WO2004103310A2 - Methods and compositions for the treatment of metabolic disorders - Google Patents

Abstract

The present invention features methods and compositions for the treatment, reduction, or prevention of metabolic disorders by administering to a mammal a nonpeptide agonist of the GLP-1 receptor. Any disorder involving an alteration in GLP- 1 receptor activity, such as a reduction in second messenger signaling, is amenable to treatment by the present invention, including, for example, diabetes and obesity.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF METABOLIC DISORDERS

Field of the Invention In general, the present invention relates to methods and compositions for treating, reducing, or preventing metabolic disorders, such as diabetes and obesity.

Background o the Invention

In an individual with normal regulation of blood glucose, the pancreatic hormone insulin is secreted in response to increased blood sugar levels. Although the level of blood glucose generally increases postprandially, the action of insulin on peripheral tissues, such as skeletal muscle and fat, causes clearance of glucose from the bloodstream. In this regard, insulin simulates cells of these peripheral tissues to actively take up glucose from the blood for storage. Loss of glucose homeostasis as a result of faulty insulin secretion or action typically results in metabolic disorders such as diabetes, which may be co-triggered or further exacerbated by obesity. Because these conditions are often fatal, strategies to reinstate proper glucose clearance from the bloodstream are required.

Glucagon-like peptide-1 (7-36) amide (GLP-1) is typically produced by the L- cells ofthe intestinal mucosa and is released in the bloodstream in response to food intake to maintain postprandial blood glucose homeostasis (Holts, Annu. Rev. Physiol 59:257-271, 1997; Drucker, Diabetes 47:159-169, 1998). In this regard, GLP-1 binds to the G-protein coupled GLP-1 receptor (GLP-1 R) on pancreatic beta cells and in turn, enhances glucose-dependent insulin secretion and proinsulin gene transcription by the up-regulation ofthe transcription factor PDX-1 (Doyle et al, Recent Prog. Horm. Res. 56:377-399, 2001). GLP-1R activation by GLP-1 also decreases glucagon secretion from islet alpha cells (Fehmann et al, Pancreas, 11 : 196-200, 1995), delays gastric emptying and influences satiety via the nervous system (Schirra et al, Proc. Assoc. Am. Physicians 109:84-97, 1997). Accordingly, the regulation of GLP-1 and GLP-1R has emerged as a possible treatment modality for metabolic disorders such as diabetes and obesity. Summary of the Invention

Using a systematic screening assay that employs a constitutively active GLP-1 receptor (GLP-1R), we have identified non-peptide agonist compounds specific for the GLP-1R. Thus, the present invention features methods and compositions useful for the treatment, reduction, or prevention of metabolic disorders such as diabetes and obesity. In particular, we have identified three non-peptide agonist compounds that can increase GLP-1 receptor activity using the screening assay ofthe invention. Based on this activity, these compounds represent therapeuticaUy useful compounds as well as leads for the optimal design of further non-peptide agonists ofthe GLP-1R that are useful for the treatment, reduction, and prevention of metabolic disorders such as diabetes and obesity. As described herein, we have also identified additional nonpeptide derivatives that also function as agonists ofthe GLP-1R.

One particular class of non-peptide agonists identified herein that may be formulated as pharmaceutical compositions are compounds having the chemical formula:

or a pharmaceutically acceptable salt thereof, such that

X represents oxygen (O), sulfur (S), or nitrogen (N)R⁸, R⁸ being hydrogen (H) or Cι_-6 alkyl. Each of R¹ and R² may be, independently, a H, substituted or unsubstituted C₆ aryl, C_1-6 alkyl, or C_1-4 alkaryl. Alternatively, R¹ and R² may together form a C₂-C₆ ring, optionally containing non-vicinal O, S, or NR⁹ linkages, R⁹ being a H or C_1-6 alkyl. Furthermore, each of R³, R⁴, R⁵, and R⁶may be, independently, a H or C_1-6 alkyl. Alternatively, R³ and R⁴ may together be =O and each of R⁵ and R⁶ may be, independently, H or C_1-6 alkyl. In yet another alternative, R³ and R⁵ do not exist, a double bond is formed between the carbons bearing R⁴ and R⁶, R⁴ is a H or C_1-6 alkyl, and R⁶ is a H, C,-C₆ alkyl, or CH=NOR¹⁰, R¹⁰ being a H or C_1-6 alkyl. R⁷ may be a H, halogen, or C_1-6 alkyl. Pharmaceutical compositions ofthe invention exclude the compound having the following formula:

Exemplary non-peptide agonists of this class include, for example:

Preferably, the non-peptide agonist is

Another class of non-peptide agonists that may be formulated as pharmaceutical compositions include compounds having the formula:

or a pharmaceutically acceptable salt thereof. X may be a O, S, or NR⁹, R⁹ being a H or Cι-₆ alkyl. Each of R and R may be, independently, a H, substituted or unsubstituted C₆ ar 'yl, C_1-6 alkyl, or Cι- alkaryl. Alternatively, R 1 and R 2 may together form a C -C ring, optionally containing non-vicinal O, S, or NR¹⁰ linkages, R¹⁰ being a H or Cι_-6 alkyl. Each of R³, R⁴, R⁵, and R⁶ may be, independently, H or C_1-6 alkyl, or, alternatively, R³ and R⁴ may together be =O and each of R⁵ and R⁶ may be, independently, H or C_1-6 alkyl. hi yet another alternative, R³ and R⁵ do not exist, a double bond is formed between the carbons bearing R⁴ and R⁶, R⁴ is a H or C_1-6 alkyl, and R⁶ is a H, Cι-C₆ alkyl, or CH=NOR¹⁰, such that R¹⁰ is H or C_1-6 alkyl. Each of R⁷ and R may be a H, halogen, or C_1-6 alkyl. Exemplary agonists of this class include, for example:

Yet another class of non-peptide agonists that may be formulated as therapeutic compositions include compounds having the formula:

or a pharmaceutically acceptable salt thereof. W may be CI, F, CF₃, CN, or CO₂R⁴, R⁴ being -β alkyl. R¹ may be a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or CH₂COR⁵, R⁵ being a C_1-6 allcoxy, C_1-4 aryloxy, or NR⁶- aryl, such that the aryl group is substituted or unsubstituted and R⁶ is H or C_1-6 alkyl. Each of R² and R³ may be, independently a H, halogen, C_1-6 alkyl, or C_1-6 alkoxy.

Alternatively, R² and R³ may together form a substituted or unsubstituted aryl ring.

Exemplary agonists of this class include, for example:

Preferably, the agonist is:

In yet another alternative, pharmaceutical compositions ofthe invention include compounds having the formula:

or a pharmaceutically acceptable salt thereof, such that Y is a halogen; Z is a substituted or unsubstituted aryl; and each of R¹ through R¹⁰ is H or C_1-6 alkyl. Preferably, this agonist class has the formula:

that each of RI and R2 may be, independently, H, Cι-C₆ alkyl, halogen, NO₂, OH, C_1-6 allcoxy, CO₂H, CO₂R¹³, NR¹⁴R¹⁵, CN, or CF₃. Each of R¹³, R¹⁴, and R¹⁵ may be a H, C_1-6 alkyl, or C_1-4 alkaryl. For example, the agonist may be:

Using the assay ofthe invention, it has been further demonstrated that the previously identified GLP-IR antagonist, T0632, is in fact an inverse agonist. Because inverse agonists and positive agonists are often structurally related, several compounds have also been identified that are structurally related to T0632 and that function as GLP-IR agonists. This class of compounds that may be formulated as pharmaceutical compositions have the formula:

or a pharmaceutically acceptable salt thereof, such that W is N or CR , X is O or S; Y is CO₂H or SO₃H; Z is a substituted or unsubstituted C₆ aryl; n is 1-3; each of R¹ - R¹⁰ is, independently, a H, Ci-Cβ alkyl, halogen, NO₂, OH, C_1-6 alkoxy, CO₂H,

CO₂R¹³, NR¹⁴R¹⁵, CN, or CF₃; and each of R¹¹ andR¹² is a H or d-C₆ alkyl. Each of

R¹³, R¹⁴, and R¹⁵ may be a H, C_1-6 alkyl, or C_1-4 alkaryl. However, the pharmaceutical compositions ofthe invention exclude the compound ofthe formula:

An exemplary non-peptide agonist derivative of T0632 is:

In all foregoing aspects ofthe invention, non-peptide agonist compounds preferably increase GLP-1 receptor activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% relative to an untreated control.

