precipitate is washed with ethyl ether and recovered by centrifuging to dryness. The derivative obtained is characterised by means of NMR and colorimetric assay with trinitrobenzene sulfonic acid to determine the free amine groups. Colorimetric assay demonstrated that the modification of the amine groups of mPEG-NH2 due to the chloride bond of the fatty acid is in excess of 95%.

Diagrams II and III show respectively the general formulae of the derivatives mPEGnkDa-palmitic acid and mPEGnKDa-stearic acid, where n is equal to 2 to 20.

Diagram II

CH30-(CH2CH20)n-NH-CO-(CH2)i4CH3 Diagram III

CH30-(CH2CH20)n-NH-CO-(CH2)i6CH3 By way of example, Fig. 1 presents the 1 H-NMR spectrum of the derivative PEG5kDa-stearic acid; δ 3,644: singlet of PEG-(CH2CH20-)n, integrates for 454 H; δ 1 ,619: multiplet, -CH2- of stearate, integrates for 2H; δ 1 ,253: broadened singlet -(CH2-)n of stearate, integrates for 28H; δ 0,879: triplet: -CH3 of stearate, integrates for 3H.

2. Preparation of PEG-tocopherol

A) Activation of the lipophilic component.

One mmol tocopherol is dissolved in 2 ml methylene chloride and 4 mmol p- nitrophenyl chloroformiate and 4 mmol triethylamine are added. The reaction is maintained under agitation in an inert nitrogen atmosphere at ambient temperature. The product thus obtained was purified over a column of silica using a 10:1 mixture of petroleum ether/ethyl ether as the eluent. The end product was concentrated in a vacuum to eliminate the organic solvent. The degree of activation, determined through UV by quantifying the p-nitrophenol released following dissolution in dimethylformamide and hydrolysis in 0.2 M NaOH proved to be in excess of 95%. The reactivity of the derivative to the amine groups was determined by using glicylglycine as a source of amine groups and determining their disappearance through reacting with para-nitrophenyl ester by means of colorimetric assay with trinitrobenzene sulfonic acid. The modification of glycylglycine using equimolar quantities of activated tocopherol and glycylglycine proved to be in excess of 90%.

Diagram IV shows the la structure of tocopherol-p-nitrophenylformiate

Diagram IV

Figure imgf000031_0001

B) Formation of the hydrophobised polymer

0.1 mmol of activated tocopherol as described in a) is reacted with 0.05 mmol methoxy-PEG-NH2 dissolved in 5 ml methylene chloride. The solution is maintained under agitation for 12 hours at ambient temperature and is then precipitated in ethyl ether. The precipitate is washed with ether and then dried in a vacuum. The product is dissolved in water and centrifuged, and the solution subjected to extensive ultrafiltration using a membrane with cut off of 1 kDa.

The hydrophobised polymer, analysed by means of colorimetric assay to evaluate the degree of binding to the NH2 group of the PEG and by NMR, demonstrates a modification of PEG in excess of 95%.

3. Preparation of methoxy-PEG-NH-(CH2)12NH-tocopherol.

Activated tocopherol as p-nitrophenyl chloroformiate (0.1 mmol) is reacted with 1 mmol dodecyldiamine dissolved in 5 ml methylene chloride. The solution is maintained under agitation for 12 hours at ambient temperature and is then precipitated in ethyl ether. The precipitate is washed with ether and then dried in a vacuum. The product is dissolved in dimethylformamide and precipitated into alkaline water. The operation is repeated three times. The precipitate is then dissolved in dimethylformamide and precipitated into water at pH 3.0. The operation is repeated three times. The end product is subjected to NMR analaysis and mass-spectrometric analysis.

Diagram V, wherein n = 12 shows the structure [Tocopherol-NH-(CH2)12NH2] Diagram V

Figure imgf000032_0001

Fig. 2 presents the mass spectrum of tocopherol functionalised with dodecyldiamine.

To a solution of tocopherol dodecyldiamine in methylene chloride (0.1 mmol in 2 ml) are added 0.1 mmol methoxy-PEG-hydroxysuccinimidyl ester. The reaction is maintained for 6 hours at ambient temperature under agitation and the modified polymer is then precipitated into ethyl ether. II precipitate is washed with ether and then dried in a vacuum. The product is so water and centre's, and the solution is subjected to extensive ultrafiltration using a membrane with cut off of 1 kDa.

