WO2014000058A1 - Method of treating glucose metabolism disorders - Google Patents

Abstract

The present disclosure provides a method of treating or preventing an abnormality of glucose metabolism in a subject, the method comprising administering an antagonist of lysosomal acid lipase (LAL) to the subject such that LAL is antagonized in a pancreatic β cell of the subject.

Description

METHOD OF TREATING GLUCOSE METABOLISM DISORDERS

RELATED APPLICATION DATA

The present application claims priority from Australian Provisional Patent Application No. 2012902788 filed on 29 June 2012 and entitled "Method of treating glucose metabolism disorders". The entire contents of that earlier application are hereby incorporated by reference.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference.

FIELD

The present disclosure relates to methods of treating or preventing an abnormality of glucose metabolism or increasing glucose-stimulated insulin secretion (GSIS).

BACKGROUND

Type 2 diabetes is a serious health concern, particularly in more developed societies that ingest foodstuffs high in sugars and/or fats. The disease is associated with blindness, heart disease, stroke, kidney disease, hearing loss, gangrene and impotence. Type 2 diabetes and its complications are leading causes of premature death in the Western world. There are an estimated 23.6 million people in the United States (7.8% of the population) with diabetes with 17.9 million being diagnosed, 90% of whom suffer from type 2 diabetes. With prevalence rates doubling between 1990 and 2005, the Center for Disease Control (CDC) in USA has characterized the increase as an epidemic. Traditionally considered a disease of adults, type 2 diabetes is increasingly diagnosed in children in parallel to rising obesity rates due to alterations in dietary patterns as well as in life styles during childhood

Generally, type 2 diabetes adversely affects the way the body converts or utilizes ingested sugars and starches into glucose. The majority of overweight and obese individuals do not develop diabetes because their pancreatic β-cells adequately respond and prevent overt hyperglycaemia through increased insulin secretion. This is known as β-cell compensation. Those who progress to type 2 diabetes do so because insulin secretion cannot match insulin demand. In this regard, type 2 diabetes is associated with a progressive decline in β-cell function, which is manifest primarily as a selective loss of GSIS. There is now good evidence for reduced β-cell mass linked with increased rates of β-cell apoptosis in people suffering from type 2 diabetes relative to weight-matched subjects without diabetes.

In most type 2 diabetes subjects, the metabolic entry of glucose into various "peripheral" tissues is reduced and there is increased liberation of glucose into the circulation from the liver. Thus, there is an excess of extracellular glucose and a deficiency of intracellular glucose. Elevated blood lipids and lipoproteins are a further common complication of diabetes. The cumulative effect of these diabetes-associated abnormalities is severe damage to blood vessels and nerves.

Many available treatments for type 2 diabetes, some of which have not changed substantially in many years, have recognized limitations. For example, while physical exercise and reductions in dietary intake of fat, high glycemic carbohydrates, and calories can dramatically improve the diabetic condition, compliance with this treatment is very poor because of well-entrenched sedentary lifestyles and excess food consumption, especially of foods containing high amounts of saturated fat.

Conventional drug-based treatments for type 2 diabetes are very limited, and focus on attempting to control blood glucose levels to minimize or delay complications. Current treatments target either insulin resistance (metformin, thiazolidinediones ("TZDs")), or insulin release from the β-cells (sulphonylureas, exenatide). Sulphonylureas, and other compounds that act by depolarizing the β-cell, have the side effect of hypoglycemia since they cause insulin secretion independent of circulating glucose levels. Other side effects of current therapies include weight gain, loss in responsiveness to therapy over time, gastrointestinal problems, and edema.

One currently approved drug, Januvia (sitagliptin, a dipeptidyl peptidase IV (DPPrV) inhibitor) increases blood levels of incretin hormones (e.g., glucagon-like peptide (GLP)-l), which can increase insulin secretion, reduce glucagon secretion and have other less well characterized effects. However, Januvia and other DPPIV inhibitors may also influence the tissue levels of other hormones and peptides, and the long-term consequences of this broader effect have not been fully investigated. For example, DPPIV is a tumor suppressor, and inhibition of this enzyme may increase the risk of some cancers, e.g., non-small cell lung cancer.

The use of clinically available agents that increase intracellular availability of GLP-1, such as orally active DPPIV inhibitors or injectable GLP-1 analogs, are also limited as a result of relatively short half-life of these agents. This means that they require frequent administration.

Another approach considered for the treatment of type 2 diabetes is the use of general lipase inhibitors to prevent fat digestion to thereby control body weight and risk of this condition. However, a weakness of this approach is that general lipase inhibitors, such as, orlistat inhibit glucose stimulated insulin secretion (GSIS; Mulder et al, Diabetes 53: 122-128, 2004). This is likely because they are required for appropriate glucose sensing in pancreatic β-cells.

It is clear from the foregoing that there is a need in the art for a method to treat or prevent or delay the onset or progression of abnormalities of glucose metabolism, e.g., type 2 diabetes.

Summary

The present disclosure is based on the inventors' finding that inhibiting a specific lipase that is expressed in pancreatic β cells is sufficient to stimulate GSIS. For example, the inventors have found that by inhibiting lysosomal acid lipase (LAL) they could stimulate GSIS in pancreatic β cells. The inventors demonstrated this effect using a small molecule inhibitor of LAL, siRNA knockdown of LAL expression and cells from mice lacking LAL expression. These findings provide the basis of inducing or improving GSIS or treating or preventing a glucose metabolism disorder. These findings also provide the basis for supplementing existing therapies for glucose metabolism disorders.

The present disclosure provides a method of treating or preventing an abnormality of glucose metabolism in a subject, the method comprising administering an antagonist of LAL to the subject such that LAL is antagonized in a pancreatic β cell of the subject.

The present disclosure provides a method of treating or preventing an abnormality of glucose metabolism in a subject, the method comprising administering an antagonist of LAL to the subject such that LAL is antagonized in a pancreatic β cell of the subject and GSIS is increased in the subject.

The present disclosure alternatively or additionally provides a method for increasing GSIS in a subject, the method comprising administering an antagonist of LAL to the subject such that LAL is antagonized in a pancreatic β cell of the subject. In one example, the subject has reduced or impaired GSIS. A subject having reduced or impaired GSIS can be readily determined by a medical practitioner based on accepted criteria at the time, e.g., as advised by the World Health Organisation or national body (eg American Diabetes Association, National Health and Medical Research Council Australia).

The present disclosure alternatively or additionally provides a method for increasing GSIS in a subject, the method comprising administering an antagonist of LAL to the subject such that LAL is antagonized in a pancreatic β cell of the subject and GSIS is increased in the subject. In one example, the antagonist is administered in an amount sufficient to increase GSIS in the subject.

In one example, the subject is in need of treatment, for example, the subject suffers from an abnormality of glucose metabolism. Exemplary conditions characterised by an abnormality of glucose metabolism are selected from the group consisting of type 2 diabetes and hyperglycaemia and combinations thereof.

In one example, the subject suffers from type 2 diabetes.

In one example, the subject does not suffer from hepatic steatosis or nonalcoholic fatty liver disease.

In one example, the subject is receiving treatment with a therapeutic compound for type 2 diabetes and/or to improve glucose tolerance or sensitivity. Suitable compounds are described herein. In this manner, a method of the disclosure can be adjunctive.

Without being bound by theory or mode of action, the inventors consider that the antagonist of LAL increases GSIS in pancreatic β cells by permitting an accumulation of substrate (e.g., triaglycerol) that would normally be degraded by LAL. Neutral lipases (e.g., adipose triglyceride lipase and/or hormone sensitive lipase activity), which are activated in the presence of glucose, then metabohze the substrate and this metabolism mediates the amplification phase of GSIS.

In one example, following administration of the antagonist of LAL, the subject retains detectable adipose triglyceride lipase and/or hormone sensitive lipase activity.

In one example, following administration of the antagonist of LAL, the level of triaglycerol in the pancreatic β cells increases compared to the level prior to administration of the antagonist.

In one example, the antagonist of LAL specifically antagonizes LAL.

In one example, the antagonist of LAL antagonizes human LAL.

Exemplary antagonists of LAL are small molecules or nucleic acids.

In some examples, the antagonist binds to LAL. In one example, the antagonist is a competitive inhibitor of LAL.

Exemplary small molecule inhibitors of LAL are thiadiazole carbamates, such as, 3,4-disubstituted thiadiazole carbamates. In one example, C(3) of the thiadiazole ring comprises a piperidine or a morpholine substituent. In one example, C(4) is a piperidine or azepane.

In one example, the antagonist of LAL has the structure:

Formula 1

Wherein,

X is CH₂ or O,

Y is CH₂ or O, and

n = 1-3.

En exemplary the antagonist of LAL has the structure of Formula 1 and wherein

(i) X is CH₂, Y is CH₂ and n=l

(ii) X is CH₂, Y is O and n=l;

(iii) X is CH₂; Y is CH₂ and n=2;

(iv) X is CH₂; Y is O and n=2;

(v) X is O; Y is CH₂ and n=2;

(vi) X is O; Y is CH₂ and n=2;

(νϋ) X is O, Y is O and n=2;

(viii) X is CH₂, Y is CH₂ and n=3; and

(ix) X is CH₂, Y is O and n=3.

For example, the antagonist of LAL has the structure of Formula 1 and wherein X is CH₂; Y is O and n=2 (also referred to herein as "lalistat"). For example, the antagonist has the structure:

Formula 2 ("lalistat")

In one example, the antagonist of LAL reduces or prevents LAL expression.

For example, the antagonist of LAL is a nucleic acid that binds to LAL encoding nucleic acid. For example, the antagonist of LAL is an antisense oligonucleotide, siRNA, RNAi, ribozyme, or DNAzyme.

In one example, the antagonist of LAL is administered in the form of a pharmaceutical composition.

In one example, the antagonist of LAL is administered a plurality of times. In this regard, the inventors have demonstrated that chronic LAL antagonism provides a greater therapeutic benefit than acute antagonism. In one example, the antagonist of LAL is administered a plurality of times such that LAL activity in pancreatic beta cells of the subject is maintained at a reduced level compared to the level in a subject suffering from a glucose metabolism disorder to who the antagonist of LAL has not been administered.

In one example, the antagonist of LAL is administered a plurality of times such that the level of triaglycerol in the pancreatic β cells increases compared to the level prior to administration of the antagonist.

In one example, the antagonist of LAL is administered concurrently with or concomitantly with another therapeutic compound. An exemplary compound is glucagon like peptide 1 (GLP-1), a GLP-1 analog, a GLP-1 receptor agonist, a DPPIV inhibitor, a sulphonurea, a meglitinide, a GPR40 agonist, a GPR119 agonist, a sodium glucose co-transporter-2 inhibitor, a thiazoHdinone, metformin or a glucokinase activator. For example:

(i) the GLP-1 analog or GLP-1 receptor agonist is selected from the group consisting of exenatide, liraglutide, exenatide LAR, taspoglutide, albiglutide and GLP1 conjugated to albumin;

(ii) the DPPIV inhibitor is selected from the group consisting of sitagliptin (JANUVIA) and vidagliptin (GALVUS);

(iii) the sulphonylurea is selected from the group consisting of glibenclamide, glyburide and gliclazide;

(iv) the meglitinide is selected from the group consisting of repaglinide and nateglinide;

(v) the GPR40 agonist is selected from the group consisting of TAK-875 and AMG- 837;

(vi) the GPR119 agonist is selected from the group consisting of PSN632408, JNJ- 38431055;

(vii) the glucokinase activator is selected from the group consisting of GKA50, piragliatin (RO4389620) and ZYGK1;

(viii) the sodium glucose co-transporter-2 inhibitor is empagliflozin; or

(ix) the thiazoHdinone is rosiglitazone, pioglitazone or troglitazone.

By improving GSIS using an antagonist of LAL, the inventors provide a manner to improve the efficacy of another compound in treating a glucose metabohsm disorder or further improving GSIS, wich can result in reduction of the dosage of the compound required to treat a glucose metabolism disorder.

Thus, the present disclosure provides a method comprising administering an antagonist of LAL to the subject such that LAL is antagonized in a pancreatic β cell of the subject, wherein the subject is receiving treatment with a compound for an abnormality of glucose metabolism or for improving GSIS.

For example, the present disclosure provides a method for improving the efficacy of a compound in the treatment of an abnormality of glucose metabolism in a subject, the method comprising administering an antagonist of LAL to the subject such that LAL is antagonized in a pancreatic β cell of the subject, wherein the subject is receiving treatment with the other compound.

The present disclosure alternatively or additionally provides a method for improving the efficacy of a compound in increasing GSIS in a subject, the method comprising administering an antagonist of LAL to the subject such that LAL is antagonized in a pancreatic β cell of the subject, wherein the subject is receiving treatment with the other compound.

The present disclosure also provides a method for reducing the dose of a compound used to treat an abnormality of glucose metabolism or for improving GSIS, the method comprising administering an antagonist of LAL to the subject such that LAL is antagonized in a pancreatic β cell of the subject.

For example, the present disclosure provides a method for reducing the dose of a compound used to treat an abnormality of glucose metabolism, the method comprising administering an antagonist of LAL to the subject such that LAL is antagonized in a pancreatic β cell of the subject, wherein the subject is receiving treatment with the other compound.

The present disclosure alternatively or additionally provides a method for reducing the dose of a compound used to increase GSIS in a subject, the method comprising administering an antagonist of LAL to the subject such that LAL is antagonized in a pancreatic β cell of the subject, wherein the subject is receiving treatment with the other compound.

Exemplary compounds are described herein and are to be taken to apply mutatis mutandis to the present examples of the disclosure.

The present disclosure also provides an antagonist of LAL for use in:

(i) treating or preventing an abnormality of glucose metabolism in a subject (e.g., wherein following administration of the antagonist of LAL to the subject, LAL is antagonized in a pancreatic β cell of the subject) (optionally, wherein the subject is receiving treatment with another compound for the abnormality of glucose metabolism);

(ii) increasing GSIS in a subject having reduced or impaired GSIS (e.g., wherein following administration of the antagonist of LAL to the subject LAL is antagonized in a pancreatic β cell of the subject) (optionally, wherein the subject is receiving treatment with another compound for the reduced or impaired GSIS);

(iii) improving the efficacy of a compound in the treatment of an abnormality of glucose metabolism in a subject;

(iv) improving the efficacy of a compound in increasing GSIS in a subject; and/or (v) reducing the dose of a compound used to treat an abnormality of glucose metabolism in a subject or to increase GSIS in a subject.

The present disclosure also provides for the use of an antagonist of LAL in the manufacture of a medicament for:

(i) treating or preventing an abnormality of glucose metabolism in a subject (e.g., wherein following administration of the antagonist of LAL to the subject, LAL is antagonized in a pancreatic β cell of the subject);

(ii) increasing GSIS in a subject having reduced or impaired GSIS (e.g., wherein following administration of the antagonist of LAL to the subject LAL is antagonized in a pancreatic β cell of the subject);

(iii) improving the efficacy of a compound in the treatment of an abnormality of glucose metabolism in a subject;

(iv) improving the efficacy of a compound in increasing GSIS in a subject; and/or

(v) reducing the dose of a compound used to treat an abnormality of glucose metabolism in a subject or to increase GSIS in a subject

The present disclosure also provides an antagonist of LAL and glucagon like peptide 1 (GLP-1), a GLP-1 analog, a GLP-1 receptor agonist, a DPPIV inhibitor, a sulphonurea, a meglitinide, a GPR40 agonist, a GPR119 agonist, a sodium glucose co- transporter-2 inhibitor, a thiazolidinone, metformin or a glucokinase activator.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A includes a series of graphical representations showing the effect of 2h acute (top panel) or 24h chronic (bottom panel) treatment of ΜΓΝ6 cells with various concentrations of lalistat on GSIS. Reference in the drawing to "2mM G" means "2 mM glucose". Similarly, reference in the drawing to "20mM G" means "20 mM glucose".

Figure IB is a graphical representation showing the effect of pretreating ΜΓΝ6 cells with 5μΜ lalistat for various periods of time ranging from 8h-48h. Following pretreatment cells were stimulated acutely at low (2mM) or high (20mM) glucose (as indicated) for quantifying GSIS. "lali" indicates results from cells treated with lalistat.

Figure 2 includes a graphical representation and copies of photographic representations showing siRNA-mediated knockdown of LAL (as verified by Western blotting shown in lower two panels) potentiates subsequent GSIS (lh) in ΜΓΝ6 beta cells, "cont si" refers to results form cells treated with a control siRNA and "lal si" refers to results from cells treated with siRNA targeting LAL encoding mRNA.

Figure 3 is a graphical representation showing the effect of chronic lalistat treatment on GSIS in isolated pancreatic islets.

Figure 4 is a graphical representation showing the effect of chronic lalistat treatment on GSIS in ΜΓΝ6 cells pretreated with palmitate for 48 h to induce a secretory defect. Results from control and palmitate cells are indicated. Reference in the drawing to "2mM G" means "2 mM glucose". Similarly, reference in the drawing to "20mM G" means "20 mM glucose", "lali" indicates results from cells treated with lalistat.

Figure 5A includes a graphical representation and copies of photographic representations showing siRNA-mediated knockdown of ATG7 (as verified by Western blotting shown in lower two panels) potentiates subsequent GSIS in ΜΓΝ6 beta cells. Results of stimulation with 2mM glucose ("2mM G") or 20mM glucose ("20mM G") are depicted. "Control si" refers to results form cells treated with a control siRNA and "Atg7 si" refers to results from cells treated with siRNA targeting Atg7 encoding mRNA.

Figure 5B is a graphical representation showing inhibition of autophagy by 24h pretreatment of isolated pancreatic islets with 5mM 4-methyladenine (3MA) results in a subsequent potentiation of GSIS. Results of stimulation with 2mM glucose ("G2") or 20mM glucose ("G20") are depicted.

Figure 6 is a series of graphical representations showing the effect of chronic lalistat treatment on neutral lipid accumulation in ΜΓΝ6 cells. Results are shown (as indicated) for diacylglcyerol (DAG), triacylglycerol (TAG), cholesterol ester (CE) and free cholesterol (FC). Reference in the drawing to "G2" means "2 mM glucose". Similarly, reference in the drawing to "G20" means "20 mM glucose", "lali" indicates results from cells treated with lalistat.

Figure 7 is a graphical representation showing expression of various lipases in ΜΓΝ6 cells as assessed by RT-PCR. Lipa, LAL; Atgl, adipose triglyceride lipase; hsl, hormone sensitive lipase; adpn, adiponutrin (PNPLA3); lpl, lipoprotein lipase.

Figure 8 is a graphical representation showing the comparison of GSIS from pancreatic islets isolated from wild-type or LAL knockout mice. KEY TO SEQUENCE LISTING

SEQ ID NO: 1 is an amino acid sequence of a human LAL.

SEQ ID NO: 2 is an amino acid sequence of a human adipose triglyceride lipase.

SEQ ID NO: 3 is an amino acid sequence of a hormone sensitive lipase. SEQ ID NO: 4 is an amino acid sequence of human GLP-1

SEQ ID NO: 5 is an amino acid sequence of exenatide

SEQ ID NO: 6 is an amino acid sequence of liraglutide

SEQ ID NO: 7 is an amino acid sequence of Taspoglutide

SEQ ID NO: 8 is a nucleotide sequence of a RNAi targeting LAL encoding mRNA SEQ ID NO: 9 is a nucleotide sequence of a RNAi targeting LAL encoding mRNA SEQ ID NO: 10 is a nucleotide sequence of a RNAi targeting LAL encoding mRNA SEQ ID NO: 11 is a nucleotide sequence of a RNAi targeting LAL encoding mRNA DETAILED DESCRIPTION

General

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

Each example of the disclosure described herein is to be applied mutatis mutandis to each and every other example unless specifically stated otherwise.

Those skilled in the art will appreciate that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, Π, and ΓΠ; DNA Cloning: A Practical Approach, Vols. I and Π (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu et al, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series; J.F. Ramalho Ortigao, "The Chemistry of Peptide Synthesis" In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany); Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R.L. (1976). Biochem. Biophys. Res. Commun. 73 336-342; Merrifield, R.B. (1963). /. Am. Chem. Soc. 85, 2149-2154; Barany, G. and Merrifield, R.B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York. 12. Wunsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Muler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer- Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer- Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474; Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); and Animal Cell Culture: Practical Approach, Third Edition (John R. W. Masters, ed., 2000), ISBN 0199637970, whole of text.

Any example of the present disclosure in respect of an antagonist of LAL shall be taken to apply mutatis mutandis to lalistat as if the example were written with the word lalistat in place of the words "antagonist of LAL".

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Selected Definitions

For the purposes of nomenclature only, and not limitation a sequence of a human lysosomal acid lipase is set forth in SEQ ID NO: 1. In one example, a reference herein to "lysosomal acid lipase" or "LAL" is a reference to human LAL. Sequences of human LAL are also set forth in NCBI RefSeqs NP_000226.2 and/or NP_001121077.1.

For the purposes of nomenclature only, and not limitation a sequence of a human adipose triglyceride lipase is set forth in SEQ ID NO: 2. In one example, a reference herein to "adipose triglyceride lipase" or "ATGL" is a reference to human ATL. Sequences of human ATGL are also set forth in NCBI RefSeq NP_065109.1.

For the purposes of nomenclature only, and not limitation a sequence of a human hormone sensitive lipase is set forth in SEQ ID NO: 3. In one example, a reference herein to "hormone sensitive lipase" or "HSL" is a reference to human ATL. Sequences of human ATL are also set forth in NCBI RefSeq NP_005348.2.

As used herein, the term "abnormality of glucose metabolism" shall be taken to mean a condition characterised by hyperglycemia and/or β-islet cell dysfunction. For example, the abnormality of glucose metabolism is type 2 diabetes.

As used herein, the term "antagonist of LAL" shall be taken to mean a compound that reduces, prevents or inhibits the activity of LAL protein and/or that reduces, prevents or inhibits expression of LAL. For example, the antagonist binds to LAL or nucleic acid encoding same, i.e., acts directly on LAL or nucleic acid encoding same. In some examples, the antagonist is specific for LAL. A compound that reduces, prevents or inhibits the activity of LAL shall be understood to act at the level of the LAL protein. A compound that reduces, prevents or inhibits expression of LAL, will necessarily reduce the LAL activity level by virtue of reducing the level of the protein, e.g., in a cell. Exemplary methods for assessing LAL activity are described herein.

By "specific for LAL" shall be understood to mean that an antagonist reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with LAL or a cell expressing same than it does with alternative proteins, e.g., other lipase proteins (e.g., ATGL or HSL or pancreatic lipase or intestinal Hpase) or cells. Specific binding does not necessarily require exclusive binding or non-detectable binding to another protein, this is meant by the term "selective binding". Generally, but not necessarily, reference to binding means specific binding, and each term shall be understood to provide explicit support for the other term.

Reference herein to a subject that "retains detectable adipose triglyceride Hpase and/or hormone sensitive lipase activity" will be understood to mean that foUowing administration of an antagonist of LAL to the subject, the levels of ATGL and/or HSL remain detectable in the pancreas (e.g., pancreatic β cells). In one example, the level of ATGL in pancreatic β ceUs is sufficient to release basal and isoproterenol-stimulated glycerol and nonesterified fatty acid (NEFA) and/or the level of HSL is sufficient to hydrolyze first fatty acid from a triacylglycerol molecule. In one example, the level is of ATGL and/or HSL is within about 50% or 60% or 70% or 80% of the level before administration of the antagonist of LAL.

Reference herein to "concurrent" administration of two compounds will be understood to mean that the compounds are administered together or the same time. This does not mean that the compounds are administered in the same solution or simultaneously.

Reference herein to "concomitant" administration of a compound will be understood to mean that the compounds are administered one after the other, such that both compounds are simultaneously active in a subject for a period of time.

As used herein, the terms "preventing", "prevent" or "prevention" in the context of preventing a condition include administering an amount of a protein described herein sufficient to stop or hinder the development of at least one symptom of a specified disease or condition.

As used herein, the terms "treating", "treat" or "treatment" include administering a therapeutically effective amount of an inhibitor(s) and/or agent(s) described herein sufficient to reduce or eliminate at least one symptom of a specified disease or condition.

As used herein, the term "subject" shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include a human or primate. For example, the mammal is a human.

Antagonists of LAL Activity

Exemplary antagonists of LAL antagonize LAL activity. For example, the antagonist binds to and antagonizes LAL activity.

In one example, the antagonist of LAL activity is a small molecule.

An exemplary small molecule antagonist of LAL is esterastin. Esterastin is also a specific antagonist of LAL, in so far as it antagonizes LAL more efficiently than it antagonizes pancreatic lipase or carboxylesterase (Imanaka et al, J Biochem, 94: 1017- is set forth in Formula 3:

Figure 3

Exemplary antagonists of LAL are disclosed in WO2007/053847. For example, an antagonist of LAL comprises the structure set forth in one of the following

Formula 4, wherein

X is O or -N(R⁷)-;

Y is N or -C(R⁸)-;

R¹ and R² represent independently alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R³ is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R² and R³ taken together form a 3-8 member ring optionally substituted with one or more of alkyl, halogen, hydroxy, alkoxy, or amino;

R⁴ is hydrogen, alkyl, cycloalkyl, aryl, or aralkyl;

R⁵ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl,

R⁶ is H or alkyl; or R5 and R6 taken together form a optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from the group consisting of O, N, and S;

R⁷ is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R¹ and R⁷ taken together form a 3-8 member ring optionally substituted with one or more of alkyl, halogen, hydroxy, alkoxy, or amino;

R⁸ and R⁹ represent independently for each occurrence H or alkyl; and n is 1 or 2;

Formula 5, wherein

X is O or -N(R⁶)-; R¹ and R² represent independently cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R³ is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R² and R³ taken together form a 3-8 member ring optionally substituted with one or more of alkyl, halogen, hydroxy, alkoxy, or amino;

R⁴ is hydrogen, alkyl, cycloalkyl, aryl, or aralkyl;

R⁵ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R⁶ is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R¹ and R⁶ taken together form a 3-8 member ring optionally substituted with one or more of alkyl, halogen, hydroxy, alkoxy, or amino;

Formula 6, wherein

R¹ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(C(R⁷)2)n-(CR7=C(R⁷)2);

R² is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or alkyl;

R³ is hydrogen, alkyl, -C0₂R⁸, or -C(0)N(R⁷)(R⁸);

R⁴ and R⁵ represent independently H or alkyl; or R⁴ and R⁵ taken together form a bond;

R⁶ and R⁷ represent independently for each occurrence H or alkyl;

R⁸ represents independently for each occurrence alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

L is a bond, -C(R⁷)₂-, or -(CR⁷=CR⁷)-; and

A¹ and A² represent independently cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR⁷=CR⁷)-aryl, or -(CR⁷=CR⁷)-heteroaryl;

Formula 7, wherein

A is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R¹ is cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR³=CR³)- aryl, or - (CR3=CR3)-heteroaryl;

R² is alkyl, cycloalkyl, heterocycloalkyl, cycloallcenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R³ represents independently for each occurrence H or alkyl; and

R⁴ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Formula 8, wherein

X is O, -N(R⁵)-, -N(R⁵)C(0)-, -C(0)N(R⁵)-, -OC(O)-, -C0₂-, or -N(R⁵)C0₂-;

Y is O, S, or -N(R⁵)-;

R¹ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, axalkyl, or heteroaralkyl;

R2 represents independently for each occurrence H or alkyl, or two R² taken together form =0;

R³ and R⁴ represent independently cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R⁵ represents independently for each occurrence H, alkyl, aryl, or aralkyl; and n is 1, 2, 3, 4, or 5;

wherein

X is O, S, or -N(R⁴)-;

R¹ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(C(R⁵)2)n-(CR5=C(R⁵)2);

R² is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR⁵=CR⁵)-aryl, or - (CR⁵=CR⁵)-heteroaryl;

R³ is H, alkyl, alkenyl, aryl, or heteroaryl; or R² and R³ taken together form an optionally substituted monocyclic or bicyclic ring having 0, 1, or 2 heteroatoms selected from the group consisting of O, N, and S;

R⁴ and R⁵ represent independently for each occurrence H, alkyl, aryl, or aralkyl; and n is 1, 2, 3, 4, or 5;

Formula 10, wherein

X is O or S;

R¹ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or - C(0)R⁵;

R² is H or alkyl;

R³ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or an optionally substituted tricyclic ring having 1 or 2 heteroatoms selected form the group consisting of O, N, and S;

R⁴ is H, alkyl, -C02R6, or -C(0)N(R⁶)₂;

R³ is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, or heteroaralkyl; or R⁵ is an aryl group optionally substituted with one or more of alkyl, halogen, -OR6, -N(R⁶)2, - C0₂R⁶, C(0)N(R⁶)₂, cyano, or nitro; and

R⁶ represents independently for each occurrence H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Formula 11, wherein

X is O or S;

R¹, R³, and A represent independently cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R² and R⁴ represent independently H or alkyl;

R⁵ is an optionally substituted monocyclic or tricyclic ring having 1 , 2, or 3 heteroatoms selected from the group consisting of O, N, and S; or

12, wherein

X¹ is -OR⁵, -SR⁵, or -N(R⁵)₂;

X² represents independently for each occurrence O, S, or -N(R⁵)-;

R¹ represents independently for each occurrence alkyl, halogen, nitro, cyano, alkoxyl, alkenyl, alkynyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl, -N(R⁵)₂, -OH, -C(0)R⁶, - C0₂R⁵, or C(0)N(R⁵);

R² and R⁴ represent independently cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R³ is H, alkyl, or halogen;

R⁵ represents independently for each occurrence H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R⁶ represents independently for each occurrence alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and n is 0, 1, 2, 3, or 4.

In one example, the antagonist of LAL has the structure:

Formula 1

Wherein,

X is CH₂ or O,

Y is CH₂ or O, and

n = 1-3.

En exemplary the antagonist of LAL has the structure of Formula 1 and wherein

(i) X is CH₂, Y is CH₂ and n=l

(ϋ) X is CH₂, Y is O and n=l;

(iii) X is CH₂; Y is CH₂ and n=2;

(iv) X is CH₂; Y is O and n=2;

(v) X is O; Y is CH₂ and n=2;

(vi) X is O; Y is CH₂ and n=2;

(vii) X is O, Y is O and n=2;

(viii) X is CH₂, Y is CH₂ and n=3; and

(ix) X is CH₂, Y is O and n=3.

For example, the antagonist of LAL has the structure of Formula 1 and wherein X is CH₂; Y is CH₂ and n=2.

For example, the antagonist of LAL has the structure of Formula 1 and wherein X is CH₂; Y is O and n=2. For example, the antagonist has the structure:

Formula 2

Antagonists of LAL Expression

In one example, an antagonist of LAL expression binds to LAL encoding nucleic acid and reduces, prevents or inhibits LAL expression.

In one example, the antagonist is a nucleic acid-based antagonist. For example, the antagonist reduces, prevents or inhibits transcription and/or translation of an LAL encoding nucleic acid, e.g., comprising a sequence encoding a protein comprising a sequence set forth in SEQ ID NO: 1. In one example, the compound is an antisense polynucleotide, a ribozyme, a PNA, an interfering RNA, a siRNA, a microRNA. Antisense Polynucleotides

The term "antisense polynucleotide" shall be taken to mean a DNA or RNA, or combination thereof that is complementary to at least a portion of a mRNA encoding LAL and capable of interfering with a post-transcriptional event such as mRNA translation. The use of antisense methods is known in the art.

An antisense polynucleotide of the disclosure will hybridize to a target polynucleotide under physiological conditions. Antisense polynucleotides include sequences that correspond to the structural genes or for sequences that effect control over gene expression or splicing. For example, the antisense polynucleotide may correspond to the targeted coding region of the genes of the disclosure, or the 5'- untranslated region (UTR) or the 3'-UTR or combination of these. It may be complementary in part to intron sequences, which may be spliced out during or after transcription, for example only to exon sequences of the target gene. The length of the antisense sequence should be at least 19 contiguous nucleotides, for example at least 50 nucleotides, and more for example at least 100, 200, 500 or 1000 nucleotides of a nucleic acid comprising a sequence encoding a protein comprising a sequence set forth in SEQ ID NO: 1 or a structural gene encoding same. The full-length sequence complementary to the entire gene transcript may be used. The degree of identity of the antisense sequence to the targeted transcript should be at least 90%, for example 95- 100%.

In one example, the antisense polynucleotide is conjugated to a pancreatic targeting peptide, e.g., as described in WO2009/08916 and/or a protein transduction domain.

Catalytic Polynucleotide

The term "catalytic polynucleotide/nucleic acid" refers to a DNA molecule or

DNA-containing molecule (also known in the art as a "deoxyribozyme" or

"DNAzyme") or an RNA or RNA-containing molecule (also known as a "ribozyme" or "RNAzyme") which specifically recognizes a distinct substrate and catalyses the chemical modification of this substrate. The nucleic acid bases in the catalytic nucleic acid can be bases A, C, G, T (and U for RNA).

Typically, the catalytic nucleic acid contains an antisense sequence for specific recognition of a target nucleic acid, and a nucleic acid cleaving enzymatic activity (also referred to herein as the "catalytic domain"). The types of ribozymes that are particularly useful in this disclosure are a hammerhead ribozyme and a hairpin ribozyme. RNA interference

RNA interference (RNAi) is useful for specifically inhibiting the production of a particular protein. This technology relies on the presence of dsRNAs that contain a sequence that is essentially identical to the mRNA of the gene of interest or part thereof, in this case an mRNA encoding an LAL protein. Conveniently, the dsRNA can be produced from a single promoter in a recombinant vector or host cell, where the sense and anti-sense sequences are flanked by an unrelated sequence which enables the sense and anti-sense sequences to hybridize to form the dsRNA molecule with the unrelated sequence forming a loop structure. The design and production of suitable dsRNA molecules for the present disclosure is within the capacity of a person skilled in the art.

The length of the sense and antisense sequences that hybridize should each be at least 19 contiguous nucleotides, for example at least 30 or 50 nucleotides, such as at least 100, 200, 500 or 1000 nucleotides. The full-length sequence corresponding to the entire gene transcript may be used. In some examples, the lengths are 100-2000 nucleotides. The degree of identity of the sense and antisense sequences to the targeted transcript should be at least 85%, for example at least 90%, such as 95-100%.

RNAi molecules targeting LAL are commercially available from Novus Biologicals, Labome and Santa Cruz Biotechnology, Inc.

Exemplary small interfering RNA ("siRNA") molecules comprise a nucleotide sequence that is identical to about 19-21 contiguous nucleotides of the target mRNA. For example, the siRNA sequence commences with the dinucleotide AA, comprises a GC-content of about 30-70% (for example, 30-60%, such as 40-60%, for example about 45%-55%), and does not have a high percentage identity to any nucleotide sequence other than the target in the genome of the mammal in which it is to be introduced, for example as determined by standard BLAST search.

siRNA molecules targeting LAL are commercially available from Santa Cruz Biotechnology, Inc. Assays

Methods for determining the ability of a compound to antagonize LAL will be apparent to the skilled person.

For example, purified LAL (e.g., human LAL) or cell lysate from cells expressing same are contacted with the compound in the presence of 4- methylumbelliferyl oleate (4MUO). Following a sufficient time for LAL to act fluorescence is measured, e.g., at 355 nm excitation/450 nm emission. A reduction in fluorescence in the presence of the compound compared to in the absence of the compound indicates that the compound is an antagonist of LAL. An exemplary assay is described in Rosenbaum et al, Biochimica et Biophysica Acta 1791 (2009) 1155— 1165.

The assay described in the previous paragraph can be adapted to assess inhibitors of LAL expression by growing cells in the presence of the compound, lysing cells and then performing the assay.

This assay can also be varied to test for specificity of an antagonist of LAL. For example, the assay is performed in the presence of a lipase other than LAL and a compound that antagonizes LAL activity but not the other lipase is considered a specific antagonist of LAL.

Other assays for measuring lipase activity, e.g., based on [¹⁴C]triolein or [¹⁴C]cholesteryl oleate are known in the art and described, for example, in Kuriyama et al, J. Lipid Res.1990. 31: 1605-1612.

An exemplary in vitro method for determining the effect of the antagonist is to contact it to β-cells (e.g., a β-cell line such as MIN6 or HC-9) with the antagonist and assessing its effect, e.g., on insulin secretion, such as in response to glucose stimulation. Exemplary assays for measuring insulin secretion are known in the art and include, for example commercially available enzyme-linked immunosorbent assays (ELISAs) as exemplified herein. An antagonist that increases insulin secretion in response to glucose is considered a an antagonist of LAL.

Alternatively, or in addition, antagonist is administered to an accepted animal model of an abnormality of glucose metabolism, e.g., type 2 diabetes. Exemplary models include high fat fed murine models of type 2 diabetes, high fat fed streptozotocin-treated rodents (Mu et al, Diabetes, 55: 1695-1704, 2006), db/db mice (commercially available), animals transgenic for islet amyloid polypeptide (e.g., as reviewed in Matveyenko et al, ILAR J. 47: 225-33, 2006) or other model as known in the art. A symptom of type 2 diabetes is then assessed, e.g., using a glucose tolerance test or GSIS assessment using islets isolated from the model to assess the effect of the antagonist.

Pharmaceutical Compositions

The antagonist of the present disclosure (syn. active ingredient) is useful for parenteral, topical, oral, or local administration, aerosol administration, or transdermal administration, for prophylactic or for therapeutic treatment.

Formulation of an antagonist to be administered will vary according to the compound, route of administration and formulation (e.g., solution, emulsion, capsule) selected. An appropriate pharmaceutical composition comprising an antagonist to be administered can be prepared in a physiologically acceptable carrier. A mixture of antagonists can also be used. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. A variety of appropriate aqueous carriers are known to the skilled artisan, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents and toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. The antagonist of this disclosure can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to art-known lyophilization and reconstitution techniques.

The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures known to the skilled artisan, and will depend on the ultimate pharmaceutical formulation desired.

The dosage ranges for the administration of the antagonist of the disclosure are those large enough to produce the desired effect. For example, the composition comprises a therapeutically or prophylactically effective amount of the antagonist.

As used herein, the term "effective amount" shall be taken to mean a sufficient quantity of the antagonist to inhibit/reduce/prevent expression and/or activity of LAL in a subject. The skilled artisan will be aware that such an amount will vary depending on, for example, the antagonist and/or the particular subject and/or the type or severity of a condition being treated. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity, e.g., weight or number of antagonists, rather the present disclosure encompasses any amount of the antagonist that is sufficient to achieve the stated purpose. In one example, the "effective amount" is sufficient to inhibit/reduce/prevent expression and/or activity of LAL in pancreatic β cells of the subject, while permitting activity of ATGL and/or HSL.

As used herein, the term "therapeutically effective amount" shall be taken to mean a sufficient quantity of the antagonist to reduce or inhibit one or more symptoms of an abnormality of glucose metabolism and/or to increase GSIS in a subject. As used herein, the term "prophylactically effective amount" shall be taken to mean a sufficient quantity of the antagonist to prevent or inhibit or delay the onset of one or more detectable symptoms of an abnormality of glucose metabolism.

The dosage should not be so large as to cause adverse side effects, such as hyper viscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication. Dosage can vary from about 0.1 mg kg to about 300 mg kg, such as from about 0.2 mg kg to about 200 mg kg, for example from about 0.5 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days.

One or more antagonists of the present disclosure can be administered to an individual by an appropriate route, either alone or in combination with (before, simultaneous with, or after) another drug or agent.

It will be appreciated by those skilled in the art that some nucleic acid antagonists (e.g., siRNA or shRNA or microRNA) of the present disclosure may be introduced into a subject by administering an expression construct of the disclosure or a cell expressing the antagonist. A variety of methods can be used for introducing a nucleic acid encoding the antagonist into a target cell in vivo. For example, the naked nucleic acid may be injected at the target site, may be encapsulated into Hposomes, or may be introduced by way of a viral vector.

Subjects to be Treated

In one example, a subject to be treated by the method of the present disclosure has impaired or inhibited GSIS. Methods for determining GSIS are known in the art and described, for example, in Fehse et al, J. Clin. Endocrinol and Metab., 90: 5991- 5997, 2005. For example, subjects are fasted for about 8 hours and then (if required) insulin is infused to achieve a "normal" plasma glucose level (e.g., about 79- lOlmg/dL). An intravenous glucose bolus is then administered and blood taken regularly to measure insulin levels. A subject that secretes less insulin in response to glucose compared to the mean level of secretion in a population of subjects known not to suffer from a glucose metabolism disorder is considered to suffer from GSIS.

In one example, the subject suffers from diabetes. For example, the subject has been diagnosed as having:

· Fasting plasma glucose level > 7.0 mmol/1 (126 mg/dl) • Plasma glucose > 11.1 mmol/1 (200 mg/dL) two hours after a 75 g oral glucose load as in a glucose tolerance test

• Symptoms of hyperglycemia and casual plasma glucose > 11.1 mmol/1 (200 mg dl)

• Glycated hemoglobin (Hb AIC) > 6.5%

In another example, the subject suffers from pre-diabetes. For example, the subject has been diagnosed as having:

• Fasting blood sugar (glucose) level of:

o 110 to 125 mg dL (6.1 mM to 6.9 mM) - WHO criteria

o 100 to 125 mg/dL (5.6 mM to 6.9 mM) - ADA criteria

· Two hour glucose tolerance test after ingesting the standardized 75 Gm glucose solution the blood sugar level of 140 to 199 mg/dL (7.8 to 11.0 mM).

• Glycated hemoglobin between 5.7 and 6.4 percent

Combinations

An example of a compound that can be administered in a method of the disclosure or formulated with an antagonist of LAL is a GLP-1 analog or a GLP-1 receptor agonist.

In one example, the compound is GLP-1 or a GLP-1 receptor agonist. For example, GLP-1 comprises a sequence set forth in SEQ ID NO: 4. In one example, the GLP-1 is conjugated to or fused with albumin.

In one example, the compound is exenatide (comprising a sequence set forth in SEQ ID NO: 5). Exenatide is a 39-amino-acid peptide, an insulin secretagogue, with glucoregulatory effects.

In one example, the compound is exenatide LAR. Exenatide LAR comprises exenatide encapsulated in microspheres made of poly (D,L) lactic-co-glycolic acid.

In one example, the compound is liraglutide comprising a sequence set forth in SEQ ID NO: 6 and having a C-16 fatty acid (palmitic acid) with a glutamic acid spacer on the lysine residue at position 26.

In one example, the compound is taspoglutide comprising a sequence set forth in SEQ ID NO: 7. Taspoglutide is a 8-(2-methylalanine)-35-(2-methylalanine)-36-L- argininamide derivative of the amino acid sequence 7-36 of human glucagon-like peptide 1.

In one example, the compound is albiglutide, which comprises two copies of a 30 amino acid sequence of human glucagon-like peptide 1 (GLP-1, fragment 7-36) that is DPP-IV resistant (by virtue of an alanine to glycine conversion at amino acid 8) fused with human albumin. In one example, the compound is dulaglutide.

In one example, the compound is a DPPIV inhibitor.

In one example, the compound is sitagliptin as set out in Formula 13:

Formula 13

as set out in Formula 14:

In one example, the compound is a sulphonylurea.

For example, the compound is ghbenclamide as set out in Formula

For example, the compound is glyburide set out in Formula 16:

For example, the compound is gliclazide as set out in Formula 17:

Formula 17

In one example, the compound is a meglitinide.

For example, the compound is repaglinide as set out in Formula 18:

Formula 18

For example, the compound is nateglinide as set out in Formula 19:

HO" ^ΝίΌ Formula 19

In one example, the compound is a GPR40 agonist.

in Formula 20:

Formula 20

For example, the compound is AMG-837 ((S)-3-(4-((4'- (trifluoromethyl)biphenyl-3-yl)methoxy)phenyl)hex-4-ynoic acid) as set out in Formula

Formula 21

In one example, the compound is a GPR119 agonist.

as set out in Formula 22:

In one example, the compound is JNJ-38431055 as set forth in Formula 23

Formula 23

In one example, the compound is a glucokinase activator.

0 as set forth in Formula 24

atin as set forth in Formula 25:

Formula 25

In one example, the compound is an inhibitor of the sodium glucose co- transporter-2 (SGLT-2). For example, the compound is empagliflozin, e.g., comprising a structure as set forth in Formula 26:

In another example, the compound is a thiazolidinone, such as, rosiglitazone, pioglitazone or troglitazone.

In another example, the compound is metformin. Kits

The present disclosure additionally comprises a kit comprising one or more of the following:

(i) an antagonist of LAL; or

(ii) an antagonist of LAL and another compound, e.g., as described herein; and/or (iii) a pharmaceutical composition of the disclosure.

In the case of a kit for therapeutic/prophylactic use, the kit can additionally comprise a pharmaceutically acceptable carrier or diluent.

Optionally a kit of the disclosure is packaged with instructions for use in a method described herein according to any example.

The present disclosure includes the following non-limiting Examples.

EXAMPLE 1: Materials and Methods

Materials:

All tissue culture media, supplements and trypsin for ΜΓΝ6 cells and isolated islets were purchased from Gibco. Protease inhibitor tablets were obtained from Roche Diagnostics. Insulin RIA kits were from Linco/Millipore. PMSF (phenylmethanesulphonylfluoride), sodium orthovanadate, DMSO

(dimethylsulphoxide), fatty acid free fraction V BSA, 1-glucose, and anti-LC3 antibody were from Sigma-Aldritch. L-pdmp (L-t¾reo-l-Phenyl-2-decanoylamino-3- morpholino-l-propanol) was from Matreya-LLC. TaqMan reverse transcription kit and TaqMan PCR probes were from Applied Biosystems. ATG7 (NM_028835) and LAL (NM_021460) ON-TARGETp/wi SMARTpool siRNA, control Non-Targeting siRNA and Dharmafect Transfection Reagent 3 were from Dharmacon. RNeasy kit was from Qiagen. The BCA (bicinchoninic acid assay) protein assay kit was from Pierce. The pre-cast NUPAGE gels, sample buffer, reducing agent, antioxidant and electrophoresis tank were from Invitrogen. The transfer system for immunoblotting, and the protein standard markers were from BioRad. 96-well conical bottom plates for islet insulin secretion assays were from Grenier Bio-one. Lalistat was a gift from Paul Helquist at the University of Notre Dame, USA. Antibodies for immunoblotting were as follows: Anti-14-3-3 from Santa-Cruz, anti-ATG7 from Cell Signalling Technologies and anti- LAL from Novus Biologicals.

Cell Culture:

The mouse insulinoma cell line ΜΓΝ6 was used herein, at passages between 26 and 35. Cells were grown at 37°C and 5% C0₂ in DMEM (Dulbecco's modified Eagle's media; 25mM glucose), supplemented with 10% FCS, lOmM HEPES, 50 units/ml of penicillin and 50μg ml streptomycin.

In some experiments, ΜΓΝ6 cells were incubated for 24 hours with 14C palmitic acid, followed by glucose stimulation (2- or 20mmol/l glucose for lh), and then subjected to subcellular fractionation to determine lipid localisation.

Lalistat and Treatment:

Cells were seeded at 3xl0⁵ cells per well in 24-well plates. For chronic lalistat treatment; cells were grown for 24 h in DMEM, then treated with 1, 5, or 10μΜ lalistat, or 1:2000 DMSO control for 24 h in DMEM and subsequent insulin secretion assays were carried out. For acute treatment, ΜΓΝ6 cells were grown for 48 h in DMEM, with media changed every 24 h, 1 or 5μΜ lalistat or 1:2000 DMSO was added during the insulin secretion assay only. siRNA transfection:

ΜΓΝ6 cells were seeded at 4xl0⁵ cells per well in 12-well plates. ATG7 ON- TARGETp/wi SMARTpool, LAL ON-TARGETp/wi SMARTpool siRNA or Non- Targeting control siRNA were transfected using Dharmafect Transfection Reagent 3. 24 h after transfection, the media was changed to DMEM (25mM glucose) and the cells incubated for 48 h, changing the media after 24 h. Cells were then subjected to immunoblotting, to verify protein knockdown, insulin secretion assays, or confocal microscopy to assess lipid droplets.

Insulin secretion Assays:

To measure insulin release from ΜΓΝ6 cells, cells were preincubated for 1 h in

Kreb's Ringer HEPES buffer (KRHB) containing 0.1% BSA and 2mM glucose. Following this, cells were incubated for 1 h at 37°C with KRHB (plus 0.1%BSA) containing either 2- or 20mM glucose. An aliquot of the buffer was taken, and insulin release measured by radioimmunoassay.

Islet Isohtion and Insulin Secretion Assays:

Islets were isolated essentially as previously described (Cantley et al, Diabetes, 58(8): 1826-34, 2009. After pancreatic digestion, islets were purified using a Ficoll gradient and finally incubated overnight in RPMI 1640 media (Roswell Park Memorial Institute 1640; llmM glucose) supplemented with 10% FCS, 0.2mM glutamine, lOmM HEPES, 50 units/ml of penicillin and 50μg/ml streptomycin. Islets were cultured for a further 48 h with 5μΜ lalistat or DMSO as control, with media changed every 24 h. To inhibit autophagy in isolated islets, following overnight culture after isolation, islets were treated with 0.92%BSA+ 3-MA (3-methyladenine) or BSA alone for 48 h. In some instances islets were also isolated from wild-type FVBN mice, or LAL knockout mice, and further maintained in tissue culture for 48h as above. Insulin secretion was then measured.

For insulin secretion assays, islets were preincubated for 1 h in KRHB with 0.1% BSA and 2mM glucose. Batches of 5 islets were picked, with at least 6 replicates per group, and insulin secretion assays carried out as per ΜΓΝ6 cells.

RNA Analysis:

Total RNA was extracted from ΜΓΝ6 cells using RNeasy minikit from Qiagen and cDNA synthesis was performed on l^g of DNA, using the TaqMan reverse transcription kit. Real time PCR was carried out on a HT-7900 PCR machine using TaqMan probes. The following probes were used: HSL LIPE (Mm_00495359_ml), DGAT1 (Mm01197412_gl), PNPLA2/ATGL (Mm_00503040_ml), PNPLA3/ADPN (Mm00504420_ml), LPL (Mm00434770_ml), ABHD5 (Mm00470731_ml), TSPO (Mm00437828) and LIPA/LAL (Mm00498820). Analysis was carried out using the standard curve method, and relative gene expression was normalised to TBP (Mm00446971) as an endogenous control.

Western Blotting:

Total cell protein from ΜΓΝ6 cells was quantified using BCA from Pierce, and equalised between treatment groups before undergoing Western blot analysis. Between 20 and 30μg protein was resolved on a pre-cast 12% SDS-PAGE gel from Invitrogen, and transferred to polyvinylidine difluoride membrane. After blocking for 1 h in 5% skim-milk, membranes were probed with the following antibodies overnight at 4°C; anti-ATG7, anti-LAL, anti-LC3, anti-pan 14-3-3. After chemiluminescent detection quantification was carried out using ImageJ.

Lipidomic Profiling using Mass Spectrometry:

Following 24h lalistat and ΜΓΝ6 cells were pelleted in ice cold PBS (2 wells of a 6-well plate per condition) and extracted using chloroform methanol (2:1, v/v) including 100 pmol of Cer 17:0, GlcCer 16:0 d3, LC) 16:0 d3 and THC 17:0 (Matreya Inc., Pleasant Gap, USA), PC 13:0/13:0, PG (phosphatidylglycerol) 17:0/17:0, PE (phosphatidylethanolamine) 17:0/17:0, PS (phosphatidylserine) 17:0/17:0, SM 12:0 (Avanti Polar Lipids, Alabaster, USA), 500 pmol of DAG (diacylglycerol) 15:0/15:0 and TAG (triacylglycerol) 17:0/17:0/17:0 (Sigma-Aldrich) and 1000 pmol of CE (cholesteryl ester) 18:0 (d6) (CDN Isotopes, Pointe-Claire, Quebec, Canada) as internal standards. Analysis was performed by electrospray ionisation-tandem MS (3) using a PE Sciex API 4000 Q/TRAP mass spectrometer with a turbo-ionspray source and Analyst 1.5 data system. Prior liquid chromatographic separation was performed on a Zorbax C18, 1.8 um, 50x2.1 mm column at 300 μΐ min using the following gradient conditions for all lipid species except DAGs and TAGs; 100% A to 0% A over 8.0 min followed by 2.5 min at 0% A, a return to 100% A over 0.5 min then 3.0 min at 100% A prior to the next injection. Solvent A and B consisted of tetrahydrofuran:methanol:water in the ratios (30:20:50) and (75:20:5) respectively, both containing 10 mM NH₄COOH. Chromatography of DAG and TAG species was performed in isocratic mode using 85% A, 15% B at 100 μΐ min. Quantification of individual lipid species was performed using scheduled multiple-reaction monitoring in positive ion mode. Individual lipid species monitored were the major species (greater than 1% of total) identified in human plasma. Multiple-reaction monitoring experiments were based on product ion of m z 369 [cholesterol-H₂0]⁺ for CE. Product ions for DAGs and TAGs were based on the neutral loss of one FA from the [M^NFLJ^"1" ion. Each ion pair was monitored for 10-50 ms with a resolution of 0.7 amu at half- peak height and averaged from continuous scans over the elution period. Lipid concentrations were calculated by relating the peak area of each species to the peak area of the corresponding internal standard. PI species were related to the PE internal standard. For DAG and TAG species containing two or three of the same FA used as a neutral loss, the signal response was corrected by dividing by the number of copies of the FA. Total lipids of each class were determined by summing the individual lipid species.

Statistical Analysis:

All data is expressed as mean± S.E.M. All statistics were performed using GraphPad Prism software, and subjected to either; two-way ANOVA (with Bonferroni post-test), one-way ANOVA (with Bonferroni post-test) or paired Student's i-test.

