Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal 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
  • A61K47/51—Medicinal 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
  • A61K47/56—Medicinal 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
  • A61K47/59—Medicinal 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
  • A61K47/60—Medicinal 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

• the present invention is related to gliicagon-like peptide- 1 (GLP-I) and analogues insulinotropic peptides, monoconjugated to biocompatible polymeric molecules by enzymatic direct and site-specific transghitamination reaction as well as their pharmaceutical formulations and delivery systems for therapeutical application in dismetabolic pathologies such as type 2 diabetes,
• Diabetes mellitus is a disease that has been known to exist for thousands of years and that currently afflicts more than 200 million people worldwide. In addition to the decrease of life expectancy and lower quality of life of individuals with diabetes, the disease and its associated complications are a major burden on health budgets.
• Type 2 diabetes or non-insulin-dependent diabetes mellitus
• non-insulin-dependent diabetes mellitus which has both genetic and environmental components and represents a multifactorial, heterogeneous group of disorders, which results from defects in insulin secretion, insulin action, or both, and leads to uncontrolled or elevated levels of blood glucose.
• Type 2 diabetes usually occurs in overweight adults over the age of 45; however. the incidence of Type 2 diabetes in overweight children (usually over the age of
• lncretins are gut-derived hormones released by nutrients that potentiate insulin secretion under elevated glycemic conditions.
• GLP-I Human giucagon-like peptide-1
• GLP-I Human giucagon-like peptide-1
• enteroendocrine L-cells of the intestine after food intake, exhibiting a potent blood glucose-lowering effect through different physiological mechanisms including the secretion of endogenous insulin in a glucose-dependent manner, the decrease of blood glucagon levels and the reduction of gastric emptying by slowing gastric motility.
• GLP-I stimulates the proliferation and differentiation of new pancreas ⁇ -cells leading to increase of ⁇ -cell mass [Hoist JJ, 2007].
• GLP-I The major form of circulating human GLP-I is a C-terminal amidated peptide of 30 aminoacid residues generally indicated as GLP-l(7-36)-amide (SEQ ID NO 1); a minor non-deamidated and C-terminally glycine extended form of 31 aminoacid residues, generally indicated as GLP-l(7-37), is also detectable in blood (SEQ ID NO 2). Both peptides display the same biological activities and are equipotent. For the purposes of the present invention, as shown by SEQ IDs NO 1 and 2, GLP-I peptides and their analogues are reported according to peptide chain numeration starting from the N-terminal histidine residue.
• GLP-I peptides and analogues that is the stimulation of insulin secretion only when plasma glucose levels are above the normal physiological value, makes these compounds potential candidates for the treatment of type 2 diabetes.
• GLP-I peptides exert their biological effects through seven transmembrane G-protein -coupled receptors expressed in the ⁇ -cell of the islets of Langerhans as well as in gastrointestinal tract and other tissues including heart, kidney, lungs and brain.
• Studies of structure-activity relationship of GLP-I peptides carried out by alanine scanning mutagenesis demostrated that positions I. 4, 6, 7. 9, 13, 15, 22. 23 and 26 are critical for receptor binding [Adelhorst K, et al., 1994: Gallwitz B et al., 1994],
• GLP-I analogues examples include exendin-3 and exendin-4.
• exendin-3 and exendin-4 Two 39- aminoacid peptides originally isolated from the venom of GiIa monster (Heloderma suspectum), which share approximately 50% sequence identity to GLP-I itself and are indeed agonists of GLP-I receptor.
• a synthetic preparation of exendin-4 (exenatide) has been approved in both USA and Europe as adjunctive therapy to for the treatment of type 2 diabetes based on two daily subcutaneous injections [Davidson MB et al., 2005], Unfortunately, the therapeutic use of GLP-I peptides is limited by a veiy short plasma half-life (for example GLP-I -amide has a ti / ?
• DPP-IV plasma dipeptidyl peptidase IV
• CD26 a serine-type protease that cleaves N-terminal dipeptides from polypeptide chains after a proline or alanine residue.
• the biological activity of natural circulating GLP-I peptides is in fact regulated by the N-terminal DPP-IV-mediated cleavage at the alanine-2 residue to give the inactive metabolite des-His-Ala-GLP-1 peptide.
