WO1997026005A1 - Igf-i in combination with hypoglycemics for treatment of diabetes - Google Patents

Abstract

A method is disclosed for better managing the progress of diabetes in mammals, particularly Type II diabetes, where IGF-I administration is combined with administering hypoglycemic agents. Preferably, the IGF-I is native sequence, mature human IGF-I and the hypoglycemic agents are insulin secretagogues such as the sulfonylurea. This combination of agents improves glycemic control in diabetes.

Description

IGF-I IN COMBINATION WITH HYPOGLYCEMICS FOR TREATMENT OF DIABETES

FIELD OF THE INVENTION This invention relates to using IGF I in combination with insulin secretagogues administered to diabetics to enhance glycemic control and better manage the progression of diabetes

BACKGROUND OF THE INVENTION

There is a need for improved therapies to treat the causes and symptoms of diabetes as it is now a major disease in the western world For example, the number of Americans with diabetes has risen almost 60 percent since 1983, and the disease rate has tripled since 1958 About 16 million Americans now have diabetes, up from 1 1 million in 1983 (figures released by American Medical Association and the American Diabetes Association, October 1995) and even this estimate is conservative m that it is estimated that up to 50% of cases go undetected

Iype II. non-ιnsulιn-dependentdιabetesmellιtus(NIDDM) which constitutesabout 80-90% of the total incidence of diabetes (Type I insulin- dependent diabetes mel tus ( IDDM ) makes up the rest), continues to grow rapidly, with respect to its incidence, prevalence, and cost Diabetic therapy has a number of goals, including relieving symptoms of hyperglycemiaand preventing long-term complications of diabetes such as microvascular and macrovascular disease The incidence and seventy of these complications in IDDM are related at least in part to the degree of glycemic control, as confirmed by the results of the Diabetes Control and Complications Trial (DCCT) There is general agreement that the same conclusion probably applies to NIDDM, especially in the case ofthe microvascular complications The medical managementof NIDDM is a progressive process Fιrstly,dιet control (hypocaloric/eucalonc) and exercise is attempted to achieve weight loss especially in obese subjects, secondly oral hypoglycemic drugs are used and finally, treatment with exogenously administered insulin

The sulfonylurea are oral hypoglycemics that are used to stimulate pancreatic insulin release Sulfonylurea are the most widely prescribed of the oral hypoglycemic agents currently approved for use in the United States Approximately 30 percent of patients initially treated with sulfonylureahave a poor response, and in the remaining 70 percent the subsequent failure rate is approximately 4 to 5 percent per year (Defronzo et al , NEJM.333.541 -549,[ 1995]) and has been reported as high as 10 percent (Geπch. NEJM 321.1231-1245,[ 1989]) This indicates that there is a clear need to improve sulfonylurea therapy

Sulfonylurea were discovered m the 1940's, by accident, when it was found that sulfonamides (used to treat infections ) brought about hypoglycemia The first sulfonylurea used clinically was I -butyl-3 sulfonylurea (carbutamide) which was replaced m the early 1950's by tolbutamide and later by about twenty newer agents of this class

Sulfonylurea are divided into two classes loosely based on their chemistry and potency All sulfonylurea are substituted arylsulfonylureas which differ by substitutions at the para position on the benzene ring and at one nitrogen residue of the urea moiety The first generation sulfonylurea include acetohexamide, chloropropamide tolazamide and tolbutamide, while the second generation drugs include glybuπde

^■ I - (glιbenclamιde),glιpιzιde and glιclazιde Second generation sulfonylureashow increased potency partly because they are larger and non - polar compared to the smaller, polar, first generation molecules Therefore, improved lipid solubility may account for the greater potency ofthe second generation sulphonylureas

As the sulfonylurea have similar qualitative activities, their pharmacokinetics may also define their potency After absoφtion from the gut sulfonylureaare all present in plasma bound greaterthan 90% to protem, especially to albumin The first generation sulfonylurea vary greatly in their serum half- lives with that of chloropropam ide being long at 24-48 hours, and that of tolbutamide being 4-7 hours Despite this variation in half- life their duration of action is uniformly short, so that they need to be given in divided daily doses In contrast the second generation molecules are 100 times as potent and even though their half-lives are short ( 1 -5 hours) they have a long duration of hypoglycemic action and thus may be administered only once daily The exact mechanisms.causmg the large discrepancy between half- life and duration of action ofthe sulphonylureas, are not known Therefore, despite this class of drug being widely used, simple concepts, such as the relationship between persistence in the body and duration of action, remain to be explained

Despite being utilized therapeutically for several decades, both the sites and the mechanism of action ofthe sulfonylurea remains in debate It is generally accepted that the sulfonylurea act, primarily, directly on the pancreas to release insulin This has been shown in in vitro studies using pancreas preparations (Nelson et al. J.Biol Chem 262.2608-2612f 19871) and in human studies (Pfeifer et alJCEM 53.1256- 1262f 198111 This predominate pancreatic effect is further substantiated by the fact that the sulfonylurea exhibit little activity in either pancreatectomized animals or in Type I diabetics who have no endogenous insulin secretion However, extra-pancreatic sites of action are suggested by human studies where with long- term sulfonylurea use, plasma insulin levels vary while blood glucose concentrationsare more consistently depressed This could be explained by the pancreatic beta cells exhibiting a maintained drug effect or because peripheral target tissues become more sensitive to insulin in response to the sulfonylurea (Kolterman.DiabetesMetab Rev 3.399-414119871) These apparent effects on peripheral targets could be due to the improved blood glucose control as chronic hyperglycemia reduces insulin secretion and impairs insulin action on extra-pancreatic target tissues Further doubts regarding the supposed extra-pancreatic effects of the sulfonylureahave been raised by the results of receptor binding studies in adipose tissue In adipose cell lines or freshly isolated pπmary adipocytes there is no high affinity binding of labeled glybuπde (Rajan et al .Endocrinology 134.1581 - 1588f 19941) These authors conclude that the anti-diabetogenic effects of sulfonylureaare not mediated by a direct action to increase peripheral glucose uptake but that the major locus of action for the sulfonylurea is the pancreatic beta cell The recent cloning of the sulfonylurea receptor (Aguilar-Bryan et al.Science 268.423 f 19951) has not yet provided direct answers to these questions

Therefore, in addition to the lack of understandingof both the pharmacokineticsand relative potencies ofthe sulfonylurea, it can also be seen that the sιte(s) of action of the sulfonylurea remains a subject of debate particularly their supposed extra-pancreatic effects

IGF-1 and 1GF-2 are growth factors with molecular weights of approximately 7500 daltons Both IGF- 1 and IGF-2 have insulin - like activities as indicated by the chosen name ofthe peptide, and are mitogenic for

-?- the cells in reproductive tissue, muscle, skeletal tissue and a wide variety of other tissues. The IGF- 1 and -2 peptides were originally named somatomedins indicative of their growth promoting or mitogenic effect.

