Methods for treating diabetes in subjects with severe insulin resistance and diabetes

Abstract

To provide a novel method of treating diabetes in a subject characterized as having severe insulin resistant diabetes.SOLUTION: A method of treating diabetes involves administering a compound of formula I or a pharmaceutically acceptable salt, solvate or prodrug thereof to subjects in need thereof, where the subject is identified as having sever insulin-resistant diabetes.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the use of AMP-activated protein kinase (AMPK) activators in the treatment of diabetes in patients particularly suitable for this treatment. Suitable patients are characterized by increased insulin resistance and high body weight. In particular, this therapy is useful for treating type 2 diabetes in patients with severe insulin resistance. [Background technology]

[0002] Diabetes mellitus (DM) is comprised of two distinct diseases: type 1 (or insulin-dependent diabetes) and type 2 (non-insulin-dependent diabetes), both of which involve dysfunction of blood glucose homeostasis. Type 2 diabetes currently affects more than 400 million people worldwide, and this number is rapidly increasing. Complications of type 2 diabetes include severe cardiovascular problems, renal failure, peripheral neuropathy, blindness, even limb loss, and ultimately death in the later stages of the disease. Type 2 diabetes is characterized by insulin resistance, and there is currently no definitive cure. Most treatments used today focus on treating dysfunctional insulin signaling, inhibiting glucose output from the liver, or inhibiting glucose reabsorption in the kidneys, but many of these therapies have several drawbacks and side effects. While improvements have been made in long-term outcomes, excess mortality and cardiovascular morbidity remain a significant challenge for healthcare systems.

[0003] The current frontline treatment for type 2 diabetes is metformin, a biguanide that lowers plasma glucose primarily by reducing hepatic glucose production. Nevertheless, there remains a need for treatments for subjects with type 2 diabetes who do not achieve glycemic control with metformin.

[0004] Diabetes is currently classified into two main forms, type 1 and type 2 diabetes, although type 2 diabetes in particular is highly variable.

[0005] Existing treatment guidelines are limited to responding to poor metabolic control as it emerges, without any means of predicting which patients will require intensive treatment. Evidence suggests that early treatment is important for preventing life-shortening complications, as target tissues appear to remember poor metabolic control even decades later (Emma Ahlqvist et al., The Lancet Diabetes Endocrinology, Vol. 6, No. 5, p. 361-369, 2018).

[0006] There remains a need to identify better classifications that would identify individuals at high risk of complications at the time of diagnosis and provide a mechanism for such patients to allow for individualized treatment regimens.

[0007] We have now discovered a new treatment that is incredibly effective for patients suffering from severe insulin-resistant diabetes.

[0008] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Summary of the Invention

[0009] According to a first aspect of the present invention there is provided a method of treating diabetes, the method comprising administering to a subject a compound of formula I: [ka] or a pharmaceutically acceptable salt, solvate or prodrug thereof to a subject in need thereof, wherein the subject has been identified as having severe insulin-resistant diabetes.

[0010] The method of the first aspect of the invention is hereinafter referred to as the "method of the invention".

[0011] The subject identified as having severe insulin resistance diabetes represents a subgroup of obese, insulin resistance and hyperinsulinemia diabetic subjects.Compared with other diabetic subjects, these subjects have 5 times higher risk of developing diabetic kidney disease.Currently, there is a lack of effective treatment, and the method of the present invention is particularly suitable for these subjects.

[0012] The pharmaceutically acceptable salt that can be mentioned includes acid addition salt and base addition salt.Such salt can be formed by conventional means, for example, by reacting the free acid or free base form of the compound of formula I with one or more equivalents of suitable acid or base, optionally in a solvent or in a medium in which the salt is insoluble, and then removing the solvent or the medium using standard techniques (for example, by lyophilization or filtration in vacuo).Salt can also be prepared by exchanging the counterion of the compound of formula I in the form of salt with another counterion, for example, by using a suitable ion exchange resin.

[0013] Examples of pharmaceutically acceptable addition salts include those derived from mineral acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid; those derived from organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, arylsulfonic acids, and those derived from metals such as sodium, magnesium, or preferably potassium and calcium.

[0014] The term "prodrug" of a related compound of Formula I includes any compound that, following oral or parenteral administration, is metabolized in vivo to form that compound in experimentally detectable amounts within a given time period (e.g., within a 6-24 hour dosing interval (i.e., 1-4 times daily)). Prodrugs of compounds of Formula I include derivatives that possess or provide the same biological function and / or activity as the related compound. For the avoidance of doubt, the term "parenteral" administration includes all forms of administration other than oral administration.

[0015] Prodrugs of compounds of Formula I can be prepared by modifying functional groups present on the compound so that the modifications are cleaved in vivo when the prodrug is administered to a mammalian subject. The modification is usually achieved by synthesizing the parent compound with a prodrug substituent. Prodrugs include compounds of Formula I in which an amino or carbonyl group in a compound of Formula I is bonded to any group that can be cleaved in vivo to regenerate the free amino or carbonyl group, respectively.

[0016] Examples of prodrugs include, but are not limited to, esters of carboxyl functional groups, N-acyl derivatives, and N-Mannich bases. General information on prodrugs can be found, for example, in Bundegaard, H., "Design of Prodrugs," p. 1-92, Elsevier, New York-Oxford (1985).

[0017] For brevity, compounds of Formula I, and pharmaceutically acceptable salts, solvates and prodrugs of said compounds, are hereinafter collectively referred to as "compounds of Formula I."

[0018] Compounds of formula I may exist as positional isomers and may also exhibit tautomerism, all tautomeric forms and mixtures thereof are included within the scope of the present invention.

[0019] Any individual feature (e.g., a preferred feature) mentioned in this specification can be interpreted alone or in combination with any other feature (including a preferred feature) mentioned in this specification (and thus a preferred feature can be interpreted in combination with or independently of other preferred features).

[0020] The compound of Formula I is a direct, universal (PAN)-AMPK activator that does not enter the brain. In preclinical models of hyperglycemia / diabetes, the compound of Formula I has been shown to increase glucose uptake into skeletal muscle, reduce insulin resistance, and promote beta-cell rest. The compound of Formula I increases energy expenditure and prevents / mitigates obesity. Similar to exercise, the compound of Formula I lowers blood pressure, increases microvascular perfusion, activates cardiac AMPK, increases cardiac glucose uptake, reduces cardiac glycogen levels, and improves left ventricular stroke volume and endurance. Furthermore, the compound of Formula I does not induce cardiac hypertrophy in mice or rats. Importantly, the compound of Formula I therefore exhibits a combination of beneficial metabolic and cardiovascular effects not observed with any other available antidiabetic drugs.

[0021] According to another first aspect of the present invention, there is provided a compound of formula I (defined above), or a pharmaceutically acceptable salt, solvate or prodrug thereof, for use in the treatment of diabetes in a subject identified as having severe insulin-resistant diabetes.

[0022] According to yet another first aspect of the present invention there is provided the use of a compound of formula I (as defined above), or a pharmaceutically acceptable salt, solvate or prodrug thereof, in the manufacture of a medicament for treating diabetes in a subject identified as having severe insulin-resistant diabetes.

[0023] Diabetes is often associated with a variety of symptoms, including polyphagia, polydipsia, polyuria, kidney damage, neurological damage, cardiovascular damage, retinal damage, lower extremity damage, fatigue, restlessness, weight loss, poor wound healing, dry or itchy skin, erectile dysfunction, cardiac arrhythmias, coma, and seizures.

[0024] The terms "treat," "treating," or "treatment" (and grammatical variations thereof) mean that the severity of a subject's condition is lessened or at least partially improved, and / or at least one clinical symptom is alleviated, alleviated, or diminished to some extent, and / or the progression of a disease or disorder is slowed. In this regard, these terms can refer to at least a partial decrease in the severity of at least one of a subject's clinical symptoms and / or a decrease in the duration of at least one of said symptoms. The terms "treat," "treating," and "treatment of" can also refer to achieving a reduction in blood glucose levels (e.g., to about 10.0 mmol / mL or less (e.g., to a level ranging from about 4.0 mmol / L to about 10.0 mmol / L), e.g., to about 7.5 mmol / mL or less (e.g., to a level ranging from about 4.0 mmol / L to about 7.5 mmol / L), or to about 6 mmol / mL or less (e.g., to a level ranging from about 4.0 mmol / L to about 6.0 mmol / L)). In certain embodiments, in the case of type 2 diabetes, the terms can refer to achieving a reduction in blood glucose levels.

[0025] A "subject in need" of the method of the invention includes a subject suffering from diabetes, particularly type 2 diabetes. Thus, in one embodiment, the method of the invention is a method of treating type 2 diabetes.

[0026] As used herein, a "therapeutically effective amount," "effective amount," or "dosage" refers to that amount of a compound, composition, and / or formulation sufficient to produce a desired effect, which may be a therapeutic and / or beneficial effect. The effective amount or dosage will vary depending on the age or general condition of the subject, the severity of the condition being treated, the particular agent being administered, the duration of treatment, the nature of any concurrent treatments, the pharmaceutically acceptable carrier used, and similar factors within the knowledge and professional judgment of one of ordinary skill in the art. Where appropriate, a "therapeutically effective amount," "effective amount," or "dosage" in any individual case can be determined by one of ordinary skill in the art by reference to relevant texts and literature and / or by conducting routine experimentation. Those skilled in the art will understand that the therapeutic effect need not be complete or curative, so long as some benefit is provided to the subject.

[0027] As used herein, the terms "disease" and "disorder" (and similarly terms condition, illness, medical problem, etc.) may be used interchangeably.

[0028] A distinct population of diabetic patients, defined as those with "severe insulin-resistant diabetes" (SIRD), lacks effective therapeutic options. The inventors have discovered that the clinical and pharmacokinetic effects observed with the compound of formula I are particularly suited to the therapeutic needs of diabetic patients with severe insulin-resistant diabetes. By administering treatment using the compound of formula I to these patients, significant clinical benefits can be realized, including reduced organ morbidity and increased survival.

[0029] The term "insulin resistance" refers to subjects with normal, or possibly increased, insulin production but significantly reduced insulin sensitivity. Subjects may be classified as having severe insulin-resistant diabetes according to the criteria set forth in Emma Ahlqvist et al., The Lancet Diabetes Endocrinology, Vol. 6, No. 5, p. 361-369, 2018. In the study described therein, subjects were assessed for six variables (glutamic acid decarboxylase antibodies, age at diagnosis, BMI (body mass index), HbA 1c Subjects were grouped based on their blood pressure (P < 0.05, P < 0.05, and two estimates of β-cell function and insulin resistance from the homeostatic model assessment) and correlated with predictive data from patient records regarding the development of complications and medication prescriptions. Subjects with severe insulin-resistant diabetes were typically those with a blood pressure of at least 30 kg / m 2 , especially at least 35 kg / m 2 Subjects also had an HbA of at least 52 mmol / mol. 1c Additionally, the subject may have a C-peptide level above the reference range at a particular test site. Determination of each of these clinical parameters can be readily accomplished using routine methods known to those skilled in the art.

[0030] It has been found that treatment with a compound of formula I can reduce body weight, improve insulin resistance, and treat hyperglycemia in mice. Administration of the compound of the present invention to diet-induced obese mice increased glucose uptake in skeletal muscle, reduced beta cell stress, and promoted beta cell resting.

[0031] Administration of this compound to humans has also been shown to have a favorable effect on glomerular microvascular perfusion. This compound reduced fasting blood glucose levels and homeostatic model assessment of insulin resistance (HOMA-IR) in a Phase IIa clinical trial in patients with type 2 diabetes (T2D) treated with metformin. This compound also improved peripheral microvascular perfusion and reduced blood pressure in both animals and patients with type 2 diabetes. Therefore, administration of the compound of Formula I, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, is expected to have beneficial effects in subjects suffering from diabetes, particularly severe insulin-resistant diabetes.

[0032] These effects may include a reduction in the subject's weight. For example, the subject's weight may be reduced to the point where the subject is no longer considered obese. A patient may have a BMI of 30 kg / m 2 If you are overweight or obese, you may be considered obese. This is defined as a BMI of 30-35 kg / m according to the WHO classification system. 2 At higher BMI levels, patients are considered severely obese (obesity class II; 35–40 kg / m²). 2 BMI), very severe obesity (obesity class III; 40–45 kg / m 2 BMI), morbid obesity (obesity class IV; 45-50 kg / m 2 Such patients may be classified as obese (BMI of 25-30 kg / m), or even more highly obese. Thus, treatment of such patients using the methods of the present invention may result in the patient being classified into a lower weight category, and may even result in the patient's BMI being reduced below the obesity threshold. 2 Patients with diabetes mellitus who are typically classified as overweight and may not be classified as having severe insulin resistance may benefit from the treatment methods described herein. In certain embodiments of the present invention, the subject loses weight.

[0033] Therefore, the method of the present invention is particularly suitable for treating obese subjects. In the context of the present invention, unless otherwise specified, the term "obese" includes subjects classified as obese class I or higher according to the WHO classification system. Thus, in an embodiment, the method is for treating obese subjects with a body weight of at least 30 kg / m 2 In another embodiment of the invention, the subject's body mass index is reduced, for example, so that the patient is classified into a less severe obesity class or is not obese at all.

[0034] As used herein, reference to a subject (or subjects) refers to a living subject being treated or receiving a prophylactic medication, including a mammalian (e.g., human) subject. In particular, reference to a subject refers to a human subject.

[0035] The method of the present invention can produce other beneficial effects in the treated subject.For example, the compound of the present invention has been shown to have a favorable effect on the renal hemodynamics of patients suffering from type 2 diabetes.The compound can cause a rapid, stable, and reversible decrease in the patient's estimated glomerular filtration rate (eGFR), which is consistent with a decrease in intraglomerular pressure.This indicates an early hemodynamic effect.Therefore, the method of the present invention can improve the renal hemodynamics of the subject.More specifically, the method of the present invention can cause a decrease in intraglomerular pressure, for example, a clinically therapeutic decrease, which can be determined through a decrease in the estimated glomerular filtration rate (eGFR) in the subject.

[0036] Other clinical benefits include reduced organ morbidity and increased chances of survival beyond the estimated time after diagnosis.

[0037] Subjects may be classified as having severe insulin-resistant diabetes, as described above. Particular subjects who may be particularly efficacious in treatment with the methods of the invention include those with an HbA of at least 52 mmol / mol. 1c This value can be determined using routine methods known in the art.

[0038] Other specific subjects that may be mentioned include those with C-peptide levels above the reference range for a particular test site. C-peptide is a short 31-amino acid polypeptide that connects the A chain of insulin to the B chain in the proinsulin molecule. In diabetes, measuring serum C-peptide levels can be used to distinguish between diseases with similar clinical features. Reference ranges may vary depending on the patient and recent activity, such as recent food intake. For example, C-peptide measurements in healthy individuals after fasting may range from 0.13 to 0.70 nmol / L. Specific elevated values ​​that may be mentioned for subjects with severe insulin-resistant diabetes include blood C-peptide concentrations of 1.4 nmol / L or higher, more specifically 1.5 nmol / L or higher.

[0039] Subjects characterized by severe insulin-resistant diabetes may be at high risk for developing diabetic kidney disease or may be diagnosed with diabetic nephropathy. They may also suffer from or be at high risk for cardiovascular disease. The methods of the present invention are believed to provide at least some organ-protective benefits to patients, particularly those described herein. As a result, treating subjects characterized by severe insulin-resistant diabetes using the methods of the present invention may result in a reduced prevalence of diabetic kidney disease during and / or after treatment. Therefore, it is preferred that the subjects treated have a high risk of susceptibility to diabetic kidney disease. Subjects already suffering from diabetic kidney disease may also benefit from the treatment methods of the present invention, for example, by reducing the rate at which the severity of diabetic kidney disease increases. In one embodiment of the present invention, the subjects treated have diabetic kidney disease.

[0040] Treatment of subjects suffering from severe insulin-resistant diabetes requires that the subject first be identified as having the condition. Some of the clinical parameters necessary for diagnosis are described elsewhere herein and in Emma Ahlqvist et al., The Lancet Diabetes Endocrinology, Vol. 6, No. 5, p361-369, 2018. Accordingly, the present invention also relates to a method for treating diabetes, the method comprising: (i) identifying a subject suffering from severe insulin-resistant diabetes; and (ii) administering to the subject a compound of Formula I (as defined elsewhere herein), or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0041] Step (i) involves a clinical evaluation of the subject, which includes an assessment of at least one of the physiological and pathological aspects described above for these subjects. A subject is considered to have severe insulin-resistant diabetes if they meet the criteria set out in Ahlqvist et al., supra. This may include, for example, one or preferably all of the following: 2 BMI of 52mmol / mol or more, HbA 1c levels, and C-peptide levels above the reference range at specific testing locations.

[0042] Step (ii) can be carried out using any suitable administration route, formulation, and administration schedule, including those described elsewhere herein. The treatment may, for example, result in the suppression of the development of systemic insulin resistance in the subject. The treatment may also result in the induction of weight loss and body fat loss, without possibly reducing the subject's food intake. Other clinical effects that may result from the treatment will be apparent from the examples.

[0043] The compound of formula I is also known as 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide.

[0044] Where a compound can exist as tautomers, the depicted structure represents one of the possible tautomeric forms, and the actual tautomer observed may vary depending on environmental factors such as solvent, temperature, or pH.

[0045] The compounds of Formula I, as well as their pharmaceutically acceptable salts, solvates, and prodrugs, can be prepared according to techniques well known to those skilled in the art, for example, as described below. For example, 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide can be prepared according to the techniques described in International Patent Application WO2011 / 004162, the entire contents of which are incorporated herein by reference.

[0046] Therefore, the compound of the present invention can be administered to a subject in any form that promotes the reduction of both fasting blood glucose level and insulin resistance (for example, by homeostatic model assessment of insulin resistance).In particular, the compound of the present invention can be administered orally, intravenously, intramuscularly, skin, subcutaneously, transmucosally (for example, sublingually or buccally), rectally, transdermally, nasally, pulmonary (for example, trachea or bronchus), topically, or by any other parenteral route, in the form of a pharmaceutical preparation containing the compound in a pharmaceutically acceptable dosage form.In certain embodiments, the compound of Formula I or its pharmaceutically acceptable salt, solvate or prodrug is administered orally, intranasally, parenterally or by inhalation.Preferably, administration is carried out orally.

[0047] The compounds of the present invention are generally administered as pharmaceutical preparations in admixture with pharmaceutically acceptable adjuvants, diluents, or carriers, which may be selected with due consideration of the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no adverse side effects or toxicity under the conditions of use. Suitable pharmaceutical preparations can be found, for example, in Remington, The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, parenterally acceptable aqueous solutions that are pyrogen-free and have the necessary pH, isotonicity, and stability can be used. Suitable solutions will be well known to those skilled in the art, as many methods have been described in the literature. A brief review of drug delivery methods may also be found, for example, in Langer, Science, 249, 1527 (1990).

[0048] In the methods of the invention described herein, the pharmaceutically active compounds can be administered in known formulations, including tablets, capsules, or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for parenteral or intramuscular administration, or via inhalation, etc. Administration by inhalation is preferably carried out using a nebulizer, for example, to deliver the compounds of the invention to small lung tissue, including the alveoli and bronchioles, preferably without causing irritation or coughing in the treated subject.

[0049] The preparation of other suitable formulations can be achieved without inventiveness by those skilled in the art using routine techniques and / or in accordance with standard and / or accepted pharmaceutical practice.

[0050] The amount of the compound of the present invention to be administered to a subject may vary depending on the condition to be treated or prevented, the severity of the condition, the subject, the route of administration, and the compound used, but can be determined by one skilled in the art without any inventive step. The compound of the present invention can be administered to patients in need thereof in various therapeutically effective doses.

[0051] Doses will vary from patient to patient, but a suitable daily dose will be in the range of about 0.1 to about 5000 mg per patient (e.g., 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mg, 1250 mg, 1500 mg, 1750 mg, 2000 mg, 2500 mg, 3000 mg, 3500 mg, 4000 mg, 4500 mg, 5000 mg, etc., or any range or value therein), administered in single or multiple doses. Administration may be continuous or intermittent (e.g., by bolus injection). The dosage may also be determined by the timing and frequency of administration. For oral or parenteral administration, the dosage preferably varies from about 1 mg to about 2000 mg per day of the compound of the present invention (or, if used, a corresponding amount of a pharmaceutically acceptable salt or prodrug thereof). In certain embodiments, the compound of Formula I, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, is administered to a subject in a daily dose ranging from about 1 to about 2000 mg.

[0052] The term "about" as used herein when referring to a measurable value such as an amount of a compound, a dose, a time, a temperature, etc., refers to a variation of 20%, 10%, 5%, 1%, 0.5%, or even as little as 0.1% of the specified amount.

[0053] In any case, the dosage administered to a mammal, particularly a human, in the context of the present invention should be sufficient to produce a therapeutic response in the mammal over a reasonable time frame.Those skilled in the art will recognize that the selection of the exact dosage and composition and the most appropriate delivery regimen will also be influenced by, among other things, the pharmacological properties of the formulation, the nature and severity of the condition to be treated, and the physical condition and mental acuity of the person receiving the medication, as well as the potency of the specific compound, the age, condition, weight, sex and response of the patient to be treated, and the stage / severity of the disease.

[0054] A practitioner or other skilled artisan will be able to routinely determine the actual dosage which will be most suitable for an individual patient. The dosages set forth above are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

[0055] Additionally, in some embodiments, the compounds of the invention are used in combination with one or more other therapeutic agents, or pharmaceutically acceptable salts, solvates or prodrugs thereof, to manufacture a medicament for the above uses (e.g., for the treatment of type 2 diabetes).

[0056] Certain other therapeutic agents that may be mentioned in this regard include sodium-glucose transport protein 2 (SGLT2) inhibitors. Those skilled in the art will understand that a sodium-glucose transport protein 2 inhibitor is a substance or agent that inhibits the activity of sodium-glucose transport protein 2. Thus, according to a second aspect of the present invention, there is provided the following combination: (A) a compound of Formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof), and (B) a sodium-glucose transport protein 2 (SGLT2) inhibitor (or a pharmaceutically acceptable salt, solvate, or prodrug thereof). Such combinations are referred to herein as "combinations of the invention."

[0057] The phrase "inhibiting the activity of sodium-glucose transport protein 2" means that a substance or agent induces a decrease in one or more functions of sodium-glucose transport protein 2, including stopping one or more functions or slowing down the rate of a particular function of sodium-glucose transport protein 2 by decreasing the function of sodium-glucose transport protein 2. A particular function that may be completely or partially inhibited is the ability of sodium-glucose transport protein 2 to act as a glucose transporter.

[0058] An SGLT2 inhibitor is a substance or drug that selectively inhibits the activity of SGLT2. By selectively inhibiting the activity of SGLT2, it is meant that the SGLT2 inhibitor selectively stops or slows down one or more functions of SGLT2 in preference to one or more functions of sodium-glucose transport protein 1 (SGLT1). For example, the level of selectivity for SGLT2 over SGLTIs may range from about 2:1 to 5000:1. For example, an SGLT2 inhibitor may have a selectivity for SGLT2 versus SGLT1 of about 10:1, about 50:1, about 100:1, about 250:1, about 500:1, about 1000:1, about 5000:1, or greater than about 5000:1. Thus, in certain embodiments, an SGLT2 inhibitor is a selective SGLT2 inhibitor. Those skilled in the art will recognize standard tests that can be performed to enable them to determine whether a substance or drug acts as a sodium-glucose transport protein 2 inhibitor.

[0059] In a preferred embodiment, the sodium-glucose transport protein 2 inhibitor is a so-called "small molecule" having a molecular weight of less than 900 Daltons (Da). Such molecules may also be called "drug-like" molecules. In a particular embodiment, the sodium-glucose transport protein 2 inhibitor present in the combination of the present invention is gliflozin. Gliflozins are a known class of small molecule sodium-glucose transport protein 2 inhibitors. Hawley et al. (Diabetes, 2016, 65, 2784-2794) and Villani et al. (Molecular Metabolism, 2016, 5, 1048-1056) recently discussed the possible mechanism of action of certain gliflozins. By inhibiting sodium-glucose transport protein 2, gliflozins reduce the extent of renal glucose reabsorption from glomerular filtrate (i.e., renal glucose reabsorption), which in turn reduces blood glucose concentrations. Any compound capable of inhibiting sodium-glucose transport protein 2 may be effective in the combination of the present invention.

[0060] Particular sodium-glucose transport protein 2 inhibitors (of the gliflozin class) that may be present in the combinations of the invention include, but are not limited to, canagliflozin, dapagliflozin, empagliflozin, ipragliflozin, tofogliflozin, sergliflozin (such as sergliflozin etabonate), remogliflozin (such as remogliflozin etabonate), ertugliflozin and sotagliflozin, and pharmaceutically acceptable salts, solvates and prodrugs thereof. In a preferred embodiment of the invention, the sodium-glucose transport protein 2 inhibitor present in the combination of the invention is canagliflozin [also known as (1S)-1,5-anhydro-1-C-(3-{[5-(4-fluorophenyl)thiophen-2-yl]methyl]}-4-methylphenyl)-D-glucitol], or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0061] The combination of the present invention may be particularly useful for treating diabetes in a subject characterized by having severe insulin-resistant diabetes. Accordingly, in a third aspect of the present invention, there is provided a method of treating diabetes in a subject characterized by having severe insulin-resistant diabetes, the method comprising administering to a subject in need thereof a combination of the present invention as defined herein.

[0062] Similarly, there is provided the use of a combination of the invention as defined herein in the manufacture of a medicament for treating diabetes in a subject characterised as having severe insulin-resistant diabetes.

[0063] Components (A) and (B) of the combination of the present invention (i.e., the compound of formula I and the SGLT2 inhibitor) can be provided as separate formulations or as a combined preparation (i.e., a single formulation containing the compound of formula I and the SGLT2 inhibitor). The compound of formula I and the SGLT2 inhibitor can be administered simultaneously or sufficiently close in time (optionally repeatedly) to allow a beneficial effect in the subject. Preferably, the beneficial effect is greater than that achievable by use of a formulation containing the compound of formula I or a formulation containing the SGLT2 inhibitor over part or the entire course of treatment, or is a beneficial effect that is not observed when treatment involves use of one but not both of the two main ingredients. Assessment of the beneficial effect of the combination of the present invention over the course of treatment depends on the condition being treated or prevented but can be accomplished by routine methods by one of skill in the art.

[0064] For example, those skilled in the art will recognize that components (A) and (B) of the combination of the present invention can be administered sequentially, separately, and / or simultaneously over the course of treatment of the relevant condition. Administration in this manner may be necessary when the two active agents have different pharmacokinetic profiles. For example, the administration frequency of one component of the combination may need to be varied independently of the administration frequency of the other component. Thus, in certain embodiments of the present invention, a compound of Formula I and a sodium-glucose transport protein 2 (SGLT2) inhibitor are administered sequentially, separately, and / or simultaneously to a subject in need thereof.

[0065] The compound of Formula I can be administered to a subject who has been treated (or will be treated) with a sodium-glucose transport protein 2 (SGLT2) inhibitor for the purpose of treating diabetes. Accordingly, in another aspect of the invention, there is provided the use of a compound of Formula I, or a pharmaceutically acceptable salt, solvate or prodrug thereof, in the manufacture of a medicament for the treatment of diabetes in a subject identified as having severe insulin-resistant diabetes, wherein the medicament is administered to a subject also being treated with a sodium-glucose transport protein 2 (SGLT2) inhibitor (e.g., as defined elsewhere herein), or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0066] Similarly, a sodium-glucose transport protein 2 (SGLT2) inhibitor can be administered to a subject who has also been treated (or will be treated) with a compound of Formula I for the purposes of treating diabetes. Accordingly, in another aspect of the present invention, there is provided the use of a sodium-glucose transport protein 2 (SGLT2) inhibitor (e.g., as defined elsewhere herein), or a pharmaceutically acceptable salt, solvate or prodrug thereof, in the manufacture of a medicament for the treatment of diabetes in a subject identified as having severe insulin-resistant diabetes, wherein the medicament is administered to a subject who has also been treated with a compound of Formula I, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0067] Sodium-glucose transport protein 2 inhibitors, such as gliflozin, and their pharmaceutically acceptable salts, solvates, and prodrugs can be prepared according to techniques well known to those skilled in the art, for example, as described below. For example, canagliflozin can be prepared according to the techniques described in International Patent Application No. WO2005 / 012326. For the avoidance of doubt, references to canagliflozin herein include canagliflozin hemihydrate, sold under the trade name Invokana®. Canagliflozin and other SGLT2 inhibitors can be administered at levels according to dosages known and generally accepted in the art.

[0068] The amount of the compound of formula I present in the combination of the present invention may be the same as or different from the amount of the existing SGLT2 inhibitor. For example, the weight ratio of the compound of formula I to the SGLT2 inhibitor present in the combination of the present invention may be about 1:1000 to about 1000:1, such as about 1:100 to 10:1 (e.g., about 1:10 to about 1:1). Specific weight ratios of the compound of formula I to the SGLT2 inhibitor that may be mentioned include 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, and 1:10.

[0069] The compound of formula I has been shown to treat overweight, insulin resistance, and hyperglycemia in mice, and has been shown to have a favorable effect on microvascular perfusion in human glomeruli.A distinct and undertreated group of human patients has been identified, characterized by severe insulin-resistant diabetes.This group is usually obese, insulin-resistant, hyperglycemic, and at high risk of diabetic kidney disease.The advantages of using the compound of formula I to treat diabetes in this group include the fact that this currently undertreated patient group can receive significantly improved therapeutic attention by targeting the treatment in this way.

[0070] The methods of the invention disclosed herein may also have the advantage that methods involving compounds of Formula I may be more effective, less toxic, longer acting, more potent, produce fewer side effects, and / or possess other useful pharmacological, physical, or chemical properties compared to methods known in the prior art as being useful for treating diabetes in such subjects. Such effects may be assessed clinically, objectively, and / or subjectively by a medical professional, the subject being treated, or an observer.

[0071] The data summarized herein for compounds of the present invention demonstrate that test compounds of Formula I effectively treat overweight, insulin resistance and hyperglycemia and have a favorable effect on microvascular perfusion in the glomerulus.

[0072] All patents, patent applications, and publications referenced herein are incorporated by reference in their entirety. In the event of a conflict of terminology, the present specification will control. Furthermore, embodiments described in one aspect of the invention are not limited to the described aspect. An embodiment may also be applied to other aspects of the invention as long as the embodiment does not prevent those aspects from operating for their intended purpose. [Brief explanation of the drawings]

[0073] The following drawings are provided to illustrate various aspects of the inventive concept and are not intended to limit the scope of the invention unless otherwise specified herein. [Figure 1](A-F). Test substance ("O304") increases p-T172 AMPK in vitro and increases p-T172 AMPK and ATP in cells. (A and B) Representative immunoblot analysis and quantification of test substance dose-dependent inhibition of PP2C-mediated dephosphorylation of p-T172 AMPK in the absence (A) (n = 8 per condition) and in the presence (B) of 1.0 mM ATP (n = 4 per condition). (C-E) Representative immunoblot analysis (C) and quantification of test substance dose-dependent increases in p-T172 AMPK and p-S79 ACC phosphorylation (n = 11 per condition) in Wi-38 human lung fibroblasts (p-T172 AMPK (D), p-S79 ACC (E)). (F) Dose-dependent increases in ATP / protein levels in Wi-38 human lung fibroblasts treated with test substance (n = 6 per condition). Data are expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<(Student's t test). [Figure 2](A-I). Test substance ("O304") prevents hyperglycemia and insulin resistance in diet-induced obese mice. (A) Weekly schedule of B6 mice fed a high-fat diet (HFD) and orally gavaged with vehicle or test substance ± metformin. (B and C) Fasting glucose (B) and fasting insulin (C) in HFD-fed B6 mice treated with vehicle (n=10), test substance (n=10), metformin (n=10), and test substance + metformin (n=10) for 6 weeks. (D) Homeostasis model assessment-insulin resistance (HOMA-IR) calculations from B and C. (E) Representative immunoblot analysis and quantification of p-T172AMPK levels in calf muscle of HFD-fed B6 mice treated with vehicle (n = 10), test substance (n = 10), metformin (n = 10), and test substance plus metformin (n = 10) for 8 weeks. (F) Relative mRNA levels of Txnip and Glut1 in calf muscle of HFD-fed B6 mice treated with vehicle (n = 10), test substance (n = 10), metformin (n = 9), and test substance plus metformin (n = 10) for 8 weeks. (G and H) Fasting blood glucose (G) and insulin (H) levels in B6 mice fed either a normal diet (RD) (n = 40) or an HFD for 7 weeks (= start; n = 10 + 10). Next, HFD-fed mice were continued on HFD for an additional 4 weeks and orally gavaged with vehicle (n = 10) or test substance + metformin (n = 10). (I) HOMA-IR calculations from G and H. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***p < 0.001 (Student's t-test). [Figure 3](A-H). Test substance ("O304") prevents diabetes in hIAPPtg diet-induced obese mice. (A) Weekly schedule of hIAPPtg mice fed a high-fat diet (HFD) and orally gavaged with vehicle or test substance. (B and C) Fasting blood glucose (B) and insulin levels (C) in HFD-fed hIAPPtg mice treated with vehicle (n=25) and test substance (n=27) for 6 weeks. (D and E) Blood glucose, plasma insulin profile, and AUC during an intraperitoneal (ip) glucose tolerance test (IPGTT) (D) and an oral glucose tolerance test (OGTT) (E) in HFD-fed hIAPPtg mice treated with vehicle (IPGTT, n=13; OGTT, n=7) and test substance (IPGTT, n=16; OGTT, n=7) for 6 weeks. (F) Plasma insulin profile and AUC during IPGTT of HFD-fed 16-week-old hIAPPtg mice treated with test substances for 6 weeks (from D, n=16) compared with 10-week-old hIAPPtg mice fed a normal diet (RD) (n=7). (G) HOMA-IR calculation from glucose and insulin levels in B and C. (H) Matsuda index calculation from IPGTT (D) and OGTT (E) in hIAPPtg mice treated with vehicle and test substances. Data are presented as mean ± SEM, *P<0.05, **P<0.01 (Student's t-test). [Figure 4](A-M). Test substance ("O304") dose-dependently prevents hyperglycemia in diet-induced obese mice and reverses diabetes in hIAPPtg diet-induced obese mice. (A) Representative immunoblot analysis and quantification of p-T172AMPK levels in calf muscle of CBA mice fed a high-fat diet (HFD) (n=10) and a test substance-HFD containing 0.4 (n=5), 0.8 (n=10), and 2 mg / g (n=10) of test substance for 7 weeks. (B-D) Fasting blood glucose (B) and insulin (C), as well as HOMA-IR (D; from B and C) in CBA mice fed a HFD (n=10) and a test substance-HFD containing 0.4 (n=5), 0.8 (n=10), and 2 mg / g (n=10) of test substance for 6 weeks. (E) Weekly schedule of hIAPPtg mice fed a HFD for 9 weeks, followed by either continuing the HFD for an additional 7 weeks or switching to a test substance-HFD (2 mg / g in FH, 0.8 mg / g in IM). (FH) Fasting blood glucose (F) and insulin (G) levels, as well as HOMA-IR (H; from F and G), were measured for hIAPPtg mice (n = 10) at baseline, 9, and 15 weeks on HFD, and for hIAPPtg mice (n = 12) at baseline, 9 weeks on HFD, and 9 weeks on HFD + 6 weeks on test substance-HFD (2 mg / g). (I and J) Changes in body weight (I) and body fat (J) were measured for hIAPPtg mice on a 15-week HFD (n = 12) or 9 weeks on HFD + 6 weeks on test substance-HFD (0.8 mg / g) (n = 7). (KM) Fasting blood glucose (K) and insulin (L) levels, and HOMA-IR (M; from K and L) in hIAPPtg mice fed a 15-week HFD (n=12) at baseline, 9 weeks, and 15 weeks, and at baseline, 9 weeks on HFD, and 9 weeks + 6 weeks on test substance-HFD (0.8 mg / g) (n=7). Data are presented as mean ± SEM, **P<0.01, **P<0.01, ***P<0.001 (Student's t-test [AD, I, and J]; paired two-tailed t-test). [Figure 5](A-E). Test substance ("O304") increases glucose uptake into skeletal muscle. (A) 2-deoxy-D-glucose (2-DG) uptake in rat skeletal L6 myotubes treated with the test substance, as indicated. (Vehicle, n=8; 2.5 μM test substance, n=6; 5.0 μM test substance, n=6; and 10.0 μM test substance, n=3). (B-D) Representative immunoblot analysis, as indicated. (B) Quantification of AMPK expression (C) (n=6) and 2-DG glucose uptake in siRNA-transfected rat skeletal L6 myotubes treated with the test substance (n=8 for each condition). (D) (E) Fluorodeoxyglucose (-FDG) levels in calf and thigh muscles of CBA mice fed a high-fat diet (HFD) (n=8) or a test substance-HFD (2 mg / g) (n=6) for 2 weeks. Data are expressed as mean ± SEM, *P<0.05, ***P<0.001 (Student's t-test). [Figure 6](A-H). Test substance ("O304") reduces amyloid formation and improves arginine-induced insulin secretion in hIAPPtg diet-induced obese mice. Representative images (A) and quantification (B) of Thio-S amyloid deposits in hIAPPtg mice (n=9) fed a high-fat diet (HFD) for 16 weeks and hIAPPtg mice (n=9) fed an HFD for 9 weeks and switched to a test substance-HFD (2 mg / g) for an additional 7 weeks. (C) Representative immunoblot analysis and quantification of test substance stimulation of p-T172AMPK in INS-1 insulinoma cells (vehicle, 2.5 μM, and 5 μM test substance, n = 9; 10 μM test substance, n = 6, respectively, per condition), mouse primary pancreatic islets (n = 6 per condition), hIAPPtg mouse primary pancreatic islets (n = 6 per condition), and human pancreatic islets (n = 8 per condition). (D and E) Representative images (D) and quantification (E) of Thio-S+ amyloid deposits in hIAPPtg islets cultured ex vivo for 96 hours in the presence of 11 mM glucose (n=36 islets), 22 mM glucose (n=45 islets), 22 mM glucose and 2.5 μM (n=42 islets), 5.0 μM (n=41 islets), and 10 μM test substance (n=36 islets), as indicated (n=3 experiments for each). (F and G) Representative images (F) and quantification (G) of Thio-S amyloid deposits in hIAPPtg islets cultured ex vivo for 96 h in 11 mM glucose (n = 43 islets), 11 mM glucose with 5.0 μM 3-MA (n = 59 islets), 22 mM glucose (n = 54 islets), 22 mM glucose with 5.0 μM test substance (35 islets), and 22 mM glucose with 5.0 μM test substance and 5.0 μM 3-MA (n = 44 islets), as indicated (n = 3 experiments each). (H) Plasma insulin profile and AUC following intraperitoneal injection of arginine (1 g / kg) into CBA mice fed a HFD (n = 10) and a test substance-HFD (0.8 mg / g) (n = 10) for 11 weeks. Data are expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). [Figure 7](A-H). Test substance ("O304") reverses established obesity under thermoneutral conditions. (A and B) Body weight change (A) and food intake (B) over time in CBA mice fed a high-fat diet (HFD) (n = 5) with test substance-HFD (2 mg / g) (n = 5) under thermoneutral conditions, as indicated. (C-E) Oxygen consumption (VO2) (C), respiratory exchange ratio (RER) (D), and energy expenditure (EE) rate (E) in CBA mice fed a HFD (n = 8) and test substance-HFD (0.8 mg / g) (n = 8) for 11 weeks. (F) Representative immunoblot analysis and quantification of ATGL and p-S406ATGL in inguinal white adipose tissue (iWAT) of CBA mice fed a HFD (n = 10) and a test substance-HFD (0.4 (n = 5), 0.8 (n = 10), and 2 mg / g (n-10)) for 7 weeks. (G) Relative mRNA levels of Atgl, Cpt1b, Ppargc1a, and Cox8b in 19-week-old CBA mice fed a HFD (n = 10) and a test substance-HFD containing 2 mg / g test substance (n = 10) for 7 weeks. (H) Relative mRNA levels of Cd36, Fas, Scd1, Acc1, and Cpt1b in brown adipose tissue (BAT) from 19-week-old CBA mice fed a HFD (n = 10) and a test substance-HFD containing 2 mg / g test substance (n = 10) for 7 weeks. Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***p < 0.001 (Student's t-test). [Figure 8](A-G). Test substance ("O304") reduces cardiac glycogen and improves stroke volume in diet-induced obese mice, but does not cause cardiac hypertrophy. (A) Cardiac glycogen content in CBA mice fed a high-fat diet (HFD) (n=10) and a test substance-HFD containing 0.8 (n=10) and 2 mg / g (n=10) of test substance for 7 weeks. (B) Fluorodeoxyglucose (-FDG) levels in the hearts of CBA mice fed an HFD (n=8) or a test substance-HFD (2 mg / g) (n=6) for 2 weeks. (C) Heart weight in CBA mice fed an HFD (n=10) and a test substance-HFD containing 0.8 (n=10) and 2 mg / g (n=10) of test substance for 7 weeks. (D-F) End-diastolic volume (EDV) (D), end-systolic volume (ESV) (E), and stroke volume (SV) (F) of 16-week-old CBA mice (n = 9) fed a normal diet (RD) and 18-week-old CBA mice fed a HFD (n = 9) and a test substance-HFD containing 0.8 mg / g (n = 10) or 2 mg / g (n = 10) of the test substance for 6 weeks. (G) Heart rate (HR) of 16-week-old CBA mice (n = 9) fed a normal diet (RD) and 18-week-old CBA mice fed a HFD (n = 9) and a test substance-HFD containing 0.8 mg / g (n = 10) or 2 mg / g (n = 10) of the test substance for 6 weeks. Data are expressed as mean ± SEM, *P<0.05, **P<0.01, ***p<0.001 (Student's t-test). [Figure 9](AF). Test substance ("O304") improves microvascular blood flow and endurance in mice. (A and B) Representative laser Doppler images (A) and quantification (B) of peripheral blood perfusion in the left hind paw of vehicle-treated (n=10) and test substance-treated (n=10) B6CBAF1 / J(F1) mice fed a high-fat diet (HFD) for 8 weeks. (C and D) Endurance test (C) and lactate levels (D) of vehicle-treated (n=14) and test substance-treated (n=14) aged lean B6 mice 30 days after test substance treatment. (E and F) Systolic (E) and diastolic (F) blood pressure in dogs receiving a single dose of vehicle or test substance at the indicated concentrations. Data are expressed as mean ± SEM, *P<0.05, #P<0.05, **P<0.01, ##P<0.01, ***P<0.001, ###P<0.001 (Student's t-test). In E and F, * indicates vehicle vs. 540 mg / kg test substance, and # indicates vehicle vs. 180 mg / kg test substance. [Figure 10](A-E). Test substance ("O304") reduces fasting blood glucose and blood pressure and increases microvascular perfusion in type 2 diabetes. Type 2 diabetes (T2D) patients receiving metformin. (A-C) Fasting plasma glucose (FPG) (A and B) and HOMA-IR (C) on days 1 and 28 in type 2 diabetes patients receiving metformin and treated with placebo (n=24) and test substance (n=25) with day 1 fasting plasma glucose (FPG) ranges of >7 and <13.3 mmol / L (>126 and <240 mg / dL). (D) Hyperemic microvascular perfusion assessed by dynamic T2*-quantification monitored by MRI in the calf muscle of type 2 diabetes patients at screening (MRI1) and days 27-29 (MRI2). The test substance and placebo groups were divided into halves based on baseline time to peak (TTP). Short TTP (placebo A [n = 14], test substance A [n = 14]) and long TTP (placebo B [n = 13], test substance B [n = 14]) represent relatively high and low rates of hyperemic perfusion, respectively. A significant reduction in TTP (P = 0.043) and an increase in Δ-T2* (P = 0.034) were observed in subjects with relatively low baseline perfusion rates (long TTP) in the test substance group (i.e., test substance B MRI1 vs. test substance B MRI2), but not in subjects with short TTP. There was also no difference between subjects with short or long TTP in the placebo group. (E) Absolute and relative changes in systolic and diastolic blood pressure from days 1 to 28 in type 2 diabetic patients receiving metformin treated with placebo (n = 27) or test substance (n = 30). Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 (Wilcoxon signed-rank test). [Figure 11] Combination therapy with compound 1 and canagliflozin. Fasting blood glucose, fasting plasma insulin, and HOMA-IR. [Example]

[0074] The test substance used in Examples 1 and 2 was 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide. This substance will be referred to hereinafter as the "test substance." The test substance used in this study was synthesized and purified by Anthem Bioscience Pvt Ltd. (Bangalore, India) for Baltic Bio AB (Umeå, Sweden) and Betagenon AB (Umeå, Sweden). 7.4.

[0075] Example 1 Here, we describe the identification and testing of a PAN-AMPK activator, termed test substance, that was found to enhance AMPK activity by inhibiting the dephosphorylation of pAMPK.

[0076] method Study design For animal studies, sample size estimates were not calculated before the study was performed. Experiments were not randomized unless otherwise specified. Researchers were not blinded to allocation during the experiments and outcome assessment, except for some measurements and quantifications (glucose tolerance test, glucose-stimulated insulin secretion, arginine-stimulated insulin secretion, amyloid quantification, echocardiography, and cardiac ultrasound). For in vivo data, each n value corresponds to one mouse. For amyloid quantification, each n value corresponds to an independent study and the total number of islets examined. For in vitro data, each n value corresponds to an independent study. When the same study was repeated, their average was considered as n = 1.

[0077] For cell culture assays, test substances were dissolved in DMSO Hybri-Max™ (Sigma, #D2650). For in vivo assays, test substances were dissolved in 2% w / v methylcellulose, 4 mM phosphate buffer pH 7.4. Metformin (Sigma #D150959) was dissolved in 2% w / v methylcellulose, 4 mM phosphate buffer pH 7.4.

[0078] The pharmacokinetics of the test substance in C57BL / 6JBomTac mice and NTac:SD rats were analyzed by UHPLC-ESI Triple Quadrupole method in plasma from non-fasted animals. The test substance (40 mg / kg test substance) was administered by oral gavage, and blood was collected 4, 8, 12, and 24 hours after administration. The test substance levels were determined via MSMS. Levels were measured in liver and brain from non-fasted control CD(SD) rats that were orally gavaged with 20 mg / kg test substance once daily for 3 weeks. Test substances were extracted with acetonitrile and levels were determined using UHPLC-ESI Triple Quad MSMS.

[0079] animal Female control:CD(SD) rats (strain #001), male and female Wistar rats (strain #003), and Zucker Crl:ZUC-Leprfa rats (strain #185) were obtained from Charles River Lab. Female NTac:SD rats were obtained from Taconic. Male C57BL / 6J (B6) mice were obtained from JAX Mice (Jax #000664). Male C57BL / 6JBomTac mice were obtained from Taconic (B6JBom). Male B6CBAF1 / J (F1) mice were obtained from JAX Mice (Jax #10011). CBA / CaCrl (CBA) mice were obtained from Charles River Lab (Charles River CBA / CaCrl). hIAPPtg mice were obtained from JAX mice (Jax#008232) and maintained by brother-sister mating and backcrossing to CBA for over 10 generations. Wild-type littermates served as controls for hIAPPtg mice.

[0080] 14-15 week old B6 males were assigned to vehicle, metformin, test substance, and metformin + test substance treatment groups (100 mg / kg, orally once daily) at 10 animals / group based on starting body weight and were fed an HFD for the entire 8 week study period.

[0081] Seven-week-old male B6 mice were fed a HFD for 7 weeks and then assigned based on body weight to test substance and metformin plus test substance treatment groups (100 mg / kg, orally once daily) and fed a HFD for an additional 4 weeks.

[0082] Twelve-week-old CBA mice were randomly assigned to HFD or one of three test substance-HFD groups (0.4 mg / g, 0.8 mg / g, and 2 mg / g) for 7 weeks. Where indicated, 16-17-week-old CBA mice fed a normal diet served as controls.

[0083] Fourteen-week-old CBA mice were randomly assigned to an HFD group or a test substance-HFD (2 mg / g) group for two weeks while housed at 22°C. The two groups were then switched from HFD to test substance-HFD, or vice versa, for an additional 4.5 weeks before being transferred from 22°C to 30°C (thermonutrality). After one week at 30°C, the diets were switched again, and core body temperatures were measured one week after the switch.

[0084] Male hIAPPtg mice (10-11 weeks old) were randomly assigned to vehicle or test substance treatment groups (100 mg / kg, orally once daily) and fed an HFD throughout the 6-week study period. Male hIAPPtg;CBA mice (10-11 weeks old) and wild-type littermates were fed an HFD for 9 weeks. After 9 weeks, mice were either sacrificed or randomly assigned to two groups and either continued on the HFD for an additional 7 weeks or switched to a test substance-HFD (2 mg / g).

[0085] Eight- to ten-week-old Wistar male and female rats were treated by oral gavage with vehicle or test substance at 100, 300, or 600 mg / kg / day for 6 months.

[0086] Animals were housed in a temperature / humidity controlled (22°C / 50% humidity) room on a 12:12 h light / dark cycle and fed ad libitum with either standard chow (Special Diet Service #801730), high-fat diet (HFD) (Research diets, Inc. #D12492) or HFD custom formulated with test substance at 2 mg / g test substance, 0.8 mg / g test substance, and 0.4 mg / g test substance, respectively (Research Diets, Inc. #D12492).

[0087] Cardiovascular safety pharmacology study using radiotelemetry in conscious beagle dogs after a single oral gavage dose Telemetry analysis was performed by CiToxLAB North America (Laval, Quebec, Canada) using a CiToxLAB system that had previously implanted a telemetry transmitter. This study was performed on adult male beagle dogs selected from the North America Dog Telemetry Colony. Arterial blood pressure, electrocardiogram, body temperature, and locomotor activity were monitored (Data Science International, model D70-PCT). All surgical procedures were performed in accordance with relevant standard operating procedures. A telemetry transmitter was placed between the aponeurosis of the internal oblique and transversus abdominis muscles in each animal. A pressure catheter was inserted into the femoral artery, and biopotential leads were extended subcutaneously in the Lead II configuration. Test substances were administered by gavage as a suspension at 60, 180, or 540 mg / kg.

[0088] Food control, weight and composition Food intake was measured weekly by providing 200 g of pellets per cage. After 1 week, the amount of pellets consumed was calculated and adjusted for the number of animals / cage. Body weight was measured weekly. Body composition was assessed using EchoMRI.

[0089] Echocardiography Left ventricular structure and function were analyzed by transthoracic high-frequency echocardiography using an MS550D transducer. Examinations were performed during light isoflurane anesthesia (1.5–2.0% in 800 mL oxygen). The anesthesia level was adjusted to maintain a respiratory rate of 80–110 breaths per minute. Left ventricular volumes were determined in B-mode using Simpson's law reconstruction. All images were analyzed offline in a blinded manner using Vevo LAB software 1.7.0. Stroke volume, cardiac output, and heart rate, as well as wall thickness and left ventricular diameter, were analyzed. Triplicate measurements per animal were performed to obtain average values.

[0090] Laser Doppler Imaging Nine-week-old F1 mice were fed an HFD for 8 weeks and treated with vehicle or test substances (40 mg / kg, orally once daily). One day before blood perfusion analysis, hair was removed from the left hind paw using Veet depilatory cream. Mice were anesthetized using isoflurane and placed on a heating pad. Blood perfusion was scanned using PeriScan PIM II Images, and images were analyzed using LDPIwin software (version 2.6.1).

[0091] Treadmill For the treadmill test, 14-month-old C57BL / 6J mice with similar running distances to exhaustion were assigned to two groups (14 mice per group) and subsequently treated with vehicle or test substance (20 mg / kg, orally once daily) for 30 days. One week before testing, mice underwent a 5-minute familiarization session on the treadmill. The protocol was as follows: 15 minutes at 18.8 m / min, 5 minutes at 24.4 m / min, and 27.1 m / min until exhaustion. At the time of exhaustion, blood lactate levels were measured using a lactate test meter (Arkray).

[0092] indirect calorimetry Twenty-one-week-old CBA mice fed a HFD or a 0.8 mg / g HFD for 11 weeks were individually housed in chambers with a 12-hour light / 12-hour dark cycle at an ambient temperature of 22°C. They were allowed to acclimate to the chambers for a minimum of 12 hours before data collection. VO2 and VCO2 rates were measured by indirect calorimetry in a TSE PhenoMaster calorimetry metabolic cage (TSE Systems GmbH) for 3 days. The respiratory exchange ratio (RER) was calculated as the ratio of VCO2 produced to VO2 consumed. An RER of 0.7 indicates fat as the primary fuel source, while an RER close to 1.0 indicates carbohydrate as the primary fuel. Energy expenditure (EE) was calculated as the product of the calorific value of oxygen (CV) [= 3.815 + (1.232 x RER)] and the volume of O2 consumed, i.e., [EE = CV x VO2 (kcal / h)], and was related to lean body mass.

[0093] Infrared Thermal Imaging Skin temperatures of unsedated Zucker rats treated with the test substance (10 mg / kg / day) or vehicle for 12 days were recorded with an infrared camera (FLIRix series ExtechIRC30, FLIR Systems Inc.) and analyzed with a specific software package (FLIRQuickReport version 1.2SP2 (1.0.1.217)). Nine rats were used per group, and the mean and maximum skin surface temperatures of each animal were measured 2 hours after the final administration.

[0094] Glucose and serum-related measurements Oral and intraperitoneal glucose tolerance tests coupled with glucose-stimulated insulin secretion were performed in 6-h-fasted, unsedated mice (Hypnorm (Veta Pharma) / Midazolam (Hamlenmice)) after intraperitoneal injection of glucose (SIGMA #G7021) (0.75 g / kg body weight). Arginine-stimulated insulin secretion was measured after intraperitoneal injection of arginine (SIGMA #A5131) (1 g / kg body weight) in unfasted, 21-week-old CBA mice fed an HFD or test substance-HFD (0.8 mg / g) for 11 weeks. Blood glucose levels were measured using a Glucometer (Ultra 2, One Touch), and plasma insulin was analyzed via an ultrasensitive mouse insulin ELISA kit (Chrystal Chem Inc. #90080). The area under the curve (AUC) was calculated according to the trapezoidal rule. The homeostatic model of insulin resistance (HOMA-IR) was calculated as follows: fasting plasma glucose (mmol / L) × fasting plasma insulin (μU / L) / 22.5. The MATSUDA index was calculated as follows: [10000 / sqrt(insulin (0 min) + glucose (0 min) + insulin mean (0-60 min) + glucose mean (0-60 min)]. Statistical significance was calculated by Student's t-test (two-tailed).

[0095] Autophagy flux assay INS-1E cells were incubated for 24 hours with or without 5 μM test substance in the presence or absence of 100 nM bafilomycin A1 (InvivoGen#tlrl-baf1) for the last 60 minutes of incubation. LC3II levels were determined and quantified by Western blot analysis. The primary and secondary antibodies used are listed in Table 1.

[0096] [Table 1]

[0097] Amyloid analysis and ex vivo islet amyloid assay Islet amyloid quantification was performed on pancreatic tissue isolated from hIAPPtg mice (n = 7 mice / n = 69 islets) fed an HFD for 16 weeks and from hIAPPtg mice (n = 5 mice / n = 48 islets) fed an HFD for 9 weeks, then switched to a test substance-HFD (2 mg / g) for 7 weeks. Isolated pancreata were frozen whole, sectioned, and amyloid content quantified by staining with thioflavin-S as previously described (2). For ex vivo analysis, islets were isolated by collagenase digestion of the pancreas (1) and cultured in RPMI medium 1640 (GIBCO #11879-0) supplemented with 11.1 or 22.2 mM glucose (GIBCO #A24940-01), 1% fetal bovine serum (GIBCO #10500), 50 U μg / ml Pen / Strep (Gibco #15140-122), 10 mM Hepes (Umea University, Laboratory Medicine), 1 mM sodium pyruvate (GIBCO #11360-039), and 0.1% 2-mercaptoethanol (Sigma #M3148). Test substances were added at 0, 2.5, 5.0, and 10 μM starting on day 0 of culture. To evaluate the effects of autophagy inhibition, 5 μM 3-methyladenine (3-MA, Aldrich #M9281) was added in combination with 5 μM test substance starting on day 0 of culture. Controls contained DMSO 1:2000. Medium and compounds were replaced every two days. After 92 hours of treatment, islets were embedded, sectioned, and amyloid content was quantified by staining with thioflavin-S as previously described (Reference 2). At least three independent experiments were evaluated.

[0098] Determination of cellular ATP content Wi-38 human lung fibroblasts were stimulated with test substances for 16 hours. ATP content was then determined using the ATP Bioluminescence Assay Kit HS II (Roche Applied Science #11699709001) according to the manufacturer's recommendations. ATP data were normalized to cellular protein determined using the BCA Protein Assay Kit (Pierce #23225).

[0099] Western blot analysis All cell lines were lysed in 0.1 M Tris-HCl, pH 6.8, 2% SDS, 10 mM sodium fluoride (SIGMA #S7920), 10 mM β-glycerophosphate (SIGMA #G6376), and 1 mM sodium vanadate (SIGMA #72060), and the supernatant was collected after 1 min at 14,000 rpm. Pancreatic islets (human and mouse) were lysed in 0.1 M Tris-HCl, pH 6.8, 2% SDS, protease inhibitors (Roche #04 693 124 001), and phosphatase inhibitors (Roche #04 906 837 001). Right calf muscle, heart, inguinal white adipose tissue (iWAT), and interscapular brown adipose tissue (BAT) were pulverized with a pestle using liquid nitrogen and homogenized in ice-cold RIPA buffer (150 mM sodium chloride (SIGMA #S7653), 1.0% NP40 (USB), 0.5% sodium deoxycholate (SIGMA #D6750), 0.1% SDS, 50 mM Tris pH 8.0, 20 mM sodium pyrophosphate (SIGMA #71515), 10 mM sodium fluoride, 10 mM β-glycerol phosphate, 1 mM sodium vanadate, and protease inhibitor cocktail (Roche #04693124001), 1 tablet / 10 ml lysis buffer). The supernatant was collected after 2 min at 14,000 rpm. This procedure was repeated at 4 °C until all fat was removed and the supernatant was clear. Samples were analyzed on a 4-15% polyacrylamide gel. The primary and secondary antibodies used are listed in Table 1. Values ​​were normalized to AMPKα, β-actin, GAPDH, or their respective non-phosphorylated counterparts.

[0100] Quantitative real-time PCR (qRT-PCR) For RNA purification, calf muscle, iWAT, interscapular BAT, and left liver lobe were pulverized with a pestle using liquid nitrogen and then transferred to the respective RNA kits. RNA from liver was prepared using the Total RNA Isolation Nucleospin II Kit (Macherey-Nagel #740955.50). RNA from adipose tissue was prepared using the RNeasy Lipid Tissue Mini Kit (Qiagen #74804). RNA from calf muscle tissue was prepared using the RNeasy Fibrous Tissue Mini Kit (Qiagen #74704). First-strand cDNA synthesis was performed using SuperScript III (First-Strand Synthesis SuperMix qRT-PCR was performed using a Qiagen RNeasy Micro Kit (Qiagen #74004) according to the manufacturer's instructions. Total RNA was prepared from isolated islets using the RNeasy Micro Kit (Qiagen #74004), and first-strand cDNA synthesis was performed using Superscript III (Invitrogen #18080-051) according to the manufacturer's instructions. Quantification of mRNA expression levels was performed essentially as previously described (reference 3). Primers used for qRT-PCR are listed in Table 2. Zeta polypeptide (YWHAS), a tyrosine 3-monooxygenase / tryptophan 5-monooxygenase-activating protein, was used to normalize expression levels, except for islets in which TBP was used.

[0101] [Table 2]

[0102] Liver lipid extraction and triglyceride determination 0.2–0.3 g of liver was homogenized in 3 ml of PBS, followed by the addition of 6 ml of chloroform / methanol (2:1). The sample was mixed until no phase separation occurred, left at room temperature for 30 minutes, and then centrifuged at 4,500 rpm for 5 minutes. The chloroform phase was transferred to pre-weighed glassware and kept at 4°C overnight. All water droplets were removed, and the chloroform was evaporated with a stream of nitrogen. The remaining solvent was then removed via a SpeedVac for 15 minutes. The glassware was reweighed, and total lipids were calculated (mg / g liver). The residue was dissolved in 35% Triton X-100 / methanol. Liver triglycerides were measured using a serum triglyceride assay kit (Sigma-Aldrich #TR0100). Analysis was performed according to the manufacturer's recommendations, with a slight modification: triglyceride measurement was performed at 560 nm instead of 540 nm.

[0103] Glycogen measurement Cardiac glycogen content was measured using a glycogen assay kit (Abcam#ab65620) according to the manufacturer's recommendations.

[0104] [1,2- to total lipids 14 [C]acetate uptake Primary human hepatocytes (65,000–130,000 cells / well in 24-well dishes) were treated with vehicle control, 0.625, 1.25, 2.5, or 5 μM test substance in serum-free Williams medium E for 2 h, followed by 0.25 μCi [1,2- 14[C]-acetate was added per well. See Table 3 for growth conditions. 200 μl of 0.5% trypsin was used to detach the cells, followed by the addition of 800 μl of chloroform / methanol (2:1) and 500 μl of 4 mM MgCl2. The samples were vortexed and spun at 14,000 rpm for 2 minutes, after which the aqueous layer was discarded. This procedure was repeated twice: first with 700 μL of chloroform / methanol (2:1) and 500 μL of 4 mM MgCl2, and then with 400 μL of chloroform / methanol (2:1) and 500 μL of 4 mM MgCl2. The organic phase was transferred to a scintillation vial and evaporated to dryness using a stream of nitrogen. The residue was dissolved in 3 ml of liquid scintillation cocktail (Optiphase HiSafe 3, Perkin Elmer #1200.437) and measured for 1 min on a Wallac 1414 beta counter (Perkin Elmer). Protein concentration was measured using 10 μl of sample before lipid extraction. C values ​​were normalized to cellular protein concentration.

[0105] [Table 3-1]

[0106] [Table 3-2]

[0107] In vivo lipogenesis Fifteen-week-old CBA mice were fed an HFD or test substance-HFD (2 mg / g) for 5 weeks, starved overnight, and then re-fed. 90 minutes later, they were injected with 1000 μCi 3H-NaOac (Perkin Elmer #NET003005MC) diluted in 0.9% NaCl. After 90 minutes, 0.2–0.3 g of liver was isolated and homogenized in 3 ml of PBS, followed by the addition of 6 ml of chloroform / methanol (2:1). The sample was mixed until no phase separation occurred, left at room temperature, and then centrifuged at 4,500 rpm for 30 minutes. The aqueous phase was removed, and 3 ml of chloroform was transferred to a scintillation vial and evaporated to dryness under a nitrogen stream while placed in a 40°C water bath. The residue was dissolved in 3 ml of optiphase HiSafe 3 (Perkin Elmer #1200.437), and 3 H was measured for 1 min on a Wallac 1414 counter. 3 H values ​​were normalized to liver weight.

[0108] Glucose uptake in L6 myotubes Rat L6 skeletal muscle cells grown in high-glucose (4.5 g / L) Dulbecco's modified Eagle's medium (Gibco #31966), 10% fetal bovine serum (Gibco #10500-064), and 25 μg / ml gentamicin (Gibco #5750) were induced to differentiate by lowering the serum concentration to 2% for 14 days, by which time the majority of myoblasts had differentiated into myotubes. Myotubes were rinsed with serum-free, low-glucose (1 g / L) DMEM (Gibco #21885), treated with vehicle control, 2.5, 5, and 10 μM test substances (serum-free, low-glucose DMEM, 0.1% DMSO) for 2 hours, rinsed with glucose-free serum-free DMEM (Gibco #11966), and then incubated for 20 minutes before the addition of 1 μCi of 2-deoxy-D-glucose (2-DG) (Perkin Elmer #NET549A250UC) for 10 minutes. Cells were rinsed three times with glucose-free serum-free DMEM and lysed in 1 ml RIPA buffer (150 mM sodium chloride, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0). 300 μl was added to 4 ml of liquid scintillation cocktail (Perkin Elmer #1200-437) and then counted for 1 minute in a Wallac 1414 beta counter. CPM was converted to arbitrary units by setting the vehicle to 1.

[0109] Six to seven days after the initiation of differentiation, L6 myotubes were transfected with siAMPKα1 and α2 (Santa Cruz Biotechnology, Inc. #sc-270142 and #sc-155985) or Silencer Negative Control siRNA (Ambion #AM4635) using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific #13778030) according to the manufacturer's instructions (forward transfection). The final siRNA concentration was set at 100 nM. The day before transfection, the medium was changed to antibiotic-free medium (high glucose, 4.5 g / L, Dulbecco's Modified Eagle Medium (Gibco #31966) and 2% fetal bovine serum (Gibco #10500-064)). The levels of AMPKα1 and α2 expression in cells transfected with siAMPKα1 and α2 and Silencer Negative Control siRNA, respectively, were quantified by Western blot. Glucose uptake was assayed 72 hours after transfection in the absence or presence of test substances (5 μM) for 4 hours. Glucose uptake induced by test substances was normalized to vehicle control uptake in cells transfected with siAMPKα1 and α2 and Silencer Negative Control siRNA, respectively.

[0110] In vivo glucose uptake Twelve-week-old CBA mice, administered a HFD or test substance-HFD (2 mg / g) for 2 weeks, were starved for 3 hours and then intravenously injected with 9 ± 1.1 MBq of clinical-grade F-fluoro-deoxy-glucose (FDG) (prepared at the Department of Nuclear Medicine, Norrlands University Hospital, Umea) in a total volume of 70–100 μL of saline under light isoflurane anesthesia (1.5–2% at 800 mL / min O2). After injection, mice were allowed to awaken and move freely within their cages. 180 minutes later, mice were sacrificed under deep isoflurane anesthesia, and blood was removed by retrograde perfusion with PBS via the aorta. When the liver was pale, tissue was collected and scanned for static uptake for 10 minutes (nanoScan PET / CT, Mediso, Hungary). The tissue was then scanned ex vivo to assess uptake in isolated tissues. Images were reconstructed to a resolution of 0.4 x 0.4 mm using 3D iterative reconstruction with four iterations and four subsets (Mediso Tera-Tomo 3D) covering an axial distance of 98 mm, using spike filtering, delay-window random correction, scatter, and CT-based attenuation correction. Volumes of interest were manually contoured in each tissue using imlook4d (www.dicom-port.com). Tracer uptake was quantified as the standardized uptake value (SUV) using the following formula: SUV = C / (I / m); where C is the measured tissue activity concentration (Bq / mL), I is the injected dose (Bq), and m is the body weight (g). C and I are attenuation-corrected at the same time.

[0111] SAMS peptide AMPK activity assay 50 ng AMPK (Upstate #14-305) was dissolved in a buffer (40 mM Hepes pH 7.45, 0.5 mM DTT, 2 mM MgCl 2、 Various combinations of 2.5, 5, or 10 μM test substance or 20 μM AMP (Sigma #A2002) in 0.1% DMSO were mixed. All settings contained 10 μg of SAMS and 0.03 μCi / μl of 32P ATP (Perkin Elmer #NEG502Z500UC) was added. The total reaction volume was 25 μl. All components were mixed on ice and incubated at 37°C for 15 minutes before being terminated with 5 μl of phosphoric acid and returned to ice. 25 μl of the reaction mixture was dried onto a Whatman P81 filter at 50°C for 2 minutes, washed three times with 250 ml of 1% phosphoric acid for 2 minutes each, then added to 4 ml of liquid scintillation cocktail (Perkin Elmer #1200-437) and counted for 1 minute in a Wallac 1414 beta counter. Radioactivity correlates with enzyme activity.

[0112] AMPK activation assay Table 3 includes the cell line origin, growth conditions, and settings for test substance-mediated AMPK activation. Human skeletal muscle cells were grown in growth medium obtained from the cell supplier for 2 days until myotube differentiation was induced in DMEM (Gibco #21885) supplemented with 2% horse serum (Gibco #26050-070). They were then treated with test substances as described in Table 3. Upon arrival, human hepatocytes were thawed at 37°C for 1 minute and then transferred to thawing medium (CHRM, Invitrogen #CM7000). After centrifugation at 100x g for 10 minutes at room temperature, the cell pellet was resuspended in Williams Medium E (Gibco #A1217601) supplemented with Hepatocyte Plating Supplement Pack (Gibco #CM3000). Cells were plated onto gelatin-coated 60 mm dishes and then incubated overnight before treatment with test substances as described in Table 3. INS-1E cells were pretreated with the medium for activation conditions (Table 3) for 4 hours before adding the test substances. All cell lines were incubated in 5% CO 2、The islets were maintained in a humidified incubator at 37°C. Table 3 shows the growth conditions and the settings for AMPK activation by test substances in mouse and human islets. After harvest, mouse islets were cultured in growth-conditioned medium at 37°C and 5% CO2 for 2 days and then treated with test substances for 2 hours. Human islets from non-diabetic and T2D (type 2 diabetes) donors were provided through the JDRF Award 31-2008-416 ECIT Islet for Basic Research program in accordance with Swedish law and the Umeå Human Research Ethics Committee (www.epn.se). Upon arrival, the islets were transferred to a 50 ml Falcon tube and allowed to rest for 5 minutes. The supernatant was removed and the islets were resuspended in medium (CMRL medium (GIBCO #21530-027), 10% fetal bovine serum (GIBCO #10500), 20 U / ml Pen:Strep (Gibco #15140-122), and 1X ATP. GlutaMax (Gibco #35050-038) was added. The islets were washed three more times with medium, transferred to a Petri dish, and allowed to recover overnight in a humidified incubator at 37°C and 5% CO2 before being treated with the test substances for 4 hours.

[0113] AMPK In Vitro Dephosphorylation Assay AMPKα2 / β1 / γ1 trimer (Life Technologies #PV4674, lot 1261361B) (1 ng / μl) was incubated with 10 μM test substance, 20 μM test substance, or 150 μM ADP (Sigma #A2754) + / - 1 mM ATP (Sigma #A1852-1VL) + / - PP2Cα (0.25-0.75 ng / μl) (Abcam ab51205-100; lot GR54133-5) and 5 mM MnCl2 in a total volume of 20 μl in buffer (40 mM Hepes, 0.5 mM DTT, 0.2 mg / ml gelatin (Sigma #G7041) and 0.4% DMSO). AMPKα2 / β1 / γ1+ / -ATP was preincubated with the test substance and ADP for 2 min at 30°C, and then PP2C / MnCl2 was added to initiate the dephosphorylation reaction, which continued for 10–15 min at 30°C. 0.17% BSA, 13 mM The reaction was stopped by adding PBS containing EDTA, 1.3x XT sample buffer, and 0.67% β-mercaptoethanol. Samples were placed on ice for 5 minutes, heated at 100°C for 5 minutes, cooled, and then run on a Western gel. All steps were performed in high-quality, low-protein-binding Eppendorf tubes. In separate experiments, 10 μM test substance, 20 μM test substance, 150 μM ADP alone, or combinations of 10 μM test substance + 150 μM ADP and 20 μM test substance + 150 μM ADP were added in buffer (40 mM Hepes, 0.5 mM DTT, 0.2 mg / ml gelatin, and 0.4% DMSO) containing + / - 0.25-0.5 ng / μL PP2Cα and 5 mM MnCl. 2、 The AMPK was incubated with 1 ng / μl of AMPKα2 / β1 / γ1 or AMPKα1 / β1 / γ1 trimers (Life Technologies #PV4672) in the presence of 5 mM MgCl2 or 5 mM MgCl2. AMPK was preincubated with test substances and ADP or a combination for 2 minutes at 30°C, followed by the sequential addition of PP2C / MnCl2 or PP2C / MgCl2 to initiate the dephosphorylation reaction. This reaction was continued for 5–15 minutes at 30°C. The reaction was then stopped and analyzed as described above.

[0114] PP2C phosphatase activity assay To measure PP2Cα activity, 3 ng / μl of PP2Cα (Abcam ab51205) and 5, 10, or 20 μM of test substance in buffer (50 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.5 mM DTT, 5 mM MgCl) were used with the Sensolyte FDP Protein Phosphatase Assay Kit (Anaspec #71100) according to the manufacturer's instructions. Fluorescence intensity was measured using BioTek Measurements were performed using a Synergy H4 multimode microplate reader.

[0115] Quantification and statistical analysis Quantification of Western blot experiments was performed using Image Lab (Bio-Rad Laboratories version 4.1 build 16) and Image-J Software (version 1.45s). Quantification of amyloid content was performed using Image-J Software (version 1.49m). All statistical analyses of in vitro and in vivo mouse data were performed by two-tailed Student's t-test. A value of P<0.05 was considered statistically significant. Patient data analysis was performed using mixed-model anova tests (two-way ANOVA for absolute changes and one-way ANOVA for percentage changes throughout) and nonparametric Wilcoxon rank-sum tests. Composite endpoints were analyzed using chi-square tests and Fisher's exact tests.

[0116] result Test substances inhibit the dephosphorylation of pAMPK in vitro and act as universal (PAN)-AMPK activators in cells. Consistent with the above, in vitro, the test substance inhibited protein phosphatase 2C-mediated (PP2C-mediated) dephosphorylation of p-T172 in human recombinant AMPK α, β, and γ trimers without inhibiting PP2C activity (Figure 1A). The test substance also protected pAMPK from dephosphorylation in the presence of excess ATP (Figure 1B) and acted additively with ADP but did not allosterically activate AMPK. Thus, the test substance mimicked the effects of ADP, but not AMP, on AMPK activity.

[0117] In non-transformed human Wi-38 lung fibroblasts, test substances dose-dependently increased levels of pAMPK, its downstream target p-S79 ACC (pACC), and the ATP / protein ratio (Figure 1C, C). Notably, test substances increased pAMPK in many different cell types, including cells involved in type 2 diabetes, such as human skeletal myotubes and hepatocytes, which preferentially express the β2 subunit, containing various different AMPK heterotrimers. Thus, test substances function as PAN-AMPK activators in cells. The mechanism of action of test substances requires that cells express the major upstream kinase LKB1. Consistent with the above, in HeLa cells, an LKB1-null phenotype, test substances failed to increase the very low basal levels of pAMPK and pACC. However, as a control, ionomycin, a Ca2+ ionophore that activates AMPK via calcium / calmodulin-dependent protein kinase kinase (CaMKK), readily activated AMPK in these cells. Thus, test substances only further increase AMPK activity in physiologically relevant cells that have intrinsic AMPK activity.

[0118] The test substance prevents insulin resistance and hyperglycemia in DIO mice. In rodents, test substances can be orally administered due to their long plasma half-life, but they do not cross the blood-brain barrier. To address whether test substances alone or in combination with metformin, as in clinical settings, can alleviate hyperglycemia and insulin resistance in vivo, mice were fed a high-fat diet (HFD), referred to as "DIO" (diet-induced obesity), and were orally administered vehicle, test substance, metformin, or test substance plus metformin (100 mg / kg / day) by gavage for 8 weeks (Figure 2A). With this regimen, test substance and test substance plus metformin, but not metformin, avoided the HFD-induced elevations in fasting blood glucose and plasma insulin levels (Figure 2B and C). As a result, compared to vehicle, DIO mice treated with test substance and test substance plus metformin did not develop insulin resistance as assessed by HOMA-IR calculations (Figure 2D). Furthermore, consistent with the potent prevention of hyperglycemia, hyperinsulinemia, and insulin resistance, test substance and test substance plus metformin, but not metformin, significantly increased pAMPK (Figure 2E), decreased Txnip mRNA levels, and increased Glut1 mRNA levels (Figure 2F) in the calf muscles of DIO mice, consistent with both insulin-dependent and insulin-independent effects. In summary, test substance increased pAMPK in calf muscles and potently protected DIO mice from hyperglycemia, hyperinsulinemia, and insulin resistance. While metformin showed no significant effect, test substance plus metformin appeared to be the most effective and significantly reduced HOMA-IR compared to test substance alone.

[0119] In patients with type 2 diabetes, test substances are used in combination with metformin to alleviate established hyperglycemia. To mimic these conditions, mice were fed an HFD for 7 weeks, resulting in hyperglycemia and insulin resistance compared to mice fed a normal diet (RD) (Figure 2, G-I). They were then treated with vehicle or test substance plus metformin for 4 weeks while continuing the HFD. While reductions in fasting insulin levels and HOMA-IR were evident after 1 week of treatment with test substance plus metformin, fasting blood glucose levels were only significantly reduced after 2 weeks of treatment with test substance plus metformin compared to vehicle (Figure 2, G-I). Longer-term treatment further reduced blood glucose levels, and after 4 weeks of treatment, fasting blood glucose levels were reduced to the level of mice fed an RD (Figure 2G). Thus, the metabolic effects of test substance plus metformin (i.e., reduction of hyperglycemia) are sufficiently similar to the effects of exercise and / or calorie restriction on hyperglycemia observed in humans.

[0120] Test substances prevent and reverse diabetes in hIAPPtgDIO mice. Because DIO mice become hyperglycemic but not overtly diabetic, we next investigated the effects of test substances in a mouse model mimicking human type 2 diabetes (i.e., insulin resistance / hyperglycemia combined with HFD-induced β-cell dysfunction). For this purpose, we used mice expressing the amyloidogenic human IAPP (hIAPP) gene under the control of the rat insulin 2 promoter (referred to as hIAPPtg mice) and fed an HFD diet for 6 weeks (Figure 3A). In hIAPPtg DIO mice, test substances administered orally by gavage at 100 mg / kg / day prevented the increase in 6-h fasting blood glucose and plasma insulin levels compared to vehicle (Figure 3B and C). Intraperitoneal (IP) (Figure 3D) and oral (Figure 3E) glucose tolerance tests (GTTs) confirmed that the test substances prevented the development of impaired glucose tolerance and compensatory hyperinsulinemia, demonstrating a relative normalization of insulin hypersecretion, which mirrored that of RD 10-week-old hIAPPtg mice (Figure 3F). Furthermore, HOMA-IR and Matsuda index models of whole-body insulin sensitivity demonstrated that the test substances suppressed the development of whole-body insulin resistance in hIAPPtg DIO mice (Figure 3G and H).

[0121] To test whether a test substance could reverse established diabetes and obesity in obese diabetic hIAPPtg mice and to avoid the potential confounding effect of oral gavage on HFD-induced obesity, we formulated an HFD containing the test substance, referred to as test substance-HFD. To test the dose-response effect of the test substance on glucose homeostasis, CBA mice were then fed either a HFD or a test substance-HFD formulated with 0.4, 0.8, and 2 mg / g of the test substance for 7 weeks. In CBA mice, the test substance-HFD dose-dependently increased pAMPK in the calf muscle (Figure 4A) and potently prevented hyperglycemia, hyperinsulinemia, and insulin resistance (Figure 4B-D). To test whether a test substance could reverse established diabetes, hIAPPtg mice were fed a HFD for 9 weeks and then switched to a test substance-HFD (2 mg / g) for 7 weeks (Figure 4E). Six weeks after the switch, the test substance-HFD reversed established hyperglycemia, hyperinsulinemia, and insulin resistance, thus reversing diabetes (Figure 4, F-H). Furthermore, the test substance induced a decrease in body weight and body fat despite increased food intake.

[0122] The strong effect of the test substance on established diabetes and obesity in hIAPPtg mice fed a HFD containing 2 mg / g of test substance leaves open the possibility that the beneficial metabolic effects observed in these mice (Figure 4, F-H) are secondary to effects on body weight and body fat. Therefore, to address this issue, we performed a diet-switching experiment in hIAPPtg mice using a lower test substance-HFD concentration. Mice were fed a HFD for 9 weeks, followed by either continuing the HFD or switching to a test substance-HFD (0.8 mg / g) for 7 weeks. With this regimen, switching to a test substance-HFD (0.8 mg / g) for 7 weeks did not result in a decrease in body weight or body fat (Figure 4, I and J). Nevertheless, 6 weeks after switching to a test substance-HFD (0.8 mg / g), glucose and insulin levels and HOMA-IR were significantly reduced (Figure 4, K-M), indicating that under these conditions, the beneficial metabolic effects of the test substance are independent of its effects on body weight and body fat loss. Taken together, these results demonstrate that the test substance potently circumvents insulin resistance, hyperinsulinemia, hyperglycemia, and overt diabetes in a mouse model of obesity-induced diabetes, type 2 diabetes.

[0123] Test substances increase glucose uptake in skeletal myotubes in vitro and in skeletal muscle in vivo. In skeletal muscle, AMPK activation is associated with both increased insulin-independent glucose uptake and reduced insulin resistance. Accordingly, in skeletal myotubes, the test substance increased 2-deoxy-D-glucose (2-DG) uptake in a dose-dependent and AMPK-dependent manner in the absence of insulin (Figure 5A-D). Furthermore, using PET analysis of tail vein injections of the radiolabeled glucose analog fluorodeoxyglucose (FDG), we observed a significant increase in FDG uptake in the calf and thigh muscles of mice fed a test substance-HFD (2 mg / g) for 2 weeks compared to mice fed a HFD (Figure 5E). This indicates that the test substance promotes glucose uptake in skeletal muscle in vivo. Collectively, these findings provide evidence that the test substance's favorable effects on glucose homeostasis are mediated, at least in part, by its stimulation of glucose uptake in skeletal muscle.

[0124] The test substance reduces beta cell stress and promotes beta cell rest. In type 2 diabetes, toxic IAPP aggregates / amyloid are associated with β-cell stress and β-cell deterioration. In hIAPPtg HFD mice switched from an HFD to a test substance-HFD (2 mg / g) for 7 weeks, the amount of islet amyloid formed was significantly reduced compared to mice that continued the HFD for 7 weeks (Figure 6, A and B). However, the reduction in amyloid observed in hIAPPtg mice fed a test substance-HFD may be a secondary effect of improving hyperglycemia and insulin resistance. However, the test substance directly increased pAMPKα in rat insulinoma INS-1 cells, isolated primary mouse WT and hIAPPtg islets, and human islets (Figure 6C). Therefore, to investigate the potential direct effect of the test substance on islet cells, we next induced amyloid formation by culturing isolated primary hIAPPtg islets at high glucose (22 mM) levels. The test substance dose-dependently attenuated amyloid formation in hIAPPtg islets cultured in 22 mM glucose (Figure 6, D and E). Basal autophagy has been shown to protect β cells from hIAPP oligomer toxicity, and AMPK activation promotes autophagy. Consistent with the above, the test substance enhanced autophagic flux in the β cell line INS-1E. In the presence of the autophagy inhibitor 3-MA, the preventive effect of the test substance on amyloid formation at 22 mM glucose was significantly attenuated (Figure 6, F and G). However, AMPK activation has also been shown to improve the function and survival of metabolically stressed β cells through the maintenance of endoplasmic reticulum (ER) function. The test substance significantly prevented the increased expression of unfolded protein response genes (i.e., indicative of ER stress) in primary mouse islets cultured in 22 mM glucose. Thus, the test substance circumvents beta cell amyloid formation in an obesity-induced type 2 diabetes mouse model, as well as in isolated mouse pancreatic islets cultured in vitro at high glucose levels.Taken together, our findings suggest that the test substance counteracts metabolically induced β-cell stress and amyloid formation in vivo by both reducing hyperglycemia and systemic insulin resistance, and by enhancing β-cell autophagy and / or endoplasmic reticulum function, although the exact mechanisms require further analysis.

[0125] The apparent ability of the test substance to reduce β-cell stress, possibly both indirectly and directly, raises the question of whether the test substance also promotes β-cell resting, which in turn maintains long-term β-cell function. Arginine stimulation of insulin secretion assesses first-phase insulin release (i.e., the pool of readily releasable granules) and provides an estimate of functional β-cell reserve capacity. Therefore, to assess the effect of the test substance on β-cell function, we next analyzed arginine stimulation of insulin secretion. Arginine stimulation of insulin secretion was increased two-fold in mice fed the test substance-HFD (0.8 mg / g) for 11 weeks compared to mice fed an HFD (Figure 6H). This provides further evidence that the test substance reduces β-cell stress, promotes β-cell resting, and then maintains / restores β-cell function.

[0126] Test substances reduce obesity and increase energy expenditure under thermoneutral conditions. To further investigate the effects of the test substance on obesity, a crossover experiment was conducted. Mice fed an HFD for 14 days rapidly gained weight, whereas mice fed a test substance-HFD (2 mg / g) consumed more food than mice fed an HFD during days 1–14, yet gained very little weight (Figure 7, A and B). When mice were switched between the HFD and test substance-HFD, mice switched from HFD to test substance-HFD on day 15 rapidly lost weight and again experienced a relative increase in food intake. Conversely, mice switched from test substance-HFD to HFD gained weight while decreasing their relative food intake (Figure 7, A and B). Next, we tested whether the test substance induces weight loss at thermoneutrality. Mice transferred from a rearing temperature of 30°C to 30°C on day 49 while continuing on the test substance-HFD still avoided weight gain, whereas mice fed an HFD continued to gain weight (Figure 7A). Furthermore, mice housed at 30°C began to lose weight rapidly when their diet was switched from HFD to test substance-HFD on day 57. Conversely, mice switched from test substance-HFD to HFD began to gain weight rapidly (Figure 7A). Under these conditions, only a small (0.2°C) non-significant increase in core temperature was observed (37.7°C ± 0.12°C in HFD-treated mice, n = 5, and 37.9°C ± 0.08°C in test substance-HFD-treated mice, n = 5). Thus, the test substance also reduces obesity at thermoneutrality.

[0127] To directly address whether the test substance circumvents obesity by increasing energy expenditure (EE), we measured oxygen consumption (VO2), respiratory exchange ratio (PER), and energy expenditure (EE) for 3 days in mice fed an HFD or a test substance-HFD (0.8 mg / g) for 11 weeks. VO2 was significantly increased during both the light and dark phases in mice fed the test substance-HFD compared to mice fed an HFD (Figure 7C). PER was significantly decreased during the light and dark phases on day 2, providing evidence that mice fed the test substance-HFD had switched their primary energy source from carbohydrates to fatty acids (FAs) (Figure 7D). As expected, EE was significantly increased during both the light and dark phases (Figure 7E). Together, these data strongly suggest that the test substance suppresses weight gain by enhancing energy metabolism.

[0128] Test substances increase adipose triglyceride lipase (ATGL) activity and the expression of genes related to fatty acid (FA) oxidation in white adipose tissue (WAT) and brown adipose tissue (BAT). Consistent with the reduction in body fat, mice fed the test substance-HFD (2 mg / g) had significantly lower inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) fat pad weights than mice fed the HFD. To reduce WAT stores, enhanced lipolysis is necessary. Desnutrin / Atgl, which encodes the rate-limiting enzyme catalyzing basal triglyceride (TG) hydrolysis, is a direct target of AMPK. AMPK-mediated phosphorylation of Atgl at S406 should increase ATGL activity and lipolysis. As described above, the test substance-HFD increased both p-S406ATGL and AtglmRNA levels in iWAT (Figure 7, F and G). Furthermore, Cpt1b, which increases mitochondrial FA uptake, and Cox8b, which increases mitochondrial activity and FA oxidation, were also increased in the iWAT of mice fed the test substance-HFD (2 mg / g) compared to mice fed the HFD (Figure 7G). The test substance dose-dependently reduced UCP1 expression in brown adipose tissue (BAT) and low levels of UCP1 expression in iWAT, arguing against ectopic expression of uncoupling protein 1 (UCP1) in WAT as the mechanism for the anti-obesity effect of the test substance. Collectively, these data provide evidence that the test substance prevents obesity, at least in part, by increasing lipolysis and FA oxidation in WAT.

[0129] Activating AMPK in BAT increases FA uptake, metabolic activity, and EE. As mentioned above, BAT fat pad weight was reduced in mice fed the test substance-HFD (2 mg / g) compared with mice fed an HFD, indicating increased BAT metabolic activity. Notably, test substance-HFD (2 mg / g) significantly increased Cd36 expression in BAT of DIO mice, indicating improved FA uptake, as well as enhanced Cpt1b expression and strongly reduced expression of genes encoding FA synthase (Fas), stearoyl-CoA desaturase 1 (Scd1), and Acc1, which, in combination, should decrease de novo lipogenesis (DNL) in BAT and increase mitochondrial FA uptake / oxidation (Figure 7H). Furthermore, recent results provide evidence that heat can be generated in brown fat without intracellular lipolysis, and that BAT can absorb and burn FA derived from lipolysis in WAT fat pads. Taken together, these findings leave open the possibility that increased activity in both WAT and BAT may combine to promote increased EE and reduced fat / body weight in DIO mice treated with test substances.

[0130] Increased lipolytic flux from WAT to the liver can cause fatty liver. However, the test substance dose-dependently suppressed lipid synthesis in human primary hepatocytes. The test substance also reduced de novo lipogenesis (DNL) in the liver by approximately 45%. In the liver of diet-induced obese (DIO) mice, Cpt1b was increased dose-dependently, and mRNA levels of Acc2, Fas, and Scd1 were reduced. Fatty liver was prevented and alleviated in DIO mice.

[0131] Test substances increase cardiac pAMPK levels, increase stroke volume, and decrease cardiac glycogen, but do not induce cardiac hypertrophy. Exercise activates cardiac AMPK, increases glucose uptake, and reduces cardiac glycogen levels. Compared to mice fed an HFD, mice fed 0.8 or 2 mg / g of the test substance-HFD for 7 weeks showed a significant increase in cardiac pAMPK levels, and cardiac glycogen content was dose-dependently reduced (Figure 8A). In a separate experiment, a significant increase in FDG uptake was observed in the hearts of mice fed a test substance-HFD (2 mg / g) for 2 weeks compared to mice fed an HFD (Figure 8B). Thus, the cardiac effects of the test substance resemble those of exercise. Compared to an HFD, 7 weeks of administration of the test substance-HFD at 0.8 or 2 mg / g did not cause an increase in heart weight or tibia length (Figure 8C). Furthermore, rats fed a RD and gavaged with 100, 300, and 600 mg / kg / day of the test substance for 6 months did not show an increase in heart or brain weight compared to vehicle. Thus, test substance-mediated AMPK activation in the heart did not cause cardiac hypertrophy.

[0132] Exercise improves cardiac function by increasing stroke volume. Therefore, we examined the effects of test substances on left ventricular (LV) function by echocardiography in mice fed RD, HFD, or test substance-HFD at 0.8 mg / g or 2 mg / kg. Compared with RD, HFD caused a significant decrease in both end-diastolic and end-systolic volumes and a small, non-significant decrease in stroke volume (Figure 8, D-F). Test substance-HFD normalized end-diastolic volume and improved it dose-dependently, but did not completely restore end-systolic volume (Figure 8, D and E). Importantly, test substance-HFD at 0.8 mg / g and 2 mg / g induced a significant increase (approximately 20%) in stroke volume compared with both RD and HFD (Figure 8F). Notably, under these anesthesia conditions, HFD caused a significant increase in heart rate compared to RD, which was normalized by test substance-HFD at 0.8 mg / g and decreased by test substance-HFD at 2 mg / g (Figure 8G). Thus, the test substance normalized the HFD-induced decrease in end-diastolic volume and induced a significant increase in stroke volume, demonstrating that the test substance mimics the beneficial effects of exercise on left ventricular (LV) function.

[0133] The test substance improves microvascular function and endurance in mice and reduces blood pressure in dogs. Decreased microvascular function and peripheral blood flow cause severe complications in type 2 diabetes. Activation of AMPK in endothelial cells and smooth muscle cells promotes vasodilation, and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) increases muscle microvascular perfusion. Therefore, to elucidate the potential effects of test substances on peripheral blood flow, we used laser Doppler imaging to monitor blood perfusion in the left hind paw of DIO mice gavaged with vehicle or test substance (40 mg / kg / day) for 8 weeks. Under these conditions, the test substance significantly increased microvascular blood flow in the hind paw compared with vehicle, without affecting body weight (vehicle-fed mice increased from 24.5 to 34 g, while test substance-fed mice increased from 25.6 to 36.5 g) (Figure 9, A and B). Supporting the notion that the test substance increases microvascular blood flow, which increases heat dissipation, skin surface temperature increased in Zucker rats treated with the test substance.

[0134] Enhanced cardiovascular function is associated with improved endurance in humans and animals. To test whether a test substance can improve endurance, we monitored distance run to exhaustion. To avoid the confounding effects of varying degrees of obesity, we used weight-matched 14-month-old mice fed a RD and gavaged with either vehicle or the test substance (20 mg / kg / day) for 30 days. Treadmill exercise reduced body weight to a similar extent in vehicle-fed mice (33.4 to 31.4 g) and test substance-fed mice (34.1 to 32.2 g). Compared to vehicle, the test substance significantly improved endurance, monitored as distance run to exhaustion (Figure 9C), and significantly reduced the increase in blood lactate levels (Figure 9D), indicating increased oxidative metabolism. Thus, consistent with the beneficial cardiovascular effects observed in DIO mice, the test substance improves endurance in lean, inactive aged mice fed a RD.

[0135] The AMPK activator AICAR has been shown to acutely reduce blood pressure and relax isolated resistance arteries in hypertensive rats. For example, as part of an investigational new drug toxicology package, telemetry studies were performed in conscious dogs after a single dose of the test substance; under these conditions, the test substance acutely reduced blood pressure (Figure 9, E and F). Thus, the test substance improves cardiac stroke volume, increases microvascular perfusion, and reduces blood pressure.

[0136] overview In DIO mice, the test substance increased glucose uptake into skeletal muscle, reduced beta cell stress, and promoted beta cell rest. The test substance improved peripheral microvascular perfusion and reduced blood pressure in the animals. It also activated cardiac AMPK, increased cardiac glucose uptake, reduced cardiac glycogen levels, and improved LV stroke volume in mice, but did not increase heart weight in mice or rats.

[0137] Example 2 - Phase IIa Clinical Trial method Clinical trial design An exploratory proof-of-concept, randomized, parallel-group, double-blind, placebo-controlled, Phase IIa 28-day study (TELLUS) of a novel AMPK activator (test substance; 1,000 mg / day) was conducted in 65 patients with type 2 diabetes who had been taking metformin for at least 3 months. The study aimed to further investigate the safety of the test substance at a single dose and its effect on fasting plasma glucose (FPG).

[0138] TELLUS is listed in the EudraCT database under protocol number 2016-002183-13. This study was conducted in accordance with ethical principles originating from the Declaration of Helsinki and consistent with the International Conference on Harmonisation (ICH) / Good Clinical Practice (GCP), the European Union (EU) Clinical Trials Directive, and applicable local regulatory requirements. The study protocol was approved by the Regional Ethics Committee in Uppsala, Sweden, project number / ID O304-2016-02. Informed consent forms were signed and personally dated by all patients and researchers before any study-related procedures were performed.

[0139] Main inclusion criteria: Male and female patients aged 18–80 years with uncomplicated type 2 diabetes mellitus who had been receiving stable type 2 diabetes treatment with metformin monotherapy for at least 3 months. HbA1c ≥ 6.5% and ≤ 9.0%, rather than FPG on day 1, was selected as the main inclusion criterion.

[0140] Key exclusion criteria were: history of myocardial infarction (MI), unstable angina, stroke, or transient ischemic attack (TIA); congestive heart failure defined as New York Heart Association (NYHA) class III-IV; clinically significant abnormalities in physical examination, echocardiogram (ECG), or clinical chemistry results as determined by the investigator.

[0141] Clinical trial compounds A 5 kg batch of test material was manufactured in compliance with Good Manufacturing Practice (GMP) by Anthem BioSciences Pvt. Ltd., Bangalore, Karnataka, India. The suspension consisted of 20 mg / ml of test material in 2% methylcellulose in phosphate buffer. A 2% methylcellulose suspension matching the color of the active product was used as a placebo. The test material and placebo suspension were manufactured, packaged, and labeled by Recipharm Pharmaceutical Development AB, Solna, Sweden.

[0142] clinical methodology Sixty-five patients were randomly assigned (1:1) to treatment with either the study substance or placebo. The screening visit (Visit 1) took place within 3 weeks before randomization and the start of investigational medicinal product (IMP). Patients were randomized on Day 1 (Visit 2) and assigned to 28 days of treatment with either the study substance or placebo (1:1). Clinical visits were conducted on Days 7, 14, 21, 28, 29, and 40 (Visits 3–8) after randomization and treatment initiation. Patients were admitted to the study clinic the night before Days 1 and 28 (Days −1 and 27, respectively) to ensure a fasting state before samples for fasting blood glucose analysis were collected. Magnetic resonance imaging (MRI) scans were performed after screening but before Day 1 and after the end of treatment according to standardized methods at the University Hospital in Uppsala, Sweden. Data analysis was performed by Antaros Medical in Uppsala. Clinical reading of the acquired scans was performed by radiologists at Antaros Medical. If any clinically significant findings were noted by the radiologists, the investigators were notified, and they were to evaluate and manage the findings according to standard medical / clinical judgment.

[0143] Any findings were reported as baseline or adverse events if they began or worsened after the first dose of study drug. The method used to assess calf muscle microvascular function (a proxy for oxygenation) included dynamic MRI T2* measurements before, during, and after reactive hyperemia (4). Sixty-five patients were randomly assigned (32 to the placebo group and 33 to the test substance group), and 59 patients completed the study (28 and 31 in the two groups, respectively). Because the MRI scans had to be performed after screening and before Day 1, an HbA1c of 6.5% to 9.0% was used as the inclusion criterion, rather than Day 1 FPG. Subsequently, a wide range of FPG values, both <7 and >13.3 mmol / l (<126 to >240 mg / dl), was observed at baseline (13.3 mmol / l (240 mg / dl) represents uncontrolled hyperglycemia), necessitating a post hoc statistical analysis of the change in FPG at day 28 compared with day 1 in type 2 diabetic patients with an FPG greater than 7 mM and less than 13.3 mM at day 1.

[0144] result The test substance improves glycemic homeostasis in type 2 diabetic patients receiving metformin. Based on the beneficial metabolic and cardiovascular effects of the test substance in preclinical animal species, the test substance was selected for clinical development and successfully completed toxicological studies and Phase I safety clinical trials in rats and dogs. Therefore, a 28-day proof-of-concept Phase IIa clinical trial, called TELLUS, was conducted in 65 patients with type 2 diabetes (T2D) stabilized on metformin. Apart from safety, FPG, insulin, and blood pressure were monitored, and microvascular perfusion of the calf muscle was examined by MRI.

