MXPA01002339A - Protein formulations - Google Patents

Abstract

The present invention discloses a stable, soluble formulation comprising a medically useful peptide or protein, a hydrophobic preservative, and nicotinamide. Said storage-stable, soluble formulation is useful as a multi-use pharmaceutical product.

Description

PROTEIN FORMULATIONS, Field of Invention The present invention is in the field of peptide and protein chemistry as it is applied to human medicine. In particular, the invention relates to the preparation of stable soluble peptide and protein formulations including nicotinamide and hydrophobic preservatives.

Background of the Invention Nicotinamide is not a widely recognized excipient in pharmaceutical formulations. For example, it is not mentioned as an excipient in the Ha n dbo ok of Ph a rma ce u t i ca l Ex c ipi en t s, 2nd edition, A. Wade & P. Séller, editors (1994). However, nicotinamide is known to increase the solubility of low molecular weight, non-protein, poorly soluble compounds, such as certain piperazide and piperazine compounds [Fawzi, et al.

Ref: 127341 J. Pharmaceut. Sci. 69: 104-106 (1980)], anti-cancer nucleoside analogues [Truelove, et al., Int. J. Pharmaceutics 19: 17-25 (1984)], paracetamol [Hamza, et al., Drug Dev. Industr. Pharmacy 11: 1577-1596 (1985)], diazepam, gr i seofulvin, progesterone, 17β-estradiol, and testosterone [Rasool, et al., J. Pharmaceut. Sci. 80: 387-393 (1991)], the phenothiazine derivative, moricizin [Hussain, et al., J. Pharmaceut. Sci. 82: 77-79 (1993)], and riboflavin [Coffman, et al., J. Pharmaceut. Sci. 85: 951-954 (1996)].

In the aforementioned formulations, nicotinamide apparently operates as a hydrotropic agent to increase the solubility of another solute when nicotinamide is added at a high concentration, this hydrotropic phenomenon is directly against the functioning of the normal solution where the addition of a second solute to a solution of a slightly soluble substance causes the less soluble substance to precipitate.

A combination of insulin and nicotinamide, optionally containing a preservative, is previously described by Jorgensen in U.S. Pat. No. 5,382,574. the formulation is reported to promote a faster absorption of insulin from an injection site. Jorgensen does not discuss any effect of nicotinamide on the stability of the formulation. On the other hand, it is possible that the effect of nicotinamide is not observed or appreciated due to the specifically recommended specification which recognizes that stabilizing agents such as phospholipids are added to stabilize the formulations. Also, it fails to mention any effect on the stability of the insulin produced by nicotylamide alone.

Molecular interactions in peptide and protein formulations are complex due to a variety of factors such as a choice of preservatives, buffer solutions, ionic strength, pH, temperature, and other excipients that are balanced to produce a relatively stable formulation suitable for manufacturing , transportation, and storage that meets the regulatory requirements for such products. The role that each contributing factor to aggregation is uncertain in view of the complexity of the given peptide or protein molecule as well as the propensity for the peptide or protein to aggregate and precipitate in formulations containing preservatives. In view of this complexity and tendency to aggregate, the effect of nicotinamide on the stability of peptide and protein formulations containing a hydrophobic preservative has not been predicted from the technique that describes the effect of the nona cotamides as a hydrophobic agent for relatively small molecules, or their apparent ability to facilitate insulin absorption from a subcutaneous injection.

Thus, the present invention provides conditions that increase the physical stability of medically useful peptides and proteins in the presence of hydrophobic preservatives and enables commercially viable multiple use soluble pharmaceuticals to treat a variety of human diseases.

Brief description of the invention.

This invention provides a stable soluble formulation, comprising a medically useful peptide or protein, a hydrophobic preservative, and nicotinamide.

The invention further provides a process for preparing said formulation, which comprises combining a peptide or protein, a hydrophobic preservative, and nicotinamide to produce said formulation.

Detailed Description of the Invention and Preferred Forms of Realization.

For the purposes of the present invention, as described and claimed herein, the following terms and abbreviations are defined as s igue: Administer: an act whose effect is to transfer a formulation of the present invention into the body of a mammal in need thereof. The administration can be by any route known to be effective by the doctor of ordinary skill. Parenteral administration is commonly understood in the medical literature as the injection of a dosage form into the body by a sterile syringe or some other mechanical device such as an infusion pump. Peripheral parenteral routes of administration include, without limitation, routes of intravenous, intramuscular, subcutaneous, and intimal administration.