Unlike most other growth factors, the IGF's are present in high concentrations in the circulation, but only a small fraction of IGF is not protein bound. The overwhelmingmajority of IGF circulates as part of a non- covalently associated ternary complex composed of IGF- 1 or IGF-2, and insulin like growth factor binding protein-3 (IGFBP-3), and a large protein termed the acid labile subunit (ALS). This complex is composed of equimolaramounts of each ofthe three components. The ternary complex of IGF plus IGFBP-3 plus ALS has a molecular weight of approximately 150,000 daltons, and it has been suggested that the function of this complex in the circulation may be to serve as a reservoir and buffer for IGF- 1 and IGF-2 preventing rapid changes of free IGF-1. Although IGF- 1 is produced in many tissues, most circulating IGF- I is believed to be synthesized in the liver.

IGF- 1 may be purified from natural sources or produced from recombinant sources. For instance. IGF- I can be purified from human serum (Rinderknecht and Humbel J Biol. Chem. 253:2769-2773 [19781). Recombinant IGF-1 processes are exhibited in Patent EPO 128,733, published in December 1984. When recombinant human IGF- 1 was first given to normal human subjects a suppression of blood insulin levels was observed (Guler et al.NEJM.3 17: 137-140[ 1987] ). The most pertinent data was demonstrated in the studies carried out in Type II diabetics by Schlach et al. (J, Clin.Metab.77: 1563- 1568f 19931) which demonstrated a fall in both serum insulin as well as a paralleled decrease in C peptide levels which indicated a fall in pancreatic insulin secretion after five days of IGF- 1 treatment. This effect has been independently confirmed by Froesch et al. fHorm.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.135:2144-2149f 19941). In addition in pancreas perfusion preparations IGF- 1 also suppresses insulin secretion (Leahy et al.. Endocrinology. 126: 1593- 1598[ 1990]) . Despite these clear in vivo inhibitory effect of IGF-1 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-1 and glucose have shown various degrees of inhibition of insulin secretion e.g. from no effect ( Sreradzcri et al.. J. Endocr.1 17 59-62 [ 1988]) to a 30% decrease in insulin release utilizing physiological levels of IGF- 1 (Van Schravendijk et al., Diabetologia. 33: 649-53 [ 1990]). In a recent study using human pancreatic islets Eizirik et al. (Eur.J. Endocr.133:248-25011995]) found no effect of IGF- I on medium insulin accumulation or on glucose -stimulated insulin release. The investigators speculate that the effect of IGF- 1 seen in vivo on insulin secretion may be secondary to the extra- pancreatic effects of IGF- 1 rather than its direct effects on the pancreas.

Therefore the mode and site of action of IGF- 1 on insulin secretion is not fully understood. The only published data showing interactions between IGF- 1 and the sulfonylurea is by Wang et al. (Diabetologia.30:797-803f l 9871 ι who studied the combination of IGF- 1 and tolazamide in vitro on the L6 skeletal muscle cell line. As discussed above, there is an ongoing debate as to the relevance of sulfonylurea action on extra- pancreatic tissues such as skeletal muscle especially due to the lack of high affinity binding sites for sulfonylu rea on such tissues. These investigators did not study the effects of sulfonylurea and IGF- 1 on pancreatic cells or explanted pancreatic tissues. Attempts have been made to measure the IGF- 1 system in patients treated with sulfonylureato discover if sulfonylurea failure in patients may be due to some difference in these patients' IGF- 1 system Grunberger evaluated insulin and IGF- 1 receptor function in Type II diabetic patients who either did or did not respond to glvbuπde treatment (J Lab Clin Med 121.534-535f l 9931) There were no parameters in the IGF-1 system that would predict whether a patient would respond or continue to respond to a sulfonylurea This study supplies no guidance as to whether or not IGF- 1 treatment could improve sulfonylurea activity or prevent sulfonylurea failure

Combinations of sulfonylurea and insulin have been widely tested in Type II diabetics but do not produce d consistent improvement in overall glycemic control (Lebovitz.In Diabetes Mel tus. 4th edition, Eds Rifkm and Porte,Elsevιer,New York,p554-574, 1990) A combination of daytime sulfonylurea (glipizide) and bedtime insulin therapy modestly improved glycemic control in a group of patients who had previously exhibited sulfonylurea failure (Shank et al .Diabetes 44.165- 172[ 1995]) but in a review of the studies designed to investigate combined insulin-sulfonylurea therapy the conclusion was that there was only modest overall improvement (Pugh et al .Diabetes Care 15.953-959[ 1992]) Furthermore this improvement was noted mainly in a sub-popu lation of diabetics, namely obese patients with high fasting C peptide levels Another review article (Peters and Davidson.Ann Int Med 1 15.45-53[ 1991 ]) concludes that combination therapy with insulin and a sulfonylurea should not be used in poorly controlled Type II diabetics This was the type of patient selected in the clinical study referred to in Example 3 of this invention Thus, there is clearly no consensus in the clinical management of diabetes that insulin combined with a sulfonylurea has a marked beneficial effect in diabetics in general and in contrast there is a belief that the combination is of no benefit in the poorly controlled situation

Thereforethe teratureon treatingwith combinationsof insulin and sulfonylureaprovides no guidance as to the efficacy of combinationsof IGF- 1 and sulfonylurea In addition, the medical management of diabetic patients who have failed sulfonylureatreatment would not be expected to be improved by combination treatment with IGF- I and sulfonylurea

SUMMARY OF THE INVENTION This invention provides a method for treating diabetes in a mammal comprising administering to the mammal an effective amount of a hypoglycemic agent, preferably an insulin secretagogue ofthe sulfonylurea class, with an effective amount of IGF- 1 so as to improve glycemic control and limit the progression of the disease

The literature describes a complex role for insulin secretagogues m controlling blood glucose in diabetes For agents of the sulfonylureaclass there is much debate as to the sιte(s) of their action in regulating insulin activity and controllingblood glucose IGF-1 is being investigated as a treatment for diabetes as it has direct hypoglycemic activity However, IGF-I has been reported to have the opposite effect to sulfonylurea on insulin secretion Sulfonylurea stimulate insulin secretion whereas IGF- 1 inhibits insulin secretion There is debate as to the site of action and the mechanism of inhibition of insulin secretion by IGF-1 This confusion provides no clear basis to predict the effect of the co-administration of insulin secretagogues and IGF- I on insulin secretion or glucose control

In many patients the insulin secretagogues either fail initially (primary failure), or fail subsequently

(secondary failure) to regulate insulin secretion and control blood glucose In such patients combination therapy with insulin has been attempted, but controversy exists as to whether there is any advantage to administering insulin secretagogues in combination with insulin It was therefore unclear whether the combination of IGF- 1 and an insulin secretagogue would be effective in either primary or secondary insulin secretagogue failure