[0145] To be included in the TELLUS study, patients with type 2 diabetes were required to undergo and pass an MRI scan before starting treatment. Therefore, HbA1c ≥ 6.5% and ≤ 9.0% at screening, rather than FPG on day 1, was used as the inclusion criteria. Therefore, a post-hoc analysis was performed for patients with an FPG range of > 7 to < 13.3 mmol / L (> 126 to < 240 mg / dL) on day 1, where 13.3 mmol / L (240 mg / dL) represents uncontrolled hyperglycemia. The mean absolute reduction in FPG on day 28 compared to day 1 was -0.10 mM in the placebo group and -0.60 mM in the test substance group (Figure 10, A and B). Wilcoxon rank-sum tests showed statistically significant absolute (P = 0.010) and relative (P = 0.018) decreases in FPG in the test substance group compared with the placebo group; two-way mixed-model ANOVA showed an absolute change of P = 0.049; and one-way mixed-model ANOVA showed a relative change of P = 0.037. Wilcoxon tests showed significant absolute (P = 0.0002) (Figure 10A) and relative (P = 0.0003) decreases in FPG on day 28 compared with day 1 in the test substance group, but not in the placebo group. In DIO mice, a significant decrease in fasting blood glucose was observed after 2 weeks of treatment with test substance plus metformin, with efficacy increasing with treatment duration (Figure 2G). Therefore, the effect of a test substance on FPG in patients with type 2 diabetes may require at least 2 weeks to be observed. As noted above, a significant decrease in FPG within the test substance group occurred between days 21 and 28 (Figure 10B), consistent with the corresponding 14-day time frame in DIO mice (Figure 2G). Furthermore, due to the long plasma t of the test substance, plasma steady-state concentrations are not reached until day 14 in type 2 diabetic patients. Notably, a Wilcoxon test showed a statistically significant decrease in both absolute (P = 0.0097) and relative (P = 0.017) HOMA-IR on day 28 compared to day 1 in the test substance group, but not in the placebo group (Figure 10C). Thus, the test substance improved glycemic homeostasis in type 2 diabetic patients receiving metformin.

[0146] Test substances increase peripheral microvascular perfusion in the calf muscle of type 2 diabetic patients receiving metformin. Because type 2 diabetes is associated with severe microvascular complications and test substances increased peripheral microvascular perfusion in mice, hyperemic microvascular perfusion was monitored in the TELLUS study by MRI and dynamic T2* quantification (the time constant of transverse relaxation caused by local magnetic field inhomogeneity) in the calf muscles of type 2 diabetic patients at screening and on days 27–29. The resulting time graphs of T2* values ​​were analyzed individually, and a series of parameters were extracted by automated curve fitting. As expected based on literature, the peripheral circulatory status of patients in the TELLUS study was not overall impaired at baseline, and no strong indication of an intervention effect was expected. Nevertheless, a two-way analysis of variance revealed a statistically significant increase in Δ-T2* (P = 0.026) in the test substance group compared with the placebo group at day 28 compared to baseline, manifested as the difference between the minimum ischemia value and the peak hyperemia value, indicating increased hyperemic perfusion. Furthermore, a Wilcoxon test revealed a significant relative increase in the T2* slope, defined as the percentage increase in hyperemic perfusion, in the test substance group but not in the placebo group on day 28 compared to baseline (P = 0.012). However, in subjects with relatively reduced peripheral circulation, the peak was poorly defined, making it difficult to correctly identify the peak characteristics associated with reactive hyperemia. Therefore, in post-hoc analyses, curve fitting was performed at the group level instead, since image noise and signal drift were averaged across many subjects. Significance testing was performed by a permutation test, a nonparametric resampling method. Under these conditions, there was a significant increase in both delta-T2* (P = 0.037) and T2* slope (P = 0.024) on day 28 compared to baseline in the test substance group compared to the placebo group. Thus, the test substance was found to increase microvascular perfusion in the calf muscle of type 2 diabetic patients receiving metformin, as assessed by changes in T2* slope and Δ-T2* at day 28 compared to baseline (Table 4).

[0147] [Table 4]

[0148] Finally, to clarify whether subjects with relatively low baseline perfusion responded to treatment, we split the test substance and placebo groups in half based on baseline time to peak (TTP). Short and long TTP represent subjects with relatively high and low rates of hyperemic perfusion, respectively. Next, we performed a permutation analysis comparing baseline MRI (MRI1) with end-of-treatment MRI (MRI2). This stratified analysis revealed a significant shortening of TTP (P = 0.043) and an increase in Δ-T2* (P = 0.034) in subjects with relatively low baseline perfusion rates (long TTP) in the test substance group. However, this was not observed in subjects with short TTP, and there was no difference between subjects with short and long baseline TTP in the placebo group (Figure 10D). Thus, the test substance preferentially increases hyperemic microvascular perfusion in the calf muscles of type 2 diabetic patients with relatively low baseline perfusion rates.

[0149] The test substance reduces blood pressure in type 2 diabetic patients receiving metformin. The microcirculation regulates peripheral vascular resistance and, in combination with cardiac output, determines arterial blood pressure. AICAR acutely reduced blood pressure in spontaneously hypertensive rats, and this test substance acutely reduced blood pressure in dogs (Figure 9, E and F). As noted above, mean absolute decreases in systolic blood pressure (-5.8 mmHg) and diastolic blood pressure (-3.8 mmHg) were observed on day 28 compared with day 1 in the test substance group, whereas slight increases of +1.2 mmHg and +0.9 mmHg, respectively, were observed in the placebo group. Wilcoxon tests within the test substance group but not the placebo group revealed statistically significant absolute decreases in both systolic blood pressure (P = 0.030) and diastolic blood pressure (P = 0.009), as well as relative decreases in systolic blood pressure (P = 0.036) and diastolic blood pressure (P = 0.014) (Figure 10E). One-way analysis of variance showed a statistically significant relative decrease in systolic blood pressure (P = 0.047) and diastolic blood pressure (P = 0.044) in the test substance group compared with the placebo group. No significant change in mean heart rate was observed in either group on day 28 compared with baseline: placebo, -0.48; test substance, -1.6 bpm. Thus, the test substance reduces systolic and diastolic blood pressure in patients with type 2 diabetes. Therefore, the effects of the test substance on fasting plasma glucose (FPG), microvascular perfusion, and blood pressure can be extrapolated from animals to patients with type 2 diabetes.

[0150] summary As described above, in a 28-day proof-of-concept Phase IIa clinical trial in patients with type 2 diabetes treated with metformin, the test substance reduced fasting plasma glucose (FPG) and homeostasis model assessment of insulin resistance (HOMA-IR), and the reduction was well tolerated. The test substance also improved peripheral microvascular perfusion and reduced blood pressure in patients with type 2 diabetes. Therefore, the major metabolic and vascular effects in animals were applicable to patients with type 2 diabetes.

[0151] References 1.Ahren B, Simonsson E, Scheurink AJ, Mulder H, Myrsen U, and Sundler F. Dissociated insulinotropic sensitivity to glucose and carbachol in high-fat diet-induced insulin resistance in C57BL / 6J mice.Metabolism.1997; 46(1):97-106. 2.Ly PT, Cai F, and Song W. Detection of neuritic plaques in Alzheimer's disease mouse model.J VisExp. 201153). 3.Steneberg P, Rubins N, Bartoov-Shifman R, Walker MD, and Edlund H. The FFA receptor GPR40 links hyperinsulinemia, hepatic steatosis, and impaired glucose homeostasis in mouse.Cell Metab.2005; 1(4):245-58. 4.Jacobi B, Bongartz G, Partovi S, Schulte AC, Aschwanden M, Lumsden AB, Davies MG, Loebe M, Noon GP, ​​Karimi S, et al.Skeletal muscle BOLD MRI: from underlying Physiological concepts to its usefulness in clinical conditions.J Magn Reson Imaging.2012; 35(6):1253-65.

[0152] Example 3 - AMPK activator + SGLT2 inhibitor Test Compound The test substances used in this study were: (A) 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide (referred to herein as "Compound 1") (synthesized and purified by Anthem Biosciences Pvt. Ltd., Bangalore, India), and (B) Canagliflozin.

[0153] Animals and Management Eight-week-old male C57BL / 6J mice were purchased from Charles River Laboratories, Jackson, Germany. All animals were housed in the Umea University animal facility (Umea Centre for Comparative Biology; UCCB) under a 12:12-h light / dark cycle (lights on at 6:00 AM) and a constant temperature of 21°C. Animals were ear-marked with unique identification numbers and housed in groups of five mice in transparent polycarbonate cages complying with the requirements of the Good Practice for the Housing and Care of Animals Used in Scientific Procedures. Wood chips were used as bedding to enrich the environment. The animals were allowed 15 weeks to acclimate to their new environment before the start of the study. Animals were allowed free access to tap water throughout the housing and study period. During the acclimatization period, animals were fed a standard pelleted diet (CRM(E) Rodent, Special Diets Services, Scanbur BK, Sweden). At the start of the study, the standard diet was changed to a very high fat diet (vHFD; Cat. No. D12492, Research Diets, Inc.), and this diet was maintained throughout the study period. All procedures were performed at the Local Dietary Laboratory in the Umeå region. Approved by the Ethics Review Committee on Animal Experiments.

[0154] Reagents and materials for biochemical analysis Ultra-sensitive Mouse Insulin ELISA Kit (Cat. No. 90080, Crystal Chem.), OneTouch® Ultra® Test Strips (LifeScan, Inc.), OneTouch® Ultra® 2 Blood Glucose Meter (LifeScan, Inc.), Microvette® CB300 Potassium-EDTA Vials (Cat. No. 16.444, Sarstedt).

[0155] Experimental setup: Twenty-three-week-old male C57BL / 6J mice were fed a HFD to promote diet-induced obesity (DIO) and were orally gavaged once daily with the following: vehicle (phosphate buffer pH 7.3, 2% w / v methylcellulose; n = 11), canagliflozin 10 mg / kg (n = 14), compound 1 75 mg / kg (n = 14), and a combination of canagliflozin 10 mg / kg and compound 1 75 mg / kg (n = 15). After 2 weeks of treatment, fasting (6-hour) blood glucose and plasma insulin levels (measured via tail vein blood samples) were analyzed. Food intake and body weight were monitored throughout the study.

[0156] biochemical analysis Blood samples were collected from the tail vein into potassium-EDTA vials, and plasma was separated by centrifugation and stored at -20°C until analysis. Plasma insulin was measured by mouse insulin ELISA (Ultrasensitive Mouse Insulin ELISA Kit). Glucose concentrations in tail vein blood were analyzed using a OneTouch® Ultra® 2 blood glucose meter (LifeScan, Inc.).

[0157] Data analysis The results shown in the figures are expressed as the mean ± standard error of the mean (SEM) of the number of animals per group. Statistical significance between the control group and the three treatment groups was analyzed by Student's t-test, and P < 0.05 was considered statistically significant.

[0158] result The results are shown in Figure 11. The combination of Compound 1 and an SGLT2 inhibitor resulted in a statistically significant (***) decrease in fasting blood glucose, fasting plasma insulin, and HOMA-IR results. Compound 1 alone showed a statistically significant (***) decrease in fasting plasma insulin and HOMA-IR results. The SGLT2 inhibitor reduced fasting blood glucose, fasting plasma insulin, and HOMA-IR results to a lesser extent.

[0159] conclusion SGLT2 inhibitors are contraindicated in type 2 diabetes patients with impaired renal function because they lack antiglycemic effects. However, the combination of compound 1 and canagliflozin was found to potently and synergistically reduce hyperglycemia, hyperinsulinemia, and insulin resistance in diet-induced obese mice (Figure 11), indicating that the combination of these two classes of compounds has the potential to both potently improve glucose homeostasis and prevent diabetic kidney disease in type 2 diabetes patients. A subgroup of type 2 diabetes patients with severe insulin-resistant diabetes (obesity (BMI ≈35), insulin resistance, and hyperinsulinemia) is at five times higher risk of developing diabetic kidney disease and currently lacks effective treatments. These patients may particularly benefit from such combination therapy with the compound of Formula I and an SGLT2 inhibitor.

Claims

1. 1. An agent for use in a method for treating obesity in a human in need thereof, comprising a compound of formula I: 【Chemical 1】 or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the method treats obesity by increasing energy expenditure through enhancing energy metabolism, reducing body weight, and / or inhibiting weight gain.

3. The human is at least 30 kg / m ２ The agent according to claim 1, having a BMI of:

4. The agent according to any one of claims 1 to 3, wherein the agent is administered orally.

5. 1. A method for reducing weight gain in a human, comprising administering to a subject a compound of formula I: 【Chemistry 2】 or a pharmaceutically acceptable salt thereof.

6. The agent according to claim 5, which suppresses weight gain by increasing energy metabolism.

7. 1. A method for reducing body weight and increasing energy expenditure in a subject in need thereof, comprising administering to said subject an amount of a compound of formula I that reduces body weight and increases energy expenditure; 【Chemistry 3】 or a pharmaceutically acceptable salt thereof.

8. The method according to claim 7, wherein the subject is obese.

9. The agent according to claim 7 or 8, wherein the subject has diabetes.

10. The agent according to any one of claims 5 to 9, wherein the agent is administered orally.

11. a compound of formula I in the manufacture of a medicament for use in a method for treating obesity in a human in need thereof; 【Chemistry 4】 or a pharmaceutically acceptable salt thereof.

12. 12. The use of claim 11, wherein the method treats obesity by increasing energy expenditure by enhancing energy metabolism, resulting in weight loss and / or inhibiting weight gain.

13. The human is at least 30 kg / m ２ The use according to claim 11, wherein the patient has a BMI of:

14. The use according to any one of claims 11 to 13, wherein the agent is administered orally.

15. a compound of formula I in the manufacture of a medicament for use in a method for inhibiting weight gain in a human; 【Chemistry 5】 or a pharmaceutically acceptable salt thereof.

16. The use according to claim 15, wherein weight gain is suppressed by increasing energy metabolism.

17. 17. The use according to claim 15 or 16, wherein the agent is administered orally.

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