WO2016001682A1

WO2016001682A1 - Treatment of hypertransaminasemia

Info

Publication number: WO2016001682A1
Application number: PCT/GB2015/051943
Authority: WO
Inventors: Jonathon Mark Tinsley; Francis Xavier Wilson; Bindu Tejura
Original assignee: Summit Therapeutics Plc
Priority date: 2014-07-04
Filing date: 2015-07-02
Publication date: 2016-01-07
Also published as: GB201412010D0

Abstract

Disclosed are compositions comprising a utrophin upregulating agent (UUR) for use in a method of treating a disease associated with loss of cellular membrane integrity in non-muscle cell tissues.

Description

Treatment of hypertransaminasemia

Technical Field This invention relates to compositions for use in the treatment of a disease associated with loss of cellular membrane integrity in non-muscle cells. In particular, the invention relates to compositions comprising a utrophin upregulator for use in a method of restoring non-muscle cell membrane integrity, for example in the treatment of hypertransaminasemia and liver diseases associated with hypertransaminasemia, including non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). The invention also relates to the corresponding therapeutic methods.

Background of the Invention DBMD and utrophin upregulation

Duchenne and Becker muscular dystrophy (DBMD) is caused by mutations in the dystrophin gene, resulting in the absence (DMD) or reduction in amount or function (BMD) of the large cytoskeletal protein dystrophin at the sarcolemma.

Dystrophin is normally present at the sarcolemma together with a group of proteins known as the dystrophin protein complex (DPC). Dystrophin binds to the intracellular cytoskeleton by associating with actin filaments at the N-terminus, while the C- terminus binds members of the DPC, including β-dystroglycan. Extracellular a- dystroglycan binds to β-dystroglycan and acts as a receptor for the extracellular- matrix protein laminin. Thus, in muscle cells, dystrophin together with the DPC links the extracellular matrix with the intracellular cytoskeleton to providing structural integrity to muscle fibers. The lack of dystrophin in muscles of DMD patients disrupts the DPC and leads to sarcolemmal instability. Transient microdisruptions in the sarcolemma also occur in DMD and lead to increased levels of intracellular Ca²⁺ and subsequent aberrant Ca²⁺- mediated signaling activating calpain-mediated proteolysis. Moreover, other signalling cascades are also probably affected, because the DPC is a multifunctional signaling complex that interacts with important additional signaling molecules such as neuronal nitric oxide synthase (nNOS), Grb2 and calmodulin. Thus, the dystrophin lesion in DBMD causes muscle to undergo repetitive cycles of degeneration followed by muscle regeneration. Over time, the regenerative potential of dystrophic muscle fibers diminishes, resulting in progressively severe muscle necrosis and wasting.

Upregulation of utrophin, an autosomal paralogue of dystrophin, has been proposed as a therapy for DMD (Perkins and Davies (2002), Neuromuscul Disord, S1 : S78- S89; Khurana & Davies (2003), Nat Rev Drug Discov 2:379-390). This treatment paradigm is based on the recognition that utrophin can functionally replace dystrophin in DMD muscle fibers: utrophin shares a high degree of sequence identity with dystrophin and also associates with members of the DPC. Moreover, when utrophin is constitutively expressed in transgenic mdx mice (the mouse model of DMD), it localizes to the sarcolemma of muscle cells and restores the components of the dystrophin protein complex (DPC), which prevents the dystrophic development and in turn leads to functional improvement of skeletal muscle. A wide variety of agents which increase utrophin activity (utrophin upregulators, referred to herein as UURs) have been described in the literature and are described in more detail below.

Hypertransaminasemia, liver disease and DBMD

Hypertransaminasemia (pathologic elevation of serum transaminase levels) is associated with liver disease. Specifically, elevated serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are associated with non-alcoholic fatty liver disease (NAFLD). NAFLD encompasses a wide spectrum of conditions ranging from simple steatosis, non-alcoholic steatohepatitis (NASH) and liver cirrhosis.

An association between DBMD and hypertransaminasemiac liver disease has recently been recognized: DBMD patients may present with NAFLD, exhibiting elevated serum levels of transaminases, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (Veropalumbo et al. (201 1) JPGN 53 (4): 463- 464).

