WO2020128529A1 - Compositions and uses thereof - Google Patents
Compositions and uses thereof Download PDFInfo
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- WO2020128529A1 WO2020128529A1 PCT/GB2019/053682 GB2019053682W WO2020128529A1 WO 2020128529 A1 WO2020128529 A1 WO 2020128529A1 GB 2019053682 W GB2019053682 W GB 2019053682W WO 2020128529 A1 WO2020128529 A1 WO 2020128529A1
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- Prior art keywords
- inhibitor
- hsd11
- sarcopenia
- bvt
- treatment
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Classifications
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- G01N2800/00—Detection or diagnosis of diseases
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- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- This invention relates to compositions and methods for the prevention, management and/or treatment of sarcopenia and related diseases and disorders in insulin sensitive individuals. More particularly, the invention relates to the treatment of sarcopenia associated with ageing, obesity and sarcopenia secondary to disease, injury, trauma or medication; to pharmaceutical compositions useful in the treatment of such conditions, and also to methods for identifying patients who are likely candidates for treatment. Related methods of screening and diagnosis are also disclosed.
- Sarcopenia (loss of muscle mass and strength) affects between 5-13% of 60-70 year olds and 11-50% of over 80 year olds. It is a major public health issue, with the number of sarcopenic patients likely to dramatically increase in the next 30 years. It has been estimated that overall prevalence rate in the elderly will rise in Europe from 11.1 %-20.2% in 2016 to 12.9%-22.3% in 2045. Sarcopenia is a strong predictor of low quality of life, mobility impairment, falls, fractures and all-cause mortality. It is the second most common cause of disability in humans. Sarcopenia is also an independent risk factor for other serious health conditions, such as cardiovascular disease.
- EWGSOP European working group on sarcopenia in older people
- diagnostic criteria which include muscle mass (appendicular lean mass divided by height squared, men:£7.23kg/m 2 , womans 5.67kg/m 2 ) and muscle function (gait speed ⁇ 0.8 m/s; or grip strength, men: ⁇ 30kg, women ⁇ 20kg).
- Sarcopenia was classified in the International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD-10) in 2016 under code M62.84.
- sarcopenia The underlying mechanism of muscle loss and function in sarcopenia is not fully understood, but studies to date have suggested that reduced muscle stem cell (satellite cell) numbers; impaired satellite cell proliferative function; loss through senescence; and ability to remain quiescent may each contribute to the loss of muscle mass and function and neuromuscular junction degeneration.
- Epigenetic changes in satellite cells may underlie their changed phenotype in injury-induced sarcopenia and age-related sarcopenia.
- Manipulation of satellite cell state and maintenance of the quiescent state is possibly a promising therapeutic strategy for sarcopenia.
- Sarcopenia is currently treated by use of resistance exercise and nutritional interventions, such as high protein diet and vitamin D. These interventions to some extent rescue satellite cell proliferative capacity and reserve numbers, but the quality of evidence for efficacy is low.
- Testosterone treatment has been shown to increase muscle size and power but it also induces undesirable side effects.
- SARM Selective androgen receptor modulators
- Androgens and SARMs are thought to increase muscle mass without improvements in muscle strength or function and have no effect on regenerative capacity (i.e. satellite cells). Accordingly, these constitute at best a symptomatic, not disease-modifying, treatment.
- Myostatin inhibition is a secreted protein that normally inhibits muscle growth. Myostatin-null animals and humans show skeletal muscle hypertrophy. Myostatin inhibitors, and inhibitors of its cognate receptor activin, have been developed. Most have shown at best only modest efficacy or a lack of long-term efficacy, and require regular injections, which has negative implications.
- the present inventors have undertaken research which has enabled them to propose a novel approach to the treatment (including prevention or management) of sarcopenia and sarcopenic diseases in insulin sensitive individuals, based on a new understanding of the mechanisms involved in these diseases.
- the present inventors have identified the significance of the observation that the decrease in proliferative capacity of muscle associated with ageing and other‘insults’ is mediated in part by cortisol.
- Cortisol is an active glucocorticoid that is convertible from the inert cortisone by action of an enzyme, cortisone reductase (also known as HSD11 B1 or I ⁇ b-HSDI).
- cortisone reductase also known as HSD11 B1 or I ⁇ b-HSDI.
- a composition comprising a HSD11 B1 inhibitor for use in the prevention, management and/or treatment of sarcopenia or a sarcopenic disease or disorder in an insulin sensitive individual.
- an inhibitor of HSD11 B1 for use in the prevention, management and/or treatment of sarcopenia or a sarcopenic disease or disorder in an insulin sensitive individual.
- a method of prevention, management and/or treatment of sarcopenia or sarcopenic disease or disorder in an individual who is sensitive to insulin comprising the administration of a therapeutically effective amount of a HSD11 B1 inhibitor in an individual in need of such prevention, management and/or treatment.
- the method may comprise first determining that the individual is sensitive to insulin prior to the administration of the HSD11 B1 inhibitor.
- an inhibitor of HSD11 B1 in the manufacture of a medicament for the prevention, management and/or treatment of sarcopenia or a sarcopenic disease or disorder in an insulin sensitive individual.
- a pharmaceutical composition comprising a HSD11 B1 inhibitor and a pharmaceutically acceptable carrier, excipient, or diluent.
- Sarcopenia primarily relates to degenerative loss of skeletal muscle mass, quality and strength associated with ageing.
- the above“sarcopenic” symptoms are also seen in other, non-age related, conditions.
- the terms“sarcopenic disease” or“sarcopenic disorder” should be understood as encompassing these.
- the invention encompasses sarcopenia associated with ageing, obesity and sarcopenia secondary to disease, injury, trauma or medication.
- glucocorticoid glucocorticoid
- sarcopenia steroid-induced, in particular glucocorticoid (GC)-induced sarcopenia, which can be seen, for example, in individuals who (for whatever reason) are subject to, often high, exposure to endogenous or exogenous glucocorticoids. In particular, this may include exposure to higher than normal levels of cortisol.
- GC glucocorticoid
- “Sarcopenic disease” also encompasses secondary sarcopenia, for example that consequent upon surgery, such as that suffered by people that are immobilised after a bone fracture, especially after hip fracture or hip replacement surgery. This is particularly prevalent in elderly patients with hip fractures, but is not restricted to this group.
- treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
- the effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or can be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
- Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting or slowing its development; and (c) relieving the disease, i.e., causing regression of the disease.
- non-human animal includes all mammals, such as non-human primates, sheep, dogs, cats, cows, horses.
- sensitivity to insulin is deemed to mean that an individual is not diabetic or insensitive to insulin.
- sensitivity to insulin may also encompass those individuals who are mildly insensitive to insulin and/or individuals who are suffering from early stage or very mild diabetes.
- the HSD11 B1 inhibitor is administered following an intermittent dosage regime.
- the intermittent dosage regime be an on/off dosing regime, such as one week of treatment followed by one week of no treatment or could be a dosing regime of less than once daily (for example a dose every 2 days, every 3 days, every 4 days, every 5 days, every 6 days or every 7 days).
- the intermittent dosing regime comprises dosing for 7 days followed by 21 days with no dosing repeated.
- HSD11 B1 inhibitors promote a dose-dependent increase in PAX7 expression, indicating an increase in MSC content in the regenerating muscle and such inhibitors would therefore be a successful approach to treating sarcopenia or a sarcopenic disease or disorder and data suggests that such an approach is particularly relevant for those individuals who are sensitive to insulin.
- the inhibitor may be a selective inhibitor which may, without competing against a substrate(s), bind or interact with a target, so as to affect the target’s ability to further bind or interact with one or more specific substrates.
- HSD11 B1 inhibition is proposed as a therapeutic strategy to treat the loss of muscle strength and regenerative capacity, including muscle wasting, associated with age (sarcopenia), injury, disease, trauma or medication (e.g. sarcopenic obesity or glucocorticoid- induced sarcopenia) in insulin sensitive individuals.
- sarcopenia associated with age
- injury, disease, trauma or medication e.g. sarcopenic obesity or glucocorticoid- induced sarcopenia
- Another aspect of the invention provides an HSD11 B1 inhibitor, method or use according to any aspect of the invention, comprised in a therapeutic strategy to treat the loss of muscle strength or muscle regenerative capacity, optionally muscle wasting, associated with age (sarcopenia), injury, disease, trauma, or medication in insulin sensitive individuals.
- sarcopenia sarcopenic obesity or glucocorticoid-induced sarcopenia.
- the invention provides an inhibitor, method or use according to any aspect of the invention wherein the prevention, management or treatment of sarcopenia or a sarcopenic disease or disorder comprises inhibiting HSD11 B1 signalling, and decreasing cortisol in the subject.
- Decreasing cortisol suitably means decreasing the level of cortisol in the blood or plasma of the subject.
- the subject in need may be identified in a test for sarcopenia and/or a sarcopenic disease or disorder and insulin sensitivity.
- the subject in need may be identified by a) measuring the level of cortisol in a biological sample taken from the subject and/or by analysing the DNA sequence, epigenetic regulation, transcript or protein levels, enzymatic activity or post-translational modification of their HSD11B1 and/or NR3C1 genes; and b) measuring the level of blood sugar in a biological sample taken from the individual and confirming that the individual is sensitive to insulin.
- insulin sensitivity the skilled addressee will appreciate that there are a range of tests available, but the most commonly used test would be a simple measurement of blood glucose levels (including levels after fasting).
- a subject in need of treatment, prevention or management of sarcopenia or a sarcopenic disease or disorder is one having an elevated cortisol level, compared to levels in a control population or a predetermined standard.
- the elevated level may be, for example, in blood, plasma or urine.
- the elevated level (in humans) may be a cortisol level of equal to or greater than 6ng/ml of sample body fluid.
- the fasting blood sugar level (in humans) may be a blood sugar level of less than or equal to 5.4 mmol/L (99 mg/dL) and will usually be between 4.0 to 5.4 mmol/L (72 to 99 mg/dL).
- HSD11 B1 inhibition is a promising therapeutic strategy for sarcopenia and sarcopenic conditions as described herein that offers advantages over the existing therapies by avoiding at least some of the problems associated with the existing therapies mentioned above.
- HSD11 B1 inhibitors for use in the invention or in the methods of treatment of the invention include any chemical or biological agent that is known or can be identified as such, or a chemical or biological agent that down-regulates cortisol or a precursor thereof, or that inactivates or reduces activation of the glucocorticoid receptor (NR3C1) or precursor or activator thereof.
- the inhibitor is a selective inhibitor of HSD11 B1 , and especially preferred are those with high potency.
- Suitable HSD11 B1 inhibitors include: I ⁇ b-HDSI inhibitors developed for the treatment of type 2 diabetes mellitus. For example, salicylate downregulates I ⁇ b-HSDI expression in adipose tissue in obese mice and hence may explain why aspirin improves glycemic control in type 2 diabetes.
- Newer compounds such as adamantyltriazoles, arylsulfonamidothiazoles, anilinothiazolones, BVT2733, INCB-13739, MK-0916 and MK-0736, are currently under investigation, and the preliminary findings from both experimental and human studies show a favourable effect on glucose and lipid metabolism, weight reduction and adipokine levels.
- Butylated hydroxyanisole is a widely used antioxidant for food preservation, and is a selective inhibitor of HSD11 B2, of the natural products, 18a-glycyrrhizic acid from the root of glycyrrhiza glabra, and also curcumin and derivatives have been identified as HSD11 B1 inhibitors.
