United States Patent [19J
`Eng
`
`[54] EXENDIN-3 AND EXENDIN-4
`POLYPEPTIDES, AND PHARMACEUTICAL
`COMPOSmONS COMPRISING SAME
`
`[76]
`
`Inventor:
`
`John Eng, 5427 Arlington Ave.,
`Bronx, N.Y. 10471
`[21] Appl. No.: 66,480
`[22] Filed:
`May 24, 1993
`
`[51]
`Int. Cl.6 ...................... A61K 38/16; C07K 14/46;
`Cl2N 15/63
`[52] u.s. Cl •........................................ 514/2; 514/866;
`435/69.1; 530/324
`[58] Field of Search ...................... 514/2, 866; 424/88;
`435/69.1; 530/324
`
`[56]
`
`References Cited
`PUBLICATIONS
`Schmidt et al. 1985. Diabetologia 28:704-707.
`J. Eng & C. Eng, Exendin-3 and -4 are Insulin Secreta(cid:173)
`gogues; Regulatory Peptides 40: 142 (1992).
`Eng, J. et al., Purification and Structure of Exendin-3,
`a New Pancreatic Secretagogue Isolated from Helo(cid:173)
`derma horridum Venom; J. Bioi. Chern. 265:20259
`(1990).
`Raufman, J.-P., Exendin-3 a Novel Peptide from Helo(cid:173)
`derma horridum Venom, Interacts with Vasoactive
`Intestinal Peptide Receptors and a Newly Described
`
`111111111111111111111111111111111111111111111111111111111111111111111111111
`US005424286A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,424,286
`Jun. 13, 1995
`
`Receptor on Dispersed Acini from Guinea Pig Pan(cid:173)
`creas; J. Bioi. Chern. 266:2897 (1991).
`Eng, J. et al., Isolation and Characterization of Exen(cid:173)
`din-4, an Exendin-3 Analogue, from Heloderma suspec(cid:173)
`tum Venom; J. Bioi. Chern. 267:7402 (1992).
`Raufmann, J.-P., et al., Truncated Glucagon-like-Pep(cid:173)
`tide-1 Interacts with Exendin Receptors on Dispersed
`Acini from Guinea Pig Pancreas; J. Bioi. Chern.
`267:21432 (1992).
`John Eng, Exendin Peptides; The Mt. Sinai J. of Med.
`59: 147 (1992).
`Gutniak, M. et al., Antidiabetogenic Effect of
`Glucagon-Like Peptide-1 (7-36) Amide in Normal
`Subjects and Patients with Diabetes Mellitus; The New
`England J. Med. 326:1316 (1992).
`
`Primary Examiner-Gamette D. Draper
`Assistant Examiner-Elizabeth C. Kemmerer
`Attorney, Agent, or Firm-Allegretti & Witcoff, Ltd.
`[57]
`ABSTRACf
`This invention encompasses pharmaceutical composi(cid:173)
`tions containing exendin-3 or exendin-4, fragments
`thereof, or any, combination thereof, and methods for
`the treatment of diabetes mellitus and the prevention of
`hyperglycemia.
`
`7 Claims, 9 Drawing Sheets
`
`SANOFI-AVENTIS Exhibit 1006 - Page 1
`
`IPR for Patent No. 8,951,962
`
`

`
`U.S. Patent
`
`June 13, 1995
`
`Sheet 1 of 9
`
`5,424,286
`
`glucose - mgIdL
`9
`CD -
`
`ID
`CD
`
`-@~glucosei
`
`time-minutes
`
`N
`
`|u1/Bu - uunsug
`
`SANOFI-AVENTIS Exhibit 1006 - Page 2
`IPR for Patent No. 8,951,962
`
`

`
`U.S. Patent
`
`June 13, 1995
`
`Sheet 2 of 9
`
`5,424,286
`
`glucose - mgIdL
`
`OOOOO
`WVMNF
`
`y¢
`
`2040 time-min
`
`Exendin-4 1nmol
`
`1nmol
`
`E.
`
`1 0
`
`‘I’
`.E
`1:
`
`II] 0.1nmol
`
`:ox
`
`0.1nmol
`
`—O—Insulin-6-glucose
`
`

`
`U.S. Patent
`
`June 13, 1995
`
`Sheet 3 of 9
`
`5,424,286
`
`glucose - mgIdL
`
`99609
`HDVNNV
`
`‘F
`.E_
`3%
`‘>"<="'
`I.l.|v'
`
`7'6
`n.E
`a“""’
`
`23
`
`‘OE
`c:
`3'1"’
`mo
`
`3
`v;'E 1
`C
`5‘__>
`0:5
`
`E N
`
`2
`
`.§’
`
`:5 L
`
`§s
`3%
`—:
`a».
