WO2008071010A1 - Polythérapies impliquant des facteurs de régulation de croissance/des hormones sélectionnés pour le diabète et les maladies apparentées - Google Patents

Polythérapies impliquant des facteurs de régulation de croissance/des hormones sélectionnés pour le diabète et les maladies apparentées Download PDF

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Publication number
WO2008071010A1
WO2008071010A1 PCT/CA2007/002339 CA2007002339W WO2008071010A1 WO 2008071010 A1 WO2008071010 A1 WO 2008071010A1 CA 2007002339 W CA2007002339 W CA 2007002339W WO 2008071010 A1 WO2008071010 A1 WO 2008071010A1
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WIPO (PCT)
Prior art keywords
glp
gastrin
agonist
receptor ligand
egf receptor
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PCT/CA2007/002339
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English (en)
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Carl Damiani
Antonio Cruz
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Waratah Pharmaceuticals Inc.
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Publication of WO2008071010A1 publication Critical patent/WO2008071010A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2207—Gastrins; Cholecystokinins [CCK]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the invention relates generally to compositions, conjugates, and methods comprising an EGF receptor ligand and a GLP-I agonist, and preferably one or both of a gastrin compound and/or gastrin agonist, and uses thereof.
  • the insulin dependent diabetic population including both Type I and Type II diabetics, is estimated to be approximately 4 million people in the United States and approximately 7-8 million people worldwide .
  • the population ranges from end stage insulin dependent diabetics (transplant, severe complications) to new onset insulin diabetics.
  • Many therapeutics and treatments have been proposed to treat and/or cure diabetes.
  • One approach is directed at enhancing islet neogenesis.
  • Islet neogenesis is the process by which islets are formed from precursor stem cells in the ducts of the developing fetal pancreas. Methods for treating diabetes based on islet neogenesis have been described in US Patent Nos. 5,885,956, 6,288,301 and 6,558,952.
  • the current invention addresses the need for additional therapies for the treatment of diabetes and related diseases, disorders, and conditions.
  • SUMMARY OF THE INVENTION Disclosed herein is the co-administration of at least one EGF receptor ligand and at least one GLP-I agonist preferably with one or both of at least one gastrin compound and at least one gastrin agonist, to prevent and/or treat diabetes and related diseases, disorders, or conditions as well as prevent and/or treat complications associated with diabetes, and related diseases, disorders, or conditions.
  • the therapeutic strategy of the present invention relates to the modification of multiple pathophysiological processes using innovative combinations of compounds that have distinct complementary, additive or synergistic mechanisms of action to provide safe and effective treatments for conditions and/or diseases disclosed herein.
  • compositions, conjugates, or methods comprising at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist employing different mechanisms to achieve maximum therapeutic efficacy, may improve tolerance to therapy with a reduced risk of side effects that may result from higher doses or longer term monotherapies (i.e., therapies with each compound alone).
  • a composition, conjugate, or method of the invention will permit the use of lower doses of the compounds with reduced adverse toxic effects of each compound.
  • a suboptimal dosage may provide an increased margin of safety, and may also reduce the cost of a drug necessary to achieve prophylaxis and therapy.
  • the increased convenience of a single combination dosage unit may result in enhanced compliance.
  • compositions, conjugates, and methods for the prevention and/or treatment of a condition and/or disease disclosed herein comprising a therapeutically effective amount of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, in particular that provide one or more beneficial effects.
  • the invention relates to compositions, conjugates, and methods for the prevention and/or treatment of a condition and/or disease disclosed herein comprising a therapeutically effective amount of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist that provide one or more beneficial effects.
  • the invention relates to compositions, conjugates, and methods for the prevention and/or treatment of diabetes in a subject receiving insulin comprising a therapeutically effective amount of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist that provide one or more beneficial effects.
  • a composition, conjugate, or method of the invention may provide sustained beneficial effects following treatment or termination of treatment. Prolonged efficacy may be evidenced by increased C-peptide production, increases in pancreatic insulin production, decreased insulin dependence, and/or about normal or reduced blood glucose levels relative to each compound alone.
  • the invention contemplates a composition, preferably a pharmaceutical composition, comprising one or both of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, in particular that provide beneficial effects relative to each compound alone, preferably sustained beneficial effects, following treatment.
  • a pharmaceutical composition may optionally comprise a pharmaceutically acceptable carrier, excipient, or vehicle.
  • the invention in another aspect, relates to a combination, such as a combined preparation or pharmaceutical composition, comprising at least one EGF receptor ligand and at least one GLP- 1 agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, and at least one additional pharmaceutically acceptable carrier, excipient, or vehicle.
  • a combination such as a combined preparation or pharmaceutical composition, comprising at least one EGF receptor ligand and at least one GLP- 1 agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, and at least one additional pharmaceutically acceptable carrier, excipient, or vehicle.
  • the invention also contemplates a pharmaceutical composition in separate containers and intended for simultaneous or sequential administration to provide one or more beneficial effects, preferably sustained beneficial effects, comprising at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, together with pharmaceutically acceptable carriers, excipients, or vehicles.
  • the invention further contemplates a conjugate comprising at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, in particular to provide one or more beneficial effects.
  • compositions and conjugates of the invention that result in compositions and conjugates with one or more beneficial effects, preferably sustained beneficial effects.
  • the invention relates to a medicinal combination of active principles, having, jointly, a complementary and/or synergistic action, this being for the treatment of diabetes, in particular of type I diabetes.
  • the invention relates to a medicinal combination of active principles, having, jointly, a complementary and/or synergistic action, this being for the treatment of diabetes, in particular of type II diabetes.
  • a method for preparing a stable pharmaceutical composition of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist adapted to provide one or more beneficial effects following treatment comprising preparing a composition comprising at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, and a pharmaceutically acceptable carrier, excipient, or vehicle effective to physically stabilize the composition.
  • a method for preparing a stable pharmaceutical composition comprising mixing at least one EGF receptor ligand and at least one GLP-I agonist preferably with one or both of at least one gastrin compound and at least one gastrin agonist, and a pharmaceutically acceptable carrier, excipient, or vehicle effective to physically stabilize the composition and in particular adapted to provide one or more beneficial effects, preferably sustained beneficial effects.
  • the invention relates to a combination treatment for preventing and/or treating a condition and/or disease disclosed herein in a subject comprising administering to the subject a therapeutically effective amount of at least one EGF receptor ligand and at least one GLP- 1 agonist preferably with one or both of at least one gastrin compound and at least one gastrin agonist, in particular to provide one or more beneficial effects.
  • a combination treatment for preventing and/or treating a condition and/or disease disclosed herein in a subject comprising administering to the subject a therapeutically effective amount of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist to provide one or more beneficial effects.
  • the invention provides a combination treatment or intervention which provides one or more sustained beneficial effects following treatment.
  • the invention further relates to the use of one or both of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, a composition, or conjugate of the invention for preventing, delaying progression of, and/or ameliorating disease severity, disease symptoms, and/or periodicity of recurrence of a condition and/or disease disclosed herein.
  • the invention relates to the prevention, delay of progression, and/or treatment, in a subject, of conditions and/or diseases using at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, a composition, or conjugate of the invention.
  • the invention provides a method for the prevention and/or intervention of a condition and/or disease disclosed herein in a subject comprising administration of at least one EGF receptor ligand and at least one GLP- 1 agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, or a composition or conjugate of the invention.
  • An EGF receptor ligand, GLP-I agonist and preferably one or both of a gastrin compound and a gastrin agonist, or a composition or conjugate may be directly administered to a subject or contacted with cells (e.g. stem cells or progenitor cells) and administered to a subject.
  • the invention provides a method for the prevention and/or intervention of a condition and/or disease disclosed herein in a subject comprising administration of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist to a subject in need thereof to provide beneficial effects.
  • the invention provides in some aspects methods for the potentiation of a gastrin compound or gastrin agonist in the treatment of a condition and/or disease in a subject, in particular diabetes and related diseases, disorders, or conditions, comprising co-administering at least one EGF receptor ligand and at least one GLP-I agonist to the subject.
  • the invention provides in some aspects methods for the potentiation of an EGF receptor ligand and a GLP- 1 agonist in the treatment of a condition and/or disease in a subject, in particular diabetes and related diseases, disorders, or conditions, comprising coadministering one or both of at least one gastrin compound and at least one gastrin agonist, with an EGF receptor ligand and GLP-I agonist to the subject.
  • the invention provides a method for the prevention and/or intervention of a condition and/or disease disclosed herein in a subject comprising coadministering at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist to a subject in need thereof.
  • the invention relates to inducing islet neogenesis in a subject comprising contacting islet precursor cells with at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, or a composition, or conjugate of the invention in a sufficient amount to increase proliferation of islet precursor cells in the subject thereby inducing islet neogenesis.
  • the invention in another aspect, relates to a method for treating diabetes mellitus in a patient in need thereof by administering at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist or a composition comprising at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, in amount(s) sufficient to effect differentiation of the patient's pancreatic islet precursor cells to mature insulin-secreting cells and/or to stimulate insulin synthesis in existing islet cells.
  • the invention provides methods for treating cells using at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, or composition or conjugate of the invention.
  • the invention relates to a method for expanding and differentiating stem cells or progenitor cells into insulin secreting cells, enhancing proliferation of insulin secreting cells, and/or sustaining islet cells or precursor cells.
  • Cells may be contacted with an EGF receptor ligand and GLP-I agonist and preferably one or both of a gastrin compound(s) and/or gastrin agonist(s) in culture or in a subject.
  • a method for treating a condition and/or disease comprising administering at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, or a composition or conjugate of the invention, with a plurality of cells to a subject in need thereof to thereby produce one or more beneficial effects, preferably sustained beneficial effects.
  • the compounds, compositions or conjugates are administered systemically.
  • the invention provides a method for treating a subject with a condition and/or disease disclosed herein comprising contacting ex vivo a plurality of cells with at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, or a composition or conjugate of the invention, optionally culturing the cells, and administering the cells to the subject in need thereof.
  • pancreatic islet cells that have been exposed in culture to a sufficient amount of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, or a composition or conjugate of the invention, to increase the number of pancreatic beta cells in the islets; optionally the population of pancreatic beta cells can be grown in culture for a time sufficient to expand the population of ⁇ -cells prior to transplantation.
  • a therapeutically effective amount of an insulin or insulin analog is also administered, separately or together with an EGF receptor ligand(s) and GLP-I agonist(s) and preferably one or both of a gastrin compound(s) and a gastrin agonist(s) to the subject.
  • a therapeutically effective amount of an immunosuppressive agent is also administered, separately or together with an EGF receptor ligand(s) and GLP-I agonist(s) and preferably one or both of a gastrin compound(s) and a gastrin agonist(s), to the subject.
  • a therapeutically effective amount of an insulin sensitivity enhancer is also administered, separately or together with an EGF receptor ligand(s) and GLP- 1 agonist(s) and preferably one or both of a gastrin compound(s) and a gastrin agonist(s), to the subject.
  • a therapeutically effective amount of glucose lowering agent is also administered, separately or together with an EGF receptor ligand(s) and GLP-I agonist(s) and preferably one or both of a gastrin compound(s) and a gastrin agonist(s), to the subject.
  • a therapeutically effective amount of an insulin secretagogue is also administered, separately or together with an EGF receptor ligand(s) and GLP- 1 agonist(s) and preferably one or both of a gastrin compound(s) and a gastrin agonist(s), to the subject.
  • a therapeutically effective amount of an antiobesity or appetite regulating agent is also administered, separately or together with an EGF receptor ligand(s) and GLP-I agonist(s) and preferably one or both of a gastrin compound(s) and a gastrin agonist(s), to the subject.
  • the invention also contemplates the use of a composition comprising a combination of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist for the preparation of one or more medicament for preventing and/or treating a condition and/or disease.
  • the invention further contemplates use of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist as a medicament or for the manufacture of a medicament for the treatment of a condition and/or disease.
  • the invention provides use of an EGF receptor ligand and a GLP-I agonist for the manufacture of a medicament for the treatment of a condition and/or disease to be used in combination with a gastrin compound and/or a gastrin agonist.
  • the invention relates to the use of additive, complementary or synergistically effective amounts of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist for the preparation of a medicament for preventing or treating a condition and/or disease.
  • the invention relates to the use of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist for the preparation of a medicament which has a protracted profile of action relative to each compound alone or any two of the compounds.
  • the invention additionally provides uses of a pharmaceutical composition and a conjugate of the invention in the preparation of medicaments for the prevention and/or treatment of conditions and/or diseases.
  • the medicaments may provide beneficial effects, preferably sustained beneficial effects following treatment.
  • the present invention relates to a method of prevention and/or treatment comprising a combination of active agents which may be administered separately or as conjugates
  • the invention also provides a kit comprising at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, and a pharmaceutical composition, or conjugate of the invention in kit form.
  • a includes a mixture of two or more compounds.
  • an EGF receptor ligand as used herein can also mean “one or more EGF receptor ligand” or “at least one EGF receptor ligand”.
  • the phrases “a GLP-I agonist”, as used herein can also mean “one or more GLP-I agonist” or “at least one GLP-I agonist”.
  • the phrase “a gastrin compound”, as used herein can also mean “one or more gastrin compound” or “at least one gastrin compound”.
  • a gastrin agonist as used herein can also mean “one or more gastrin agonist” or "at least one gastrin agonist”.
  • Selected compounds described herein contain one or more asymmetric centers and may give rise to enantiomers, diasteriomers, and other stereoisomeric forms which may be defined in terms of absolute stereochemistry as (R)- or (S)-. Therefore, the invention includes all such possible diasteriomers and enantiomers as well as their racemic and optically pure forms.
  • Optically active (R)- and (S)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
  • the compounds described herein contain centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and A geometric isomers. All tautomeric forms are intended to be included within the scope of the invention.
  • administering and “administration” refer to the process by which a therapeutically effective amount of compounds or a composition or conjugate contemplated herein is delivered to a subject for prevention and/or treatment purposes.
  • Compositions are administered in accordance with good medical practices taking into account the subject's clinical condition, the site and method of administration, dosage, patient age, sex, body weight, and other factors known to physicians.
  • subject and “patient” refer to an animal including a warmblooded animal such as a mammal, which is afflicted with or suspected of having or being pre-disposed to a condition and/or disease as disclosed herein.
  • the terms refer to a human.
  • the terms also include domestic animals bred for food, sport, or as pets, including horses, cows, sheep, poultry, fish, pigs, cats, dogs, and zoo animals.
  • the methods herein for use on subjects/individuals/patients contemplate prophylactic as well as curative use.
  • Typical subjects for treatment include persons susceptible to, suffering from or that have suffered a condition and/or disease disclosed herein.
  • a carrier, excipient, or vehicle refers to a medium which does not interfere with the effectiveness or activity of an active ingredient and which is not toxic to the hosts to which it is administered.
  • a carrier, excipient, or vehicle includes diluents, binders, adhesives, lubricants, disintegrates, bulking agents, wetting or emulsifying agents, pH buffering agents, and miscellaneous materials such as absorbants that may be needed in order to prepare a particular composition.
  • a carrier, excipient, or vehicle is selected to stabilize an EGF receptor ligand, GLP-I agonist, gastrin compound, and/or gastrin agonist.
  • “Pharmaceutically acceptable salt(s),” means a salt that is pharmaceutically acceptable and has the desired pharmacological properties.
  • pharmaceutically acceptable salts is meant those salts which are suitable for use in contact with the tissues of a subject or patient without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are described for example, in S. M. Berge, et al., J. Pharmaceutical Sciences, 1977, 66:1.
  • Suitable salts include salts that may be formed where acidic protons in the compounds are capable of reacting with inorganic or organic bases.
  • Suitable inorganic salts include those formed with alkali metals, e.g. sodium and potassium, magnesium, calcium, and aluminum.
  • Suitable organic salts include those formed with organic bases such as the amine bases, e.g. ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Suitable salts also include acid addition salts formed with inorganic acids (e.g. hydrochloride and hydrobromic acids) and organic acids (e.g. acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benezenesulfonic acid). When there are two acidic groups present, a pharmaceutically acceptable salt may be a mono-acid-mono-salt or a di-salt; and similarly where there are more than two acidic groups present, some or all of such groups can be salified.
  • organic bases such as the amine bases, e.g. ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.
  • Suitable salts also include
  • prevention and/or treating refers to the administration to a subject of biologically active agents either before or after onset of a condition and/or disease.
  • a treatment may be either performed in an acute or chronic way.
  • prevention includes the management and care of a subject at risk of developing a condition and/or disease disclosed herein prior to the clinical onset of the condition and/or disease.
  • Treatment or intervention refers to the management and care of a subject at diagnosis or later.
  • An objective of prevention, treatment, or intervention is to combat the condition and/or disease and includes administration of the active compounds to prevent or delay the onset of the symptoms or complications, or alleviating the symptoms or complications, or eliminating or partially eliminating the condition and/or disease.
  • a “beneficial effect” refers to an effect of a combination of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist, or composition or conjugate thereof that is greater than the effect of the compounds or selected combinations alone.
  • the beneficial effect includes favorable pharmacological and/or therapeutic effects, and improved pharmacokinetic properties and biological activity.
  • a beneficial effect may be an additive effect, complementary or synergistic effect.
  • beneficial effects include but are not limited to the following: reduced or absent islet inflammation, decreased disease progression, increased survival, or elimination or partial elimination of a condition and/or disease.
  • the beneficial effect is a "sustained beneficial effect" where the beneficial effect is sustained for a prolonged period of time after termination of treatment.
  • one or more of the aforementioned effects are sustained for a prolonged period of time after termination of treatment.
  • a beneficial effect may be sustained for at least about 2, 4, 6, 8, 10, 2 to 4 weeks, 2 to 6 weeks, 2 to 8 weeks, 2 to 12 weeks, 2 to 24 weeks, 2 weeks to 12 months, and 2 weeks to 18 months following treatment.
  • the period of time a beneficial effect is sustained may correlate with the duration and timing of the treatment.
  • a subject may be treated continuously for about 2 to 8 weeks, 2 to 12 weeks, 2 to 16 weeks, 2 weeks to 6 months, 2 weeks to 12 months, or periodically.
  • a sustained beneficial effect may manifest as one or more of increased C-peptide production, increased pancreatic insulin production, about normal or reduced blood glucose levels for a prolonged period following treatment, decreased insulin dependence or delivery, increased beta cell production and/or inhibition of programmed cell death (apoptosis), or reduction in insulin use in a subject.
  • the beneficial effect may be a statistically significant effect in terms of statistical analysis of an effect of the the compounds versus the effects of each of the compounds or two compounds alone.
  • "Statistically significant” or “significantly different” effects or levels with the compounds compared with each compound or selected combinations (eg. two compounds) alone may represent levels that are higher or lower than a standard. In embodiments of the invention, the difference may be 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 times higher or lower compared with the effect obtained with each compound or two compounds alone.
  • An "additive effect" of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist refers to an effect that is equal to the sum of the effects of the individual compounds
  • a "synergistic effect" of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist refers to an effect that is greater than the additive effect which results from the sum of the effects of the individual compounds.
  • Combination treatment means that the active ingredients are administered concurrently to a patient being treated.
  • each component may be administered at the same time, or sequentially in any order at different points in time. Therefore, each component may be administered separately, but sufficiently close in time to provide the desired effect, in particular a beneficial, complementary, additive, or synergistic effect.
  • the first compound may be administered in a regimen which additionally comprises treatment with a second, third, or fourth, etc. compound.
  • the term refers to administration of one or more EGF receptor ligand and one or more GLP-I agonist with preferably one or more of a gastrin compound and a gastrin agonist to a patient within 1 week, 2 weeks, 3 weeks, 4 weeks, 8 weeks, 12, weeks, 18 weeks, or one year, including separate administration of the medicaments each containing one of the compounds as well as simultaneous administration whether or not the compounds are combined in one formulation or whether they are three separate formulations.
  • a “medicament” refers to a pharmaceutical composition suitable for administration of a pharmaceutically active compound(s) (e.g.
  • EGF receptor ligand and one or more GLP-I agonist preferably one or more gastrin compound and/or one or more gastrin agonist
  • ' 'Therapeutically effective amount relates to the amount or dose of active compounds
  • compositions or conjugates of the invention that will lead to one or more desired beneficial effects, preferably one or more sustained beneficial effects.
  • a "therapeutically effective amount” can provide a dosage which is sufficient in order for prevention and/or treatment of a condition and/or disease in a subject to be effective compared with no treatment.
  • “Synergistically effective amount” relates to the amount of dose of active compounds (e.g. EGF receptor ligand and GLP-I agonist and one or both of a gastrin compound and a gastrin agonist), compositions or conjugates of the invention that will provide a synergistic effect, in particular a synergistic beneficial effect.
  • active compounds e.g. EGF receptor ligand and GLP-I agonist and one or both of a gastrin compound and a gastrin agonist
  • compositions or conjugates of the invention that will provide a synergistic effect, in particular a synergistic beneficial effect.
  • the expression “complementary action” or “complementary effect” refers to the pharmacological action of one, two, three or more different compounds making it possible to act on the same pathology via different pharmacological mechanisms, for example the combined use of at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound
  • Potentiation refers to an increase of a corresponding pharmacological activity or therapeutic effect. Potentiation of one component of a combination or composition of the present invention by co-administration of the other components according to the present invention means that an effect is being achieved that is greater than that achieved with one component alone.
  • Suboptimal dose or suboptimal dosage refers to a dose or dosage of one or more active compound which is less than the optimal dose or dosage for that compound when used in monotherapy.
  • association refers to any physical association between molecules.
  • the terms preferably refer to a stable association between molecules due to, for example, electrostatic, hydrophobic, ionic, hydrogen-bond interactions, or covalent interactions.
  • nucleic acid or fragment thereof, or polypeptide indicates that, when optimally aligned with another nucleic acid or fragment or polypeptide there is a percent sequence identity in at least about 50%, more preferably 60% of the nucleotide bases or amino acid residues, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95-98% of the nucleotide bases or amino acid residues.