- Epigallocatechin gallate from green tea can also potently inhibit this enzyme, green tea is a complex mixture of various phenolics with contents varying with production and processing.
- Potent and selective inhibitors of the HSD11 B1 enzyme also include those known as AZD4017, ABT384, ABT305, INCB-13739, BVT.3498, BVT 116429, bezafibrate; CRx- 401 , diflunisal; BMS-823778; UE2343; and carbenoxolone, respectively.
- AZD4017 2-[(3S)-1-[5-(cyclohexylcarbamoyl)-6-propylsulfanylpyridin-2-yl]piperidin-3-yl]acetic acid.
- AZD4017 is a selective, orally bioavailable inhibitor of the enzyme 11 -b- hydroxysteroid dehydrogenase type 1 , with potential protective activity.
- AZD4017 has been used in clinical trials for a number of indications: Idiopathic Intracranial Hypertension (400 mg oral tablet twice daily for 12 weeks); Obesity (oral suspensions 1200 mg once daily for 10 days); Diabetes Mellitus Type 2 (1st trial: oral suspension, ascending multiple doses starting at 75 mg once daily; 2nd trial: 400 mg oral tablet twice daily for 35 days); Iatrogenic Cushing's Disease (400 mg twice daily for 7 days with 20 mg prednisolone); Raised Intraocular Pressure (Europe: 200 mg oral tablet once daily for 28 days; USA: 2 x 200 mg oral tablets twice daily for 28 days).
- ABT384 is a potent, selective inhibitor of I I-b-hydroxysteroid dehydrogenase type 1.
- ABT384 has been used in clinical trials for adults with mild to moderate Alzheimer’s disease with subjects receiving 10 mg or 50 mg of ABT384 once daily for 12 weeks.
- INCB-13739 is an orally available small molecule inhibitor of 1 1 -b- hydroxysteroid dehydrogenase type 1. INCB-13739 has been used in clinical trials for adults with Type 2 Diabetes, with a maximum dose of 200 mg per day (either as one dose, combined with metformin or 100 mg twice daily, alone).
- BVT3498 3-chloro-2-methyl-N-[4-[2-(3-oxomorpholin-4-yl)ethyl]-1 ,3-thiazol-2- yljbenzenesulphonamide (BVT 3498).
- BVT3498 is a highly selective inhibitor of the enzyme I I -b-hydroxysteroid dehydrogenase type 1 .
- BVT.3498 has been used in clinical trials for adults with Type 2 Diabetes, completing phase I trials in 2002 and entering phase II trials. The trials were stopped in 2005.
- BVT 116429 is a selective inhibitor of the enzyme I I-b-hydroxysteroid dehydrogenase type 1. Studies have been performed on diabetic mice using up to 30 mg/kg once daily for 10 days.
- CRx-401 2',4'-difluoro-4-hydroxybiphenyl-3-carboxylic acid
- CRx-401 diflunisal
- CRx-401 is a novel insulin sensitizer designed to provide anti-diabetic activity. It consists of a low dose of diflunisal in conjunction with a modified-release therapeutic dose of bezafibrate. It has been used in clinical trials for patients with Type II Diabetes taking metformin. Patients were given CRx-401 consisting of 400 mg bezafibrate sustained-release and 250 mg diflunisal once daily.
- Bezafibrate is an antilipemic agent that lowers cholesterol and triglycerides. It is an approved drug for patients with mixed hyperlipidaemia if statin is not tolerated. The dose is 200 mg orally three times daily using immediate-release medicines and 400 mg orally once daily using modified-release medicines.
- Acute Myocardial Infarction 400 mg every 24 hours
- Mitochondrial Diseases 200 mg - 600 mg three times daily for 12 weeks
- Moderate to severe cholestatic itch 400 mg once daily
- Adrenomyeloneuropathy 400 mg once daily until week 12, and subsequently use 800 mg once daily until week 24
- Carnitine Palmitoyltransferase II Deficiency (3 x 200 mg per day)
- Bipolar Disorder 400 mg once daily
- 5-(2,4-difluorophenyl)-2-hydroxybenzoic acid is a salicylate derivative, is a nonsteroidal anti-inflammatory agent (NSAIA) with pharmacologic actions similar to other prototypical NSAIAs. It is commonly prescribed for pain relief and used in conditions including osteoarthritis, rheumatoid arthritis, renal impairment and hepatic impairment in maximum doses of 1500 mg per day. It is also undergoing clinical trials for the conditions of: HIV Infection (500 mg twice daily for 4 weeks); and Familial Amyloidosis (250 mg twice daily for 24 months).
- HIV Infection 500 mg twice daily for 4 weeks
- Familial Amyloidosis 250 mg twice daily for 24 months.
- BMS-823778 is an orally available potent and selective inhibitor of 1 1 -b- hydroxysteroid-dehydrogenase 1 . It has been used in clinical trails for the conditions of: Atherosclerotic Cardiovascular Disease (2 mg - 15 mg orally once daily for 1 year); Hypertension (2 mg - 15 mg orally once daily for 12 weeks); Dyslipidemia (2 mg - 20 mg orally once daily for 28 days); and Diabetes Mellitus Type 2 (2 mg - 20 mg orally once daily for 28 days, with metformin).
- UE2343 (3-hydroxy-3-pyrimidin-2-yl-8-azabicyclo[3.2.1]octan-8-yl)-[5-(1 H-pyrazol-4-yl)thiophen-3- yljmethanone (UE2343).
- UE2343 is a potent, orally bioavailable, brain-penetrant 1 1-b- hydroxysteroid-dehydrogenase 1 inhibitor.
- Clinical trials have evaluated the safety, tolerability of efficacy of XanamemTM (comprising UE2343) in subjects with mild dementia due to Alzheimer’s Disease. In the trial, oral XanamemTM 10 mg capsules were given once daily.
- carbenoxolone is a hemisuccinate derivative of glycyrrhetinic acid and is frequently used in the treatment of peptic ulcers and the topical treatment of mouth ulcers. It is also a non- selective I I-b-hydroxysteroid-dehydrogenase 1 inhibitor that also inhibits 1 1 -b- hydroxysteroid-dehydrogenase 2. It has also been used in clinical trials for Type 2 diabetes, with a dosage of 100 mg three times per day.
- UE2343 (3-hydroxy-3-pyrimidin-2-yl-8-azabicyclo[3.2.1 ]octan-8-yl)-[5-(1 H-pyrazol-4-yl)thiophen-3- yl]methanone (UE2343).
- UE2343 is a potent, orally bioavailable, brain-penetrant 11-b- hydroxysteroid-dehydrogenase 1 inhibitor.
- Clinical trials have evaluated the safety, tolerability of efficacy of XanamemTM (comprising UE2343) in subjects with mild dementia due to Alzheimer’s Disease. In the trial, oral XanamemTM 10 mg capsules were given once daily.
- carbenoxolone is a hemisuccinate derivative of glycyrrhetinic acid and is frequently used in the treatment of peptic ulcers and the topical treatment of mouth ulcers. It is also a non- selective 11-b-hydroxysteroid-dehydrogenase 1 inhibitor that also inhibits 11-b- hydroxysteroid-dehydrogenase 2. It has also been used in clinical trials for Type 2 diabetes, with a dosage of 100 mg three times per day. Included in the scope of the above-described inhibitors are derivatives thereof well-known for use in the pharmaceutical art.
- the inhibitors referred to herein may be provided as any pharmaceutically acceptable derivative, selected from, but not limited to, any one or more of the following: pharmaceutically acceptable salts, pharmaceutically acceptable solvates, pharmaceutically acceptable enantiomers, pharmaceutically acceptable hydrates, pharmaceutically acceptable polymorphs, pharmaceutically acceptable esters and pharmaceutically acceptable prodrugs.
- the inhibitors for use according to this invention include any metabolites thereof that are therapeutically active.
- the inhibitors for use according to this invention include any prodrugs thereof. Prodrugs are compounds that are converted to therapeutically active compounds as they are being administered to a patient or after they have been administered to a patient.
- the inhibitors for use according to this invention are selective inhibitors of the I ⁇ b-HSDI (or HSD11 B1) enzyme.
- I ⁇ b-HSDI or HSD11 B1 enzyme.
- glucocorticoid concentrations are modulated by two enzymes of I ⁇ b-HSD (namely, IIb-HSDI and I Ib-H802), which have differing cofactor requirements and substrate affinities, and in vitro studies have shown that I Ib-HSDI is capable of acting as both a reductase and a dehydrogenase.
- selectivity for the type 1 isoenzyme, 11 b-HSDI is preferred.
- the invention provides a method of screening for an agent capable of inhibiting HSD11 B1 levels in a patient in need thereof comprising the steps of:
- step (c) comparing the level of cortisol or precursor thereof determined in step (b) with a cortisol or precursor level in a control population of cells which has not been contacted with the candidate inhibitor.
- the method is an in vitro method.
- the candidate inhibitor is optionally comprised in a library of candidate inhibitors.
- the invention provides an inhibitor for decreasing cortisol levels or levels of a cortisol precursor in a patient, wherein the inhibitor has been identified by the method of screening of the invention.
- level of cortisol refers to the amount of cortisol that is found in a sample, e.g. of serum or plasma. The amount of may be determined directly or indirectly. Direct methods of determining cortisol include chromatographic methods, radioimmunoassay, enzyme- linked immunoassays and non-isotopic immunoassays. Liquid chromatography based mass spectrometry (LC-MS/MS) are commonly used to measure serum and plasma cortisol levels from multiple time points with high sensitivity. The effect of the candidate inhibitor on cortisol levels may be assessed as a function of time, by carrying out repeated measurements over a particular time-course.
- LC-MS/MS Liquid chromatography based mass spectrometry
- the present invention also provides for the case when the HSD11 B1 inhibitor is used, administered or formulated together with vitamin D.
- the HSD11 B1 inhibitor is used, administered or formulated together with vitamin D.
- together with is not meant to imply only an intimate admixture of the two active ingredients, but also encompassed is separate, simultaneous or sequential administration.
- the vitamin D may be administered by oral, parental, sub-lingual, sub-cutaneous, transdermal or intra-nasal administration. It may be administered as an oral vitamin D supplement or a probiotic supplement.
- the vitamin D may, for example, be in the form of a nutritional composition or supplement, or a diet product.
- the patient may have previously been determined to be vitamin D deficient.
- the invention provides a method of maintaining or increasing muscle function and/or mass and/or preventing or reducing muscle wasting in a patient in need thereof, particularly an ageing or elderly patient, comprising administering to the patient the inhibitor and vitamin D.
- the vitamin D maintains or increases muscle mass; alternatively, the vitamin D substantially prevents or reduces a reduction in muscle mass.
- the invention provides for the use or method according to the invention of a combined preparation of an inhibitor and vitamin D, wherein the inhibitor and vitamin D are for simultaneous, combined, sequential or separate administration to a patient.
- HSD11 B1 inhibitor instead of or as well as combining an inhibitor with vitamin D, other combinations comprising an HSD11 B1 inhibitor are within the scope of this invention, such as a diet product, as mentioned above in relation to patient identification/diagnosis, and/or an exercise regime may be combined with the inhibitor to maintain or increase muscle function and/or mass.