The hydrophobised polymer, analysed by colorimetric assay to evaluate the degree of binding to the NH2 group of the PEG and by NMR: demonstrates modification of the PEG in excess of 95%.

4. Synthesis of PEG-colanic acid (amide).

A) Activation of colanic acid

10 mmol thionyl chloride was added to a solution of 1 mmol colanic acid in 1 ml methylene chloride. The organic solution is maintained under reflux for 3 hours in a nitrogen atmosphere and the product is then distilled to separate the chloride of the colanic acid. An aliquot part of the product obtained is dissolved in methylene chloride and ethyl amine is added and maintained for one hour at ambient temperature. The derivative is then analysed by mass spectrometry to confirm formation of the chloride of colanic acid.

B) Formation of the hydrophobised polymer. To a solution of 2 ml methylene chloride containing 0.08 mmol methoxy-PEG-NH2, 0.24 mmol colanic acid prepared as described in A) and 0.16 mmol triethylamine or added under agitation. The reaction mixture is maintained under agitation at ambient temperature for 12 hours. At the end of the reaction the organic solution is filtered and added dropwise to 50 ml ethyl ether. The precipitate is washed with ethyl ether, recovered by centrifugation and dried. The dried product is then dissolved in acidic water and centrifuged. The solution is finally lyophilised and the derivative of taking is characterised by NMR and colorimetric assay with trinitrobenzene sulfonic acid for determination of the amine groups. Colorimetric assay indicates that the reaction has led to modification of more than 95% of the amine groups of the PEG. By way of example, Fig. 3 shows the 1 H-NMR spectrum of the derivative PEG5kDa-colanic acid; δ 0.636: singlet of -CH3 of the colanic acid in Y on the carbonyl, integrates for three; δ 0.908 doublet of the -CH3 groups of colanic acid integrates for six; δ 3.637 singlet of (CH2CH20-)n of the PEG, integrates for 454.

5. Synthesis of methoxy- PEG-NH-fCHgVigNH-colanic acid.

A) synthesis of the intermediate dodecylamino-colanic acid

To a solution of 2 ml methylene chloride containing 2.4 mmol dodecyldiamine are added under agitation 0.24 mmol colanic acid chloride and 4.8 mmol triethylamine. The reaction mixture is maintained under agitation at ambient temperature for 12 hours. At the end of the reaction the organic solution is filtered to dryness in a current of nitrogen. The residue is solubilised in 2 ml dimethylformamide and then precipitated into acidic water at pH 3.0. The precipitate is separated by centrifugation and washed another two times in acidic water, and finally lyophilised. The product thus obtained is subjected to NMR analysis and mass spectrometry. By way of example, Fig. 4 presents the mass spectrum of the derivative colanic acid-NH-(CH2)i2NH2.

B) Formation of the hydrophobised polymer

0.1 mmol methoxy-PEG-N-hydroxysuccinimmide is added to 0.1 mmol colanic acid functionalised with dodecylamine in 2 ml methylene chloride. The reaction is maintained under agitation at ambient temperature for 4 hours and then the end product is precipitated into ethyl ether washed with ether and dried in a vacuum. The end product is characterised by colorimetric assay for determination of the free, unreacted amine groups and by means of NMR analysis.

6. Synthesis of methoxy-PEG-O-colanic acid (ester).

0.24 mmol colanic acid chloride and 0.16 mmol triethylamine are added under agitation to a solution of 2 ml methylene chloride containing 0.08 mmol of methoxy-PEG-OH. The reaction mixture is maintained under agitation at ambient temperature for 2 hours. At the end of the reaction the organic solution is filtered and added dropwise to 50 ml ethyl ether. The precipitate is washed with ethyl ether and recovered by centrifuging to dryness. The dry product was then dissolved in acidic water and centrifuged. Finally, the solution is lyophilised and the derivative obtained is characterised by NMR.

