WO2000061177A9 - Composition based on oppositely-charged polypeptides - Google Patents

Abstract

A composition is disclosed that comprises a mixture of polypeptides of opposite charge and an excipient selected from the group consisting of arginine, lysine, glutamic acid, sodium dodecyl sulfate, beta-hydroxy cyclodextrin, and beta-cyclodextrin sulfobutyl ether.

Description

COMPOSITION BASED ON OPPOSITELY-CHARGED POLYPEPTIDES

Background of the Invention Field of the Invention

This invention relates to formulations contaming mixtures of oppositely-charged polypeptides such as insulin-like growth factor (IGF-I) and msulm In particular, this mvention entails a formulation contammg selected excipients that enable the mixing of oppositely-charged proteins m the same formulation the excipients preventing the interaction of the proteins that normally would make them precipitate from the solution Description of Related Art

Human IGF-I is a 7649-dalton polypeptide (Rinderknecht and Humbel, Proc Natl Acad Sci USA. 73 2365 (1976), Rinderknecht and Humbel, J Biol Chem . 253 2769 (1978) belongmg to a family of somatomedins with insulin-like and mitogenic biological activities that modulate the action of growth hormone (GH) Van Wyk p.tal . Recent Prog Horm Res .30 259 (1974), Binoux, Ann Endocnnol .41 157 (1980), Clemmons and Van Wyk, Handbook EXD Pharmacol , 57 161 (1981), Baxter, Adv Clin Chem .25 49 (1986), U S Pat No 4,988,675, WO 91/03253, and WO 93/23071 IGF-I contains three disulfide bonds, and has a pi of 8 65 and molar absorptivity of 0 645 at 276 nm IGF-I naturally occurs in human body fluids, for example, blood and human cerebral spinal fluid

Most tissues and especially the liver produce IGF-I together with specific IGF-binding proteins Like GH IGF-I is a potent anabolic protein See Tanner et al , Acta Endocnnol . 84 681-696 ( 19771. Uthne e? al J dm Endocnnol Metab .39 548-554 (1974) See also Ross et al . Intensive Care Med . 19 Suppl 2 S54-57 (1993), which is a review of the role of insulin, growth hormone, and IGF-I as anabolic agents in the cntically ill IGF-I may be punfied from natural sources, e g , human serum (Rinderknecht and Humbel. J Biol Chem , supra), or made recombmantly (e g , EP 123,228 and 128,733) Vanous methods for formulating IGF-I have been descnbed These mclude, for example, EP 440,989, which discloses a method for prepaπng a dned composition of IGF-I, which comprises drying a solution contammg IGF-I together with a strong acid, WO 91 / 18621 on formulating IGF-I m a citrate buffer at pH 6, US Pat No 5,374,620 on formulating IGF-I and GH in a growth-promoting composition, US Pat No 5,681 ,814 on formulating IGF-I m an acetate buffer, PCT/SE94/00010 on a stable solution contammg IGF-I m a phosphate buffer m an amount of 50 mmol or less, giving a pH of 5 5 to 6 5, which is isotonic and suitable for injection, and WO 95/34318 on a solution compnsing IGF-I in an aqueous solution with a reduced concentration of oxygen

IGF-I has hypoglycemic effects in humans similar to insulin when administered by intravenous bolus injection, but also promotes positive nitrogen balance Underwood et al . Hormone Research, 24 166 (1986)

IGF-I is known to exert glucose-lowering effects in both normal (Guler et al , N Engl J Med , 317 137-140 (1987)) and diabetic individuals (Schoenle et al , Diabetologia. 34 675-679 (1991), Zenobi et al , J Clm Invest . 90 2234-2241 (1992)) (see also Sherwin et al , Hormone Research 4J. (Suppl 2) 97-101 (1994), Takano et al , Endocnnol Japan, 37 309-317 (1990). Guler et al , Acta Paediatr Scand (Suppl ).367 52-54 (1990)) with a time course descnbed as resembling Regular msulm See also Kerr et al , "Effect of Insulin-like Growth Factor 1 on the responses to and recognition of hypoglvcemia " Amencan Diabetes Association (ADA) 52nd Annual Meeting. San Antonio, Texas, June 20-23, 1992 which reported an increased hypoglycemia awareness following rhIGF-1 administration In addition, single administration of rhIGF-I reduces overnight GH levels and insulin requirements in adolescents with IDDM Cheetham et al Clin Endocnnol .40 515-555 (1994), Cheetham et al . Diabetologia 36 678-681 (1993)

Recombinant human IGF-I administered to Type II diabetics as reported by Schalch et al , J Clin Metab , 77 1563-1568 (1993) demonstrated a fall m both serum insulin as well as a paralleled decrease in C peptide levels which indicated a reduction in pancreatic insulin secretion after five days of IGF-I treatment. This effect has been independently confirmed by Froesch et al, Horm. Res.. 42: 66-71 (1994). In vivo studies in normal rats also illustrate that IGF-I infusion inhibits pancreatic insulin release. Fursinn et al, Endocrinology. 135: 2144-2149 (1994). In addition, in pancreas perfusion preparations IGF-I also suppresses insulin secretion. Leahy et al, Endocrinology. 126: 1593-1598 (1990). Despite these clear in vivo inhibitory effects of IGF-I on insulin secretion in humans and animals, in vitro studies have not yielded such uniform results.

In vitro studies using multiple concentrations of both IGF-I and glucose have shown various degrees of inhibition of insulin secretion, e.g., from no effect (Sreradzeri et al, J. Endocrinol., 117: 59-62 (1988)) to a 30% decrease in insulin release utilizing physiological levels of IGF-I. Van Schravendijk et al. , Diabetologia, 33: 649- 653 (1990). In a recent study using human pancreatic islets, Eizirik et al, Eur. J. Endocr.. 133: 248-250 (1995) found no effect of IGF-I on medium insulin accumulation or on glucose-stimulated insulin release. The investigators speculate that the effect of IGF-I seen in vivo on insulin secretion may be secondary to the extra- pancreatic effects of IGF-I rather than its direct effects on the pancreas. Therefore, the mode and site of action of IGF-I on insulin secretion are not fully understood. A number of biochemical changes induced by short-term rhIGF-I administration are described in the literature. Prominent among these is a phosphate and potassium lowering effect of recombinant human IGF-I (rhIGF-I) reported in healthy subjects during euglycemic clamp. Boulware et al, "Phosphate and potassium lowering effects of insulin-like growth factor I in humans: comparison with insulin" The Endocrine Society, 74th Annual Meeting, San Antonio, Texas, 1992, June 24-27. See also Guler et al. Acta Paediatr. Scand. (Suppl.). 367. supra.

Recombinant human IGF-I (rhIGF-I) has the ability to improve insulin sensitivity. For example, rhIGF-I (70 μg kg bid) improved insulin sensitivity in non-diabetic, insulin-resistant patients with myotonic dystrophy. Vlachopapadopoulou et al. J. Clin. Endo. Metab.. \_: 3715-3723 (1995). Saad et al, Diabetologia. 37: Abstract 40 (1994) reported dose-dependent improvements in insulin sensitivity in adults with obesity and impaired glucose tolerance following 15 days of rhIGF-I treatment (25 μg and 100 μg/kg bid). RhIGF-I also improved insulin sensitivity and glycemic control in some patients with severe type A insulin resistance (Schoenle et al, Diabetologia. 34: 675-679 (1991); Morrow et al., Diabetes. 42 (Suppl.): 269 (1993) (abstract); Kuzuya et al., Diabetes.42: 696-705 (1993)) or others with non-insulin dependent diabetes mellitus. Schalch et al, "Short-term metabolic effects of recombinant human insulin-like growth factor I (rhIGF-I) in type II diabetes mellitus", in: Spencer EM. ed.. Modern Concepts of Insulin-like Growth Factors (New York: Elsevier: 1991)pp.705-715; Zenobi et al, J. Clin. Invest.. 90: 2234-2241 (1993).

Though insulin resistance has not been considered a prominent feature of type I diabetes, it is clearly present in some individuals and may be most clinically important during adolescence. As GH has well known anti- insulin effects, the elevated GH levels during adolescence may mediate much of this insulin resistance. Press et al, supra; Defeo et al, supra; Campbell et a/.. N. Eng. J. Med.. supra, Campbell et al, Metabolism, supra; Arias et al, supra; Davidson et al, supra.