Unexpectedly, when tested directly on isolated, cultured pancreatic islets of Langerhans, IGF- 1 did not oppose the insulin release caused by an insulin secretagoguesuch as a sulfonylurea Even more surprisingly, when islets were cultured in the setting of long- term high glucose exposure in the presence of a sulfonylurea, IGF- I had the opposite effect in that it actually stimulated rather than inhibited insulin secretion Further. IGF- l was tested during secretagogue failure in animals and in human diabetics In animals, in a situation where glucose tolerance was worsened by sulfonylurea, the co-administrationof IGF- 1 was found to prevent the failure and maintain glucose tolerance In human studies, diabetics who were poorly controlled while on either sulfonylurea or insulin therapy and were thus deemed treatment failures, were then treated with IGF- 1 In the patients previously treated with an insulin secretagogue, in this case a sulfonylurea, there was a surprising!) large, dramatic, and highly significant improvement in glycemic control compared to that seen in patients previously treated with insulin

Thus, IGF-1 therapy can be combined with insulin secretagogue therapy to improve the ongoing regulation of blood glucose during the progression of diabetes Additionally, when the insulin secretagogue exhibits a failure to adequately regulate glucose, IGF- 1 can be used eitherto prevent this failure from occurring or to restore glycemic control if failure has already occurred

BRIEF DESCRIPTION OF THE DRAWINGS FIG 1 showsa bar graph ofthe effect of glucose and tolbutamide on insulin secretion over a 24 hour period by isolated rat pancreatic islets (means +/-SE)

FIG 2 showsa bar graph ofthe effect of glucose and IGF- 1 on insulin secretion over a 24 hour period by isolated rat pancreatic islets (means+/-SE)

FIG 3 shows a bar graph ofthe effect of the combination of IGF- 1 and tolbutamideon insulin secretion, over a 24 hour period (means +/-SE) FIG 4 representsthe % change in blood glucose over 90 minutes following a bolus administration of glucose after a fast (a glucose tolerance test) in one group of normal rats pretreated with alcohol as placebo (control) or in a group of rats pretreated with tolbutamide at either 25 or 125 mg kg (means+/-SE)

FIG 5 represents the % change in blood glucose over 90 minutes in normal rats, following a glucose tolerance test, either pretreated with alcohol as placebo (control), or pretreated with tolbutamide at 25 mg kg (means +/-SE) FIG 6 represents the % change in blood glucose over 90 minutes in normal rats follow ing a glucose tolerance test either pretreated with alcohol as placebo (control) or pretreated with tolbutamide at 50 mg/kg (means+/-SE)

FIG 7 represents the % change in blood glucose over 90 minutes in normal rats, following a glucose tolerance test, in a group pretreated with normal rat chow (control) or a group that were pretreated with glyburide mixed in their food for 6 days (means+/-SE , * represents statistical significance, p<0 05)

FIG 8 representsthe serum insulin concentrationstaken during the glucose tolerance test performed in the control and glyburide treated rats depicted in FIG 7 (means +/SE)

FIG 9 show s a bar graph representing basal serum blood glucose (mg%) and insulin concentration (ng/m l) in four groups of rats, control group (placebo continuously administered-no oral glyburide), IGF-1 treated group no glybuπde.a glyburide treated group with no IGT-l treatment and a group treated with both glyburide and IGF- 1 (means+/-SE)

FIG 10 representsthe %change in blood glucose over 120 minutes, following a glucose tolerance test, in the four groups of rats depicted in FIG 9 (means+/-SE) after 3 days ofthe study FIG 1 1 represents the serum insulin concentration over the same 120 minutes following the glucose tolerance test given to the four groups of rats depicted in FIG 9 (means+/-SE) after 3 days of study

FIG 12 representsthe %change in blood glucose over 120 minutes, following a glucose tolerance test given to four groups of normal rats, a control group (placebo continuously administered for 14 days-no oral glipizide), a des( l -3)IGF-l treated group with no glipizide, a glipizide treated group with no des( l -3)IGF- l treatment and a group treated with both des( l -3)IGF- I and glipizide after 3 days of treatment (means+/-SE)

FIG 13 represents the serum insulin concentration over the same 120 minutes following the glucose tolerance test given to the four groups of rats depicted in FIG 12 (means+/-SE) after 3 days of study

FIG 14 represents the blood glucose over 120 minutes, following a glucose tolerance test, in all four groups of rats depicted in FIG 12 (means+/-SE) after 7 days of study FIG 15 represents the change in Hemoglobin A l c(%) over 12 weeks in Typell Diabetic patients being treated with IGF-I subsequent to a period of treatment with an oral hypoglycemic agent (HA).group p is the placebo treated group, groups 1, 2, 4 and 8 are the IGF- 1 treated groups, (being 10,20,40,80 μg/kg,respectι vely) (means+/-SE)

FIG 16 represents the change in Hemoglobin A lc(%) over 12 weeks in Typel I diabetic patients being treated with IGF-lsubsequentto a period of treatment with insulin (mean+/-SE) Groupdesignationsare the same as those depicted in FIG 15

DESCRIPTION OF THE PREFFRRFD EMBODIMENTS A Definitions

As used herein, "mammal" signifies humans as well as other mammals, and includes animals of economic importance such as bovine, ovine, and porcine animals The preferred mammal herein is a human As used herein, "IGF-1 " refers to insulin-like growth factor- 1 from any species, including bovine, ovine, porcine equine and preferably human, in native-sequence or in variant form, and from any source, whether natural, synthetic, or recombinant Preferred herein for animal use is IGF-I from the particular species being treated, such as porcine IGF-I to treat pigs, ovine 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 methionine, prepared, e g , by the process described in EP 230,869 published Aug 5, 1987, EP 128,733 pub shed Dec 19, 1984 or EP 288,451 published Oct 26, 1988 More preferably, this native-sequence IGF-1 is recombinantly produced and is available from Genentech, Ine , South San Francisco, CA for clinical investigations

The most preferred IGF-I variants are those described in PCT WO 87/01038 published Feb 26, 1987 and in PCT WO 89/05822 published June 29, 1989, i e , those wherein at least the glutamic acid residue is absent at position 3 from the N-terminus ofthe mature molecule or those that have a deletion of up to five amino acids at the N-terminus The most preferred vaπant has the first three amino acids from the N-termmus deleted

(variously designated as brain IGF, tIGF-I, des(l-3)-IGF-l or des-IGF-I)

As used herein, "treatment"refers to therapeutic and prophylactic treatment Those in need of treatment include those already with the disorder as well as those in which treatment ofthe disorder has failed As used herem,"dιabetιc"refers to a progressive disease of carbohydrate metabolism involving inadequate production or utilization of insulin and is characterized by hyperglycemia and glycosuπa