Alkylparaben: refers to an alkylparaben Cl up to C4, or mixtures thereof. Preferably, the alkylparaben is methylparaben, et Iparaben, propylparaben, or but Iparaben.

Cresol: refers to meta-cresol, ortho-cresol, para-cresol, chloro-sun, or mixtures of the same.

Hydrophobic preservative: refers to a hydrophobic compound that is added to a pharmaceutical formulation to act as an anti-microbial agent. A parenteral formulation will meet the guidelines for an effective preservative to be a commercially viable multiple use product. Among the preservatives known in the art to be acceptable in the formulations are alkylarabienes, phenolic preservatives, benzyl alcohol, chlorobutylene, benzoic acid, and various mixtures thereof. See, for example, Wallhauser, K.-H., Develop. , Biol .. standard. 24, pages 9-28 (Basel, S. KragerA 1974).

Isotonicity agent: refers to a compound that is physiologically tolerated and imparts an appropriate tonicity to the formulation to prevent the total flow of water through the membranes of the cells. Compounds, such as glycerin, are commonly used for such purposes at known concentrations. Other possible isotonicity agents include salts, for example, NaCl, and sugars, for example, dextrose, mannitol, and sucrose.

Nicotinamide: a compound of the formula Pharmaceutically acceptable buffer: the pH of the formulation can be quenched with a pharmaceutically acceptable buffer, such as, without limitation, sodium acetate, sodium phosphate, sodium citrate, sodium tartrate, TRIS or a basic amino acid, such as , histidine, lysine or arginine. Other pharmaceutically acceptable buffer solutions are known in the I technique. , The selection and concentration of 1 buffer solution is known in the art.

Phenolic preservative: refers to phenol and cresol Soluble: refers to the relative absence of I added protein that is visually perceptible. The degree of aggregation of proteins in a formulation can be inferred by measuring the turbidity of the formulation. The greater the turbidity of the formulation, the greater the extent of the aggregation of the protection in the formulation. Turbidity is commonly determined by nephelometry, and is measured in phanerous Neural Turbidity Units (UTN).

Stable: a "stable" formulation is one in which the protein or peptide remains soluble for an extended period of time under storage conditions.

Treatment: as used herein: describes the management and care of a patient for the purpose of fighting a disease, condition, or disorder and includes the administration of a formulation of the present invention to prevent the onset of symptoms or complications, alleviate symptoms or complications, or eliminate the disease, condition, or disorder. The treatment, as used herein, includes the administration of the protein for cosmetic purposes. A cosmetic purpose will seek to take control of the weight of a mammal to improve its body appearance.

In one embodiment, the invention relates to formulations containing a hydrophobic preservative, nicotinamide and a medically useful peptide or protein, which are defined to include native hormones and functional analogues (excluding insulin and insulin analogs and leptin and leptin analogs), native cytokines and functional analogues, soluble protein vaccines, and antibodies and antibody fragments of all forms.

The following list of medically useful peptides and proteins is provided for illustrative purposes and is in no way to limit the scope of the medically useful peptides and proteins that are consistent with the invention: oxytocin, vasopressin, adrenocorticotropin hormone and the like, factor of epidermal growth, growth factor derived from platelets, prolactin, luteini s inga hormone releasing hormone, growth hormone, growth hormone releasing hormone, somat os t at ina, glucagon, GLP-1 related compounds, IL- 2, IL-10, IL-15, m er f eron-a, ß,?, Tet raga st rata gastrina, pent aga st rebña, urogastrina, secretin, calcitonin, encafalinas, endorphins, angiot ens inas, hormone liberador de la thyrotropin, tumor necrosis factor, nerve growth factor, granulocyte colony stimulating factor, macrophage granulocyte colony stimulating factor, est factor imulant of the macrophage colony, renin, bradykinin, bacitracins, polymyxins, colistins, tirocidins, gramicidins.

A more preferred group of medically useful peptides and proteins, defined as "Group I polypeptide" for the purposes of this specification, consists of acylated insulins, particularly C6-C20 acylations of the epsilon amino group of Lys in B-chain insulin, especially insulin human LysB28ProB29 acylated C14, mammalian growth hormone, growth hormone releasing hormone, compounds related to GLP-1, erythrocyte progenitor hormone (EPO), hormone and parathyroid fragments, especially as described in US Pat. Nos. 4,086,196 and 5,208,041, ß-lipotropin, fibroblast growth factor 8 and analogous, os t eopr oter ina -2 and 3, int leucin-10 and 15 and their analogs, vascular endothelial growth factor and follicle stimulating hormone (FSH) and variants.