A general scheme for the etiology of some clinical phenotypes which give rise to insulin resistance and the possible effects of administration of IGF-I on selected representative subjects is given in several references. See, e.g., Elahi et al, "Hemodynamic and metabolic responses to human insulin-like growth factor- 1 (IGF-I) in men," in: Modern Concepts of Insulin-Like Growth Factors. (Spencer, EM, ed.), Elsevier, New York, pp. 219-224

(1991); Quinn et αt. New Engl. J. Med., 323: 1425-1426 (1990); Schalch et al, "Short-term metabolic effects of recombinant human insulin-like growth factor 1 (rhIGF-I) in type 11 diabetes mellitus," in: Modern Concepts of Insulin-Like Growth Factors. (Spencer, EM, ed ), Elsevier, New York, pp 705-714 (1991), Schoenle et al , Diabetologia, 34 675-679 (1991), Usala ef a/ . N Eng J Med , 327 853-857 (1992), Lieberman et al , J Clin Endo Metab , 75 30-36 (1992), Zenobi e α/ . J Clin Invest .90 2234-2241 (1992), Zenobi ef al . J Clin Invest , 89 1908-1913 (1992). Kerre a/ . J Clin Invest .91 141-147 (1993) WO 94/16722 discloses a method of chronic modification of cell barrier properties by exposmg a cell to a modification-effective amount of IGF-I for at least about seven days and a method of chronic amelioration or reversal of insulin resistance However, when IGF-I was used to treat type II diabetes patients in the clinic at a dose of 120-160 μg/kg twice daily, the side effects outweighed the benefit of the treatment Jabn et al . Diabetes, 43 369-374 (1994) See also Wilton, Acta Paediatr , 383 137-141 (1992) regarding side effects observed upon treatment of patients with IGF-I US Pat No 4,988,675 describes treatment of type II diabetics with IGF-I, US Pat No 5,466,670 descnbes treatment of type I diabetics with IGF-I, WO 91/03253 reports use of IGF-I to treat severe msuhn-resistant diabetics, and WO 96/01124 descnbes use of IGF-I to prevent diabetes, delay clinical onset of diabetes, and provide a protective effect against diabetes

The treatments of choice m type II diabetes have become combmation therapies These combinations histoncally involved the use of multiple forms of insulin, short-acting msulm, intermediate-acting, and long-acting msulms Review articles on insulin formulations mclude Kissel and Volland, Deutsche Apotheker-Zeitung. 134 25 (1994) and Campbell, Pharmacy Times. 59 40 (1993) More recently, combinations of msulm with other anti- diabetic drugs, which are taken orally such as sulphonylureas and biguamdes, have become commonplace

As to combinations of IGF and insulin, Genn et al , Biochem Arch .5 53-59 (1989) discloses the anabolic effect of insulin and IGF-II Jacob et al , Am J Phvsiol .260 E262 -E268 (1991) discloses the metabolic effects of IGF-I and msulm m spontaneously diabetic BB/w rats, see also US Pat No 4,876,242 Furthermore, the stimulation of cardiac protem synthesis after treatment with insulin and IGF is disclosed by Fuller et al , Biochem Soc Trans . 19 277 S (1991) The experiments have been performed in vitro with freshly isolated cardiac myocytes The effects on protein metabolism after treatment with insulin and IGF on dogs which have been starved overnight are reported by Umplebyefa/ . Eur J Clin Invest , 24 337-344 (1994) Shojaee-Moradie e/α/ discloses a companson of the effects of IGF-I, insulin, and combined infusions thereof on glucose metabolism in dogs Randazzo and Jarett, Exp Cell Res . 190 (1) 31-39 (1990) discloses charactenzation of the growth of munne fibroblasts that express human insulin receptors and the effect of IGF-I and insulin on DNA synthesis thereof Tomas et al , Diabetes.45 170- 177 ( 1996) discloses the effects of joint IGF-I and insulin infusion on diabetic rats Dunger et al , Metabolism. 44 119-123 (1995) suggests that IGF-I in conjunction with insulin may provide an additional approach to management of IDDM during adolescence Mathe, Biomedicine and Pharmacotherapy.49 221-224 (1995) discloses the role of IGF's in their relation with msulm for treating diabetes mellitus

As to the patent literature, US Pat 4,988,675 discloses a combination of IGF-I with a lower amount of insulin than normal to treat Type II diabetes WO 96/01125 published 18 January 1996 discloses the use of a combmation of insulin and an IGF-I m the manufacture of a medicament for counteracting a decrease m nitrogen balance and for counteracting a decrease in protein synthesis and which can be used for treatment of a protein catabo sm due to glucocorticoid excess U S Pat No 5,091,173 discloses a composition suitable for topical application to mammalian skin or hair comprising a cell-free supernatant from a culture of dermal papilla cells sufficient to increase hair growth comprising one or more members of the IGF family selected from IGF-I, IGF-II, and insulin

There are various forms of human insulin on the market that differ in the duration of action and onset of action Jens Brange. Galenics of Insulin, The Physico-chemical and Pharmaceutical Aspects of Insulin and Insulin Preparations (Springer- Verlag, New York, 1987), page 17-40. Regular insulin is a clear neutral solution that contains hexameric insulin. It is short acting, its onset of action occurs in 0.5 hour after injection and duration of action is about 6-8 hours. NPH (Neutral Protamine Hagedorn) insulin, also called Isophane Insulin, is a crystal suspension of insulin-protamine complex. These crystals contain approximately 0.9 molecules of protamine and two zinc atoms per insulin hexamer. Dodd et al. Pharmaceutical Research. 12: 60-68 (1993). NPH-insulin is an intermediate- acting insulin; its onset of action occurs in 1.5 hours and its duration of action is 18-26 hours. 70/30 insulin is composed of 70% NPH-insulin and 30% Regular insulin. There are also Semilente insulin (amorphous precipitate of zinc insulin complex), UltraLente insulin (zinc insulin crystal suspension), and Lente insulin (a 3:7 mixture of amorphous and crystalline insulin particles), as well as Humalog insulin (rapid- acting monomeric insulin solution, as a result of reversing the Lys (B28) and Pro(B29) amino acids on the insulin B-chain) that was recently introduced into the market by Eli Lilly and Company.

NPH-, 70/30, and Regular insulin are the most widely used insulins, accounting for 36%, 28%, and 15%, respectively, of the insulin prescriptions in 1996. These three forms of insulin add up to 79% of all insulin prescriptions. It was therefore determined that the IGF-I formulation needs to be mixable with Regular, NPH-, and 70/30 insulin.

Patients with Type I or Type II diabetes typically take two to four subcutaneous injections of insulin daily to control their blood sugar. The use of an injectable drug other than insulin to treat diabetes, such as IGF-I, is naturally limited due to the desire of diabetics to administer a minimum number of injections. Adding two more subcutaneous injections daily, for IGF-I administration, to regimens that already require several injections per day of insulin is not practical. Further, when combining two proteins such as IGF-I and insulin, it would be necessary to have the resulting formulation stable and well absorbed by the patient, as well as having intermediate-acting insulin. An intermediate-acting insulin regulated in a time- and target-tissue-dependent manner in response to changing demands of the metabolic environment is described by Lewitt et al, Endocrinology. 129: 2254-2265 (1991). U.S. Pat. No. 5,788,959 discloses a drug delivery device comprising a single-phase matrix solution of oppositely-charged water-soluble polymers such as polypeptides wherein the matrix solution has dispersed therein a pharmaceutically-active ingredient different from the polymers. Further Burgess et al. , J. Pharm. Pharmacol..43: 232-236 (1991) discloses complex coacervation between oppositely-charged albumin and acacia mixtures, with coacervation being a common method of microencapsulation. Mauk and Mauk, Biochemistry. 21 : 4730-4734 (1982) discloses complex formation between purified human methemoglobin and the tyrptic fragment of bovine liver cytochrome b₅ and report a model for interaction between these molecules in which each hemoglobin subunit binds one cytochrome b₅ by means of complementary charge interactions between oppositely-charged groups on the two proteins. Furthermore, EP 615,444 discloses a peroral administration form for peptidic medicaments containing the medicament, such as insulin, distributed in a gelatin or gelatin derivative matrix of opposite charge. EP 313,343 discloses a method of purifying a crude protein from its impurities by ion-exchange chromatography at a pH such that the protein and impurities have an opposite charge so that selective binding occurs.

Presently, diabetics mix NPH-insulin (intermediate-acting neutral protamine hagedorn insulin) with Regular insulin. It would be desirable to mix oppositely-charged polypeptides such as insulin and IGF-I, each from separate vials in the same syringe or other delivery vessel, and to inject or otherwise deliver the mixture immediately. U.S. Patent No. 5,783,556 discloses a formulation of mixed NPH-insulin and IGF-I. U.S. Pat. No.

5,756,463 discloses a combination of IGF-I and insulin and its use in counteracting a decrease in nitrogen balance and a decrease in protein synthesis. U.S. Patent No. 4,608,364 discloses an active-compound combination of an insulin derivative and an unmodified insulin or a specific analog thereof for treating diabetes. It would be desirable to mix all types of insulin with IGF-I for this purpose, as well as to mix other polypeptides such as protamine and insulin, which are cunently sold as a precipitating complex.

Summary of the Invention Upon mixing, two polypeptides that are oppositely charged tend to associate with each other and form an aggregate or precipitate out of solution. Use of certain formulation excipients can prevent aggregation and precipitation of such polypeptides when mixed, including IGF-I with insulin and protamine with insulin. Many excipients such as salts, buffers, neutral amino acids, polyols, sugars, and detergents were not effective in preventing precipitation. Accordingly, the present invention provides, in one embodiment, a composition comprising a mixture of pharmaceutically-active polypeptides of opposite charge and an excipient selected from the group consisting of arginine, lysine, glutamate (glutamic acid), sodium dodecyl sulfate, and a combination of beta-hydroxy cyclodextrin and arginine, wherein the polypeptides are soluble in the composition.