In a further aspect, the invention also features a method for treating, reducing, or preventing a metabolic disorder by administering to a mammal a therapeutically effective amount of a pharmaceutical composition containing a non-peptide agonist of GLP-IR. Such agonist compounds include any ofthe agonists described above. Furthermore, the mammal may also be administered:

or any compound in U.S.P.N. 5,807,883, hereby incorporated by reference. Preferably, the metabolic disorder involves an alteration in GLP-IR activity (such as a reduction in GLP-IR activity) and includes disorders such as diabetes (e.g., diabetes type I, diabetes type II, and gestational diabetes) and obesity. In addition to humans, any mammal (e.g., dog, cat, horse, baboon, or cow) may be treated according to the present invention.

Optionally, the mammal administered the non-peptide agonist ofthe invention may also receive a second therapeutic regimen, which may include a second therapeutic agent. According to this invention, examples of second therapeutic agents include nateglinide, metformin, thiazolidinediones, alpha-glucosidase inhibitors, repaglinide, nateglinide, sulfonylurea, miglitol, or insulin. Other agents may include agents that increase insulin production (e.g., amaryl, glucotrol, glucotrol XL, micronase, diabeta, Glynase, prandin, and Starlix), agents that increase sensitivity to insulin (e.g., glucophage, avandia, and Actos), or agents that delay absorption of glucose from the gut (e.g., precose and glyset). If desired, the second therapeutic agent and the non-peptide agonist may be formulated in the same composition. If the agents are present in different formulations, the second therapeutic agent may be administered within 24 hours, 2 days, 4 days, 7 days, or 14 days (before or after) the non-peptide agonist. In addition to a second therapeutic agent, administration ofthe non-peptide GLP-IR agonist ofthe present invention may also be complemented by a low-fat diet, controlled glucose diet, low sodium diet, stress management, physical exercise, reduction in alcohol intake, or reduction in smoking.

By "agonist" is meant any chemical substance that combines with a receptor so as to initiate an activity ofthe receptor; for a peptide hormone receptor (e.g., GLP- 1R), the agonist preferably alters a second messenger signaling activity. An

"agonist," as used herein, is therefore a compound that enhances or increases an activity, e.g., a second messenger signaling activity, of a receptor. Preferably, the agonist ofthe invention increases the activity ofthe GLP-1 receptor by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or even more than 100% relative to an untreated control. Thus, a "full agonist" refers to an agonist capable of activating the receptor to the maximum level of activity, e.g., a level of activity, which is induced by a natural, i.e., an endogenous, peptide hormone. A "partial agonist" refers to a positive agonist with reduced intrinsic activity relative to a full agonist.

By "a therapeutically effective amount" is meant an amount of a compound, alone or in a combination, required to treat, reduce, or prevent a disorder, for example, a disorder involving an alteration in GLP-IR, in a mammal. The effective amount of active compound(s) varies depending upon the route of administration, age, body weight, and general health ofthe subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen.

By "a metabolic disorder" is meant any pathological condition resulting from an alteration in a mammal's metabolism. Such disorders include those resulting from altered GLP-1 receptor activity. According to this invention, an alteration in GLP-1 receptor activity is typically a reduction in GLP-1 receptor activity by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% relative to GLP-1 receptor activity in a healthy individual. Metabolic disorders include, for example, diabetes (e.g., diabetes type I, diabetes type II, and gestational diabetes) and obesity. The term "pharmaceutical composition" is meant any composition that contains at least one therapeutically or biologically active agent and is suitable for administration to a patient. Any of these formulations can be prepared by well-known and accepted methods ofthe art. See, for example, Remington: The Science and Practice of Pharmacy, 20^th edition, (ed. AR Gennaro), Mack Publishing Co., Easton, PA, 2000.

By "second messenger signaling activity" is meant the production of an intracellular stimulus (e.g., cAMP, cGMP, ppGpp, inositol phosphate, or calcium ion) in response to activation ofthe receptor, or to activation of a protein in response to receptor activation, including but not limited to a kinase, a phosphatase, or to activation or inhibition of a membrane channel.

By "treating, reducing, or preventing a metabolic disorder" is meant ameliorating such condition before or after it has occurred. As compared with an equivalent untreated control, such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique. A patient who is being treated for diabetes or obesity, for example, is one who a medical practitioner has diagnosed as having such a condition. Diagnosis may be by any suitable means known in the art. Methods for diagnosing diabetes, for example, by measuring glucose tolerance are described, for example, in U.S. Patent No. 6,537,806, hereby incorporated by reference. Diagnosis and monitoring may also employ urine tests (urinalysis) measuring glucose and ketone levels (products of the breakdown of fat); measurements of blood levels of glucose; glucose tolerance test; or measurements of Hemoglobin Ale (HbAlc) levels. An individual is considered obese when their weight is 20% (25% in women) or more over the maximum weight desirable for their height. An adult who is more than 100 pounds overweight, is considered to be morbidly obese. Obesity is also defined as a body

I ₂ mass index (BMI) over 30 kg/m . A patient in whom the development of diabetes or obesity is being prevented may or may not have received such a diagnosis. One in the art will understand that these patients may have been subjected to the same standard tests as described above or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors (e.g., family history, hypertension, high cholesterol levels, or having a pathological condition predisposing to obesity or diabetes). As used herein, the terms "alkyl" and the prefix "allo" are inclusive of both straight chain and branched chain saturated or unsaturated groups, and of cyclic groups, i.e., cycloalkyl and cycloalkenyl groups. Unless otherwise specified, acyclic alkyl groups are from 1 to 6 carbons. Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 8 ring carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclopentyl, cyclohexyl, and adamantyl groups. The alkyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halogen, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. By "aryl" is meant a carbocyclic aromatic ring or ring system. Unless otherwise specified, aryl groups are from 6 to 18 carbons. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl, and indenyl groups. By "heteroaryl" is meant an aromatic ring or ring system that contains at least one ring hetero-atom (e.g., O, S, N). Unless otherwise specified, heteroaryl groups are from 1 to 9 carbons. Heteroaryl groups include furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, oxatriazolyl, pyridyl, pyridazyl, pyrimidyl, pyrazyl, triazyl, benzofuranyl, isobenzofuranyl, benzothienyl, indole, indazolyl, indolizinyl, benzisoxazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, naphtyridinyl, phthalazinyl, phenanthrolinyl, purinyl, and carbazolyl groups.

By "heterocycle" is meant a non-aromatic ring or ring system that contains at least one ring heteroatom (e.g., O, S, N). Unless otherwise specified, heterocyclic groups are from 1 to 9 carbons. Heterocyclic groups include, for example, dihydropyrrolyl, tetrahydropyrrolyl, piperazinyl, pyranyl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophene, tetrahydrothiophene, and morpholinyl groups. Aryl, heteroaryl, or heterocyclic groups may be unsubstituted or substituted by one or more substituents selected from the group consisting of C_1-6 alkyl, hydroxy, halo, nitro, C_1-6 alkoxy, Cι_-6 alkylthio, trifluoromethyl, trifluoromethoxy, Cι_-6 acyl, arylcarbonyl, heteroarylcarbonyl, nitrile, C_1-6 alkoxycarbonyl, arylalkyl (wherein the alkyl group has from 1 to 6 carbon atoms) and heteroarylalkyl (wherein the alkyl group has from 1 to 6 carbon atoms).

By "halide" or "halogen" or "halo" is meant bromine, chlorine, iodine, or fluorine.

By "alkoxy" is meant a chemical substituent ofthe formula -OR, where R is an alkyl group. By "aryloxy" is meant a chemical substituent ofthe formula -OR , where R is an aryl group. By "alkaryl" is meant a chemical substituent ofthe formula -RR , where R is an alkyl group and R is an aryl group, By "all ieteraryl" is meant a chemical substituent ofthe formula RR , where R is an alkyl group and R is a heteroaryl group.

By "non-vicinal O, S, or NR" is meant an oxygen, sulfur, or nitrogen heteroatom substituent in a linkage, wherein the heteroatom substituent does not form a bond to a saturated carbon that is bonded to another heteroatom. The present invention provides significant advantages over standard therapies for the treatment and prevention of diabetes and obesity. Administration of a nonpeptide agonist ofthe GLP-IR according to the present invention differs from standard therapies by modifying the injury process, rather than merely mitigating the symptoms. Furthermore, non-peptide GLP-IR agonists have significantly more favorable pharmacokinetic properties relative to peptide agonists, such as derivatives of GLP-1 or exendin-4. These latter agonists, even if protected from rapid enzymatic degradation by modification ofthe peptide molecule, cannot be effectively administered via enteral routes.