- a diet product as mentioned above in relation to patient identification/diagnosis, and/or an exercise regime may be combined with the inhibitor to maintain or increase muscle function and/or mass.
- one or more other pharmacologically active agent(s) may be combined with an inhibitor, including selective androgen receptor modulators (SARMs), such as ostarine or myostatin blockers (e.g. myostatin antibodies, activin receptor antibodies and activin receptor-Fc), such as LY2495655 or Bimagrumab, or beta2 receptor agonists such as formoterol, or ghrelin receptor agonists such as anamorelin, or anabolic catabolic transforming agents (ACTA), such as MT-102, or other compounds mentioned hereinabove.
- SARMs selective androgen receptor modulators
- ostarine or myostatin blockers e.g. myostatin antibodies, activin receptor antibodies and activin receptor-Fc
- beta2 receptor agonists such as formoterol
- ghrelin receptor agonists such as anamorelin
- anabolic catabolic transforming agents (ACTA) such as MT-102, or other compounds mentioned herein
- the therapeutic ingredient(s), such as the inhibitors referred to herein may be administered at any suitable pharmacological dose, it being understood that the exact amounts (i.e. the therapeutically effective amounts) will depend upon the nature of the inhibitor and the condition to be treated.
- suitable doses may comprise a daily dosage of from about 0.1 milligram to about 100 milligrams per kilogram of animal body weight, preferably given as a single daily dose or in divided doses two to six times a day, or in sustained release form.
- the total daily dosage is preferably from about 1 milligram to about 1000 milligrams, more preferably from about 1 milligram to about 350 milligrams, especially from about 1 mg to about 100 mg.
- the total daily dose is preferably in the range of from about 7 milligrams to about 350 milligrams.
- such doses may be in the range of from about 50 to 500 mg per day, such as about 240mg or about 100mg per day.
- This dosage regimen may be adjusted, as known by those skilled in the art, to provide the optimal therapeutic response.
- the present invention further provides a use or method as described herein, wherein one or more therapeutic ingredients, including the inhibitor referred to herein, may be comprised in a pharmaceutical formulation, optionally together with one or more pharmaceutically acceptable carriers therefor.
- the one or more pharmaceutically acceptable carriers of the/each therapeutic ingredient may be the same or different.
- the pharmaceutical formulation may further comprise an inhibitor of inflammatory signalling, including those described below.
- the HSD11 B1 inhibitor may be for administration separately, sequentially or simultaneously with one or more pharmaceutically active ingredients.
- compositions suitable for oral, rectal, topical, parenteral including subcutaneous, intramuscular, and intravenous, ocular (ophthalmic), pulmonary (nasal or buccal inhalation), or nasal administration (such as, for example, in the form of liquid drops or spray), although the most suitable route in any given case will depend on the nature and severity of the conditions being treated and on the nature of the therapeutic ingredient(s), e.g. the nature of the HSD11 B1s inhibitors, and/or other active ingredients present.
- the HSD11 B1 inhibitor and optionally any other therapeutic ingredient(s), is/are preferably in a form suitable for intramuscular administration.
- compositions of the present invention may be conveniently presented in unit dosage form (e.g. a fixed dosage form) and prepared by any of the methods well-known in the art of pharmacy.
- Intramuscular dosage forms represent an advantageous dosage form as they allow fast absorption of large volumes directly to a target area, and as such, they may be preferred.
- the therapeutic ingredient(s), such as the inhibitors referred to herein, can be brought into an intimate physical admixture with one or more pharmaceutical carriers according to conventional pharmaceutical formulating techniques.
- the carrier(s) may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intramuscular and intravenous), preferably intramuscular.
- any of the usual pharmaceutical excipients may be employed, such as, for example, diluents of a solid or liquid nature, flavouring agents, preservatives, colouring agents, and the like.
- Liquid preparations may be in the form of, for example, suspensions, elixirs and solutions. Such liquid preparations may comprise one or more of: sucrose as a sweetening agent, methyl and/or propylparabens as preservatives, a dye and flavouring.
- Oral solid preparations are preferred over oral liquid preparations and are preferably in the form of, for example, powders, hard and soft capsules and tablets.
- the one or more pharmaceutical carriers may include one or more of: starches, sugars, microcrystalline cellulose, solid or liquid diluents, granulating agents, lubricants, binders, disintegrating agents and the like.
- the tablets, pills, capsules, and the like may also contain one or more if: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin.
- a dosage unit form is a capsule, it may contain, in addition to materials of the above type, comprise a liquid carrier such as a fatty oil.
- tablets and capsules represent an advantageous oral dosage unit form.
- tablets may be coated by standard aqueous or non-aqueous techniques.
- tablets may be coated with shellac, sugar or both.
- Such formulations, compositions and preparations will preferably contain at least 0.1 % of the therapeutic ingredient(s), such as the inhibitors referred to herein (e.g. the HSD11 B1s inhibitors).
- the percentage of the therapeutic ingredient(s), such as the inhibitors referred to herein in these formulations/compositions may, of course, be varied and may conveniently be between about 2% to about 60% by weight of the unit dose.
- the amount of the therapeutic ingredient(s), such as the inhibitors referred to herein in such therapeutically useful compositions is such that an effective dosage (i.e. the therapeutically effective amount) will be obtained.
- Various other materials may be present to act as coatings or to modify the physical form of the dosage unit.
- the pharmaceutical forms include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
- Solutions or suspensions of these active inhibitors can be prepared in water for injections, optionally suitably mixed with a surfactant such as hydroxypropylcellulose.
- Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils.
- the pharmaceutical carrier(s) can be a solvent or dispersion medium containing, for example, water, an alcohol (e.g. ethanol), a polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycols), suitable mixtures thereof, and vegetable oils, and combinations thereof.
- an alcohol e.g. ethanol
- a polyol e.g. glycerol, propylene glycol and liquid polyethylene glycols
- suitable mixtures thereof e.g. glycerol, propylene glycol and liquid polyethylene glycols
- the invention also provides a method of identifying a human or non-human animal suitable for or in need of the prevention, management or treatment, of sarcopenia or a sarcopenic disease or disorder by use or administration of a HSD11 B1 inhibitor, which method of identifying comprises:
- “therapeutically effective amount” refers to the amount of the HSD11 B1 inhibitor that, when administered to a subject for cosmetic, preventative or therapeutic treatment, is sufficient to effect the desired treatment (e.g. of the disease).
- The“therapeutically effective amount” will vary, for example, depending on the inhibitor(s) used, the state of the disease and its severity and the age, weight, etc., of the subject to be treated, as described above.
- the HSD11 B1 inhibitor, or formulations thereof as described above may be administered to a subject using any one of the available methods and routes suitable for drug delivery, as described herein.
- the HSD11 B1s inhibitor or a formulations thereof is administered by injection and/or delivery, for example, to a site in a muscle.
- the HSD11 B1 inhibitor or formulation thereof are administered by direct delivery in to a muscle prior to surgery or anticipated prolonged immobility.
- the HSD11 B1 inhibitor of a formulation thereof is administered by direct delivery in to a muscle after surgery or prolonged immobility.
- the HSD11 B1 inhibitor or a formulation thereof may be administered in a single dose or in multiple doses.
- a suitable frequency of administration may be at least once per day, every other day, once per week, once every two, three, or four weeks, once every month, two months, or once every three to six months.
- the HSD11 B1 inhibitor may be administered over a period of at least a week, at least a month, at least three to six months, at least one, two, three, four, or five years, or over the course of the disease, or the lifetime of the subject.
- the invention may particularly applicable to ageing subjects, for example, a human subject over the age of 30, or of an age in the range of from 30 to 100 years old.
- the muscle referred to herein is or includes skeletal muscle.
- Suitable tests for assessing muscle function include grip strength assessment using a dynamometer; one repeat maximum on leg press, chest press or leg extension; gait speed; 6 min walk test; time up and go; short physical performance battery; Fried frailty criteria; and stair climbing time assessments.
- Muscle mass (which may equate with muscle volume, muscle thickness or myofibre/muscle fibre size) may be measured by dual-energy X-ray absorptiometry (DXA) or bio-impedance tests.
- DXA dual-energy X-ray absorptiometry
- MRI may be used for assessing muscle volume and ultra-sound may be used for assessing muscle thickness and pennation angle.
- the subject referred to in any one of the aspects of the present invention is a mammal; humans are preferred.
- both human and veterinary subjects are within the scope of the invention.
- the inhibitor may be artificially generated. That is to say that it is not naturally occurring.
- the inhibitor may however be a naturally occurring molecule whose concentration and formulation in a medicament or pharmaceutical preparation enables it to be used for the prevention, amelioration or treatment of a brain cancer, whereas otherwise it would have no or limited efficacy.
- the inhibitor comprises a small molecule.
- the small molecule may be any appropriate organic molecule that inhibits HSD11 B1.
- the inhibitor may comprise an antibody or antibody mixture.
- antibody or antibodies may be polyclonal or may be monoclonal.
- the monoclonal antibodies may be obtained by any suitable method. Suitable methods will be known to a person skilled in the art.
- the monoclonal antibodies may be obtained using hybridoma technology, such as fusing antibody producing cells of antigen-immunised mammals with mammalian myeloma cells to produce a hybridoma cell line.
- the hybridoma cell line is produced by first immunising a mammal with an immunising antigen to produce an immunised mammal. Suitable immunising antigens are as defined above.
- the mammal may be immunised by any suitable method such as, for example, by intraperitoneal, subcutaneous, intravascular, intramuscular or intrasplenic injection or by oral administration.
- the immunising antigen may be administered as a suspension or solution in a buffer, such as phosphate buffered saline (PBS), optionally with an adjuvant, such as Freund’s complete adjuvant.
- a buffer such as phosphate buffered saline (PBS)
- an adjuvant such as Freund’s complete adjuvant.
- Any suitable mammal may be used such as, for example, mice, rats, rabbits, sheep or goats. It will be appreciated by a person skilled in the art that the mammal should typically be chosen so as to be compatible with the myeloma cells used in the subsequent cell fusion step.
- the immunising antigen is administered to the mammal several times, such as 2, 3, 4 or more times, at 4 to 21 day intervals.
- the antibody producing cells of the immunised mammal may be splenic cells.
- the splenic cells of the immunised mammal may be collected and fused with mammalian myeloma cells.
- the mammalian myeloma cells may be from any suitable source such as, for example, mice, rats or rabbits.
- the mammalian myeloma cells may be from the same mammalian source as the mammal immunised with the immunising antigen.
- the myeloma cells may be from mice.
- the mammalian myeloma cells are selected so as to have a hypoxyanthine-guanine- phosphoribosyltransferase deficiency (HGPRT) and/or a thymidine kinase deficiency (TK ).
- HGPRT hypoxyanthine-guanine- phosphoribosyltransferase deficiency
- TK thymidine kinase deficiency
- the mammalian myeloma cells may be mouse P3/NS1/1-Aq4-1 cells.
- the splenic cells may be fused to the mammalian myeloma cells by any suitable method. Suitable methods will be known to a person skilled in the art.
- the splenic cells may be fused to the mammalian myeloma cells using electrofusion, optionally in the presence of a fusion promoter such as, for example, polyethylene glycol (PEG) or hemagglutinating virus of Japan (HVJ).
- a fusion promoter such as, for example, polyethylene glycol (PEG) or hemagglutinating virus of Japan (HVJ).