In a preferred aspect of this embodiment, the composition may further comprise a buffer such as histidine, at a pH of about 7 to 7.5.

In a further embodiment, the invention provides a kit comprising:

(a) a container comprising the above composition comprising effective amounts of the polypeptides; and

(b) instructions for using the above composition to treat a disorder against which the composition is effective. If the polypeptides are IGF-I and an insulin, the disorder is preferably a hyperglycemic disorder.

In yet another aspect, the invention supplies a method for preparing the above composition comprising mixing together as component (a) a first polypeptide in an excipient selected from the group consisting of arginine, lysine, glutamic acid, sodium dodecyl sulfate, beta-hydroxy cyclodextrin, and beta-cyclodextrin sulfobutyl ether; and as component (b) a second polypeptide having an opposite charge from the first polypeptide. In a preferred aspect, the method may further comprise the step of incubating the mixture for a period of time at about 30-40°C, preferably for about 15 minutes at 37°C, wherein the composition further comprises phosphate-buffered saline. In still another embodiment, the invention provides a kit for preparing the above composition comprising:

(a) a container comprising a first polypeptide in an excipient selected from the group consisting of arginine, lysine, glutamic acid, sodium dodecyl sulfate, and a combination of beta-hydroxy cyclodextrin and arginine, and;

(b) a container comprising a second polypeptide having an opposite charge from the first polypeptide; and

(c) instructions for combining the contents of containers (a) and (b). Preferably, the resulting mixture is a pharmaceutically acceptable formulation.

In a still further aspect, the invention provides a method for treating a hyperglycemic disorder such as diabetes in a mammal comprising administering to the mammal, preferably by either injection or infusion, an effective amount of the above composition.

Mixing of two polypeptides of opposite charge such as IGF-I and an insulin requires the physical and chemical properties of both polypeptides to remain unchanged after mixing. Further, it is preferred that the pharmacokinetic and glucose pharmacodynamic profiles remain the same before and after mixing. If one of the polypeptides is IGF-I, it is preferred that the formulation have a minimum of a two-year shelf life. To enable mixing with another polypeptide, the formulation of the first polypeptide can be a clear liquid formulation, or a suspension formulation in which the polypeptide (such as IGF-I) is in the form of crystals, amorphous precipitate, or protein dry powder. An Example herein describes the development of a clear liquid IGF-I formulation that is mixable with insulin.

If the polypeptides are IGF-I and insulin, the targeted IGF-I dose will be 10, 20 or 40 μg/kg/injection (or 1, 2 or 4μl/kg/injection for a 10 mg/ml IGF-I formulation). From the insulin dosage used by Type I and Type II patients in phase II clinical trials, it can be calculated that maximum potential mixing ratio can reach 6: 1 (vokvol) for Regular insulin:IGF-I. The minimum potential mixing ratio can reach 1 : 15 (vol: vol) for NPH- insulin:IGF-I. Therefore, in the Example herein, mixing ratios ranging from 1 :6 to 6: 1 (vol: vol) were studied for mixing Regular insulin with IGF-I; mixing ratios ranging from 1:15 to 15:1 (vol: vol) were smdied for mixing NPH-insulin with IGF-I; and mixing ratios ranging from 1:15 to 6:1 (vokvol) were studied for mixing 70/30 insulin with IGF-I. Brief Description of the Drawings

Figure 1 shows the acidic pH reversed-phase HPLC chromatogram of an IGF-I sample.

Figure 2 shows the acidic pH reversed-phase HPLC chromatogram of Regular insulin.

Figure 3 shows the acidic pH reversed-phase HPLC chromatogram for an IGF-I and insulin mixture.

Figure 4 shows a graph of solution clarity of Regular insulin (triangles) or IGF-I in an acetate-buffered formulation used as a control herein (circles) as a function of solution pH, which was adjusted by adding 0. IN HCI or NaOH.

Description of the Preferred Embodiments A. Definitions

As used herein "polypeptide" or "polypeptide of interest" refers generally to peptides and proteins having more than about ten amino acids. Examples of mammalian polypeptides include molecules such as, e.g., renin, a growth hormone, including human growth hormone; bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; αl-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; thrombopoietin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial naturietic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor- alpha and -beta; enkephalinase; a serum albumin such as human serum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as beta-lactamase; DNase; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; integrin; protein A or D; rheumatoid factors; a neurotrophic factor such as brain-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-β; cardiotrophins (cardiac hypertrophy factor) such as cardiotrophin- 1 (CT- 1 ); platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-βl , TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(l-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19; erythropoietin; osteoinductive factors; immuno toxins; a bone morphogenetic protein (BMP); protamine; an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL- 10; anti-HER-2 antibody; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; antibodies; and fragments of any of the above-listed polypeptides. The prefened polypeptides of mterest are mammalian polypeptides, most preferably human polypeptides Examples of such mammalian polypeptides include t-PA, VEGF, gpl20, antι-HER-2, anti-CDl la, antι-CD18, DNase, IGF-I, IGF-II, msulm, protamme, bram IGF-I, growth hormone, relaxin chains, LHRH analogues, cholecystokιnιn-8 analogues, growth hormone releasing factor, insulin chams or pro-msulm, urokinase, lmmunotoxins, neurotrophins, and antigens Especially preferred mammalian polypeptides are those combmations that are administered to mammals sufficiently frequently that it would be desirable to give one shot or dosage contammg both Particularly preferred such combinations include, e g , insulin and an IGF, most preferably IGF-I, or protamine and insulin, or growth hormone and an IGF such as IGF-I, or a LHRH analogue such as leuprohde and a cholecystokιnιn-8 analogue such as CCK-8 As used herein, "oppositely-charged polypeptides" or "polypeptides of opposite charge" means that one polypeptide is negatively charged and one is positively charged at a given pH The charges can be determined, for example, based on pK_a values of the lomzable groups of the polypeptide, or by its isoelectnc point (pi), as determined, for example, by gel electrofocusmg Generally, the negatively-charged polypeptide has a net negative charge at about pH 6 to 8, or has more than about five negatively-charged residues For a polypeptide to be used as a negatively-charged polypeptide, it must have a higher number of negative charges compared to the number of positive charges Conversely, the positively-charged polypeptide generally has a net positive charge at about pH 6 to 8 or has more than about five positively-charged residues For a polypeptide to be used as a positively-charged polypeptide, it must have a higher number of positive charges compared to the number of negative charges For example, IGF-I and msulm have pi values of 8 7 and 5 4, respectively, at a solution pH of 7 2, and therefore are oppositely-charged Further, at solution pH 7 4, leuprohde (a LHRH analogue)ιs positively charged, whereas a cholecystokιnιn-8 analogue (CCK-8) is negatively charged due to a pi value of less than 4 The charges on the two oppositely-charged polymers are sufficient to bring about an electrostatic interaction Other examples of negatively- charged polypeptides mclude hepann, albumin (bovme serum albumin has a pi of 4 8, with a net negative charge of -18 at pH 7 1), and beta-lactoglobu n (pi of 5 1 with a net negative charge of -5 at pH 7 1) Oppositely-charged polypeptides herein may be native polypeptides or those that are deπvatized or synthetic, providing that they are "pharmaceutically active " By "pharmaceutically active" is meant a polypeptide that has efficacy m one or more biological assays, including immunological assays and mitogenic assays For example, pharmaceutically-active polypeptides include antigens as well as antibodies, and receptors as well as ligands, which act m an immunological or biological sense As used herein, "IGF-I" refers to insulin-like growth factor from any species, including bovme, ovme, porcme, equine, and preferably human, m native-sequence or in variant form, and from any source, whether natural, synthetic, or recombmant Preferred herein for animal use is that form of IGF-I from the particular species being treated, such as porcme IGF-I to treat pigs, ovme IGF-I to treat sheep, bovine IGF-I to treat cattle, etc Preferred herein for human use is human native-sequence, mature IGF-I, more preferably without a N-terminal methiomne, prepared, e g , by the process described m EP 230,869 published August 5, 1987, EP 128J33 published December

19, 1984, or EP 288,451 published October 26, 1988 More preferably, this native-sequence IGF-I is recombinantly produced and is available from Genentech, Inc , South San Francisco, CA for clinical investigations

The preferred IGF-I vanants are those described in US Pat Nos 5,077,276, 5,164,370, or 5,470,828, or m WO 87/01038, t e , those wherein at least the glutamic acid residue is absent at position 3 from the N-terminus of the mature molecule or those having a deletion of up to five amino acids at the N- terminus The most preferred vanant has the first three ammo acids from the N-terminus deleted (variously designated as brain IGF, tIGF-I, des(l-3)-IGF-I, or des-IGF-I) As used herein, "msulm" refers to any type of insulin from any species, including bovine, ovine, porcme, equine, and preferably human, and from any source, whether natural, synthetic, or recombmant All insulin drugs reported, for example, in Diabetes Mellitus - Theory and Practice, fourth edition, Harold Rifkin, MD, Ed (Elsevier, New York, 1990), Chapter 29, and U S Pharmacist. 18 (Nov Suppl ) p 38-40 (1993) are suitable herem All the vanous forms of human insulm on the market are included, such as those mentioned in Jens Brange, Galemcs of