EXAMPLE 2

Viscosity study

Solutions of various polymers hydrophobised at various concentrations are prepared in 20 mM phosphate buffer, 0.15 M NaCI and pH 7.2. The solutions are left to agitate gently for two hours and then analysed by means of a Haake RS 00 rotational viscometer with a DC60/20 double cone system. The operating temperature is 25°C. By way of example, Fig. 5 presents the viscosity profile of the PEG5000-colanic acid derivative as a function of the polymer concentration and the correlated functional group.

EXAMPLE 3

Determination of the critical micellar concentration

10 μΙ of a 1 mg/ml solution of fenantrene in chloroform are placed in an Eppendorf and and drawn off dry in a vacuum. 1 ml of solution of the various hydrophobised polymers in mQ water at various concentrations is added to the samples thus obtained. The mixture is mixed in a vortex and then in a thermostatically controlled bath heated to 36 °C for 12 hours under agitation. The solutions are then centrifuged at 4000 rpm for two minutes and analysed by means of fluorimetry using an excitation wavelength of 294 nm and an emission wavelength of 365 nm. The fluorescence intensity is plotted graphically as a function of the log of the concentration of each polymer. The equations of the two lines obtained are calculated and the point of intersection corresponding to the CMC is determined [An Y.J. , Carraway E.R., Schlautman M.A., Solubilization of polycyclic aromatic hydrocarbons by perifluorinated surfactant micelles, Water Res. 36:300-308, 2003].

Fig. 6 shows, by way of example, the critical micellar association profile obtained with the series mPEG2000-colanic acid [PEG5kDa-colanic acid (·) PEG-iokDa-colanic acid (o), PEG5kDa-colanic acid (□)-], whilst Table I above shows the critical micellar concentrations (CMC) calculated for some polymers.

EXAMPLE 4

As stated in Example 4, the dimensions of the polymeric micelles forming were also determined, by means of dynamic light scattering analysis (PCS, photon correlation spectroscopy), demonstrating that the sizes of the micelles forming with the polymers under investigation are within the interval 15-40 nm. By way of illustration, the profile (light scattering) of size distribution of the mPEG-5000- colanic acid is presented in Fig. 7, while Table II summarises the size values obtained with the various polymers..

Analysis of micellar size

Analyses of dynamic light scattering were conducted using solutions of polymer in water at the concentration of 300-600 Dg/ml. the determinations were carried out using a Nicomp 170 with Spectraphysics control supplied by Pacific Scientific. By way of example, a size profile of mPEG5kDa-colanic acid is presented in Fig. 7. Size-related data for the micelles obtained with a number of polymers are shown in Table II above.

EXAMPLE 5

Determination of the association with peptides or proteins.

This example illustrates the association of macromolecular drugs used as a model protein, recombinant human growth (r-h-GH), recombinant human granulocyte colony-stimulating factor (r-h-GCSF), and of the peptide drug GLP-1 with polymers based on mPEG modified with appropriate lipophilic molecules.

Two solutions are prepared in phosphate buffer 20 mM phosphate buffer, 0.15 M NaCI and pH 7.2, one of protein (rec-h-GH or rec-h-GCSF), at 2 mg/ml and one of hydrophobised polymer at 40 mg/ml. To aliquot parts of 40 μΙ of protein solution, 5, 10, 20, 30, 40, 60 and 80 μΙ of polymeric solution, and the volume is finally made up to 120 μΙ with the same phosphate buffer. The samples are maintained for one night at ambient temperature under gentle agitation and then analysed by means of gel-filtration chromatography in HPLC, using a BioGel SEC 40 XL column with 20-ml loop, eluted with phosphate buffer 63 mlW 3% isopropanol pH = 7.2, flow 0.6 ml/min. The elution profile was determined using a UV detector at 280 nm. The area of the peaks corresponding to the free (non-associated) protein and to the protein associated with the polymer was evaluated. From the area of the peaks under consideration, the areas are subtracted of the peaks obtained by means of chromatography of samples prepared in an identical way to those before, but using a buffer solution devoid of protein. The areas of the peaks relating to the associated and non-associated proteins were processed on the basis of calibration lines previously obtained with protein alone in various concentrations.

Fig. 8 A-E present, by way of example, the elution profiles

obtained by incubating r-h-GH with different quantities of mPEG2kDa-stearic acid. In particular, the elution profile of rh-GH (A) and of a solution wherein 80 mg of protein is incubated with 400 mg (B), 800 mg (C), 1200 mg (D) and 2400 mg (E) of PEG2kDa-stearic acid are illustrated.

Figs. 9 and 10 present the chromatographic calibration lines relating to r-G-CSF and r-h-GH, respectively.

By way of example, Figs. 11-18 show the protein/polymer interaction profiles obtained with r-h-GH and r-h-G-CSF with a number of hydrophobised polymers, wherein the quantity of associated and non-associated protein is given as a function of the quantity of polymer added in the assay.

Specifically:

Fig. 11 illustrates the association profile of rh-GH with PEG2kDa-stearic acid. Shown on the ordinate is the quantity of protein associated (·) or non-associated (o) in the 20 ml assayed as a function of increasing quantities of polymer,

Fig. 12 illustrates the association profile of rh-GH with PEG5kDa-stearic acid. Shown on the ordinate is the quantity of protein associated (·) or non-associated (o) in the 120 ml assayed as a function of increasing quantities of polymer, Fig. 13 illustrates the association profile of rh-GH with PEG2kDa-colanic acid. Shown on the ordinate is the quantity of protein associated (·) or non-associated (o) in the 120 ml assayed as a function of increasing quantities of polymer,

Fig. 14 illustrates the association profile of rh-GH with PEG5i<Da-colanic acid. Shown on the ordinate is the quantity of protein associated (·) or non-associated (o) in the 120 ml assayed as a function of increasing quantities of polymer,

Fig. 15 illustrates the association profile of rh-GH with PEGiokDa-colanic acid. In ordinata viene riportata la quantita di proteina associated (·) o non-associated (o) nei 120 ml di saggio in funzione di increasing quantities di polymer.

Fig. 16 illustrates the association profile of rh-GH with PEG2okDa-colanic acid. Shown on the ordinate is the quantity of protein associated (·) or non-associated (o) in the 120 ml assayed as a function of increasing quantities of polymer,

Fig. 17 illustrates the association profile of rh-GCSF with PEG5kDa-colanic acid ester. Shown on the ordinate is the quantity of protein associated (·) or non- associated (o) in the 120 ml assayed as a function of increasing quantities of polymer,

Fig. 18 illustrates the association profile of rh-GCSF with PEG5kDa-NH(CH2) 2NH- colanic acid ester. Shown on the ordinate is the quantity of protein associated (·) or non-associated (o) in the 120 ml assayed as a function of increasing quantities of polymer,

Table III above presents the mean charging values, expressed as milligrams of rh- GH or of rh-GCSF associated with 100 milligrammi of hydrophobised polymer [mg/100 mg].

EXAMPLE 6

Scatchard and Klotz analysis

The association values obtained from the experiments described in Example 5 were processed by means of Scatchard analysis (Scatchard G., The attractions of proteins for small molecules and ions, Ann. N.Y. Acad. Sci. 51 : 660-672, 1949) to determine the parameters of association kD (dissociation constant, M-1 ) and Bmax. By way of example, Fig. 19 shows the plots (Scatchard analysis) obtained with the association of mPEG5000-colanic acid with r-h-G-CSF. Table IV summarises the values for kD and Bmax calculated for the association of a number of polymers hydrophobised with r-h-GH and r-h-G-CSF.

The results obtained from the association studies with the various hydrophobised polymers and r h-GH or r h-G CSF were worked up further by means of Klotz analysis [; Boborvnik S.A. Ligand-receptor interactions: a new method for determining the binding parameters, J. Biochem. Biophys. Meth. 55:71-86, 2003], which confirmed the results obtained.

EXAMPLE 7

Studies of correlations between the degree of charging and the phvsicochemical properties of the polymers

The structural characteristics of the PEG-modifying agents were determined by means of MOE computational analysis (Molecular Operating Environment MOE 2004.03 Chemical Computing Group Inc., Montreal, Canada), which enabled a number of molecular descriptors to be obtained, such as polar surface area, van der Waals surface area, molecular volume, etc.