The follicle-stimulating hormone "FSH", which is produced recombinantly or isolated, and the variants of the follicle-stimulating hormone "FSH variants" as defined herein are well known in the art. FSH as used herein refers to FSH produced as a full-length mature protein that includes, but is not limited to, human FSH or "hFSH," which are produced recombinantly or isolated from human sources (see Shome). B., et al., J. Prot. Chem., 7: 325-339, 1998; Saxena BB and Rathnam P., J. Biol. Chem., 251: 993-1005, 1976; Watkins et al., DNA, 6 : 205-212, 1987; Shome B. and Parlo AF, J. Clin Endocrinol, Me tab., 39 (1): 203-205, 1974; and Bec, et al., DNA, 4:76, 1985; 5,405,945, and US 5,639,640), each citation incorporated for reference. Additionally, several variant FSHs are known or are understood from the art (see Shome, J. Clin Endocrin, Metab 39: 187 (1974), Saxena, J. Biol .. Chem 251 (4): 993-1005 (1976)).; 1978; Sairam et al., Biochem J. 197: 541 Sairam 1981 publication identifies conserved amino acid sequences reported by the publications of Saxena et al., Shome et al., Closset et al., And Fujiki et al., Sairam, Biochem J 197: 541, 551 (1981) The prior art (1) evidences a preference for the chain sequence beta FSH of Saxena over that of Shome; (2) directs the publication of the carboxy terminal heterogeneity; (3) states that portions of the molecule are affected by differences between species that are not essential for the activity of the hormone and (4) leads the guide drawn from the homologies between the species and between the beta chains of the three human glycoprotein hormones, FSH, LH and TSH.

C terminal homogeneity is reported for all published sequences except for that of porcine FSH-s, in which glutamic acid is only the C terminal residue. For position 27, Saxena allocates a tryptophan residue for this position which also finds support in the evaluated conservation for a tryptophan at position 24 for FSH-B, among all the prior art species. For positions 44 and 46, Saxena shows that, at position 44, the residue can be arginine instead of lysine and, at position 46, lysine instead of arginine. The porcine, equine and ovine sequences also reflect an evolutionary pressure to conserve arginine at position 44. Variations at three positions, 21, 22 and 44 involve structurally conservative or evolutionarily conserved substitutions ("homologs"), each of which has biological activity.

Each of the references of Sairam, Shome, and Closset, describe isoleucine residues, serine in positions 21-22, while Saxena describes leucine, threonine and Fujiki describes isoleucine, threonine in these positions.

Each of these descriptions is not only an evolutionary conservative substratum, but also a conservative structural substitution.

The variation at position 41 between the aspartic acid described by each of Sairam, Shome, Closser, and Fujiki and asparagine described by Saxena, Closset and Sairam involve two evolutionarily conservative residues, each of which provide biological activity. These descriptions of conservative substitutions and evolutionarily conservative substitutions guide the skilled technician to distinguish the beta chain FSH variants, within the hFSH-B chain genre.

A more preferred group of medically useful peptides and proteins consists of compounds related to GLP-1 and growth hormone in native mammals.

A more highly preferred group of peptides consistent with the present invention is glucagon-like peptide 1, its analogs and derivatives as defined herein.

The water soluble copolymers and polymer conjugates of the aforementioned peptides and proteins are also consistent with the present invention, and include, for example, polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol , polyvinyl pyrrolidone, poly i-1,3-dioxane, poly-1,3,6-trioxane, maleic anhydride / copolymer, polyamino acids (either homopolymers or random or non-random copolymers), and dextran or poly (n) -vinyl pir rol idona) pol ie ti len glycol, propylene glycol polypropylene oxide homopolymers / ethylene oxide copolymers, polyoxyethylated polyols, polyvinyl polyvinyl toy alcohol. Polyethylene glycol propionaldehyde is preferred.

The term "GLP-1" refers to a peptide 1 similar to human glucagon whose sequences and structures are known in the art. See U.S. Patent No. 5,120,712, incorporated herein by reference. These are two native forms of human GLP-1, GLP-1 (7-37) OH and GLP-1 (7 -36) NH2, which are distinguished only when necessary.