This link between dystrophin function and liver disease implies a role for one or more of the various dystrophin isoforms in liver function. While full-length dystrophin (muscle or M-dystrophin) is absent in hepatocytes, a 71 kDa isoform (liver or G- dystrophin) is known to be expressed in the liver and so may play a role in signalling and/or or membrane integrity in the liver analogous to that of the M-dystrophin isoform in muscle.

Summary of the Invention

It has now been discovered that UURs can reduce the levels of serum

transaminases, including AST and ALT, and therefore find utility in the treatment of hypertransaminasemia and liver diseases associated therewith, including nonalcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

Without wishing to be bound by any theory, it appears that the increase in utrophin protein levels and/or function produced on administration of UURs can stabilize not only the dystrophin deficient sarcolemmal membrane of DBMD muscle fibres but also the cell membranes of non-muscle cells in both DBMD and non-DBMD subjects, including hepatocyte membranes.

Thus, UURs may be used to treat diseases associated with loss of cellular membrane integrity in non-muscle cell tissues. The discovery of a ubiquitous membrane stabilizing role for utrophin also permits novel treatments for diseases caused by loss of cellular membrane integrity. Such diseases may be characterized by the presence of elevated levels of cytosolic proteins and/or enzymes in the blood. Thus, the invention finds particular application in therapeutic methods for reducing the leakage of transaminases from the liver (including aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) thereby alleviating hypertransaminasemia and so providing a novel mechanism of action for the treatment of liver diseases associated with hypertransaminasemia (including NAFLD and NASH).

Detailed description Definitions

A "pharmaceutical composition" is a solid or liquid composition in a form,

concentration and level of purity suitable for administration to a patient (e.g. a human or animal patient) upon which administration it can elicit the desired physiological changes. Pharmaceutical compositions are typically sterile and/or non-pyrogenic. The term non-pyrogenic as applied to the pharmaceutical compositions of the invention defines compositions which do not elicit undesirable inflammatory responses when administered to a patient. The pharmaceutical compositions of the invention preferably comprise a pharmaceutically acceptable carrier,

pharmaceutically acceptable excipient, physiologically acceptable carrier or physiologically acceptable excipient. As used herein, and unless otherwise specified, the term "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," "physiologically acceptable carrier," or "physiologically acceptable excipient" refers to a

pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications,

commensurate with a reasonable benefit/risk ratio. See, Remington: The Science and Practice of Pharmacy, 21 st Edition, Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th Edition, Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition, Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical P reformulation and Formulation, 2nd Edition, Gibson Ed., CRC Press LLC: Boca Raton, FL, 2009.

As used herein, and unless otherwise specified, the term "therapeutically effective amount" are meant to include the amount of a compound or composition that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disorder, disease, or condition being treated. As used herein, and unless otherwise specified, the terms "treat," "treating," and "treatment" are meant to include preventing, reducing the incidence of, curing, alleviating or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or alleviating or eradicating the cause(s) of the disorder, disease, or condition itself.

The term "disease" is intended to define any disorder, disease or condition, preferably of the human body. As used herein, and unless otherwise specified, the term "subject" refers to an animal, including but not limited to, a primate (e.g., human), cow, pig, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms "subject" and "patient" are used interchangeably herein in reference, for example, to a mammalian subject, such as a human subject, in one embodiment, a human.

All references to particular chemical compounds herein are to be interpreted as covering the compounds per se, and also, where appropriate, pharmaceutically acceptable N-oxides, salts, hydrates, solvates, complexes, bioisosteres, metabolites or prodrugs thereof.

UURs for use according to the invention

UURs as a class upregulate utrophin. This upregulation may be effected at the level of utrophin expression (for example transcriptionally or post-transcriptionally), utrophin stability (either per se or in the context of the utrophin protein complex, UPC) or at the level of the recruitment of utrophin to the UPC. As explained above, the present inventors have discovered that utrophin upregulation can stabilize cellular membranes ubiquitously, and so may be used to improve cell membrane integrity in non-muscle cells including hepatocytes.