Description of the Figures FIGURE 1 is a schematic diagram showing the relationship between a full or partial agonist, an inverse agonist, and an antagonist.

FIGURE 2 A is a schematic showing the alignment ofthe consensus sequences in the PTH-1 receptor and the GLP-1 receptor.

FIGURE 2B is a bar graph showing cAMP production by the wild type form of the GLP-1 receptor and the "51" mutant (distinguished from the wild type GLP-IR by substitution ofthe cysteine at amino acid position 347 (C347) with QQYR) in the presence or absence ofthe GLP-1 ligand. FIGURE 2C is a graph showing a concentration-response experiment of GLP-1 with respect to the wild type form ofthe GLP-1 receptor (open circles) and the "51" mutant (filled circles).

FIGURES 3A-3B show a series of graphs representing basal (squares) and GLP-1 stimulated (circles) cAMP production following an increase in surface expression of wild type GLP-1 receptors (open) and the "51" mutant (closed).

FIGURE 4 shows a diagram representing an amino acid alignment of GLP-1 receptor peptide ligands.

FIGURE 5 is a bar graph representing receptor activation ofthe GLP-1 receptor and the "51" mutant receptor by various GLP-1 receptor peptide ligands. FIGURES 6A-6B show a series of graphs representing radioligand binding studies using the agonist ligand GLP2 (D9E) and the antagonist ligand (exendin9-39) with respect to the wild-type GLP-1 receptor (open circles) and the "51" mutant (closed circles). FIGURE 7 is a table showing peptide and non-peptide ligand affinities at the wild-type GLP- 1 receptor and the "51 " mutant. '

FIGURE 8 A shows the chemical structures of T0632, A (=G0), and B (=G9) compounds. FIGURE 8B shows a bar graph representing cAMP production by COS-7 cells expressing empty vector (left block), wild type GLP-1 receptor (middle block), and the "51 " mutant (right block) in the absence (black bar) or presence (open bars) ofthe T0632, A (=G0), and B (=G9) compounds.

FIGURE 9 is a table showing binding affinities for GLP-1 and Exendin 9-39 on un-tagged and tagged wild-type GLP-IR and the "51" mutant.

FIGURE 10 is a bar graph showing basal, GLP-1 stimulated, and Exendin 3-39 stimulated cAMP production in COS-7 cells transfected with un-tagged and tagged wild-type GLP-IR and "51" mutant

FIGURES 11 A-l 1H show a series of pictures representing transmission light and confocal microscopy images of cells expressing tagged wild-type GLP-1 receptor and "51" mutant.

FIGURE 12 shows a bar graph representing an HA-tag-based ELISA assay measuring receptor expression levels of tagged wild-type GLP-1 receptor and "51" mutant. FIGURES 13A and 13B show a series of bar graph representing basal- and ligand- stimulated cAMP production of tagged wild-type GLP-IR and "51" mutant with and without an overnight (ON) incubation with T0632.

FIGURE 14 is a bar graph showing the expression levels of tagged wild-type GLP-1 receptor and the "51" mutant following the long-term (24 hours) incubation with different agonists and putative antagonists.

FIGURES 15A-15H show a series of pictures representing transmission light and confocal microscopy images of COS-7 cells expressing tagged wild-type GLP-1 receptor and the "51" mutant with and without long-term (24 hours) incubation with T0632. FIGURE 16 is a bar graph showing Exendin 9-39 binding of un-tagged and tagged wild-type GLP-1 receptor and "51" mutant with and without long-term (24 hours) incubation with T0632. FIGURE 17 is a bar graph showing the surface expression ofthe tagged "51" GLP-1 receptor mutant following long-term incubation with T0632, followed by the removal of T0632.

FIGURE 18 is a bar graph showing cAMP production in GLP-1 receptor mutant "51 " and its N-terminally tagged derivative mutant "286" in the absence or presence of various ligands.

FIGURES 19A-19E represent the chemical structures ofthe T0632, A, B, and C compounds and non-peptide agonist derivatives thereof.

FIGURE 20 shows a series of bar graphs representing cAMP activation in cells transfected with the constitutively active GLP- 1 receptor or an empty vector following treatment with compound A, B, and C and non-peptide agonist derivatives thereof.

Detailed Description Glucagon-like peptide 1 (GLP-1) is an enteroendocrine peptide hormone that enhances the growth and insulin-secretory capacity of pancreatic beta cells. Due to its central function in the regulation of glucose clearance from the bloodstream, the interaction between GLP-1 and the GLP-1 receptor (GLP-IR) represents a potential source for the development of treatment modalities for metabolic disorders such as diabetes and obesity.

Using a screening assay that employs a constitutively active GLP-1 receptor, we have identified three small molecule non-peptide GLP-IR agonists, namely compounds A, B, and C (see FIGURES 8 A and 19E). Administration of these agonists to a mammal increases the activity ofthe GLP-IR (e.g., second messenger signaling) (FIGURES 8B, 13, 18 and 20). In addition to their therapeutic potential, these agonists are also useful as lead compounds for the optimal and rational design of further small molecule agonists ofthe GLP-IR. Using this latter strategy, we have identified additional non-peptide agonist derivatives of compounds A, B, and C (as shown in FIGURES 19B-19E) that are also useful for the treatment of metabolic disorders. In addition, using the screening assay ofthe invention, we have further identified the previously known GLP-IR antagonist, T0632 compound (FIGURE 8A), as an inverse agonist. Because inverse agonists and positive agonists are often structurally related, we have also identified non-peptide agonist derivatives of T0632. Exemplary non-peptide agonist derivatives of T0632 are provided in FIGURE 19A and U.S.P.N. 5,807,883, hereby incorporated by reference.

Desirably, the non-peptide agonist ofthe invention increases GLP-IR activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) relative to an untreated control such that metabolic disorders involving alterations in GLP-IR activity (e.g., diabetes and obesity) are treated, prevented, or reduced.

The methods ofthe present invention may also be used prophylactically. For example, a mammal may be treated without having been diagnosed with any ofthe disorders amenable to treatment according to the present invention, but presenting risk factors for such disorders (e.g., family history of diabetes, obesity, age, ethnicity, delivering a baby of greater than nine pounds, hypertension, high blood levels of tryglycerides, or high blood cholesterol levels).

Diagnosis of any ofthe disorders amenable to treatment according to this invention may be performed using any method known in the art, and is described in detail, for example in U.S.P.N. 6,537,806, hereby incorporated by reference.

Disorders involving alterations in GLP-IR activity are typically characterized by a reduction in GLP-IR activity (e.g., second messenger signaling) in a mammal by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% relative to GLP-IR activity in a healthy individual, as measured by any method known in the art. Diabetes may be diagnosed, for example, by urine tests (urinalysis) measuring glucose and ketone levels (products ofthe breakdown of fat); measurements of blood levels of glucose; glucose tolerance test; or by the detection of molecular markers characteristic of diabetes in a biological sample collected from the mammal, such as blood, serum, or urine (e.g., measurements of Hemoglobin Ale (HbAlc) levels). Type I diabetes is usually manifested in childhood and is characterized by symptoms such as increased thirst, increased urination, weight loss in spite of increased appetite, fatigue, nausea, and vomiting. Type I diabetes is normally diagnosed by high levels of glucose and ketones in urine or blood. Type II diabetes typically occurs in adulthood and is normally manifested by such symptoms as increased thirst, increased urination, increased appetite, fatigue, blurred vision, slow-healing infections, and impotence in men. Type II diabetes is diagnosed when fasting glucose levels are higher than 126 mg/dL on two occasions or when random glucose levels are higher than 200mg/dL and are accompanied by symptoms such as increased thirst, increased urination and fatigue. An individual is considered obese when their weight is 20% (25% in women) or more over the maximum weight desirable for their height. An adult who is more than 100 pounds overweight, is considered to be morbidly obese. Obesity is also defined as a body mass index (BMI) over 30 kg/m .