The term "GLP-1 analog" is defined as a molecule having one or more substitutions, withdrawals, inversions, or amino acid additions, compared to GLP-1. Many GLP-1 analogs are known in the art and include, for example, GLP-I (7-34) and GLP-I (7-35), GLP-I (7-36), Val8-GLP-1 ( 7-37), Gln9-GLP-1 (7-37), D-Gln9-GLP-1 (7-37), Thr16-Lys18-GLP-1 (7-37), and Lys 18-GLP- 1 - (7 -37). The preferred GLP-1 analogs are GLP-I (7-34) and GLP-I (7-35), which are described in U.S. Pat. No. 5,118,666, incorporated herein by reference.

The term "GLP-1 derivative" is defined as a molecule having the amino acid sequence of GLP-1 or a GLP-1 analog, but additionally having a chemical modification of one or more of its amino acid side groups, atoms α-carbon, terminal amino group, or terminal carboxylic acid group. A chemical modification includes, but is not limited to, adding chemical portions, creating new bonds, and removing chemical portions. Modifications in the side groups of amino acids include, without limitation, the acylation of e-amino lysine groups, the N-alkylation of arginine, histidine, or lysine, the alkylation of glutamic or aspartic carboxylic acid groups, and the deamination of glutamine or asparagine. Modifications of the terminal amino include, without limitation, modification of des-amino, lower N-alkyl, N-di-lower alkyl and N-acyl modifications, modifications of the terminal carboxy group include, without limitation, modifications to the amide, alkyl amide lower, dialkyl amide, and lower alkyl ester. Lower alkyl is C? -C alkyl. Additionally, one or more side groups, or terminal groups, can be protected by protecting groups known to the ordinarily skilled protein chemist. The α-carbon of an amino acid can be mono- or dimethyl side.

The term "GLP-1 molecule" means GLP-1, GLP-1 analog, or GLP-1 derivative.

Another preferred group of GLP-1 analogs is defined by the formula: Rl-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp- Leu-Val-Lys-Gly-Arg-R2 SEQ ID NO: 1 and pharmaceutically acceptable salts thereof, wherein: Ri is selected from the group consisting of L-histidine, D-histidine, deamino-histidine, 2-amino-his t idine, b-hydroxy-hi stine, homohistidine , to fa -f luoromet il-hi st idina, and alpha-methyl-hist idina; X is selected from the group consisting of Ala, Gly, Val, Thr, lie, and alpha-met il-Ala; And it is selected from the group consisting of Glu, Glu, Ala, Thr, Ser, and Gly; Z is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly; and R2 is selected from the group consisting of NH2, and Gly-OH; with the condition of when Ri is His, Z is Ala, Y is Glu, and Z is Glu, R2 should be NH2 • Yet another preferred group of GLP-1 compounds consistent with the present invention is described in WO 91/11457 (US Patent No. 5, 545,618, incorporated herein by reference) and consists essentially of GLP-1 (7-34) , GLP-1 (7-35), GLP-1 (7-36), or GLP-1 (7-37), or the amide forms thereof, and pharmaceutically acceptable salts thereof, which have at least one Selected modification of the group consisting of: (a) substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, laucine, methionine, phenylalanine, arginine, or D-lysine for lysine at position 26 and / or position 34; or substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, laucine, methionine, pheonine 1, lysine, or a D-arginine for arginine at position 36; b) substitution of an oxidation-resistant amino acid for tryptophan at position 31; (c) substitution of at least one of: tyrosine for valine at position 16; lysine for serine in position 18; aspartic acid for glutamic acid in position 21; serine for glycine in position 22; arginine for glutamine in position 23; arginine for alanine in position 24; and glutamine for lysine at position 26; Y (d) substitution of at least one of: glycine, serine, or cysteine for alanine in position 8; aspartic acid, glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine for glutamic acid in the position 9; serine, cysteine threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine or phenylalanine for glycine in position 10; and glutamic acid for aspartic acid in position 15; Y (e) substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine, or the D- or N-acylated form of histidine alkylated for histidine in the position 7; wherein, in the substitutions is (a), (b), (d), and (e), the amino acid substituents can optionally be in the D form and the amino acids substituted in the 7-position can optionally be in the N-acylated or N-alkylated form.

Still other GLP-1 molecules consistent with the present invention are also described in WO 98/08871, and include a lymphophilic substituent linked to the N-terminal or C-terminal amino acid residue, wherein the substituent is an alkyl group or a group having an omega carboxylic group.