The charging data obtained in the association studies previously reported were correlated with the CMC values obtained with the various hydrophobised polymers. The correlation obtained with r-h-GH is presented in Fig. 20. Specifically, the data displayed represent the correlation between the degree of charging and CMC obtained with rh-GH and PEG modified with linear chains (·) and PEG modified with cyclic lipophilic compounds (o).

EXAMPLE 8

Determination of the association pharmacokinetic profile of r-h-G-

CSF/mPEG5kDa-colanic acid

This experiment investigated the long-term effect of the association non-covalent r-h-G-CSF/ mPEG5kDa-colanic acid (1 :21 p/p) administered by the subcutaneous route in rats, by means of evaluation of the serum levels of the protein over time.

Two groups, each of 4 male Sprague-Dawley rats weighing approximately 300-

350 g, were respectively treated with r-h-G-CSF and with the non-covalent association r-h-G-CSF/mPEG5kDa-colanic acid (1 :21 p/p) by means of subcutaneous injection administered dorsally. Each of the four animals in the first group received r-h-G-CSF dissolved in saline solution buffered to pH 5 withIO mM acetate buffer, at a dose of 0,1mg/Kg.

Each of the four animals in the second group received r-G-CSF/mPEG5kDa- colanic acid (21 :1 p/p), dissolved in saline solution buffered to pH 5 with 10mM acetate buffer, at the equivalent dose of 0.1 mg di r-h-G-CSF/Kg.

In rats of the first group (treated with r-h-G-CSF), samples were obtained at time 0 and after 15 minutes, 30 minutes, and 1 , 2, 4, 8 and 24 hours after administration. In rats of the second group (treated with r-h-G-CSF/mPEG5kDa-colanic acid) samples of 0.5 ml of blood were obtained at time 0 and after 1 , 2, 4, 8, 24, 32, 48 and 72 hours after administration. The blood was treated in such a way as to obtain the serum on which the immunological determinations of the protein were carried out by means of the ELISA test (Elisa Kit: Human G-CSF Assay Kit; cod. JP27131 ; IBL Co. Ltd.), using r-h-G-CSF as the reference standard.

Using the serum concentration values obtained, the pharmacokinetic profiles depicted in Fig. 21 were calculated and the AUC (area under the curve), Tmax and Cmax (time and concentration of the peak ), and the t ½ (half-life) were determined.

The results obtained are displayed in Fig. 21 (pharmacokinetic profile) and in Table V above (pharmacokinetic parameters). Specifically, the data displayed represent the pharmacokinetic profile in the rat of the non-covalent association rh- GCSF/PEG5kDa-colanic acid (1 :21 p/p) ( A ), compared with that of native rh-GCSF nativo (·).

EXAMPLE 9

Determination of the pharmacodynamic profile of the association r-h-G- CSF/mPEG5kDa-colanic acid

This experiment studies the activity in vivo, that is, the pharmacodynamic profile, of the non-covalent association r-h-G-CSF/mPEG5kDa-colanic acid (1 :21 p/p) administered subcutaneously in mice, by measuring the neutrophil count per ml of blood over time.

Four groups of C3H/HeN male mice were treated respectively with a single administration of 3 mg/kg of r-h-G-CSF/mPEG5kDa-colanic acid (group 1 , 7 animals), with a single administration of its carrier (group 2, 4 animals), with

4 administrations, repeated daily, of 0.25 mg/kg of r-h-G-CSF (group 3, 7 animals) and with 4 administrations, repeated daily, of the corresponding carrier (group 4, 4 animals).

Samples of blood were collected from the tail of the animals at the following times: · Time 0, 6, 24, 48, 72, 96, 120 hours after the treatment in groups 1 and 2 Time 0, 6, 30, 54, 78, 96, a 120 hours after the treatment in groups 3 and 4

5 microlitres of blood sampled were appropriately diluted in a solution of EDTA (20 microlitrres), and it was on these that the white blood cell and neutrophil counts were performed using a Vet abc automated haematological analyser (Scil Animal Care).