• GLP-I derivatives are extensively reported in both scientific and patent literature; they can be obtained, among others, according to the following approaches: a) by selective amino acids substitutions in GLP-I peptidic chain that confer DPP-IV resistance as, for example, by replacement of the second N-terminal amino acid L-alanine with D-alanine or serine resulting in GLP-I analogues which maintain the insulinotropic activity with a significant increase of plasma stability and in vivo half-life [Uckaya G et aL 2005; Ritzel U et aL 1998; Buckley DI et al., US5545618] b) by attaching of one or more lipophilic substituents to the side chains of aminoacid residues of GLP-I and GLP-I analogues where the lipophilic substituents comprise 4 to 40 carbon atoms and are chemically conjugated to aminoacids both directly or through a spacer.
• GLP-I analogues derivatized with lipophilic moieties showed a protracted profile of action in vivo and higher persistance in plasma compared to natural GLP-I (Knudsen L et al., EP0944 648) c) by exploiting naturally occurring insulinotropic peptides such as, for example, exendin-3 and exendin-4 (or its corresponding synthetic form exenatide), two 39 amino acid peptides originally isolated from salivary glands of lizards [Eng J et al., 1992] which were found to be potent GLP-I receptor agonists.
• Exenatide whose therapeutic efficacy was also demonstrated in patients with type 2 diabetes, shares about 50% sequence identity with GLP-I while differs in N-terminal position 2 where alanine is substituted by a glycine residue, which confers DPP- IV-resistance and a comparatively longer half-life without reducing biological activity.
• Site specific monopegylated GLP- ⁇ peptides could be prepared by selective chemical pegyiation with propionaldehyde functionalized monometoxy-PEG (m-PEG) carried out at pH 4.5 on the ⁇ -amino group of N-terminal His 1 residue; however the resulting mono-pegylated GLP-I , although resistant to DPP-IV degradation, was shown to have little biological activity in vitro [Lee SH et al. 2005].
• m-PEG propionaldehyde functionalized monometoxy-PEG
• GLP-I -amide monopegylated on Lys 20 residue was prepared by a multi-step chemical synthesis including maleic anhydride protection of lysine ⁇ -amino groups, chromatographic separation of Lys 20 - and Lys 28 -protected GLP-I isomers, chemical pegyiation carried out independently on each isomer by reaction of free lysine ⁇ -amino group with 2 kDa m-PEG functionalized with succinimidy!
• acylated derivatives of human GLP-I or its analogues as for example liraglutide or ⁇ Arg 28 -Lys 20 -N-[ ⁇ -( ⁇ -Glu ⁇ N- ⁇ -hexadecanoyl ⁇ )]-GLP4 ⁇ in which the acyl moiety promotes in vivo serum albumin binding and prolongs the half-life to about 13-15 hours without reduction of potency [Knudsen LB et ai., 2000].
• the present invention relates to polymer-conjugated incretin mimetic peptides, such as GLP-I peptides, and their analogues and derivatives characterized, with respect to natural peptides, by maintenance of therapeutical useful biological activity, resistance to peptidase degradation and longer circulating half-life.
• the term incret ⁇ n mimetic peptides means a compound that mimics the action of the naturally occurring incretin hormone GLP-I by enhancing glucose-dependent insulin secretion and related glucose-lowering actions such as, for example, inhibition of glucagon release following meals, slowing the rate of gastric emptying and promotion of satiety. It is well-known that the conjugation of peptide or protein molecules to high molecular weight biocompatible hydrophilic polymers modifies the physico- chemical properties of the resulting conjugated complexes largely maintaining the biological function of the original non-conjugated molecule, such as for example the capability of receptors recognition.
• conjugation of proteins and peptides with a polymeric moiety may hamper physical contact between the protein and both specific and non specific proteolytic enzymes preventing or reducing the enzymatic proteolysis.
• Well-known polymeric moieties that have been used in the conjugation of therapeutic proteins are linear or branched poly(ethylene glycol) (PEG) chains with molecular weight between about 2 kDa and 60 IcDa.
• Covalent conjugation of such polymers to one or more residues in the protein molecule modifies a number of functional aspects of the conjugated complexes including, for example, the renal clearance thanks to the increase of apparent molecular size, the stability towards enzymatic degradation as well as the reduction of immunogenicity due to the masking of immunogenic epitopes on the protein surface.