- PEG polyethylene glycol
- HVJ hemagglutinating virus of Japan
- the splenic cells and the mammalian myeloma cells may be mixed at a ratio of 1 :1 to 10:1.
- the fused cells may be cultured and screened to selectively obtain hybridomas.
- the fused cells may be cultured in any suitable medium.
- the fused cells may be screened for hybridomas by any suitable method. Suitable methods will be known to a person skilled in the art.
- the fused cells may be screened for hybridomas using enzyme immunoassay, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or surface plasmon resonance (SPR).
- ELISA enzyme-linked immunosorbent assay
- ELISA enzyme-linked immunosorbent assay
- the fused cells may be screened for hybridomas by screening for binding to HSD11 B1. More preferably, the fused cells may be screened for hybridomas by screening for binding to HSD11 B1 , using enzyme-linked immunosorbent assay (ELISA).
- ELISA enzyme-linked immunosorbent assay
- the monoclonal antibodies produced by the cultured hybridomas may be obtained by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the monoclonal antibodies produced by the cultured hybridomas may be obtained by centrifugation of the supernatant of the culture.
- the obtained hybridomas when the monoclonal antibodies are produced by culturing the obtained hybridomas in the abdominal cavities of a suitable mammal such as, for example, a mouse, the obtained hybridomas may be intraperitoneally administered to the mammal such as, for example, mouse.
- the monoclonal antibodies produced by the culture in the abdominal cavities of a suitable mammal such as, for example, a mouse may then be obtained by collecting the fluid in the peritoneal cavity.
- the monoclonal antibodies produced by culturing the obtained hybridomas in a suitable medium or in the abdominal cavities of a suitable mammal such as, for example, a mouse may be used directly or may be purified.
- the monoclonal antibodies may be purified.
- the monoclonal antibodies may be purified by any suitable method. Suitable methods will be known to a person skilled in the art.
- the monoclonal antibodies may be purified by ammonium sulphate precipitation, ion exchange chromatography or an anti-lgG antibody column.
- the antibody of the present invention may be polyclonal.
- the polyclonal antibodies may be produced by any suitable method. Suitable methods will be known to a person skilled in the art.
- the polyclonal antibodies may be produced by immunising a mammal with an immunising antigen to induce the production of antibodies specific for the said immunising antigen. Suitable immunising agents are as defined above. Any suitable mammal may be used such as, for example, mice, rats or rabbits.
- the polyclonal antibodies produced by the immunised mammal may be obtained by any suitable method. Suitable methods will be known to a person skilled in the art.
- the polyclonal antibodies may be obtained by collecting the serum of the immunised mammal.
- the polyclonal antibodies produced by immunising a mammal with an immunising antigen and collecting the serum of the immunised mammal may be used directly or may be purified.
- the monoclonal antibodies may be purified.
- the polyclonal antibodies may be purified by any suitable method. Suitable methods will be known to a person skilled in the art.
- the polyclonal antibodies may be purified by ammonium sulphate precipitation, ion exchange chromatography or an anti-lgG antibody column.
- the polyclonal antibodies may be screened for binding to HSD11 B1 using any suitable method. Suitable methods will be known to a person skilled in the art.
- the polyclonal antibodies may be screened for binding to HSD11 B1 using enzyme immunoassay, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or surface plasmon resonance (SPR).
- enzyme immunoassay such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or surface plasmon resonance (SPR).
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- SPR surface plasmon resonance
- the polyclonal antibodies may be screened for binding to the HSD11 B1 using enzyme-linked immunosorbent assay (ELISA).
- the antibody of the present invention may be any suitable isotype.
- the antibody is of isotype IgG.
- antibodies of the isotype IgG typically comprise four peptide chains, of which two are heavy chains and two are light chains, and have two fragment antigen-binding (Fab) regions.
- the Fab regions comprise complementary determining regions (CDRs) which are the part of the antibody which bind to the antigen.
- the inhibitor or inhibitors may comprise a whole antibody or whole antibodies, it may comprise an antibody fragment or a modified form thereof.
- Suitable examples of whole antibodies include, but are not limited to, monovalent or divalent antibodies.
- Suitable examples of antibody fragments include, but are not limited to, Fab, F(ab’) 2 , Fv, Fab/c having one Fab and complete Fc, and single chain Fv (scFv) having heavy (H) or light (L) chain Fvs connected by a suitable linker.
- Other antibody scaffold proteins may be employed, such as Nanobodies RTM (these constructs, marketed by Ablynx (Belgium), comprise synthetic single immunoglobulin variable heavy domain derived from a camelid (e.g.
- Domain Antibodies (marketed by Domantis (Belgium), comprising an affinity matured single immunoglobulin variable heavy domain or immunoglobulin variable light domain), UniBodies (marketed by Genmab, UniBodies are modified fully human lgG4 antibodies where the hinge region of the antibody has been eliminated), Trifunctional Antibodies (monoclonal antibodies with binding sites for two different antigens), Affibodies (marketed by Affibody, Affibodies are based on a 58-amino acid residue protein domain, a three helix bundle domain, derived from one of the IgG- binding domains of staphylococcal protein A), Anticalins (antibody mimetics synthesised from human lipocalins, which can also be formatted as dual targeting proteins, so-called Duocalins) or DARPins (Designed Ankyrin Repeat Proteins) (which are another example of antibody mimetic based on repeat proteins, such as anky
- the antibody may be optimised or may be humanised.
- Optimised and like terms as used herein, means that the amino acid sequence of the antibody is adapted, such as by mutation or modification including, for example, glycosylation, so as to be suitable for use in the patient to which it is to be administered.
- Humanised and like terms as used herein, means that the amino acid sequence of the antibody is adapted, such as by mutation or modification including, for example, glycosylation, to reduce the composition of non-human amino acid sequences in the antibody.
- the antibody when humanised, may be partially humanised or may be substantially fully humanised.
- partially humanised is mean that part of the amino acid sequence of the antibody has been adapted, such as by mutation or modification including, for example, glycosylation, to be the same as the amino acid sequence of the human antibody.
- the antibody when partially humanised, may be partially humanised in any region of the antibody.
- the antibody when partially humanised, may be partially humanised in one or more of the variable fragment antigen-binding (Fab) regions of the antibody.
- substantially fully humanised is meant that substantially all of the amino acid sequence of the antibody has been adapted, such as by mutation or modification including, for example, glycosylation, to be the same as the amino acid sequence of the human antibody.
- the antibody is substantially fully humanised.
- the optimised and/or humanised antibody and derivatives may be produced by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the optimised and/or humanised antibody and derivatives thereof may be produced using genetic engineering technology, chimaeric technologies, CDR grafting or veneering.
- a polynucleotide encoding the antibody may be isolated and cloned into an expression vector to obtain a recombinant plasmid, transforming a host organism with the obtained recombinant plasmids, culturing the transformants and causing expression of the polynucleotide encoding the antibody.
- each polynucleotide may be cloned into the same or different expression vectors. Any suitable host organism may be transformed with the obtained recombinant plasmids.
- the host organism may be prokaryotic, such as E.coli, bacilli including Bacillus subtilis and enterobacteriaceae including Salmonella typhimurium, or eukaryotic, such as yeast including Saccharamyces cerevisiae.
- prokaryotic such as E.coli
- bacilli including Bacillus subtilis and enterobacteriaceae including Salmonella typhimurium
- eukaryotic such as yeast including Saccharamyces cerevisiae.
- yeast Saccharamyces cerevisiae.
- the optimised and/or humanised antibody may bind to any suitable antigen.
- the optimised and/or humanised antibody binds to HSD11 B1.
- the antibody or antibody mixtures may be present in the composition at any suitable concentration.
- the antibody may be present at a concentration of about 0.1 nanograms (ng) to 100 micrograms (mg), more preferably about 1 ng to 50 mg, most preferably about 10 mg to 50 mg.
- the inhibitor or inhibitors comprise a peptide or peptide mimetic thereof, or C-terminal amidated peptide thereof.
- peptide and“peptides” include compounds that have amino acid residues (H- Ca-[side chain]) but which may be joined by peptide (-CO-NH-) or non-peptide linkages.
- Peptides may be synthesised by the Fmoc-polyamide mode of solid-phase peptide synthesis. Reagents for peptide synthesis are readily commercially available.
- Purification of the peptides may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and (principally) reverse-phase high performance liquid chromatography. Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis.
- FAB fast atom bombardment
- the peptide may contain at least one D-amino acid residue, such as 1 , 2 or 3 or 4 or 5 or 6 or 7 or 8 D-amino acids.
- the composition of the invention may contain 0, 1 , 2 or 3 D-amino acids.
- the presence of D-amino acids in the composition of the invention may be useful in preventing degradation of the compound by proteases.
- Other methods for making peptides resistant to proteolytic degradation include blocking the N- and/or C- terminal amino acid residues. Thus, in some embodiments the N- and/or C-terminal amino acid residues are blocked. Suitable blocking methods include acetylation of the N- terminus or incorporating a pyroglutamate residue at the N-terminus.
- the peptide may be a peptide aptamer.
- Peptide aptamers typically consist of short, 5-20 amino acid residues long sequences that can bind to a specific target molecule.
- Retro-inverso peptidomimetics in which the peptide bonds are reversed, can be synthesised by methods known in the art. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Retro-inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are more resistant to proteolysis.
- the peptide may be linear. Although, it may be advantageous to introduce a cyclic moiety into a peptide-based framework. The cyclic moiety restricts the conformational space of the peptide structure and this may lead to an increased efficacy. An added advantage of this strategy is that the introduction of a cyclic moiety into a peptide may also result in the peptide having a diminished sensitivity to cellular peptidases.
- the peptide may be joined to another moiety.
- Convenient moieties to which the peptide may be joined include polyethylene glycol (PEG) and peptide sequences, such as TAT and antennapedia which enhance delivery to cells.
- PEGylation is a method well known to those skilled in the art wherein a (peptide or other compound) is modified such that one or more polyethylene glycol (PEG) molecules are covalently attached to the side chain of one or more amino acids. It is one of the most important molecule altering structural chemistry techniques (MASC). Other MASC techniques may be used; such techniques may improve the pharmacodynamic properties of a compound, for example extending its serum half-life in vivo.
- a PEG-peptide conjugate is formed by first activating the PEG moiety so that it will react with, and couple to, the compound of the invention.
- PEG moieties vary considerably in molecular weight and conformation, with the early moieties (monofunctional PEGs; mPEGs) being linear with molecular weights of 12kDa or less, and later moieties being of increased molecular weights.
- mPEGs monofunctional PEGs
- PEG2 a recent innovation in PEG technology, involves the coupling of a 30kDa (or less) mPEG to a lysine amino acid (although PEGylation can be extended to the addition of PEG to other amino acids) that is further reacted to form a branched structure that behaves like a linear mPEG of much greater molecular weight. Methods that may be used to covalently attach the PEG molecules to the peptides.
- PEGylation of the compound of the invention includes reduced renal clearance which, for some products, results in a more sustained adsorption after subcutaneous administration as well as restricted distribution, possibly leading to a more constant and sustained plasma concentrations and hence an increase in clinical effectiveness. Further potential advantages include reduced immunogenicity of the therapeutic compound, and lower toxicity.
- the inhibitor or inhibitors is/are pro-drugs of the peptide.
- a pro drug is a compound which is metabolised in vivo to produce the molecule, such as a protein.