Insulin. The Physico-chemical and Pharmaceutical Aspects of Insulin and Insulin Preparations (Spnnger- Verlag, New York, 1987), page 17-40 These include Regular insulm, NPH (Neutral Protamine Hagedorn) insulin, also called Isophane Insulin, 70/30 msulm, composed of 70% NPH-insulm and 30% Regular msulm, Semilente insulin, UltraLente msulm, Lente insulin, and Humalog insulm Preferred herem for animal use is that form of msulm from the particular species being treated, such as human insulin to treat humans

As used herein, the term "disorder" in general refers to any condition that would benefit from treatment with the oppositely-charged polypeptides ingredients in the composition herein, including any disease or disorder that can be treated by effective amounts of these polypeptides This mcludes chronic and acute disorders, as well as those pathological conditions which predispose the mammal to the disorder in question Non-lrmitmg examples of disorders to be treated herem mclude benign and malignant tumors, leukemias and lymphoid malignancies, neuronal, g al, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal, and blastocoehc disorders, hematopoiesis-related disorders, tissue-growth disorders, skin disorders, desmoplasia, fibrotic lesions, hyperglycemic disorders, kidney disorders, bone-related disorders, trauma such as burns, incisions, and other wounds, catabohc states, testicular-related disorders, and inflammatory, angiogemc, and lmmunologic disorders, including artenosclerosis An example of a disorder that can benefit from treatment with IGF-I and an insulin mcludes diabetes

As used herein, the term "hyperglycemic disorders" refers to all forms of diabetes, such as type I and type II diabetes, as well as hypennsulmemia and hyperlipidemia, e g , obese subjects, and insulin-resistant diabetes, such as Mendenhall's Syndrome, Werner Syndrome, leprechaunism, hpoatrophic diabetes, and other hpoatrophies The preferred hyperglycemic disorder is diabetes, especially type I and type II diabetes "Diabetes" itself refers to a progressive disease of carbohydrate metabolism involving inadequate production or utilization of msulm and is charactenzed by hyperglycemia and glycosuna

As used herein, the term "treating" refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment mclude those already with the disorder as well as those prone to having the disorder or diagnosed with the disorder or those in which the disorder is to be prevented Consecutive treatment or administration refers to treatment on at least a daily basis without interruption in treatment by one or more days Intermittent treatment or administration, or treatment or administration in an intermittent fashion, refers to treatment that is not consecutive, but rather cyclic in nature The treatment regime herein can be either consecutive or intermittent, but preferably is consecutive when both proteins are formulated and administered together As used herem, "mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic, and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, sheep, pigs, cows, etc The preferred mammal herein is a human The term "non-adult" refers to mammals that are from pennatal age (such as low-birth- weight infants) up to the age of puberty, the latter being those that have not yet reached full growth potential As used herein, the term "hypoglycemic agent" refers to secretagogues, preferably oral agents, excluding msulm, which cause the secretion of insulin by the pancreas More preferred herein for human use are the sulfonylurea class of oral hypoglycemic agents Examples include glybuπde, ghpizide, and ghclazide In addition, agents that enhance insulin sensitivity, such as biguamdes, are within this definition, and also are preferred

As used herem, "complexed" in the context of polypeptides means that they are covalently bonded or otherwise have a b ding affinity that is greater than about 1 (mμ) Examples would include a complex of IGF-I and one or more of its binding protems, or of a ligand and its receptor, or of methemoglobin and the tryptic fragment of bovme liver cytochrome b, or complex coacervation, as with albumm and acacia

As used herem, "soluble" refers to polypeptides that, when in aqueous solutions, are completely dissolved, resulting in a clear to slightly opalescent solution with no visible particulates, as assessed by visual inspection A further assay of the turbidity of the solution may be made by measuring UV absorbances at 320 to 360 nm with a 1-cm pathlength cell (Eckhardt e/ α/ J Pharmaceutical Science and Technology. 48 64-70 (1994))

A "stabilizer" is any compound that functions to preserve the active polypeptides in the formulation, e g , msulm and IGF-I, so that they do not degrade or otherwise become inactive over a reasonable penod of tune or develop pathogens or toxins that prevent their use Examples of stabilizers mclude preservatives that prevent bacteria, viruses, and fungi from proliferating in the formulation, anti-oxidants, or other compounds that function m vanous ways to preserve the stability of the formulation

A "buffer" as used herein is any suitable buffer that is GRAS and generally confers a pH from about 4 8 to 8, preferably from about 7 to 7 5, most preferably about 7 2, if the polypeptides are IGF-I and insulin Examples include acetic acid salt buffer, which is any salt of acetic acid, including sodium acetate and potassium acetate, succmate buffer, phosphate buffer, citrate buffer, histidine buffer, or any others known to the art to have the desired effect The most prefened buffer is histidine for a pH of about 7 to 7 5

B Modes for Carrying Out the Invention

Generally, the formulations are prepared by mixing the polypeptides of opposite charge, each at the desired degree of punty, uniformly and intimately with one another and with one or more of the following excipients arginine, lysme, glutamate, or sodium dodecyl sulfate, or with a combmation of the two excipients beta-hydroxy cyclodextrin and arginine Optionally, the composition may also contain, for parenteral administration, a pharmaceutically or parenterally acceptable earner, I e , one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other mgredients of the formulation For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides Preferably the carrier is a parenteral earner, more preferably a solution that is lsotomc with the blood of the recipient Examples of such carrier vehicles include water, saline or a buffered solution such as phosphate- buffered saline (PBS), Ringer's solution, and dextrose solution Most preferably, the carrier vehicle is PBS

The preferred excipients depend on the types of polypeptides bemg employed, the molar ratio of the two polypeptides m the composition, the presence, types, and amounts of other ingredients, etc For example, if the polypeptides are IGF-I and msulm, the preferred excipient is arginine in a concentration of 100-200 mM, more preferably about 160 mM for a 1 1 vol/vol ratio of IGF-I and insulin Further, if the insulm is NPH-msulm, it cannot have the beta-cyclodextnn sulfobutyl ether as excipient, alone or in combination with arginine, because it causes re-solubi zation of insulin m the NPH portion, thereby dissociating NPH-msulm complex

The composition preferably also contains a buffer that brings the pH to about 7-7 5, such as histidine, if the polypeptides are insulin and IGF-I The concentration of excipient employed also depends on the type and the ratio of the polypeptides For example, if the excipient is arginine and the polypeptides are Regular insulin and IGF-I at an insulin concentration of 3 8 mg/ml and an IGF-I concentration of 10 mg/ml, the maximum ratio of insulin to IGF-I is 0 85 1 if the concentration of argimne is 150 mM However, as the concentration of arginine is mcreased, the ratio of Regular insulin to IGF-I is increased, such that at 230 mM argmme, the maximum ratio is 2 5 1 When lysine or glutamate is employed as excipient at 280 mM, the maximum ratio is 1 1 Furthermore, the mixing ratio can be 1 1 to 6 1 when 0 5% SDS is employed Mixmg can be done at less than a 1 4 1 ratio when 5% beta-hydroxy cyclodextnn and 150 mM arginine are employed, less than 6 1 when 5% beta-hydroxy cyclodextnn and 230 mM arginine are employed, less than 0 25 1 if 1% beta-cyclodextπn sulfobutyl ether is used, less than 1 2 1 if 1% beta-cyclodextrm sulfobutyl ether and 150 mM argmme are used, and less than 10 1 if 5% beta-cyclodextnn sulfobutyl ether and 230 mM argmme, or 2 5% of such ether with 230 mM argmme, or 2% of such ether with 150 mM arginine are used The volume volume ratio of the polypeptides depends mainly on the types of polypeptides, concentration of polypeptides, type(s) of excιpιent(s), and concentratιon(s) of excιpιent(s) For NPH-insulin, the ratio of insulin IGF-I generally ranges from about 1 15 to 15 1 vol /vol , preferably about 1 1 to 15 1 For Regular insulm, the ratio generally varies from about 1 6 to 6 1 , preferably from about 1 1 to 1 6 For 70/30 insulin, the preferred ratio range is from about 1 1 to about 6 1

The polypeptides are typically formulated m such vehicles at a pH of from about 4 5 to 8, dependmg mainly on the pi of the polypeptides, preferably m the presence of a buffer that mamtams the pH level If the polypeptides are IGF-I and insulin, preferably the IGF-I is formulated at about pH 7 to 7 5, more preferably about 7 2, usmg histidine as buffer, before mixmg with the msulm The final preparation is a stable liquid