Because the enzyme, dipept id 1 -pept idase IV (DPP IV), may be responsible for observing a rapid in vivo deactivation of the administered GLP-1 (see, for example, Mentlein, R., et al., Eur. J. Biochem., 214: 829-835 (1993)], the administration of GLP-1 analogs and derivatives that are protected from the DPP activity of Iv is preferred, and the administration of Gly8-GLP-1 (7-36) NH2, Va 18-GLP-1 (7-37) OH, α-me ti 1 -Al a8-GLP-1 (7-36) H2, and Gly8-Gln21-GLP-1 (7-37) OH, or pharmaceutically acceptable salts thereof, is more preferred.

The use in the present invention of a molecule claimed in U.S. Patent No. 5,188,666, incorporated herein by reference, is preferred. Such a molecule is selected from the group consisting of a peptide having the amino acid sequence: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp- Leu-Val-X SEQ ID NO: 2 wherein X is selected from the group consisting of Lys and Lys-Gly; and a derivative of said peptide, wherein said peptide is selected from the group consisting of a pharmaceutically acceptable acid addition salt of said peptide; a pharmaceutically acceptable carboxylate salt of said peptide; a pharmaceutically acceptable lower alkyl ester of said peptide; and a pharmaceutically acceptable amide of said peptide selected from the group consisting of amide, lower alkyl amide, and lower dialkyl amide.

Another preferred group of GLP-1 molecules for use in the present invention, consists of compounds described in U.S. Pat. No. 5,512, 549, incorporated herein by reference, having the general formula 1: R ^ Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Xaa-Glu-Phe-Ile-Ala ' Trp-Leu-Val-Lys-Gly-Arg-R3 I R2 SEQ ID NO: 3 and pharmaceutically acceptable salts thereof, wherein R1 is selected from the group consisting of 4-zolpropionylimide, 4-zoacetide or 4-imido imidazo-a, dimethyl-1-acetyl; R2 is selected from the group consisting of unbranched acyl C? -C? O, or is absent; R3 is selected from the group consisting of Gly-OH or NH2; and, Xaa is Lys or Arg, can be used in the present invention.

The most preferred compounds of formula 1 for use in the present invention are those in which Xaa is Arg and R2 is unbranched C6-C10 acyl.

The highly preferred compounds of formula 1 for use in the present invention are those in which Xaa is Arg, R 2 is unbranched acyl C 6 -C 0, and R 3 is Gly-OH.

The most highly preferred compounds of formula 1 for use in the present invention are those in which Xaa is Arg, R 2 is unbranched acyl C 6 -C 0, R 3 is Gly-OH, and R 1 is 4-imidazopropionyl.

The most preferred compounds of formula 1 for use in the present invention are those in which Xaa is Arg, R 2 is unbranched acyl C 6 -C 10, R 3 is Gly-OH, and R 1 is 4-imido zopropioni lo.

The use of Val-GLP-1 (7-37) OH or a pharmaceutically acceptable salt thereof, as claimed in U.S. Pat. 5,705,483, incorporated herein by reference, in the present invention is highly preferred.

Methods for preparing GLP-1, GLP-1 analogs, or GLP-1 derivatives useful in the present invention, are well known in the art and are easily understood by the chemist or biochemist in proteins of ordinary skill. The amino acid portion of the active compound used in the present invention, or a precursor thereof, can be made either by synthetic solid phase chemistry, purification of GLP-1 molecules from natural sources, or recombinant DNA technology. Synthetic organic routine techniques allow the alkylation and acylation of GLP-1 derivatives.

The term "compound related to GLP-1" refers to any compound that falls within the definition of GLP-1, GLP-1 analog, or GLP-1 derivatives.

The unexpected effect of nicotinamide on the stability of the formulation is demonstrated by preparing formulations of the present invention, and comparing its turbidity with the turbidity of the controls lacking nicotine nicotine.

The parenteral formulations of the present invention can be prepared using conventional mixing and dissolving methods. One of ordinary skill in the formulation sciences will recognize that the order of addition of a medically useful, hydrophobic conservative peptide or protein, and nicotinamide, will vary without compromising the stability of the resulting formulations.