As used herein, "hypoglycemic agent" is a secretagogue, preferably an oral agent, excluding insulin, which causes the secretion of insulin by the pancreas More preferred herein for human use are the sulfonylurea class of oral hypoglycemic agents Examples include glyburide, glipizide and gliclazide B Modes for carrying out the invention

The IGF-I is directly administered to the mammal by any suitable technique, including parenterally, intranasal ly, orally, or by any other effective route Examples of parenteral administration include subcutaneous, intramuscular, intravenous, intraarterial, and intraperitonealadministration Most preferably, the administration is by continuous infusion (using, e g , minipumps such as osmotic pumps), or by injection (using e g , intravenous or subcutaneous means) Preferably, the administration is subcutaneous and by injection for IGF-1 The administration may also be as a single bolus or by slow-release or depot formulation

In addition, the IGF-1 is suitably administered together with one of its binding proteins for example, IGFBP-3. which is described m WO 89/09268 published Oct 5, 1989 and by Martin and Baxter. J Biol Chem 261 8754-8760 ( 1986) The IGF-I may also be suitably coupled to a receptor or antibody or antibody fragment for administration

The treatment regimen or pattern of administration of the agents may be one of simultaneous administration with the hypoglycemic agent and the IGF-1 In addition, the treatment regimen may be phasic with an alternating pattern of administration of one agent followed at a later time by the administration ofthe second agent Phasic administration includes multiple administrations of one agent followed by multiple administrationsof the second agent The sequence that the agents are administered in and the lengths of each period of administration would be as deemed appropriate by the practitioner

As a general proposition, the total pharmaceutically effective amount of IGF-I administered parenterally per dose will be in the range of about 10 μg/kg/day to 200 μg/kg/day of patient body weight. although this will be subject to therapeutic discretion If given continuously, the IGF-I is typically administered at a dose rate of about 0 5μg/kg/hour to about 10 g/kg/hour, either by 1 -2 injections per day or by continuous subcutaneous release, for example, using a minipump, patch, implant, or a depot formulation The IGF- 1 is also suitably admintsteredby sustained- release systems Suitable examples of sustained- release compositions include semi-permeable polymer matrices in the form of shaped articles, e g films, or microcapsules Sustained-release matrices include polylactides (U S Pat No 3,773,919), copolymers of L- glutamic acid and gamma-ethyl-L-glutamate (Sidman et al , Biopolvmers 22 547-556[ 1983]). poly (2- hydroxyethylmethacrylate) (Langer et al J Biomed Mater Res 15 167- 277 [ 1981 ]), ethylene vinyl acetate (Langer et al ,sιψrά) or poly-D-(-)-3 hydroxybutyπc acid (EPl 33,988) Sustained-release IGF- 1 compositions also include posomally entrapped IGF-1 Liposomes are prepared by methods known per se DE3,218, 121 ,U S Pat Nos 4,485,045 and 4,545,545 Ordinarily, the liposomes are of small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol % cholesterol, the selected proportion being adjusted for the optimal IGF- 1 therapy

For parenteral administration, in one embodiment, the IGF- 1 is formulated by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, l e ,one that is non toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation I he formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides

Generally, the formulations are prepared by contacting the IGF- 1 uniformly and intimately with liquid carriers or finely divided solid carriers or both Then, if, necessary, the product is shaped into the desired formulation Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient Examples include water, saline, Ringers solution, and dextrose solution Non- aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes

The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability Such mateπalsare non- toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts, antioxidants such as ascorbic acid, low molecular weight (less than about ten residues) polypeptides, e g , polyarginine or tπpeptides, proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as poly-vinyl -pyrrolidone, amino acids, such as glycine, glutamic acid, aspartic acid, or arginine, monosaccharides, disacchaπdes, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, counterions such as sodium, and or nonionic surfactants such as po!ysorbates,poloxmers,or PEG The IGF- 1 is typically formulated in such vehicles at a concentration of about 0 1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8 Full length IGF- 1 is generally stable at a pH ofno more than about 6, des( 1-3 )-IGF- l is stable at about 3 2 to 5 It will be understood that use of certain ofthe foregoing excipients, carriers, or stabilizers will result in the formation of IGT- 1 salts

In addition, the IGF- 1 , preferably the full- length IGF- 1 , is suitably formulated in an acceptable carrier vehicleto form a pharmaceutical composition, preferably one that does not contain cells In one embodiment, the buffer used for formulation will depend on whether the composition will be employed immediately upon mixing or stored for later use If employed immediately, the full length IGF- 1 can be formulated in mannitol glycine. and phosphate at pH 7 4 If this mixture is to be stored, it is formulated in a buffer at a pH of about 6 such as surfactant that increases the solubility of the IGF-I at this pH, such as 0 1% polysorbate20 or poloxamer 188 1 he final preparation may be a stable liquid or a lyophilized solid

IGF- 1 to be used for therapeutic use must be sterile Sterility is readily accomplished by filtration through sterile filtration membranes (e g 0 2 micron membranes) Therapeutic IGF- 1 compositions generally are placed into a container having a sterile access port, for example, a vial having a stopper pierceable by a hypodermic injection needle IGF-1 ordinaπlywill be stored in unit or multi-dosecontainers, for example, sealed ampoules 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-1 solution, and the resulting mixture is lyophilized The infusion solution is prepared by reconstituting the lyophilized IGF- I in bactenostatic Water- for-Injection The insulin secretagogue or hypoglycemic agent is administered to the mammal by any suitable technique including parentally, intranasally, orally, or by any other effective route Most preferably, the administration is by the oral route For example M1CRONASE™ 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 1 25 to 20 mg per day, which may be given as a single dose or divided throughout the day as deemed appropriate (PDR.1995.2563-5 ) Other examples of glyburide based tablets available for prescription are GLYNASE™ (Upjohn) drug and DIABETA™ (Hoechst- Roussel) drug GLUCOTROL™ ( Pratt ) is the trademark for a glipizide ( l-cyclohexyl-3-[p-[2-(5- methylpyrazine carboxamιdo)ethyl] phenyl]sulfonyl]urea) 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 sulfonylurea (PDR.1995.1902-3)

The invention will be more fully understood by reference to the following examples They should not, however, be construed as limiting the scope ofthe invention All literature and patent citations are expressly incoφorated by reference

EXAMPLE I Protocol

Rat islets of Langerhans were freshly isolated from pancreata of normal male Sprague Dawley rats (SD) by collagenase digestion followed by batch incubation in a modified RPMI media using a shaking water bath at 37°C under 5% COi - 95% 0₂ After recovery for an hour in media without glucose, islets were incubated in different concentrationsof glucose, recombinant human IGF- 1 (rhIGF- 1 ) or the sulfonylurea, tolbutamide The media were changed hourly, before the experiment, to obtain a basal release rate and then during the experiment (at 1, 2, 3. 20, 21, 22, 23, and 24 h) and insulin secretion was determined by assaying the insulin concentration by RIΛ (Linco Research) in these samples Data are means ± SEM (n^~3/gφ) Results

In the first set of experiments (Figure 1 ) a high dose ( I mM) ofthe insulin secretagogue tolbutamide was added to cultures maintained at physiological glucose concentrations (5mM) and at diabetic glucose concentratιons( 16 mM) The data show the expected initial large insulin secretory response to the high glucose followed by desensitization after 23 h of continual stimulation At this time when the islets were desensitized to glucose, they were challenged maximally with 22mM glucose + 2 5 μM forskolin (cAMP generating agent) As expected, high glucose ( 16 mM) and tolbutamide (ImM) increased insulin secretion Tolbutamide greatly enhanced insulin secretion at 5 mM glucose but had a lesser effect at high glucose concentrations Therefore m this incubation system the expected effects of glucose and secretagogues were observed In a second set of similar experiments (Fιgure2) rhIGF-I ( 13nM and 65nM) had no effect on glucose- stimulated insulin secretion over the first 3 h of incubation These two doses of IGF- 1 (approximately 100 ng/ml and 500 ng/ml) were chosen to cover the range of IGF- 1 concentrations achieved after treatment with effective IGF- 1 doses in vivo However, when the islets were desensitized (shown here at 23 h) to the continuous glucose stimulation. rhIGF-I at 65nM more than doubled glucose ( 16 mM) stimulated insulin secretion ( 1 22=-O 33 vs 2 87±0 42 p<0 05) After 24 h when the islets were challenged with 22 mM glucose and 2 5 μM forskolin, rhIGF-I at both 13nM and 65nM enhanced 5 mM and 16 mM glucose stimulated insulin secretion, reaching significance for high dose rhlGF-I (3 6±0 8 and 10 l±l 34 for 5 and 16mM glucose respectively, vs 6 1±0 96 and 15 3± 1 75 ng /islet, for 5 and 16mM glucose plus rhIGF-1, respectively) I herefore acute exposure to rhlGF- I did not affect glucose- stimulated insulin secretion Unexpectedly, chronic exposure to rhIGF-I, even when islets were desensitized by high glucose, resulted in an increased release of insulin

In a third set of experiments (Figure 3) the combination of tolbutamide and rhIGF- 1 was tested at low and high glucose concentrations The tolbutamide data shown in Figure 1 was confirmed as during the first 3 h of incubation tolbutamide potentiated insulin secretion (p<0 05 at 5 mM glucose) The initial phase of maintained insulin secretion (during the first 3 h) that was stimulated by tolbutamide was not inhibited by the addition of rhIGF- 1 However, after 23 h tolbutamιde+ rhIGr- 1 ( 13 and 65nM) tended to stimulate rather than to inhibit insulin secretion with the combination of 16 mM glucose + 65nM rhlGF- l + 1 mM tolbutamide maintaining greater insulin secretion in these desensitized islets (p<0 05 vs glucose alone) After 24 h, 22mM glucose + 2 5 μM forskolin stimulated greater (p<0 05) insulin secretion from islets treated with tolbutamide + rhlGF- 1 compared to those treated with glucose alone In this experiment the islet insulin content at the end ofthe expenmentwas not significantlydifferentfor the experimental groups, yet there were clear and significant effects of rhIGF- 1 on insulin release during the experiment This suggests that the increased release of insulin caused by IGF- 1 may have been associated with increased net insulin synthesis

It had been suggested, largely on the basis of in vivo studies, that IGF- 1 suppresses insulin secretion by acting directly on the pancreas However, the above data indicate that rhIGF-I at concentrations ( 100-500 ng/ml) in the physiologicalrange, or in the range reached by rhlGF- 1 injections in humans, does not affect acute glucose-stimulatedinsulin secretion Furthermore, rhIGF-I does not antagonize tolbutamide-stimulated insulin secretion at either 5mM or 16mM glucose In addition, rhIGF-I assisted insulin secretion in islets that were desensitized by chronic hyperglycemia This situation mimics, in vιtιo,tiιe diabetic state In this situation rhlGF- 1 also enhanced insulin secretion induced by a maximal stimulation

In the next example these in vitro studies with tolbutamide in normal islets and in islets desensitized by chronic hyperglycemia were extended to animal studies These six animal studies include studies with the hypoglycemic agents tolbutamide, glybeπde and glipizide and full length IGF- 1 and a truncated IGF- 1 (des( l-3) IGF- 1)

EXAMPLE 2

Studies in vivo in the rat

Tolbutamide Studies These experiments studied the short-term effect of tolbutamide administration on blood glucose concentrations before and after a glucose tolerance test (GTT) These studies were designed to extend the in vitro observations and to discover the effect in vivo of tolbutamide administration in the rat

Study 1 Protocol Sixteen male SD rats (average body weight of 250 g) were randomized on body weight into 3 groups and given 2 oral gavages of either, a) 40% alcohol, b) 62 5 mg'ml or 125 mg/kg tolbutamide in 40% alcohol, or c) 12 5 mg ml or 25 mg/kg tolbutamide in 40% alcohol The gavages were given using a gavage needle and a one ml syringe on day zero at 4 PM and the next morning at 9 AM Food was then withdrawn from the animals, to reduce fluctuations in glucose homeostasis due to changes in food intake Two to three hours later, at 1 1 - 12 AM, the rats were anesthetized with ketamme/xylazme The jugular vein was cannulated using MRE-040 tubing and the cannula attached to an automated blood sampling machine Anesthesia was maintained using further doses of ketamine/xylazine After 3 basal blood samples (at - 15, - 10 and -5 minutes) the rats received, at time zero, 1 0 ml of a glucose solution of 500 mg/ml (2 gm/kg) as an intraperitoneal GTT Blood samples were then taken via the cannula using the automated sampling machine at plus 5, 10, 15, 20, 25, 30, 45, 60, 75 and 90 minutes, the samples were centrifuged and plasma was obtained The glucose concentration in the plasma was subsequently determined by a coupled hexokmase procedure using Technicon reagents (Tarrytown New York) on a Chem i Plus clinical chemistry analyzer (Bayer/Miles/Technicon) Results

In the 3 basal blood samples, before glucose administration, the blood glucose concentrations tended to be low er for the groups pre-treated with tolbutamide Following the glucose challenge blood glucose values tended to be lower for the groups pre-treated with tolbutamide, especially for the group treated with the low dose of tolbutamide (25 mg/kg) The low dose had a significant effect at 90 minutes using the absolute glucose concentratιons,and for the percentage change data this difference was statistically significant 45 minutes after the GTT However for the high- dose group no significant effect was seen The data is shown plotted as percentage change in glucose in Figure 4 Two oral gavages of tolbutamidetended to improve glucose clearance as measured by a glucose tolerance test, but this effect showed no clear dose relationship and was only significant at one time point for the low dose of tolbutamide

Study 2 The object of this experiment was to give the low dose of tolbutamide ( 25 mg/kg ), as this dose seemed to cause a response in Study 1 This dose was comparedto a group of rats given only the vehicle Once again, the object was to study the short-term effect of tolbutamideadministrationon blood glucose concentrations and its effects on the glucose concentrations after a glucose tolerance test Protocol Sixteen male SD rats (average body weight of 250 g) were randomized on body weight into 2 groups and given 2 oral gavages of either a) 40% alcohol.or b) 25 mg/kg tolbutamide in 40% alcohol

The gavages were given on day zero at 4 PM and the next morning at 9 AM Food was then withdrawn from the animals to reduce fluctuations in glucose homeostasis due to changes in food intake Two to three hours later at 1 1-12 AM the rats were anesthetizedwith ketamme/ xylazine, and ajugular vein was cannulated using

MRE-040 tubing which was attached to an automated blood sampling machine Anesthesia was maintained using further doses of ketamine/xylazine After 3 basal blood samples (at - 15, - 10 and -5 minutes) the rats received, at time zero, 1 ml of a glucose solution of 500 mg/ml, at 2 g /kg as an intraperitoneal GTT Blood samples were then taken at plus 5, 10, 15, 20, 25, 30, 45, 60, 75 and 90 minutes, the samples were centrifuged and plasma was obtained Glucose was then measured in the samples as described above

Results

There was no clear effect of tolbutamide on the concentrations of glucose in the blood either before or after the GTT The data are shown as absolute blood glucose concentrations in Figure 5 Using two administrations of tolbutamide, given approximately 16 hours apart, there was no consistent effect on blood glucose In Study 1 there was a significant improvement in glucose clearance, whereas in Study 2 using a larger number of animals there was no clear effect of the 25 mg/kg dose of tolbutamide

Study 3 This experiment used a higher dose of tolbutamide (50 mg/kg) than the 25 mg/kg dose used in Study 2 The object was to study the short-term effects of tolbutamideadministrationon blood glucose and the effects on the glucose concentrations after a glucose tolerance test

Protocol

Sixteen male SD rats (average body weight of 200 g) were randomized on body weight into 2 groups

They were given 2 oral gavages of 0 3 ml of either a) 40% alcohol,or b) 50 mg kg tolbutamide in 40% alcohol

The gavages were given on day zero at 4 PM and the next morning at 9 AM Food was then withdrawn from the animals, to reduce fluctuations in glucose homeostasis due to changes in food intake Two to three hours later, at 1 1 - 12 AM. the rats were anesthetized with ketamine/xylazine, the jugular vein was cannulated using MRJΞ-040 tubing and the cannula attached to an automated blood sampling machine Anesthesia was maintained using further doses of ketamine/xylazine After 3 basal blood samples (at - 15, - 10 and -5 minutes) the rats received, at time zero, 1 ml of a glucose solution of 500 mg/ml, at 2 gm/kg as an intraperitoneal GTT Blood samples were taken at plus 5, 10, 15, 20, 25, 30, 40, 50, 60, and 90 minutes, the samples were centrifuged and plasma was obtained Glucose was then measured in the samples as described above Results

There was a clear effect of tolbutamide, with blood glucose concentrationsbemg higher after a glucose challenge in the tolbutamide- treated rats The tolbutamide- treated rats showed a worsening of their glucose tolerance which was statistically significant (p<0 05 by t Test) after 30 and 45 minutes The change in blood glucose (mg/dl) from the 3 basal values is shown in Fig 6

Therefore, at a higher dose than in Study 2, a clear worsening of glucose tolerance was found using two administrations of tolbutamide, and giving a dose of 50 mg/kg

Three separate studies in male rats measured the effects on blood glucose of administering two oral gavages of tolbutamide At low doses there was some evidence of an improvement in glucose clearance whereas at higher doses there was either no effect of tolbutamide or at 50 mg/kg a worsening of glucose tolerance This suggests that in the rat high doses of tolbutamide induce a rapid worsening of glucose control In humans sulfonylurea induces a short-term improvement in glucose tolerance, but in most patients "sulfonylurea failure" then occurs It is possible that the rat very rapidly, in hours rather than years, also exhibits sulphonlyurea failure, modeling the human situation To further investigatethese findings longer-term sulfonylureaadministration was then studied

Glyburide Studies To study the long-term administration of a sulfonylurea, the best experimental approach was to incoφorate the drug into the rat chow so that the rats dosed themselves when eating ad libitum By incoφorating a known amount of drug into the food and measuring food intake it was possible to administer known doses over long periods with much less stress than frequent oral gavages However tolbutamide, is relatively insoluble except at high concentrations of alcohol Therefore, the more potent sulfonylurea glyburide was used

Study 4 First, the effect on blood glucose and insulin of feeding glyburide for six days, mixed in the chow of rats at a dose of 2 5 mg/kg day, was studied Protocol

Eighteen SD rats were housed in individual cages and their intake of po dered food (diet 5001 ) was measured For 9 ofthe rats glyburide was incoφorated into their food so they ate 2 5 mg/kg/day of glyburide The 9 other rats served as the control group as they received the diet without glyburide Every day the rats were weighed and food intake was measured for the 6 days Then after a 6 hour fast while they were conscious the rats were given a glucose tolerance test While the rats were gently restrained blood was sampled from a tail vein and serum separated Glucose and insulin levels were measured as described above Results

Compared to the control group, those receiving glyburide in their food ate a similar amount of chow and gained a similar amount of body weight Blood glucose before the glucose tolerance test tended to be lower in the glyburide- treated group (143 ± 5 mg/dl vs 132 ± 6 mg/dl, p =0 14) However after a glucose tolerance test the glyburide treated rats showed a very large rise in blood glucosecompared to the untreated control group indicating that glyburide caused a clear worsening of glucose tolerance For absolute blood glucose this difference was statistically significant after 30 and 60 minutes, while on a percentage change basis this difference was significant at 30, 60, 90 and 120 minutes The percent change in blood glucose is shown in Figure 7 Insulin concentrations were also measured in all the blood samples Figure 8 shows the insulin concentrations before and after the glucose tolerance test