`
`|Lu[6u'- uunsug
`
`SANOFI-AVENTIS Exhibit 1006 - Page 4
`IPR for Patent No. 8,951,962
`
`

`
`U.S. Patent
`
`June 13, 1995
`
`Sheet 4 of 9
`
`5,424,286
`
`SANOFI-AVENTIS Exhibit 1006 - Page 5
`
`IPR for Patent No. 8,951,962
`
`

`
`U.S. Patent
`
`June 13, 1995
`
`Sheet 5 of 9
`
`5,424,286
`
`glucose - mg/dL
`
`9I
`
`D
`
`Exendin-4 1nmol
`
`+Exendin(9-39)'42nmol
`
`7-
`-E0
`
`'55%
`
`x?
`LU
`
`Exer.Idin-4 1nmol
`
`time-min
`
`|uJ/Bu - uunsug
`
`SANOFI-AVENTIS Exhibit 1006 - Page 6
`IPR for Patent No. 8,951,962
`
`

`
`U.S. Patent
`
`June 13, 1995
`
`Sheet 6 of 9
`
`5,424,286
`
`""" I
`
`CD
`I
`
`:E
`C)
`
`0 -:::,. ~
`lc •
`·- C)
`"'CS ·-
`c LL
`CD
`>< CD
`
`C") .-I
`
`~------~--------~------~------~~
`o•
`0
`0 co
`0
`CD
`N
`
`naMJ6u - unnsu1
`
`SANOFI-AVENTIS Exhibit 1006 - Page 7
`
`IPR for Patent No. 8,951,962
`
`

`
`U.S. Patent
`
`June 13, 1995
`
`Sheet 7 of 9
`
`5,424,286
`
`glucose mg/dL
`
`3 E: oc
`
`o 0 E Ea
`
`s
`
`5
`
`time-minutes
`
`3:‘
`"?
`a:T
`
`E'
`
`5cox6
`
`|LU/5U — uunsug
`
`SANOFI-AVENTIS Exhibit 1006 - Page 8
`IPR for Patent No. 8,951,962
`
`

`
`U.S. Patent
`
`June 13, 1995
`
`Sheet 8 of 9
`
`5,424,286
`
`
`
`
`
`Y31exendln-4(1-31)amide,1nmol
`
`-e—glucosef
`
`time-minutes
`
`|uu6u - ugmsug
`
`SANOFI-AVENTIS Exhibit 1006 - Page 9
`IPR for Patent No. 8,951,962
`
`

`
`U.S. Patent
`
`June 13, 1995
`
`Sheet 9 of 9
`
`5,424,286
`
`glucose -mgldL
`
`120
`
`3
`
`:3
`as
`as
`Em
`W
`
`8m
`g
`"_
`.5
`$2’
`
`LI.
`
`on
`E
`oi:
`G”
`
`=
`E
`
`C Q=
`
`A3
`2 1
`=
`8
`:21
`C
`2+?
`0g
`
`_l
`
`E‘
`EC
`
`S
`1"I
`a.
`_l
`(D
`
`o
`
`-so
`
`gun/Bu - uunsug
`
`SANOFI-AVENTIS Exhibit 1006 - Page 10
`IPR for Patent No. 8,951,962
`
`

`
`EXENDIN-3 AND EXENDIN-4 POLYPEPTIDES,
`AND PHARMACEUTICAL COMPOSffiONS
`COMPRISING SAME
`
`BACKGROUND OF THE INVENTION
`
`1
`
`5,424,286
`
`LICENSE RIGHTS
`This U.S. Government has a paid-up license in this
`invention and the right in limited circumstances to re(cid:173)
`quire the patent owner to license others on reasonable
`terms as provided for by the terms of the agreement
`with the Department of Veterans Affairs, reference
`number 0241, GPB No. 20-560.
`
`2
`In 1981, it was discovered that Gila monster (Helo(cid:173)
`derma suspectum) venom stimulated pancreatic enzyme
`secretion in vitro (Raufman, J. P., et al., Gastroenterol(cid:173)
`ogy 80:1257 abst. (1981); Raufman, J. P., et al., Am. J.
`s PhysioL 242: G470-G474 (1982)). Several peptides have
`been isolated from the venom that can stimulate in(cid:173)
`creased cAMP and amylase release from dispersed pan(cid:173)
`creatic acinar cells. These structural analogs to the
`mammalian peptides VIP (vasoactive intestinal peptide)
`10 and secretin
`include helospectin-1, helospectin-11
`(Parker, D. S. et al., J. Bioi. Chern. 259:11751-11755
`(1984)),and helodermin (Hoshino, M. et al., FEBS Lett.
`178:233-239 (1984)). Recently, we discovered another
`peptide that increases cAMP and stimulated the release
`IS of amylase in dispersed acinar cells. This peptide was
`found in Heloderma horridum venom and was termed
`1. Field of the Invention
`This invention is in the field of the prevention and
`exendin-3 (Eng, J. et al., J. Bioi. Chern. 265:
`20259-20262 (1990). Exendin-3 shares homology with
`treatment of diabetes mellitus.
`VIP, secretin, helospectin-1 and -11, and helodermin.