• poly(ethylene glycol)-conjugated (pegylated) therapeutic proteins because of their improved stability and their more favorable pharmacokinetics profile enabling lower dose-frequency than their non-pegylated counterparts [Greenwald RB et al., 2003].
• therapeutic proteins commercially available as pegylated derivatives are, among others, PEG-Interferon-alpha, used for the treatment of patients infected with the hepatitis C virus, PEG-Filgrastim (PEG-granulocyte-colony stimulating factor), that stimulates the granulocytes production and differentiation in bone marrow, and Pegvisomant, a pegylated human growth hormone receptor antagonist which is indicated for the treatment of acromegaly [Parveen S and Sahoo SK, 2006].
• PEG-Interferon-alpha used for the treatment of patients infected with the hepatitis C virus
• PEG-Filgrastim PEG-granulocyte-colony stimulating factor
• Pegvisomant a pegylated human growth hormone receptor antagonist which is indicated for the treatment of acromegaly [Parveen S and Sahoo SK, 2006].
• Pegylated therapeutic proteins should ideally be homogeneous products with wel! defined structural and functional characteristics.
• chemical pegylation of proteins are based on non specific reactions with nucleophilic residues (most commonly the ⁇ -amino group of surface lysine residues or the side-chain carboxylic groups) and produces different extent of conjugation and/or mixtures of pegylated positional isomers each of them could give rise to variations in characteristics relevant to clinical application including biological activity and appearance of side-effects.
• a potential way to overcome the limitations of the above reported chemical approaches and to prepare site-specific mono conjugated GLP-I peptides and GLP-I derivatives and analogues with biocompatible polymers is to exploit the application of the selective transglutaminase catalyzed conjugation between biocompatible polymers functionalized with a primary amino group and a single glulamine residue contained in GLP-I like peptides.
• Transglutaminases E. C. 2.3.2.13; protein-glutamine ⁇ -glutamyltransferase; Tgase
• Tgase enzymes of both eukaryotic and prokaryotic origin which catalyze an acyl transfer reaction between a ⁇ -carboxyamido group of protein-bound glutamine residue and an ⁇ -ami ⁇ ogroup of a lysine residue or a variety of primary amines, in particular straight chain alkyfamines, according to the following reaction scheme:
• -CONH 2 is the ⁇ -carboxamide group of a protein-bound glutamine residue acting as acy! donor and R-NH 2 represents a variety of primary amines (including an ⁇ -aminogroups of lysine residues in the protein chain) acting as acyl acceptor.
• the currently known eukaryotic transglutaminases include, among others, the blood coagulation factor XIIIa, the keratinocyte Tgase (type 1 Tgase) and the ubiquitous tissue type Tgase (type 2 Tgase).
• the various eukaryotic Tgases belong to a family of isoenzymes composed of several subunits and sharing a high degree of both sequence similarity and functional properties, with molecular masses of about 75-90 kDa and a similar Ca 2+ -dependent catalytic mechanism of action [Esposito C and Caputo I, 2004],
• M-Tgases microbial Tgases
• Both mammalian and microbial Tgases act with a specific selectivity on side- chain of glutamine residues of proteins representing the acyl donor mojety while much lower or no selectivity was displayed towards the primary amino group representing the acyl acceptor moiety.
• both Tgases react not only with the ⁇ -amino groups of lysine residues on protein chains, but also with compounds bearing a primary amino group or with aliphatic alkylamines, preferably with primary aliphatic amines on a linear chain of at least four carbon atoms [Ohtsuka T et al., 2000].
• these Tgase catalyzed conjugation reactions were earned out in the presence of a significant excess of the primary amino group functionalized m-PEG substrates (which were present in about 100-1000 fold molar excess) with respect to the glutamine-containing protein substrates.
• GLP-I peptides and insulinotropic GLP-I analogues and derivatives bearing a single glutamine residue can be conjugated directly to aikylamino functionalized polymers with high yields; the term "directly” is herein used to indicate that the covalent bond occurs “directly” between the GLP-I peptide and the aikylamino functionalized polymer, that is without the need of any intermediate linker.
• these alkylamines can be linear or branched m- PEG of molecular weight from 2 to 50 IcDa, preferably from 5 to 40 IcDa, more preferably from 10 to 30 kDa, even more preferably from 15 to 25 IcDa; in the preferred embodiment, the m-PEG has a molecular weight of about 20 kDa.