To assess whether the present invention is useful to treat, reduce, or prevent a disorder involving an alteration in GLP-IR activity, any method known in the art may be used. Accordingly, any method described above is useful for this purpose and may include, for example, measuring GLP-1 receptor activation before and following treatment. Measurements of hemoglobin Ale (HbAlc) levels is also an indication of average blood glucose during the previous two to three months and is typically used to monitor a patient's response to diabetes treatment. Furthermore, a medically desirable result may be a reduction of any ofthe symptoms described above.

Pharmaceutical Formulations

As described above, we have identified various non-peptide agonists ofthe GLP-1 receptor, namely compounds A, B, and C (FIGURES 8 A and 19E) using a screening strategy that employs a constitutively active GLP-1 receptor. These compounds have the ability to significantly stimulate the basal activity ofthe constitutively active receptor (FIGURES 8B and 20). Using these compounds as leads, we have further identified non-peptide agonist derivatives that also function to increase GLP-IR activity (see FIGURES 19B-19E that also shows the generic formula of non-peptide agonist derivatives of compound A, B, and C, as well as FIGURE 20). We further show that the previously identified GLP-IR antagonist, T0632 (FIGURE 8A), functions as an inverse agonist. Using this compound as a lead, we have identified several non-peptide agonist derivatives (see FIGURE 19A, which also shows the generic formula of non-peptide agonist derivatives of T0632). According to this invention, the administration of non-peptide agonists of GLP-IR to a mammal increases the level of activity of GLP-IR by at least 10%>, 20%o, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% relative to an untreated control such that disorders involving alterations in GLP-IR activity (e.g., a reduction in GLP-IR), including for example, diabetes (e.g., type I, type 11, or gestational diabetes) and obesity are treated, prevented, or reduced, hi most instances, an effective daily dosage ofthe non-peptide agonist will be in the range of from about 0.001 mg/kg to 0.01 mg/ kg to about 200 mg/kg, e.g., from about 0.1 mg/kg to about 100 mg/kg of body weight, administered in single or divided doses. However, it may also be necessary to use dosages outside such limits.

The pharmaceutical composition including the non-peptide agonist ofthe invention can be provided systemically or locally, by injection (e.g., intrasmuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, or intraoccular), as well as by oral, topical (e.g., ointment, or patch), or transdermal administration. Alternatively, these compositions may be provided by inhalation, or by suppository. Compositions according to the invention may also be provided to mucosal tissue, by lavage to vaginal, rectal, urethral, buccal, and sublingual tissue, for example.

Second therapeutic agents

According to the present invention, the non-peptide agonist ofthe GLP-1 receptor is delivered to the mammal in a pharmaceutically acceptable carrier, alone or in combination with one or more therapeutic agents. When the second therapeutic agent is present in a different pharmaceutical composition, different routes of administration may be used. For example, the second therapeutic agent may be administered orally, or by intravenous, intramuscular, or subcutaneous injection. If desired, more than one therapeutic agent may be administered with the non-peptide agonist and concentrations known to be effective for such therapeutic agents can be used. Desirably, the non-peptide agonist and the second therapeutic agent are administered in a single pharmaceutical composition consisting of an effective amount in a pharmaceutically acceptable carrier. Alternatively, the non-peptide agonist ofthe invention and the second therapeutic agent are administered in separate formulations within at least 1, 2, 4, 6, 10, 12, 18, or more than 24 hours apart. These reagents can be combined and used with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers. A pharmaceutical carrier can be any compatible, non-toxic substance suitable for the administration ofthe compositions ofthe present invention to a mammal. Pharmaceutically acceptable carriers include, for example, water, saline, buffers and other compounds described for example in the Merck Index, Merck & Co., Rahway, New Jersey. Slow release formulation or a slow release apparatus may be also be used for continuous administration.

Second therapeutic agents may include, for example, insulin, nateglinide, metformin, thiazolidinediones, alpha-glucosidase inhibitors, repaglinide, nateglinide, sulfonylurea, or miglitol. Other second therapeutic agents may also include agents that increase insulin production (e.g., amaryl, glucotrol, glucotrol XL, micronase, diabeta, Glynase, prandin, and Starlix), agents that increase sensitivity to insulin (e.g., glucophage, avandia, and Actos), and agents that delay absorption of glucose from the gut (e.g., precose and glyset).

Concentrations of non-peptide agonist and the second therapeutic agent necessary to treat, reduce, or prevent the disorder involving an alteration in GLP-1 receptor activity, diabetes, or obesity will depend upon different factors, including means of administration, target site, physiological state ofthe mammal, and other medication admimstered. Thus treatment dosages may be titrated to optimize safety and efficacy and is within the skill of an artisan ofthe art. Determination ofthe proper dosage and administration regime for a particular situation is within the skill of the art.

In addition to second therapeutic agents, the administration ofthe non-peptide agonist ofthe invention may also include a modification to the lifestyle ofthe patient being treated. Such changes may include low-fat diet, controlled glucose consumption, low sodium diet, stress management, physical exercise, reduction in alcohol intake, or reduction in smoking.

The pharmaceutical compositions according to the present invention are formulated according to the mode of administration to be used, hi cases where pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen-free and particulate free. An isotonic formulation is preferably used. Generally, additives for isotonicity can include for example sodium chloride, dextrose, mannitol, sorbitol and lactose. Stabilizers may also be used and include, for example, gelatin and albumin.

Constitutively Active GLP-1 Receptors

The screening assay used to identify the non-peptide GLP-IR agonists ofthe invention employs a constitutively active GLP-IR. These receptors may be generated by any method, for example, the methods described in U.S.P.N. 6,376,198 and 6,566,080 and U.S.S.N. 10/441,787, all of which are hereby incorporated by reference. In one approach, constitutively active receptors are generated by the introduction of mutations in the C-terminal portion ofthe third intracellular loop of the wild-type GLP-1 receptor. For example, the cysteine residue at amino acid position 347 may be substituted with glutamine-glutamine-tyrosine-arginine (QQYR) residues. Other mutations that also result in constitutive activation ofthe GLP-IR include, for example, deletion of Cys347, deletion of He 345, or substitution of

Cys347 with GGY. These constitutively active receptors, which have a higher basal activity than the corresponding wild-type GLP-1 receptor, are useful for screening of agonist compounds for GLP-IR. The sensitivity ofthe screening method ofthe invention can further be improved by introducing a tag, such as an HA-tag or a T7 tag in the N-terminal portion ofthe receptor, following the leader sequence. For example, the tag maybe inserted between amino acid positions 23 and 24 ofthe wild type receptor. If desired, the assay ofthe invention may also be performed following the long-term exposure ofthe constitutively active GLP-IR to the inverse agonist T0632 for a period ranging between twelve and twenty- four hours. According to this invention, long-term treatment with T0632 increases the expression ofthe mutant receptor at the cell surface, hi addition to synthetic mutant receptors, examples of enhanced receptors include naturally occurring mutant receptors, which cause a disease phenotype by virtue of their enhanced receptor activity. The constitutively active GLP-IR ofthe invention is preferably human but may also be a non-human receptor (e.g., rat, mouse, mastomys, Xenopus, canine receptors, or hybrid variants thereof), which amplifies an agonist signal to a greater extent than does the corresponding wild-type human receptor. Identification of Agonists, Inverse Agonists, and Antagonists

To determine whether a candidate compound is an agonist or an inverse agonist ofthe GLP-1 receptor, the compound is exposed to a constitutively active form ofthe GLP-1 receptor (or enhanced receptor), which has a greater or an enhanced ability to amplify the intrinsic activity of an agonist, hi one particular approach, the second messenger signaling activity ofthe enhanced receptor is measured in the presence ofthe candidate compound and compared to the second messenger signaling activity ofthe enhanced receptor in the absence ofthe candidate compound. A change in second messenger signaling activity indicates that the candidate compound is an agonist. Thus, an increase in second messenger signaling activity indicates that the compound is either a full or partial positive agonist while a decrease in second messenger signaling activity indicates that the compound is an inverse (also termed a 'negative') agonist.