Thus, any UUR may be used according to the invention, and suitable UURs include various classes of small-molecules which act at the level of utrophin gene expression. These have been extensively described in the literature, and include 2- arylbenzoxazoles, 2-arylbenzotriazoles and 2-arylindazoles. This class is exemplified by SMT C1100 (5-(ethylsulfonyl)-2-(naphthalen-2-yl)benzo[d]oxazole), a small molecule utrophin upregulator currently in Phase 1 b clinical trials. Other UURs upregulate the activity of utrophin act by protein stabilisation or enhancing translational efficiency. For example, recombinant human biglycan (rhBGN) upregulates utrophin and can recruit it to the sarcolemma (see e.g. Amenta et al. (2011) PNAS 108(2): 762-767), while another component of the DPC, sarcospan (SSPN), improves cell surface expression of utrophin-glycoprotein complexes (Marshall and Crosbie- Watson (2013) Skeletal Muscle 3: 1).

Many other proteins and compounds are known to increase utrophin activity at the level of the abundance of utrophin glycoprotein complex (UGC) at the sarcolemma, including CT GalNAc transferase (Galgt2), ADAM12, heregulin, L-arginine, activated calcineurin-A alpha, N-acetylcysteine, activated Akt, GW501516, artificial utrophin gene transcription factors (e.g. the artificial zinc finger factors UtroUp and Jazz - see e.g. Onori et al. (2013) BMC Molecular Biology 2013, 14:3) and α7β1 integrin. Thus, other suitable UURs for use according to the invention include these proteins and compounds together with upregulators of their expression/production in vivo.

UURs which stimulate utrophin overexpression Suitable for use according to the invention are agents which stimulate the overexpression of utrophin at the transcriptional level. These include any of a large number of small molecule UURs which have been extensively described in the literature, including 2-arylbenzoxazoles, 2-arylbenzotriazoles and 2-arylindazoles described in more detail below.

2-arylbenzoxazoles

UURs of the 2-arylbenzoxazole class have the formula:

They are described in Chancellor et al. (2011) J. Med. Chem., 201 1 , 54 (9): 3241- 3250, and in WO2007/091106 (the contents of each of which is hereby incorporated by reference). 2-arylbenzoxazole UURs include 5-(ethylsulfonyl)-2-(naphthalen-2- yl)benzo[d]oxazole (SMT C1 100), a small molecule utrophin upregulator that has the potential to be a universal treatment for DMD and is currently in Phase 1 b clinical trials:

5-(ethylsulfonyl)-2-(naphthalen-2-yl)benzo[d]oxazole (SMT C1100) The synthesis and therapeutic use of this compound is described in our earlier WO2007/091106, while its various polymorphic forms and processes for the production of such forms are described in WO2009/021748 and WO2009/021749. The compound acts in synergy with corticosteroids, including prednisone, prednisolone and deflazacort to reduce exercise-induced fatigue in mouse models of DMD (see our earlier WO2009/019504).

2-arylbenzotriazoles

UURs of the 2-arylbenzoxazole class have the formulae:

They are described in DeMoor et al. (2011) Bioorganic & Medicinal Chemistry Letters, 21 (16): 4828^1831 and WO2007/091107 (the contents of each of which i hereby incorporated by reference).

2-arylindazoles

UURs of the 2-arylindazole class have the formulae:

They are described in DeMoor et al. (201 1) Bioorganic & Medicinal Chemistry Letters, 21 (16): 4828-4831 (the content of which is hereby incorporated by reference). WO2009/021747 (the content of which is also hereby incorporated by reference) describes related compounds of formulae:

Suitable UURs may also be selected from those of the general formulae:

Such compounds are described in our earlier WO2009/021750; WO2009/013477; WO2009/121623; WO2009/141159; WO2010/020432; WO2010/057833;

WO2010/069684; W02010/086040; WO2010/1 12093; WO/2010/1 12092 and WO/2010/112091 (the contents of each of which is hereby incorporated by reference). The identity of the various substituents shown in the above formulae are defined in the references provided. UURs which stimulate utrophin overexpression for use according to the invention may be identified in the following predictive assay.