• Such polymers are conjugated by a direct enzymatic reaction catalyzed by a transglutaminase, preferably a microbial transglutaminase (M-Tgase), with a molar ratio between aikylamino functionalized polymers and GLP-I like peptides comprised between 1 :1 and 1 :100, preferably between 1:5 and 1 :35, more preferably between 1 :15 and 1:25; according to a most preferred embodiment, it is about 1 :20.
• a transglutaminase preferably a microbial transglutaminase (M-Tgase)
• M-Tgase microbial transglutaminase
• an amino functionalized biocompatible polymer such as for example linear or branched alkylamino m-PEG of molecular weigth from 2 to 60 kDa
• GLP-I peptides monoconjugated by M-Tgase-catalyzed direct reaction with biocompatible polymers on the single glutamine residue naturally present in the position 17 of GLP-I -amide or C -terminal glycine extended GLP-I or on the single glutamine residue naturally present in the position 13 of exenatide.
• GLP-I peptides and analogue insulinotropic peptides have no defined structure in aqueous solution [Thornton K and Gorensteiii DG, 1994] with the reported capability of Tgases to recognize as substrate glutamine residues located on solvent accessible, flexible and locally unfolded region of the protein chain [Fontana A et aL 2008] one could expect that glutamine residues introduced into the peptidic chain of Q 17N-GLP-1 -peptides and Q17N-GLP-1 analogues would provide alternative substrates for the direct M-Tgase catalyzed monoconjugation reaction with biocompatible polymers.
• Another embodiment of the present invention is represented by monoconjugaled Q17N-GLP-l-amide and insulinotropic Q 17N-GLP-1 -peptide analogues obtained by direct M-Tgase catalyzed reaction with biocompatible polymers on a single glutamine residue introduced in substitution of any residue which is both: a) not involved in receptor binding of the GLP-I -peptides, that is to say any residue but the ones in position 1 , 4, 6.
• GLP-I peptides and GLP-I peptide analogues and derivatives which are used according to the present invention as substrates for the site-specific direct M- Tgase catalyzed mono-pegylation reaction can be prepared by conventional chemical methods well known in the art, such as by solid-phase peptide synthesis or by condensation reaction of peptide fragments as previously disclosed (see for example Me ⁇ field B, 1986 and Kaiser ET, 1989 which are incorporated by reference herein).
• GLP-I peptides and GLP-I peptide analogues and derivatives can also be prepared by recombinant DNA technologies according to methods well known in the art and previously disclosed (see for example Sambrock J et al., 1989 which is hereby incorporated by reference).
• the direct enzymatic reactions of the present invention are carried out by dissolving GLP-I peptides or GLP-I peptide analogues and derivatives in a suitable aqueous buffer at pli between 4 and 9, more preferably at pH between 5 and 8 in a concentration between 50 -1000 ⁇ M, more preferably between 100 and 500 ⁇ M and adding less than 50, and preferably less than 25 fold molar excess of linear or branched alkylamino m-PEG with molecular mass between 2 and 60 IeDa and 0, 1 to 2 U/ml, more preferably 0.2 to 1 LVmL of M-Tgase and leaving the reaction mixture at temperature between 10 and 50 0 C, more preferably between 15 and 30 0 C, for 10 hours or longer.
• M-Tgase can be, for example, a preparation of Activa WM commercially available from Ajinomoto or a preparation of any natural or recombinant microbial derived M-Tgase with equivalent activity. It is well known that the kinetics of the M-Tgase catalyzed reaction, as of any other enzymatic reaction, can be modulated by temperature and by the amount of enzyme and consequently the reaction time of the above described reactions can be reduced by choosing any convenient combination of enzyme concentration and temperature compatible with the maintenance of catalytic activity.
• the site-specific monopegylated GLP-I analogues of the present invention were purified by traditional methods as salting out, dialysis, ultrafiltration, isoelectric precipitation, column chromatography or by their combination and characterized according to methods known in the art and described, for example, in the experimental section. As above reported, no pegylation was obtained when the Tgase catalyzed reactions between GLP-I analogues and amino functionalized m- PEG were performed in the presence of a preparation of a mammalian Tgase, as shown in tables 1 and 2,
• the site-specific monopegylated GLP-I -peptides and analogues showed improved resistance towards enzymatic degradation by DPP-IV studied at 37°C in vitro, compared to the unmodified peptide.