- pro-drugs One of skill in the art will be familiar with the preparation of pro-drugs.
- the peptide may be a peptide mimetic.
- a peptide mimetic is an organic compound having similar geometry and polarity to the molecules defined herein, and which has a substantially similar function.
- a mimetic may be a molecule in which the NH groups of one or more peptide links are replaced by CH 2 groups.
- a mimetic may be a molecule in which one or more amino acid residues is replaced by an aryl group, such as a napthyl group.
- an inhibitor or inhibitors comprise nucleic acid, such as single stranded DNA or RNA, which is capable of binding to and inhibiting HSD11 B1. It is envisaged that the same targets on HSD11 B1 are also suitable for targeting with peptides and peptide aptamers will also be suitable for targeting with RNA or modified RNA aptamers. Nucleic acids such as single stranded DNAs and RNAs may be provided that bind to and inhibit HSD11 B1 expression by binding to mRNA or DNA. Typically, the nucleic acids are single stranded and have from 100 to 5000 bases.
- Figure 1 are graphs showing the effect of p38 inhibition on the regeneration of cardiotoxin- injured muscle as detailed in Example 1
- A Relative mRNA expression of Pax7; ** p ⁇ 0.05, cardiotoxin vs. cardiotoxin + Losmapimod 12mg/Kg (t-Test).
- B Number of Pax7 positive cells per mm 2 in Tibialis anterior muscle sections; #p ⁇ 0.01 .vehicle+vehicle vs. cardiotoxin+vehicle: *p ⁇ 0.05, cardiotoxin+vehicle vs. cardiotoxin+ Losmapimod 12mg/Kg;
- Figure 2 are graphs showing the average absolute numbers of Pax7 positive cells and the effect in MyoD, MyoG and fusion index after treatment with DMSO vehicle, 100 nM or 1 mM losmapimod with donors stratified as insulin resistant normal, insulin resistant sarcopenic, insulin sensitive normal and insulin sensitive sarcopenic, as detailed in Example 1.
- Left stages in the development of MSC to myotubes, showing their phenotypic markers; green squares indicate the cell type assessed in the plots displayed to the right.
- Pax7 +cells including satellite stem cells, satellite cells and myoblasts
- Pax7+ MyoD+ cells including satellite stem cells, satellite cells and myoblasts
- MyoD+ cells including myoblasts and myocytes
- MyoD single positive cells myocytes
- fused myotubes asserted in three different ways with MyoG, MHC and fusion index
- Figure 3 are graphs showing the effect of p38 inhibition on the development from MSC to fused myotubes after treatment (fold-change of Pax7 positive cells and the effect in MyoD, MyoG and fusion index after treatment with vehicle or 100 nM and 1 mM losmapimod, relative to their respective DMSO control at each timepoint for insulin sensitive normal and insulin sensitive sarcopenic).
- Top left stages in the development of MSC to myotubes, showing their phenotypic markers; green squares indicate the cell type assessed in the plots displayed to the right.
- Pax7 +cells satellite stem cells, satellite cells and myoblasts
- Pax7+ MyoD+ cells myoblasts
- MyoD+ cells myoblasts and myocytes
- MyoD single positive cells myocytes
- fused myotubes assessed by MHC and fusion index
- Figure 4 are graphs showing the effect of BEN2427 (ABT384) on a control, Sar c + DMSO, Sar c + inh 100nm, Sar c + inh 1 mM and Sar c + inh 10 mM.
- PAX7 is essential for the maintenance and function of MSCs - in particular, PAX7-expressing muscle satellite cells are indispensable for adult skeletal muscle regeneration. Thus, PAX7 is widely used as a marker for MSCs.
- the effect of p38 MAPK inhibition on the regeneration of cardiotoxin-injured muscle was examined in three groups of mice. C57BI/6J mice were treated with a single dose of cardiotoxin in both Tibilialis anterior (TA) muscles (intramuscular, 50 pi of 10 mM solution) and then administered with vehicle for Losmapimod for 21 days (p.o., 10 ml/kg, BID), starting one day after the injury. Losmapimod was dosed at 1 and 12 mg/kg per day and the expression of the MSC marker Pax7 determined by qPCR ( Figure 1A) and immunofluorescence ( Figure 1 B).
- the expression of mouse MyoG and Pax7 was evaluated with qPCR with the 7500 Real-Time PCR System (Applied Biosystems) using the relative standard curve method and TaqMan probe-based assays (Applied Biosystems). The data was analysed using the 7500 software v.2.0.5, Microsoft Excel 2013 and GraphPad Prism v.7.00. Outliers were detected using Grubb’s test and significant outliers were excluded. The results were normalized to the geometrical mean of the housekeeping genes Rps18 and Hprt (see Figure 1A).
- the following exemplary immunohistochemical test methods were used to detect biological markers of interest of the present invention. For example, increased proliferation of muscle satellite cells and/or inhibition of differentiation of muscle satellite cells in a subject can be established by determining whether there is an increase in PAX7 expression in the muscle of the subject by using one of these methods. Suitable modifications of the methods described below, or other suitable methods known to those skilled in the art, may be applied to stain, isolate and/or detected/visualise markers of interest.
- DSHB Monoclonal Mouse Anti-Human MF-20
- DSHB Monoclonal Mouse Anti-Human MF-20
- X0944, Dako isotype specific negative control
- Goat anti-mouse IgG secondary antibodies (abeam 150113) labelled with Alexa flour 488 dye were used for visualization.
- PAX7 a similar procedure was used using a rabbit polyclonal anti-human PAX7 monoclonal antibody and an anti-rabbit IgG labelled with Alexa 647. Using the abovementioned protocol, the content of PAX7 positive cells was assessed ( Figure 1 B).
- losmapimod promotes a dose-dependent increase in PAX7 expression, correlated with an increase in Pax7+ MSC content in the regenerating muscle.
- p38MAPK inhibition increases both the expression of pax7 and the MSC content in regenerating cardiotoxin injured muscle.
- HSD11 B1 inhibition also increases the number of pax7 expressing satellite cells derived from human muscle biopsies (both normal and sarcopenic) and that the inhibition does not have any effect on human satellite cells derived from a patient with insulin resistance/diabetes.
- This initial data suggests that HSD11 B1 inhibitors would appear to be suitable for treating sarcopenia and especially those individuals who are insulin sensitive.
- BEN2427 (a HSD11 B1 inhibitor (ABT384) was assessed at day 10 at 100nM, 1 mM and 10 mM. Table 1 below shows the results.
- losmapimod increases the pool of satellite cells in sarcopenic myoblasts to more than in normal cells when compared to the corresponding DMSO controls at each timepoint.
- Differentiation to myotube-forming cells, assessed by MyoG, MHC and fusion index was found to be reduced during treatment and recovered from day 7 onwards, when treatment was removed.
- formulations are provided below along with suggested dosage regimes. It will be understood that these are for illustrative purposes and these would be optimized during further experimentation, which may include clinical trials. For simplicity, the formulations do not stipulate any non-active components (such as pharmaceutically acceptable carriers or excipients etc.)
- Formulation 2C INCB-13739 - Oral Tablet for the Treatment of Sarcopenia in Insulin Sensitive Patients
- Formulation 2D Bezafibrate - Oral Tablet for the Treatment of Sarcopenia in Insulin Sensitive Patients
- Formulation 2E Diflunisal - Oral Tablet for the Treatment of Sarcopenia in Insulin Sensitive Patients
- HSD11 B1 inhibitors could also be employed in place of those outlined above.
- the therapeutically effective doses will of course depend on the activity and format of the chosen inhibitor.
- Organism Homo sapiens (Human) ( " from http://www.uniprot.org/uniprot/P28845):
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Abstract
The present invention relates to therapeutic compositions comprising a HSD11B1 inhibitor for use in the prevention, management and/or treatment of sarcopenia or a sarcopenic disease or disorder in individuals who are sensitive to insulin, and optionally following intermittent dosing.
Description
Compositions and Uses Thereof
Technical Field of the Invention
This invention relates to compositions and methods for the prevention, management and/or treatment of sarcopenia and related diseases and disorders in insulin sensitive individuals. More particularly, the invention relates to the treatment of sarcopenia associated with ageing, obesity and sarcopenia secondary to disease, injury, trauma or medication; to pharmaceutical compositions useful in the treatment of such conditions, and also to methods for identifying patients who are likely candidates for treatment. Related methods of screening and diagnosis are also disclosed.
Background to the Invention
Sarcopenia (loss of muscle mass and strength) affects between 5-13% of 60-70 year olds and 11-50% of over 80 year olds. It is a major public health issue, with the number of sarcopenic patients likely to dramatically increase in the next 30 years. It has been estimated that overall prevalence rate in the elderly will rise in Europe from 11.1 %-20.2% in 2016 to 12.9%-22.3% in 2045. Sarcopenia is a strong predictor of low quality of life, mobility impairment, falls, fractures and all-cause mortality. It is the second most common cause of disability in humans. Sarcopenia is also an independent risk factor for other serious health conditions, such as cardiovascular disease. The European working group on sarcopenia in older people (EWGSOP) defined diagnostic criteria, which include muscle mass (appendicular lean mass divided by height squared, men:£7.23kg/m2, womans 5.67kg/m2) and muscle function (gait speed <0.8 m/s; or grip strength, men: <30kg, women <20kg). Sarcopenia was classified in the International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD-10) in 2016 under code M62.84.
The underlying mechanism of muscle loss and function in sarcopenia is not fully understood, but studies to date have suggested that reduced muscle stem cell (satellite cell) numbers; impaired satellite cell proliferative function; loss through senescence; and ability to remain quiescent may each contribute to the loss of muscle mass and function and neuromuscular junction degeneration. Epigenetic changes in satellite cells may underlie their changed phenotype in injury-induced sarcopenia and age-related sarcopenia. Manipulation of satellite cell state and maintenance of the quiescent state is possibly a promising therapeutic strategy for sarcopenia.
Sarcopenia is currently treated by use of resistance exercise and nutritional interventions, such as high protein diet and vitamin D. These interventions to some extent rescue satellite cell proliferative capacity and reserve numbers, but the quality of evidence for efficacy is low.
Testosterone treatment has been shown to increase muscle size and power but it also induces undesirable side effects.
Selective androgen receptor modulators (SARM) have been trialled for sarcopenia and show some efficacy, but SARMs have a limiting side-effect profile and potential for abuse. Androgens and SARMs are thought to increase muscle mass without improvements in muscle strength or function and have no effect on regenerative capacity (i.e. satellite cells). Accordingly, these constitute at best a symptomatic, not disease-modifying, treatment.
Another therapeutic approach to sarcopenia is myostatin inhibition. Myostatin is a secreted protein that normally inhibits muscle growth. Myostatin-null animals and humans show skeletal muscle hypertrophy. Myostatin inhibitors, and inhibitors of its cognate receptor activin, have been developed. Most have shown at best only modest efficacy or a lack of long-term efficacy, and require regular injections, which has negative implications.
There therefore remains, an urgent need for an improved treatment for sarcopenia and sarcopenic diseases and disorders. Demographic changes are likely to increase the need for effective and safe treatments for these diseases, particularly in an ageing population.