A diagram explaining the difference between full and partial agonists, inverse agonists, and antagonists is shown in FIGURE 1 (see also Milligan et al, TIPS,

16:10-13, 1995). In FIGURE 1, the position ofthe equilibrium between an inactive state R and an active state R varies with individual receptors and is altered by the presence of receptor ligands. Agonists function by stabilizing R while inverse agonists preferentially stabilize R. A continuum of ligands between full agonists (at the extreme right-hand side ofthe see-saw as these move the equilibrium furthest to the right) and full inverse agonists (at the extreme left-hand side ofthe see-saw) is expected to exist. Antagonists, which do not alter the position ofthe equilibrium, define the position of the fulcrum. An antagonist may be, for example, a competitive or a non-competitive inhibitor. Using the assay described in the present invention, we have identified agonists that are useful therapeutically and that can also serve as lead compounds for further pharmaceutical optimization. In particular, to optimize these compounds systematic chemical modifications can be made, and their effects can be further assessed using enhanced receptors according to the method ofthe invention. Exemplary derivatives of agonists that can also increase GLP-IR activity are provided in FIGURES 19A- 19E. By following such a development strategy the intrinsic activity of new agonists can be optimized so as to be useful therapeutically against a metabolic disorder, for example, one that can be mitigated by stimulating the GLP-IR. Knowing that a particular ligand functions as a positive or inverse agonist, as opposed to an antagonist, facilitates the identification of ligand species most likely to achieve a given physiological effect, or to achieve a physiological effect absent an unwanted side effect.

Agonists of the GLP-1 Receptor

A candidate compound identified by the present invention is a ligand that binds with specificity to the GLP-IR, e.g., a peptide, peptoid, or non-peptide ligand, preferably a non-peptide ligand. A candidate compound is shown to be a positive or inverse agonist by the screening assay disclosed herein. Exemplary candidate compounds include peptoid compounds ((see, e.g., Horwell et al, Eur. J. Med. Chem., 30 Suppl.:537S-550S, 1995; Horwell et al, J. Med. Chem., 34:404-14, 1991); dipeptoid analogues (see, e.g., Horwell et al. J. Med. Chem. , 34:404-14, 1991); cyclic nucleotides and modified amino acids (see, e.g., Dethloff et al, DrugMetab., 24:261- 93, 1992); and benzodiazepine derivatives, e.g.., the compounds described in Bock et al, J. Med. Chem., 33:450-55, 1990, or a derivative thereof. Additional benzodiazepine derivatives having a peptide hormone receptor agonist activity are described in the following patents and patent applications, each of which is hereby incorporated by reference: EPA 167919, EPA 284256, EPA 434360, EPA 434364, EPA 434369, EPA 514125, EPA 51426, EPA 514133, EPA 508796, EPA 508797, EPA 508798, EPA 508799, EPA 523845, EPA 523846, EPA 559170, EPA 549039, EPA 667,334, WO 9211246, WO 93032078, WO 9308175, WO 9307131, WO 9317011, WO 9319053, WO 9308175, WO 9413648, WO 9403437, WO 9611689, and U.S. Patent No. 5,521,175. Also encompassed are those compounds described in Henke et al, J. Med. Chem., 39:2655-58, 1996; or in Willson et al, J. Med. Chem., 39:3030-34, 1996 (both hereby incorporated by reference), which are shown to be inverse agonists. Exemplary candidate compounds that function as GLP-IR agonists are further described, for example, in U.S. Patent Numbers 5,750,353, 6,376,198, and 6,566,080 as well as U.S. Serial Numbers 08/718,047 and 10/127,940, all of which are hereby incorporated by reference.

Any of these or other GLP-1 receptor agonists may be optimized according to the methods described herein or may be administered alone or in conjunction with the therapeutic compounds ofthe invention for the treatment of metabolic disorders. Identification of Novel Constitutively Active GLP-1 Receptors and Use for Identification of GLP-1 Receptor Agonists

As indicated above, the agonists ofthe present invention were identified using constitutively active GLP1 receptors. We noted that a considerable difference existed in the length of the third intracellular loop of the GLP- IR relative to the PTH receptor (FIGURE 2A), such that the loop of GLP-IR was much shorter than that ofthe PTH receptor. Using site-directed mutagenesis, a mutant of GLP-IR was generated to compensate for this difference in length. The cysteine residue at position 347 ofthe wild-type GLP-IR was therefore replaced with the glutamine-glutamine-tyrosine- arginine (QQYR) sequence found in the PTHR (the C347-QQYR mutant will also be referred to as "51"). This mutation resulted in the constitutive activation ofthe GLP- IR. When transiently expressed in COS-7 cells and assessed for its ability to induce cAMP production, the C₃₄₇QQYR mutant or "51" receptor showed a significant higher level of basal activity (approximately 3-fold) relative to cells transfected with the empty vector, pcDNAl (FIGURE 2B). Although the mutant receptor can be further stimulated with the endogenous ligand (e.g., GLP-1), the maximal level of stimulation was much lower than that ofthe wild type receptor, perhaps reflecting a defect in receptor processing or expression. When assessed with homologous competition binding, the expression level ofthe C₃₄ QQYR mutant was found to be 6.87 x 10 ₅ receptors / cell versus 4.24 ' x 107 receptors / cell with the wild type. A concentration-response experiment showed that the potency (=50%> curve inflection point) of GLP-1 at the "51" mutant (filled circles, 88pM) was shifted three-fold to the left in comparison with wild type (open circles, 274 pM), consistent with an established hallmark criterion of constitutively active receptors (FIGURE 2C). hi addition to the C347-QQYR substitution, we have generated similar constitutively active mutants by modifications ofthe same region (e.g., by deleting the cysteine residue at amino acid position 347 or the isoleucine residue at amino acid position 345, or alternatively by inserting additional neutral or charged amino acids in the position normally occupied by cysteine 347). Increasing the number of expressed wild type receptors (by modulating the amount of transfected cDNA) also led to a parallel increase in GLP-1 stimulated cAMP production in these cells (FIGURE 3 A, open circles). The level of receptor expression was quantified by ¹²⁵I exendin radioligand binding studies (x-axis). Compared with the wild-type receptor, the level of expression ofthe "51" mutant (FIGURE 3 A, closed circles) even at the highest cDNA transfection levels was low, and the low level of GLP-1 induced signaling observed with this mutant was commensurate with this. Increasing the expression level ofthe wild type receptor (open squares) did not result in an increase in basal cAMP production, which was close to zero at each condition (FIGURE 3B, open squares), hi contrast, despite its low expression level (closed squares), the "51" mutant still showed an elevated level of basal signaling, thus confirming the constitutive activity ofthe mutant receptor relative to the wild type receptor. In order to characterize the C_{3 7}QQYR mutant, a range of peptides was employed (FIGURE 4). These ligands were chosen to include a selection of peptides with known and previously unknown efficacy at the GLP-IR (e.g., exendin-4 (2-39 - 6F), a potent antagonist; Exendin 4 (2-39, D9E), a weak partial agonist; and GLP -2 (E9D), a strong partial agonist). The level of second messenger signaling triggered by each ligand at the wild type and the C₃₄₇QQYR mutant receptor was measured