In one embodiment for treating diabetes, the composition comprises IGF-I and NPH-insulin in a volume ratio of insulin IGF-I of from about 15 1 to 1 15 (v/v) The more preferred amounts of IGF-I and insulm m this composition are from about 1 to 10 mg IGF-I and from about 0 2 to 2 mg insulin The composition of claim 7 wherem the volume volume ratio of msulm IGF-I is from about 0 2 1 to about 1 1

The composition herein also may contam a stabilizer For example, quaternary ammonium salts are useful stabilizers in which the molecular structure includes a central nitrogen atom jomed to four organic (usually alkyl or aryl) groups and a negatively- charged acid radical These salts are useful as surface-active germicides for many pathogenic non-sporulating bactena and fungi and as stabilizers Examples include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldrmethylammonium chlondes m which the alkyl groups are long-cham compounds), and benzethomum chlonde Other types of stabilizers include aromatic alcohols such as phenol and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, and m-cresol The preferred stabilizer herein is phenol or benzyl alcohol, and the most preferred is phenol

The stabilizer is included in a stable liquid form of the insulin and IGF-I formulation, but not in a lyophihzed form of the formulation In the latter case, the stabilizer is present m the bacteriostatic water for injection (BWFI) used for reconstitution

One preferred composition contammg IGF-I and an insulin contams at least argmme as well as a buffer that bnngs the pH to about 7 to 7 5, most preferably histidine, and a phenol, optionally with PBS More preferably, the arginine is present m a concentration of about 100 to 300 mM and the ratio of insulin IGF-I is from about 0 1 1 to 10 1, more preferably about 0 2 1 to about 1 1 In another preferred embodiment, this formulation with 100 to 300 mM argimne, as well as buffer such as histidine and a phenol also contains beta-cyclodextrm sulfobutyl ether as an excipient in a concentration of about 1 - 10% A more preferred composition of this type comprises about 5-20 mg/ml IGF-I, about 2-10 mg/ml insulin, about 100-200 mM arginine, about 5-20 mM histidine at pH about 7-7 5, and about 1-5 mg/ml phenol The most preferred composition comprises about 10 mg/ml IGF-I, about 3-4 mg/ml msulm, about 160 mM arginine, about 10 mM histidine, and about 3 mg/ml phenol, at pH about 7 2 Another prefened composition containing IGF-I and an insulin contains sodium dodecyl sulfate as excipient in a concentration of about 1-10% and has a ratio of insulin:IGF-I of about 1 :1 to 6:1.

The final formulation, if a liquid, is preferably stored at a temperature of from about 2 to 8 °C for a suitable time period. Alternatively, the formulation can be lyophilized and provided as a powder for reconstitution with water for injection that is stored as described for the liquid formulation.

The polypeptides to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutic compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The polypeptide composition ordinarily will be stored in unit or multi-dose containers, for example, sealed ampules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-mL vials are filled with 5 mL of sterile-filtered 1% (w/v) aqueous IGF-I solution, and the resulting mixture is lyophilized. The subcutaneous injection solution is prepared by reconstituting the lyophilized insulin using bacteriostatic Water-for-Injection. This solution is then mixed with a similarly reconstituted insulin solution or a liquid insulin solution.

The formulation containing both the IGF-I and insulin can be made by many different methods. One method comprises mixing insulin with an IGF-I-containing composition (having the ingredients as described below).

The IGF-I-containing solution useful for administering with the insulin solution as described above preferably contains arginine, more preferably contains arginine and a stabilizer, still more preferably contains arginine, a stabilizer, and a buffer, and more preferably is as follows: About 5-20 mg/ml IGF-I, about 100-200 mM arginine, about 5-20 mM buffer at about pH 7-7.5, and about 1 -5 mg/ml phenol. The most preferred composition for this purpose comprises about 10 mg/ml IGF-I, about 160 mM arginine, about 10 mM histidine at about pH 7.2, and about 3 mg/ml phenol. Kits are also contemplated for this invention. A typical kit would comprise a container, preferably a vial, for a first polypeptide such as IGF-I in an excipient as described above; a container, preferably a vial, comprising a second polypeptide of the opposite charge, and instructions, such as a product insert or label, directing the user to combine the contents of the two containers, i.e., the two formulations. This would preferably provide a pharmaceutical formulation. Preferably, if the polypeptides are IGF-I and insulin, the pharmaceutical formulation is for treating diabetes. Also, preferably the container with IGF-I additionally comprises a stabilizer such as benzyl alcohol or phenol, or both, in the buffer at a pH of from about 7.0 to 1.5. Preferably, the user will be instructed to combine the contents of the containers, i.e., the two formulations, in a syringe for immediate injection.

Another typical kit is one where the composition is already prepared such that it is contained in one container and the kit also has instructions for using the composition to treat an appropriate disorder. The composition of polypeptides is directly administered to the mammal by any suitable technique, including infusion, injection, and pulmonary and transdermal administration. The specific route of administration will depend, e.g., on the medical history of the patient, including any perceived or anticipated side effects using either polypeptide alone, the types of polypeptides employed, and the particular disorder to be corrected. Examples of parenteral administration include subcutaneous, intramuscular, intravenous, intraarterial, and intraperitoneal administration of the composition.

One preferred method of delivery for systemic-acting drugs involves administration by continuous infusion (using, e.g. , slow-release devices or minipumps such as osmotic pumps or skin patches), or by injection (using, e.g. , intravenous or subcutaneous means, including single-bolus administration) For example, if the polypeptides are insulm and IGF-I, delivery of the composition by injection will be the prefened form of administration for treatmg diabetes

Another preferred route of administration is lontophoretic transdermal delivery either for localized or systemic therapy Iontophoresis is a means of enhancing the flux of ionic drugs across skin by the applicatino of an electrochemical potential gradient Drugs suitable for this method of delivery include LHRH analogues, insulin, growth hormone, and cholecystokιnιn-8 analogues See, for example, Snmvasan et al , J Pharm Sci .79 588-591

(1990)

The composition to be used in the therapy will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clmical condition of the mdividual patient (especially the side effects of treatment with the polypeptides as smgle agents), the site of delivery of the composition, the types of polypeptides employed, the method of administration, the schedulmg of administration, and other factors known to practitioners The "effective amounts" of each component for purposes herein are thus determined by such considerations and must be amounts that result in bioavailabihty of the drugs to the mammal As a general proposition, the total pharmaceutically effective amount of the polypeptides admmistered parenterally per dose will be m the range of from about 10 μg/kg/day to about 1 mg/kg/day based on kg of patient body weight For the combmation of IGF-I and insulin, the parenteral amount per dose ranges from about 10 to 200 μg/kg/day of IGF-I based on kg of patient body weight, and from about 0 5 to 500 units/day of insulin, although, as noted above, this will be subject to a great deal of therapeutic discretion Preferably for treatment of diabetes m humans, the dose of IGF-I is from about 1 to 10 mg twice per day, more preferably from about 20 to

80 μg/kg/m ection (I e , from about 1 5 to 6 mg) twice a day subcutaneously, and the dose of insulin is from about 5 to 50 units/injection (l e , from about 0 2 to 2 mg) twice a day subcutaneously The ratio of insulin to IGF-I m this formulation by volume is that referred to above

Although injection is preferred, an infusion device may also be employed for continuous SC infusions An intravenous bag solution may also be employed The key factor m selecting an appropnate dose is the result obtained, for example, m the case of diabetes as measured by decreases m blood glucose so as to approximate the normal range, or by other cntena for measunng treatment of diabetes as defined herein as are deemed appropnate by the practitioner Further information on dosmg insulin can be found in Diabetes Mellitus - Theory and Practice. supra, Chapters 29 and 30 Also, the formulation herein is suitably administered along with other agents that produce the desired pharmacological effect, for example, m the case of IGF-I and insulin, an IGF binding protem, for example, one of those currently known, t e , IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, or IGFBP-6, or with the ALS of the IGF bmding complex The preferred binding protein for IGF-I herein is IGFBP-3, which is described m U S Pat No 5J58J87 and by Martin and Baxter. J Biol Chem , 261 8754-8760 (1986) This glycosylated IGFBP-3 protein is an acid-stable component of about 53 Kd on a non-reducing SDS-PAGE gel of a 125-150 Kd glycoprotein complex found in human plasma that carries most of the endogenous IGFs and is also regulated by GH

The administration of the IGF binding protein with IGF-I and insulin may be accomplished by the method described in U S Pat No 5,187,151 Briefly, the IGF-I and IGFBP are administered in effective amounts by subcutaneous bolus injection m a molar ratio of from about 0 5 1 to 3 1 , preferably about 1 1 , the insulin is already present with the IGF-I Furthermore, the formulation is suitably administered along with an effective amount of a hypoglycemic agent such as a sulfonylurea. The hypoglycemic agent is administered to the mammal by any suitable technique including parenterally, intranasally, orally, or b any other effective route. Most preferably, the administration is by the oral route. For example, MICRONASE™ Tablets (glyburide) marketed by Upjohn in 1.25, 2.5, and 5 mg tablet concentrations are suitable for oral administration. The usual maintenance dose for Type II diabetics, placed on this therapy, is generally in the range of from about 1.25 to 20 mg per day, which may be given as a single dose or divided throughout the day as deemed appropriate (Physician's Desk Reference. 2563-2565 (1995)). Other examples of glyburide-based tablets available for prescription include GLYNASE™ brand drug (Upjohn) and DIABETA™ brand drug (Hoechst-Roussel). GLUCOTROL™ (Pratt) is the trademark for a glipizide (1- cyclohexyl-3-(p-(2-(5-methylpyrazine carboxamide)ethyl)phenyl)sulfonylurea) tablet available in both 5 and 10 mg strengths and is also prescribed to Type II diabetics who require hypoglycemic therapy following dietary control or in patients who have ceased to respond to other sulfonylureas (Physician's Desk Reference. 1902-1903 (1995)). Other hypoglycemic agents than sulfonylureas, such as the biguanides (e.g., metformin and phenformin) or troglitozones, or other drugs affecting insulin action may also be employed. The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. All literature and patent citations mentioned herein are expressly incorporated by reference.