Summary of the Invention
The present inventors have undertaken research which has enabled them to propose a novel approach to the treatment (including prevention or management) of sarcopenia and sarcopenic diseases in insulin sensitive individuals, based on a new understanding of the mechanisms involved in these diseases. In particular, the present inventors have identified the significance of the observation that the decrease in proliferative capacity of muscle associated with ageing and other‘insults’ is mediated in part by cortisol.
Cortisol is an active glucocorticoid that is convertible from the inert cortisone by action of an enzyme, cortisone reductase (also known as HSD11 B1 or I ΐ b-HSDI).
In accordance with an aspect of the present invention, there is provided a composition comprising a HSD11 B1 inhibitor for use in the prevention, management and/or treatment of sarcopenia or a sarcopenic disease or disorder in an insulin sensitive individual.
In accordance with a related, but alternative, aspect of the present invention, there is provided an inhibitor of HSD11 B1 for use in the prevention, management and/or treatment of sarcopenia or a sarcopenic disease or disorder in an insulin sensitive individual.
In accordance with a related, but yet further, aspect of the present invention, there is provided a method of prevention, management and/or treatment of sarcopenia or sarcopenic disease or disorder in an individual who is sensitive to insulin, wherein said method comprises the administration of a therapeutically effective amount of a HSD11 B1 inhibitor in an individual in need of such prevention, management and/or treatment. The method may comprise first determining that the individual is sensitive to insulin prior to the administration of the HSD11 B1 inhibitor.
In accordance with a related, but further, alternative aspect of the present invention, there is provided use of an inhibitor of HSD11 B1 in the manufacture of a medicament for the prevention, management and/or treatment of sarcopenia or a sarcopenic disease or disorder in an insulin sensitive individual.
In accordance with a related, but yet alternative, aspect of the present invention, there is provided a pharmaceutical composition, comprising a HSD11 B1 inhibitor and a pharmaceutically acceptable carrier, excipient, or diluent.
Sarcopenia primarily relates to degenerative loss of skeletal muscle mass, quality and strength associated with ageing. However, the above“sarcopenic” symptoms are also seen in other, non-age related, conditions. Accordingly, in the description of the present invention, the terms“sarcopenic disease” or“sarcopenic disorder” (which are often used interchangeably) should be understood as encompassing these. In particular, the invention encompasses sarcopenia associated with ageing, obesity and sarcopenia secondary to disease, injury, trauma or medication. This includes steroid-induced, in particular glucocorticoid (GC)-induced sarcopenia, which can be seen, for example, in individuals who (for whatever reason) are subject to, often high, exposure to endogenous or exogenous glucocorticoids. In particular, this may include exposure to higher than normal levels of cortisol. “Sarcopenic disease” also encompasses secondary sarcopenia,
for example that consequent upon surgery, such as that suffered by people that are immobilised after a bone fracture, especially after hip fracture or hip replacement surgery. This is particularly prevalent in elderly patients with hip fractures, but is not restricted to this group.
As used herein, the terms "treatment", "treating",“treat” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or can be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting or slowing its development; and (c) relieving the disease, i.e., causing regression of the disease.
The terms“subject” or“individual” used herein includes any human or non-human animal. The term “non-human animal” includes all mammals, such as non-human primates, sheep, dogs, cats, cows, horses.
The terms“sensitive to insulin”,“insulin sensitive” and“sensitivity to insulin” are deemed to mean that an individual is not diabetic or insensitive to insulin. Of course, sensitivity to insulin may also encompass those individuals who are mildly insensitive to insulin and/or individuals who are suffering from early stage or very mild diabetes.
Also preferably the HSD11 B1 inhibitor is administered following an intermittent dosage regime. The skilled addressee will understand that the intermittent dosage regime be an on/off dosing regime, such as one week of treatment followed by one week of no treatment or could be a dosing regime of less than once daily (for example a dose every 2 days, every 3 days, every 4 days, every 5 days, every 6 days or every 7 days). Preferably, the intermittent dosing regime comprises dosing for 7 days followed by 21 days with no dosing repeated.
Until the presently disclosed invention, it was not clear which particular pathway(s) or target compound(s) or, indeed combinations thereof, should be modulated to manipulate MSC state and population to provide an effective medicine for a subject suffering from sarcopenia or sarcopenic disease or disorder and especially individuals who are sensitive to insulin.
The present inventors have identified that modulation of HSD11 B1 , via inhibition, can prevent, inhibit and/or even reverse reduction of MSC populations, and affect the state of MSC (e.g. prevent, inhibit and/or even reverse MSC senescence). The present inventors have surprisingly shown that HSD11 B1 inhibitors, promote a dose-dependent increase in PAX7 expression, indicating an increase in MSC content in the regenerating muscle and such inhibitors would therefore be a successful approach to treating sarcopenia or a sarcopenic disease or disorder and data suggests that such an approach is particularly relevant for those individuals who are sensitive to insulin.
Without wishing to be bound by any particular theory, the inhibitor may be a selective inhibitor which may, without competing against a substrate(s), bind or interact with a target, so as to affect the target’s ability to further bind or interact with one or more specific substrates.
In one aspect of the invention, HSD11 B1 inhibition is proposed as a therapeutic strategy to treat the loss of muscle strength and regenerative capacity, including muscle wasting, associated with age (sarcopenia), injury, disease, trauma or medication (e.g. sarcopenic obesity or glucocorticoid- induced sarcopenia) in insulin sensitive individuals.
Another aspect of the invention provides an HSD11 B1 inhibitor, method or use according to any aspect of the invention, comprised in a therapeutic strategy to treat the loss of muscle strength or muscle regenerative capacity, optionally muscle wasting, associated with age (sarcopenia), injury, disease, trauma, or medication in insulin sensitive individuals. This includes, for example, sarcopenic obesity or glucocorticoid-induced sarcopenia.
In a further aspect, the invention provides an inhibitor, method or use according to any aspect of the invention wherein the prevention, management or treatment of sarcopenia or a sarcopenic disease or disorder comprises inhibiting HSD11 B1 signalling, and decreasing cortisol in the subject. Decreasing cortisol suitably means decreasing the level of cortisol in the blood or plasma of the subject.
According to an aspect of the invention, the subject in need may be identified in a test for sarcopenia and/or a sarcopenic disease or disorder and insulin sensitivity. In an aspect, the subject in need may be identified by a) measuring the level of cortisol in a biological sample taken from the subject and/or by analysing the DNA sequence, epigenetic
regulation, transcript or protein levels, enzymatic activity or post-translational modification of their HSD11B1 and/or NR3C1 genes; and b) measuring the level of blood sugar in a biological sample taken from the individual and confirming that the individual is sensitive to insulin. For, insulin sensitivity, the skilled addressee will appreciate that there are a range of tests available, but the most commonly used test would be a simple measurement of blood glucose levels (including levels after fasting).
In a preferred aspect of the invention, a subject in need of treatment, prevention or management of sarcopenia or a sarcopenic disease or disorder is one having an elevated cortisol level, compared to levels in a control population or a predetermined standard.
The elevated level may be, for example, in blood, plasma or urine. In one aspect, the elevated level (in humans) may be a cortisol level of equal to or greater than 6ng/ml of sample body fluid. In another aspect, the fasting blood sugar level (in humans) may be a blood sugar level of less than or equal to 5.4 mmol/L (99 mg/dL) and will usually be between 4.0 to 5.4 mmol/L (72 to 99 mg/dL).
HSD11 B1 inhibition is a promising therapeutic strategy for sarcopenia and sarcopenic conditions as described herein that offers advantages over the existing therapies by avoiding at least some of the problems associated with the existing therapies mentioned above.
HSD11 B1 inhibitors for use in the invention or in the methods of treatment of the invention include any chemical or biological agent that is known or can be identified as such, or a chemical or biological agent that down-regulates cortisol or a precursor thereof, or that inactivates or reduces activation of the glucocorticoid receptor (NR3C1) or precursor or activator thereof. Preferably, the inhibitor is a selective inhibitor of HSD11 B1 , and especially preferred are those with high potency.
Suitable HSD11 B1 inhibitors include: I ΐ b-HDSI inhibitors developed for the treatment of type 2 diabetes mellitus. For example, salicylate downregulates I ΐ b-HSDI expression in adipose tissue in obese mice and hence may explain why aspirin improves glycemic control in type 2 diabetes.
Natural products and older agents such as thiazolidinediones and fibrates seem to exert an inhibitory effect on I ΐ b-HSDI , ameliorating the cardiometabolic profile. Newer compounds, such as adamantyltriazoles, arylsulfonamidothiazoles, anilinothiazolones, BVT2733, INCB-13739, MK-0916 and MK-0736, are currently under investigation, and the
preliminary findings from both experimental and human studies show a favourable effect on glucose and lipid metabolism, weight reduction and adipokine levels. Butylated hydroxyanisole (BHA) is a widely used antioxidant for food preservation, and is a selective inhibitor of HSD11 B2, of the natural products, 18a-glycyrrhizic acid from the root of glycyrrhiza glabra, and also curcumin and derivatives have been identified as HSD11 B1 inhibitors.
Epigallocatechin gallate from green tea can also potently inhibit this enzyme, green tea is a complex mixture of various phenolics with contents varying with production and processing. Potent and selective inhibitors of the HSD11 B1 enzyme also include those known as AZD4017, ABT384, ABT305, INCB-13739, BVT.3498, BVT 116429, bezafibrate; CRx- 401 , diflunisal; BMS-823778; UE2343; and carbenoxolone, respectively.
2-[(3S)-1-[5-(cyclohexylcarbamoyl)-6-propylsulfanylpyridin-2-yl]piperidin-3-yl]acetic acid (AZD4017). AZD4017 is a selective, orally bioavailable inhibitor of the enzyme 11 -b- hydroxysteroid dehydrogenase type 1 , with potential protective activity. AZD4017 has been used in clinical trials for a number of indications: Idiopathic Intracranial Hypertension (400 mg oral tablet twice daily for 12 weeks); Obesity (oral suspensions 1200 mg once daily for 10 days); Diabetes Mellitus Type 2 (1st trial: oral suspension, ascending multiple doses starting at 75 mg once daily; 2nd trial: 400 mg oral tablet twice daily for 35 days); Iatrogenic Cushing's Disease (400 mg twice daily for 7 days with 20 mg prednisolone); Raised Intraocular Pressure (Europe: 200 mg oral tablet once daily for 28 days; USA: 2 x 200 mg oral tablets twice daily for 28 days).
4-[[2-methyl-2-[4-[5-(trifluoromethyl)pyridin-2-yl]piperazin-1- l]propanoyl]amino]adamantane-1-carboxamide (ABT-384). ABT384 is a potent, selective inhibitor of I I-b-hydroxysteroid dehydrogenase type 1. ABT384 has been used in clinical trials for adults with mild to moderate Alzheimer’s disease with subjects receiving 10 mg or 50 mg of ABT384 once daily for 12 weeks.
4-[[2-methyl-2-[[4-(trifluoromethyl)phenyl]methoxy]propanoyl]amino]adamantane-1- carboxamide (ABT-305)
(3S)-1-[(3-Chloro-2-methylphenyl)sulphonyl]-N-cyclohexyl-3-piperidinecarboxamide (INCB-13739). INCB-13739 is an orally available small molecule inhibitor of 1 1 -b- hydroxysteroid dehydrogenase type 1. INCB-13739 has been used in clinical trials for adults with Type 2 Diabetes, with a maximum dose of 200 mg per day (either as one dose, combined with metformin or 100 mg twice daily, alone).