  • Percent sequence identity refers to the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues in a polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid or nucleic acid sequence identity can be achieved in various conventional ways, for instance, using publicly available computer software including the GCG program package (Devereux J.
  • an “analog” refers to a polypeptide wherein one or more amino acid residues of a parent or wild-type polypeptide have been substituted by another amino acid residue, one or more amino acid residues of a parent or wild-type polypeptide have been inverted, one or more amino acid residues of the parent or wild-type polypeptide have been deleted, and/or one or more amino acid residues have been added to the parent or wild-type polypeptide.
  • Such an addition, substitution, deletion, and/or inversion may be at either of the N-terminal or C- terminal end or within the parent or wild-type polypeptide, or a combination thereof.
  • an analog is a peptide wherein 6 or less amino acids have been substituted and/or added and/or deleted from the parent or wild-type peptide, more preferably a peptide wherein 3 or less amino acids have been substituted and/or added and/or deleted from the parent or wild-type polypeptide, and most preferably, a peptide wherein one amino acid has been substituted and/or added and/or deleted from the parent or wild-type polypeptide.
  • Mutations may be introduced into a polypeptide by standard methods, such as site- directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions can be made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which an amino acid residue is replaced with an amino acid residue with a similar side chain.
  • Amino acids with similar side chains are known in the art and include amino acids with basic side chains (e.g. Lys, Arg, His), acidic side chains (e.g. Asp, GIu), uncharged polar side chains (e.g. GIy, Asp, GIu, Ser, Thr, Tyr and Cys), nonpolar side chains (e.g.
  • Mutations can also be introduced randomly along part or all of the native sequence, for example, by saturation mutagenesis. Following mutagenesis the variant polypeptide can be recombinantly expressed.
  • a "derivative" refers to a polypeptide in which one or more of the amino acid residues of a parent polypeptide have been chemically modified.
  • Derivatives may be obtained by chemically modifying one or more amino acid residues of the parent polypeptide or analog thereof, for instance by alkylation, acylation, glycosylation, pegylation, ester formation, deamidation, amide formation, or by introducing a lipophilic functionality.
  • a derivative designates a peptide or analogue thereof which is chemically modified by introducing an ester, alkyl or lipophilic functionality on one or more amino acid residues of the peptide or analogue thereof.
  • a "chimeric polypeptide” comprises all or part (preferably biologically active) of a selected polypeptide operably linked to a heterologous polypeptide (i.e., a polypeptide other than the selected polypeptide).
  • a heterologous polypeptide i.e., a polypeptide other than the selected polypeptide.
  • the term "operably linked” is intended to indicate that a selected polypeptide and the heterologous polypeptide are fused in- frame to each other.
  • the heterologous polypeptide can be fused to the N-terminus or C-terminus of a selected polypeptide.
  • Chimeric and fusion proteins can be produced by standard recombinant DNA techniques.
  • Bioavailability refers to the extent to which a drug or metabolite is absorbed into the general circulation of a subject and becomes available at the site of action of the drug in the subject.
  • the term, "receptor ligand” as used herein in connection with a receptor for a particular ligand shall mean any composition that binds to, interacts with, or stimulates the receptor.
  • a receptor ligand includes within the scope of the definition a receptor agonist for the receptor for an EGF receptor ligand or GLP-I agonist, whether or not the agonist is structurally related to the ligand or agonist.
  • EGF receptor ligand encompasses any compound, including peptides and non-peptide compounds, which fully or partially associate with and/or activate the EGF receptor.
  • an EGF receptor ligand is selected that has a suitable IC 5 O, for example an IC 50 of about ⁇ 0.7 nM, at an EGF receptor, as measured by methods known in the art (see Singh et al (1995) J. Biol. Chem. 270: 8429-8438, and Kopin et al (1995) J. Biol. Chem. 270: 5019-5023 describing in vitro cell growth assays, and receptor binding assays as described in Singh et al (1995) J. Biol.
  • An EGF receptor ligand may also be selected for particular applications in the present invention based on one or more of the following characteristics: ability of the EGF receptor ligand to bind to its receptor, preferably with an affinity constant K d less than about 1 ⁇ M, more preferably less than about 10OnM; ability to initiate a signal transduction pathway resulting in insulinotropic activity; insulinotropic activity; stimulation of beta cell proliferation/differentiation; resistance to DPP IV cleavage; and, an in vivo half-life, in particular, an in vivo half-life of at least about 15 minutes to 24 hours, preferably 2 to 10 hours or 2 to 8 hours in humans using conventional methods (see for example, the method described in US 2003/0144206).
  • the term encompasses compounds that in combination with a GLP-I agonist, and preferably one or both of a gastrin compound and a gastrin agonist provide at least one beneficial effect.
  • an EGF receptor ligand is selected to stimulate the EGF receptor such that when a GLP- 1 agonist, gastrin compound and/or gastrin agonist in the same or adjacent tissue or in the same individual is also present, neogenesis of insulin-producing pancreatic islet cells is induced.
  • the term includes any EGF receptor ligands that demonstrate additive, synergistic, or complementary activity with a GLP- 1 agonist and preferably one or both of a gastrin compound and a gastrin agonist.
  • EGF receptor ligand in particular EGF or TGF ⁇ . See also, Carpenter and Wahi, Chapter 4, in Peptide Growth Factors (Eds. Sporn and Roberts, Springer Verlag, 1990).
  • an EGF receptor ligand includes a polypeptide that shares a characteristic EGF-like domain defined by 6 cysteine residues that generate 3 peptide loops through the formation of disulphide bonds (Dunbar AJ and Goddard C, 2000 Int J. Biochem. Cell Biol. 32, 805-815).
  • an EGF receptor ligand includes a polypeptide that shares substantial sequence similarity with a mammalian EGF and possesses some or all of the biological activity of a wild-type EGF.
  • the EGF receptor ligand is a polypeptide that shares substantial sequence similarity with full length, wild-type human epidermal growth factor (EGF; see SEQ ID NO: 36), a 53 amino acid protein with a molecular weight of 6217 daltons (Karnes, W., Epidermal growth factor and transforming growth factor alpha, 1994, Raven Press, New York).
  • the group of compounds that comprises the EGF receptor ligands includes modified EGF receptor ligands which include variants of normal or wild type EGF, TGF- ⁇ etc. Modifications may affect one or more biological activity such as the rate of clearance of EGF receptor ligand.
  • the term includes peptides having an amino acid sequence with substantially similarity or identity to that of human EGF receptor ligand (e.g., EGF or TGF- ⁇ ), for example, with one or a few amino acid substitutions at various residue positions.
  • the term includes modified forms of an EGF receptor ligand which vary from wild-type EGF in chain length and amino acid sequence. Modifications can be made that affect both a biological activity and the rate of clearance of a wild-type EGF receptor ligand.
  • an EGF receptor ligand includes a polypeptide that shares substantial sequence similarity with a mammalian TGF ⁇ and possesses some or all of the biological activity of a wild-type TGF ⁇ .
  • Amino acid sequences of human TGF- ⁇ are shown in SEQ ID NOs: 42 and 43 and in GenBank Accession No. NP_003227.
  • TGF- ⁇ shares cysteine disulfide bond structures with a family of TGF- ⁇ related polypeptides such as vaccinia growth factor, amphiregulin precursor, betacellulin precursor, betacellulin, heparin binding EGF-like growth factor, epiregulin (rodents), HUS 19878, myxomavirus growth factor (MGF), Shope fibroma virus growth factor (SFGF), and schwannoma derived growth factor.
  • TGF- ⁇ related polypeptides such as vaccinia growth factor, amphiregulin precursor, betacellulin precursor, betacellulin, heparin binding EGF-like growth factor, epiregulin (rodents), HUS 19878, myxomavirus growth factor (MGF), Shope fibroma virus growth factor (SFGF), and schwannoma derived growth factor.
  • TGF- ⁇ related polypeptides including without limitation amphiregulin, vaccinia growth factor, myxomavirus growth factor (MGF), pox virus growth factor, Shope fibroma virus growth factor (SFGF), and heparin-binding EGF-like growth factor (HB-EGF) are also useful in the methods of the invention.
  • amphiregulin vaccinia growth factor
  • MEF myxomavirus growth factor
  • SFGF Shope fibroma virus growth factor
  • HB-EGF heparin-binding EGF-like growth factor
  • the invention also provides a class of peptides, including EGF and peptides smaller than the 53 amino acid human EGF, yet retaining some or all EGF biological activity, which are useful as pharmacologic and therapeutic agents.
  • EGF receptor ligands include without limitation full length EGF, which is EGF 1-53, and further include EGF 1-48, EGF1-49, EGF1-52, and fragments and active analogs thereof (see for example US Patent No. 5,434,135).
  • the invention also provides a class of peptides, including TGF - ⁇ and peptides smaller than the 50 amino acid human TGF- ⁇ [see SEQ ID NOs. 42 and 43, GenBank Accession No. NP_003227], yet retaining some or all TGF- ⁇ biological activity, which are useful as pharmacologic and therapeutic agents.
  • TGF- ⁇ and peptides smaller than the 50 amino acid human TGF- ⁇ [see SEQ ID NOs. 42 and 43, GenBank Accession No. NP_003227]
  • TGE- ⁇ forms TGF- ⁇ l-48, TGF- ⁇ l-47, TGF ⁇ l-51, and TGF- ⁇ 57 (US 6,815,418).
  • EGF forms have been genetically engineered to have alterations in structure and activities, for example, EGF having a methionine at position 21 replaced by a leucine residue has been described (U.S. Patent number 4,760,023).
  • Recombinant human EGF (hEGF) having 51 residues, i.e., lacking the two C-terminal residues at positions 52 and 53 of hEGF, and having a neutral amino acid substitution at position 51 retain EGF activity and are more resistant to protease degradation during a microbial production process, and following administration to a subject.
  • a series of nucleic acid molecules have been described that encode a family of proteins that have significant similarity to EGF and TGF ⁇ (WO 00/29438).
  • EGF muteins having histidine at residue 16 replaced with a neutral or acidic amino acid have been described (WO 93/03757), such forms retaining activity at low values of pH.
  • Chemical analogs and fragments of EGF and TGF ⁇ can retain their ability to bind various members of the EGF receptor family (U.S. patent number 4,686,283).
  • Various modifications of EGF or TGF ⁇ confer advantageous properties affecting one or more of recombinant protein production, in vitro and in vivo stability, and in vivo activity.
  • a recombinant modified EGF receptor ligand comprising a C-terminus deleted form of human EGF of 51 amino acids in length, having asparagine at position 51 (referred to herein as EGF5 IN), which retains substantial activity in the treatments described herein, and has in vivo and/or in vitro stability that is at least about as great or greater than normal or wild type hEGF (See International Application No. PCT/US02/33907).
  • an EGF receptor ligand is a recombinant EGF having a methionine at position 21 replaced by a leucine residue (U.S. Patent No. 4,760,023); a recombinant hEGF with an aspartyl residue at position 1 1 replaced by an isoaspartyl (George- Nascimento et al., Biochemistry, 29, 9584-9591, 1990); an EGF mutein having histidine at residue 16 replaced with a neutral or acidic amino acid (WO 93/03757); or, a chemical analog or fragment of wild-type EGF and TGF-alpha that binds to members of the EGF receptor family (See U.S. Patent No. 4,686,283).
  • an EGF receptor ligand is a polypeptide that has substantial sequence similarity or identity to EGF and TGF-alpha and is described in WO 00/29438.
  • an EGF receptor ligand is a wild-type EGF receptor ligand with modifications to the C-terminus amino acid residues at position 48 to position 53. Examples of such modified EGF receptor ligands are described in US Published Application No. 20030171269 and WO 03040310. In other aspects, an EGF receptor ligand is a modified EGF receptor ligand including without limitation a wild-type EGF receptor ligand with amino acid residues at position 48 to position 53 deleted or replaced.
  • an EGF receptor ligand is a wild-type EGF receptor ligand with the following amino acids deleted or replaced: the basic amino acids at positions 48 (lysine in wild-typeEGF) and 53 (arginine), the aromatic amino acids at positions 49 (tryptophan) and 50 (tryptophan), and/or the aliphatic amino acid at position 52 (leucine).
  • an EGF receptor ligand is represented by A-B wherein A comprises an amino acid sequence with substantial similarity or identity to amino acids 1 to 47, 1 to 48, 1 to 50, or 1 to 53 of SEQ ID NOs: 36 through 40, and B is between 1 to 10 amino acids.
  • B is a single neutral, hydrophobic or charged amino acid.
  • B is a single amino acid excluding glutamate.
  • B is a single neutral, hydrophobic or charged amino acid.
  • B is a hydrophobic amino acid such as alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, or valine.
  • B is a charged amino acid such as aspartic acid, glutamic acid, arginine, or lysine.
  • B is a neutral amino acid such as asparagine, glutamine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, alanine, threonine, tryptophan, tyrosine, or serine, especially asparagine, glutamine, alanine, or serine.
  • an EGF receptor ligand represented by A-B is selected to provide increased resistance to proteolysis compared with that of wild-type EGF and/or a biological activity that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared with that of wild-type EGF.
  • the biological activity can be mitogenesis, cytoprotection, inhibition of acid secretion, growth of a tissue precursor cell, differentiation of a tissue precursor cell, and/or growth and differentiation of a tissue precursor cell.
  • Mitogenesis can be determined by the effect of the amino acid sequence on rate of mitosis of epithelial cells.
  • Acid secretion can be determined in a gastric fistulated animal. Differentiation can be determined by islet neogenesis or mucosal cell formation
  • an EGF receptor ligand wherein A comprises amino acids 1-50 of SEQ ID NO: 36 wherein at least one of residues 1-50 is substituted with at least one conservative amino acid substitution, especially one, two or three amino acid substitutions, in the sequence as shown in SEQ ID NO: 36.
  • a conservative amino acid substitution refers to the replacement of an amino acid with a chemically similar amino acid. Examples of conservative amino acid substitution are: a charged amino acid replaced by a different amino acid of the same charge, such as Asp replaced by GIu, or an aromatic hydrophobic amino acid, e.g., Trp, replaced by a different aromatic hydrophobic amino acid, e.g., Phe (see U.S. Patent No. 6,207,154, issued Mar. 27, 2001).
  • An EGF receptor ligand can be utilized wherein A comprises amino acids 1 to 47, 1 to 48, 1 to 50, or 1 to 53 of SEQ ID NOs: 36 through 40, wherein amino acids 1 to 47, 1 to 48, 1 to 50, or 1 to 53 comprise a deletion of at least one amino acid residue from positions 1-5 of SEQ ID NOs: 36 through 40.
  • Such an EGF receptor ligand may have at least 50% of a biological activity of human wild-type EGF as shown in SEQ ID NO: 36.
  • an EGF receptor ligand is utilized wherein A comprises amino acids 1-53 of SEQ ID NO: 36 and wherein at least one amino acid is replaced or deleted at positions 48-53 of the carboxy terminus, the amino acid sequence being more stable to proteolysis than that of SEQ ID NO: 36.
  • an EGF receptor ligand is utilized wherein A comprises amino acids 1-50 of SEQ ID NO: 36 and B is aspargine (i.e., EGF 1-51 glu 51 asn Asn 51 -hEGF51, or EGF5 IN) (see SEQ ID NO: 37 and the DNA sequence of SEQ ID NO: 41 encoding an EGF 5 IN).
  • an EGF receptor ligand is utilized wherein A comprises amino acids 1 -50 of SEQ ID NO: 36 and B is alanine (EGF51 A - SEQ ID NO: 38). In a further particular embodiment, an EGF receptor ligand is utilized wherein A comprises amino acids 1-50 of SEQ IDNO: 36 and B is glutamine (EGFQ - SEQ IDNO: 39).
  • an EGF receptor ligand is utilized wherein A comprises amino acids 1-50 of SEQ ID NO: 36 and B is serine (EGF51S - SEQ ID NO: 40).
  • An EGF receptor ligand can be prepared using methods known in the art, in particular using recombinant techniques.
  • an EGF receptor ligand is produced using recombinant techniques in an appropriate host cell for example the organism Pichiapastoris.
  • an EGF receptor ligand has a purity of greater than 80%, 83%, 85%, 86%, 90%, 92%, 94%, 95%, 98%, or 99%, a concentration of about 1.0 to 1.5 mg/ml, and/or a molecular weight of about 5932 ⁇ 3 Da.
  • GLP-I agonist is understood to refer to any compound, including peptides and non-peptide compounds, which fully or partially activate a GLP- 1 receptor (e.g, human GLP- 1 receptor), or fully or partially mimic or increase a reaction, activity, or function of glucagon- like peptide 1 receptor ligands or initiate such reaction, activity, or function, or reduce or prevent inhibition of any reaction, activity or function of glucagon-like peptide 1 receptor ligands.
  • GLP- 1 receptor e.g, human GLP- 1 receptor
  • the "GLP-I agonist” is any peptide or non-peptide small molecule that binds to a GLP-I receptor, preferably with an affinity constant (K D ) or a potency (EC 50 ) of below 1 ⁇ M, e.g., below 100 nM as measured by methods known in the art (see e.g. WO 98/08871) and exhibits insulinotropic activity, where insulinotropic activity may be measured in in vivo or in vitro assays known to those of ordinary skill in the art.
  • the GLP-I agonist may be administered to an animal and the insulin concentration measured over time.
  • the term includes analogs, derivatives, isomers, chimeric polypeptides, fragments and modifications of a GLP-I agonist
  • WO 93/19175 NovoNordisk A/S
  • suitable GLP-I analogues and derivatives which can be used according to the present invention include those referred to in WO 99/43705 (Novo Nordisk A/S), WO 99/43706 (Novo Nordisk A/S), WO 99/43707 (Novo Nordisk A/S), WO 98/08871 (Novo Nordisk A/S), WO 99/43708 (Novo Nordisk A/S), WO 99/43341 (Novo Nordisk A/S), WO 87/06941 (The General Hospital Corporation), WO 90/11296 (The General Hospital Corporation), WO 91/11457 (Buckley et al.), WO 98/43658 (Eli Lilly & Co.), EP 0708179- A2 (Eli Lilly & Co.), EP 0699686-A2 (Eli Lilly & Co.), and WO 01/
  • a GLP-I agonist comprises a parent polypeptide of the formula GLP-I (7-R) wherein R is 36, 37, 38, 39, 40, 41 , 42, 43, 44, and 45, and wherein optionally up to 5, 10, or 15 amino acid residues are replaced with any ⁇ -amino acid residue.
  • a GLP-I agonist comprises or is selected from the group of glucagon-like peptide 1 receptor ligands consisting of GLP-I (7-36)-amide, GLP-l(7-37), a GLP-l(7-36)- amide analogue, a GLP-l(7-37) analogue, or a derivative of any of these.
  • a GLP-I agonist is a GLP-I (7-36)-amide or Ty r '-exendin ⁇ l- 31)-amide.
  • the GLP- 1 agonist is a naturally truncated GLP- 1 polypeptide (GLP- 1(7-36) or ((GLP- 1(7-37)), or an analogue or derivative thereof.
  • the sequences of these naturally occurring truncated GLP- 1 agonists are represented in SEQ ID NOs: 18, 19, and 20.
  • a GLP- 1 agonist may have the amino acid sequence of SEQ ID NOs: 16, 17, 18, or 19 modified so that amino acid residues at positions 1-20, preferably 1-15, more preferably 1 - 10, most preferably 1 -5 differ from the sequences of SEQ ID NOs: 17, 18 or 19.
  • the GLP-I agonist is an analogue of GLP- 1(7-37) or GLP-l(7-36) which has less than 10 amino acid residues that are different from those in GLP-I (7-37) or GLP-I (7-36), less than 5 amino acid residues that are different from those in GLP-I (7-37) or GLP-I (7-36), less than 3 amino acid residues that are different from those in GLP-I (7-37) or GLP-I (7-36), preferably only one amino acid residue that is different from a sequence of GLP-I (7-37) or GLP-I (7-36).
  • GLP-I agonists that may have specific utility in the present invention include polypeptides where one or more amino acids have been added to the N-terminus and/or C- terminus of GLP-l(7-37) or GLP-l(7-36). Preferably, about one to six amino acids may be added to the N-terminus and/or from about one to eight amino acids may be added to the C- terminus. In certain applications GLP-I agonists are selected that have up to 39 amino acids. Amino acids at positions 1-6 of an extended GLP-I agonist may be selected so that they are the same or are conservative substitutions of the amino acids at the corresponding positions of the parent GLP- 1 (7-37) or GLP- 1 (7-36). Amino acids at positions 38-45 of an extended GLP- 1 agonist may be selected so that they are the same or conservative substitutions of the amino acids at the corresponding positions of exendin-3 or exendin-4 (SEQ ID NOs: 22 and 23, respectively).
  • a GLP-I agonist comprising a position 8 analogue wherein the backbone for such analogs or fragments thereof contains an amino acid other than alanine.
  • the amino acid at position 8 may be selected from glycine, valine, leucine, isoleucine, serine, threonine, or methionine.
  • a GLP-I agonist is an insulinotropic analogue of GLP-l(l-37), for example, Met 8 -GLP-l(7-37), wherein the alanine in position 8 has been replaced by methionine and the amino acid residues in position 1 to 6 have been deleted, and Arg 34 -GLP- 1 (7-37) wherein the valine in position 34 has been replaced with arginine and the amino acid residues in position 1 to 6 have been deleted.
  • GLP-I agonists are selected comprising the sequence GLP-
  • the amino acid at position 8 may be selected from glycine, valine, leucine, isoleucine, serine, threonine, or methionine, preferably valine or glycine.
  • the analogs may additionally contain (a) an amino acid at position 22 selected from glutamic acid, lysine, aspartic acid, arginine, and preferably glutamic acid or lysine; (b) an amino acid at position 30 selected from glutamic acid, aspartic acid, serine, or histidine; (c) an amino acid at position 37 selected from lysine, arginine, threonine, glutamic acid, aspartic acid, serine, tryptophan, tyrosine, phenylalanine, or histidine; and/or (d) an amino acid at position 27 selected from alanine, lysine, arginine, tryptophan, tyrosine, phenylalanine, or histidine.