EXAMPLE I Materials and Methods Recombinant human IGF-I was obtained from Genentech, Inc. Regular insulin (HUMULIN™ R) and

70/30 insulin (HUMULIN™ 70/30) were obtained from Eli Lilly and Company. The 0.5-cc and 1-cc insulin syringes were obtained from Becton Dickinson, and PD10 columns (SEPHADEX™ G25M) were obtained from Pharmacia (catalog #17-0851-01).

Table 1 lists formulation dosage forms for Regular, NPH-, and 70/30 insulin. Table 1

Insulin Formulation Dosage Forms

HUMULIN™ R zinc-insulin, 100 USP units, 3.8 mg/ml

(neutral Regular 10μg-40μg zinc / 100 USP units insulin) 2.5 mg/ml m-cresol 16 mg/ml glycerine pH~7.2

HUMULIN™ NPH 100 USP units/ml, 3.8 mg/ml

(neutral protamine 3-6 mg protamine Hagedorn) 16 mg/ml glycerine 1.6 mg/ml m-cresol 10-40 μg zinc/100 USP 0.65 mg/ml phenol phosphate, pH~7.2

HUMULIN™ 70/30 100 USP units/ml, 3.8 mg/ml (70% NPH, 30% Regular approximately 2 5 mg protamine msulm) phosphate, pH~7 2 m-cresol zmc phenol

Preparation of IGF-I for formulation screen

IGF-I in 0 2M citrate, 26 mg/ml, was used as the starting material Citrate was difficult to remove from IGF-I solutions Therefore, a two-step diafiltration process was designed to remove citrate and at the same time mcrease IGF-I concentration Diafiltration was accomplished using a tangential flow filtration unit The process steps were as follows

(a) The IGF-I solution contammg 200mM citrate was first diafiltered into 200mM NaCl, 230mM arginine and lOmM histidine at pH 72

(b) The IGF-I solution was then diafiltered into 230mM argmme, lOmM histidine, pH 7 2 IGF-I was concentrated to 28 6 mg/ml Argimne was used in both steps due to its ability to keep IGF-I in solution at high concentrations at pH

7 2 Thus, removing citrate and concentratmg IGF-I could be accomplished without precipitating IGF-I The above IGF-I solution was then buffer- exchanged into vanous testing formulations usmg PD-10™ columns Assay Methods (1) Visual inspection color, appearance and clanty, (2) pH,

(3) IGF-I concentration determined by UV absorbance at 276 nm using an absorptivity of 0 646 cm (mg/ml) ,

(4) solution turbidity determined by UV absorbance at 340 to 360 nm,

(5) quantitation of IGF-I and insulm m solution determined by the acidic pH reversed-phase HPLC (rp-HPLC) method Column VYDAC™, C18, 30θA, 25cm

Flow rate 0 5 ml/mmute

Injection volume 25 μl/injection

Detection wavelength 214nm

Column temperature 50°C Solvent A 0 1% tnfluroacetic acid in H,0

Solvent B 0 1 % tnfluroacetic acid in acetonitrile

Gradient tune %A

0 72

20 72 25 70 5

40 61 5

50 40

51 72 60 72 Figure 1 shows the acidic reversed-phase HPLC of an IGF-I sample Figure 2 shows the acidic reversed- phase HPLC of Regular insulm Figure 3 shows the acidic reversed-phase HPLC of a 1 1 (vol vol ) mixture of Regular insulin IGF-I It is evident from comparing and reviewing these Figures that IGF-I and Regular insulin are well separated by this HPLC method Quantitation of both IGF-I and insulin can be achieved by utilizing the peak areas of IGF-I and msulin peaks For example, the percentage of IGF-I remaining m solution can be obtamed by comparing the IGF-I peak areas before and after IGF-I is mixed with msulm Procedure for mixing IGF-I and insulin

(1) Draw air mto an insulm synnge equal to dosage of IGF-I Insert needle into IGF-I vial, and inject air into bottle Remove needle/syrmge from the IGF-I vial without withdrawing any IGF-I solution

(2) For NPH and 70 30 msulm, gently invert msulm vial several times before use No mixing is required for Regular insulm Inject air into the msulin vial (3) With the needle still m the msulin vial, turn msulm bottle and synnge upside down

(4) Make sure the tip of the needle is in solution, and withdraw correct volume of msulin into synnge

(5) Before removing needle from the insulin vial, check the syrmge for air bubbles If air bubbles are present, hold syrmge straight up and tap its side, until bubbles float to the top Push them out with plunger and withdraw conect msulin dose (6) Remove needle from the msulm vial and insert it into the IGF-I vial Turn IGF-I bottle and synnge upside down Make sure the tip of the needle is in the solution, and withdraw desired amount of IGF-I into synnge

(7) Remove needle/synnge from the IGF-I vial, and inject msulin/IGF-I mixture into a clean glass centnfuge tube Analysis Procedures for the Insulin / IGF-I mixture The IGF-I/ιnsulιn mixture was assayed for soluble IGF-I and insulm content either after filtration through a 0 2 μm filter or after centnfugation at 2000 r p m for 10 mmutes In some cases, the IGr-I and msulm mixture was first incubated with phosphate buffered saline at 37°C for 10 minutes before the mixture was filtered or centnfuged for analysis

Results and Discussion Mixability of the acetate-buffered IGF-I formulation with insulin:

The acetate-buffered IGF-I formulation used as a standard herem is a clear liquid and contains 10 mg/ml

IGF-I, 100 mM sodium chlonde, 2 mg/ml polysorbate 20, 9 mg/ml benzyl alcohol, 50 mM acetate, pH 5 4 This product is mtended for multi-use purposes for up to 28 days of use Shelf life was set at 60 months at 2-8°C storage

The mixability of the acetate-buffered IGF-I formulation with insulin was first evaluated It would be most desirable if this IGF-I formulation could be used to mix with insulin during administration

Table 2 shows results of mixing the acetate-buffered IGF-I formulation with Regular insulm At 1 1 (vol vol) mixing ratio, the solution turned very cloudy upon mixing 43% of insulin and 14% of IGF-I precipitated out of solution

Table 2 IGF-I m the acetate formulation mixed with

Regular insulm

The appearance of Regular insulin and IGF-I in the acetate formulation was monitored as the pH of these solutions was adjusted by adding 0 IN HCI or NaOH The solution was characterized as either "clear" or "turbid", and solution clarity vs. pH is plotted in Figure 4. Regular insulin at pH 7.2 is a clear solution, and it remains clear above pH 6.4; the Regular insulin solution turns cloudy at pH 6.32 or below. IGF-I in the acetate formulation at pH 5.4 is a clear solution and remains clear below pH 6.34. IGF-I solution turns cloudy above pH 6.39, and the precipitated IGF-I subsequently turns into a clear gel. These observations are consistent with the general understanding that protein solubility decreases as pH approaches the protein' s isoelectric point (pi). The isoelectric points of IGF-I and insulin are 8.7 and 5.4, respectively. Data shown in Figure 4 suggest that no suitable pH range exists at which both IGF-I and Regular insulin can stay in solution. Therefore, it is not possible simply to adjust the pH of the acetate IGF-I formulation to produce a clear IGF-I/insulin mixture.

Table 3 shows the result of mixing the acetate IGF-I formulation with NPH-insulin at various mixing ratios. At NPH:IGF-I mixing ratios of 1 : 1 and 14:1 (vokvol), both NPH-insulin and IGF-I were unchanged before and after mixing. All IGF-I remained in solution and all NPH remained as NPH crystals with no soluble insulin in solution. However, when NPH and IGF-I were mixed at a 1:14 (vokvol) ratio, 100% of the insulin was released from the NPH crystals, although IGF-I remained unchanged.