3-chloro-2-methyl-N-[4-[2-(3-oxomorpholin-4-yl)ethyl]-1 ,3-thiazol-2- yljbenzenesulphonamide (BVT 3498). BVT3498 is a highly selective inhibitor of the enzyme I I -b-hydroxysteroid dehydrogenase type 1 . BVT.3498 has been used in clinical trials for adults with Type 2 Diabetes, completing phase I trials in 2002 and entering phase II trials. The trials were stopped in 2005.
(5S)-2-[[(1S)-1-(2-fluorophenyl)ethyl]amino]-5-methyl-5-(trifluoromethyl)-1 ,3-thiazol-4-one (BVT 116429) and its methyl analogue, (5S)-2-[[(1 S)-1-(2-methylphenyl)ethyl]amino]-5- methyl-5-(trifluoromethyl)-1 ,3-thiazol-4-one. BVT 116429 is a selective inhibitor of the enzyme I I-b-hydroxysteroid dehydrogenase type 1. Studies have been performed on diabetic mice using up to 30 mg/kg once daily for 10 days.
2-(4-{2-[(4-chlorobenzoyl)amino]ethyl}phenoxy)-2-methylpropanoic acid (CRx-401 , bezafibrate)
2',4'-difluoro-4-hydroxybiphenyl-3-carboxylic acid (CRx-401 , diflunisal). CRx-401 is a novel insulin sensitizer designed to provide anti-diabetic activity. It consists of a low dose of diflunisal in conjunction with a modified-release therapeutic dose of bezafibrate. It has been used in clinical trials for patients with Type II Diabetes taking metformin. Patients were given CRx-401 consisting of 400 mg bezafibrate sustained-release and 250 mg diflunisal once daily.
2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]phenoxy]-2-methylpropanoic acid (Bezafibrate) is an antilipemic agent that lowers cholesterol and triglycerides. It is an approved drug for patients with mixed hyperlipidaemia if statin is not tolerated. The dose is 200 mg orally three times daily using immediate-release medicines and 400 mg orally once daily using modified-release medicines. It is also undergoing clinical trials for the conditions of: Acute Myocardial Infarction (400 mg every 24 hours); Mitochondrial Diseases (200 mg - 600 mg three times daily for 12 weeks); Moderate to severe cholestatic itch (400 mg once daily); Adrenomyeloneuropathy (400 mg once daily until week 12, and subsequently use 800 mg once daily until week 24); Carnitine Palmitoyltransferase II Deficiency (3 x 200 mg per day); and Bipolar Disorder (400 mg once daily).
5-(2,4-difluorophenyl)-2-hydroxybenzoic acid (Diflunisal) is a salicylate derivative, is a nonsteroidal anti-inflammatory agent (NSAIA) with pharmacologic actions similar to other prototypical NSAIAs. It is commonly prescribed for pain relief and used in conditions including osteoarthritis, rheumatoid arthritis, renal impairment and hepatic impairment in maximum doses of 1500 mg per day. It is also undergoing clinical trials for the conditions of: HIV Infection (500 mg twice daily for 4 weeks); and Familial Amyloidosis (250 mg twice daily for 24 months).
2-[3-[1-(4-chlorophenyl)cyclopropyl]-[1 2 4]triazolo[4,3-a]pyridin-8-yl]propan-2-ol
(BMS-823778). BMS-823778 is an orally available potent and selective inhibitor of 1 1 -b- hydroxysteroid-dehydrogenase 1 . It has been used in clinical trails for the conditions of: Atherosclerotic Cardiovascular Disease (2 mg - 15 mg orally once daily for 1 year); Hypertension (2 mg - 15 mg orally once daily for 12 weeks); Dyslipidemia (2 mg - 20 mg orally once daily for 28 days); and Diabetes Mellitus Type 2 (2 mg - 20 mg orally once daily for 28 days, with metformin).
(3-hydroxy-3-pyrimidin-2-yl-8-azabicyclo[3.2.1]octan-8-yl)-[5-(1 H-pyrazol-4-yl)thiophen-3- yljmethanone (UE2343). UE2343 is a potent, orally bioavailable, brain-penetrant 1 1-b- hydroxysteroid-dehydrogenase 1 inhibitor. Clinical trials have evaluated the safety, tolerability of efficacy of Xanamem™ (comprising UE2343) in subjects with mild dementia due to Alzheimer’s Disease. In the trial, oral Xanamem™ 10 mg capsules were given once daily.
(33)-3-[(3-carboxypropanoyl)oxy]-1 1-oxoolean-12-en-30-oic acid (carbenoxolone). carbenoxolone is a hemisuccinate derivative of glycyrrhetinic acid and is frequently used in the treatment of peptic ulcers and the topical treatment of mouth ulcers. It is also a non- selective I I-b-hydroxysteroid-dehydrogenase 1 inhibitor that also inhibits 1 1 -b- hydroxysteroid-dehydrogenase 2. It has also been used in clinical trials for Type 2 diabetes, with a dosage of 100 mg three times per day.
(3-hydroxy-3-pyrimidin-2-yl-8-azabicyclo[3.2.1 ]octan-8-yl)-[5-(1 H-pyrazol-4-yl)thiophen-3- yl]methanone (UE2343). UE2343 is a potent, orally bioavailable, brain-penetrant 11-b- hydroxysteroid-dehydrogenase 1 inhibitor. Clinical trials have evaluated the safety, tolerability of efficacy of Xanamem™ (comprising UE2343) in subjects with mild dementia due to Alzheimer’s Disease. In the trial, oral Xanamem™ 10 mg capsules were given once daily.
^)-3-[(3-carboxypropanoyl)oxy]-11-oxoolean-12-en-30-oic acid (carbenoxolone). carbenoxolone is a hemisuccinate derivative of glycyrrhetinic acid and is frequently used in the treatment of peptic ulcers and the topical treatment of mouth ulcers. It is also a non- selective 11-b-hydroxysteroid-dehydrogenase 1 inhibitor that also inhibits 11-b- hydroxysteroid-dehydrogenase 2. It has also been used in clinical trials for Type 2 diabetes, with a dosage of 100 mg three times per day.
Included in the scope of the above-described inhibitors are derivatives thereof well-known for use in the pharmaceutical art. For use according to any aspect of the present invention, the inhibitors referred to herein, may be provided as any pharmaceutically acceptable derivative, selected from, but not limited to, any one or more of the following: pharmaceutically acceptable salts, pharmaceutically acceptable solvates, pharmaceutically acceptable enantiomers, pharmaceutically acceptable hydrates, pharmaceutically acceptable polymorphs, pharmaceutically acceptable esters and pharmaceutically acceptable prodrugs.
The inhibitors for use according to this invention include any metabolites thereof that are therapeutically active. The inhibitors for use according to this invention include any prodrugs thereof. Prodrugs are compounds that are converted to therapeutically active compounds as they are being administered to a patient or after they have been administered to a patient.
Preferably, the inhibitors for use according to this invention are selective inhibitors of the I ΐ b-HSDI (or HSD11 B1) enzyme. This is because glucocorticoid concentrations are modulated by two enzymes of I ΐ b-HSD (namely, IIb-HSDI and I Ib-H802), which have differing cofactor requirements and substrate affinities, and in vitro studies have shown that I Ib-HSDI is capable of acting as both a reductase and a dehydrogenase. Hence, selectivity for the type 1 isoenzyme, 11 b-HSDI, is preferred.
In another aspect, the invention provides a method of screening for an agent capable of inhibiting HSD11 B1 levels in a patient in need thereof comprising the steps of:
(a) contacting a population of cells with a candidate inhibitor;
(b) determining the level of cortisol or a precursor thereof in the population of cells; and
(c) comparing the level of cortisol or precursor thereof determined in step (b) with a cortisol or precursor level in a control population of cells which has not been contacted with the candidate inhibitor.
Preferably, the method is an in vitro method.
The candidate inhibitor is optionally comprised in a library of candidate inhibitors.
In another aspect, the invention provides an inhibitor for decreasing cortisol levels or levels of a cortisol precursor in a patient, wherein the inhibitor has been identified by the method of screening of the invention.
The term "level of cortisol" refers to the amount of cortisol that is found in a sample, e.g. of serum or plasma. The amount of may be determined directly or indirectly. Direct methods of determining cortisol include chromatographic methods, radioimmunoassay, enzyme- linked immunoassays and non-isotopic immunoassays. Liquid chromatography based mass spectrometry (LC-MS/MS) are commonly used to measure serum and plasma cortisol levels from multiple time points with high sensitivity. The effect of the candidate inhibitor on cortisol levels may be assessed as a function of time, by carrying out repeated measurements over a particular time-course.
The present invention also provides for the case when the HSD11 B1 inhibitor is used, administered or formulated together with vitamin D. By together with is not meant to imply only an intimate admixture of the two active ingredients, but also encompassed is separate, simultaneous or sequential administration.
The vitamin D may be administered by oral, parental, sub-lingual, sub-cutaneous, transdermal or intra-nasal administration. It may be administered as an oral vitamin D supplement or a probiotic supplement. The vitamin D may, for example, be in the form of a nutritional composition or supplement, or a diet product.
The patient may have previously been determined to be vitamin D deficient.
In another aspect, the invention provides a method of maintaining or increasing muscle function and/or mass and/or preventing or reducing muscle wasting in a patient in need thereof, particularly an ageing or elderly patient, comprising administering to the patient the inhibitor and vitamin D. Preferably, the vitamin D maintains or increases muscle mass; alternatively, the vitamin D substantially prevents or reduces a reduction in muscle mass.
In another aspect, the invention provides for the use or method according to the invention of a combined preparation of an inhibitor and vitamin D, wherein the inhibitor and vitamin D are for simultaneous, combined, sequential or separate administration to a patient.
Instead of or as well as combining an inhibitor with vitamin D, other combinations comprising an HSD11 B1 inhibitor are within the scope of this invention, such as a diet product, as mentioned above in relation to patient identification/diagnosis, and/or an
exercise regime may be combined with the inhibitor to maintain or increase muscle function and/or mass.
Furthermore, one or more other pharmacologically active agent(s) may be combined with an inhibitor, including selective androgen receptor modulators (SARMs), such as ostarine or myostatin blockers (e.g. myostatin antibodies, activin receptor antibodies and activin receptor-Fc), such as LY2495655 or Bimagrumab, or beta2 receptor agonists such as formoterol, or ghrelin receptor agonists such as anamorelin, or anabolic catabolic transforming agents (ACTA), such as MT-102, or other compounds mentioned hereinabove.
For use according to any one of the aspects of the present invention, the therapeutic ingredient(s), such as the inhibitors referred to herein may be administered at any suitable pharmacological dose, it being understood that the exact amounts (i.e. the therapeutically effective amounts) will depend upon the nature of the inhibitor and the condition to be treated. For example, suitable doses may comprise a daily dosage of from about 0.1 milligram to about 100 milligrams per kilogram of animal body weight, preferably given as a single daily dose or in divided doses two to six times a day, or in sustained release form.
For most large mammals, the total daily dosage is preferably from about 1 milligram to about 1000 milligrams, more preferably from about 1 milligram to about 350 milligrams, especially from about 1 mg to about 100 mg. In the case of a 70 kg adult human, the total daily dose is preferably in the range of from about 7 milligrams to about 350 milligrams. Typically, such doses may be in the range of from about 50 to 500 mg per day, such as about 240mg or about 100mg per day. This dosage regimen may be adjusted, as known by those skilled in the art, to provide the optimal therapeutic response.