(FIGURE 5; open and closed bars correspond to activity at the wild type and mutant receptors, respectively). Using the mutant receptor, we were able to detect trace activities of some compounds that were previously thought to lack activity. Thus, when tested at saturating concentrations, the activities of ligands at the "51" mutant (closed bars) were systematically amplified in comparison with the corresponding wild type values (open bars). For example, exendin (9-39), a classical antagonist, was found to have a low level of activity. Another proposed antagonist, GLP-1 (9-36) was found to be a partial agonist at the mutant receptor. Exendin-4 (2-39, D9E) was designed to parallel the antagonist defined for the glucagon receptor, glucagon (2-29, D9E), and both of these compounds were found to have activity at the C₃₄ QQYR mutant. Notably, ligands that had previously been defined as partial agonists for the GLP-IR, exendin (3-39) and GLP -2, were found to have amplified activities that exceeded the activity ofthe full agonist, GLP-1. No major differences were observed in the activities of previously defined full agonists. These findings reinforce the fact that the mutant "51" is a systematic amplifier, which makes it a useful tool for detecting trace activities of compounds in drug screening processes that would otherwise be overlooked. Such compounds provide valuable leads for further optimization into full agonists at the wild type GLP-1 receptor, thereby providing candidate drugs for the treatment of metabolic disorders, such as diabetes and obesity. In addition to the increase in activity observed with the C₃₄ QQYR mutant receptor, significant shifts in ligand binding affinity were also seen (FIGURES 6 and 7). Radioligand binding studies revealed increased affinity of an agonist ligand (e.g., GLP2D9E) at the "51" mutant (closed circles) relative to the wild type GLP-1 receptor (open circles). Glucagon, a known full agonist with weak affinity for the GLP-IR, showed the most significant change. For glucagon, the IC₅₀ value of radioligand competition (which is inversely related to affinity) decreased from 564 nM at the wild type receptor to 4.6 nM at the mutant receptor (FIGURE 7). In contrast, known antagonists ofthe receptor, such as exendin (9-39), showed very little change in the binding affinity for the two receptors (FIGURES 6 and 7). Overall, binding affinity at the mutant receptor versus the wild type receptor was found to increase for agonists but not for antagonists, a characteristic that is also applicable to constitutively active class A receptors. The increased ligand affinity together with amplified ligand function further contributes to the utility ofthe "51" mutant as a sensitive tool for the identification of non-peptide agonists according to the present invention. We assessed ligand-induced cAMP production by several non-peptide compounds at the wild type and C ₄₇QQYR mutant receptors. Two non-peptide compounds, GO and G9 (also named compounds A and B) were found to stimulate the mutant receptor to a significant level. FIGURE 8B shows cAMP production in COS- 7 cells expressing either the empty vector (no receptor, left block), the wild type GLP- 1 receptor (middle block), or the "51" mutant (right block) in the absence (black bar) or the presence of either of these compounds (open bars). Another previously reported GLP-1 receptor non-peptide ligand, T0632 (or T6) did not detectably alter basal receptor activity. Given the proven correlation between compound activity at the wild type GLP-1 receptor and the "51" mutant, these findings make compounds A and B strong candidates for further optimization into small non-peptide agonist drugs, a goal that has been hitherto illusive. Agonist activity was also found in several derivatives of compounds A and B, as shown in FIGURES 19B-D and 20. HA-tags or T7-tags were next introduced into the N-terminal part ofthe human wild type as well as the mutant GLP-IR, directly after the predicted leader sequence. To determine this position, we used the online program "SignalP VI.1", which is available at http://www.cbs.dtu.dk/services/SignalP. According to this calculation, the GLP-IR leader sequence is 23 amino acids long and the most convenient cleavage site appeared to be between amino acid position 23 and 24 ofthe GLP-IR: LLLLGMVGRAGP - RPQGATVSLWET. By introducing the HA-tag (YPYDVPDYA) or the T7-tag (MASMTGGQQMG) at this position, no alteration of the predicted position of cleavage occurred. Using these constructs, we demonstrated that the binding affinities for GLP-1 and Exendin4 (9-39) on the tagged GLP-IRs (wt and mutant) were not different from their un-tagged counterparts (FIGURE 9). Competition binding studies using ¹²⁵I Exendin4 (9-39) resulted in Kj values of about 2.0 nM for Exendin4 (9-39) for all tagged and un-tagged wild type and mutant receptors (Goke et al, JBiol Chem 268:19650-19655, 1993, Thorens et al, Diabetes 42:1678-1682, 1993). The Ki value of 1.4 nM for GLP-1 on the wild type GLP-IR was also unchanged, compared to previously published data. The previously reported ten-fold higher affinity for GLP-1 (Ki = 0.15 nM) on the mutant receptor could also be detected in these tagged mutant GLP-IRs (Doran et al, supra). The functionality ofthe tagged GLP-IRs - measured as cAMP production in transfected COS7 cells - also remained unchanged. While cAMP production of tagged wild type GLP-IRs after stimulation with the full agonist GLP-1 or partial agonist Exendin4 (3-39) lead to comparable results with their un-tagged templates, some differences could be found in the tagged mutant GLP-IR. The general trend was still the same as in the un-tagged mutant GLP-IR (including the increased basal activity and the finding that partial agonists act like full agonists). In comparison to the un-tagged mutant GLP-IR, their tagged counterparts had a five- fold higher basal activity (FIGURE 10), whereas the total activity after GLP-1 stimulation was not significant higher than the corresponding un-tagged GLP-IR (although there was a tendency to slightly higher levels). Because of differences in total amounts of cAMP produced by the wild type GLP-IR or the mutant receptor after addition of GLP-1, we assumed that this could be due to a lower (cell surface) expression levels of mutant GLP-IRs. This theory was also supported by comparing the total amounts of bound ¹²⁵I-labeled ligand in binding studies on wild-type GLP-IRs or mutant GLP-IRs. Confocal microscopy was next used to investigate the distribution of tagged receptors. While virtually all tagged receptors in wild type GLP-IR transfected COS7 cells were found at the cell surface (compare FIGURE 1 IB (surface expression) with FIGURE 1 ID (total expression)), most ofthe tagged mutant receptors were detected inside the cells (FIGURE 1 IF vs. 11H). To further investigate these differences in GLP-IR distribution, a whole-cell based ELISA technique was developed, which allowed a quantitative determination of total (permeabilized) and cell surface bound (non-permeabilized) receptor levels. This method revealed that the total receptor amounts of wild type and mutant GLP-IRs produced by transfected COS7 cells were almost the same (FIGURE 12, total ODU around 2.0). However, while the wild-type GLP-IR surface expression reached more than 90% (ODU = 1.9), the surface expression ofthe tagged mutant GLP-IR was below 10%o (ODU = 0.17) of its total expression.

Since low counts in binding studies and reduced cAMP production could be linked to low mutant GLP-IR surface expression levels, further investigations focused on whether and how the amount of mutant receptors found on the cell surface could be influenced by incubation with various GLP-IR ligands. Surprisingly, we found that long-term incubation with T0632, a non-peptide compound - formerly described as a GLP-IR specific antagonist, which binds to W ₃ at the N-terminus ofthe receptor - further increased basal activity ofthe tagged mutant GLP-IR (FIGURE 13A). In addition, as shown in FIGURE 13 A, long-term incubation with T0632 further amplified the partial agonist activity of compounds such as GO and Exendin (9-39). The ability of T0632 to enhance the receptor activity ofthe "51" receptor but not the wild-type receptor (FIGURE 13B) is likely attributable to the inverse agonist properties of this compound (demonstrated in FIGURE 18 as a compound-induced reduction in basal receptor activity during a short-term incubation (1 hour)), hi this regard, long-term incubation with cognate inverse agonists may increase the expression of constitutively active G-protein coupled receptors at the cell surface as a result ofthe induction of a less active receptor conformation that is also less prone to desensitization or internalization mechanisms. To further investigate the long-term effect of T0632 incubation- and its possible connection to cell surface expression - a more quantitative and sensitive peroxidase based ELISA was used. A 24-hour incubation with T0632, known peptide agonists (e.g., GLP-1 or exendin-4), or a putative antagonist (exendin 9-39) resulted in the discovery that T0632 was the only compound which was able to increase cell surface expression ofthe mutant GLP-IR (FIGURE 14). hi contrast, long-term incubation with T0632 did not influence (neither increased nor decreased) surface expression ofthe wild type GLP-IR.

The apparent ability of T0632 to enhance surface expression ofthe constitutively active GLP-IR mutant was also verified by confocal microscopy (FIGURE 15). Data are only shown for the mutant GLP-IR, since no changes in expression levels were detectable in wild type GLP-IRs. FIGURES 15B (cell surface expression) and 15D (total expression) show untreated COS7 cells transfected with the T7-tagged mutant GLP-IR, while FIGURES 15F (cell surface expression) and 15H (total expression) show tagged mutant receptors, which were incubated with T0632 for 24 hours. Comparing FIGURES 15B with 15F, the effect of an increased surface expression level after long-term incubation with T0632 is clearly visible. Comparison of FIGURES 15D and 15H revealed that long-term incubation with T0632 induced a shift from intracellular to cell surface expression ofthe mutant GLP- IR.

The subcellular receptor redistribution induced by T0632 could also be confirmed by competition binding studies using ¹²⁵I Exendin4 9-39. If T0632 really increases cell surface expression ofthe GLP-IR mutant, higher amounts of bound ¹²⁵I Exendin4 9-39 should be observed (FIGURE 16). Again, while T0632 did not influence binding to the wild type GLP-IR, there was an almost 400%o increase in bound ligand to the mutant receptors.

To further explore the kinetics of T0632-induced redistribution of mutant GLP- 1 receptors from the intracellular space to the cell surface, the time dependency ofthe compound-induced increase in receptor expression was investigated. The level of mutant GLP-IR/ expression was gradually increased during the 24-hour incubation (FIGURE 17). However, this increase was preceded by a lag of 12 hours following the addition of T0632. At this time, the level of mutant GLP-IR in cells that were incubated with T0632 was double the amount found on T0632 untreated cells. After an additional 6 hours, a second doubling in receptor expression in T0632 treated versus untreated cells was observed. At the end ofthe 24-hour incubation with T0632, more than 60% ofthe total mutant receptor amount could be detected on the cell surface. No additional increase was observed by further prolonging incubation time. Complementary experiments revealed that the T0632-mediated effect on receptor surface expression was reversible after washing away the compound. The increased level of mutant GLP-IR on the cell surface persisted for approximately one hour after this washout and dropped significantly three hours following the removal of T0632 (FIGURE 17). This decrease continued over the next six hours, and the level of receptor surface expression as observed in untreated cells was reached 12 hours after removing T0632.

FIGURE 18 shows cAMP production in cells transfected with the GLP-1 receptor mutant "51" or its N-terminally tagged derivative mutant "286" in the absence or presence of various ligands. Receptor-expressing cells were incubated with or without ligand for one hour prior to measuring levels of accumulated cAMP. The enhanced basal activity as observed after tagging the mutant receptor (construct 286) was a prerequisite for demonstrating the inverse agonist properties of T0632 (significantly reduces basal receptor activity).

Because inverse agonists and positive agonists compounds are often structurally related, we have identified non-peptide derivatives of T0632 that function as agonists ofthe GLP-IR. Exemplary non-peptide agonist derivatives of T0632 are provided in FIGURE 19A and U.S.P.N 5,807,883, hereby incorporated by reference.

The above experiments were carried out using the following materials and methods.

Materials ,

Tissue culture medium and fetal bovine serum were purchased from Life Technologies, h e. and from fritergen Company (Purchase, NY). Bolton-Hunter labeled ¹²⁵I Exendin4 (9-39) and ¹²⁵I GLP-1 (both at 2,200 μCi/mmol) were obtained from Perkin Elmer Life Sciences. Non-radioactive Exendin4, Exendin9, and GLP-1 (7-36 amide) were purchased from American Peptide Company, Inc. (Sunnyvale, CA). The truncated peptide Exendin4 (3-39) was provided by the Tufts University Peptide Synthesis Core Facility (Boston, MA), using an automated solid-phase synthesizer (Applied Biosystems, model 431 A) and was purified by HPLC (Waters, model LC4000). The molecular weights of these peptides were verified by linear matrix-assisted laser desorption /ionization mass spectrometry (Perspective Biosystems, model Voyager). All primary (anti T7-tag and anti HA-tag) and secondary (anti mouse TexasRed conjugated and anti mouse peroxidase-conjugated) antibodies were obtained from Santa Cruz Biotechnology, CA.

Receptors

The human GLP-1 receptor cDNA was obtained by polymerase chain reaction based on a published sequence. The construct was sub-cloned into pcDNAl

(Invitrogen, Carlsbad, CA) and analyzed using an Applied Biosystems 373 automated DNA sequencer. The resulting amino acid sequence from the protein-coding region was identical to the published sequence as found in the Swiss-Prot Database (P43220).

Mutagenesis

HA-tag (YPYDVPDYA - tatccatatgacgtaccagactatgca) and T7-tag (MASMTGGQQMG - atggctagcatgacaggaggacagcagatggga) insertion mutants were generated using a slightly altered method of Kunkel's (Bebenek et al, Nucleic Acids Res 17:5408, 1989, and Kunkel et al, Proc. Natl. Acad. Sci. USA 82:488-492, 1985) method for oligonucleotide-directed, site-specific mutagenesis as described previously (Beinborn et al, Nature 362:348-350, 1993). Oligonucleotides were synthesized at the Tufts University DNA Synthesis Core Facility (Boston, MA). Restriction digests and dideoxynucleotide sequencing were used to verify the introduction of desired mutations into the GLP-1 receptor.

Cell Culture and Transfection

COS-7 cells (1*10⁶ cells/10 cm dish) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with heat-inactivated fetal bovine serum (10%), v/v), 26mM sodium bicarbonate, and penicillin G sodium/streptomycin sulfate (100 units/ml and 100 μg/ml, respectively). After an overnight incubation in a humidified atmosphere with 5 % CO₂, cells were transiently transfected using the diethylaminoethyl-dextran method, adding 5 μg cDNA encoding the un-tagged and tagged wild type or mutant GLP-1 receptors, as described previously (Kopin et al, Proc Natl Acad Sci USA 89:3605-3609, 1992).

Radioligand binding assays

One day after rransfection, COS-7 cells containing the wild type or mutant GLP-1 receptors were cultured on 24-well plates (10,000 cells/well or 50,000 cells/well, respectively) and cells were allowed to attach overnight in DMEM. Radioligand binding assays were carried out in Hank's balanced salt solution, supplemented with 25 mM HEPES, pH 7.4, 0.25%> bovine serum albumin and 15 mM phenylmethylsulfonyl fluoride. Competition binding assays were done using 15 pM of I Exendin4 (9-39) in absence or presence of increasing concentrations of unlabeled peptide or non-peptide ligands. Equilibrium binding occurred after incubation of cells at 37°C for 100 minutes. Cell monolayers were washed three times with lml/well Hank's balanced salt solution (see above) and then hydrolyzed in 0.75 ml of IN NaOH for analysis in a Beckman Gamma 5500B counter.

Measurement of cAMP Formation

24 hours after transfection, COS-7 cells containing the wild type and mutant receptors were seeded on 24-well plates (100,000 cells/well) and allowed to attach for an additional 24 hours. Cells were incubated at room temperature for one hour in Dulbecco's modified Eagle's medium supplemented with 1% bovine serum albumin, ImM isobutyl-methylxanthine, 0.4 μM Pro-Boro-Pro, and 25 mM HEPES, pH 7.4. After removal ofthe supernatant, cells were incubated with 0.1N HC1 and lysed by freeze-thawing in liquid nitrogen. After neutralization, cAMP levels were determined by radio-immunoassay (acetylation method) using a FlashPlate^® kit (Perkin Elmer Life Sciences). Plate-bound radioactivity was measured using a Packard Topcount^® proximity scintillation counter. Confocal Microscopy

50,000 COS7 cells were transfected with either T7-tagged wild type or mutant GLP-IR were seeded on cover slips, which were placed into six well plates. After cells adhered, 2 ml of DMEM was added and cells were cultured overnight at 37°C with 5% CO₂. To detect the total amount of receptors (surface plus cytoplasm), cells were washed with 2 ml PBS and fixed using 3.7% formaldehyde in PBS for 20 min at room temperature followed by an incubation with 0.1% TRITON X-100 for 2 min at room temperature to permeabilize cell membranes. After two washes with PBS, samples were incubated with 10%o FBS in PBS for an additional 20 min at room temperature to block nonspecific binding, followed by an overnight incubation at 4°C with the primary antibody: mouse anti-T7 tag (1:10,000). After two washes with 10% FBS in PBS, samples were incubated with a secondary goat anti mouse Texas Red®- conjugated antibody (1 : 1,000) at 4°C overnight. Samples were washed three times with PBS, before mounting on cover slips. For detection of cell surface receptor expression, cells were washed with 10% FBS in PBS - followed by an overnight incubation at 4°C with the primary antibody: mouse anti-T7 tag (1 : 10,000). After two washes with 10%> FBS in PBS, cells were fixed using 3.7%> formaldehyde in PBS for 20 min at room temperature, followed by another washing step with 10%> FBS in PBS. Samples were next incubated with a secondary goat anti mouse Texas Red®- conjugated antibody (1:1000) at 4°C overnight, washed and mounted as described above.

Whole cell ELISA

2 X 10⁴ COS7 cells transfected with either HA-tagged wild type or mutant GLP-IR were seeded in 250 μl DMEM on 96-well plates and cultured overnight at 37°C with 5% CO₂. To estimate the total amount of receptors, cells were washed with 200 μl PBS and fixed with 100 μl 3.7% formaldehyde in PBS for 20 min at room temperature. Cells were next incubated with 0.1 %> TRITON X-100 for 2 min at room temperature to permeabilize cell membranes, and subsequently washed twice with PBS followed by an overnight incubation at 4°C with the primary antibody: mouse anti HA-tag (1:400). To estimate to number of receptors on the surface, cells were washed with 200 μl 10% FBS in PBS, followed by an overnight incubation at 4°C with the primary antibody: mouse anti HA-tag (1 :400). After two washes with 10% FBS in PBS, cells were fixed using 3.7%> formaldehyde in PBS for 20 min at room temperature. Following the incubation with the primary antibody, all wells were washed twice with 10% FBS in PBS and samples were incubated with a secondary goat anti mouse peroxidase-conjugated antibody (1:1,600) at 4°C overnight. Following two washing steps with 10%> FBS in PBS, all wells were washed once with pure PBS. Following the removal of all remaining liquids, 100 μl ofthe peroxidase substrate TMB (BioFX™, Owings Mills, MD) was added and kept in the dark at room temperature for about 15 min. Absorbance at 650 nm was measured using a 96-well plate reader. Values for unspecific binding (only secondary antibody) were subtracted to calculate true receptor expression levels.