`2. Description of the Prior Art
`Diabetes mellitus (DM) is a major chronic illness 20 The venom of Heloderma suspectum was examined and
`found in humans with many consequences. Some com-
`another peptide was purified from it. This peptide
`called exendin-4 is an analogue of exendin-3 with an
`plications arising from long-standing diabetes are blind-
`ness, kidney failure, and limb amputations. Insulin-
`identical sequence except for substitutions in residues 2
`dependent diabetes mellitus (IDDM) accounts for 10 to
`and 3 from the amino terminus (Eng, J. et al.,J. Bioi.
`15% of all cases of diabetes mellitus. The action of 25 Chern. 267:742-7405-(1992)). Experiments were done to
`IDDM is to cause hyperglycemia (elevated blood glu-
`establish that the exendins could stimulate cAMP activ-
`cose concentration) and a tendency towards diabetic
`ity in dispersed pancreatic acinar cells, and a specific
`ketoacidosis (DKA). Currently treatment requires
`antagonist, exendin(9-39) amide, which can inhibit the
`chronic administration of insulin. Non-insulin depen-
`effects of the exendins, was identified. (Raufman, J.P. et
`dent diabetes mellitus (NIDDM) is marked by hyper- 30 al., J. BloL Chern. 266: 2897-2902 (1991 )) Experiments
`were performed to establish that GLP-1 could interact
`glycemia that is not linked with DKA. Sporadic or
`persistent incidence of hyperglycemia can be controlled
`with possible exendin receptors in dispersed pancreatic
`by administering insulin. Uncontrolled hyperglycemia
`acinar cells in vitro (Raufman, J. P. et al., J. BloL Chern.
`can damage the cells of the pancreas which produce
`267:21432-21437 (1992)).
`insulin (the .8-islet cells) and in the long term create 35
`SUMMARY OF THE INVENTION
`greater insulin deficiencies. Currently, oral sulfonylu-
`This invention encompasses pharmaceutical composi-
`reas and insulin are the only two therapeutic agents
`tions containing exendin-3 or exendin-4, or any combi-
`available in the United States. for treatment of Diabetes
`nation thereof, and methods for the treatment of diabe-
`mellitus. Both agents have the potential for producing
`hypoglycemia as a side effect, reducing the blood glu- 40 tes mellitus and the prevention of hyperglycemia.
`The compositions of the invention will normalize
`cose concentration to dangerous levels. There is no
`generally applicable and consistently effective means of
`hyperglycemia through glucose-dependent,
`insulin-
`maintaining an essentially normal fluctuation in glucose
`dependent and insulin-independent mechanisms. There-
`fore they will be useful as primary agents for the treat-
`levels in DM. The resultant treatment attempts to mini-
`mize the risks of hypoglycemia while keeping the glu- 45 ment of type II diabetes mellitus and as adjunctive
`agents for the treatment of type I diabetes mellitus. The
`cose levels below a target value. The drug regimen is
`combined with control of dietary intake of carbohy-
`invention specifically provides for exendin-4(1-39) as an
`drates to keep glucose levels in control.
`insulinotropic agent.
`A fragment of human peptide molecule called, gluca-
`The use of an effective amount of exendins as a treat-
`gon-like peptide-I (GLP-1) has been found to be a glu- so ment for diabetes mellitus has the advantage of being
`more potent than other insulinotropic peptides. The
`cose-dependent insulinotropic agent (Gutniak, M., et al.
`N. Engl. J. Bled. 1992; 326:1316-1322). GLP-1 is itself a
`present invention is especially suited for the treatment
`fragment of the human pro glucagon molecule. Another
`of patients with diabetes, both type I and type II, in that
`the action of the peptide is dependent on the glucose
`active fragment, glucagon-like insulinotropic peptide
`(GLIP), corresponds to GLP-1(7-36). It was reasoned ss concentration of the blood, and thus the risk of hypo-
`glycemic side effects are greatly reduced over the risks
`that since GLIP is the naturally active form found in
`humans after a meal, this peptide may aid in glucose
`in using current methods of treatment. Thus the use of
`regulation in IDDM and NIDDM.
`insulinotropic peptides such as exendin-3 and exendin-4,
`In normal subjects, the infusion of GLIP significantly
`has many advantages in the treatment of diabetes melli-
`lowered the meal-related increases in blood glucose 60 tus over current methods.
`The present invention also provides for inhibitory
`concentration, and the plasma concentrations of insulin
`and glucagon. In patients with NIDDM, the treatment
`agents derived from the exendins. In particular, exen-
`reduced the requirement for insulin by 8 fold. In pa-
`din-4(9-39) as an inhibitor of exendin-4 and GLP-1 in-
`tients with IDDM, the GLIP treatment lowered the
`sulinotropic activity.
`insulin required by one half. This glucose-dependent 65
`The present invention also provides for a method for
`activity is a very desirable characteristic for a therapeu-
`treating diabetes mellitus in an individual, wherein said
`tic agent that can be used to treat DM avoiding tile
`method comprises providing an amount of an insulino-
`complications of hypoglycemic side effects.