EXAMPLE 2: Results

As shown in Figure 1A, chronic treatment with lalistat (i.e., for about 24 hours) dose-dependently augmented a subsequent acute (lh) glucose-stimulated insulin secretion from ΜΓΝ6 beta cells. As is shown in Figure IB, this beneficial effect is seen after as little as 8 hours treatment with lalistat and is maintained during chronic treatment. For example, treatment of cells with 5μΜ lalistat resulted in a 1.6 fold increase in GSIS compared to control (p<0.001). A similar effect was also seen when LAL expression was knocked down in ΜΓΝ6 cells by siRNA (as shown in Figure 2).

Similarly, treatment of isolated pancreatic β cells with lalistat (5μΜ for 48 hours) augments subsequent GSIS (Figure 3).

When ΜΓΝ6 cells were pretreated with palmitate for 48 hours to induce an insulin secretion defect, lalistat still augmented insulin secretion, albeit to a lesser degree than in the absence of palmitate pretreatment (see Figure 4). These data indicate that the effect of lalistat can augment GSIS even in pancreatic β-like cells that are defective in insulin secretion.

The foregoing data indicate that inhibition of LAL activity or expression potentiates or augments GSIS. Thus, these data indicate that antagonism of LAL provides a means to improve GSIS in subjects in which this response is deficient (e.g., subjects suffering from glucose metabolism disorders).

The results obtained with lalistat are reminiscent of those obtained by inhibiting autophagy by knocking down expression of atg7 using siRNA in ΜΓΝ6 cells or by inhibiting autophagy in mouse β cells using 3MA. For example, inhibiting expression of atg7 resulted in a 1.5 fold increase in GSIS (see Figure 5 A), and treating cells with 3MA resulted in a 2 fold increase in GSIS (see Figure 5B).

To further characterize how antagonism of LAL may be providing a therapeutic effect, the effect of this compound on lipid profile of ΜΓΝ6 cells was assessed. As is shown in Figure 6, chronic lalistat (24hour, 5μΜ) augments neutral lipid accumulation in ΜΓΝ6 cells. For example, lalistat potentiates accumulation of diacylglcyerol (DAG; about 2 fold) and mobilization of triacylglycerol (TAG; about 1.7 fold) in response to acute glucose exposure, and also augments cholesterol ester (CE; about 6.7 fold) but not free cholesterol (FC). Moreover, DAG was further increased about 2 fold following 20mM glucose treatment.

Without being bound by theory or mode of action, the data presented above indicate that by inhibiting LAL (e.g., using lalistat or siRNA) lipophagy is inhibited, thereby leading to accumulation of a pool of TAG such that can be mobilized in response to acute glucose stimulation (mediated by neutral lipases, ATGL and HSL). In this regard, as is shown in Figure 7, ATGL and HSL are both expressed in pancreatic β cells.

Similarly, subsequent GSIS was augmented from LAL knockout versus wild- type islets when assayed following 48 in tissue culture (Figure 8).

Claims

CLAIMS:

1. A method of treating or preventing an abnormality of glucose metabolism in a subject, the method comprising administering an antagonist of lysosomal acid lipase (LAL) to the subject such that LAL is antagonized in a pancreatic β cell of the subject.

2. A method for increasing glucose stimulated insulin secretion in a subject having reduced or impaired GSIS, the method comprising administering an antagonist of lysosomal acid lipase (LAL) to the subject such that LAL is antagonized in a pancreatic β cell of the subject.

3. The method of claim 1 or 2, wherein the subject suffers from an abnormality of glucose metabolism.

4. The method of any one of claims 1 to 3, wherein the subject suffers from a condition selected from the group consisting of type 2 diabetes, hyperglycaemia and combinations thereof.

5. The method of any one of claims 1 to 3, wherein the subject suffers from type 2 diabetes.

6. The method of any one of claims, wherein the subject retains detectable adipose triglyceride lipase and/or hormone sensitive lipase activity following administration of the antagonist of LAL.

7. The method of any one of claims 1 to 6, wherein the antagonist of LAL specifically antagonizes LAL.

8. The method of any one of claims 1 to 7, wherein the antagonist of LAL antagonizes human LAL.

9. The method of any one of claims 1 to 8, wherein the antagonist of LAL is a small molecule or a nucleic acid.

10. The method of any one of claims 1 to 9, wherein the antagonist binds to LAL.

11. The method of claim 10, wherein the antagonist is a thiadiazole carbamate.

12. The method of claim 11, wherein the antagonist is a 3,4-disubstituted thiadiazole carbamate.

13. The method of claim 11 or 12, wherein the antagonist of LAL has the structure:

Formula 1

Wherein,

X is CH₂ or O,

Y is CH₂ or O, and

n = l-3.

14. The method of claim 13, wherein the antagonist of LAL has the structure of Formula 1 and wherein

(i) X is CH₂, Y is CH₂ and n=l

(ii) X is CH₂, Y is O and n=l;

(iii) X is CH₂; Y is CH₂ and n=2;

(iv) X is CH₂; Y is O and n=2;

(v) X is O; Y is CH₂ and n=2;

(vi) X is O; Y is CH₂ and n=2;

(νϋ) X is O, Y is O and n=2;

(viii) X is CH₂, Y is CH₂ and n=3; and

(ix) X is CH₂, Y is O and n=3.

15. The method of claim 13 or 14, wherein the antagonist of LAL has the structure of Formula 1 and wherein X is CH₂; Y is O and n=2.

16. The method of any one of claims 1 to 9, wherein the antagonist of LAL reduces or prevents LAL expression.

17. The method of claim 16, wherein the antagonist of LAL is a nucleic acid that binds to LAL encoding nucleic acid.

18. The method of any one of claims 1 to 17, wherein the antagonist of LAL is administered in the form of a pharmaceutical composition.

19. The method of any one of claims 1 to 18, wherein the antagonist of LAL is administered a plurality of times.

20. The method of claims 19, wherein the antagonist of LAL is administered a plurality of times such that LAL activity in pancreatic beta cells of the subject is maintained at a reduced level compared to the level in a subject suffering from a glucose metabolism disorder to who the antagonist of LAL has not been administered.

21. The method of any one of claims 1 to 20, wherein the antagonist of LAL is administered concurrently with or concomitantly with another therapeutic compound.

22. The method of claim 21, wherein the other therapeutic compound is glucagon like peptide 1 (GLP-1), a GLP-1 analog, a GLP-1 receptor agonist, a dipeptidyl peptidase 4 (DPPIV) inhibitor, a sulphonurea, a meglitinide, a GPR40 agonist, a GPR119 agonist, a sodium glucose co-transporter-2 inhibitor, a thiazolidinone, metformin or a glucokinase activator.

23. The method of claim 21 or 22, wherein the:

(i) the GLP-1 analog or GLP-1 receptor agonist is selected from the group consisting of exenatide, liraglutide, exenatide LAR, taspoglutide, albiglutide, dulaglutide and GLP1 conjugated to albumin;

(ii) the DPPIV inhibitor is selected from the group consisting of sitagliptin (JANUVIA) and vidagliptin (GALVUS);

(iii) the sulphonylurea is selected from the group consisting of glibenclamide, glyburide and gliclazide;

(iv) the meglitinide is selected from the group consisting of repaglinide and nateglinide;

(v) the GPR40 agonist is selected from the group consisting of TAK-875 and AMG- 837;

(vi) the GPR119 agonist is selected from the group consisting of PSN632408, JNJ- 38431055;

(vii) the glucokinase activator is selected from the group consisting of GKA50, piragliatin (RO4389620) and ZYGK1; (viii) the sodium glucose co-transporter-2 inhibitor is empagliflozin; or

(ix) the thiazolidinone is rosiglitazone, pioglitazone or troglitazone.

24. A composition comprising an antagonist of lysosomal acid lipase (LAL) and glucagon Hke peptide 1 (GLP-1), a GLP-1 analog, a GLP-1 receptor agonist, a dipeptidyl peptidase 4 (DPPIV) inhibitor, a sulphonurea, a meglitinide, a GPR40 agonist, a GPR119 agonist, a sodium glucose co-transporter-2 inhibitor, a thiazolidinone, metformin or a glucokinase activator.