  • a group of GLP-I analogs and derivatives for use in the present invention comprises the GLP- 1 agonists described in U. S . Patent No. 5,545,618 and US Patent Application Serial No. 20040018975.
  • the analogs include active GLP-I peptides, 7-34, 7-35, 7-36 and 7-37 having amino acid substitutions at positions 7-10 and/or are truncations at the C-terminus and/or contain various other amino acid substitutions in the basic peptide.
  • Preferred analogs include those with D-amino acid substitutions in the 7 and 8 positions and/or N-alkylated or N-acylated amino acids in the 7 position since they are particularly resistant to degradation in vivo.
  • a GLP-I agonist comprises a peptide comprising or selected from the group consisting of GLP-I (1-38); GLP-I (1-39), GLP-I (1-40), GLP-I (1- 41), GLP-I (7-38), GLP-I (7-39), GLP-I (7-40), and GLP-I (7-41).
  • At least one amino acid of a GLP-I agonist has at least one substituent attached directly or indirectly (e.g. via a spacer such as ⁇ -Glu or ⁇ -Ala).
  • a substituent is generally selected to make the profile of action of the parent GLP-I agonist more protracted, make the GLP-I agonists more metabolically and physically stable, and/or increase solubility of the GLP-I agonist.
  • An example of a particular substituent is an amide, a carbohydrate, and a lipophilic substituent.
  • a lipophilic substituent includes but is not limited to an alkyl group, a group which has an ⁇ -carboxylic acid group, an acyl group of a straight- chain or branched fatty acid or alkane such as tetradecanoyl and hexadecanoyl.
  • Particular compositions, conjugates and treatments of the invention use GLP-I agonists with lipophilic substitutents such as those described in WO 99/43341 (Novo Nordisk) and US 2003/0119734A1 (Novo Nordisk).
  • a GLP-I agonist is a GLP-I (7-36)-amide or Tyr 31 -exendin-4(l-3 l)-amide.
  • the GLP-I agonist is a derivative of GLP- 1(7-36)-amide, GLP- 1 (7-37), a GLP- 1 (7-36)-amide analogue or a GLP- 1 (7-37) analogue, which comprises at least one lipophilic substituent.
  • the GLP- 1 derivative has three lipophilic substituents, two lipophilic substituents, or one lipophilic substituent attached to the parent peptide (i.e., GLP- l(7-36)-amide, GLP- 1(7-37), a GLP-l(7-36)-amide analogue or a GLP-I (7-37) analogue), where each lipophilic substituent(s) preferably has 4-40 carbon atoms, more preferably 8-30 carbon atoms, even more preferably 8-25 carbon atoms, even more preferably 12-25 carbon atoms, and most preferably 14-18 carbon atoms.
  • GLP-I agonist that is a derivative of GLP-I (7-36) or GLP-I (7-37) comprising a lipophilic substitutent.
  • the GLP-I agonist is Arg 34 Lys 26 (N ⁇ ( ⁇ -Glu(N ⁇ -hexadecanoyl)))-GLP-l (7-37).
  • the lipophilic substituent comprises a partially or completely hydrogenated cyclopentanophenathrene skeleton. In another embodiment, the lipophilic substituent is a straight-chain or branched alkyl group.
  • the lipophilic substituent is an acyl group of a straight-chain or branched fatty acid.
  • the lipophilic substituent is an acyl group having the formula CH 3 (CH 2 ) n CO-, wherein n is an integer from 4 to 38, preferably an integer from 12 to 38, and most preferably is CH 3 (CH 2 ) I2 CO-, CH 3 (CH 2 ) 14 CO-, CH 3 (CH 2 ) 16 CO-, CH 3 (CH 2 ) 18 CO-, CH 3 (CH 2 ) 20 CO- and CH 3 (CH 2 ) 22 CO-.
  • the lipophilic substituent is tetradecanoyl.
  • the lipophilic substituent is hexadecanoyl.
  • the lipophilic substituent has a group which is negatively charged such as a carboxylic acid group.
  • the lipophilic substituent may be an acyl group of a straight-chain or branched alkane ⁇ , ⁇ -dicarboxylic acid of the formula HOOC(CH 2 VCO-, wherein m is an integer from 4 to 38, preferably an integer from 12 to 38, and most preferably is HOOC(CH 2 ) 14 CO-, HOOC(CH 2 ) 16 CO-, HOOC(CH 2 ) I8 CO-, HOOC(CH 2 ) 20 CO- or HOOC(CH 2 ) 22 CO-.
  • the lipophilic substituent(s) may contain a functional group which can be attached to one of the following functional groups of an amino acid of the parent GLP-I peptide:
  • a lipophilic substituent is attached to the carboxy group of the R group of any Asp and GIu residue.
  • a lipophilic substituent is attached to the carboxy group attached to the alpha-carbon of the C-terminal amino acid.
  • a lipophilic substituent is attached to the epsilon-amino group of any Lys residue.
  • the lipophilic substituent is attached to the parent GLP-I peptide by means of a spacer.
  • a spacer must contain at least two functional groups, one to attach to a functional group of the lipophilic substituent and the other to a functional group of the parent GLP-I peptide.
  • the spacer is an amino acid residue except Cys or Met, or a dipeptide such as Gly-Lys.
  • a dipeptide such as Gly-Lys means any combination of two amino acids except Cys or Met, preferably a dipeptide wherein the C-terminal amino acid residue is Lys, His or Trp, preferably Lys, and the N-terminal amino acid residue is Ala, Arg, Asp, Asn, GIy, GIu, GIn, He, Leu, VaI, Phe, Pro, Ser, Tyr, Thr, Lys, His and Trp.
  • an amino group of the parent peptide forms an amide bond with a carboxylic group of the amino acid residue or dipeptide spacer
  • an amino group of the amino acid residue or dipeptide spacer forms an amide bond with a carboxyl group of the lipophilic substituent
  • Preferred spacers are lysyl, glutamyl, asparagyl, glycyl, beta-alanyl and gamma- aminobutanoyl, each of which constitutes an individual embodiment. Most preferred spacers are glutamyl and beta-alanyl.
  • the spacer is Lys, GIu or Asp
  • the carboxyl group thereof may form an amide bond with an amino group of the amino acid residue
  • the amino group thereof may form an amide bond with a carboxyl group of the lipophilic substituent.
  • a further spacer may in some instances be inserted between the ⁇ -amino group of Lys and the lipophilic substituent.
  • such a further spacer is succinic acid which forms an amide bond with the ⁇ -amino group of Lys and with an amino group present in the lipophilic substituent.
  • such a further spacer is GIu or Asp which forms an amide bond with the ⁇ -amino group of Lys and another amide bond with a carboxyl group present in the lipophilic substituent, that is, the lipophilic substituent is an N ⁇ -acylated lysine residue.
  • the spacer is an unbranched alkane ⁇ , ⁇ -dicarboxylic acid group having from 1 to 7 methylene groups, which spacer forms a bridge between an amino group of the parent peptide and an amino group of the lipophilic substituent.
  • the spacer is succinic acid.
  • the lipophilic substituent with the attached spacer is a group of the formula CH 3 (CH 2 ) p NH-CO(CH 2 ) q CO-, wherein p is an integer from 8 to 33, preferably from 12 to 28 and q is an integer from 1 to 6, preferably 2.
  • the lipophilic substituent with the attached spacer is a group of the formula CH 3 (CH 2 ) r CO-NHCH(COOH)(CH2) 2 CO-, wherein r is an integer from 4 to 24, preferably from 10 to 24.
  • the lipophilic substituent with the attached spacer is a group of the formula CH 3 (CH 2 ) S CO-NHCH((CH 2 )2COOH)CO-, wherein s is an integer from 4 to 24, preferably from 10 to 24.
  • the lipophilic substituent is a group of the formula COOH(CH 2 ) t CO- wherein t is an integer from 6 to 24.
  • the lipophilic substituent with the attached spacer is a group of the formula -NHCH(COOH)(CH 2 ) 4 NH-CO(CH 2 ) U CH 3 , wherein u is an integer from 8 to 18.
  • the lipophilic substituent with the attached spacer is a group of the formula CH 3 (CH 2 ) V CO-NH-(CH 2 ) Z -CO, wherein v is an integer from 4 to 24 and z is an integer from 1 to 6.
  • the lipophilic substituent with the attached spacer is a group of the formula -NHCH(COOH)(CH 2 ) 4 NH-COCH((CH 2 ) 2 COOH)NH-CO(CH 2 ) W CH 3 , wherein w is an integer from 10 to 16.
  • the lipophilic substituent with the attached spacer is a group of the formula -NHCH(COOH)(CH 2 ) 4 NH-CO(CH 2 ) 2 CH(COOH)NHCO(CH 2 ) X CH 3 , wherein x is zero or an integer from 1 to 22, preferably 10 to 16.
  • the GLP- 1 agonist is Arg 34 , Lys 26 (N ⁇ -( ⁇ -Glu(N ⁇ -hexade- canoyl)))-GLP-l(7-37).
  • the GLP- 1 agonist comprises or is selected from the group consisting of Gly 8 -GLP- 1(7-36)-amide, Gly 8 -GLP- 1(7-37), Val 8 -GLP-l(7-36)-amide, VaI 8 - GLP-l(7-37), Val 8 Asp 22 -GLP-l(7-36)-amide, Val 8 Asp 22 -GLP-l(7-37) , Val 8 Glu 22 -GLP-l(7- 36)-amide , Val 8 Glu 22 -GLP-l(7-37), Val 8 Lys 22 -GLP-l(7-36)-amide, VaI 8 LyS ⁇ -GLP- 1(7-37), Val 8 Arg 22 -GLP- 1 (7-36)-amide, Val 8 Arg 22 -GLP- 1 (7-37), Val 8 His 22 -GLP-l (7-36)-amide, Val 8 His 22 -GLP-l(7-37),
  • the GLP-I agonist comprises or is selected from the group consisting of Arg 26 -GLP- 1(7-37); Arg 34 -GLP-l(7-37); Lys 36 -GLP-l(7-37); Arg 26 ' 34 Lys 36 -GLP- 1(7-37); Arg 26 ' 34 -GLP-l(7-37); Arg 26 ' 34 Lys 40 -GLP-l(7-37); Arg 26 Lys 36 -GLP-l(7-37);
  • the GLP-I agonist comprises or is selected from the group consisting of: Gly 8 -GLP-l(7-36)-amide, Gly 8 -GLP-l (7-37), Val 8 -GLP-1 (7- 36)-amide, Val 8 -GLP-l (7-37), Val 8 Asp 22 -GLP-l (7-36)-amide, Val 8 Asp 22 -GLP- 1 (7-37), Val 8 Glu 22 -GLP- l (7-36)-amide, Val 8 Glu 22 -GLP-l (7-37), Val 8 Lys 22 -GLP- l (7-36)-amide, Val 8 Lys 22 -GLP- l (7-36)-amide, Val 8 Lys 22 -GLP- l (7-37) -Val 8 Arg 22 -GLP- l (7-36)-amide, Val 8 Arg 22 -GLP-l(7-37),Val 8 His 22 -GLP-l(7-36)-amide, -Val 8
  • a GLP-I agonist comprises or is selected from the group consisting of Val 8 -GLP-1(7-37)OH, Gly 8 -GLP-1(7-37)OH, Glu 22 -GLP-1(7-37)OH, Lys 22 - GLP-I (7-37)OH, Val 8 -Glu 22 -GLP-1(7-37)OH, VaI 8 - Lys 22 -GLP-1(7-37)OH, Gly 8 -Glu 22 -GLP- 1(7-37)OH, Gly 8 -Lys 22 -GLP-1(7-37)OH, Glu 22 -GLP-1(7-36)NH 2 , Lys 22 -GLP- 1(7-3 O)NH 2 , Val 8 -Glu 22 -GLP- 1 (7-36)NH 2 , Val 8 -Lys 22 -GLP- 1 (7-36)NH 2 , Gly 8 -Glu 22 -GLP- 1 (7-36)NH 2 ,
  • the GLP- 1 agonist comprises or is selected from the group consisting of Gly 8 -GLP-l(7-37), Val 8 GLP-l(7-37), Val 8 Asp 22 GLP-l(7-37), VaI 8 GIu 22 GLP-I (7-37), VaI 8 LyS 22 GLP- 1(7-37), and VaI 8 HiS 22 GLP-l(7-37), and analogs and derivatives thereof.
  • the GLP-I agonist comprises or is selected from the group consisting of Val 8 Trp 19 Glu 22 -GLP-l(7-37), VaI 8 GIu 22 VaF-GLP- 1(7-37), Val 8 Tyr 16 Glu 22 - GLP-l(7-37), Val 8 Trp 16 Glu 22 -GLP-l(7-37), Val 8 Leu 16 Glu 22 -GLP-l(7-37), Val 8 Tyr 18 GIu 22 - GLP-l(7-37), Val 8 Glu 22 His 37 -GLP- 1(7-37), Val 8 Glu 22 Ile 33 -GLP- 1(7-37),
  • the GLP-I agonist is a stable GLP-I analogue/derivative.
  • a "stable GLP-I analogue/derivative” includes a GLP-I analogue or a derivative of a GLP-I analogue which exhibits an in vivo plasma elimination half-life of at least 10 hours in man, as determined by the method described below. Examples of stable GLP-I analogue/derivatives can be found in WO 98/08871 and WO 99/43706. The method for determination of plasma elimination half-life of a compound in man is summarized as follows. The compound is dissolved in an isotonic buffer, pH 7.4, PBS or any other suitable buffer.
  • the dose is injected peripherally, preferably in the abdominal or upper thigh.
  • Blood samples for determination of active compound are taken at frequent intervals, and for a sufficient duration to cover the terminal elimination part (e.g. Pre-dose, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24 (day 2), 36 (day 2), 48 (day 3), 60 (day 3), 72 (day 4) and 84 (day 4) hours post dose).
  • Pre-dose 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24 (day 2), 36 (day 2), 48 (day 3), 60 (day 3), 72 (day 4) and 84 (day 4) hours post dose.
  • Determination of the concentration of active compound is performed as described in Wilken et al., Diabetologia 43(51):A143, 2000.
  • Derived pharmacokinetic parameters are calculated from the concentration-time data for each individual subject by use of non-compartmental methods, using the commercially available software WinNonlin Version 2.1 (Pharsight, Cary, NC, USA).
  • the terminal elimination rate constant is estimated by log-linear regression on the terminal log-linear part of the concentration-time curve, and used for calculating the elimination half-life.
  • GLP-I analogues and derivatives are disclosed in WO 98/08871 (analogues with lipophilic substituent) and in WO 02/46227 (analogues fused to serum albumin or to an Fc portion of an Ig).
  • the GLP-I agonist is formulated so as to have a half-life in man, as discussed above, of at least 10 hours. This may be obtained by sustained release formulations known in the art.
  • the GLP-I agonist is an exendin including exendin agonists.
  • An exendin includes naturally occurring exendin peptides that are found in the salivary secretions of the Gila-monster and the Mexican Bearded Lizard, reptiles that are endogenous to Arizona and Northern Mexico.
  • An "exendin agonist” is a compound that fully or partially mimics or increases the effects, activity or reactions of an exendin, or reduces or prevents inhibition of any effect, activity or reaction of an exendin.
  • Exendins also include analogues and derivatives of a naturally occurring exendin peptide, in particular a stable analogue or derivative of a naturally occurring exendin peptide.
  • Exendins include without limitation exendin-4, exendin-3, or analogs or derivatives thereof.
  • Exendin-3 is present in the salivary secretions of Heloderma horridum (Mexican Beaded Lizard) (Eng, J., et al., J. Biol. Chem., 267:7402-05, 1992).
  • a sequence for an exendin-3 peptide is in SEQ ID NO: 22.
  • Exendin-4 is a novel peptide from Heloderma suspectum (GiIa monster) venom, having 53% homology with GLP-I (7-36)amide (J Biol. Chem., 267:7402-05, 1992).
  • Exendin-4 functions as a long-acting potent agonist of the glucagon-like peptide 1 (GLP- 1) receptor, as it is resistant to degradation by DDP-IV. Exendin-4 has properties similar to GLP-I, and regulates gastric emptying, insulin secretion, food intake, and glucagon secretion.
  • GLP-1 glucagon-like peptide 1
  • exendin-4 examples include exenatide (synthetic form also known as AC2993 or ByettaTM, Amy Hn) ; exenatide LAR (long acting form) ; ZP 10 (modified exendin 4 having addition of six lysine residues, Aventis/Zealand Pharma), and APlO (long acting formulation, Alkermes, Cambridge MA).
  • exendin-4 is exendin-3.
  • the exendin is exendin-4, in particular exenatide, more particularly ByettaTM.
  • analogues and derivatives include an exendin-4(l-39) analogue or a derivative of an exendin-4(l-39).
  • exendins as well as analogues, derivatives, fragments, and agonists thereof are disclosed in WO 97/46584, US Patent No. 5,424,286, WO 01/04156, US Patent Nos. 6,956,026, 6,924,264, 6,902,744, 6,528,486, and 6,506,724 and references therein.
  • US 5,424,286 describes a method for stimulating insulin release with an exendin polypeptide.
  • WO 97/46584 describes truncated versions of exendin peptide(s). The disclosed peptides increase secretion and biosynthesis of insulin, but reduce those of glucagon.
  • WO 01/04156 describes exendin-4 analogues and derivatives as well as the preparation of these molecules. Exendin-4 analogues stabilized by fusion to serum albumin or Fc portion of an Ig are disclosed in WO 02/46227.
  • an exendin has the empirical formula C IS4 H 282 N 5O O OO S and a molecular weight of 41.86.6 daltons, and comprises the following amino acid sequence: H- His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg- Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 [SEQ ID NO: 24].
  • an exendin has an amino acid sequence comprising His- Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu- Phe-Ile-Glu-T ⁇ -Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser [SEQ IDNO: 23].
  • exendins include the following: [SEQ ID NO 25: His GIy GIu GIy Thr Phe Thr Ser Asp Leu Ser Lys GIn Met GIu GIu GIu Ala VaI Arg Leu Phe lie GIu Trp Leu Lys Asn GIy GIy], exendin-4 (1-30) amide [SEQ ID NO: 26: His GIy GIu GIy Thr Phe Thr Ser Asp Leu Ser Lys GIn Met GIu GIu GIu Ala VaI Arg Leu Phe He GIu Trp Leu Lys Asn GIy GIy- NH 2 ], exendin-4 (1-28) amide [SEQ ID NO 31 : His GIy GIu GIy Thr Phe Thr Ser Asp Leu Ser Lys GIn Met GIu GIu GIu Ala VaI Arg Leu Phe He GIu Trp Leu Lys Asn-NH 2 ], 14 Leu, 25 Phe
  • an exendin precursor in particular Met-Lys-Ile-Ile- Leu-T ⁇ -Leu-Cys-Val-Phe-Gly-Leu-Phe-Leu-Ala-Thr-Leu-Phe-Pro-Ile-Ser-T ⁇ -Gln-Met-Pro- Val-Glu-Ser-Gly-Leu-Ser-Ser-Glu-Asp-Ser-Ala-Ser-Ser-Glu-Ser-Phe-Ala-Ser-Lys-Ile-Lys-Arg- His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Asn-Met-Glu-Glu-Glu-Glu-Ala-Val-Arg- Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Thr-Leu-P
  • the exendin comprises exendin-4.
  • the pharmaceutical composition comprises a peptide selected from: exendin-4 (1-30), exendin-4 (1-30) amide, exendin-4 (1-28) amide, 14 Leu, 25 Phe exendin-4 amide, 14 Leu, 25 Phe exendin-4 (1-28) amide, and 14 Leu, 22 Ala, 25 Phe exendin-4 (1-28) amide.
  • exendin analogs include without limitation exendin agonist compounds such as those described in U.S. Provisional Application US20060035836A1, US20050267034A1, US6989366, US6956026, and publications and patents and patent applications referenced therein.
  • the GLP-I agonist is a stable exendin-4 analogue/- derivative.
  • stable exendin-4 analogue/derivative refers to an exendin-4(l -39) analogue or a derivative of an exendin-4(l -39) analogue which exhibits an in vivo plasma elimination half- life of at least 10 hours in man, as determined by the method described above for a "stable GLP-I analogue/derivative".
  • the GLP-I agonist is Ser 38 ,Lys 39 ' 40 ' 41 ' 42 ' 43 ' 44 -Exendin-4(l- 39)amide.
  • the GLP-I agonist is selected from the non-peptide small molecule GLP-I agonists disclosed in WO 00/42026.
  • An amino acid portion of a GLP-I agonist can be prepared by a variety of methods known in the art such as solid-phase synthesis, purification of GLP-I agonists from natural sources, recombinant technology, or a combination of these methods. See for example, United States Patent Nos. 5,188,666, 5,120,712, 5,523,549, 5,512,549, 5,977,071, 6,191,102, Dugas and Penney 1981, Merrifield, 1962, Stewart and Young 1969, and the references cited herein.
  • GLP-I agonist derivatives can be produced by appropriate derivatization of an appropriate backbone produced, for example, by recombinant DNA technology or peptide synthesis (e.g. Merrifield-type solid phase synthesis) using methods known in the art of peptide synthesis and peptide chemistry.
  • GLP-I agonists are listed in Table 1. GLP-I agonists are in each case generically and specifically disclosed in the referenced patent document or publication. Any of the substances disclosed in the patent documents and publications referenced in Table 1 are considered potentially useful as GLP-I agonists to be used in carrying out the present invention.
  • Exemplary GLP-I agonist compositions include: BIM 51077 (GLP-I analog resistant to DPP-IV digestion, available from Begon Ipsen); AC2592 (GLP-I, from Amylin, San Diego CA); Byetta® (Eli Lilly and Amylin); ThGLP-I (GLP-I, modified amino acids and fatty acid attachment, from Theratechnologies, Saint-Laurent, Quebec, Canada); DAC:GLP-1 (Conjuchem, Montreal, Quebec, Canada); CJC-1131 or DACjGLP-I (GLP-I analog engineered for covalent coupling to albumin, Conjuchem), LY315902 and sustained release LY315902 (DDP-IV resistant GLP-I analog from Eli Lilly, Indianapolis, IN); low molecular weight GLP-I mimetic, Albugon (albumin: GLP-I fusion peptide from Human Genome Sciences, Rockville, MD); Liraglutide or NN2211 (long acting GLP-I derivative that is obtained by acy
  • GLP-I agonist includes substances that decrease inactivation or increase activity, function or reactions of glucagon-like peptide 1 receptor ligands. Examples of such substances include DPP-IV inhibitors. GLP-I agonists may be produced by methods known in the art and they may be administered at therapeutically effective doses known in the art for the compounds.