`tropic composition sufficient to treat said diabetes; said
`
`SANOFI-AVENTIS Exhibit 1006 - Page 11
`
`IPR for Patent No. 8,951,962
`
`

`
`5,424,286
`
`4
`min. GLP-l's insulinotropic activity is inhibited by
`exendin (9-39) amide.
`
`3
`compositiOn contammg an insulinotropic molecule;
`wherein said molecule is selected from the group con(cid:173)
`sisting of:
`DETAILED DESCRIPTION OF THE
`(a) a peptide having the amino acid sequence substan(cid:173)
`INVENTION
`tially identicle to the sequence of exendin-3 or 5
`The present invention provides for novel polypep-
`exendin-4 or fragments thereof; and
`(b) a derivative of said peptide (a), wherein said de-
`tides which are unexpectedly useful as insulinotropic
`rivative is selected from the group consisting of:
`agents. Insulinotropic agents being agents which can
`stimulate, or cause the stimulation of, the synthesis or
`(1) a pharmaceutically acceptable acid addition salt
`10 expression of the hormone insulin. The polypeptides of
`of said peptide;
`the present invention are termed exendin-3 and exendin-
`(2) a pharmaceutically acceptable carboxylate salt
`4. These peptides were originally isolated from the
`of said peptide;
`(3) a pharmaceutically acceptable lower alkyl ester
`venom of Heloderma horridum and Heloderma suspec-
`tum respectively. In one embodiment of the invention,
`of said peptide; and,
`(4) a pharmaceutically acceptable amide of said 15 polypeptides corresponding to the amino acid sequence
`peptide wherein said pharmaceutically accept-
`of exendin-3 and exendin-4 are synthesized by the solid
`able amide is selected from the group consisting
`phase method as previously described (Merrifield, J.
`of amide, lower alkyl amide and lower dialkyl M., Chern. Soc. 85: 2149 (1962); Stewart and Young,
`amide; wherein said molecule has an insulino-
`Solid Phase Peptide Synthesis, Freeman, San Francisco,
`tropic activity which exceeds the insulinotropic 20 1969, pp. 27-66). In addition, it is also possible to isolate
`activity of exendin-3 or exendin-4 or fragments
`naturally occuring polypeptides from venom samples in
`thereof.
`a fashion similar to the original isolation of exendins 3
`Thus the invention provides for the peptides or pep-
`and 4. It is further possible to obtain the desired poly-
`tide fragments, made synthetically or purified from
`peptides by using
`recombinant DNA
`techniques
`natural sources, which embody the biological activity 25 (Maniatis, T. et al., Molecular Biology: A Laboratory
`of the exendins, or fragments thereof, as described by Manual, Cold Spring Harbor, N.Y., 1982). The inven-
`the present specification.
`tion encompasses polypeptides which are insulinotropic
`and can be derived from naturally-occuring amino acid
`BRIEF DESCRIPTION OF THE DRAWINGS
`sequences. These proteins consist of the following
`FIG. 1 is a graph showing exendin stimulated insulin 30 amino acid sequences:
`secretion in a dog. Endogenous insulin secretion stimu-
`Exendin-3
`[SEQ
`ID No:1] HSDGTFfSDL
`lated by exendin-3 (200 nmol) in a conscious dog. Exen-
`SKQMEEEA VR LFIEWLKNGG PSSGAPPPS
`din-3 was given as a bolus injection into a leg vain at
`Exendin-4
`[SEQ
`ID No:2] HGEGTFTSDL
`time 0. Plasma was measured by radioimmunoassay.
`SKQMEEEA VR LFIEWLKNGG PSSGAPPPS
`FIG. 2a & 2b & 2c are graphs showing the serial 35
`The invention also encompasses the insulinotropic
`injection of GLP-1 and Exendin-4. FIG. 2a, Dog #1.
`fragments of exendin-4 comprising the amino acid se-
`FIG. 2b, Dog #2. FIG. 2c, Dog #3. Serial injections of
`quentes:
`GLP-1(7-36) amide alternating with exendin-4 into the
`Exendin-4(1-31) [SEQ ID No:3] HGEGTFTSDL
`left atrium via a chronically indwelling catheter. GLP-
`SKQMEEA VR LFIEWLKNGG P
`1(7-36) amide was given at time 0 (0.1 nmol) and at 40 40 y31 Exendin-4(1-31 ) [SEQ ID No:4] HGEGTFTSDL
`min (1 nmol). Exendin-4 was given at 20 min (0.1 nmol)
`SKQMEEEA VR LFIEWLKNGG Y
`and at 60 min (1 nmol). In 2c, the rise and fall in the
`The invention also encompasses the inhibitory [rag-
`baseline insulin between time 0 and 60 min is unex-
`ment of exendin-4 comprising the amino acid sequence:
`plained.
`Exendin-4(9-39) [SEQ ID No:S] DL SKQMEEEA VR
`FIG. 3 is a graph illustrating the effect of exendin 45
`LFIEWLKNGG PSSGAPPPS
`with and without antagonist. Insulin response in a nor-
`The invention further encompasses a method for the
`mal dog to exendin-4 with or without exendin(9-39)
`enhancement of insulin production or expression which
`amide. Glucose was infused at 100 mg/min. Exendin-4
`comprises the steps of providing to a mammalian beta
`(1 nmol) was given as an intravenous bolus at 60, 120
`type pancreatic islet cell an effective amount of the
`and 180 min. Exendin(9-39) amide, 42 nmol, was given 50 insulinotropic peptides disclosed above.
`together with exendin-4 at 120 min. The first phase of
`Also provided for by the present invention are those
`insulin release is greatly reduced and the second phase is
`amino acid sequences in the above peptides which are
`abolished by this ratio of antagonist to agonist.
`capable of functioning as insulinotropic hormones. In
`FIG. 4 is a graph illustrating the effect of exendin on
`addition, the invention also provides for the addition of
`cultured beta cells. /3TC-3 cell insulin response to exen- 55 amino acids to enhance attachment to carrier molecules,
`din-4, insulin mg/well vs. exendin-4 logM.
`or added to enhance the insulinotropic effect.
`FIG. 5 is a graph demonstrating the effect of exendin
`A material is said to be "substantially free of natural
`antagonist on glucose -stimulated increase in insulin.
`contaminants" if it has been substantially purified from
`Conscious dog infused with glucose at 200 mg/min
`materials with which it is normally and naturally found.
`beginning at time 0. A bolus injection of exendin(9-39) 60 Examples of natural contaminants of exendin-3 or exen-
`amide was made at 60 minutes.
`din-4 are: other peptides, carbohydrates, glycosylated
`FIG. 6 is a graph illustrating the effect ofY31 exen-
`peptides, lipids, membrane, other venom components
`din-4(1-31) amide. Conscious, fasted dog injected with a
`etc. A material is also said to be substantially free of
`natural contaminants if these contaminants are substan-
`bolus of Y31 exendin-4(1-31) amide at time 0.
`FIG. 7 is a graph illustrating the effect ofGLP-1 with 65 tially absent of from a sample of the material.
`and without antagonist. Insulin responses in a fasted dog
`The compounds of the present invention can be for-
`to GLP-1 (1 nmol) injected either alone at time 0 or
`mulated according to known methods to prepare phar-
`together with exendin(9-39) amide (180 nmol) at time 60
`maceutically useful compositions. In these composi-
`
`SANOFI-AVENTIS Exhibit 1006 - Page 12
`
`IPR for Patent No. 8,951,962
`
`

`
`5,424,286
`
`6
`The examples which follow are illustrative of specific
`embodiments of the invention, and various uses thereof.
`They are set forth for explanatory purposes only, and
`are not to be taken as limiting the invention.
`
`Example 1
`The exendins are insulinotropins
`Naural or synthetic exendin-3 and exendin-4 were
`tested in several biological systems, including conscious
`dog, anesthetized dog with chronic indwelling left atrial
`catheters, and beta TC-3 insulinoma cell line (described
`in D'Ambra et al., Endocrinology 126:2815-2822 (1990))
`in cell culture. FIG. 1 shows an insulin secretory re(cid:173)
`sponse to bolus injection of exendin-3 in a conscious dog
`with a seven-fold increase in insulin concentration
`above basal levels. Similar results are obtained using
`exendin-4. Since exendin-4 does not interact with VIP
`receptors and acts solely on exendin receptors, it has
`been used for subsequent studies.
`
`5
`tions, exendin-3 and or exendin-4, or their functional
`derivatives are combined in admixture with a pharma(cid:173)
`ceutically acceptable carrier vehicle. Suitable vehicles
`and their formulations, inclusive of other human prote(cid:173)
`ins, e.g. human serum albumin, are well known. In order 5
`to form an effective pharmaceutical composition, the
`composition will contain an effective amount of the
`exendin-3 or exendin-4, or functional derivatives to(cid:173)
`gether with a suitable amount of carrier vehicle. Other
`compositions may combine exendin-3 and exendin-4, or 10
`their functional derivatives with other effective drugs
`that may treat other symptoms, or the same symptoms.
`The use of exendin-3 and 4 in compositions that may
`be injected intravenously, intramuscularly, subcutane(cid:173)
`ously, or intraperitoneally, would call for dosages of 15
`about 0. 1 pg!kg to 1,000 mg/kg body weight depend(cid:173)
`ing on many individual factors such as age, severity of
`disease, total body weight, sex and other mitigating
`factors.
`The insulinotropic properties of a compound may be 20
`determined by in vitro or in vivo assay. The compound
`in question may be administered to animals and moni(cid:173)
`toring the release of insulin. It is possible to monitor the
`increase in insulin production in cell culture as well.
`The sequences of the invention also provide a means 25
`for identifying any specific mamalian analogs to the
`exendins. This can be accomplished by direct compari(cid:173)
`son of amino acid sequences, or by the translation of
`RNA or DNA sequences which may encode for the
`amino acid sequences of the invention, or by inhibition 30
`of activity by the specific exendin inhibitor, exendin
`(9-39) amide.
`The sequences of the invention also provides a means
`for generating antibodies specific for the exendins, and
`further for the production of monoclonal antibodies for 35
`the exendins and fragments thereof. Thus the invention
`provides a means for purifying mammalian or other
`analogs to the exendins by the method of affinity chro(cid:173)
`matography.
`
`Example 2
`Exendin-4 insulin secretagogue activity is glucose de(cid:173)
`pendent
`Dogs with glucose concentrations clamped at graded
`levels show a glucose-dependent insulinotropic re(cid:173)
`sponse to exendin-4. Dosages of exendin-4 which do not
`stimulate insulin release at fasting glucose concentra(cid:173)
`tions of50-75 mgldL (such as 0.1 nmol exendin-4 given
`as a bolus) are able to produce a peak insulin response of
`one-fold above basal when given to dogs in a clamped,
`hyperglycemic state.
`Exendin-4 stimulates a greater insulin secretory re(cid:173)
`sponse than GLP-1
`Synthetic exendin-4 was compared with GLP-1 (pur(cid:173)
`chased from Peninsula Labs, Belmont, Calif.) by alter(cid:173)
`nating injections of bolus doses into dogs with chronic
`indwelling left atrial catheters. Since GLP-1 and exen(cid:173)
`din-4 are glucose dependent in their insulinotropic re(cid:173)
`sponse, paired equimolar doses of GLP-1 and exendin-4
`40 were given with GLP-1 administered first to avoid the
`possibility that falling glucose levels in the animals
`cause a diminished insulinotropic response to GLP-1
`relative to exendin-4. Dogs #1 and #2 in FIGS. 2a and
`2b maintained constant fasting glucose concentrations
`throughout the experiments in a range between 60 and
`80 mgldL. FIGS. 2a, 2b and 2c show a comparison of
`insulinotropic responses to alternating bolus injections
`of GLP-1 and exendin-4 at 20 minute intervals and at
`increasing doses ranging from 0.1 nmol to 10 nmol ad-
`
`Specific Examples
`Testing was done to establish if exendin-3 or exendin-
`4 could stimulate pancreatic insulin secretion in mam(cid:173)
`mals. Since both exendin-3 and exendin-4 peptides have
`about 50% homology with glucagon and GLP-1(7-36) 45
`(glucagon-like peptide-1), and GLP-1(7-36) was found
`to bind to exendin receptors, it was thought possible
`that exendins could act in similar fashion as GLP-1 on
`other receptors.
`
`Exendin-3 HSDG'IFI'SDL
`Exendin-4 HGEG'IFI'SDL
`GLP-1
`HAEG'IFI'SDV
`Glucagon HSQG'IFI'SDY
`
`TABLE1
`SKQMEEEA VR
`SKQMEEEA VR
`SSYLEGOAAK
`SKYLDSRRAQ
`
`LFIEWLKNGG
`LFIEWLKNGG
`EFIA WL VKGR
`DFVQWLMNT
`
`PSSGAPPPS
`PSSGAPPPS
`
`ministered through chronic indwelling left atrial cathe-
`Polypeptides corresponding to the amino acid se-
`ters into anesthetized dogs.
`quence of exendin-3 and exendin-4 were synthesised by
`In contrast to the euglycemia present in the first two
`the solid phase method as previously described (Merri-
`field, J. M., Chem. Soc. 85:2149 (19625; Stewart and 60 dogs, the third dog in FIG. 3c was exceptionally hyper-
`glycemic, probably as a result of an infected catheter.
`Young, Solid Phase Peptide Synthesis, Freeman, San
`Francisco, 1969, pp. 27-66). It is also possible to isolate
`Several points are illustrated by this experiment. First,
`euglycemic dogs normally do not respond to 0.1 nmol
`naturally occuring polypeptides from venom samples in
`a fashion similar to the original isolation of exendins 3
`of either GLP-1 or exendin-4 with an insulin secretory
`and 4. It is further possible to obtain the desired poly- 65 resonse as illustrated by the first two dogs, whereas the
`techniques
`hyperglycemic dog bad clear insulinotropic responses
`peptides by using recombinant DNA
`(Maniatis,T. et al., Molecular Biology: A Laboratory
`to this lower dose of peptide. Second, the rapid normal-
`ization of hyperglycemia to euglycemic levels follow-
`Manual, Cold Spring Harbor, N.Y., 19825).