In one embodiment of the invention, nicotinamide can be added to a solution of purified peptide or protein and then lyophilized without adversely affecting chemical or physical stability in the solid state. During reconstitution with a diluent containing a hydrophobic preservative and not containing nicotinamide, the protein formulation exhibits superior physical stability attributed to the presence of nicotinamide in the formulation. Conversely, a stable formulation of the present invention can be prepared by dissolving a measured mass of pure lyophilized protein in water, and then adding measured volumes of nicotinamide and hydrophobic preservative solutions in amounts sufficient to provide the desired concentrations. Optional compounds may also be added, such as an isotonicity agent of a pharmaceutically acceptable buffer. The pH of the formulation can be adjusted using, for example, hydrochloric acid or sodium hydroxide solutions. once prepared the formulations of the present invention are generally sterilized by filtration before administration. The formulations of the present invention can be prepared by many other processes that are readily apparent to one of ordinary skill in the art. For example, the manner and conditions under which the components are combined, the type of acid or base used to adjust the pH, and the method for sterilizing the formulations can be optimized by someone of ordinary skill.

The hydrophobic preservative and nicotinamide used in the formulations of the present invention are readily available from commercial suppliers in a quality sufficient to meet the regulatory requirements for administration to humans.

The formulations of the present invention may optionally contain other compounds in addition to the medically useful peptide or protein, hydrophobic preservative and nicotinamide. For example, pharmaceutically acceptable solubilizers such as Tween 20 (polyoxiet i len (20) sorbitan monolaurate) Tween 40 (monopalmitate of polyoxiet i len (20) sorbitan), Tween 80 monooleate of polyoxyethylene (20) sorbitan), Pluronic F68 (polyoxypropylene polyoxyethylene copolymer blocks), and PEG (polyethylene glycol), can optionally be added to the formulation to reduce aggregation. These additives are particularly useful in a pump or plastic container used to administer the formulation. The pharmaceutically acceptable surface active agent can also reduce protein aggregation. An agent of isotonicity, preferably glycerin, optionally can be added to the parenteral, soluble formulation. The concentration of the isotonicity agent is in the range known in the art for parenteral formulations, preferably around 1 to 25 mg / ml, more preferably about 8 to 16 mg / ml or about 16 mg / ml to about 25 mg / ml , and still more preferably around 16 mg / ml. A pharmaceutically acceptable buffer solution may optionally be added to control the pH.

As noted above, the invention provides soluble formulations comprising a medically useful peptide or protein, a hydrophobic preservative, and nicotinamide. Preferably, the concentration of nicotinamide is between 0.01 and 2 moles. Other preferred ranges of nicotinamide concentration are: between 0.05 moles and 1.5 moles; between 0.1 moles and 1.0 moles; between 0.1 moles and 0.5 moles; between 0.5 moles and 1.0 moles; and between 0.15 moles and 0.25 moles. By adding nicotinamide to the formulation, the peptides and proteins remain in solution in the presence of certain preservatives, assuming a multi-use parenteral formulation that is relatively free of protein aggregation.

Phenolic preservatives, used simply or in combination, are preferred preservatives for use in the formulations of the present invention. Another group of preferred preservatives are the alkyl paraben preservatives. The benzyl alcohol and benzoic acid are other preferred preservatives. The most highly preferred preservatives are phenol and m-cresol, used simply or in combination. When the molar ratio of phenol to m-cresol is used in combination in the formulations, it is preferably between 3 to 1, and 1 to 3, while the total concentration of preservative is preferably between about 1 mg / mL and 10 mg. / mL. The concentration of preservative required to maintain the effectiveness of preservation, which, again, may depend on the preservative, its solubility, the temperature and the pH of the formulation, among other variables. Generally, the amount of necessary conservator can be found in, see, for example, WALLHAUSER, K.-H., DEVELOP. BIOL. STANDARD. 24, pp. 9-28 (Basel, S. Krager, 1974).

In general, the concentration of the medically useful peptide or protein may be in the range of from about 0.01 to about 100 mg / mL depending on the pharmacology of the given protein or peptide. For example, the concentration of human growth hormone is from about 0.25 mg / mL to about 40 mg / mL. Preferably, the concentration is from about 0.25 mg / mL to about 25 mg / mL. More preferably, the concentration is from about 0.5 mg / mL to about 10 mg / mL. Other preferred ranges of concentration are from about 0.5 mg / mL to about 15 mg / mL.

The concentrations of molecules related to GLP-1 are in the range from about 0.01 mg / mL to 10 mg / mL, preferably from about 0.1 mg / mL to about 5 mg / mL, more preferably from about 0.25 mg. / mL up to about 1 mg / mL. More preferably, the concentration is from about 0.5 mg / mL to about 1.0 mg / mL.