Data Analysis

GraphPad Prism software version 3.0 (GraphPad, San Diego, CA) was used to calculate radioligand competition binding and agonist/antagonist concentration- response curves from at least three independent experiments. Statistical comparisons were made by one-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparisons post-tests, using InStat software version 3.0 for Windows (GraphPad).

Other Embodiments

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light ofthe teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope ofthe appended claims.

What is claimed is:

Claims

Claims

1. A pharmaceutical composition comprising a non-peptide agonist ofthe GLP-1 receptor, wherein said agonist increases GLP-1 receptor activity by at least 10%) relative to an untreated control, and wherein said agonist is a compound of formula (I):

(I) or a pharmaceutically acceptable salt thereof, wherein

W is N or CR¹¹;

X is O or S;

Y is CO₂H or SO₃H;

Z is a substituted or unsubstituted C₆ aryl; n is 1-3; each of R¹ - R¹⁰ is, independently, a H, Cι-C₆ alkyl, halogen, NO₂, OH, C_1-6 allcoxy, CO₂H, CO₂R¹³, NR¹⁴R¹⁵, CN, or CF₃, wherein each of R¹³, R¹⁴, and R¹⁵ is H, C_1-6 alkyl, or C_1-4 alkaryl; and each of R and R is, independently, H or Ci-Cβ alkyl, except that said agonist is not a compound of formula:

2. The pharmaceutical composition of claim 1, wherein said agonist is

3. A pharmaceutical composition comprising a non-peptide agonist ofthe GLP-1 receptor, wherein said agonist increases GLP-1 receptor activity by at least 10%) relative to an untreated control, and wherein said agonist is a compound of formula (II):

(II), or a pharmaceutically acceptable salt thereof, wherein

X is O, S, or NR⁸, wherein R⁸ is H or Cι_-6 alkyl; each of R¹ and R² is, independently, a H, substituted or unsubstituted C₆ aryl,

' 1 9

C_1-6 alkyl, C_1-4 alkaryl, or R and R together form a C -C₆ ring, optionally containing non-vicinal O, S, or NR⁹ linkages, wherein R⁹ is H or C_1-6 alkyl; each of R³, R⁴, R⁵, and R⁶ is, independently, H or Cι_-6 alkyl, or R³ and R⁴ together are =O and each of R and R is, independently, H or C_1-6 alkyl, or both of R³ and R⁵ do not exist, a double bond is formed between the carbons bearing R⁴ and R⁶, R⁴ is H or C_1-6 alkyl, and R⁶ is H, Cι-C₆ alkyl, or CH=NOR¹⁰, wherein R¹⁰ is H or Cι-₆ alkyl; and

R⁷ is a H, halogen, or C_1-6 alkyl, except that said agonist is not a compound of formula:

4. The pharmaceutical composition of claim 3, wherein said agonist is selected from the group consisting of:

5. The pharmaceutical composition of claim 4, wherein said agonist is

6. A pharmaceutical composition comprising a non-peptide agonist ofthe GLP-1 receptor, wherein said agonist increases GLP-1 receptor activity by at least 10%) relative to an untreated control, and wherein said agonist is a compound of formula (III):

(III) or a pharmaceutically acceptable salt thereof, wherein

X is O, S, or NR⁹, wherein R⁹ is a H or C_1-6 alkyl;

1 each of R and R is, independently, a H, substituted or unsubstituted C₆ aryl, C1-6 alkyl, C_1-4 alkaryl, or R¹ and R² together form a C₂-C₆ ring, optionally containing non- vicinal O, S, or NR¹⁰ linkages, wherein R¹⁰ is H or C_1-6 allcyl; each of R³, R⁴, R⁵, and R⁶ is, independently, H or C_1-6 alkyl, or R³ and R⁴ together are -O and each of R⁵ and R⁶ is, independently, H or C_1-6 allcyl, or both of R³ and R⁵ do not exist, a double bond is formed between the carbons bearing R⁴ and R⁶, R⁴ is H or C_1-6 allcyl, and R⁶ is H, C C₆ allcyl, or CH=NOR¹⁰, wherein R¹⁰ is H or C_1-6 alkyl; and each of R and R is a H, halogen, or C_1-6 alkyl.

7. The pharmaceutical composition of claim 6, wherein said agonist is:

8. A pharmaceutical composition comprising a non-peptide agonist ofthe GLP-1 receptor, wherein said agonist increases GLP-1 receptor activity by at least 10%) relative to an untreated control, and wherein said agonist is a compound of formula (IV):

γ^N R²

R¹ S X NX R³ (IV), or a pharmaceutically acceptable salt thereof,

wherein

W is CI, F, CF₃, CN, or CO₂R⁴, wherein R⁴ is C_1-6 alkyl;

R¹ is a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or CH₂COR⁵, wherein R⁵ is C_1-6 alkoxy, C_1-4 aryloxy, or NR⁶-aryl, wherein the aryl group is substituted or unsubstituted and R⁶ is H or C_1-6 allcyl; and each of R² and R³ is, independently a H, halogen, C_1-6 alkyl, or C1.6 alkoxy, or

R and R together form a substituted or unsubstituted aryl ring.

9. The pharmaceutical composition of claim 8, wherein said agonist is selected from the group consisting of:

10. The pharmaceutical composition of claim 9, wherein said agonist is:

11. A pharmaceutical composition comprising a non-peptide agonist ofthe GLP- 1 receptor, wherein said agonist increases GLP-1 receptor activity by at least 10% relative to a control, and wherein said agonist is a compound of formula (V):

or a pharmaceutically acceptable salt thereof, wherein

Y is a halogen;

Z is a substituted or unsubstituted aryl; and each of R¹ through R¹⁰ is, independently, a H or Cι_-6 allcyl.

12. The pharmaceutical composition of claim 11, wherein said agonist has the formula:

wherein each of RI and R2 is, independently, a H, Cι-C₆ allcyl, halogen, NO , OH, C_1-6 alkoxy, CO₂H, CO₂R¹³, NR¹⁴R¹⁵, CN, or CF₃, wherein each of R¹³, R¹⁴, and R¹⁵ is, independently, a H, C_1-6 alkyl, or Cι_-4 alkaryl.

13. The pharmaceutical composition of claim 12, wherein said agonist is:

14. The pharmaceutical composition of any one of claims 1,3, 6, 8, or 11, wherein said agonist increases GLP-1 receptor activity by at least 20%.

15. The pharmaceutical composition of any one of claims 1,3, 6, 8, or 11, wherein said GLP-1 receptor activity is cAMP production.

16. A method for treating, reducing, or preventing a metabolic disorder in a mammal, said method comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition of any one of claims 1-15.

17. A method for treating, reducing, or preventing a metabolic disorder in a mammal, said method comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising a non-peptide agonist, wherein said agonist is:

18. The method of claim 16 or 17, wherein said metabolic disorder is diabetes or obesity.

19. The method of claim 18, wherein said diabetes is Type I diabetes, Type II diabetes, or gestational diabetes.

20. The method of claim 16 or 17, wherein said method further comprises administering to said mammal a second therapeutic agent.

21. The method of claim 20, wherein said second therapeutic agent is selected from the group consisting of insulin, nateglinide, metformin, thiazolidinediones, alpha-glucosidase inhibitors, repaglinide, nateglinide, sulfonylurea, miglitol, amaryl, glucotrol, glucotrol XL, micronase, diabeta, Glynase, prandin, Starlix, glucophage, avandia, Actos, precose, and glyset.

22. The method of claim 16 or 17, wherein said mammal further undergoes a therapeutic regimen selected from the group consisting of a low-fat diet, controlled glucose diet, low sodium diet, stress management, physical exercise, reduction in alcohol intake, and reduction in smoking.