`
`SANOFI-AVENTIS Exhibit 1006 - Page 13
`
`IPR for Patent No. 8,951,962
`
`

`
`7
`ing modest doses of the two peptides reflects the great
`potential for use of these peptides in treatment of dia(cid:173)
`betic states. Third, despite the rapid normalization of
`the hyperglycemia, hypoglycemia does not occur. This
`class of therapeutic agents might be termed "euglyce- 5
`mic" agents. The potential for hypoglycemia caused by
`overdosages of these agents is minimized. Hypoglyce(cid:173)
`mia is avoided even when the agents are given in the
`euglycemic state. Fourth, despite the administration of
`exendin-4 following an equivilent dose of GLP-1 in the 10
`setting of decreasing glucose levels, the insulin response
`as defmed by area under the curve, is consistently 2-3
`fold greater for exendin-4 compared to GLP-1. The
`greater response to exendin-4 holds true for t~e t":o
`euglycemic animals as well. A summary of the msulm 15
`responses is shown in Table 2.
`TABLE2
`AUC(GLP-1) AUC (EX-4) EX-4/GLP-1
`2.0
`4.1
`2.1
`7.3
`2.8
`2.6
`12.9
`2.6
`5.0
`14.2
`2.5
`5.7
`
`Dog
`
`#I
`#2
`#3
`
`Dose
`lnmol
`lnmol
`lnmol
`!Onmol
`
`20
`
`5,424,286
`
`8
`pletely inhibited while the first phase is more resistant to
`complete inhibition. This fmding suggests a differential
`sensitivity to inhibition between the first and second
`phases of insulin release. A pathological condition
`which may correlate to this phenomenon is a loss of first
`phase insulin secretion in type 2 diabetes.
`When exendin(9-39) amide is given together with
`GLP-1 at molar ratio of 180:1 there is substantial inhibi(cid:173)
`tion of the insulin secretory response, as shown in FIG.
`7.
`
`Example 4
`Exendin-4 acts directly on the beta cell
`Beta TC-3 cells were obtained through Norman
`Fleischer (Diabetes Research and Training Center,
`Albert Einstein College of Medicine, N.Y.) and cul(cid:173)
`tured in serum-containing media in 48-well culture
`dishes to confluency. Fresh media was added 24 hours
`before the cells were tested. The cells were tested in
`Earle's balanced salt solution containing IBMX, BSA
`and 16.7 mM glucose with graded concentrations of
`exendin-4 for 1 hour at 3 7• C. before collection of media
`supernate and assay for insulin concentrations. FIG. ~
`shows a dose response curve to exendin-4 indicating
`that exendin-4 acts directly on beta cells to stimulate
`insulin secretion.
`
`Example 5
`Exendin-4 reduces the hyperglycemic state in a diabetic
`animal model
`The db/db mouse is a genetically obese and diabetic
`strain of mouse. The db/db mouse develops hypergly(cid:173)
`cemia and hyperinsulinemia concomitant with its devel(cid:173)
`opment of obesity and thus serves as a model of obese
`type 2 diabetes (NIDDM). Five 11-week old db/db
`mice purchased from The Jackson Laboratories (Bar
`Harbor, Me.) had sub-orbital sinus blood samples tal<en
`before and 60 minutes after intraperitoneal injection of
`exendin-4 at 10 nmol each animal (1 microgram/gram
`body weight). Blood glucose measurements were made
`with a glucose meter (YSI 1500 glucose analyzer, Yel(cid:173)
`low Springs, Ohio). The blood glucose levels in the
`animals were (average±standard error, in mg/dL glu(cid:173)
`cose) 310±37before and 181±37 one hour after admin(cid:173)
`istration of exendin-4. Thus, exendin-4 was able to re(cid:173)
`duce the diabetic levels of blood glucose by 40% in
`these animals.
`
`Example 6
`We have compared the effect of COOH-terminal
`truncations on the insulinotropic activity of exendin-4.
`The y31 mutation ofexendin-4( 1-31) amide has a TYR
`for PRO substitution at position 31 from the amino
`terminus. This mutant was shown to have insulinotropic
`activity when infused into dog. FIG. 6 shows this result.
`This result indicates that the amino acids in the exendin-
`4 sequence located between residues 1 and 31 are impor(cid:173)
`tant for the insulinotropic activity.
`This invention thus provides for compounds that are
`an unexpectedly efficient means of stimulating insulin
`production in vitro and in vivo that will be useful for
`the treatment of diabetes mellitus, as well as specific
`inhibitors therof.
`
`Table 2 shows the relative ratio of insulin secretion 25
`stimulated by serial injections of GLP-1 (7-36) amide
`and exendin-4 expressed as area under the curve (AUC).
`AUC=T-B where T=total insulin secreted (sum of
`concentrations at times 2,4,6, 10 mad 14 min. and
`B=baseline insulin=average of insulin concentrations 30
`at times 0 and 20 min. Multiplied by a factor of 5.
`
`35
`
`Example 3
`Exendin(9-39) amide inhibits endogenous, exendin-4,
`and GLP-1 insulinotropic activity.
`Antagonistic peptides can arise by a number of mech(cid:173)
`anisms. The gene encoding the exendins may also en(cid:173)
`code for related peptides which have antagonistic activ(cid:173)
`ity. The production of antagonistic peptides may then
`be either initiated or suppressed by differential cleavage 40
`of the pre-propeptide. The antagonistic peptides may
`also arise through post-translational modification of the
`agonist peptide, specifically through differential cleav(cid:173)
`age to produce extended or truncated forms of the ago(cid:173)
`nist peptide. In our studies of the structure-function 45
`relationship of exendin peptide sequences, the NH2-ter(cid:173)
`minally truncated exendin analog, exendin(9-39) amide,
`was shown to have potent antagonistic activity against
`exendin-3 and exendin-4 in a pancreatic acinar cell sys(cid:173)
`tem measuring cAMP activity. (Raufman et al., J. Bioi. 50
`Chem 266:2897-2902 (1991); Eng et al., J. Bioi. Chem
`267:7402-7505 (1992)). FIG. 5 shows the effect ofexen(cid:173)
`din(9-39) amide when administered alone on circulating
`insulin levels while glucose levels are clamped at ap(cid:173)
`proximately 100% above fasting levels. Following in- 55
`jection of the antagonist there is a rapid decrease in
`circulating insulin levels to 60% of the maximum con(cid:173)
`centration. This result indicates that the antagonist in(cid:173)
`hibited an endogenous insulinotropin that accounted for
`a substantial portion of the insulin secretory response to 60
`hyperglycemia.
`.
`When exendin(9-39) amide is given together With
`exendin-4 at a molar ratio of 40:1, there is substantial
`inhibition of the insulin secretory response, as shown in
`FIG. 3. The second phase of insulin release is com-
`
`SEQUENCE LISTING
`
`SANOFI-AVENTIS Exhibit 1006 - Page 14
`
`IPR for Patent No. 8,951,962
`
`

`
`9
`
`5,424,286
`
`-continued
`
`10
`
`( I ) GENERAL INFORMATION:
`
`( i i i ) NUMBER OF SEQUENCES: 7
`
`( 2 ) INFORMATION FOR SEQ ID NO:!:
`
`( i ) SEQUENCE CHARACJ'ERISTICS:
`( A ) LENG'IH: 39 amino acids
`( B ) TYPE: amino acid
`( C ) STRANDEDNESS: single
`( D ) TOPOLOGY: linear
`
`( i i ) MOLECULE TYPE: peptide
`
`( i X) FEATURE:
`( A ) NAME/KEY: Peptide
`(B) LOCATION: 1..39
`(D) O'IHER INFORMATION: Jlabel=Exendin-3
`
`( x i ) SEQUENCE DESCRIPTION: SEQ ID NO: I:
`
`His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu
`I
`5
`I 0
`I 5
`
`Glu Ala Val Arg Leu Phe lie Glu Trp Leu Lys Asn Gly Gly Pro Ser
`20
`25
`30
`
`Ser Gly Ala Pro Pro Pro Ser
`3 5
`
`( 2 ) INFORMATION FOR SEQ ID N0:2:
`
`( i ) SEQUENCE CHARACJ'ERISTICS:
`( A ) LENG'IH: 39 amino acids
`( B ) TYPE: amino acid
`( C ) STRANDEDNESS: single
`( D ) TOPOLOGY: linear
`
`( i i ) MOLECULE TYPE: peptide
`
`( i x ) FEATURE:
`( A ) NAME/KEY: Peptide
`( B ) LOCATION: 1 .. 39
`(D) O'IHER INFORMATION: Jlabel=Exendin-4
`
`( x i ) SEQUENCE DESCRIPTION: SEQ ID N0:2:
`
`His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu
`I
`5
`I 0
`I 5
`
`Glu Ala Val Arg Leu Phe lie Glu Trp Leu Lys Asn Gly Gly Pro Ser
`20
`30
`25
`
`Ser Gly Ala Pro Pro Pro Ser
`3 5
`
`( 2 ) INFORMATION FOR SEQ ID N0:3:
`
`( i ) SEQUENCE CHARACTERISTICS:
`( A ) LENG'IH: 31 amino acids
`( B ) TYPE: amino acid
`( C ) STRANDEDNESS: single
`( D ) TOPOLOGY: linear
`
`( i i ) MOLECULE TYPE: peptide
`
`( i x) FEATURE:
`( A ) NAME/KEY: Peptide
`( B ) LOCATION: 1..31
`(D) O'IHER INFORMATION: /label=Exendin-1-31
`I note="Exendin-4(1-31)"
`
`( x i ) SEQUENCE DESCRIPTION: SEQ ID N0:3:
`
`His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Met Glu Glu
`5
`I 0
`I 5
`
`Glu Ala Val Arg Leu Phe lie Glu Trp Leu Lys Asn Gly Gly Pro
`20
`25
`30
`
`SANOFI-AVENTIS Exhibit 1006 -