The solubility of the medically useful peptide or protein in the present formulations is such that the turbidity of the formulation is less than 50 UTN. More preferably, the turbidity is less than 20 UTN. Still more preferably, the turbidity is less than 10 UTN.

Parental, peripheral administration is preferred. The formulations prepared according to the present invention can be administered using a syringe, injector, pump, or any other device recognized in the art for parenteral administration. The amount of a formulation of the present invention that is to be administered to treat a given disease will depend on a number of factors, including, without limitation, the sex of the patient, weight and age, the root causes of the disease, the route of administration and biological availability, and the potency of the given peptide or protein. When the administration is intermittent, the amount per administration also takes into account the interval between doses, and the biological availability of the protein of the formulation. The administration of the formulation of the present invention will be continuous. This is within the ability of the ordinary physician to title the dose and the range or frequency of administration of the formulation of the present invention to execute the desired clinical result.

The present invention can be better understood with reference to the following examples. These examples are intended to be representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.

Example 1 The following concentrated solutions were prepared and used to make the test formulations.

V-GLP-1 (7-37) OH, as described in U.S. Pat. No. 5,705,483, is biologically synthesized in recombinant E. coli, purified to homogeneity, and then lyophilized. A sample of the lyophilized peptide is dissolved in water which has been previously adjusted to a pH of 9 with 5 N NaOH for a final concentration of 2 mg / mL. This concentrated solution is then adjusted to a pH of 7.8 using 10% HCl. 1 M Nicotinamide in water. 100 mM sodium phosphate in water, pH 7 phenol at 2% (w / v) in water. m-cresol at 1.2% (w / v) in water. Pure benzyl alcohol. Sterile water to irrigate.

All the formulations shown below are prepared at a final concentration of 5mM phosphate buffer and 0.5mg / mL of V8-GLP-1 (7-37) OH. The concentrated solutions of phosphate buffer, nicotinamide, and V8-GLP-1 (7 -37) OH are filtered through a 0.22 μm filter before use. The concentrated solutions and reagents are mixed in appropriate ratios to obtain the formulations shown below. The order of addition was conservative, nicotinamide, buffer, sterile water, V8-GLP-1. The final pH of the formulations is checked and adjusted to 7.8 if necessary. The clarity of the samples is measured using a HACH 2100AN turbidimeter.

UTN UTN preservative after 3 days initial nicot inamide @ 5 ° C 1 0 mM 0.3% m-cresol 48,495 98.14 2 100 mM 0.3% m-cresol 3.6365 48.89 3 200 mM 0.3% m-cresol 0.099 32.65 4 300 mM 0.3% m-cresol 0.015 3.83 400 mM 0.3% m-cresol 0.0995 0.05 6 O mM 0.5% phenol 0.1765 44.19 100 mM 0.5% phenol 0.5965 32.48 200 mM 0.5% phenol 0.2115 20.19 9 300 mM 0.5% phenol 0.044 0.47 400 mM 0.5% phenol 0.215 0.47 1.5% alcohol 11 0 mM -0.1415 35.12 of benzyl 1.5% alcohol 12 100 mM -0.055 22.89 of benzyl 1.5% alcohol 13 200 mM -0.0165 -0.5 of benzyl 1.5% alcohol 14 300 mM 0.1465 0.42 of benzyl 1.5% alcohol 15 400 mM 0.07; 028 of benzyl E jmplo 2 A concentrated solution containing 5 mg / mL of GLP-1 (7.37) OH in 70 mM of phosphate buffer at a pH of 7.4 was prepared and sterile filtered through a 0.22 μm filter. A concentrated solution of 1% m-cresol in water was prepared and sterile filtered at through a 0.22 μm filter. A concentrated 2 M nicotinamide solution was prepared in water and filtered sterile through a 0.22 μm filter. The concentrated solutions were mixed as follows: Concentra¬ Solution Solution Final solution concentr concentrated concentrate Nicotine DI water GLP gives m-amide cresol nicotinamide 0.800 mi 1,200 mi 0 mi 2,000 mi 0 mM 0. 800 mi 1,200 mi 0.500 mi 1,500 mi 250 mM 0. 800 mi 1,200 mi 1,000 mi 1,000 mi 500 mM 0. 800 mi 1,200 mi 1,500 mi 0.500 mi 750 mM 0. 800 mi 1,200 mi 2,000 mi 0 mi 1000 mM All final formulations were prepared at a volume of 4.0 ml and contained 1 mg / ml of GLP-I (7.37) OH, 14 mM of phosphate buffer at a pH of 7.4 with 0.3% m-cresol as conservative. Please note: nicotinamide and protein were always mixed before the addition of the preservative. The LPG formulations were aged at 25 ° C. All the nicotinamide contained in the formulations shows good physical stability during 4 days and a measurement of less than 10 UTN. All non-nicotinamide formulations measured more than 65 UTN.