  • DPP-IV inhibitor is an antagonist of a dipeptidylpeptidase- IV (DPP-IV) or another member of the family of serine peptidases that includes quiescent cell proline dipeptidase, DPP8, and DPP9. It exhibits inhibition of the enzymatic activity of DPP- IV and functionally related enzymes, such as from 1-100% inhibition, and especially preserves the action of substrate molecules, including but not limited to GLP- 1 , GIP, peptide histidine methionine, and other similar molecules.
  • a DPP-IV inhibitor may indirectly affect the levels of GLP-I (Hughes, T. et al., 2002, Am I Diabetes Assoc Abstract 272) by inhibiting an enzyme involved in its integrity.
  • a DPP-IV inhibitor can be a peptidic or a non-peptidic compound, in particular aspects of the invention the DPP-IV inhibitor is a non-peptidic compound.
  • DPP IV inhibitor is also intended to comprise active metabolites and prodrugs of a DPP-IV inhibitor, such as active metabolites and prodrugs of DPP-FV inhibitors.
  • a “metabolite” refers to an active derivative of a DPP-IV inhibitor produced when the DPP- IV inhibitor is metabolized.
  • a “prodrug” refers to a compound that is either metabolized to a DPP-IV inhibitor or is metabolized to the same metabolite(s) as a DPP-IV inhibitor.
  • the inhibitors also include the corresponding stereoisomers as well as the corresponding polymorphs, e.g., crystal modifications, which are disclosed in the cited patent documents.
  • DPP-IV inhibitors are listed in Table 2 and in WO 05049088 and references therein. DPP-IV inhibitors are in each case generically and specifically disclosed in the referenced patent document or publication. Any of the substances disclosed in the patent documents and publications referenced in Table 2 are considered potentially useful as DPP-IV inhibitors to be used in carrying out the present invention.
  • the DPP-IV inhibitor is sitagliptin, vildagliptin, PSN9301, saxagliptin, N-(N'-substituted glycyl)-2-cyanopyrrolidines, L-threo-isoleucyl thiazolidine (P32/98), L-allo-isoleucyl thiazolidine, L-threo-isoleucyl pyrrolidine, isoleucine thiazolidide, valine purrolidide, NVP-DPP738 (Novartis, Cambridge, MA), LAP237 (Novartis), P32/98 (Probiodrug AG, Halle, Germany), P93/01 (Probiodrug), and L-allo- isoleucyl pyrrolidine described in U.S.
  • a DPP IV inhibitor can be prepared by a variety of methods known in the art.
  • DPP-IV inhibitors for the combinations, uses, methods, and kits of the present invention are l- ⁇ 2-[(5-cyanopyridin-2-yl) amino]ethylamino ⁇ acetyl-2 (S)-cyano- pyrrolidine dihydrochloride (DPP728), especially the dihydrochloride thereof; (S)-l-[(3- hydroxy- 1 -adamantyl)amino]acetyl-2-cyano-pyrrolidine (LAF237); L-threo-isoleucyl thiazolidine (compound code according to Probiodrug: P32/98); MK-0431 ; GSK23 A; BMS- 477118; 3-(aminomethyl)-2-isobuthyl-l-oxo-4-phenyl-l,2-dihydro-6-isoquinoline carboxamide and 2- ⁇ [3-(aminomethyl)-2-isobuthyl-4-phenyl- 1 -o
  • a “gastrin/CCK receptor” refers to a member of the G-protein-coupled receptor family that displays a characteristic binding affinity for a cholecystokinin (CCK) including without limitation CCK-8, desulfated CCK-8, CCK-33, CCK-4, or gastrins including without limitation desulfated or sulfated gastrin- 17, or pentagastrin, or other CCK or gastrin analogues or family members.
  • CCK/gastrin receptor proteins are CCK A and CCK ⁇ /gastrin receptors, in particular CCK ⁇ /gastrin receptor.
  • a “gastrin compound” refers to any compound, including peptides and non-peptide compounds, which fully or partially associate with and/or activate a gastrin/CCK receptor and/or increase gastrin secretion.
  • a gastrin compound is selected that has a suitable IC 50 , for example an IC 50 of about - 0.7 nM, at a gastrin/CCK receptor, as measured by methods known in the art (see Singh et al (1995) J. Biol. Chem.270: 8429-8438, and Kopin et al (1995) J. Biol. Chem.
  • a gastrin compound may also be selected based on other criteria such as activity, half-life etc.
  • Gastrin compounds that may be used in the present invention include without limitation one or more of a gastrin compound including a cholecystokinin or a cholecytokinin agonist.
  • neuropeptide compound encompasses compounds that in combination with an EGF receptor ligand and GLP-I agonist and optionally a gastrin agonist provide at least one beneficial effect.
  • a gastrin compound is selected to stimulate an EGF receptor ligand or GLP-I agonist so that neogenesis of insulin-producing pancreatic islet cells is induced.
  • the term includes any gastrin compound that demonstrates additive, synergistic, or complementary activity with a growth/hormone regulatory factor and a gastrin agonist.
  • a gastrin compound includes a polypeptide that shares substantial sequence similarity with a mammalian gastrin and possesses some or all of the biological activity of a mammalian gastrin.
  • a gastrin compound may be an active analog, fragment or other modification which, for example, shares amino acid sequence similarity with an endogenous mammalian gastrin, for example, shares 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity
  • a “gastrin compound” includes, without limitation, the various forms of gastrin, such as gastrin 71, gastrin 52, gastrin 34 (big gastrin), gastrin 17 (little gastrin), gastrin 14, and gastrin 8 (mini gastrin), pentagastrin, tetragastrin, and fragments, analogs, and derivatives thereof. Sequences for gastrins including big gastrin-34 (Bonato et al, 1986, Life Science 39:959) and small gastrin-17 (Bentley et al (1966) Nature 209:583) are known in the art, and some are shown in SEQ ID NOs: 1 to 9.
  • sequences for gastrins include gastrin 71 of SEQ ID NO: 5 (amino acid residues 22 to 92), gastrin 52 of SEQ ID NO: 6, gastrin 34 (big gastrin) of SEQ ID NOs: 1 or 2, gastrin 17 (little gastrin) of SEQ ID NO: 3 or 4, gastrin 14 of SEQ ID NO: 7, and gastrin 6 of SEQ ID NO: 8 or 9.
  • Gastrin-34 is essentially an extension of an amino acid sequence at the N-terminal end of gastrin-17. Big gastrin is cleaved in vivo to release gastrin-17.
  • GIp at the N-terminal end of a gastrin is pyroglutamate, which is a naturally cyclized form of glutamate. GIp may be present or absent in a gastrin compound. In various embodiments, where cysteine or lysine is added to a terminus of gastrin having a pyroglutamate, the pyroglutamate is replaced with a glutamate, or the pyroglutamate is deleted.
  • a gastrin 34 or gastrin- 17 may be used in the invention where there is a methionine or a leucine at position 15, as shown in SEQ ID NOs: 1-4 herein.
  • gastrin compounds examples include the compounds disclosed in U.S. Patent No. 6,288,301.
  • a gastrin compound may be selected that is a peptide or non-peptide agonist or partial agonist of the gastrin receptor such as A71378 (Lin et al., Am. J. Physiol. 258 (4 Pt 1): G648, 1990).
  • a gastrin compound may be selected that is a gastrin/CCK ⁇ receptor ligand including but not limited to cholecystokinin (CCK) such as CCK 58, CCK 33, CCK 22, CCK 12 and CCK 8; and the like.
  • CCK cholecystokinin
  • a “gastrin compound” includes a modified form of a gastrin, including but not limited to a modified form of gastrin 71 [SEQ ID NO: 5, amino acid residues 22 to 92], gastrin 52 [SEQ ID NO: 6], gastrin 34 (big gastrin) [SEQ ID NO: 1 or 2], gastrin 17 (little gastrin) [SEQ ID NO: 3 or 4], gastrin 14 [SEQ ID NO: 7], gastrin 8, gastrin 6 [SEQ ID NO: 8 or 9], pentagastrin, and tetragastrin.
  • a modified gastrin preferably comprises TrpMetAspPhe-NH 2 [SEQ ID NO: 13] or TrpLeuAspPhe-NH 2 [SEQ ID NO: 14].
  • a modified gastrin comprises at least amino acids 1 -34, 18-
  • a gastrin compound used in aspects of the methods, compositions, and conjugates of the invention may comprise gastrin 17 and analogs and derivatives thereof.
  • the gastrin compound is synthetic human gastrin 1 having 17 amino acid residues with a Leu residue at amino acid position 15 [SEQ ID NO: 4].
  • a gastrin compound used in the methods, compositions and conjugates of the invention may comprise gastrin 34 and analogs and derivatives thereof.
  • the gastrin compound is a synthetic human gastrin 34 with methionine or leucine at position 32 [SEQ ID NO: l or 2].
  • Modified gastrin compounds for use in the present invention comprise the modified gastrin compounds described in PCT/CA03/01778, US Serial No. 10/719,450 and U.S. Application Serial No. 60/519,933 incorporated in their entirety by reference.
  • a modified gastrin can be a gastrin derivative or analogue comprising a minimal sequence of 6 amino acids (from the C-terminal end) of a gastrin, in particular amino acid residues 1 to 34, 18 to 34 or 29-34 of SEQ ID NOs: 1 or 2, or amino acid residues 1-17,
  • a reactive group is generally capable of linking a gastrin sequence, directly or indirectly via a crosslinking agent and/or spacer region, to a carrier.
  • a reactive group may be introduced by adding or substituting an amino acid comprising a reactive group, for example by adding a cysteine or lysine. Therefore, a modified gastrin may comprise a gastrin sequence (e.g. gastrin-34 or gastrin 17) wherein at least one reactive amino acid (e.g. cysteine or lysine) is added or substituted.
  • the addition of a reactive amino acid can be at a terminal region, in particular an N-terminal region.
  • a modified gastrin may also optionally comprise a spacer.
  • a spacer can interact with a reactive group, for example, an amino acid comprising a reactive group.
  • a spacer can be one or more amino acids, peptides, peptidomimetics, or small organic molecules.
  • a spacer can comprise at least one amino acid, preferably at least two, three, four or five amino acids and in certain embodiments it is a sequence of several amino acids, including without limitation alanine or glycine.
  • a spacer can comprise alternating amino acids (e.g. glycine and/or alanine), non-alternating amino acids, a random sequence or a particular sequence.
  • a spacer can be synthesized as part of, or may be chemically attached to an amino acid of a gastrin sequence.
  • a modified gastrin may optionally comprise a cross-linking agent.
  • a cross-linking agent may comprise a homobifunctional or heterobifunctional portion for interaction directly or indirectly with a gastrin, spacer and/or a reactive group.
  • a cross-linking agent may interact with a gastrin sequence or a spacer, or it may be added to a reactive group at the end (in particular N-terminus) of a modified gastrin.
  • a cross-linking agent can be any agent that can link a gastrin sequence and a carrier directly or via a spacer.
  • homobifunctional crosslinking agents include without limitation amino group directed homobifunctional cross-linking reagents such as bisimidates (e.g. methyl acetimidate-HCl), bifunctional aryl halides (e.g. l,5-dichloro-2,4-dinitrobenzene), bifunctional acylating agents (e.g. diisocyanates), bifunctional sulfonyl halides (e.g. phenol- 2,4-disulfonyl-chloride), bifunctional acylazides (e.g. tartryl diazide), dialdehydes (e.g. glutaraldehyde), and diketones (e.g.
  • amino group directed homobifunctional cross-linking reagents such as bisimidates (e.g. methyl acetimidate-HCl), bifunctional aryl halides (e.g. l,5-dichloro-2,4-dinitrobenzene),
  • heterobifunctional crosslinkers include amino and sulfhydryl group directed bifunctional reagents (e.g. N- succinimidyl-3-(2-pyridyldithio propionate), carboxyl and either sulfhydryl or amino group directed bifunctional reagents (e.g. p-nitrophenyl diazoacetate), and carbonyl and sulfhydryl group directed bifunctional reagents (e.g. l-(aminooxy)-4-[3-nitro-2-pyridyl)dithio)]butane).
  • amino and sulfhydryl group directed bifunctional reagents e.g. N- succinimidyl-3-(2-pyridyldithio propionate
  • carboxyl e.g. p-nitrophenyl diazoacetate
  • carbonyl and sulfhydryl group directed bifunctional reagents e.g. l-(aminooxy)
  • a modified gastrin can optionally comprise a carrier which may be a polymer.
  • a carrier may be a polymer of amino acids (proteins), sugars (polysaccharides), nucleosides, synthetic polymers and mixtures thereof.
  • a protein carrier may be a protein found in the circulatory system. Examples of protein carriers found in the circulatory system, in particular the human circulatory system, include without limitation plasma components such as serum, purified serum proteins such as albumin (in particular human serum albumin), transferrin, or an immunoglobulin, red blood cell proteins such as glycophorin A and AE- 1 , sugar binding proteins such as a lectin, inactivated enzymes, phosphate and sulphate binding proteins, and lipid binding proteins.
  • Suitable polymeric carriers include without limitation cellulose and derivatives thereof, starch and derivatives thereof, heparin and derivatives thereof, and synthetic polymers such as polyethylene glycol (PEG) and dextran, and derivatives thereof.
  • Carriers may be attached to a gastrin or spacer by way of reactive groups on, or introduced to, the carrier, gastrin, and/or spacer.
  • carriers can be covalently attached to reactive groups (such as thiol groups, alpha and epsilon amino groups, carboxyl groups or aromatic groups) on a gastrin or spacer which may be present or added by chemical modification of the gastrin or spacer.
  • a modified gastrin can comprise a gastrin of SEQ
  • a group of modified gastrin compounds include compounds having an amino acid sequence comprising from the amino terminus Z-Y m -X n -AAi-AA 2 -AA 3 -AA 4 -AAs-AA 6 , wherein AAi is Tyr or Phe, AA 2 is GIy, Ala, or Ser, AA 3 is Tip, VaI, or He, AA 4 is Met or Leu, AA 5 is Asp or GIu, and AA 6 is Phe or Tyr and wherein AA 6 is optionally amidated;
  • Z is a carrier, in particular a polymer and when the polymer is a protein Z is an amino acid sequence;
  • Z is a protein, in particular a protein of the circulatory system, more particularly a serum protein, still more particularly albumin, most particularly human serum albumin.
  • X is one or more amino acid residues from position 18 to position 28 of SEQ ID NO: 1. Therefore, the gastrin compounds by virtue of the presence of X can have any of gastrin sequences from positions 18-28, 19-28, 20-28, 21-28, etc.
  • the gastrin compound optionally contains an amino acid spacer (Y) of length m, and m is 0 to about 20 residues.
  • X is one or more amino acid residues from position 1 to 11 or 2 to 11 of SEQ ID NOs: 3 or 4. Therefore, the gastrin compounds by virtue of the presence of X can have any of gastrin sequences from positions 2 to 11 , 3 to 11 , 4 to 11 , 5 to 11 , etc.
  • the gastrin compound optionally contains an amino acid spacer (Y) of length m, and m is 0 to about 20 residues.
  • a gastrin compound includes a modified gastrin compound of the formula X n -AA 1 - AA 2 -AA 3 -AA 4 -AA 5 -AA 6 , where there is no spacer (Y) and m is 0, which may further comprise a bifunctional cross-linking agent for interaction or linkage to a carrier Z, where Z further comprises a non-proteinaceous polymer such as dextran or PEG.
  • a modified gastrin compound particularly described herein may further comprise an amino terminal cysteine or lysine residue.
  • the gastrin component contains at least amino acid residues 29-34 of SEQ ID NOs: 1 or 2, and it is associated with a polymer, a lipid or a carbohydrate.
  • the polymer may be a synthetic or naturally occurring polymer.
  • the term polymer includes a protein polymer of amino acids, and is not limited to a synthetic polymer.
  • the polymer may be a polyethylene glycol (PEG) or a dextran.
  • a modified gastrin compound can be based on SEQ ID NOs: 1 or 2 or "big" gastrin-34 and have a residue at position 32 which is a methionine or a leucine, respectively.
  • Another preferred modified gastrin compound comprises a structure C- Y m -X, wherein C is Cys or Lys, Y m is an optional spacer region comprising m amino acid residues of a small neutral amino acid, and X is at least six amino acid residues comprising at least positions 12- 17 of gastrin- 17 (SEQ ID NOs: 3 or 4) or at least positions 29-34 of gastrin-34 (SEQ ID NOs: 1 or 2).
  • This modified gastrin compound can further comprise a bifunctional cross-linking agent wherein one reactive portion of the cross-linking agent is covalently linked to C, and the other reactive portion is covalently linked to a polymer or protein.
  • AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 in a modified gastrin compound is Tyr-Gly-Trp-Met-Asp-Phe [SEQ ID NO: 10] or Tyr-Gly-Trp-Leu-Asp- Phe [SEQ ID NO: H].
  • a gastrin compound used in the methods, compositions and conjugates of the invention is gastrin 34 or gastrin 17 or portions thereof, directly or indirectly interacting or associated with a serum protein, in particular albumin or an immunoglobulin, more particularly human serum album.
  • a gastrin compound comprises synthetic human gastrin 34 having 2-34 amino acid residues of SEQ ID NO: 1 or 2, and optionally anN-terminal cysteine and/or a carrier; synthetic human gastrin having 1-17 amino acid residues with a Leu residue at amino acid position 15 [SEQ ID NO: 4] and optionally an N-terminal cysteine residue; and a synthetic human gastrin having amino acid residues 2 to 17 or 5- 17 of SEQ ID NO: 3 or 4, optionally with an N-terminal cysteine residue and/or a carrier (e.g. PEG or human serum albumin) linked via a spacer [e.g.
  • a carrier e.g. PEG or human serum albumin
  • Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala i.e. (GA) 5 ] [SEQ ID NO: 12], in particular, a synthetic human gastrin having amino acid residues 2 to 17 or 5-17 of SEQ ID NOs: 3 or 4, with a human serum albumin (HSA) polymer linked via a GIy- Ala-Gly- Ala-Gly-Ala-Gly-Ala-Gly- Ala [i.e., (GA) 5 ] spacer, and optionally an N-terminal cysteine residue.
  • HSA human serum albumin
  • Gastrin compounds may be synthesized by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart). The synthesis may be performed using manual procedures or by automation. Automated synthesis may be carried out, for example, using an Applied Biosystems 43 IA peptide synthesizer (Perkin Elmer). Gastrin compounds may also be obtained from commercial sources. For example, synthetic human gastrin 17 with methionine or leucine at position 15 are available from Bachem AG, Bubendorf, (Switzerland), and from Research Plus Inc (New Jersey, USA).
  • the gastrin compound is a leucine substituted gastrin 17 of SEQ ID NO: 3.
  • a gastrin compound may also be characterized by the following properties: isoelectric point of about 3.4; purity of at least about 80%, 85%, 90%,
  • a "gastrin agonist” refers to any substance that fully or partially mimics a reaction, activity, or function of a gastrin compound or initiates such reaction, activity, or function, or reduces or prevents inhibition of any reaction, activity or function of a gastrin compound.
  • a gastrin agonist is a gastrin secretagogue.
  • a gastrin agonist is selected that provides, in combination with an EGF receptor ligand and a GLP-I agonist and optionally a gastrin compound, therapeutically effective amounts of gastrin in a subject (e.g,. a diabetic subject).
  • a gastrin agonist is selected that provides, in combination with an EGF receptor ligand and a GLP-I agonist and optionally a gastrin compound, an about 10 to 1000 fold, 10 to 500 fold, 10 to 100 fold, 10 to 50 fold, 5 to 500 fold, 5 to 100 fold, or 5 to 50 fold increase in plasma gastrin.
  • gastrin agonists are proton pump inhibitors and histamine-2 receptor antagonists.
  • a “proton pump inhibitor” and “PPI” are used interchangeably herein and include a substance which inhibits gastric acid secretion by blocking the proton pump and/or increasing gastrin secretion.
  • it refers to any acid labile pharmaceutical agent possessing pharmacological activity as an inhibitor of H 4 TK + - ATPase. More particularly it contemplates substances which covalently bind to H+/K+- ATPase, the enzyme responsible for gastric acid secretion.
  • a PPI includes compounds comprising a 2-[(2-pyridinyl) methyl sulphinyl]-lH-benzimidazole skeleton or a related skeleton, which may optionally be substituted in various forms.
  • a proton pump inhibitor may, if desired, be in the form of a free base, free acid, salt, ester, solvates (in particular hydrates), anhydrate, amide, enantiomer, isomer, tautomer, prodrug, polymorph, derivative, or the like, provided that the free base, salt, ester, hydrate, amide, enantiomer, isomer, tautomer, prodrug, or any other pharmacologically suitable derivative is therapeutically active.
  • proton pump inhibitors include but are not limited to: soraprazan (Altana); ilaprazole (U.S. Patent No. 5,703,097) (Il-Yang); AZD-0865 (AstraZeneca); hydroxyomeprazole; dontoprazole; dontoprazole; dontoprazole; dontoprazole; dontoprazole; perprazole; ransoprazole; pariprazole; YH- 1885 (PCT Publication WO 96/05177) (SB-641257) (2-pyrimidinamine, 4-(3,4-dihydro-l- methyl-2(lH)-isoquinolinyl)-N-(4-fluorophenyl)-5,6-dimethyl-monohydro- chloride)
  • the proton pump inhibitor is selected from the group consisting of 5- methoxy-2-[(4-methoxy-3,5-dimethyl-2-pyridinyl)-methylsulphinyl]-lH-benzimidazole (omeprazole), 5-methoxy-2-[(S)-[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl] sulphinyl]- 1 H-benzimidazole (esomeprazole), 2-[3-methyl-4-(2,2,2-trifluoroethoxy)-2-pyridinyl) methylsulphinyl]-lH-benzimidizole (lansoprazole), 2- ⁇ [4-(3-methoxypropoxy)-3- methylpyridin-2-yl]methylsulphinyl ⁇ - 1 H-benzimidazole(rabeprazole), 5-difluoromethoxy-2- [(3 ,4-di-methoxy-2-pyridin)-
  • a proton pump inhibitor is in the form of a salt.