• Table 4 shows that while GLP-I -amide was completely inactivated by cleavage of the N-terminal dipeptide after S hours at 37 0 C. its monopegylated derivative still maintained about 70 per cent of active structure even after 24 hour incubation, with GLP-I -amide conjugated to higher molecular weight PEG chains being more resistant to DPP- IV cleavage, as shown in table 4 and figure 1.
• the monopegylated derivative of Q17N/A24Q-GLP-1 mutant also displayed a similar resistance to in vitro DPP-IV degradation as shown in table 4 and figure 1.
• Table 4 shows that
• Glucose related parameters calculated for the first 180 minutes after glucose administration show that both GLP-I-PEG products, in particular GLP-I - PEG 3 QkDa 5 have an activity similar to that of Exenatide, probably due to a comparable DPP-IV resistance.
• the glucose-stabilizing activity displayed by 20 kDa and 30 IcDa monopegylated GLP-I -amide up to 8 hours after an oral glucose load is possibly due to a combination of increased proteolytic stability and clearance reduction with a final biological efficacy comparable or better than the one displayed by exenatide.
• monopegylated GLP-I analogues are present in a concentration from 0.1 to 50 mg/ml and wherein said formulations have a pH from 5 to 9, more preferably from 7 to 8.
• These formulations may further comprise buffer systems with or without addition of tonicity and chelating agents as well as preservatives, stabilizers and surfactants known in the art.
• such formulations are aqueous solutions and/or aqueous suspensions while in a further embodiment they are powdered preparations obtained by any acceptable methods, preferably by liophylization.
• Systems for long term delivery of site-specific monoconjugated derivative of GLP-I like peptides described in the present invention include, among others, organic or inorganic polymeric nano- and micro- particles, liposomes and liposome-like vescicles, lipid nanoparticles, hydrogel- based microparticles, thermo- and pH-responsive polymers and microemiilsions known in the art as exemplified by the following basic references: Kohane DS, 2007; Lee KY and Yuk SH, 2007; Singh S et al., 2007; Jorgensen L et al., 2006; Schmaljohann D, 2006; Muiler RH and Keck CM, 2004; Shina VR and Trehan A, 2003, herein incorporated by reference.
• a sustained release formulation of monopegylated GLP-I like peptides with incretin mimetic properties can be prepared by incorporating the active principle in Poloxamer 407 dissolved in pH 4 acetate buffer at 4°C.
• the kinetic of in vitro release from a 22% w/w Poloxamer 407 solution containing 0.3 mg/ml of GLP-I -amide Q17- PEG 20 kDA, studied by incubating 1 ml of formulated gel at 37 0 C with 1 ml of acetate buffer 0.2 M pH 4.0 is shown in figure 3.
• Poloxamer-based formulations being able to undergo a transition to a gel phase above 18 0 C, represent a suitable system for in situ gelation upon subcutaneous injection which is followed by a slow release in the circulation of the monopegylated GLP-I -peptides.
• GLP-I glucagon-like peptide- 1
• GLP-I analogues mean the 30 amino acid C -terminally amidated GLP- 1 (SEQ ID No 1), the 31 amino acid C-terminally glycine extended GLP-I (SEQ ID No 2) and their biologically active forms with deletion, addition or substitution of one or more amino acid residues as well as synthetic or natural insulinotropic peptides with at least 40% sequence identity with respect to human 31 amino acid C-terminally glycine extended GLP-I of SEQ ID No 2.
• the term biologically active forms means that the products act as incretin mimetics in that they are able to potentiate glucose-induced insulin secretion when administered in vivo.
• Q17N-GLP-1 analogues (where Q and N are respectively the one letter code notation for glutamine and asparagine) according the use of the present invention mean GLP-I -amide or C-terminally glycine extended -GLP-I containing an asparagine residue in position 17 and one single glutamine residue in other positions of the peptidic chain.
• nano- and micro-particles mean any inorganic and organic polymeric material with dimensions ranging from
• thermoresponsive polymer means any polymeric material whose transition from sol to gel is triggered by an increase in temperature to form an in situ depot drug delivery system as described, for example, by Singh S et al. (2007), Schmalijohann D (2006), herein incorporated by reference.