After six days treatment with glyburide, basal insulin concentrations tended to be lower, although not significantly so However, after the GTT, insulin concentrations in the glyburide- treated rats failed to rise to the degree seen in the control group After 30 minutes the concentrations in the control group were 8 6 ± 1 2 ng/ml vs 5 5 ± 0 5 ng/ml in the glyburide group, this difference was significant, p=0 025 Therefore, using the second generation sulfonylurea.glybuπde, similar effects were seen to those using tolbutamide Glyburide was given in the food so that the exposure was more continuous than the bolus-dose exposures with the gavages used in Studies 1 to 3 Despite this difference in the pattern of exposure, a similar effect on glucose control was seen In addition, an explanation for the worsening of glucose tolerance was found in that the ability ofthe pancreas to release insulin was found to be impaired An inappropriately low amount of insulin was secreted for the imposed glucose load from the GTT Once again, a state of sulfonylurea failure was induced in this example in rats treated with glyburide

Study 5 Now that a dose-regimen, a dose and length of exposure needed to induce sulfonylurea failure in the rat had been established, concomitant IGr- 1 treatment was tested Protocol

Twenty-eight SD rats (250 g) were housed in individual cages and their intake of powdered food (diet 5001) was measured Glyburide was incoφorated into the food to give a dose of 2 5 mg/kg/d of glyburide, or the rats were fed the 5001 diet without glyburide Osmotic minipumps (Alzet 2001™pumps, Alza Palo Alto CA) were implanted sub-cutaneously into the rats to deliver IGF- 1 at a dose of 670 μg/rat/d or 2 5 mg/kg/d, while other pumps were filled with the IGF-1 excipient There were 4 groups of rats,

1 ) Excipient in food, excipient in pumps,

2) Excipient in food. IGF-I in pumps,

3) Glyburide in food excipient in pumps and 4) Glyburide in food, IGF-1 in pumps

For 7 days the rats were weighed daily and food intake was measured Then after 3 and 7 days the rats, while conscious, were given a GTT after a 6 hour fast Blood was sampled from a tail vein while the rats were gently restrained Serum insulin levels were measured by RIA (Linco Research) and glucose levels measured as described above

Results

After 3 days the blood glucose concentrations before the GTT were lower in the glyburide treated rats ( 158 ± 5 mg/dl in control vs 133 ± 3 mg/dl, p<0 05). were not affected by IGF- 1 , and tended to be lower in the group treated with both drugs ( 141± 13 mg/dl) The low blood glucose before the GTT in the glyburide rats was associated with a higher blood insulin concentration (control, 3 4 ± 1 0 vs glyburide, 5 4 ± 0 4 ng ml p<0 05) The basal blood glucose and insulin concentrationsare shown in Figure 9 Figure 10 shows the GTT data, and Figure 1 1 the insulin concentrations after the GTT given 3 days following the beginning of drug dosing Glucose tolerance was worsened by glyburide 30 and 60 minutes after the GTT The insulin concentrations in the glyburide treated rats did not rise with a glucose challenge By itself. IGF- 1 had little effect on glucose tolerance However when IGF- I and glyburide were given in combination, IGF- 1 reversed the deterioration in glucose tolerance caused by glyburide Therefore, although IGF- 1 caused a reduction in basal insulin levels, it restored insulin secretion in response to a glucose load in glyburide- treated rats The GTT was then repeated after 7 days of treatment By this time the response to the failure of glyburide was more evident in terms of glucose clearance Basal insulin secretion was significantly suppressed by IGF- 1 alone but not by the combination of IGF- 1 and glyburide After the GTT, although glyburide allowed insulin secretion, glucose tolerance was dramatically impaired This suggests that the sulfonylurea failure is not totally dependent on a pancreatic mechanism Glucose clearance was not affected by IGF- 1 compared to that of controls, but the combination of glybuπdeand IGF- 1 showed an improvement in glucose clearance compared to glyburide alone

This experiment produced suφrising results Insulin secretion was stimulated by glyburide after a 6- hour food withdrawal and blood glucose concentrations were reduced IGF-1 suppressed basal insulin concentrations m the presence of glyburide, but by itself had no effect on basal blood glucose Despite the rise in basal insulin concentrations, indicating that glybuπdewas stimulating insulin secretion, glyburide suppressed the insulin secretory response to a simulated meal (the GTT) and compared to control animals worsened glucose tolerance IGF- 1 plus glyburide, despite suppressing basal insulin levels, allowed an insulin response to the GTT and improved glucose tolerance compared to treatment with glyburide alone It was suφrising that the sulfonylureas would stimulate insulin release in the fasted state yet would not allow release after a glucose challenge IGF- 1 , which is generally thought to inhibit insulin secretion and did so in the fasted state, equally suφrisingly allowed insulin secretion to be recovered in response to the GTT both in the presence and the absence of the sulfonylurea

Study 6 The activity of glipizide, another second- generation sulfonylurea, and of another form of IGF- 1 , the truncated IGF-1 des ( 1-3) IGF- 1 , were tested alone or together in the rat Protocol

Twenty-eight SD rats (250 g) were housed in individual cages and their intake of powdered food (diet 5001 ) was measured Glipizide was incoφorated into the food at a concentration to give 2 5 mg/kg/d of glipizide or the rats were fed the 5001 diet without glipizide Osmotic minipumps (A et 2002™pumps, Alza Palo Alto CA) were implanted sub-cutaneously into the rats to deliver des( l -3)IGF- l at a dose of 270 μg/rat/d or i I mg/kg/d, while other pumps were filled with the IGF- 1 excipient T he analog of IGF- 1 , des(l -3)IGF- l , was used at a lower dose than was used for IGF- I in Study 5, as des( l -3)IGF- l is more potent than IGF- 1 There were 4 groups of rats, I ) Excipient in food, excipient in pumps,

2) Excipient in food, des(l-3)lGF-l in pumps,

3) Glipizide in food, excipient in pumps, and

4) Glipizide in food, des(l-3)IGF-l in pumps

For 14 days the rats were weighed daily and food intake was measured At 3, 7 and 14 days after a 6 hour fast, while conscious the rats were given a GTT and blood was sampled from a tail vein while the rats were gently restrained Serum insulin levels were measured by RIA (Linco Research) and glucose levels measured as described above Results

After 3 days the blood glucose concentrations before the GTT were lower in the glipizide- treated rats ( 171±7 mg/dl in control vs 133 ± 4 mg/dl, p<0 05), were not affected by des( l -3)IGF- l , and were not reduced in the group treated with both drugs (167± 12 mg/dl) The low blood glucose before the GTT in the glipizide rats was not associated with a change blood insulin concentration (control, 1 4 ± 0 14 vs glipizide, 1 3 ± 0 14 ng ml). but des( l-3)IGF- l reduced basal insulin levels

Figure 12 shows the GTT data, and Figure 13 the insulin concentrations after the GTT given 3 days following the beginning of drug dosing Glucose tolerance was worsened by glipizide at 30 and 60 minutes

By ιtselfdes( l -3)IGF-l had little effect on glucose tolerance However when des(l -3)IGF- l and glipizide were given in combination .IGF- I reduced the deterioration in glucose tolerance caused by glipizide The insulin concentrations in the glipizide- and des( 1 -3 )1G F- 1 treated rats did not rise with a glucose challenge Although