  • a salt of a proton pump inhibitor may be prepared for example from formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, ftimaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, stearic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, embonic, methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, cyclohexylaminosulfonic, algenic, ⁇ -hydroxybutyric, galactaric and galacturonic acids.
  • acid addition salts are prepared from the free base of a proton pump inhibitor using conventional methods involving reaction of the free base with a suitable acid.
  • suitable acids for preparing acid addition salts include without limitation organic acids, such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • an acid addition salt is converted to a free base by treatment with a suitable base.
  • an acid addition salt is a halide salt, which is prepared using hydrochloric or hydrobromic acids.
  • the basic salt is an alkali metal salt, such as a sodium salt or copper salt.
  • salts of proton pump inhibitors include without limitation: a sodium salt form such as esomeprazole sodium, omeprazole sodium, rabeprazole sodium, pantoprazole sodium; or a magnesium salt form such as esomeprazole magnesium or omeprazole magnesium described in U.S. Patent No. 5,900,424; a calcium salt form; or a potassium salt form such as the potassium salt of esomeprazole described in U.S. Patent No. 6,511,996.
  • Other salts of esomeprazole are described in U.S. Patent Nos. 4,738,974 and 6,369,085. Salt forms of pantoprazole and lansoprazole are disclosed in U.S. Patent Nos. 4,758,579 and 4,628,098, respectively.
  • esters of proton pump inhibitors are utilized.
  • An ester may be prepared by functionalization of hydroxyl and/or carboxyl groups which may be present within the molecular structure of the drug.
  • the esters are acyl- substituted derivatives of free alcohol groups, such as moieties derived from carboxylic acids of the formula -RCOOR 1 where R 1 is an alkyl group in particular a lower alkyl group.
  • An ester can be converted to a free acid, if desired, by using conventional procedures such as hydrogenolysis or hydrolysis.
  • a proton pump inhibitor or its salts can be in a crystalline form. Crystals of a proton pump inhibitor may contain variable amounts of solvent. Therefore, the term "proton pump inhibitor” includes all solvates, in particular all hydrates, of the proton pump inhibitors and their salts.
  • the proton pump inhibitor is a salt or hydrate including without limitation pantoprazole- sodium sesquihydrate [pantoprazole-sodium ⁇ l.5 H 2 O], (-)-pantoprazole-sodium sesquihydrate, pantoprazole-magnesium dihydrate, omeprazole-magnesium, omeprazole-magnesium tetrahydrate, esomeprazole-magnesium and esomeprazole-magnesium tetrahydrate.
  • the proton pump inhibitor is a substituted bicyclic aryl-imidazole, wherein the aryl group may be, for example, a pyridine, a phenyl, or a pyrimidine group which is attached to the 4- and 5-positions of the imidazole ring.
  • Proton pump inhibitors comprising a substituted bicyclic aryl-imidazole include, but are not limited to, omeprazole, hydroxyomeprazole, esomeprazole, lansoprazole, pantoprazole, rabeprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontop
  • Substituted bicyclic aryl-imidazole compounds as well as their salts, hydrates, esters, amides, enantiomers, isomers, tautomers, polymorphs, prodrugs, and derivatives may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry. See, e.g., March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience, 1992); Leonard et al. , Advanced Practical
  • a tautomer of a substituted bicyclic aryl-imidazole includes without limitation tautomers of omeprazole such as those disclosed in U.S. Patent Nos. 6,262,085; 6,262,086;
  • An example of an isomer of a substituted bicyclic aryl-imidazole is an isomer of omeprazole including but not limited to an isomer disclosed in: Oishi et al., Acta
  • An amide of a bicyclic aryl-imidazole compound may be prepared using techniques known to those skilled in the art or described in the pertinent literature.
  • an amide may be prepared from esters, using suitable amine reactants, or they may be prepared from an anhydride or an acid chloride by reaction with an amine group e.g., ammonia or a lower alkyl amine.
  • Suitable polymorphs include but are not limited to the polymorphs described in PCT
  • a proton pump inhibitor suitable for use in the invention is a benzimidazole compound, for example, a benzimidazole compound described in the following patent documents: U.S. Patent Nos. 4,045,563; 4,255,431 ; 4,359,465; 4,472,409;
  • EP-A-295603 EP-A-166287; EP-A-519365; EP5129; EP 174,726; EP 166,287; GB 2,163,747; and JP-A-59181277.
  • a proton pump inhibitor comprises or is selected from the group consisting of omeprazole, hydroxyomeprazole, esomeprazole, tenatoprazole, lansoprazole, pantoprazole, rabeprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontopraz
  • a proton pump inhibitor comprises or is selected from the group consisting of tenatoprazole, lansoprazole, pantoprazole, rabeprazole, dontoprazole, Vietnameseprazole, periprazole, ransoprazole, pariprazole, leminoprazole; or a free base, free acid, salt, hydrate, ester, amide, enantiomer, isomer, tautomer, polymorph, or prodrug thereof.
  • H-2 receptor antagonist refers to a compound which blocks H-2 receptors, but does not have meaningful activity in blocking histamine- 1 receptors.
  • Selective H-2 antagonists include compounds which are disclosed in US Patent Nos. 5,294,433, 5,364,616, and US Patent Application No. 20050042283, including without limitation cimetidine [Merck Index, 11th edition (1989), p. 354 (entry no. 2279) and Physicians' Desk Reference, 46th edition (1992), p. 2228]; etintidine (U.S. Patent No. 4,112,234); ranitidine or its hydrochloride salt (AH-19065) [U.S.
  • immunosuppressive agent refers to any agent which inhibits or prevents an immune response.
  • immunosuppressive agents are listed in Table 3. Immunosuppressive agents are in each case generically and specifically disclosed in the referenced documents or publications. Any of the substances disclosed in the documents and publications referenced in Table 3 are considered potentially useful as immunosuppressive agents to be used in carrying out the present invention.
  • the agents may be produced by methods known in the art and they may be administered at therapeutically effective doses known in the art for the compounds.
  • An exemplary immunosuppressive agent is a drug, for example, a rapamycin; a corticosteroid; an azathioprine; mycophenolate mofetil; a cyclosporine; a cyclophosphamide; a methotrexate; a 6-mercaptopurine; FK506; 15-deoxyspergualin; an FTY 720; a mitoxantrone; a 2-amino- 1,3 -propanediol; a2-amino-2[2-(4- octylphenyl)ethyl]; propane-1,3- diol hydrochloride; a 6-(3 dimethyl-aminopropionyl) forskolin; and a demethimmunomycin.
  • a drug for example, a rapamycin; a corticosteroid; an azathioprine; mycophenolate mofetil; a cyclosporine; a
  • an immunosuppressive agent is a protein, for example, a protein comprising an amino acid sequence of an antibody.
  • the immunosuppressive agent may be at least one of: hut 124; BTI-322, allotrap-HLA 15 B270; OKT4A; Enlimomab; ABX-CBL; OKT3; ATGAM; basiliximab; daclizumab; thymoglobulin; ISAtx247; Medi-500; Medi-507; Alefacept; efalizumab; infliximab; and an interferon.
  • the immunosuppressive agent is dexamethasone, cyclosporin A, azathioprine, brequinar, gusperimus, 6-mercaptopurine, mizoribine, rapamycin, tacrolimus (FK-506), folic acid analogs (e.g., denopterin, edatrexate, methotrexate, piritrexim, pteropterin, Tomudex®, trimetrexate), purine analogs (e.g., cladribine, fludarabine, 6-mercaptopurine, thiamiprine, thiaguanine), pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine, emitefur, enocitabine, floxuridine, fluorouracil, gemcitabine, tegafur), fluocinolone, triaminolone,
  • insulin sensitivity enhancer or "insulin resistance deblocker” refers to a substance that restores impaired insulin receptor function to deblock insulin resistance thereby enhancing insulin sensitivity.
  • Exemplary insulin sensitivity enhancers are pioglitazone [Fujita et al., Diabetes, 32, 804-810, 1983, JP-A S55(1980)-22636 (EP-A 0008203), JP-A S61(1986> 267580 (EP-A 193256)], CS-045, PPAR( ⁇ ) antagonists (fibrates), rexinoids, protein tyrosine kinase inhibitors ⁇ 3 adrenergic receptor antagonists, thiazolidinedione derivatives and substituted thiazolidinedione derivatives which may be used in combination with insulin [JP- A H4(1992)-66579, JP-A H4(1992)-69383, JP-A H5(1993)-202042]. Insulin sensitivity enhancers may be produced
  • insulin sensitivity enhancers include 5-[[3,4- dihydro-2-(phenylmethyl)-2H-l-benzopyran-6-yl]methyl]-2,4-thiazolidinedione (generic name: englitazone) or its sodium salt; 5-[[4-[3-(5-methyl-2-phenyl-4-oxazolyl)-l- oxopropyl]phenyl]methyl]-2,4-thiazalidinedione (generic name: darglitazone/CP-86325) or its sodium salt; 5-[2-(5-methyl-2-phenyl-4oxazolylmethyl)benzofuran-5ylmethyl]-2,4- oxazolidinedione(CP-92768); 5-(2-naphthalenylsulfonyl)-2,4-thiazolidinedione (AY-31637); 4-[(2-naphthalenyl)methyl]-3H-l,2,3,5-oxa
  • Certain thiazolidinedione insulin sensitisers are also disclosed in European Patent Applications Publication Numbers: 0008203, 0139421, 0032128, 0428312, 0489663, 0155845, 0257781, 0208420, 0177353, 0319189, 0332331, 0332332, 0528734, 0508740; International Patent Application Publication Numbers 92/18501, 93/02079, 93/22445 and U.S. Patent Nos. 5,104,888 and 5,478,852.
  • an insulin sensitivity enhancer is a thiazolidinedione insulin sensitiser, in particular a thiazolidinedione insulin sensitiser including compounds comprising a 2,4-thiazolidinedione moiety.
  • the insulin sensitiser is a thiazolidinedione insulin sensitiser including without limitation (+)-5-[[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H- 1 -benzopyran-2- yl)methoxy]phenyl]methyl]-2,4-thiazolidine dione (or troglitazone), 5-[4-[(l- methylcyclohexvl)methoxy]benzyl]thiazolidine-2,4-dione (or ciglitazone), 5-[4-[2-(5- ethylpyridin-2-yl)ethoxy]benzyl]thiazolidine-2,4-dione (
  • Acyclic insulin sensitisers which have insulin sensitiser activity may also be utilized in the present invention. These sensitisers are illustrated in International Patent Application Publication Numbers WO93/21166 and WO94/01420, and in U.S. Patent No. 5,232,945 and International Applications Nos. WO92/03425 and WO91/19702. Other exemplary insulin sensitisers are those disclosed in European Patent Application Publication Number 0533933, Japanese Patent Application Publication Number 05271204 and U.S. Patent No. 5,264,451.
  • glucose lowering agent refers to a substance having one or more of the following actions: stimulates anaerobic glycolysis, increases the sensitivity to insulin in the peripheral tissues, inhibits glucose absorption from the intestine, suppresses hepatic gluconeogenesis, and inhibits fatty acid oxidation.
  • the glucose lowering agent may be produced by methods known in the art and they may be administered at therapeutically effective doses known in the art for the compounds.
  • Glucose lowering agents that may be used in the present invention include biguanide compounds, thiazolidinediones, and ⁇ -glucosidase inhibitors.
  • Biguanide compounds include but are not limited to N-dimethylbiguanides, substituted or otherwise, and for example metformin, but also other pharmaceutical compounds, for example buformin or fenformin, or a salt thereof with a therapeutically compatible mineral acid or organic acid.
  • a glucose lowering agent is metformin hydrochloride. Metformin is commercially available in 500 mg, 850 mg and 1000 mg tablets under the GLUCOPHAGE® tradename from Bristol Meyers Squibb.
  • Metformin hydrochloride may be administered in humans at an initial daily dose of from 500 mg to about 800 mg and increased, as needed, to a maximum daily dosage of 2550 mg.
  • ⁇ -glucosidase inhibitors including without limitation acarbose (Precose®) and miglitol (Glycet®), inhibit ⁇ -glucosidase enzymes resulting in the reduction of glucose concentrations in the blood.
  • insulin secretagogue is a compound which promotes increased secretion of insulin by the pancreatic beta cells.
  • an insulin secretagogue' s action is initiated by binding to and closing a specific sulfonylurea receptor (an ATP-sensitive K + channel) on pancreatic ⁇ -cells which decreases K + influx, leading to depolarization of the membrane and activation of a voltage-dependent Ca 2+ channel.
  • Increased Ca 2+ flux into the ⁇ -cell activates a cytoskeletal system that causes translocation of insulin to the cell surface and its extrusion by exocytosis.
  • Insulin secretagogues include sulfonylureas, meglinitides and amylin compounds or modulators.
  • a sulfonylurea useful for the methods and combinations of this invention may be a glyburide (DIABETATM), glipizide (GLUCOTROLTM, GIBENESETM, MONODIABTM), glipizide (XL) (GLUCOTROL XLTM), glimepiride (AMARYLTM), glibenclamide (Daonil, Euglucon), gliclazide (Diamicron), gliquidone (Glurenorm), glibormuride, glisoxepide, glisentide, glisolamide, glyclopyamide, glycylamide, chlorpropamide (DIABINESETM), carbutamide, acetohexamide, tolbutamide, or tolazamide, or a pharmaceutically acceptable salt form of these agents.
  • DIBINESETM glyburide
  • glipizide GLUCOTROLTM
  • GIBENESETM glipizide
  • a combination of glyburide and metformin hydrochloride can also be commercially obtained under the GLUCOV ANCETM tradename (Bristol Meyers Squibb). Further suitable insulin secretagogues include repaglinide.
  • GLUCOV ANCETM tradename Bristol Meyers Squibb
  • Further suitable insulin secretagogues include repaglinide.
  • Each of these agents may be produced by methods known in the art. These agents may also be administered at the pharmaceutically or therapeutically effective dosages or amounts known in the art for these compounds such as those described in the Physician's Desk Reference 2001, 55 Edition, Copyright 2001, published by Medical Economics Company, Inc.
  • Meglinitides useful for the methods and combinations of this invention include repaglinide (Prandin®) and nateglinide (Starlix®).
  • An "amylin compound” refers to amylin and modulators thereof, in particular amylin agonists.
  • the term “amylin” includes compounds such as those defined in U.S. Patent No. 5,234,906 and U.S. Patent No. 5,367,052.
  • it includes the human peptide hormone referred to as amylin and secreted from the beta cells of the pancreas, and species variations of it. The hormone is secreted along with insulin from the beta cells of the pancreas in response to a meal.
  • the invention contemplates the use of a 37 amino acid amylin protein hormone that includes the sequence KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY (SEQ. ID. NOs: 44 and 45), and sequences that share substantial sequence similarity thereto.
  • amylin agonist refers to a compound that binds to or otherwise directly or indirectly interacts with an amylin receptor or other receptor or receptors with which amylin itself may interact to elicit a biological response, a compound that mimics the function, activity, or effects of amylin, and/or peptide analogues of amylin useful as agonists of amylin.
  • An amylin agonist may be a peptide or a non-peptide compound, and includes amylin agonist analogs.
  • Amylin agonists useful in this invention include amylin agonist analogs disclosed and claimed in U.S. Patent No. 5,686,411, U.S. Patent No. 5,175,145, U.S. Patent No.
  • amylin agonists include calcitonins and peptides or their equivalents having similar amino acid sequences to known calcitonins and having one or more of the known biological activities, in particular, the ability to increase circulating glucose concentration in humans.
  • the amylin agonist is Symlin®.
  • Amylin compounds may be produced by methods known in the art and they may be administered at therapeutically effective doses known in the art for the compounds.
  • an “antiobesity agent” or “appetite regulating agent” refers to any substance that may be used in the treatment or prevention of obesity or to modulate appetite in a subject.
  • the agents may be produced by methods known in the art and they may be administered at therapeutically effective doses known in the art for the compounds.
  • antiobesity and appetite regulating agents include without limitation anorectic agents such as bromocryptine, dexfenfluramine and the like, monoamine reuptake inhibitors such as sibutramine and the like, sympathomimetics such as phendimetrazine and the like, fatty acid uptake inhibitors such as orlistat or the like, thyromimetics such as triiodothyronine or the like, CART (***e amphetamine regulated transcript) agonists, catecholaminergic agents (e.g.
  • NPY neuropeptide Y
  • MC 4 meianocortin 4
  • MC 3 melanocortin 3
  • TNF tumor necrosis factor
  • CRF corticotropin releasing factor
  • CRF BP corticotropin releasing factor binding protein
  • urocortin agonists melanin concentrating hormone antagonists
  • B 3 adrenergic receptor agonists MSH (melanocyte-stimulating hormone) agonists or mimetics
  • INCH melanocyte- concentrating hormone
  • thyromimetic agents dehydroepiandrosterone or an analog thereof
  • glucocorticoid receptor agonist or antagonist ciliary neurotrophic factors
  • human agouti-related protein antagonists CCK (cholec)
  • ⁇ 3 -adrenergic receptor agonists include without limitation ⁇ 4-[2-(2-[6 aminopyridin-3- yl]-2(R)-hydroxyethylamino)ethoxy]phenyl ⁇ acetic acid, ⁇ 4-[2-(2-[6-aminopyridin-3-yl]-2(R)- hydroxyethylamino)ethoxy]phenyl ⁇ benzoic acid, ⁇ 4-[2-(2-[6-aminopyridin-3-yi]-2(R)- hydroxyethylamino)ethoxy]phenyl ⁇ propionic acid, and ⁇ 4-[2- (2-[6- aminopyridin-3-yl]-2(R)- hydroxyethylamino) ethoxy]phenoxy ⁇ acetic acid.
  • the appetite regulating agent is an amphetamine-related appetite suppressant in particular phentermine or phentermine hydrochloride (see U. S. Patent No. 2,408,345).
  • the appetite regulating agent is the gut hormone peptide W (PW) (Batterham RL, Bloom SR, Ann N Y Acad Sci. (2003 Jun); 994: 162-8), in particular the gut hormone fragment peptide YY3-36 peptide ( W336).
  • the antiobesity agent is leptin. In a further aspect of the invention the antiobesity agent is dexamphetamine or amphetamine.
  • the antiobesity agent is a serotonin agonist, in particular fenfluramine or dexfenfluramine or dexfenfluramine hydrochloride, in particular fenfluramine and dexfenfluramine (see U.S. Patent No. 3,198,834).
  • the anti-obesity agent is a monamine reuptake inhibitor, in particular sibutramine or its hydrochloride salt (see U.S. Patent No. 4,929,629), preferably in the form of MeridiaTM.
  • the antiobesity agent is a dopamine agonist, in particular bromocriptine (see U.S. Patent Nos. 3,752,814 and 3,752, ⁇
  • the antiobesity agent is a lipase inhibitor, in particular dexfenfluramine hydrochloride or orlistat (see U.S. Patent No. 4,598,089 and U.S. Patent No. 6,004,996; orlistat is commercially available under the trade name XenicalTM).
  • the antiobesity agent is mazindol or phentermine.
  • Phentermine is commercially available under the trade name IonaminTM.
  • the antiobesity agent is phen-fen, which is a combination of fenfluramine or its hydrochloride and phentermin.
  • the antiobesity agent is phendimetrazine (BontrilTM,
  • X-TrozineTM or its tartrate salt, diethylpropion (TenuateTM) or its hydrochloride salt, fluoxetine, sertaline or its hydrochloride salt, ephedrine or its sulphate salt, bupropion, topiramate, benzphetamine or its hydrochloride salt, phenylpropanolamine or its hydrochloride salt, or ecopipam.
  • an antiobesity agent is selected from the group consisting of phentermine, leptin, bromocriptine, dexamphetamine, amphetamine, fenfluramine, dexfenfluramine, sibutramine, orlistat, dexfenfluramine, mazindol, phentermine, phendimetrazine, diethylpropion, fluoxetine, bupropion, topiramate, diethylpropion, benzphetamine, phenylpropanolamine or ecopipam, ephedrine, pseudoephedrine and pharmaceutical salts thereof.
  • “Insulin” includes fast-, intermediate-, and long-acting insulins.
  • fast- acting insulins include regular insulins and prompt insulin zinc suspensions (semilente insulins); intermediate-acting insulins include isophane insulin suspensions (NPH insulins, isophane insulin) and the insulin zinc suspensions (lente insulins); and the long-acting insulins include protamine zinc insulin suspensions, and extended insulin zinc suspensions (ultralente insulins).
  • the preparations may be available as either porcine or bovine insulins.
  • the term includes recombinant human insulin available as regular and isophane insulins and as insulin zinc suspensions, as well as modified fast-acting insulin [Lys(B28), Pro(B29) human insulin analog, created by reversing the amino acids at positions 28 and 29 on the insulin B-chain].
  • insulins include without limitation the fast-acting insulins available from Eli Lilly such as (a) Iletin® I (Regular); (b) Regular Iletin® II (Pork, 100 Units); (c) Regular Iletin® II (Concentrated, Pork, 500 Units); (d) Humalog® Injection
  • DNA origin 100 Units
  • the fast-acting insulins available from Novo Nordisk such as (a) Novolin® R (Regular, Human Insulin Injection (recombinant DNA origin) 100 Units); (b) Novolin® R PenFill 1.5 ml Cartridges (Regular, Human Insulin Injection (recombinant DNA origin) 100 Units); (c) Novolin® R PrefilledTM (Regular, Human Insulin Injection (recombinant DNA origin) in a 1.5 ml Prefilled Syringe, 100 units/ml); (d) Regular Purified Pork Insulin (100 Units/ml); and (e) Velosulin® BR (Buffered Regular Human Insulin Injection, 100 Units/ml); the intermediate-acting insulins available from Eli Lilly such as (a) Humulin® 50/50 (50% human insulin isophane suspension and 50% human insulin injection (rDNA origin), 100 Units); (b) Humuline® 70/30 (70% human insulin isophane suspension and 30% human insulin injection (r
  • agents identified by generic or tradenames herein may be taken from the standard compendium "The Merck Index'" or from databases such as PubMed (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi), and patent databases (http://www.uspto.gov/ patft/index.html; http://patentsl .ic.gc.ca/intro-e.html; http://register.epoline.org/espacenet/ ep/en/srch-reg.htm).
  • PubMed http://www.ncbi.nlm.nih.gov/entrez/query.fcgi
  • patent databases http://www.uspto.gov/ patft/index.html
  • http://register.epoline.org/espacenet/ ep/en/srch-reg.htm A person skilled in the art using these references
  • the agents may also be administered at the pharmaceutically or therapeutically effective dosages or amounts known in the art for these compounds, such as those described in the Physician's Desk Reference 2001, 55 Edition, Copyright 2001 , published by Medical Economics Company, Inc.
  • "Condition(s) and/or disease(s)” refers to one or more pathological symptoms or syndromes for which an EGF receptor ligand and a GLP-I agonist and preferably one or both of a gastrin compound and a gastrin agonist provide a beneficial or therapeutic effect.
  • the condition and/or disease may require reduction of blood glucose levels, inhibition of gastric acid secretion, inhibition of apoptosis of ⁇ -cells, stimulation of proliferation or differentiation of ⁇ -cells, and reduction of body weight or insulin dependence.
  • conditions and/or diseases include but are not limited to dyslipidemia, hyperglycemia, severe hypoglycemic episodes, stroke, left ventricular hypertrophy, arrhythmia, bacteraemia, septicaemia, irritable bowel syndrome, functional dyspepsia, diabetes, catabolic changes after surgery, stress induced hyperglycemia, respiratory distress syndrome, gastric ulcers, myocardial infarction, impaired glucose tolerance, hypertension, chronic heart failure, fluid retentive states, metabolic syndrome and related diseases, disorders, or conditions, obesity, diabetic complications as well as symptoms of other diseases in which tissue is damaged due to elevated glucose levels, including Alzheimer's Disease, Parkinson's Disease, and other age-related, tissue-degenerative diseases, as well as the artherogenic effects of elevated leptin, for example in patients with impaired glucose tolerance and obese non-diabetic patients.
  • diabetes means any manifested symptoms of diabetes in any mammal including experimental animal models, and including human forms such as Type I and Type II diabetes, early stage diabetes, and a pre-diabetic condition characterized by mildly decreased insulin or mildly elevated blood glucose levels.
  • a "pre-diabetic condition” describes a subject demonstrating a symptom in terms of insulin or glucose level, and/or demonstrating a susceptibilty to diabetes or a related condition due to family history, genetic predisposition, or obesity in the case of Type II diabetes, and includes a subject who has previously had diabetes or a related disease, disorder, or condition and is subject to risk of recurrence.
  • Type II diabetes Diseases, disorders, and conditions related to diabetes, in particular Type II diabetes, include without limitation, diabetic nephropathy, diabetic retinopathy and diabetic neuropathy, macular degeneration, coronary heart disease, myocardial infarction, diabetic cardiomyopathy, myocardial cell death, coronary artery diseases, peripheral arterial disease, stroke, limb ischemia, vascular restenosis, foot ulcerations, endothelial dysfunction and/or atherosclerosis.
  • a condition and/or disease may be selected from the group consisting of (a) Type I or Type II diabetes mellitus and related diseases, disorders or conditions (including but not limited to diabetic nephropathy, diabetic retinopathy and diabetic neuropathy); (b) insulin resistance and syndrome X, obesity and related diseases, disorders or conditions (including but not limited to Insulin Resistance, Type II Diabetes Mellitus, Reproductive Disorders, Cardiovascular Disease, Pulmonary Disease, Gallstones and Fasting-induced cholecystitis, Cancers and Cutaneous Disease), Cushing's Syndrome, Hypothyroidism, Insulinoma, Craniopharyngioma and Other Disorders Involving the Hypothalamus; (c) congestive heart failure, left ventricular hypertrophy, survival post myocardial infarction (Ml), coronary artery diseases, atherosclerosis, angina pectoris, thrombosis, (d) hypertension including hypertension in the elderly, familial dyslipidemichypertension and
  • Insulinotropic activity refers to an ability of a substance to stimulate insulin secretion in response to elevated glucose levels to produce or increase glucose uptake by cells and decreased serum glucose or blood glucose levels. Methods known in the art can be employed to assay for insulinotropic activity. For example, in vitro and in vivo methods may be used that measure GLP-I receptor binding activity or gastrin receptor binding activity, receptor activation (see the methods described in EP 619,322 to Gelfand et al and US Patent No. 5,120,712), and/or insulin or C-peptide levels.
  • Compounds, compositions or conjugates described herein have insulinotropic activity if islet cells secrete insulin in the presence of the compounds, compositions, or conjugates above background levels or levels in the absence of the compounds, compositions, or conjugates.
  • a compound may be administered to an animal and the insulin concentration can be monitored over time.
  • Islet neogenesis means formation of new beta cells by differentiation, which may or may not have the characteristics of stem cells which have the ability to reproduce in an unlimited manner.
  • the invention is related to compositions, conjugates, and methods that utilize at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist.
  • the invention relates to compositions, conjugates, and methods for the prevention, intervention and/or treatment of a condition and/or disease discussed herein comprising at least one EGF receptor ligand and at least one GLP-I agonist and preferably one or both of at least one gastrin compound and at least one gastrin agonist to provide one or more beneficial effect.
  • compositions, conjugates and methods of the invention provide enhanced beneficial effects, in particular sustained beneficial effects relative to an EGF receptor ligand, GLP-I agonist, gastrin compound, and/or gastrin agonist alone.
  • the beneficial effects may be additive, complementary or synergistic effects.
  • beneficial effects in particular sustained beneficial effects of a composition, combination treatment, or conjugate of the invention may manifest as one or more of the following: a) An increase in pancreatic insulin levels relative to the levels measured in the absence of the active compounds or for each compound alone after administration to a subject with symptoms of diabetes. Pre ferably the compounds together induce at least about a 0.05%. 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%, 35%, 40%, 45%, or 50% increase in pancreatic insulin levels in a subject. b) Prevention or reduction of an absence of symptoms of islet inflammation after administration to a subject with symptoms of diabetes.
  • the compounds induce at least about a 1 %, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decrease in blood glucose levels. Most preferably, the compounds yield blood glucose levels about or close to the levels common in a normal subject.
  • the compounds together induce at least about a 0.05%,
  • the compounds provide at least about a 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in destruction of beta-cells.
  • beta-cell function An increase in beta-cell function.
  • the compounds induce at least about a 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% increase in beta-cell function, i) A decrease in insulin delivery or usage compared with the absence of the compounds or for each compound alone in diabetic subjects.
  • the compounds provide at least about a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 100%, 30-100%, 30-80%, or 35-75%, reduction in insulin delivery or usage.
  • the compounds induce at least about a 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decrease in fasting blood glucose levels.
  • a formulation or dosage form comprising the active compounds yields fasting blood glucose levels about or close to the levels common in a normal subject.
  • a formulation or dosage form comprising the active compounds results in at least about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 26%, 30%, 33%, 34%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% increase in the insulinogenic index.
  • m) A reduction in proinsulin levels relative to the levels measured in the absence of the active compounds or each compound alone in subjects with symptoms of Type 2 diabetes.
  • the active compounds induce at least about a 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decrease in proinsulin levels.
  • a formulation or dosage form comprising the active compounds yields proinsulin levels about or close to the levels common in a normal subject.
  • n) A reduction of development of conditions related to diabetes or diabete-related complications including diabetic nephropathy, diabetic retinopathy and diabetic neuropathy, macular degeneration, coronary heart disease, myocardial infarction, diabetic cardiomyopathy, myocardial cell death, coronary artery diseases, peripheral arterial disease, stroke, limb ischemia, vascular restenosis, foot ulcerations, endothelial dysfunction and/or atherosclerosis.
  • o) A reduction, prevention, or slowing of the rate of disease progression in a subject with diabetes.
  • p) A reduction or prevention of the development of severe hyperglycemia and ketoacidosis with symptoms of diabetes.
  • q) An increase in survival in a subject with symptoms of diabetes.
  • the therapeutic effects comprise or consist essentially of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen of a) through q).
  • therapeutic effects of a gastrin compound or treatment of the invention can manifest as a) and b); a), b), and c); a), b), c), and d); a), b), c), d), and e); a), b), c), d), e), and f); a), b), c), d), e), f), and g); a), b), c), d), e), f), g), and h); a), b), c), d), e), f), g), and h); a), b), c), d), e), f), g), h), and i); a), b), c), d), e), f), g), h), and i);
  • One or more of these beneficial effects or sustained beneficial effects can be demonstrated in a diabetic subject or disease model, for example a non-obese (NOD) mouse with symptoms of diabetes, using standard methods known to the skilled artisan.
  • NOD non-obese
  • Commercially available methods and kits may also be used to assay pancreatic insulin levels, glucose levels, C-peptide levels and hemoglobin AIc.
  • An EGF receptor ligand may be selected for particular applications in the present invention based on one or more of the following characteristics: ability of the EGF receptor ligand to bind to its receptor, preferably with an affinity constant K d less than about 1 ⁇ M, more preferably less than about 10OnM; ability to initiate a signal transduction pathway resulting in insulinotropic activity; insulinotropic activity; stimulation of beta cell proliferation/differentiation; resistance to DPP IV cleavage; and, an in vivo half-life in particular an in vivo half-life of at least about 15 minutes to 24 hours, preferably 2 to 10 hours or 2 to 8 hours in humans using conventional methods (see for example, the method described in US 2003/0144206).
  • the EGF receptor ligand comprises a sequence of SEQ ID NO: 36 and sequences with substantial sequence similarity thereto, and fragements, derivatives, analogs, or modifications thereof.
  • an EGF receptor ligand is represented by A-B wherein A comprises an amino acid sequence with substantial similarity to amino acids 1 to 47, 1 to 48, 1 to 50, or 1 to 53 of SEQ ID NOs: 36 through 40, and B is between 1 to 10 amino acids.
  • B is a single neutral, hydrophobic or charged amino acid.
  • B is a single amino acid excluding glutamate.
  • B is a single neutral, hydrophobic or charged amino acid.
  • an EGF receptor ligand is utilized wherein A comprises amino acids 1-50, 1-47, or 1-48 of SEQ ID NO: 36 and B is a neutral amino acid.
  • an EGF receptor ligand is EGF 1-51 glu51asn or Asn 51 -hEGF51.
  • an EGF receptor ligand is represented by A-B wherein A comprises amino acids 1-53 of SEQ ID NO: 36 and wherein at least one amino acid is replaced or deleted at positions 48-53 of the carboxy terminus, the amino acid sequence being more stable to proteolysis than that of SEQ ID NO: 36.
  • a GLP-I agonist may be selected for particular applications in the present invention based on one or more of the following characteristics: ability of the GLP-I agonist to bind to a GLP-I receptor, preferably with an affinity constant Ka less than about 1 ⁇ M, more preferably less than about 10OnM; insulinotropic activity; stimulation of beta cell proliferation/differentiation; resistance to DPP IV cleavage; and, bioavailability and in vivo half-life.
  • the GLP-I agonist is GLP- 1(7-36) or GLP-I (7-37) or an analog or derivative thereof.
  • a GLP-I agonist comprises or is selected from the group consisting of Val 8 -GLP-1(7-37)OH, Gly 8 -GLP-1(7-37)OH, Glu 22 -GLP-1(7-37)OH, Lys 22 - GLP-I (7-37)OH, Val 8 -Glu 22 -GLP-1(7-37)OH, VaI 8 - Lys 22 -GLP-1(7-37)OH, Gly 8 -Glu 22 -GLP- 1(7-37)OH, Gly 8 -Lys 22 -GLP-1(7-37)OH, Glu 22 -GLP-1(7-36)NH 2 , Lys 22 -GLP-1(7-36)NH 2 , Val 8 -Glu 22 -GLP-1(7-36)NH 2 , Val 8 -Lys 22 -GLP-1(7-36)NH 2 , Gly 8 -Glu 22 -GLP-1(7-36)NH 2 , G
  • the GLP- 1 agonist comprises or is selected from the group consisting of Gly 8 -GLP- 1(7-37), Val 8 GLP-l(7-37), Val 8 Asp 22 GLP-l(7-37), VaI 8 GIu 22 GLP- 1(7-37), VaI 8 LyS 22 GLP- 1(7-37), and VaI 8 HiS 22 GLP-l(7-37), and analogs and derivatives thereof.
  • the GLP-I agonist comprises or is selected from the group consisting of Gly 8 -GLP- 1(7-36) amide, VaI 8 GLP-I (7-36) amide, Val 8 Asp 22 GLP-l(7- 36) amide, VaI 8 GIu 22 GLP- 1(7-36) amide, VaI 8 LyS 22 GLP- 1(7-36) amide, and VaI 8 HiS 22 GLP- 1(7-36) amide, and analogs and derivatives thereof.
  • a GLP-I agonist is a derivative of GLP-I (7-36) or GLP-I (7-37) comprising a lipophilic substitutent.
  • the GLP-I agonist is Arg 34 Lys 26 (N ⁇ ( ⁇ -Glu(N ⁇ -hexadecanoyl)))-GLP-l(7-37).
  • the GLP-I agonist comprises or is selected from the group consisting of Gly 8 -GLP-l(7-37), VaI 8 GLP- 1(7-37), Val 8 Asp 22 GLP-l(7-37), VaI 8 GIu 22 GLP- 1(7-37), VaI 8 LyS 22 GLP- 1(7-37), VaI 8 HiS 22 GLP- 1(7-37), Arg 34 Lys 26 (N ⁇ ( ⁇ - Glu(N ⁇ -hexadecanoyl)))-GLP- 1(7-37), Gly 8 -GLP- 1(7-36) amide, Val 8 GLP-l(7-36) amide, VaI 8 ASp 22 GLP- 1(7-36) amide, VaI 8 GIu 22 GLP- 1(7-36) amide, VaI 8 LyS 22 GLP- 1(7-36) amide, and VaI 8 HiS 22 GLP-I (7-36)
  • a GLP-I agonist is exendin (e.g. exendin 3 or exendin 4) or an analog, derivative, or fragment thereof.
  • the GIp-I agonsist is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGX [SEQ ID NO: 25] wherein X-P or Y, and HX 1 X 2 GTFITSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS [SEQ IDNO: 26] wherein X 1 X 2 ⁇ SD (exendin-3) or GE (exendin-4).
  • a GLP-I agonist is an insulinotropic analogue of exendin-4(l -39), in particular Ser 2 Asp 3 -exendin-4(l-39) wherein the amino acid residues in position 2 and 3 have been replaced with serine and aspartic acid, respectively (this particular analogue is also being known in the art as exendin-3, SEQ ID NO: 22).
  • Aib 8 ' 35 GLP-I (7-36) amide or Ser 38 ,Lys 39>40 ' 4M2 ' 43 ' 44 -Exendin-4(l-39)amide are utilized.
  • the GLP-I agonist is exenatide (Amylin Pharmaceuticals Inc/Eli Lilly & Co), CJC-1131 (ConjuChem Inc.) or liraglutide (NN-2211; Novo Nordisk A/S/Scios Inc).
  • the GLP-I agonist is a stable GLP-I agonist in particular a stable GLP-I analogue or derivative, or a stable exendin-4 or exendin-3 analogue or derivative, more particularly ByettaTM.
  • a gastrin compound may be selected for particular embodiments in the present invention and to provide a specific beneficial effect(s) based on characteristics including its insulinotrophic activity, the ability to augment the activity of an EGF receptor ligand and/or a GLP-I agonist, and/or increase the physical or chemical stability of an EGF receptor ligand and/or a GLP-I agonist.
  • a gastrin compound can also be selected based on its ability to stimulate proliferation/differentiation of beta cells, and its in vivo half-life.
  • a gastrin compound comprises a sequence of one of SEQ ID NOs: 1 to 9 or modifications thereof.
  • a gastrin compound used in aspects of the methods, compositions, and conjugates of the invention is gastrin 17 and analogs and derivatives thereof.
  • the gastrin compound is synthetic human gastrin having 17 amino acid residues with a Leu residue at amino acid position 15 [SEQ ID NO: 4].
  • a gastrin compound used in the methods, compositions and conjugates of the invention is gastrin 34 and analogs and derivatives thereof.
  • the gastrin compound is a synthetic human gastrin 34 with methionine or leucine at position 32 [SEQ ID NOs: 1 or 2].
  • a gastrin compound used in the methods, compositions and conjugates of the invention is gastrin 34 or gastrin 17 or portions thereof, directly or indirectly interacting or associated with a serum protein, in particular albumin or an immunoglobulin, more particularly human serum album.
  • a gastrin compound comprises an amino acid sequence comprising, from the amino terminus, Z-Y 01 -X n -AA 1 -AA 2 -AA 3 -AA 4 -AAs-AA 6 , wherein AA 1 is Tyr or Phe, AA 2 is GIy, Ala, or Ser, AA 3 is Trp, VaI, or He, AA 4 is Met or Leu, AA 5 is Asp or GIu, and AA 6 is Phe or Tyr; Z is a polymer and when the polymer is a protein Z is an amino acid sequence; Y m is an optional spacer region comprising m amino acid residues of a small neutral amino acid including but not limited to serine and alanine, and X is any consecutive portion of residues 1-28 of SEQ ID NOs: 1 or 2, or residues 1-17 of SEQ ID NOs: 3 or 4, preferably AA I -AA 2 -AA 3 -AA 4 -AA S -AA 6 is Tyr-Gly-
  • the gastrin agonist is a proton pump inhibitor.
  • the proton pump inhibitor is leminoprazole, nepaprazole, tenatoprazole, omeprazole, esomeprazole, lansoprazole, rabeprazole, pantoprazole, pariprazole, (-)pantoprazole, soraprazan, ilaprazole, AZD-0865, hydroxyomeprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontop
  • the gastrin agonist is one or more of omeprazole, hydroxyomeprazole, esomeprazole, tenatoprazole, lansoprazole, pantoprazole, rabeprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontop
  • the gastrin agonist is one or more of tenatoprazole, lansoprazole, pantoprazole, rabeprazole, dontoprazole, Vietnameseprazole, periprazole, ransoprazole, pariprazole, leminoprazole; or a free base, free acid, salt, hydrate, ester, amide, enantiomer, isomer, tautomer, polymorph, metabolite or prodrug thereof.
  • compositions of the invention can be selected that provide beneficial effects, in particular statistically significant beneficial effects or sustained beneficial effects, compared with one or two of an EGF receptor ligand, a GLP-I agonist, a gastrin compound, and a gastrin agonist.
  • beneficial effects in respect to a diabetic condition may be evidenced by one or more of the beneficial effects described herein, in particular one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all of the beneficial effects described above in a) through q) above.
  • a pharmaceutical composition comprising at least one EGF receptor ligand and at least one GLP-I agonist, and one or more of at least one gastrin compound and at least one gastrin agonist.
  • the EGF receptor ligand is represented by
  • A-B wherein A comprises amino acids 1-50, 1-48, or 1-47 of SEQ IDNOs: 36 to 40 and B is a neutral amino acid, in particular aspargine (EGF 1-51 glu 51 asn or Asn 51 -hEGF51), and the GLP-I agonist is selected from the group consisting of Gly 8 -GLP-l(7-37), VaI 8 GLP- 1(7-37), VaI 8 ASp 22 GLP- 1(7-37), VaI 8 GIu 22 GLP- 1(7-37), VaI 8 LyS 22 GLP-I (7-37), VaI 8 HiS 22 GLP-I (7- 37), Arg 34 Lys 26 (N ⁇ ( ⁇ -Glu(N ⁇ -hexadecanoyl)))-GLP-l(7-37), Gly 8 -GLP-l(7-36) amide, VaI 8 GLP-I (7-36) amide, Val 8 Asp 22 GLP-l(7-36) amide,
  • a pharmaceutical composition in particular with a beneficial effect(s), in particular statistically significant beneficial effect(s) or sustained beneficial effect(s), is provided comprising an EGF receptor ligand represented by A-B wherein A comprises amino acids 1-53 of SEQ ID NO: 36 and wherein at least one amino acid is replaced or deleted at positions 48-53 of the carboxy terminus, the amino acid sequence being more stable to proteolysis than that of SEQ ID NO: 36, [preferably A comprises amino acids 1-50 of SEQ ID NO: 36 and B is aspargine (i.e., EGF 1-51 glu51asn or Asn 51 -hEGF51)], a GLP-I agonist selected from the group consisting of Asn 51 -hEGF51, Gly 8 -GLP- 1(7-37), Val 8 GLP-l(7-37), Val 8 Asp 22 GLP-l(7-37), VaI 8 GIu 22 GLP- 1(7-37), VaI 8 LyS 22 GLP- 1(7-
  • compositions with beneficial effects comprising EGF 1-51 glu51asn, GLP-l(7-36) [SEQ ID NO: 20] or an exendin-4 (e.g.
  • exenatide and one or both of at least one gastrin- 17(leu) [SEQ ID NO: 4] and at least one gastrin agonist selected from the group consisting of esomeprazole, tenatoprazole, lansoprazole, pantoprazole, rabeprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole
  • a pharmaceutical composition with beneficial effects comprising EGF 1-51 glu5 lasn, Aib 8 ' 35 GLP-l(7-36) amide or Ser 38 ,Lys 39 ' 40 ' 41>42 ' 43 ' 44 -Exendin- 4(l-39)amide, and one or more of at least one gastrin of any one of SEQ ID NOs.