E j us 3 A concentrated solution containing 20 mg / ml hGH in 20 mM citrate buffer at a pH of 7.5 was prepared and sterile filtered through a 0.22 μm filter. A concentrated 1.5% m-cresol solution was prepared in water and sterile filtered through a 0.22 μm filter. The concentrated solutions were mixed as follows: Solution Solution Nicotinamide Concentration concentrates final aggregate concentration of hGH da of m-nicot inamide cresol 5.0 mi 2.0 mi 0 mg 0 mM . 0 mi 2.0 mi 611 mg 500 mM . 0 mi 2.0 mi 916 mg 750 mM . 0 mi 2.0 mi 1221 mg 1000 mM All final formulations were prepared at a volume of 10 ml and contained 10 mg / ml hGH, 10 mM citrate buffer at a pH of 6.0 with 0.3% m-cresol as a preservative . Please note: nicotinamide and protein were always mixed before the addition of the preservative. The results are shown in Figure 1. The UTN 200 value for the formulation without nicotinamide represents the limit of detection by the instrument used. The current value is probably much higher.

The principles, preferred embodiments, and modes of operation of the present invention are described in the aforementioned specification. The invention that is tried to protect in the present, nevertheless, is not constructed as limiting for the particular forms described, since these are appreciated as illustrative instead of restrictive. Variations and changes can be made by those skilled in the art without departing from the spirit of the invention. It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (13)

Claims

1. A soluble formulation, characterized in that it comprises a medically useful peptide or protein, a hydrophobic preservative, and nicotinamide.

2. The soluble formulation according to claim 1, characterized in that medically useful peptide or protein is selected from a group 1 polypeptide.

3. The soluble formulation as claimed in any of claims 1 to 2, characterized in that medically useful peptide or protein, is selected from the group consisting of GLP-1 (7-37) OH, GLP-1 (7-36) NH 2, Gly8GLP-l (7-37) OH, Val8GLP-l (7-37) OH, and Asp8GLP (7-37) OH.

4. The soluble formulation as claimed in any of claims 1 to 3, characterized in that the concentration of the medically useful peptide or protein is between 0.01 mg / ml and 100 mg / ml.

5. The soluble formulation as claimed in any of claims 1 to 4, characterized in that the preservative is selected from the group consisting of phenol, cresol, aliparabiene, benzyl, alcohol, benzoic acid, chlorobutanol and mixtures thereof.

6. The soluble formulation as claimed in any of claims 1 to 5, characterized in that the preservative is phenol, m-cresol, methylparaben, propylparaben, chlorocresol, benzyl alcohol, or mixtures thereof.

7. The soluble formulation as claimed in any of claims 1 to 6, characterized in that the concentration of nicotinamide is greater than 0.01 mol and less than 2.0 mol.

8. The soluble formulation as claimed in any of claims 1 to 7, characterized in that the concentration of mcotinamide is greater than 0.1 mol and less than 1.0 mol.

9. The soluble formulation as claimed in any of claims 1 to 8, characterized in that it also comprises an ionicity agent.

10. The soluble formulation as claimed in any of claims 1 to 9, characterized in that it further comprises a pharmaceutically acceptable buffer solution.

11. A process for preparing a soluble formulation as claimed in any of claims 1 to 10, characterized in that it comprises combining a medically useful peptide or protein, a hydrophobic preservative, and nicotinamide to form the soluble formulation as claimed in any one of claims 1 up to 10.

12. A method for treating diabetes in a mammal in need thereof, characterized in that it comprises administering to said mammal the soluble formulation of claim 3.

13. A manufacturing article, characterized in that it comprises a packaging material and a bottle comprising a solution of the formulation according to claim 1, wherein the packaging material comprises a mark indicating that such a solution can be used to treat a disease. "•:» ...- .., 9: