United States Patent r19J
`Young et al.
`
`111111111111111111111111111111111111111111111111111111111111111111111111111
`US005234906A
`5,234,906
`[11J Patent Number:
`[45] Date of Patent: Aug. 10, 1993
`
`[54] HYPERGLYCEMIC COMPOSITIONS
`Inventors: Andrew Young, San Diego; Garth J.
`[75]
`S. Cooper, Solana Beach, both of
`Calif.
`{73] Assignee: Amylin Pharmaceuticals, Inc., San
`Diego, Calif.
`[21] Appl. No.: 640,478
`Jan. 10, 1991
`[22) Filed:
`[51) Int. Cl.5 .............................................. A61K 37/28
`[52] U.S. Cl .......................................... 514/12; 514/21
`[58) Field of Search ................... 514/12, 21, 808, 866,
`514/884
`
`(56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`4,826,763 5/1989 Norris et al. ....................... 435/69.4
`
`FOREIGN PATENT DOCUMENTS
`309100 3/1989 European Pat. Off ..
`
`OTHER PUBLICATIONS
`Leighton et al, "Pancreatic Amylin and Calcitonin
`Gene-Related Peptide Cause Resistance to Insulin ...
`", Nature 335: 632-35 (1988).
`Yamaguchi et al, "Calcitonin Gene-Related Peptide·
`and Induction of Hyperglycemia ... ", Diabetes 39:
`168-'74 (Feb. 1990).
`Molina et al, "Induction of Insulin Resistance In Vivo
`by Amylin and CGRP", Diabetes 39: 260-65 (Feb.
`1990).
`Cooper et al., Biochem. Biophys. Acta 1014:247-58
`(1989).
`Cooper et al., Proc. Nat'l. Acad. Sci., USA 85:7763-66
`(1988).
`.
`Leighton et al., Diab. Med. 6: Suppl. 2, Al4 (1989).
`Ciraldi et al., Diabetes 39:149A (1990).
`
`Journal of Biological Chemistry
`
`Kreutter et al., Diabetes 39:121A (1990).
`Molina et al., Diabetes 39:260-65 (1990).
`Koopmans et al., Diabetes 39:l0lA (1990).
`Young et-al., Diabetes 39:ll6A (1990).
`V. Marks, "Glucagon in the Diagnosis and Treatment
`of Hypoglycemia," Chapter 55 of Handbook of Experi(cid:173)
`mental Pharmacology, vol. 66/11, P. J. Lefebvre (Ed.)
`(Springer-Verlag 1983).
`Young et al., Am. J. Physiol. 259:E457-61 (1990).
`Leighton et al., Biochem. J. 269-19-23 (1990).
`Yamaguchi et al. Diabetes 39:168-74 (1990).
`Leighton et al., TIBS 15:295-99 (1990).
`Ahren et al., Int'!. Journal of Pancreatology 6: 1-15
`(1990).
`Nishi et al.,
`265:4173-76.
`Clark, Diab. Med. 6:561-67 (1989).
`Cooper et al., Diabetes 1988, pp. 493-496, Larkins,
`Zimmet, and Chisholm (Eds.), (Elsevier Science Pub(cid:173)
`lishers B.V. 1989).
`Cooper et al., Progress in Growth Factor Research
`1:99-105 (1989).
`Johnson et al., New England Journal of Medicine
`321:513-18 (1989).
`Primary Examiner-Jeffrey E. Russel
`Attorney, Agent, or Firm-Lyon & Lyon
`~STRACT
`[57]
`Compositions having amylin or an amylin agonist and a
`glucagon compound, particularly peptide compounds,
`for the control of glucose production in mammals are
`provided. The compositions are useful in the treatment
`of hypoglycemia, including acute hypoglycemic condi(cid:173)
`tions such as those brought on by insulin overdose and
`the overuse of oral hypoglycemic agents.
`
`24 Claims, 7 Drawing Sheets
`
`Gl.UClt,IJi 100µ.g AT <Nr, Alffi.lN KIO,-.g 1J Sir
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`IO.IRS Af1ER IIITRAVENC\JS INJEtB
`
`APOTEX EXHIBIT 1062
`Apotex v. Alkermes
`IPR2025-00514
`
`

`

`U.S. Patent
`
`Aug. 10, 1993
`
`Sheet 1 of 7
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`5,234,906
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`

`

`U.S. Patent
`
`Aug. 10, 1993
`
`Sheet 2 of 7
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`5,234,906
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`

`

`U.S. Patent
`
`Aug. 10, 1993
`
`Sheet 3 of 7
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`5,234,906
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`
`

`

`U.S. Patent
`
`Aug. 10, 1993
`
`Sheet 4 of 7
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`5,234,906
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`

`

`U.S. Patent
`
`Aug. 10, 1993
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`Sheet 5 of 7
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`5,234,906
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`U .s.· Patent
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`Aug. 10, 1993
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`

`

`U.S. Patent
`
`Aug. 10, 1993
`
`Sheet 7 of 7
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`5,234,906
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`

`

`1
`
`HYPERGLYCEMIC COMPOSITIONS
`
`5,234,906
`
`FIELD OF THE INVENTION
`The field of the invention is biology and, more partic(cid:173)
`ularly, the biology of diabetes. The invention relates to
`compositions which comprise amylin or an amylin ago(cid:173)
`nist and a glucagon compound, which are preferably
`polypeptides, for use in the treatment of acute hypogly(cid:173)
`cemia and other hypoglycemic conditions.
`BACKGROUND
`Glucagon was discovered in 1923, two years after the
`discovery of insulin. Chemically unrelated to insulin,
`glucagon is a single-chain polypeptide hormone con- 15
`taining 29 amino acid residues and having a molecular
`weight of nearly 3500. In contrast to insulin, glucagon
`contains no cysteine and, consequently, no disulfide
`linkages. The structure of human glucagon is identical
`to porcine, bovine, and rat glucagon and many current 20
`glucagon preparations are extracted from beef and pork
`pancreas.
`Glucagon secretion, like that of insulin, is controlled
`by the interplay of gastrointestinal food products, hor(cid:173)
`mones, and other factors. Glucagon is secreted from 25
`pancreatic a-cells in response to stimuli which include
`(i) falling blood glucose levels, (ii) the physiological
`increments in amino acids which follow a protein meal,
`(iii) vigorous exercise, (iv) starvation, and (v) hypogly(cid:173)
`cemia. It was discovered as a hyperglycemic factor, 30
`present in pancreatic extracts, which stimulated hepatic
`glycogenolysis (the so-called 'hyperglycemic glycogen(cid:173)
`olytic factor'). Glucagon is reported to exert major
`effects on liver glucose metabolism to increase hepatic
`glucose production, at least through cAMP-mediated 35
`actions, which are exerted both directly, to release glu(cid:173)
`cose from glycogen through stimulation of glycogenol(cid:173)
`ysis, and indirectly, through inhibition of glycogen
`synthesis. During relative hypoinsulinemia, glucagon
`can also stimulate gluconeogenesis. Glucagon is not 40
`considered to. exert physiologically significant effects
`on carbohydrate metabolism in muscle.
`Glucose is physiologically the most important regula(cid:173)
`tor of glucagon. A rise in plasma glucose concentration
`leads to an inhibition of glucagon secretion and vice 45
`versa. Unger, R. H. and Orci, L., "Glucagon and the A
`Cell," N. Eng. J. Med. 304:1518-1524 and 1575-1580
`(1981). Both insulin and somatostatin inhibit the secre(cid:173)
`tion of glucagon.
`The role of glucagon and, in general, its actions are 50
`reported to be antagonistic to those of insulin. Insulin
`serves as a hormone of fuel storage while glucagon is
`reported to serve as a hormone of fuel mobilization.
`Following a carbohydrate meal, pancreatic ~-cells se(cid:173)
`crete insulin and pancreatic a-cell secretion of glucagon 55
`is suppressed; this allows cells to store fuels such as
`glucose in liver, muscle, and adipose tissue. Conversely,
`during starvation, stimulation of glucagon secretion and
`suppression of insulin secretion direct breakdown and
`efficient utilization of fuels stored intracellularly, ini- 60
`tially liver glycogen, and subsequently adipose tissue
`fat, to meet the energy needs of the brain and other
`tissues. A regulated role for glucagon as the hormone of
`injury and insult (catabolic illness) has been proposed.
`For example, impaired glucose tolerance and hypergly- 65
`cemia noted with infection are associated with in(cid:173)
`creased concentrations of plasma glucagon. Similar
`increases are seen in patients with myocardial infarc-
`
`2
`tions, burns, and after major trauma. In these situations,
`glucagon is said to stimulate gluconeogenesis and pro(cid:173)
`vide the glucose needed under conditions of insult.
`Glucagon, therefore, has generally accepted physio-
`5 logical
`roles as a counterregulatory
`(anti-hypo(cid:173)
`glycemic) hormone, and a major regulator of fuel me(cid:173)
`tabolism during starvation. Because of its effect to in(cid:173)
`crease blood glucose levels in individuals with extant
`hepatic glycogen stores, glucagon is widely used clini-
`10 cally in the acute management of severe hypoglycemia
`complicating insulin replacement therapy of insulin(cid:173)
`dependent (type I) diabetes mellitus. Glucagon is par(cid:173)
`ticularly useful in the treatment of insulin-induced hy-
`poglycemia when dextrose (glucose) solution is not
`available or, for example, when a patient is convulsing
`or recalcitrant and intravenous glucose cannot be ad-
`ministered. Glucagon is effective in small doses, and no
`evidence of toxicity has been reported with its use.
`When given, glucagon may be administered intrave(cid:173)
`nously, intramuscularly, or subcutaneously, typically in
`a dose of 1 milligram. Once glucagon is introduced for
`hypoglycemic coma induced by either insulin or oral
`hypoglycemic agents, a return to consciousness should
`be observed within 20 minutes; otherwise, intravenous
`glucose must be administered as soon as possible. Good(cid:173)
`man and Gillman's The Pharmacolooic Basis of The(cid:173)
`raueutics, p. 1510-1512 (7th Ed. 1985).
`Hypoglycemic reactions may occur in any diabetic
`subject treated with insulin or with an oral hypoglyce(cid:173)
`mic agent. Rea_ctions are frequently seen in the labile
`form of the disease, a form characterized by unpredict(cid:173)
`able spontaneous reductions in insulin requirement. In
`other instances, precipitating causes are responsible,
`such as a failure to eat, unaccustomed exercise, and
`inadvertent administration of too large a dose of insulin.
`Frequently, however, there is no discernible cause.
`When the rate of fall in blood glucose is rapid, the early
`symptoms are those brought on by the compensating
`secretion of epinephrine, which includes sweating,
`weakness, hunger, tachycardia, and "inner trembling."
`When the concentration of glucose falls slowly, the
`symptoms and signs are primarily related to the brain
`and include headache, blurred vision, diplopia, mental
`confusion, incoherent speech, coma, and convulsions. If
`the fall in blood glucose is rapid, profound, and persis-
`tent, all such symptoms may be present.
`The majority of the signs and symptoms of insulin
`hypoglycemia are the results offunctional abnormalities
`of the central nervous system, since hypoglycemia de(cid:173)
`prives the brain of the substrate (glucose) upon which it
`is almost exclusively dependent for its oxidative metab(cid:173)
`olism. During insulin coma, oxygen consumption in
`human brain decreases by nearly half. The reduction in
`glucose consumption is disproportionately greater,
`which indicates that the brain is utilizing other sub-
`strates. After prolonged fasting in man the brain adapts,
`and the bulk of the fuel utilized is made up of ketone
`bodies. A prolonged period of hypoglycemia causes
`irreversible damage to the brain. Goodman and Gil/(cid:173)
`man's The Pharmaco/ogic Basis of Therapeutics, p.
`1502-1503 (7th Ed. 1985).
`The symptoms of hypoglycemia yield almost immedi(cid:173)
`ately to the intravenous injection of glucose unless hy(cid:173)
`poglycemia has been sufficiently prolonged to induce
`organic changes in the brain. If the patient is not able to
`take a soluble carbohydrate or a sugar-containing liquid
`such as fruit juice orally and if glucose is not available
`
`

`

`3
`for intravenous injection, glucagon may be given. It
`will be understood, however, that the utility of gluca(cid:173)
`gon in treating hypoglycemia is limited by its inaction
`or ineffectiveness in patients with depleted liver glyco(cid:173)
`gen stores. Since glucagon acts only on liver (but not on
`skeletal muscle) glycogen by converting it to glucose, it
`has no therapeutically useful hyperglycemic effect in
`patients with depleted liver glycogen, a condition
`which cannot be determined in the fitting patient. Thus,
`in the convulsing or comatose patient, glucagon treat- 10
`ment will not alleviate hypoglycemia if the patient has
`no or insufficient liver glycogen to be mobilized. In
`addition to states of starvation, it is also understood that
`glucagon is of little or no help in other states in which
`liver glycogen is depleted such as adrenal insufficiency 15
`or chronic hypoglycemia. Normally, then, intravenous
`glucose must be given if the patient fails to respond to
`glucagon.
`SUMMARY OF THE INVENTION
`The present invention is directed to methods of con(cid:173)
`trolling glucose production in mammals, and to meth(cid:173)
`ods of treating acute hypoglycemia and other hypogly(cid:173)
`cemic conditions, by the co-administration of a gluca- 25
`gon compound and amylin or an amylin agonist. In
`particular, the method comprises the administration of a
`preferred composition comprising a combined glucagon
`and amylin pharmaceutical composition for treatment
`of hypoglycemic conditions. These compositions are 30
`particularly useful in treating those hypoglycemic con(cid:173)
`ditions where the effect of glucagon or amylin alone
`may not be predicted with certainty. In instances of
`severe hypoglycemia, especially with an unconscious or
`comatose patient or animal, it is important to reliably 35
`alleviate hypoglycemia without the necessity of inquiry
`into nutritional status or the presence or absence of
`hepatic glycogen stores.
`The invention also provides for pharmaceutical com(cid:173)
`positions comprising a glucagon compound and amylin 40
`or an amylin agonist together in a pharmaceutically
`acceptable carrier in therapeutically effective amounts.
`
`20
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`The invention will be further described with refer- 45
`ence to the accompanying drawings in which:
`FIG. 1 shows the plasma glucose response (mean±(cid:173)
`SEM, n=6 for each curve) of rats infused with somato(cid:173)
`statin (3.4 nmol/hr) and injected with 66 nmol/kg amy-
`Iin (open circle), peptide control (open square), or phen- 50
`tolamine in a regimen that replicated the blood pressure
`response to amylin (BP control, open triangle). Aster(cid:173)
`isks above the symbols indicate differences between
`amylin-treated and peptide control groups. Asterisks
`below the symbols indicate differences between the 55
`amylin treated and BP control groups.
`FIG. 2 shows the plasma lactate response (mean±(cid:173)
`SEM, n=6 for each curve) for groups described in
`FIG. 1. Symbols and asterisks have the same meaning as
`in FIG. 1.
`FIG. 3A shows the mean arterial blood pressure
`response (2-second means±S.E. indicated by shading)
`for rats injected with amylin (66 nmol/kg), peptide
`control or phentolamine in a schedule designed to repli(cid:173)
`cate the amylin blood pressure response. Subacute 65
`blood pressure response is shown in FIG. 3B as mean
`arterial pressure (30-second means± S.E.). Symbols,
`error bars and asterisks have the same meaning as in
`
`60
`
`5,234,906
`
`4
`FIG. 1. In addition, the acute blood-pressure response is
`plotted at the time of injection.
`FIG. 4 shows isotopically determined, non-steady(cid:173)
`state endogenous (hepatic) glucose production in rats
`5 injected intravenously with 25.5 nmol amylin (open
`circle), peptide control (open square) or phentolamine
`as described for the above figures (open triangle). Sam(cid:173)
`ple numbers and the meaning of symbols, bars and aster(cid:173)
`isks are the same as in FIGS. 1 and 2.
`FIGS. SA and SB show the effects of an intravenous
`injection of 100 micrograms glucagon (0 hours) fol(cid:173)
`lowed by an intravenous injection of 100 micrograms
`amylin (6 hours) on plasma arterial levels of glucose
`(5A) and lactate (5B) in 18-hour fasted rats.
`FIG. 6 shows the effects of an intravenous injection
`of 100 micrograms glucagon (0 hours) followed by an
`intravenous injection of 100 micrograms amylin (6
`hours) on plasma arterial levels of glucose and lactate in
`(-0 -) fed and (-0-) fasted (20±1 hour) rats.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`Diabetes mellitus is a metabolic disorder which is
`defined by the presence of chronically elevated levels of
`blood glucose (hyperglycemia). Dietary carbohydrate
`is absorbed into the bloodstream mainly in the form of
`glucose. The pancreatic hormone insulin stimulates the
`rapid clearance of glucose from the blood by stimulat(cid:173)
`ing glucose oxidation, the conversion of glucose to
`glycogen in skeletal muscle and triacylglycerol in liver
`and adipose tissue and also by suppression of hepatic
`glucose production. Insulin, therefore, plays a funda(cid:173)
`mental role in maintaining blood glucose levels within
`the physiological range.
`1) diabetes mellitus
`Insulin-dependent
`(Type
`("IDDM") results from an autoimmune-mediated de(cid:173)
`struction of pancreatic /3-cells with consequent loss of
`insulin production, which results in hyperglycemia.
`People with Type l diabetes have an absolute require(cid:173)
`ment for insulin replacement therapy in order to ensure
`survival. In marked contrast, non-insulin-dependent
`(Type 2) diabetes mellitus ("NIDDM") is often charac(cid:173)
`terized by hyperglycemia in the presence of higher(cid:173)
`than-normal levels of plasma insulin (hyperinsulinemia).
`Thus, in Type 2 diabetes, tissue processes which control
`carbohydrate metabolism are believed to have de(cid:173)
`creased sensitivity to insulin. Progression of the Type 2
`diabetic state is associated with increasing concentra(cid:173)
`tions of blood glucose and coupled with a relative de(cid:173)
`crease in the rate of glucose-induced insulin secretion.
`The primary aim of treatment in both forms of diabe(cid:173)
`tes mellitus is the same, namely, the reduction of blood
`glucose levels to as near normal as possible. The treat(cid:173)
`ment of Type 1 diabetes necessarily involves the admin(cid:173)
`istration of replacement doses of insulin, which is ad(cid:173)
`ministered by the parenteral route. In contrast, the
`treatment of Type 2 diabetes frequently does not re(cid:173)
`quire the administration of insulin. For example, initial
`therapy of Type 2 diabetes may be based on diet and
`lifestyle changes augmented by therapy with oral hypo(cid:173)
`glycemic agents such as the sulfonylureas. If after an
`adequate trial of diet and lifestyle modification, fasting
`hyperglycemia persists in the Type 2 diabetic patient, a
`diagnosis of "primary diet failure" may be made, and
`either a trial of oral hypoglycemic therapy or direct
`institution of insulin therapy may be required to pro(cid:173)
`duce control of hyperglycemia in an attempt to mini(cid:173)
`mize the complications of the disease.
`
`

`

`5,234,906
`
`5
`Treatment with oral hypoglycemic agents such as the
`sulfonylureas may lead to hypoglycemic reactions, in(cid:173)
`cluding coma, four or more hours after meals. These
`hypoglycemic episodes may last for several days so that
`prolonged or repeated glucose administration is re- 5
`quired. Such hypoglycemic reactions are unpredictable
`and may occur after as little as one dose, after several
`days of treatment, or after months of drug administra(cid:173)
`tion. Most hypoglycemic reactions are observed in pa(cid:173)
`tients over 50 years of age, and are most likely to occur 10
`in patients with impaired hepatic or renal function.
`Over-dosage or inadequate or irregular food intake may
`initiate such hypoglycemic reactions. Other drugs can
`increase the risk of hypoglycemia from sulfonylureas;
`these include other hypoglycemic agents, sulfonamides, 15
`propranolol, salicylates, phenylbutazone, probenecid,
`dicumarol, choloramphenacol, monoamine oxidase in(cid:173)
`hibitors, and alcohol.
`It is noteworthy that, notwithstanding the above(cid:173)
`noted avenues of treatment, insulin therapy remains a 20
`treatment of choice for many patients with Type 2 dia(cid:173)
`betes, especially those who have undergone primary
`diet failure and are not obese or those who have under(cid:173)
`gone both primary diet failure and secondary oral hy(cid:173)
`perglycemic failure. Nevertheless, insulin therapy must 25
`be combined with a continued effort at dietary control
`and lifestyle modification, and in no way can be thought
`of as a substitute for these. In order to achieve optimal
`results, insulin therapy should be combined with self
`blood glucose monitoring and appropriate estimates of 30
`glycosylated blood proteins.
`As with the sulfonylurea agents, hypoglycemia is the
`major adverse effect of insulin therapy and is a primary
`factor preventing the achievement of euglycemic con(cid:173)
`trol in the insulin therapy of Type 1 diabetes. Hypogly- 35
`cemia is by far the most serious and common adverse
`reaction to the administration of insulin, and can result
`in substantial morbidity and even death. Thus, it will be
`understood that the major barrier in striving for eugly(cid:173)
`cemia with intensified regimens of insulin treatment is 40
`the increased risk ·of severe hypoglycemia. Zinman, B.,
`"The Physiologic replacement of insulin. An elusive
`goal," N.Engl. J. Med., 321:363-370 (1989).
`Insulin-induced hypoglycemia is experienced at some
`time by virtually all Type 1 diabetics. In some studies, 45
`severe hypoglycemia (necessitating hospitalization or
`assistance from another person) has been observed in
`25% of all diabetic patients over a one year period. In
`addition, hypoglycemia is reported to account for about
`3-7% of deaths in patients with Type 1 diabetes. Sha- 50
`frir, E., et al. in Felig, P., et al., "Endocrinology and
`Metabolism," pages 1043-1178 (2nd edition 1987). Al(cid:173)
`though rates of hypoglycemic incidents vary among
`individuals, patients undergoing conventional insulin
`therapy suffer an average of about one episode of symp- 55
`tomatic hypoglycemia per week, whereas those practic(cid:173)
`ing intensive insulin therapy suffer about two to three
`such episodes per week. Thus, over a time frame of
`forty years of Type 1 diabetes, the average patient can
`be projected to experience two thousand to four thou- 60
`sand episodes of symptomatic hypoglycemia. Approxi(cid:173)
`mately 10% of patients undergoing conventional insulin
`therapy suffer at least one episode of severe hypoglyce(cid:173)
`mia, i.e., requiring assistance from others, including
`hyperglycemic treatment such as glucose or glucagon 65
`administration and episodes with seizure or loss of con(cid:173)
`sciousness, in a given year. The yearly incidence of
`severe hypoglycemic episodes rises to about 25%
`
`6
`among patients undergoing intensive therapy. Cryer, P.
`IDDM" Diabetes
`E., et al., "Hypoglycemia
`in
`38:1193-1198 (1989).
`The brain has only an extremely limited ability to
`store carbohydrate in the form of glycogen and is al(cid:173)
`most entirely dependent on glucose as its source of
`energy; thus, it is very sensitive to hypoglycemia. Hy(cid:173)
`poglycemia is defined as a blood-glucose level of below
`40 mg/mf; symptoms of cerebral dysfunction rarely
`occur until the glucose content of the cerebral arterial
`blood falls below this level. However, symptoms of
`hypoglycemia may occur even though the blood(cid:173)
`glucose is normal or only minimally reduced, if there
`has been a rapid fall from a much higher level. Severe
`or recurrent episodes of hypoglycemia may result in
`permanent cerebral damage. Thus, treatment of the
`hypoglycemic state represents a medical emergency.
`Amylin is the major protein constituent of the islet
`amyloid which is reported to be found in patients with
`type 2 diabetes mellitus. Human amylin has a somewhat
`unusual amino acid composition in that it contains no
`acidic residues. Amylin is a 37 amino acid peptide hav(cid:173)
`ing two post translational modifications, a Cys2-Cys7
`intramolecular disulfide bond and a carboxy-terminal
`amide group. It has been reported that the presence of
`both of these post-translational modifications in the
`peptide structure of the synthetic molecule yield the
`greatest biological activity to inhibit glycogen synthesis
`in skeletal muscle. Cooper, G. J. S., Willis, A. C., Clark,
`A., Turner, R. C., Sim, R. B. & Reid, K. B. M. Proc.
`Natl. Acad. Sci. USA 84:8628-8632 (1987); Cooper, G.
`J. S., Roberts, A. N., Todd, J. A., Sutton, R., Day, A. J.,
`Willis, A. C., Reid, K. B. M. & Leighton, B. in Diabetes
`1988, ed. Larkins, R., Zimmet, P. & Chisholm, D. (El(cid:173)
`sevier, Amsterdam), pp. 493-496 (1989).
`Human amylin has 43-46% sequence identity with
`human CGRP-1 and CGRP-2 (calcitonin gene-related
`peptides 1 and 2) respectively. Human amylin also has
`weaker sequence similarities with calcitonin, insulin, the
`relaxins, and the insulin-like growth factors (IGFs).
`This observation concerning sequence similarities sup(cid:173)
`ports the determination that there is a peptide hormone
`superfamily which includes calcitonin, the CGRPs,
`amylin, and the A-chain related region of the relaxin,
`insulin and the IGFs. Amylin is reported to be the prod(cid:173)
`uct of a single gene present on chromosome 12 in hu(cid:173)
`mans. This gene has typical features of one encoding a
`polypeptide hormone, including prepro- and proamylin
`sequences, typical 5' and 3' dibasic processing signals,
`and a Gly residue 3' to the codon for the carboxytermi(cid:173)
`nal Tyr, which constitutes an amidation signal. Roberts,
`A. N., et al., Proc. Nat. Acad. ScL U.S.A. 86:9662-9666
`(1989). There is a high degree of interspecies conserva(cid:173)
`tion between both the amylins and the CGRPs, particu(cid:173)
`larly in the region of the amino- and carboxy-termini.
`These regions of strong conservation correspond to the
`structural regions within the molecules which contain
`the post-translational modifications necessary for: at
`least some of their biological activities. The variable
`sequence in the mid-portion of the amylin molecule
`contains the region said to be primarily responsible for
`amyloid formation.
`Amylin is synthesized in the islets (Leffert, J. D.,
`Newgard, C. B., Okamoto, H., Milburn, J. L. & Luskey,
`K. L, Proc. Natl. Acad. Sci. USA 86:3127-3130 (1989)
`and Roberts, A. N., Leighton, B., Todd, J. A., Cock(cid:173)
`burn, D., Sutton, R., Boyd, Y., Holt, S., Day, A. J.,
`Foot, E. A., Willis, A. C., Reid, K. B. M. & Cooper, G.
`
`

`

`5,234,906
`
`7
`J. S., Proc. Natl Acad. Sci. USA 86:9662-9666 (1989)),
`from which it is secreted along with insulin in response
`to nutrient secretagogues. Ogawa, A., Harris, V.,
`Mccorkle, S. K., Unger, R. H. & Luskey, K. L., J. Clin.
`Invest. 85, 973-976 (1990). Deposition of amylin in islet 5
`amyloid correlates well with the loss of islet /3-cells and
`defective insulin secretion found in type 2 diabetics.
`Gepts, W., The Islets of Lanoerhans, ed. Cooperstein, S.
`J. & Watkins, D. (Academic Press, New York, NY), pp.
`321-356 (1980), Fehmann, H. C., Weber, V., Goke, R., 10
`Goke, B. & Arnold, R., FEBS Lett. 262:279-281 (1990)
`and Cooper, G. J. S., Day, A. J., Willis, A. C., Roberts,
`A. N., Reid, K. B. & Leighton, B., Biochim. Biophys.
`Acta 1014, 247-258 (1989). Amylin's ability to cause
`insulin resistance in many model systems, combined 15
`with its presence in human islet amyloid, supports the
`determination that it is central to the pathogenesis of
`non-insulin dependent diabetes mellitus. Cooper, G. J.
`S., Day, A. J., Willis, A. C., Roberts, A. N., Reid, K. B.
`& Leighton, B. Biochim. Biophys. Acta 1014:247-258 20
`(1989) and Leighton, B. & Cooper, G. J. S., Nature
`(Lond) 335:632-635 (1988).
`.
`In skeletal muscle in vitro, amylin has been reported
`to modulate several key pathways of carbohydrate me(cid:173)
`tabolism, including incorporation of glucose into glyco- 25
`gen (Leighton, B. & Cooper, G. J. S., Nature (Lond)
`335:632-635 (1988) and Cooper, G. J. S., Leighton, B.,
`Dimitriadis, G. D., Parry-Billings, M., Kowalchuk, J.
`M., Howland, K., Rothbard, J.B., Willis, A. C. & Reid,
`K. B. M., Proc. Natl. A cad. Sci. USA 85:7763-7766 30
`(1988)), glycogenolysis (Leighton, B., Foot, E. A. &
`Cooper, G. J. S. (1989) Diab. Med. 6: Suppl. 2, A4
`(1989)), and glucose uptake. Ciaraldi, T. P., Cooper, G.
`J. S. & Stolpe, M., Diabetes 39, 149A. (1990) and Kreut(cid:173)
`ter, D., Orena, S. J. & Andrews, G. C., Diabetes 39, 35
`(Suppl. l):121A (1990). The effects ofamylin in skeletal
`muscle depend upon distribution of fiber type. Leigh(cid:173)
`ton, B., Foot, E. A. & Cooper, G. J. S. (1989) Diab.
`Med. 6: Suppl. 2, A4 (1989). While amylin was reported
`to inhibit glycogen synthesis in both red (soleus) and 40
`white (extensor digitorum longus) muscle, it was re(cid:173)
`ported to stimulate glycogenolysis (and subsequent
`lactate production) only in white muscle. Leighton, B.,
`Foot, E. A. & Cooper, G. J. S., Diab. Med. 6, Suppl.
`2:A4 (1989). White (type II) muscle fibers constitute the 45
`bulk of muscle mass in most mammals surveyed.
`Ariano, M. A., R. B. Armstrong, and V. R. Edgerton, J.
`Histochem. Cytochem. 21:51-55 (1973).
`The effects of amylin on glycogen synthesis in iso(cid:173)
`lated red muscle (soleus) were reported equipotent with 50
`those of the pure /3-adrenergic agonist, isoprenaline.
`Leighton, B. & Cooper, G. J. S., Nature (Lond)
`335:632-635 (1988). In L6 myocytes, maximal reduction
`of glucose uptake has been reported at 10 pM. Ciaraldi,
`T. P., Cooper, G. J. S. & Stolpe, M., Diabetes 39:149A 55
`(1990) and Kreutter, D., Orena, S. J. & Andrews, G. C.,
`Diabetes 39 (Suppl. 1): 121A (1990). These effects occur
`at the physiological concentrations of the hormone as
`measured and set forth in the below Examples.
`Amylin also has been reported to produce marked 60
`effects on pathways of glucose metabolism in animals in
`vivo. In experiments utilizing the euglycemic, hyperin(cid:173)
`sulinemic glucose clamp, amylin reversed insulin(cid:173)
`mediated suppression of hepatic glucose output in rats.
`Molina, J.M., Cooper, G. J. S., Leighton, B. & Olefsky, 65
`J.M., Diabetes 39:260-265 (1990) and Koopmans, S. J.,
`vanMansfeld, A. D. M., Jansz, H. S., Krans, H. M. J.,
`Radder, J. K., Frolich, M., deBoer, S. F., Kreutter, D.
`
`8
`K., Andrews, G. C. & Maassen, J. A., Diabetes 39:!0lA
`(1990). Amylin also decreased peripheral uptake of
`glucose. Molina, J.M., Cooper, G. J. S., Leighton, B. &
`Olefsky, J.M., Diabetes 39:260-265 (1990), Koopmans,
`S. J., vanMansfeld, A. D. M., Jansz, H. S., Krans, H. M.
`J., Radder, J. K., Frolich, M., deBoer, S. F., Kreutter,
`D. K., Andrews, G. C. & Maassen, J. A., Diabetes
`39:!0lA (1990) and Young, D. A., Deems, R. 0., McIn(cid:173)
`tosh, R.H., Deacon, R. W. & Foley, J.E., Diabetes 39
`(Suppl. 1 ): 116A (1990).
`As noted above, glucagon owes its place in the treat(cid:173)
`ment of hypoglycemia almost entirely to its ability to
`liberate glucose from the liver by initiating glycogenol(cid:173)
`ysis through activation of liver phosphorylase. Its abil(cid:173)
`ity to accelerate gluconeogenesis, which is probably
`more important in glucose homeostasis, plays little part
`in this action, nor is glucagon reported to have signifi(cid:173)
`cant effects upon peripheral glucose utilization (except
`possibly to accelerate it secondarily to glucagon(cid:173)
`stimulated insulin secretion). The hyperglycemic effect
`of glucagon is abolished or diminished when, for any
`reason, the quantity of glycogen in the liver is reduced
`or is otherwise unavailable for conversion into glucose.
`V. Marks, "Glucagon in the Diagnosis and Treatment
`of Hypo