  • gastrin agonist selected from the group consisting of esomeprazole, tenatoprazole, lansoprazol
  • compositions with beneficial effects comprising an EGF receptor ligand represented by A-B wherein A comprises amino acids 1- 50, 1-48, or 1-47 of SEQ ID NOs: 36 to 40 and B is a neutral amino acid, in particular aspargine (e.g.
  • EGF 1-51 glu 51 asn or Asn 51 -hEGF51 exendin-4, in particular exenatide, and one or both of at least one gastrin of any one of SEQ ID NOs: 1 to 9, in particular gastrin- 34(leu) [SEQ ID NO: 2] or gastrin- 17(leu) [SEQ ID NO: 4], and at least one gastrin agonist selected from the group consisting of esomeprazole, tenatoprazole, lansoprazole, pantoprazole, rabeprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontop
  • a pharmaceutical composition comprising at least one EGF receptor ligand, at least one GLP-I agonist, and at least one gastrin agonist.
  • compositions with statistically significant beneficial effects or sustained beneficial effects comprising EGF 1-51 glu51 asn,
  • Arg 34 Lys 26 (N ⁇ ( ⁇ -Glu(N ⁇ -hexadecanoyl)-GLP- 1 (7-37) or an exendin-4 (e.g. exenatide), and at least one gastrin agonist selected from the group consisting of esomeprazole, tenatoprazole, lansoprazole, pantoprazole, rabeprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazo
  • a pharmaceutical composition comprising at least one EGF receptor ligand, at least one GLP-I agonist, and at least one gastrin compound.
  • a pharmaceutical composition with statistically significant beneficial effects or sustained beneficial effects comprising EGF 1-51 glu51asn, Arg 34 Lys 26 (N ⁇ ( ⁇ -Glu(N ⁇ -hexadecanoyl)-GLP-l(7-37) or exendin-4 (e.g. exenatide), and of any one of SEQ ID NOs: 1 to 9, in particular gastrin- 17(leu) [SEQ ID NO: 4].
  • pharmaceutically acceptable salts of an EGF receptor ligand, GLP-I agonist, gastrin compound, and/or a gastrin agonist are utilized.
  • the invention in particular aspects provides a pharmaceutical composition which has been adapted for administration to a subject to provide sustained beneficial effects to treat a condition and/or disease, preferably diabetes.
  • the composition is in a form such that administration to a subject results in blood glucose levels that are about normal or lower and that persist in the subject for a prolonged period of time after cessation of treatment.
  • This invention provides a conjugate comprising at least one EGF receptor ligand linked to or interacting with a GLP-I agonist, and optionally a gastrin agonist or gastrin compound wherein the interaction is for example, via an amino or a carboxyl group.
  • the invention also relates to isolated covalent conjugates of the invention, and compositions comprising covalent conjugates of the invention.
  • An EGF receptor ligand and GLP-I agonist, and optionally a gastrin agonist or gastrin compound may be conjugated to a species via an ester bond between an OH and a COOH of a molecule.
  • Conjugates may be conjugated with an intermediate spacer or linker.
  • a suitable spacer or linker may be a mono- or disaccharide, an amino acid, a sulfate, a succinate, an acetate, or an oligomeric polymeric spacer or linker comprising one or more of such moieties.
  • the invention also provides methods of preparing conjugates with improved pharmacokinetic properties, biological activity, and beneficial effects.
  • the methods comprise incubating an EGF receptor ligand, GLP-I agonist, and one or more of a gastrin agonist and a gastrin compound under conditions that allow formation of a covalent linkage between the compounds.
  • the invention therefore contemplates a process for preparing a covalent conjugate comprising an EGF receptor ligand covalently bonded or linked to a GLP-I agonist, and optionally a gastrin compound and/or a gastrin agonist the process comprising: incubating an EGF receptor ligand and a GLP-I agonist and optionally a gastrin agonist and/or gastrin compound under conditions and at a pH and for a time sufficient for formation of a covalent bond or linkage between the EGF receptor ligand and GLP-I agonist and optionally gastrin agonist and/or gastrin compound; and isolating the covalent conjugate.
  • the above process for preparing a conjugate provides a conjugate with a substantial amount of an EGF receptor ligand covalently linked to a GLP-I agonist.
  • N-terminal or C-terminal fusion proteins or chimeric proteins comprising an EGF receptor ligand conjugated with a GLP-I agonist and optionanlly gastrin compound and/or a gastrin agonist, optionally with a spacer or linker, may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal sequence of an EGF receptor ligand and the sequence of a GLP- 1 agonist, and optionally a gastrin compound and/or a gastrin agonist.
  • the invention relates to a conjugate prepared by a process described herein.
  • the invention also relates to a pharmaceutical formulation or composition comprising conjugates of the invention and a pharmaceutically acceptable carrier, excipient, or vehicle.
  • the invention further relates to a pharmaceutical formulation or composition of substantially pure covalent conj ugates comprising an EGF receptor ligand covalently linked to a GLP-I agonist and optionally a gastrin compound and/or a gastrin agonist which provides beneficial effects preferably sustained beneficial effects compared to the compounds alone.
  • a pharmaceutical formulation consisting essentially of covalent conjugates comprising an EGF receptor ligand covalently linked without an intermediate spacer or linker to a GLP-I agonist and optionally a gastrin compound and/or a gastrin agonist.
  • a pharmaceutical formulation consisting essentially of covalent conjugates comprising an EGF receptor ligand covalently linked with an intermediate spacer or linker to a GLP-I agonist and optionally a gastrin compound and/or a gastrin agonist.
  • a composition or conjugate comprising at least one EGF receptor ligand and at least one GLP-I agonist and optionally at least one gastrin compound and/or at least one gastrin agonist have greater sustained insulinotropic activity following treatment compared with the activity of the compounds alone.
  • the invention provides methods for the prevention, treatment and/or intervention of a condition and/or disease in a subject comprising administering at least one EGF receptor ligand and at least one GLP-I agonist and optionally at least one gastrin compound and/or at least one gastrin agonist, or a pharmaceutical composition of the invention to provide a beneficial effect, in particular a sustained beneficial effect.
  • gastrin compound of any one ofSEQ IDNOs: 1 to 9 or modifications thereof, in particular gastrin-34(leu) [SEQ ID NO: 2] gastrin- 17(leu) [SEQ ID NO: 4] and a gastrin agonist selected from the group consisting of esomeprazole, tenatoprazole, lansoprazole, pantoprazole, rabeprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, dontoprazole, donprazole, periprazole, ransoprazole, pariprazole and leminoprazole; or a free base, free acid, salt, hydrate, ester, amide, enantiomer, isomer, tautomer, polymorph, metabolite or prodrug thereof.
  • gastrin compound of any one ofSEQ IDNOs: 1 to 9 or modifications thereof, in particular gastrin-34(
  • GLP-I agonist and at least one gastrin agonist are administered.
  • At least one EGF receptor ligand, at least one GLP-I agonist, and at least one gastrin compound are administered.
  • exenatide and a gastrin of any one of SEQ ID NOs: 1 to 9, in particular gastrin- 17(leu) [SEQ ID NO: 4], are administered.
  • EGF 1-51 glu51asn, Arg 34 Lys 26 (N ⁇ ( ⁇ -Glu(N ⁇ -hexadecanoyl)-GLP-l(7-37) or exenatide, and gastrin- 17(leu) [SEQ ID NO: 4] are administered.
  • EGF 1-51 glu 51 asn a GLP-I agonist selected from the group consisting of Gly 8 -GLP-l(7-37), VaI 8 GLP- 1(7-37), VaI 8 ASp 22 GLP- 1(7-37), VaI 8 GIu 22 GLP- 1(7-37), VaI 8 LyS 22 GLP- 1(7-37), VaI 8 HiS 22 GLP- 1(7- 37), Arg 34 Lys 26 (N ⁇ ( ⁇ -Glu(N ⁇ -hexadecanoyl)))-GLP- 1(7-37), Gly 8 -GLP-l(7-36) amide, Val 8 GLP-l(7-36) amide, Val 8 Asp 22 GLP-l(7-36) amide, VaI 8 GIu 22 GLP- 1(7-36) amide, Val 8 Lys 22 GLP-l(7-37),
  • the invention provides a method for the prevention and/or intervention of a condition and/or disease discussed herein in a subject comprising administration of at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist.
  • the compounds may be directly administered to a subject or contacted with cells (e.g. stem cells or progenitor cells) and administered to a subject.
  • the invention also provides a combination treatment for preventing and/or treating a condition and/or disease discussed herein in a subject comprising administering to the subject a therapeutically effective amount of at least one EGF receptor ligand and at least one GLP- 1 agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist to provide beneficial effects.
  • the invention provides a combination treatment or intervention which provides sustained beneficial effects following treatment.
  • the invention provides a combination treatment for treating or preventing a condition and/or disease in a subject comprising administering to the subject a therapeutically effective amount of at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist to produce beneficial effects, preferably sustained beneficial effects.
  • the invention also relates to a method of treatment comprising administering a therapeutically effective amount of at least one EGF receptor ligand and at least one GLP- 1 agonist in combination with the administration of at least one gastrin agonist and/or at least one gastrin compound which upon administration to a subject with symptoms of diabetes produces beneficial effects, preferably sustained beneficial effects, manifested as reduced blood glucose levels and/or increased pancreatic insulin.
  • therapeutically effective amounts of at least one EGF receptor ligand and at least one GLP-I agonist, and one or more of at least one gastrin compound and at least one gastrin agonist are combined prior to administration to a subject.
  • therapeutically effective amounts of at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist are mixed at a physiologically acceptable pH.
  • the invention provides a method for stimulating pancreatic islet beta cell proliferation in a subject comprising administering a therapeutically effective amount of a composition or conjugate of the invention, or administering in combination at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist.
  • the invention provides a method for increasing the number and/or size of pancreatic islet beta cells in a subject comprising administering a therapeutically effective amount of a composition or conjugate of the invention, or administering in combination at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist.
  • the invention provides a method for preventing or treating Type I or Type II diabetes comprising administering a therapeutically effective amount of a composition or conjugate of the invention, or administering in combination at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist.
  • the invention provides a method for amelioriating progression of disease or obtaining a less severe stage of disease in a person suffering from Type II diabetes comprising administering a therapeutically effective amount of a composition or conjugate of the invention, or administering in combination at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist.
  • the invention relates to a method of delaying the progression of impaired glucose tolerance or non-insulin requiring Type II diabetes to insulin requiring Type II diabetes comprising administering a therapeutically effective amount of a composition or conjugate of the invention, or administering in combination at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist.
  • the invention also relates to a method of increasing the insulin synthesis capability of a subject comprising administering a therapeutically effective amount of a composition or conjugate of the invention, or administering in combination at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist.
  • the invention further relates to inducing islet neogenesis in a subject comprising contacting pancreatic islet precursor cells with at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist, or a composition or conjugate of the invention in a sufficient amount to increase proliferation of pancreatic islet precursor cells in the subject thereby inducing islet neogenesis.
  • the invention contemplates a method of expanding a functional beta cell mass of pancreatic islet transplants in a diabetic patient, the method comprising administering to the patient a therapeutically effective amount of at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist, or a composition or conjugate of the invention.
  • the invention provides methods for treating diabetes mellitus in a patient in need thereof by administering a composition comprising at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist, in an amount sufficient to effect differentiation of the patient's pancreatic islet precursor cells to mature insulin-secreting cells and/or to stimulate insulin synthesis in existing islet cells.
  • composition can be administered systemically or expressed in situ by host cells containing one or more nucleic acid construct in an expression vector wherein the nucleic acid construct comprises a coding sequence for at least one EGF receptor ligand and at least one GLP-I agonist, and optionally one or more of at least one gastrin compound and at least one gastrin agonist, together with transcriptional and translational regulatory regions functional in pancreatic islet precursor cells.
  • Pancreatic islet precursor cells may be characterized as cells originating from insulin-producing islets with substantial proliferative potential, and capable of doubling in number about every 60 hours.
  • the pancreatic islet precursor cells may also be characterized as cells expressing one or more marker protein including CK19, CK7, Ck8, CkI 8, nestin, carbonic anhydrase II, DU-P AN2, carbohydrate antigen 19-9 and mucin MUCl.
  • the pancreatic islet precursor cells may comprise cells from one or more sources including pancreas, umbilical cords, embryos and established stem cell lines.
  • the pancreatic islet precursor cells may comprise a plurality of stem cells and/or ductal epithelial cells.
  • the pancreatic islet precursor cells may be immortalized precursor cells prepared using methods known to those skilled in the art, for example by transformation with hTERT.
  • the invention provides methods for treating cells (e.g. pancreatic islet precursor cells), preferably cells in culture using at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist, or compositions, or conjugates of the invention.
  • the invention also provides cell based treatment methods using at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist, or compositions, or conjugates of the invention. See PCT/CA03/33595 for a description of general culture and cell based treatment methods.
  • the invention relates to a method for expanding and differentiating stem cells or progenitor cells into insulin secreting cells comprising contacting the stem cells or progenitor cells with at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist, or a composition or conjugate of the invention in sufficient amounts to expand and differentiate stem cells or progenitor cells.
  • the amount of expansion and differentiation may be significantly different compared with that achieved in the absence of the compounds, composition or conjugate, in particular the amount may be significantly greater compared with an amount achieved with the compounds alone.
  • the stem cells or progenitor cells are contacted with the compounds, composition, or conjugate in culture.
  • the stem cells or progenitor cells are contacted with the compounds, compositions, or conjugates in a subject.
  • the compounds, compositions or conjugates may be administered to a subject before, during, or after implantation of stem cells in the subject to expand and differentiate the stem cells in the subject.
  • the stem cells may be obtained from pancreatic islets, umbilical cords, embryos, or stem cell lines.
  • the method may additionally comprise administering an immunosuppressive agent.
  • the invention also relates to a method for enhancing proliferation of insulin secreting cells in culture comprising contacting the cells with at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist, composition or conjugate of the invention in sufficient amounts to enhance proliferation of the cells.
  • the amount of proliferation may be significantly different compared with that achieved in the absence of the compounds, compositions or conjugates. In an embodiment, the amount of proliferation is significantly greater compared with the compounds alone
  • the invention further relates to a method for sustaining pancreatic islet cells or pancreatic islet precursor cells in culture comprising culturing the cells in the presence of at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist, or a composition or conjugate of the invention in an amount sufficient to sustain the cells in culture.
  • the cells may be sustained in culture for a significantly longer period of time compared with cells cultured in the absence of the compounds, compositions or conjugates, or in the presence of at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist.
  • Culturing cells in the presence of at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist or a composition or conjugate of the invention will be particularly useful in preparing and maintaining cells intended for transplantation.
  • the invention provides a method of treating a condition and/or disease comprising administering at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist, or a composition or conjugate of the invention with a plurality of cells (e.g. pancreatic islet precursor cells, mature pancreatic islet cells, pancreatic ductal cells) to a subject in need thereof to thereby produce a beneficial effect, preferably a sustained beneficial effect.
  • a plurality of cells e.g. pancreatic islet precursor cells, mature pancreatic islet cells, pancreatic ductal cells
  • a method for treating a subject with a condition and/or disease described herein comprises contacting ex vivo a plurality of cells (e.g., pancreatic islet precursor cells, mature pancreatic islet cells, pancreatic ductal cells) with at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist, or a composition or conjugate of the invention, optionally culturing the cells, and administering the cells to the subject in need thereof.
  • a plurality of cells e.g., pancreatic islet precursor cells, mature pancreatic islet cells, pancreatic ductal cells
  • the cells are pancreatic ductal cells and the amount of compounds/composition/conjugate used in the method is generally effective to increase the amount of insulin secreting cells in the subject.
  • the cells may be autologous (i.e. from the same subject), or may be from another individual of the same species, or from a different species.
  • the cells are pancreatic islet precursor cells or mature pancreatic islet cells.
  • the invention also contemplates a method for treating diabetes in a subject comprising transplanting a pancreatic islet preparation into the subject and administering a therapeutically effective amount of at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist, or a composition or conjugate of the invention.
  • the number of cells administered to an individual afflicted with a condition and/or disease will vary according to the severity of the condition and/or disease, the mode of administration, and/or the site of administration.
  • a therapeutically effective amount of cells is a safe and effective amount, and in particular an amount necessary to provide one or more beneficial effect, in particular a sustained beneficial effect, or a synergistic effect.
  • Cells can be administered to subjects using a variety of means apparent to those of skill in the art. Suitable methods include injection of the cells into a target site in a subject. Cells may be inserted into a delivery device to facilitate injection or implantation into the subjects.
  • Examples of delivery devices include tubes, e.g., catheters, for injecting cells and fluids into the body of a subject.
  • Cells can be prepared for delivery in a variety of different forms.
  • the cells may be suspended in a solution or gel, or mixed with a pharmaceutically acceptable carrier, excipient, or diluent in which the cells remain viable.
  • Pharmaceutically acceptable carriers, excipients, and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art.
  • the solution is generally sterile, and will often be isotonic.
  • a solution of cells is preferably selected that is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms through the use of, for example, parabens, chlorobutanol, phenol, scorbic acid, thimerosal, and the like.
  • Modes of administration of cells include without limitation systemic intracardiac, intracoronary, intravenous, intradermal, or intra-arterial injection and injection directly into the tissue or organ at the intended site of activity, or in proximity to the site of activity.
  • a cell preparation can be administered by any convenient route, for example by infusion or bolus injection and can be administered together with other biologically active agents. Administration in some aspects is preferably systemic.
  • a cell preparation can be administered by any convenient route, for example by infusion or bolus injection and can be administered together with other biologically active agents.
  • Methods of the invention may further comprise measuring or monitoring one or more of the following markers: blood glucose, serum glucose, blood glycosylated haemoglobin, pancreatic beta cell mass, serum insulin, pancreatic insulin levels, morphometrically determined beta cell mass, C-peptide, amount of insulin secreting cells, and glucose responsiveness of insulin secreting cells.
  • the invention also contemplates the use of a combination of at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist as a medicament or for the preparation of a medicament, in particular providing beneficial effects, preferably sustained beneficial effects in treating a condition and/or disease.
  • the invention relates to the use of a therapeutically effective amount of at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist as a medicament for providing beneficial effects, preferably sustained beneficial effects, in treating a condition and/or disease.
  • the invention provides the use of at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist as a medicament or for the preparation of a medicament for increasing (preferably sustained increase) the number and/or size of beta cells (e.g. pancreatic islet precursor cells or mature pancreatic islet cells) in a subject after treatment.
  • the invention provides the use of at least one EGF receptor ligand and at least one GLP- 1 agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist as a medicment or for the preparation of a medicament for stimulation (preferably sustained stimulation) of beta cell (e.g.
  • the invention provides the use of at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist as a medicament or for the preparation of a medicament for treatment of Type I or Type II diabetes.
  • Therapeutic efficacy and toxicity of compounds, compositions and conjugates of the invention may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals such as by calculating a statistical parameter such as the ED 50 (the dose that is therapeutically effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
  • the therapeutic index is the dose ratio of therapeutic to toxic effects and it can be expressed as the ED 50 /LD 50 ratio.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
  • the invention additionally provides uses of a pharmaceutical composition and a conjugate of the invention in the preparation of medicaments for beneficial effects, preferably sustained beneficial effects, in the treatment of conditions and/or diseases.
  • compositions of the present invention or fractions thereof typically comprise suitable pharmaceutical diluents, excipients, vehicles, or carriers selected based on the intended form of administration, and consistent with conventional pharmaceutical practices.
  • the carriers, vehicles etc. may be adapted to provide an additive, complementary, synergistically effective or therapeutically effective amount of the active compounds.
  • Suitable pharmaceutical diluents, excipients, vehicles, and carriers are described in the standard text, Remington: The Science and Practice of Pharmacy, 21 st Edition. University of the Sciences in Philadelphia (Editor), Mack Publishing Company.
  • the active components can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, methyl cellulose, magnesium stearate, glucose, calcium, sulfate, dicalcium phosphate, mannitol, sorbital, and the like.
  • an oral, non-toxic pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, methyl cellulose, magnesium stearate, glucose, calcium, sulfate, dicalcium phosphate, mannitol, sorbital, and the like.
  • the drug components may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
  • Suitable binders e.g. gelatin, starch, corn sweeteners, natural sugars including glucose; natural and synthetic gums, and waxes
  • lubricants e.g.
  • disintegrating agents e.g. starch, methyl cellulose, agar, bentonite, and xanthan gum
  • flavoring agents, and coloring agents may also be combined in the compositions or components thereof.
  • a pharmaceutical composition has a pH from about 7 to 10.
  • Formulations for parenteral administration of a composition of the invention may include aqueous solutions, syrups, aqueous or oil suspensions and emulsions with edible oil such as cottonseed oil, coconut oil, almond oil, or peanut oil.
  • Dispersing or suspending agents that can be used for aqueous suspensions include synthetic or natural gums, such as tragacanth, alginate, acacia, dextran, sodium carboxymethylcellulose, gelatin, methylcellulose, and polyvinylpyrrolidone.
  • compositions for parenteral administration may include sterile aqueous or nonaqueous solvents, such as water, isotonic saline, isotonic glucose solution, buffer solution, or other solvents conveniently used for parenteral administration of therapeutically active agents.
  • a composition intended for parenteral administration may also include conventional additives such as stabilizers, buffers, or preservatives, e.g. antioxidants such as methylhydroxybenzoate or similar additives.
  • a solid form pharmaceutical composition is provided (e.g. tablets, capsules, powdered, or pulverized form) comprising a crystalline or amorphous EGF receptor ligand and GLP-I agonist, and optionally a gastrin compound and/or gastrin agonist.
  • the invention relates to a liquid drug formulation comprising pharmaceutically acceptable salts of an EGF receptor ligand, a GLP- 1 agonist and preferably a gastrin compound, and/or a gastrin agonist, and to lyophilized drug formulations that can be reconstituted to provide suspensions that are stable and suitable for parenteral administration.
  • the invention relates to an aqueous composition
  • an aqueous composition comprising pharmaceutically acceptable salts of an EGF receptor ligand, a GLP-I agonist, and preferably a gastrin compound and/or a gastrin agonist, and a solvent system which effects solubilization.
  • the invention also provides a drug comprising an aqueous formulation of pharmaceutically acceptable salts of an EGF receptor ligand, a GLP- 1 agonist, and preferably one or more of a gastrin compound, and a gastrin agonist, with at least one solubilizer.
  • a composition of the invention may be sterilized by, for example, filtration through a bacteria retaining filter, addition of sterilizing agents to the composition, irradiation of the composition, or heating the composition.
  • the compounds, conjugates, and compositions of the present invention may be provided as sterile solid preparations e.g. lyophilized powder, which are readily dissolved in sterile solvent immediately prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labelled for treatment of an indicated condition.
  • labelling would include amount, frequency, and method of administration.
  • compositions can also be formulated as a depot preparation.
  • long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • the fractions may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil), or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions of the invention and components thereof may comprise soluble polymers as targetable drug carriers.
  • the compounds, compositions, medicaments, and conjugates of the present invention can be administered by any means that produce contact of the active agent(s) with the agent's sites of action in the body of a subject or patient.
  • the active ingredients can be administered simultaneously or sequentially, and in any order at different points in time, to provide the desired beneficial effects.
  • Each active ingredient may be independently administered any effective number of times, including more than once, as may be indicated by a physician or veterinarian.
  • compositions can be formulated for sustained release, for delivery locally or systemically. It lies within the capability of a skilled physician or veterinarian to select a form and route of administration that optimizes the effects of the compositions, conjugates, and treatments of the present invention.
  • the compositions may be administered in oral dosage forms such as tablets, capsules
  • compositions of the invention may be administered by intranasal route via topical use of suitable intranasal vehicles, or via a transdermal route, for example using conventional transdermal skin patches.
  • a dosage protocol for administration using a transdermal delivery system may be continuous rather than intermittent throughout the dosage regimen.
  • a particular route of administration is parenteral administration, preferably peripheral parenteral administration.
  • Parenteral administration is generally understood to refer to the injection of a dosage form into the body by a sterile syringe or some other mechanical device such as an infusion pump.
  • parenteral routes include intravenous, intramuscular, subcutaneous, and intraperitoneal routes of administration.
  • the compounds or conjugates described herein may be combined with distilled water at an appropriate pH.
  • the present invention includes combination treatments providing additive or synergistic activity, delivering an additive or synergistically effective amount, or an amount to provide a therapeutically effective amount of at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist, or a conjugate or composition of the invention. Therefore, pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an additive, complementary, synergistically effective amount or a therapeutically effective amount.
  • the dosage regimen of the invention will vary depending upon known factors such as the pharmacodynamic characteristics of the agents and their mode and route of administration; the species, age, sex, health, medical condition, and weight of the patient, the nature and extent of the symptoms, the kind of concurrent treatment, the frequency of treatment, the route of administration, the renal and hepatic function of the patient, and the desired effect.
  • the effective amount of a drug required to prevent, counter, or arrest progression of a condition can be readily determined by an ordinarily skilled physician or veterinarian.
  • a composition, medicament, or treatment of the invention may comprise a unit dosage of at least one EGF receptor ligand, a unit dosage of at least one GLP-I agonist, and preferably a unit dosage(s) of one or more of at least one gastrin compound and/or gastrin agonist.
  • a pharmaceutical composition of the invention can comprise a therapeutically effective suboptimal dosage of at least one EGF receptor ligand and at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist, that are more effective at decreasing or reducing glucose levels for a sustained period following treatment compared with a dosage of any of the compounds alone.
  • a pharmaceutical composition or treatment comprising at least one EGF receptor ligand and at least one GLP- 1 agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist in doses that are equal to or at least 1.1 , 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold lower than the doses of each compound required to provide beneficial effects, preferably sustained beneficial effects, to treat a condition and/or disease.
  • the invention provides a pharmaceutical composition or treatment comprising between 0.01 to 500, 0.01 to 1000, 0.01 to 2000, 0.01 to 6000, 0.1 to 500, 0.1 to 1000, 0.1 to 2000, 0.1 to 6000, 0.5 to 6000, 1 to 100, 1 to 200, 1 to 500, 1 to 1000, 1 to 2000, 10 to 100, 10 to 500, 10 to 1000, 10 to 2000, 15 to 50, 15 to 100, 15 to 500, 15 to 1000, 15 to 1500, 20 to 30, 20 to 5o, 20 tolOO, 20 to 500, 20 to 1000, 100 to 1500, 100 to 6000, 1000 to 6000, 2000 to 6000, and 3000 to 6000 micrograms of an EGF receptor ligand per dosage unit, in particular single dosage unit and 0.05 to 10, 0.05 to 25, 0.05 to 50, 0.05 to 100, 0.05 to 500, 0.05 to 1000, 0.05 to 1500, 0.05 to 2000, 0.1 to 1000, 0.1 to 2000, 0.1 to 2500, 0.1 to 3000, 0.1 to 3500, 0.1 to 0.1
  • the amount of a gastrin in a unit dosage form, in particular a single unit dosage form may be from about 50 to 2000 ⁇ g, 50- 3000 ⁇ g, 50-4000 ⁇ g, 50-5000 ⁇ g, 50-6000 ⁇ g, 100 to 2000 ⁇ g, 100-3000 ⁇ g, 100-4000 ⁇ g, 100-5000 ⁇ g, 100-6000 ⁇ g, 200 to 2000 ⁇ g, 200-3000 ⁇ g, 200-4000 ⁇ g, 200-5000 ⁇ g, 200-6000 ⁇ g, 300 to 2000 ⁇ g, 300-3000 ⁇ g, 300-4000 ⁇ g, 300-5000 ⁇ g, 300-6000 ⁇ g, 400 to 2000 ⁇ g, 400-3000 ⁇ g, 400-4000 ⁇ g, 400-5000 ⁇ g, 400-6000 ⁇ g, 500 to 2000 ⁇ g, 500-3000 ⁇ g, 500- 4000 ⁇ g, 5OO-5OOO ⁇ g, or 500-6000 ⁇ g, 600 to 2000 ⁇ g, 600-3000 ⁇ g, 600-4000
  • the invention provides a pharmaceutical composition or treatment comprising between 0.1 to 1, 0.1 to 5, 0.1 to 10, 0.1 to 20, 0.1 to 30, 0.1 to 40, 0.1 to 50, and 0.1 to 60 micrograms/kg/day of an EGF receptor ligand; 0.1 to 10, 0.1 to 15, 0.1 to 20, 0.1 to 30, 0.1 to 40, 0.1 to 50, or 0.1 to 60 micrograms/kg/day of a GLP-I agonist; and, preferably 10 to 50, 20 to 50, 10 to 40, 20 to 40, 10 to 35, 15 to 35, 20 to 35, 28 to 35, 29 to 35, 30 to 35, 28 to 31, 28 to 32, 28 to 33, 28 to 34, 28 to 35, 29 to 31, 29 to 32, 29 to 33, 29 to 34, 29 to 35 or 30 micrograms/kg/day of a gastrin compound that is a form of gastrin.
  • an EGF receptor ligand and a GLP-I agonist, and optionally a gastrin agonist and/or gastrin compound may be in ratios selected to augment the activity of one or more of the compounds to produce a beneficial effect, in particular a sustained beneficial effect, and/or to produce an additive, complementary or synergistic effect.
  • the ratio of an EGF receptor ligand to a GLP-I agonist may be from 1 :1 to 1:200, 1 :1 to 1 : 150, 1;1 to 1 :100, 1 :1 to 1 :110, 1 :1 to 1:100, 1 :1 to 1 :75, 1 :1 to 1 :50, 1:1 to 1 :25, 1 :1 to 1 :10, 1 :1 to 1 :5, and 1 : 1.
  • the ratio of a GLP-I agonist to an EGF receptor ligand may be from 1 :1 to 1 :200, 1 :1 to 1: 150, 1;1 to 1 :100, 1 :1 to 1 :110, 1 :1 to 1 :100, 1 :1 to 1:75, 1 :1 to 1 :50, 1 :1 to 1 :25, 1:1 to 1 :10, and 1 :1 to 1 :5.
  • a composition of the invention or components thereof may be administered to a subject continuously for 2 weeks to 12 months, 2 weeks to 6 months, 2-16 weeks, 2 weeks to 12 weeks, and/or 2-8 weeks, or periodically.
  • the present invention also includes compositions, conjugates, and treatments of the invention in combination with one or more additional therapeutic agents including without limitation immunosuppressive agents, antidiabetic agents including without limitation insulin sensitivity enhancers, glucose lowering agents, insulin secretagogues, and insulin, antiobesity agents and appetite regulating agents, antihypertensive agents, agents for the treatment and/or prevention of complications resulting from or associated with a condition and/or disease, in particular diabetes and obesity, anti-nausea medications, anti-headache medications, and general medications that treat or prevent side effects.
  • additional therapeutic agents including without limitation immunosuppressive agents, antidiabetic agents including without limitation insulin sensitivity enhancers, glucose lowering agents, insulin secretagogues, and insulin, antiobesity agents and appetite regulating agents, antihypertensive agents, agents for the treatment and/or prevention of complications resulting from or associated with a condition and/or disease, in particular diabetes and obesity, anti-nausea medications, anti-headache medications, and general medications that treat or prevent side
  • the present invention relates to a method of treatment comprising a combination of active agents which may be administered separately or as conjugates
  • the invention also provides a kit comprising at least one EGF receptor ligand and at least one GLP-I agonist, and one or more of at least one gastrin compound and at least one gastrin agonist, or a pharmaceutical composition or conjugate in kit form.
  • the invention also relates to a pharmaceutical kit comprising one bottle with an EGF receptor ligand, another bottle with a GLP-I agonist, and preferably a bottle(s) with a gastrin compound and/or gastrin agonist in one box.
  • a kit may comprise a package which houses a container which contains a conjugate or composition of the invention or components thereof and also houses instructions for administering the conjugate or composition to a subject.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of a pharmaceutical composition of the invention to provide a beneficial effect, in particular a sustained beneficial effect.
  • Associated with such container(s) can be various written materials such as instructions for use, or a notice in the form prescribed by a governmental agency regulating the labeling, manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration.
  • the invention relates to a "kit-of-parts", for example, the components to be combined according to the present invention can be dosed independently or by use of different fixed combinations with distinguished amounts of the components, i.e. simultaneously or at different time points.
  • the parts of the kit can then be administered simultaneously or chronologically staggered, that is, at different time points and with equal or different time intervals for any part of the kit.
  • Parts of a kit maybe administered simultaneously or chronologically staggered, i.e., at different points in time and with equal or different time intervals for any component of a kit.
  • Time intervals can be selected such that the effect on the condition and/or disease in the combined use of the parts is larger than the effect that would be obtained by use of only any one of the components.
  • the invention provides a kit-of-parts comprising: (a) an amount of an EGF receptor ligand or a pharmaceutically acceptable salt thereof in a first unit dosage; (b) an amount of a GLP-I agonist or a pharmaceutically acceptable salt thereof in a second unit dosage; and preferably (c) an amount of a gastrin compound and/or gastrin agonist or a pharmaceutically acceptable salt thereof in a third unit dosage, in the form of one, two or three or more separate units of the components (a) to (c).
  • the invention further relates to a commercial package comprising at least one EGF receptor ligand, at least one GLP-I agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist, together with instructions for simultaneous, separate or sequential use.
  • a commercial package comprising as active ingredients at least one EGF receptor ligand, at least one GLP- 1 agonist, and preferably one or more of at least one gastrin compound and at least one gastrin agonist, is provided in the form of two, three or more separate units of the components, together with instructions for simultaneous, separate or sequential use, or any combination thereof, in the delay of progression or treatment of a condition and/or disease disclosed herein.
  • NOD mice spontaneously develop insulin-dependent diabetes as a result of autoimmune destruction of pancreatic islet ⁇ -cells.
  • This study will be aimed at treating diabetes in NOD mice by regenerating islet ⁇ -cells or reducing insulin dependence using EGF, GLP-I, and optionally gastrin and/or a PPI.
  • mice ages 12-16 weeks will be treated for 18 days only, with either (i) a vehicle alone as a control, (ii) an EGF receptor ligand (EGF 1-51 glu 51 asn) alone; (iii) a GLP- 1 agonist alone ⁇ GLP-l(7-36) [SEQ ID NO: 19], Arg 34 Lys 26 (N ⁇ ( ⁇ -Glu(N ⁇ -hexadecanoyl)- GLP- 1(7-37), or an exenatide (e.g.
  • mice will be monitored daily for urine glucose levels and weekly for FBG levels during the treatment and for an additional 6 weeks after the treatment is stopped.
  • pancreatic insulin levels will be determined in each group as well as histological analysis of the pancreatic tissue will be performed. Pancreatic tissues will be fixed and stained for insulin producing cells. The beta cell mass will be determined by morphometric analysis.
  • Example 2 Effects of a Combination of an EGF Receptor Ligand and a GLP-I Agonist Optionally with Gastrin and/or a PPI in Diabetic Rats
  • STZ diabetic rats of weight 200-250 g will be administered once daily for 12 days either (i) a vehicle alone as a control, (ii) an EGF receptor ligand (EGF 1-51 glu 51 asn) alone; (iii) a GLP-I agonist alone ⁇ GLP- 1(7- 36) [SEQ IDNO. 19], Arg 34 Lys 26 (N ⁇ ( ⁇ -Glu(N ⁇ -hexadecanoyl)-GLP-l(7-37), or an exenatide (e.g.
  • IPGTT intraperitoneal glucose tolerance tests
  • Diabetic STZ rats will be prepared as described in Example 2 using rats of body weight greater than 30Og. These rats will be treated by subcutaneous administration (subcutaneous continuous infusion) as follows. Rats will be treated for two weeks with either (i) a vehicle alone as a control, (ii) an EGF receptor ligand (EGF 1-51 glu 51 asn) alone; (iii) a GLP-I agonist alone ⁇ GLP-l(7-36) [SEQ ID NO: 19], Arg 34 Lys 26 (N ⁇ ( ⁇ -Glu(N ⁇ - hexadecanoyl)-GLP-l(7-37), or an exenatide (e.g.
  • Example 4 A Phase 1/2 randomized, double-blind, controlled clinical trial to evaluate the safety, tolerability, pharmacokinetic profile and effects of repeated subcutaneous doses of EGF in combination with a GLP-I Agonist and Gastrin in patients with Type I diabetes. Study Design
  • a total of 20 patients with Type I diabetes will be randomized on Day 1 of the Treatment Phase.
  • Fifteen (15) patients will be randomized to receive active study medication and 5 patients will be randomized to receive vehicle control. Additional patients may be entered into this study to ensure that 20 patients receive at least 21 days of treatment.
  • This study has a 2 week Baseline period, 4 week Treatment period, and 6 month (non investigational drug treatment period). After undergoing Screening procedures the potential patients will enter a 14 day Baseline Phase where baseline data will be collected. During this period and throughout the study, patients will remain on their insulin regimen and will record insulin intake and blood glucose levels daily through the use of a daily diary.
  • patients will enter the Treatment Phase where they will be randomized to receive either once daily sc injections of El, GLP-I agonist plus a gastrin, as separate injections or once daily sc injections of vehicle control (as separate injections to mimic active treatment). Patients will receive once daily doses in the morning after breakfast for a period of 28 days. Patients randomized to active treatment will receive treatment according to the following schedule: During the first week of treatment, patients will receive 0.3 ⁇ g/kg El and a GLP-I agonist (e.g., Byetta in recommended doses). Some patients will also receive gastrin (30 ⁇ g/kg). If patients are not able to tolerate the first dose level, they will be stepped-down, in a blinded fashion to a lower dose.
  • GLP-I agonist e.g., Byetta in recommended doses
  • EGF Epidermal Growth Factor
  • the study is a randomized 2:1 treatment to vehicle control, double-blind design. A total of 30 patients with Type II diabetes will be randomized on Day 1 of the Treatment Phase. Twenty (20) patients will be randomized to receive active study medication and 10 patients will be randomized to receive vehicle control. This study has a 2 week Baseline period, a 4 week Treatment period, and a 6 month
  • patients will enter the Treatment Phase where they will be randomized to receive either once daily sc injections of EGF, GLP-I Agonist, and a gastrin as separate injections or once daily sc injections of vehicle control (as separate injections to mimic active treatment). Patients will receive once daily doses in the morning after breakfast for a period of 28 days. Patients randomized to active treatment will receive treatment according to the following schedule: During the first week of treatment, patients will receive 0.3 ⁇ g/kg EGF and ByettaTM at recommended doses, and some patients will also receive 30 ⁇ g/kg of a gastrin. If patients are not able to tolerate the first dose level, they will be stepped-down, in a blinded fashion.
  • the dose will be escalated for example to 0.5 ⁇ g/kg EGF and 30 ⁇ g/kg of gastrin during the second week of treatment and for the duration of the treatment period. Patients not tolerating the second dose level will be stepped-down, in a blinded fashion, to the lower dose.
  • Type II diabetes patients requiring Metformin and/or Thiazolidinedione therapy male or female, ages 30 - 60 years inclusive.
  • EGF Epidermal Growth Factor
  • Gastrin analogue [gastrin- 17(leu) of SEQ ID NO: 4], 4 mg/mL Vehicle Control - 0.9 % normal saline
  • Beta cell function as defined by the change in insulin secretion

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Abstract

L'invention concerne des compositions, des conjugués et des procédés pour la prévention et/ou le traitement d'une affection et/ou d'une maladie, qui comprennent une quantité thérapeutiquement efficace d'au moins un ligand de récepteur EGF, au moins un agoniste GLP1 et, de préférence, un ou plusieurs d'au moins un composé de gastrine et d'au moins un agoniste de gastrine. La combinaison d'au moins un ligand de récepteur EGF, d'au moins un agoniste GLP-1 et d'un ou de plusieurs d'au moins un composé de gastrine et d'au moins un agoniste de gastrine permet d'obtenir des effets bénéfiques, en particulier des effets bénéfiques soutenus, dans la prévention et/ou le traitement d'affections et/ou de maladies comprenant, toutefois sans caractère limitatif, le diabète, l'hypertension, l'insuffisance cardiaque chronique, les états de rétention de fluide, l'obésité, le syndrome métabolique et les maladies et troubles associés. Des combinaisons d'au moins un ligand de récepteur EGF, d'au moins un agoniste GLP-1, et de préférence d'un ou de plusieurs d'au moins un composé de gastrine et d'au moins un agoniste de gastrine, peuvent être sélectionnées pour produire des effets additifs, complémentaires ou synergiques inattendus.
PCT/CA2007/002339 2006-12-12 2007-12-12 Polythérapies impliquant des facteurs de régulation de croissance/des hormones sélectionnés pour le diabète et les maladies apparentées WO2008071010A1 (fr)

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WO2009039964A3 (fr) * 2007-09-11 2009-08-13 Mondobiotech Lab Ag Utilisation d'un peptide en tant qu'agent thérapeutique
WO2009039964A2 (fr) * 2007-09-11 2009-04-02 Mondobiotech Laboratories Ag Utilisation d'un peptide en tant qu'agent thérapeutique
WO2011134471A1 (fr) * 2010-04-27 2011-11-03 Zealand Pharma A/S Conjugués peptidiques d'antagonistes du récepteur de glp-1 et de gastrine, et leur utilisation
CN103003300A (zh) * 2010-04-27 2013-03-27 西兰制药公司 Glp-1受体激动剂和胃泌素的肽缀合物及其用途
US9089538B2 (en) 2010-04-27 2015-07-28 Zealand Pharma A/S Peptide conjugates of GLP-1 receptor agonists and gastrin and their use
EA023925B1 (ru) * 2010-04-27 2016-07-29 Зилэнд Фарма А/С Пептидные конъюгаты агонистов рецептора glp-1 и их применение
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CN103003300B (zh) * 2010-04-27 2017-06-09 西兰制药公司 Glp‑1受体激动剂和胃泌素的肽缀合物及其用途
US10406207B2 (en) 2010-04-27 2019-09-10 Zealand Pharma A/S Peptide conjugates of GLP-1 receptor agonists and gastrin and their use
US9861706B2 (en) 2011-11-03 2018-01-09 Zealand Pharma A/S GLP-1 receptor agonist peptide gastrin conjugates
US11795204B2 (en) 2012-07-23 2023-10-24 Zealand Pharma A/S Glucagon analogues
US10253081B2 (en) 2012-09-17 2019-04-09 Zealand Pharma A/S Glucagon analogues
US9975939B2 (en) 2012-09-17 2018-05-22 Zealand Pharma A/S Glucagon analogues
US11884713B2 (en) 2013-10-17 2024-01-30 Zealand Pharma A/S Acylated glucagon analogues
US10457714B2 (en) 2013-10-17 2019-10-29 Zealand Pharma A/S Acylated glucagon analogues
US11091528B2 (en) 2013-10-17 2021-08-17 Zealand Pharma A/S Acylated glucagon analogues
US11034747B2 (en) 2013-10-17 2021-06-15 Zealand Pharma A/S Glucagon analogues and methods of use
US10131702B2 (en) 2013-11-06 2018-11-20 Zealand Pharma A/S Glucagon-GLP-1-GIP triple agonist compounds
US10093713B2 (en) 2013-11-06 2018-10-09 Zealand Pharma A/S GIP-GLP-1 dual agonist compounds and methods
US11008375B2 (en) 2013-11-06 2021-05-18 Zealand Pharma A/S GIP-GLP-1 dual agonist compounds and methods
US11111285B2 (en) 2013-11-06 2021-09-07 Zealand Pharma A/S Glucagon-GLP-1-GIP triple agonist compounds
US10253078B2 (en) 2014-10-29 2019-04-09 Zealand Pharma A/S GIP agonist compounds and methods
US11001619B2 (en) 2014-10-29 2021-05-11 Zealand Pharma A/S GIP agonist compounds and methods
US11814417B2 (en) 2014-10-29 2023-11-14 Zealand Pharma A/S GIP agonist compounds and methods
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