Table 3

IGF-I in the acetate formulation mixed with NPH-insulin

Development of new IGF-I formulation mixable with insulin:

Due to the inability of the acetate IGF-I formulation to mix with insulin, a new formulation was developed that meets the following criteria: (1 ) A clear IGF-I solution formulated at pH 7.2. This serves to prevent drastic pH shifts when IGF-I and insulin are mixed at wide ranges of mixing ratios. (2) An IGF-I formulation that prevents insulin and IGF-I interaction upon mixing, so to avoid precipitation of insulin and or IGF-I. (3) An IGF-I formulation that will not dissociate insulin from NPH crystals upon mixing. During the development of such an IGF-I formulation, various excipients such as salts, buffers, metal ions, sugars, amino acids, polyols, surfactants, and cyclodextrins were screened as listed in Table 4.

Table 4

Excipients Screened for the Development of a New IGF-I Formulation for Mixing with Insulin

The control acetate-buffered IGF-I formulation turned cloudy when the pH was adjusted to above 6 4 Many of the excipients listed in Table 4 enabled IGF-I to be a clear solution at pH 7 2 These excipients included ammo acids (glycine, lysme, argmme, histidine, glutamate, aspartate), salts (sodium chloride, sulfate, phosphate, citrate, bicarbonate, ascorbate, succmate), cyclodextnn denvatives (β-hydroxy cyclodextnn and β-cyclodextnn sulfobutyl ether), SDS, and glycerol However, upon mixmg of IGF-I and Regular msulm, most of the excipients above were not able to prevent insulin and IGF-I precipitation

The excipients that proved to be effective in preventmg precipitation of Regular insulm and IGF-I upon mixing were arginine, lysine, glutamate, SDS, β-hydroxy cyclodextnn and β-cyclodextnn sulfobutyl ether Mixmg results using these excipients are shown in Table 5 0 5% SDS was very effective in achieving a clear insulin and

IGF-I mixture However, when 0 5% SDS was added to NPH-insulin at a 1 1 (vol vol) ratio, the solution clarified, which indicated the dissolution of NPH crystals Both lysine and glutamate when used at a concentration of 280 mM prevented precipitation upon 1 1 (vol vol) mixing of IGF-I and Regular insulin

Table 5 Excipient Effects on the Appearance of IGF-I/Regular Insulin Mixture (a)

(a) In addition to the excipients listed in the Table, all IGF-I formulations contain 10 mg/ml IGF-I and 10 mM histidine at pH 1.2.

(b) Regular insulin was slowly added to 200 ml of 10 mg/ml IGF-I in 150 mM arginine, 10 mM histidine at pH 7.2. The solution remained clear until 170 ml of Regular insulin was added. 170 mlJOO ml = 0.85: 1 mixing ratio.

(c) Mixing was done similarly as described in (b). (d) Under these conditions, Regular insulin was slowly added to 200 ml of IGF-I. The mixed solution remained clear even after 2 ml of Regular insulin was added.

Arginine was more effective than lysine or glutamate in keeping both insulin and IGF-I in solution. As shown in Table 5, at 150 mM arginine concentration, Regular insulin and IGF-I could be mixed at mixing ratios up to 0.85:1 (vokvol). When mixing ratios exceeded 0.85: 1, precipitation of both IGF-I and insulin occurs. At 230 mM arginine concentration, more Regular insulm could be mixed with IGF-I to form a clear mixture, up to a mixing ratio of 2 5 1 (vol vol)

Without being limited to any one theory, the protection against precipitation offered by arginine is believed to be due to the fact that at pH 7 2 msulin molecules are negatively charged, and the positively-charged argmme molecules interact with negatively-charged msulin, thereby shielding the mteraction and subsequent precipitation between positively-charged IGF-I and negatively-charged msulm High concentrations of arginine are required for mixing at a high insulm IGF-I ratio

As shown in Table 5, 1% β-cyclodextπn sulfobutyl ether allows mixmg of Regular insulin and IGF-I at a mixmg ratio below 0 25 1 Use of 5% ^β-hydroxy cyclodextnn did not allow mixing at any ratio Use of both cyclodextnn and arginine is extremely effective m preventing insulin and IGF-I precipitation upon mixing β- cyclodextnn sulfobutyl ether used at a concentration of 2% to 5% together with argmme used at a concentration of 150 mM to 230 mM allowed Regular msulin and IGF-I to be mixed at a very wide range of mixing ratios, up to 10 1 (msulm vol IGF-I vol) Without being limited to any one theory, it is believed that this is due to the fact that at pH 7 2, positively-charged argimne interacts with negatively-charged insulin, and negatively-charged β- cyclodextnn sulfobutyl ether interacts with positively-charged IGF-I, thereby preventing msulm and IGF-I mteraction and subsequent precipitation However, in the presence of β-cyclodextnn sulfobutyl ether, NPH- insulm crystals dissolved Without limitation to any one theory, this is presumably due to the strong interaction of β- cyclodextnn sulfobutyl ether with positively-charged protamine, which breaks the protamine-insuhn complex Therefore, although the use of both β-cyclodextnn sulfobutyl ether and or SDS and argmme in the IGF-I formulation proves to be extremely effective m mixing with Regular insulin, it can not be used to mix with NPH- msulm

The above excipient screenmg study results identified arginine to be the preferred choice of excipient for an IGF-I formulation to be mixed with both Regular and NPH-msulm A study was then earned out to evaluate the effect of argmme concentration on the ability of IGF-I to mix with msulin The IGF-I formulation tested in this study contained 10 mg/ml IGF-I, 10 mM histidine, pH 7 2, 3 mg/ml phenol, and varying amounts of argmme

Insulin and IGF-I were mixed at vanous mixing ratios Percent soluble IGF-I and insulin were assayed by the acidic reversed-phase HPLC method Results m Table 6 showed that for mixing with Regular msulm, a high concentration of arginine is required At 80 mM of argimne concentration, 39% of IGF-I and 68% of insulin precipitated when mixed at a 1 1 (vol vol) ratio At 230 mM of argmme concentration, all msulm and all IGF-I remamed in solution when mixed at a 1 1 ratio However, at 230 mM arginine concentration, when mixed at 5 1 (vol vol) IGF-I NPH ratio, nearly 50% of NPH dissolved into soluble insulin 160 mM arginine seemed to produce the best results for mixing with both Regular and NPH-msulm At a 1 1 (R- insulin IGF-I) mixing ratio, essentially none of Regular insulm and IGF-I remained m solution At a 1 1 (NPH-insulin IGF-I) mixing ratio, nearly 100% of NPH-insulm was in solution and all NPH crystals remamed mtact Table 6

Effect of Arginine concentration on the mixability of IGF-I(a) with Regular and NPH-insulin

(a) The IGF-I formulation tested in this study contained 10 mg/ml IGF-I, 10 mM histidine, pH 7.2, 3 mg/ml phenol, and varying amounts of arginine.

(b) To simulate subcutaneous depot condition, after mixing of IGF-I and Regular insulin, 1 ml of the mixture was first incubated with 2 ml of phosphate-buffered saline at 37°C for 15 min before the amounts of soluble insulin and IGF-I were analyzed.

(c) If IGF-I and Regular insulin are fully mixable, the resulting mixture should have 100% soluble IGF-I and 100% soluble insulin.

(d) If IGF-I and NPH-insulin are fully mixable, the resulting mixture should have 100% soluble IGF-I and 0% soluble insulin.

A preferred IGF-I formulation for mixing with insulin was therefore defined to be 10 mg/ml IGF-I, 160 mM arginine, 10 mM histidine at pH 7.2 and 3 mg/ml phenol. The mixability of this formulation with Regular, NPH-, and 70/30 insulin at various mixing ratios was assessed and results are tabulated in Tables 7, 8 and 9, respectively.

Table 7 Mixing Results for the New IGF-I Formulation Mixed with Regular Insulin (a)

(a) The new IGF-I formulation contained 10 mg/ml IGF-I, 160 mM arginine, 10 mM histidine at pH 7.2 and 3 mg/ml phenol.

(b) To simulate subcutaneous depot condition, after mixing of IGF-I and Regular insulin, 1 ml of the mixture was first incubated with 2 ml of phosphate buffered saline at 37°C for 15 min before the amount of soluble insulin and IGF-I were analyzed.