Luciferase reporter assay (murine H2K cells) The cell line used for the screen is an immortalized mdx mouse H2K cell line that has been stably transfected with a plasmid containing ^«8.4kb fragment of the Utrophin A promoter including the first untranslated exon linked to a luciferase reporter gene. Under conditions of low temperature and interferon containing media, the cells remain as myoblasts. These are plated into 96 well plates and cultured in the presence of compound for three days. The level of luciferase is then determined by cell lysis and reading of the light output from the expressed luciferase gene utilising a plate luminometer.

The H2K/mdx/Utro A reporter cell line cells were plated out into 96 well plates (Falcon 353296, white opaque) at a density of approximately 5000 cells/well in 190 μΙ normal growth medium. The plates were then incubated at 33°C in the presence of 10% C0₂ for 24 hrs.

Compounds were dosed by adding 10 μΙ of diluted compound to each well giving a final concentration of 10 μΜ (where a different final concentration was required, the amount of compound solution added was amended accordingly). The plates were then incubated for a further 48 hrs. Cells were then lysed in situ following the manufacture's protocols (Promega Steady-Glo Luciferase Assay System (E2520)) and then counted for 10 seconds using a plate luminometer (Victor1420).

Therapeutic applications As explained above, the present inventors have discovered that utrophin upregulation can stabilize cellular membranes ubiquitously, and so may be used to improve cell membrane integrity in non-muscle cells.

The invention therefore finds application in the treatment of any disease caused by impaired cellular membrane integrity in non-muscle cells and in methods for restoring non-muscle cell membrane integrity in vivo.

Diseases caused by impaired cellular membrane integrity in non-muscle cells which may be treated according to the invention may be identified by the presence of elevated levels of cytosolic proteins and/or enzymes in the blood. For example, in one embodiment the invention may be used in the treatment of hypertransaminasemia characterized by the presence of elevated serum levels of liver transaminases (including aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT)).

Thus, the invention finds particular application in therapeutic methods for reducing the leakage of transaminases from the liver (including aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) thereby alleviating

hypertransaminasemia and so providing a novel mechanism of action for the treatment of liver diseases associated with hypertransaminasemia (including NAFLD and NASH).

In particular, the invention relates to compositions comprising a utrophin upregulator for use in a method of restoring non-muscle cell membrane integrity, for example in the treatment of hypertransaminasemia and liver diseases associated with

hypertransaminasemia, including for example non-alcoholic fatty liver disease

(NAFLD) and non-alcoholic steatohepatitis (NASH).

The above methods and therapeutic treatments are of general application, but may find particular application in the treatment of DBMD subjects, and in particular to the treatment of DBMD subjects exhibiting hypertransaminasemia.

Pharmaceutical Compositions The compositions of the invention are administered in the form of a pharmaceutical composition comprising a UUR and one or more pharmaceutically acceptable excipients.

The pharmaceutical compositions can be provided in solid, semisolid, or liquid dosage forms for oral administration. As used herein, oral administration also includes buccal, lingual, and sublingual administration. Suitable oral dosage forms include, but are not limited to, tablets, fastmelts, chewable tablets, capsules, pills, strips, troches, lozenges, pastilles, cachets, pellets, medicated chewing gum, bulk powders, effervescent or non-effervescent powders or granules, oral mists, solutions, emulsions, suspensions, wafers, sprinkles, elixirs, and syrups. The pharmaceutical compositions can be administered parenterally by injection, infusion, or implantation, for local or systemic administration. Parenteral

administration, as used herein, include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, intravesical, and subcutaneous administration.

The pharmaceutical compositions provided herein for parenteral administration can be formulated in any dosage forms that are suitable for parenteral administration, including solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and solid forms suitable for solutions or suspensions in liquid prior to injection. Such dosage forms can be prepared according to conventional methods known to those skilled in the art of pharmaceutical science (See, Remington: The Science and Practice of Pharmacy, supra). The pharmaceutical compositions provided herein can be administered topically to the skin, orifices, or mucosa. The topical administration, as used herein, includes

(intra)dermal, conjunctival, intracorneal, intraocular, ophthalmic, auricular, transdermal, nasal, vaginal, urethral, respiratory, and rectal administration. A preferred formulation of SMT C1 100 is described in WO/2013/167737 (the content of which is hereby incorporated by reference).

Examples A Phase 1 b study in 12 ambulatory DMD patients aged 5 to 1 1 years old was carried out with an oral aqueous suspension of SMT C1 100. Three escalating dose cohorts (4 patients per cohort) were dosed at 50 mg/kg BID, 100 mg/kg BID and 100 mg/kg TID. 10 days or oral dosing occurred within 10 minutes of consuling food. As shown in Figure 1 (which shows a plot of the average reduction from baseline (Estimate, square) with upper and lower 95% confidence limits (diamond) for each time point after dosing (Day 7, Day 12) and follow-up (Follow-up, 3 days after completion of dosing). Enzyme levels of creatine phosphokinase (CPK), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were significantly elevated in the subjects. During dosing with SMT C1100, a statistically significant reduction in blood plasma levels of the enzymes creatine phosphokinase ('CPK'), aspartate aminotransferase ('AST') and alanine aminotransferase ('ALT') was observed when compared to pre-dose levels. The plasma levels of these enzymes moved towards baseline after dosing had stopped.

The levels of other enzyme markers of liver function such as gamma-glutamyl transferase ('GGT'), alkaline phosphatase ('ALK') and albumin ('ALB') would be expected to remain stable over time. Indeed, during dosing with SMT C1100, the plasma levels of these three markers showed littlechange.

Equivalents

The foregoing examples are presented for the purpose of illustrating the invention and should not be construed as imposing any limitation on the scope of the invention. It will readily be apparent that numerous modifications and alterations may be made to the specific embodiments of the invention described above and illustrated in the examples without departing from the principles underlying the invention. All such modifications and alterations are intended to be embraced by this application.

Claims

CLAIMS:

1. A composition comprising a utrophin upregulating agent (UUR) for use in a method of treating a disease associated with loss of cellular membrane integrity in non-muscle cell tissues.

2. The composition of claim 1 for use in a method of treating hypertransaminasemia.

3. The composition of claim 2 for use in a method of treating liver disease associated with hypertransaminasemia.

4. The composition of claim 3 for use in a method of treating non-alcoholic fatty liver disease (NAFLD).

5. The composition of claim 3 for use in a method of treating non-alcoholic

steatohepatitis (NASH).

6. A composition comprising a utrophin upregulating agent (UUR) for use in a method of treating non-alcoholic fatty liver disease (NAFLD).

7. A composition comprising a utrophin upregulating agent (UUR) for use in a method of treating non-alcoholic steatohepatitis (NASH).

8. A method for treating a disease as defined in any one of the preceding claims comprising administering a therapeutically effective amount of a composition comprising a UUR to a subject in need thereof.

9. Use of a utrophin upregulating agent (UUR) in the manufacture of a medicament for use in a method of treatment as defined in any one of the preceding claims.

10. The composition, method or use of any one of the preceding claims wherein said UUR stimulates utrophin expression.

1 1. The composition, method or use of claim 10 wherein said UUR is a utrophin transcriptional activator.

12. The composition, method or use of claim 10 or claim 11 wherein said UUR is selected from: 2-arylbenzoxazoles, 2-arylbenzotriazoles, 2-arylindazoles, Jazz and UtroUp.

13. The composition, method or use of claim 12 wherein said UUR is SMT C1100.

14. The composition, method or use of any one of claims 1-10 wherein said UUR stabilizes the UGC or recruits utrophin to the UGC.

15. The composition, method or use of any one of claims 1-10 or 14 wherein said UUR is selected from: biglycan (for example recombinant human biglycan (rhBGN)); sarcospan; CT Gal N Ac transferase (Galgt2); ADAM 12; heregulin; L-arginine; activated calcineurin-A alpha; N-acetylcysteine; activated Akt; GW501516 and α7β1 integrin.

16. The composition, method or use of any one of the preceding claims wherein said composition is a pharmaceutical composition.

17. The composition, method or use of any one of the preceding claims wherein said treatment or method is applied to a subject suffering from DMD or BMD.

18. The composition, method or use of any one of the preceding claims wherein said treatment or method is applied to a subject not suffering from DMD or BMD.