The pharmacodynamic parameters (Tmax, ANCmax, AUCANC) obtained are displayed in Table VI above, while the pharmacodynamic profile (ANC vs. time) is shown in Fig. 22. The results obtained demonstrate the long-term pharmacological effects of the association r-h-G-CSF/mPEG5kDa-colanic acid. Specifically, the data displayed in Fig. 22 represent the pharmacodynamic profile in mice of the non-covalent association rh-GCSF/PEG5kDa-colanic acid (1 :21 p/p) ( A ), compared with that of rh-GCSF (·). The broken lines show the course of the neutrophils in mice treated with the carrier. EXAMPLE 10

Preparation of the supramolecular complex GLP-1 /mPEG5kDa-colanic acid and determination of the increase in its stability to enzymatic hydrolysis by the dipeptidyl peptidase (DPP IV)

This experiment studies the preparation and in-vitro stability of the supramolecular association GLP-1 /mPEG5KDa-colanic acid to the action of the dipeptidyl peptidase IV by comparison with the non-associated peptide.

0,6 mg GLP-1 was added to a solution of PEG-colanic acid 5kDa prepared by dissolving 5 mg of hydrophobised polymer in 1 ml of PBS at pH 7.3. The solution was left overnight under gentle agitation and then analysed in SEC-HPLC. The analysis demonstrated that all the GLP-1 was associated with the polymer.

0,6 mg GLP-1 is then solubilised in 1 ml of PBS at pH 7.3.

50 μΙ of each of the two solutions were analysed by means of RP-HPLC (time 0).

To each of the two solutions 0.005 IU of DPP IV was added and then they were diluted PBS until a concentration in GLP-1 equal to 100 g/ml was obtained. The solutions were placed in an incubator at +37°C.

At 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 24 and 32 hours of incubation, 50 μΙ samples were taken from each of the two solutions to which 2.5 μΙ of an aqueous solution of 10% trifluoroacetic acid (TFA) was added, and the samples were analysed RP-HPLC to determine the concentration of the dipeptide Hys-Ala formed from the GLP-1 by action of the DPPIV.

The results of the analysis presented in Table VII above.

From the results obtained, the degradation profiles of the two compounds were traced, which are displayed in the graph in Fig. 23, where it is demonstrated that the molecular complex deriving from the association of GLP-1 with the polymer mPEG5KDa-Colanic acid (association 1 :25 (p/p) confers stability upon GLP-1 against the hydrolysing action of DPP IV).

Indeed, following 8 hours of incubation, whereas the solution of native GLP-1 is virtually completely hydrolysed, in the solution of GLP-1 in association with the polymer only 5% of GLP-1 is hydrolysed. Even after 1 .5 days of incubation of the the GLP-1/mPEG5KDa-colanic acid, majority of the peptide is still intact (approximately 84%). To be specific, Fig. 23 displays the in-vitro stability profiles of native GLP-1 (■) and of GLP-1 associated with PEG5KDa-colanic acid ( A ) by means of DPP IV in aqueous solution.

The text of the description plus claims of Patent Application MI2011A001866 of 13 October 2011 is included here in its entirety for reference.