(c) If IGF-I and Regular insulin are fully mixable, the resulting mixture should have 100% soluble IGF-I and 100% soluble insulin. Table 8 Mixing Results for the New IGF-I Formulation Mixed with NPH-msulm (a)

(a) The new IGF-I formulation contams 10 mg/ml IGF-I, 160 mM arginine, 10 mM histidine atpH 7 2 and 3 mg/ml phenol

(b) If IGF-I and NPH-insulin are fully mixable, the resulting mixture should have 100% soluble IGF-I and 0% soluble insulm

Table 9 Mixing Results for the New IGF-I Formulation Mixed with 70 30 Insulm (a)

20

(a) The new IGF-I formulation contained 10 mg/ml IGF-I, 160 mM argmme, 10 mM histidine at pH 7 2 and 3 mg/ml phenol

(b) If IGF-I and 70 30 msulm are fiilly mixable, the resulting mixture should have 100% soluble IGF-I and 22% to 30% of soluble msulin 70/30 msulm theoretically should contain 30% soluble msulm However, due to adsorption of soluble insulin onto NPH-insulin, the actual amount of soluble insulm is 22% by the acidic reversed-phase HPLC method

When the prefened IGF-I formulation was mixed with Regular msulin at a 1 1 (vol vol) ratio, the solution turned turbid 65% of IGF-I and 93% of msulm were in solution when assayed directly after mixmg However, after this mixmre was incubated m phosphate-buffered salme at 37°C for 15 minutes to simulate subcutaneous depot condition, the solution clanfied and 95% of IGF-I and 99% of insulin were in solution Table 7 shows IGF-I and Regular insulin mixing results after the mixtures were incubated with phosphate-buffered saline at 37°C for 15 minutes When mixed at 1 1, 2 1, 6 1 (Regular insulin IGF-I) ratios, precipitation occurred initially However, the mix clarified upon incubation with phosphate-buffered saline and the percent soluble IGF-I and msulm was nearly 90% or above Therefore, the pharmacokinetic profiles of IGF-I and insulm may not result in much change before and after mixmg

When the preferred IGF-I formulation was mixed with NPH-insulin at a mixing ratio from 1 15 to 15 1 (vol vol) ratio, the solution remained cloudy, indicating the presence of intact NPH-insulin As shown in Table 8, at mixing ratios between 1 1 to 15 1 (NPH IGF-I), all IGF-I was m solution and all NPH remained as lnsuhn- protamine complex At low NPH IGF-I mixing ratios, a sufficient amount of argmme in the preferred formulation started to dissociate NPH The amount of NPH dissociation was relatively low (24%) even at the extreme of 1 15 mixing ratio Therefore, at most of the mixing ratios, the pharmacokinetic profiles of IGF-I and insulin may not result in much change before and after mixing Table 9 shows the result of mixing the preferred IGF-I formulation with 70/30 insulin Theoretically, the 70/30 msulm should contain 30% soluble insulin However, soluble insulin tended to adsorb onto the NPH-insulin crystals The actual amount of soluble insulin in the 70/30 insulin was 22% by the acidic reversed-phase HPLC method If IGF-I and 70/30 msulm were fully mixable, the resulting mixture should have 100% soluble IGF-I and 22% to 30% of soluble insulm Results in Table 9 show that the percent soluble IGF-I and insulin did not change when 70/30 and IGF-I were mixed at ratios of 1 : 1 to 6: 1 (vokvol). At low 70/30: IGF-I mixing ratios, a portion of the NPH dissolved into soluble insulin.

In all of the above studies, no chemical degradation of either IGF-I or insulin was observed as a result of mixing by the acidic reversed-phase HPLC assay. Conclusions

To reduce the frequency of injections and improve patient compliance, it is desirable to mix polypeptides of opposite charges and then co-administer them. In this Example, the compatibility of IGF-I in the control acetate- buffered formulation and insulin was first evaluated. Immediately after mixing Regular insulin with IGF-I in the acetate formulation, the mixture turned from a clear solution into a cloudy suspension, indicating precipitation of both IGF-I and insulin. When mixed with NPH-insulin, the acetate-buffered IGF-I formulation dissociated NPH- insulin-protamine complex at low NPH/IGF-I mixing ratios.

Excipient screening studies were conducted to increase the compatibility between IGF-I and insulin. A preferred IGF-I formulation was developed for mixing with insulin. It contains 10 mg/ml IGF-I, 160 mM arginine, 10 mM histidine at pH7.2 and 3 mg/ml phenol. The ability of this arginine formulation to mix with Regular, NPH-, and 70/30 insulin at various mixing ratios was assessed. When this formulation was mixed with Regular insulin, percent soluble IGF-I and insulin were nearly 90% or above for all potential mixing ratios. Mixing of this IGF-I formulation and NPH-insulin at most of the mixing ratios resulted in no change in IGF-I and NPH before and after mixing. At very low NPH/IGF-I mixing ratios, portions of NPH-insulin-protamine complex dissociated.

Excipients such as arginine, lysine, glutamate, SDS, and/or certain cyclodextrins were shown to be effective in preventing IGF-I and insulin electrostatic interaction and subsequent precipitation upon mixing. The use of SDS and β-cyclodextrin sulfobutyl ether together with arginine in the IGF-I formulation proved to be extremely effective in allowing IGF-I and Regular insulin to be mixed at a very wide range of mixing ratios. However, they can not be used to mix with NPH-insulin due to the dissociation of NPH-insulin-protamine complexes in the presence of SDS and β-cyclodextrin sulfobutyl ether.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising a mixture of pharmaceutically-active polypeptides of opposite charge and an excipient selected from the group consisting of arginine, lysine, glutamate, sodium dodecyl sulfate, and a combination of beta-hydroxy cyclodextrin and arginine, wherein the polypeptides are soluble in the composition.

2. The composition of claim 1 wherein the excipient is arginine.

3. The composition of claim 1 wherein two or more of said excipients are used.

4. The composition of claim 3 wherein the excipients are arginine, sodium dodecyl sulfate, and beta- cyclodextrin sulfobutyl ether and the polypeptides are IGF-I and an insulin that is not NPH-insulin.

5. The composition of claim 1 wherein the polypeptides are IGF-I and insulin.

6. The composition of claim 5 wherein the volume:volume ratio of insulin:IGF-I is from about 1 : 15 to 15: 1.

7. The composition of claim 6 wherein the volume:volume ratio of insulin:IGF-I is from about 1 :6 to 6: 1.

8. The composition of claim 7 wherein the volume:volume ratio of insulin:IGF-I is from about 0.2: 1 to about 1:1.

9. The composition of claim 1 further comprising a buffer.

10. The composition of claim 9 wherein the buffer is at a pH of about 7 to 7.5.

11. The composition of claim 10 wherein the buffer is histidine.

12. The composition of claim 1 further comprising phosphate-buffered saline.

13. The composition of claim 1 further comprising a stabilizer.

14. The composition of claim 5 wherein the excipient is arginine and additionally comprising histidine and a phenol.

15. The composition of claim 5 wherein the excipient is arginine in a concentration of about 100 to 300 mM and the ratio of insulin:IGF-I is from about 0.1 : 1 to 10: 1.

16. The composition of claim 15 further comprising beta-cyclodextrin sulfobutyl ether as excipient in a concentration of about 1-10%.

17. The composition of claim 5 wherein the excipient is sodium dodecyl sulfate in a concentration of about 1-10% and the ratio of insulin:IGF-I is about 1 :1 to 6:1.

18. The composition of claim 6 comprising about 5-20 mg/ml IGF-I, about 2-10 mg/ml insulin, about 100-200 mM arginine, about 5-20 mM histidine at pH about 7-7.5 and about 1-5 mg/ml phenol.

19. The composition of claim 7 comprising about 10 mg/ml IGF-I, about 3-4 mg/ml insulin, about 160 mM arginine, about 10 mM histidine at pH 7.2 and about 3 mg/ml phenol.

20. The composition of claim 1 wherein the polypeptides are not complexed.

21. A kit comprising:

(a) a container comprising the composition of claim 1 comprising effective amounts of the polypeptides; and

(b) instructions for using the above composition to treat a disorder against which the composition is effective.

22. The kit of claim 21 wherein the polypeptides are IGF-I and an insulin and the disorder is a hyperglycemic disorder.

23. A method for preparing the composition of claim 1 comprising mixing together as component (a) a first polypeptide in an excipient selected from the group consisting of arginine, lysine, glutamic acid, sodium dodecyl sulfate, beta-hydroxy cyclodextrin, and beta-cyclodextrin sulfobutyl ether; and as component (b) a second polypeptide having an opposite charge from the first polypeptide.

24. The method of claim 23 further comprising incubating the mixture for aperiod of time at about 30-40°C, wherein the composition further comprises phosphate-buffered saline.

25. The method of claim 24 wherein the mixture is incubated for about 15 minutes at 37°C.

26. A kit for preparing the composition of claim 1 comprising: (a) a container comprising a first polypeptide in an excipient selected from the group consisting of arginine, lysine, glutamic acid, sodium dodecyl sulfate, and a combination of beta-hydroxy cyclodextrin and arginine, and;

(b) a container comprising a second polypeptide having an opposite charge from the first polypeptide; and (c) instructions for combining the contents of containers (a) and (b).

27. The kit of claim 26 wherein when the polypeptides are combined, the composition is a pharmaceutically acceptable formulation.

28. The kit of claim 27 wherein the pharmaceutically acceptable formulation is for treating a hyperglycemic disorder.

29. The kit of claim 26 wherein the first polypeptide is IGF-I and the second polypeptide is an insulin.

30. The kit of claim 26 wherein the excipient is arginine.

31. The kit of claim 30 wherein container (a) comprises about 5-20 mg/ml IGF-I, about 100-200 mM arginine, about 5-20 mM histidine at about pH 7-7.5 and about 1-5 mg/ml phenol.

32. The kit of claim 31 wherein container (a) comprises about 10 mg/ml IGF-I, about 160 mM arginine, about 10 mM histidine at about pH 7.2 and about 3 mg/ml phenol.