• a GLP-I peptide is dissolved in a 10 mM. pH 8.0 sodium dihydrogen citrate solution at the concentration of 0.5 mg peptide / ml, corresponding to a concentration of about 150 ⁇ M.20 kDa m-PEG-arnine (Methoxypolyethylene glycol amine 20000. Iris Biotech) is then added to the peptide solution to achieve a 20:1 PEG : GLP-I molar ratio. To the reaction mixture, microbial transglutaminase (Activa WM, Ajinomoto), to a final concentration of 0.25 U /ml, is then added. Reaction is earned out under agitation for 16 hours at room temperature.
• microbial transglutaminase Activa WM, Ajinomoto
• the solution is diluted in a 20 mM, pH 4.0, sodium acetate buffer and purified by ion-exchange column chromatography (Macrocap SP), eluting with a NaCI linear gradient (from 0 to 500 mM in 10 column volumes).
• the fractions pool containing monopegylated GLP-I is concentrated, desalted and liophylized.
• the yield of direct enzyme catalyzed monopegylation of GLP-I peptides is around 60%.
• GLP-I -amide is dissolved in a 50 mM, pH 7.5 TRIS solution at a concentration of 0.5 mg peptide / ml, corresponding to a concentration of about 150 ⁇ M.
• 20 IdDa m-PEG-amine Metalethylene glycol amine 20000, Fluka
• 0.3 U/ml of guinea pig transglutaminase (Sigma) and 10 mM CaCIi are then added and the mixture maintained under agitation for 16 hours at room temperature.
• RP-HPLC perforated on the starting and final reaction mixture only showed the presence of unreacted GLP-I -amide with no traces of pegylated derivative.
• S 12Q/Q1 W-GLP-I -amide is dissolved in a 10 mM, pH 8.0 sodium dihydrogen citrate solution at the concentration of 0.5 mg peptide / ml, corresponding to a concentration of about 150 ⁇ M.
• Q17N/A24Q-GLP-l-amide is dissolved in a 10 mM, pH 8.0 sodium dihydrogen citrate solution at the concentration of 0.5 mg peptide / ml, corresponding to a concentration of about 150 ⁇ M.
• 20 kDa m-PEG-amine Metaloxypolyethylene glycol amine 20000, Iris Biotech
• microbial transglutaminase Activa WM, Ajinomoto
• Reaction is carried out under agitation for 16 hours at room temperature.
• the solution is diluted in 20 mM, pH 4.0 sodium acetate buffer and purified by column chromatography (Macrocap SP), eluting with a NaCl linear gradient (from 0 to 500 mM in 10 column volumes).
• the fractions pooS containing monopegylated GLP-I mutant is concentrated, desalted and liophylized.
• the extent of pegylation reaction as well as the purity of the final monopegylated GLP-I peptide mutant are checked by RP-HPLC.
• the yield of direc enzyme catalyzed monopegylation of GLP-I mutants is around 60%.
• This assay is based on the release of insulin, in the presence of glucose and different concentrations of GLP-I peptides and their monopegylated derivatives, from RIN-m5f cells derived from insulinoma of rat beta cells. Briefly, 2 x 10 D cells are plated in 6 well plates in complete RPMI medium and incubated at 37°C for 2 days; cells are washed, incubated 4 more hours with medium with lower glucose concentration (0.2%), then washed again and incubated for 1 hour with a 10 mM solution of GLP-I peptides or their monopegylated derivatives. Supernatant is harvested and title of released insulin is determined by ELISA assay.
• a 27 % solution of Poloxamer 407 was prepared by slowly dissolution of the polymer in 0.2 M acetate buffer pH 4.0 at 4 0 C.
• GLP-l-amide-Q17 PEG 20 IcDa was added to obtain a final concentration of 0.3 mg/ml of GLP-I -amide-Q 17 PEG 20 IcDa and 22% Poloxamer and the solution was stored at 4 ⁇ C.
• a dissolution test was performed by incubating 1 ml of formed gel at 37 0 C with 1 ml of acetate buffer 0.2 M pH 4.0. Solution was collected and replaced by an equivalent volume of buffer at different time point, in order to evaluate the release of GLP-I -amide-Q17-PEG 2OkDa by RP-HPLC analysis. Results of dissolution test are shown in figure 3 REFERENCES
• Escherichia coli in vitro refolding and characterization of the refolded form.
• PEGylated glucagon-like peptide- 1 isomers as type 2 anti-diabetic treatment. lnsul inotropic activity, glucose-stabilizing capability and proteolytic stability.

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