Claims

1. A polymer based on modified polyethylene glycol (PEG) of formula (I), Y- (OCH2CH2)n-O-CH2-CH2-X-A (I)
wherein
(OCH2CH2)n- is a polyoxyethylene chain wherein n varies from 100 to 9000, preferably from 500 to 5000,
Y is -CH3 or -H,
X is
a) a saturated or unsaturated C C2o linear alkyl group, having each end functionalised with a group selected from hydroxyl, amino or thiol,
b) a saturated or unsaturated C3-C5 branched alkyl group, having each end functionalised with a group selected from hydroxyl, amino or thiol,
c) a heteroatom selected from N, S, or an NH group;
A is a lipophilic group selected from
i) a saturated or unsaturated aliphatic carboxylic acid comprising from 8 to 36 atoms of carbon, preferably from 0 to 20C, or
ii) a polycyclic group selected from colanic acid, cholic acid, chenodeoxychoiic acid, deoxycholic acid and/or their analogues and derivatives.
2. Polymer based on modified polyethylene glycol (PEG) according to
Claim 1 , characterized in that X is
- NH - (CH2)m -NH -,
wherein m is from 1 to 20, preferably from 8 to 14, or
- R - CO - (CH2)p - CO - R - wherein p is from 1 to 20, preferably from 8 to 14, and
wherein R, if present, is S or another residue deriving from a carboxyl activating group.
3. Polymer based on modified polyethylene glycol (PEG) according to
Claim 1 wherein
Y is -CH3 or -H,
X is NH,
A is a polycyclic group selected from colanic acid, cholic acid, chenodeoxychoiic acid, deoxycholic acid and preferably is colanic acid.
4. Polymer based on modified polyethylene glycol (PEG) according to
Claim 1 , characterized in that said X group is N and said A group is a residue of colanic acid.
5. Use of a polymer based on modified polyethylene glycol (PEG) according to any one of Claims 1-5 as a carrier for biologically active macromolecules or biopharmaceuticals.
6. Supramolecular complex comprising at least a polymer of modified polyethylene glycol (PEG) of formula (I) according to any one of Claims 1-5 and at least a biologically active macromolecule.
7. Complex according to Claim 5, characterized in that said biologically active macromolecule is a polypeptide, protein or oligonucleotide which is biologically and/or pharmaceutically active.
8. Complex according to Claim 5 or 6, characterized in that said biologically active macromolecule is the growth hormone (h-GH), the growth factor of granulocites colonies (G-CSF) or the GLP-1 hormone (Glucagon-like Peptide - 1 ) or their homologous or derivative having a homology degree corresponding at least to 90% or maintaining al least 80% of biological activity.
9. Use of a supramolecular complex according to any one of Claims 6-8 for the sustained release of the biologically active macromolecule.
10. Use according to Claim 9 wherein said biologically active macromolecule is selected from the growth hormone (h-GH), the growth factor of granulocites colonies (G-CSF) or the GLP-1 hormone (Glucagon-Like Peptide-1 ) or their homologous or derivative having a homology degree corresponding at least to 90% or maintaining at least 80% of biological activity.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5904936A (en) 1995-03-28 1999-05-18 Flamel Technologies Particles based on polyamino acid(s) and capable of being used as delivery vehicles for active principle(s) and method for preparing them
US6180141B1 (en) 1996-03-15 2001-01-30 Flamel Technologies Composite gel microparticles as active principle carriers
WO2001037809A1 (en) 1999-11-23 2001-05-31 Flamel Technologies Colloidal suspension of submicronic particles as vectors for active principles and method for preparing same
US20010051189A1 (en) 1996-07-29 2001-12-13 Universidade De Santiago De Compostela Application of nanoparticles based on hydrophilic polymers as pharamaceutical forms
WO2002028521A1 (en) 2000-10-06 2002-04-11 Flamel Technologies Colloidal suspension of submicronic particles for carrying active principles and their mode of preparation
WO2007112794A1 (en) 2006-03-30 2007-10-11 Bio-Ker Srl New activated poly(ethylene glycols) and related polymers and their applications

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6309663B1 (en) * 1999-08-17 2001-10-30 Lipocine Inc. Triglyceride-free compositions and methods for enhanced absorption of hydrophilic therapeutic agents
WO2004078121A3 (en) * 2003-02-28 2004-10-28 Alza Coporation Liposome composition for reduction of liposome-induced complement activation
US20050164952A1 (en) * 2004-01-23 2005-07-28 Vital Pharmaceuticals, Inc. Delivery system for growth hormone releasing peptides

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5904936A (en) 1995-03-28 1999-05-18 Flamel Technologies Particles based on polyamino acid(s) and capable of being used as delivery vehicles for active principle(s) and method for preparing them
US6180141B1 (en) 1996-03-15 2001-01-30 Flamel Technologies Composite gel microparticles as active principle carriers
US20010051189A1 (en) 1996-07-29 2001-12-13 Universidade De Santiago De Compostela Application of nanoparticles based on hydrophilic polymers as pharamaceutical forms
WO2001037809A1 (en) 1999-11-23 2001-05-31 Flamel Technologies Colloidal suspension of submicronic particles as vectors for active principles and method for preparing same
WO2002028521A1 (en) 2000-10-06 2002-04-11 Flamel Technologies Colloidal suspension of submicronic particles for carrying active principles and their mode of preparation
WO2007112794A1 (en) 2006-03-30 2007-10-11 Bio-Ker Srl New activated poly(ethylene glycols) and related polymers and their applications