IGF- 1 and glipizide caused a reduction in insulin levels, only glipizide reduced glucose clearance glipizide and des( 1 -3)IGF- 1 plus glipizide (control, 154 ± 7 mg%, des( 1 -3)1GF- 1 , 148± 6 mg%, glipizide, 127± 6 mg%, IGF- 1 + glipizide, 131 ± 7 mg%) At this time there was no effect of the treatments on basal insulin secretion After the GTT, although glipizide allowed insulin secretion, glucose tolerance was dramatically impaired Once more this suggests that the sulfonylurea failure is not totally dependent on a pancreatic mechanism Glucose clearance was not affected by des(l-3)IGr-l compared to that of controls, but insulin levels were suppressed This experiment confirms and extends the above findings using tolbutam ide, glyburide and full-length IGF-1

EXAMPLE 3

Clinical data Treatment of Type II diabetic Patients with rhIGF- 1 following treatment with insulin or a hypoglycemic agent Two hundred and twelve Type II diabetic patients who were in poor glycemic control (Hemoglobin

A lc [HbAlc] average of 10) were taken off all other drug treatments for fourteen days (prior treatment wash out period) They were then randomly assigned to receive either placebo (n=43) or one of four doses of rhlGF- 1 The doses were 10 μg/kg (n =41 ) or 20 μg/kg (n=44), 40μg/kg/day (n=41 ) or 80 μg/kg/day (n=43) twice a day by s/c injection The treatment period was twelve weeks Patients had either previously been treated with insulin or with a hypoglycemic agent, in most cases glyburide The numbers of patients previously treated with a hypoglycemic agent were, placebo(n=24), 10 g/kg/day (n=21 ), 20 g/kg/day(n=26), 40μg/kg/day(n=17), 80μg/kg/day(n=22) The numbers of patients previously treated with insulin were, placebo (n= 19) 10μg/kg/day (n=20), 20 μg/kg/day (n=l 8), 40μg kg/day (n=24) and 80μg/kg/day (n=21 ) Drop- out rates during the course of the study were about equivalent between the pretreatment groups, although there was a dose related difference

Results indicate that the patients pretreated with an oral hypoglycemic agent (Figure 15) followed by IGF-I treatment were in better glycemic control than the group that had received insulin (Figure 16) followed by IGF- 1 treatment This is depicted by a dose- related fall in hemoglobin A I C, the best indicatorof successful medical management of diabetes (Crofford Ann Rev Med 46.267-279[ 1995]) In the high- dose group (80 μg/kg rhlGF- 1 per day) and the placebo group there was a high drop- out rate due to an increased incidence of undesirable side effects, whereas at the lower doses of IGT-I drop- out rates were low, indicative of the beneficial effects of treatment Improved glycemic control would be a desired effect in patients currently on hypoglycemictherapy in that it would prolong this period of management of their diabetes before resorting to exogenous insulin treatment by daily injections In patients exhibiting failure of therapy such as those selected for this study, therapy with IGF- 1 is clearly beneficial A direct result of this beneficial effect would be a reduction in the required oral hypoglycemicdose, thus reducing dose- related side effects as well as potentially delaying failure of oral hypoglycemic treatment

Epidemiologic data show that long-term hyperglycemia increases the risk that diabetic subjects will develop complications Thus, improvement of glycemic control, as measured by a fall in the hemoglobin A le level in this example, would be predicted to decrease the risk of the complications of diabetes The Diabetes Control and Complications Trial (DCCT) reviewed by Crofford. Annu Rev Med 46 267-79 ( 1995), demonstrated that intensive treatment of patients with insulin-dependent diabetes mellitus can substantially reduce the onset and progression of diabetic complications such as retinopathy, nephropathy, and neuropathy Therefore, the use of the combination of IGF-1 and hypoglycemic agents would be expected to have such beneficial effects in patients with diabetes Conclusions The preceding Examples, which include studies in vitro, in animals, and in humans, consistently show the unexpected advantages of the combination of IGT- l and hypoglycemic agents

In Example I , using cultured pancreatic islets of Langerhans, IGF- 1 did not oppose the insulin release caused by an insulin secretagogue sulfonylurea In fact, when the islets were cultured in the setting of high glucose exposure and in the presence of a sulfonylurea, to mimic the diabetic state, IG F- 1 had the opposite effect to that predicted in that it stimulated rather than inhibited insulin secretion The in vitro data would therefore predict that the combination treatment of IGF-1 and an insulin secretagogue would improve glycemic control in diabetic patients In Examples 2 and 3 IGF-1 was tested during secretagogue failure in animals and in humans In animals, a situation of secretagogue failure was induced in that glucose tolerance was worsened by a sulfonylurea In this situation the co- administration of IGI - I with a sulfonylurea was found to ameliorate the failure and maintain glucose tolerance These data in animals predict that combination treatment with IGF- I and an insulin secretagogue would improve glycemic control in diabetic patients

In Example 3 a study in Type II diabetic humans produced very compelling evidence supporting the use ofthe combination of IGF- 1 and oral hypoglycemics In this human study diabetics who were being treated with either sulfonylureaor insulin and were poorly controlled, and therefore were considered treatment failures, were then treated with IGF-1 In the patients previously treated with sulfonylurea there was a dramatic and suφrisingly large improvement in glycemic control compared to that in patients previously treated with insulin Thus, IGF- 1 therapy can be combined with insulin secretagogue therapy to improve the regulation of blood glucose in diabetes Additionally, when the insulin secretagogue shows a failure to regulate glucose, IGF- 1 can be used either to prevent this failure from occurring or to restore glucose regulation if failure has already occurred Thus treating diabetics with IGF- 1 and an insulin secretagogue would be an improvement on current medical practice

Claims

WHAT IS CLAIMED

1 A method for treating diabetes in a mammal comprising administering to the mammal an effective amount of a hypoglycemic agent and of insulin-like growth factor- 1 (IGF- 1 )

2 The method of claim 1 wherein the mammal is human

3 The method of claim 1 wherein the effective amount of IGF-I administered on a daily basis is about 1 O g/kg/day to 200μg/kg/day given as a single or twice daily injection or by utilizing a continuous delivery device typically administered at a dose rate of about 0 5μg/kg/hr to about l Oμg/kg hr

4 The method of claim 1 wherein the hypoglycemic agent is a sulfonylurea

5 The method of claim 1 wherein the hypoglycemicagent is administered separately from the IGF-I

6 The method of claim 1 wherem the hypoglycemicagent is administeredconcurrently with the IGF-1

7 The method of claim 1 wherein the mammal, prior to the administration of IGF- 1 , was treated unsuccessfully with a hypoglycemic agent alone

8 The method of claim 1 wherein the IGF-I is the analog des( l -3) IGF-1