Non-Patent Citations (18)

* Cited by examiner, † Cited by third party
Title
"Brayden Controlled release technologies for drug delivery", DRUG DISCOV, TODAY, vol. 8, 2003, pages 976 - 978
"Molecular Operating Environment MOE", March 2004, CHEMICAL COMPUTING GROUP INC.
AN Y.J.; CARRAWAY E.R.; SCHLAUTMAN M.A.: "Solubilization of polycyclic aromatic hydrocarbons by perifluorinated surfactant micelles", WATER RES., vol. 36, 2003, pages 300 - 308
BLANCHETTE J. ET AL.: "Principles of transmucosal delivery of therapeutic agents", BIOMED. PHARMACOTHER., vol. 58, 2004, pages 142 - 151
BOBORVNIK S.A.: "Ligand-receptor interactions: a new method for determining the binding parameters", J. BIOCHEM. BIOPHYS. METH., vol. 55, 2003, pages 71 - 86
CHIRINO A. J. ET AL.: "Minimizing the immunogenicity of protein therapeutics", DRUG DISCOV. TODAY, 2004, pages 82 - 90, XP002395255
CLELAND J. ET AL.: "Emerging protein delivery methods", CURR OPIN. BIOTECHNOL, vol. 12, 2001, pages 212 - 219, XP001205967, DOI: doi:10.1016/S0958-1669(00)00202-0
ELISA KIT: "Human G-CSF Assay Kit", IBL CO. LTD
EUR. J. PHARM. BIOPHARM, vol. 68, 2008, pages 656 - 666
JEONG J.H.; KIM S.W; PARK T.G.: "A new antisense oligonucleotide delivery system based on self-assembled ODN-PEG hybrid conjugate micelles", J. CONTROL. REL., vol. 94, 2004, pages 1 - 14
LAVASANIFAR A; SAMUEL J.; KNOW G.S.: "The effect of alkyl-core structure on micellar properties of poly(ethyleneoxyde)-block-poly(L-aspartamide) derivatives", COLL. SURF. B BIOINTERF., vol. 22, 2001, pages 115 - 126
ORIVE G. ET AL.: "Drug delivery in biotechnology: present and future", CURR. OPINION BIOTECHNOL, vol. 14, 2003, pages 659 - 664
PANYAM J.; LABHASETWAR V.: "Biodegradable nanoparticles for drug and gene delivery to cells and tissue", ADV. DRUG DEL. REV., vol. 55, 2003, pages 329 - 347, XP008096954, DOI: doi:10.1016/S0169-409X(02)00228-4
RAZZACKI S.Z.; THWAR P.K.; YANG M.; UGAZ V.M.; BURNS M.A.: "Integrated microsystems for controlled drug delivery", ADV. DRUG DEL. REV, vol. 56, 2004, pages 185 - 198, XP002615035, DOI: doi:10.1016/j.addr.2003.08.012
SCATCHARD G.: "The attractions of proteins for small molecules and ions", ANN. N.Y. ACAD. SCI., vol. 51, 1949, pages 660 - 672, XP008000889, DOI: doi:10.1111/j.1749-6632.1949.tb27297.x
SCHELLEKENS H.: "Immunogenicity of therapeutic proteins: clinical implications and future prospects", CLIN.THER, vol. 24, 2002, pages 1720 - 1740, XP002465634, DOI: doi:10.1016/S0149-2918(02)80075-3
VERONESE F.M.; HARRIS J.M.: "Introduction and overview of peptide and protein pegylation", ADV. DRUG DEL. REV., vol. 54, 2002, pages 453 - 456, XP002464348, DOI: doi:10.1016/S0169-409X(02)00029-7
WANG W.: "Protein aggregation and its inhibition in biopharmaceutics", INT. J. PHARM., vol. 289, 2005, pages 1 - 30, XP004967405, DOI: doi:10.1016/j.ijpharm.2004.11.014

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