Accordingly, the present invention further provides a use or method as described herein, wherein one or more therapeutic ingredients, including the inhibitor referred to herein, may be comprised in a pharmaceutical formulation, optionally together with one or more pharmaceutically acceptable carriers therefor. The one or more pharmaceutically acceptable carriers of the/each therapeutic ingredient may be the same or different.
Optionally, other therapeutic ingredients may be included in the pharmaceutical formulations of the present invention described herein.
For example, in accordance with any one of the aspects of the present invention described herein, the pharmaceutical formulation may further comprise an inhibitor of inflammatory signalling, including those described below. Accordingly, in accordance with any one of the aspects of the present invention described herein, the HSD11 B1 inhibitor may be for administration separately, sequentially or simultaneously with one or more pharmaceutically active ingredients.
The formulations include compositions suitable for oral, rectal, topical, parenteral, including subcutaneous, intramuscular, and intravenous, ocular (ophthalmic), pulmonary (nasal or buccal inhalation), or nasal administration (such as, for example, in the form of liquid drops or spray), although the most suitable route in any given case will depend on the nature and severity of the conditions being treated and on the nature of the therapeutic ingredient(s), e.g. the nature of the HSD11 B1s inhibitors, and/or other active ingredients present.
In accordance with any one of the aspects of the present invention described herein, the HSD11 B1 inhibitor, and optionally any other therapeutic ingredient(s), is/are preferably in a form suitable for intramuscular administration.
The formulations of the present invention may be conveniently presented in unit dosage form (e.g. a fixed dosage form) and prepared by any of the methods well-known in the art of pharmacy. Intramuscular dosage forms represent an advantageous dosage form as they allow fast absorption of large volumes directly to a target area, and as such, they may be preferred.
In practical use, the therapeutic ingredient(s), such as the inhibitors referred to herein, can be brought into an intimate physical admixture with one or more pharmaceutical carriers according to conventional pharmaceutical formulating techniques. The carrier(s) may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intramuscular and intravenous), preferably intramuscular.
In preparing the formulations/compositions in their dosage form for administration, any of the usual pharmaceutical excipients may be employed, such as, for example, diluents of a solid or liquid nature, flavouring agents, preservatives, colouring agents, and the like. Liquid preparations may be in the form of, for example, suspensions, elixirs and solutions.
Such liquid preparations may comprise one or more of: sucrose as a sweetening agent, methyl and/or propylparabens as preservatives, a dye and flavouring. Oral solid preparations are preferred over oral liquid preparations and are preferably in the form of, for example, powders, hard and soft capsules and tablets. When in solid form, the one or more pharmaceutical carriers may include one or more of: starches, sugars, microcrystalline cellulose, solid or liquid diluents, granulating agents, lubricants, binders, disintegrating agents and the like.
The tablets, pills, capsules, and the like may also contain one or more if: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin. When a dosage unit form is a capsule, it may contain, in addition to materials of the above type, comprise a liquid carrier such as a fatty oil.
Because of their ease of administration, tablets and capsules represent an advantageous oral dosage unit form. If desired, tablets may be coated by standard aqueous or non-aqueous techniques. For example, tablets may be coated with shellac, sugar or both.
Such formulations, compositions and preparations will preferably contain at least 0.1 % of the therapeutic ingredient(s), such as the inhibitors referred to herein (e.g. the HSD11 B1s inhibitors). The percentage of the therapeutic ingredient(s), such as the inhibitors referred to herein in these formulations/compositions may, of course, be varied and may conveniently be between about 2% to about 60% by weight of the unit dose. The amount of the therapeutic ingredient(s), such as the inhibitors referred to herein in such therapeutically useful compositions is such that an effective dosage (i.e. the therapeutically effective amount) will be obtained.
Various other materials may be present to act as coatings or to modify the physical form of the dosage unit.
For parenteral administration, the pharmaceutical forms include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Solutions or suspensions of these active inhibitors can be prepared in water for injections, optionally suitably mixed with a surfactant such as
hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils.
Accordingly, the pharmaceutical carrier(s) can be a solvent or dispersion medium containing, for example, water, an alcohol (e.g. ethanol), a polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycols), suitable mixtures thereof, and vegetable oils, and combinations thereof. In all cases, the form must be sterile and must be fluid to enable administration via a syringe. It must be stable under the conditions of manufacture and storage. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
Individual risk for sarcopenia in old age is variable and seems to be determined in significant part by adverse early life environmental exposures.
In a further aspect, therefore, the invention also provides a method of identifying a human or non-human animal suitable for or in need of the prevention, management or treatment, of sarcopenia or a sarcopenic disease or disorder by use or administration of a HSD11 B1 inhibitor, which method of identifying comprises:
(a) determining whether the human or non-human animal is at risk from, or suffering from sarcopenia or a sarcopenic disease or disorder;
(b) determining whether said human or non-human animal is sensitive to insulin; and
(c) selecting those humans or non-human animals which are at risk from, or suffering from sarcopenia or a sarcopenic disease or disorder animals and which are sensitive to insulin.
In any one of the methods of treatment of the present invention,“therapeutically effective amount” refers to the amount of the HSD11 B1 inhibitor that, when administered to a subject for cosmetic, preventative or therapeutic treatment, is sufficient to effect the desired treatment (e.g. of the disease). The“therapeutically effective amount” will vary, for example, depending on the inhibitor(s) used, the state of the disease and its severity and the age, weight, etc., of the subject to be treated, as described above.
In any one of the methods of preventing, inhibiting or treating muscle loss or atrophy in a subject according to the invention, the HSD11 B1 inhibitor, or formulations thereof as
described above may be administered to a subject using any one of the available methods and routes suitable for drug delivery, as described herein.
Optionally, the HSD11 B1s inhibitor or a formulations thereof, is administered by injection and/or delivery, for example, to a site in a muscle. Optionally, the HSD11 B1 inhibitor or formulation thereof, are administered by direct delivery in to a muscle prior to surgery or anticipated prolonged immobility. Alternatively, the HSD11 B1 inhibitor of a formulation thereof, is administered by direct delivery in to a muscle after surgery or prolonged immobility.
The HSD11 B1 inhibitor or a formulation thereof, may be administered in a single dose or in multiple doses. A suitable frequency of administration may be at least once per day, every other day, once per week, once every two, three, or four weeks, once every month, two months, or once every three to six months. The HSD11 B1 inhibitor may be administered over a period of at least a week, at least a month, at least three to six months, at least one, two, three, four, or five years, or over the course of the disease, or the lifetime of the subject.
The invention may particularly applicable to ageing subjects, for example, a human subject over the age of 30, or of an age in the range of from 30 to 100 years old.
Preferably, the muscle referred to herein is or includes skeletal muscle. Suitable tests for assessing muscle function include grip strength assessment using a dynamometer; one repeat maximum on leg press, chest press or leg extension; gait speed; 6 min walk test; time up and go; short physical performance battery; Fried frailty criteria; and stair climbing time assessments. Muscle mass (which may equate with muscle volume, muscle thickness or myofibre/muscle fibre size) may be measured by dual-energy X-ray absorptiometry (DXA) or bio-impedance tests. Similarly, MRI may be used for assessing muscle volume and ultra-sound may be used for assessing muscle thickness and pennation angle.
Preferably, the subject referred to in any one of the aspects of the present invention is a mammal; humans are preferred. However, both human and veterinary subjects are within the scope of the invention. For veterinary applications, the age of the animal would be scaled from the human situation using the average lifespan for calibration.
The inhibitor may be artificially generated. That is to say that it is not naturally occurring. The inhibitor may however be a naturally occurring molecule whose concentration and formulation in a medicament or pharmaceutical preparation enables it to be used for the prevention, amelioration or treatment of a brain cancer, whereas otherwise it would have no or limited efficacy.
In embodiments, the inhibitor comprises a small molecule. The small molecule may be any appropriate organic molecule that inhibits HSD11 B1.
The inhibitor may comprise an antibody or antibody mixture. Such antibody or antibodies may be polyclonal or may be monoclonal.
The monoclonal antibodies may be obtained by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the monoclonal antibodies may be obtained using hybridoma technology, such as fusing antibody producing cells of antigen-immunised mammals with mammalian myeloma cells to produce a hybridoma cell line. Preferably, the hybridoma cell line is produced by first immunising a mammal with an immunising antigen to produce an immunised mammal. Suitable immunising antigens are as defined above. The mammal may be immunised by any suitable method such as, for example, by intraperitoneal, subcutaneous, intravascular, intramuscular or intrasplenic injection or by oral administration. Preferably, the immunising antigen may be administered as a suspension or solution in a buffer, such as phosphate buffered saline (PBS), optionally with an adjuvant, such as Freund’s complete adjuvant. Any suitable mammal may be used such as, for example, mice, rats, rabbits, sheep or goats. It will be appreciated by a person skilled in the art that the mammal should typically be chosen so as to be compatible with the myeloma cells used in the subsequent cell fusion step. Preferably, the immunising antigen is administered to the mammal several times, such as 2, 3, 4 or more times, at 4 to 21 day intervals.
Typically, the antibody producing cells of the immunised mammal may be splenic cells. Preferably, the splenic cells of the immunised mammal may be collected and fused with mammalian myeloma cells. The mammalian myeloma cells may be from any suitable source such as, for example, mice, rats or rabbits. Preferably, the mammalian myeloma cells may be from the same mammalian source as the mammal immunised with the immunising antigen. Preferably, the myeloma cells may be from mice. Preferably, the mammalian myeloma cells are selected so as to have a hypoxyanthine-guanine- phosphoribosyltransferase deficiency (HGPRT) and/or a thymidine kinase deficiency (TK ). For example, the mammalian myeloma cells may be mouse P3/NS1/1-Aq4-1 cells.
The splenic cells may be fused to the mammalian myeloma cells by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the splenic cells may be fused to the mammalian myeloma cells using electrofusion, optionally in the presence of a fusion promoter such as, for example, polyethylene glycol (PEG) or hemagglutinating virus of Japan (HVJ). Preferably, the splenic cells and the mammalian myeloma cells may be mixed at a ratio of 1 :1 to 10:1.
Typically, the fused cells may be cultured and screened to selectively obtain hybridomas. The fused cells may be cultured in any suitable medium. The fused cells may be screened for hybridomas by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the fused cells may be screened for hybridomas using enzyme immunoassay, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or surface plasmon resonance (SPR). Preferably, the fused cells may be screened for hybridomas using enzyme-linked immunosorbent assay (ELISA). It will be appreciated by a person skilled in the art that it is the supernatant of the culture of the fused cells that is typically screened. Preferably, the fused cells may be screened for hybridomas by screening for binding to HSD11 B1. More preferably, the fused cells may be screened for hybridomas by screening for binding to HSD11 B1 , using enzyme-linked immunosorbent assay (ELISA).
The monoclonal antibodies produced by the cultured hybridomas may be obtained by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the monoclonal antibodies produced by the cultured hybridomas may be obtained by centrifugation of the supernatant of the culture.
In certain alternative embodiments, when the monoclonal antibodies are produced by culturing the obtained hybridomas in the abdominal cavities of a suitable mammal such as, for example, a mouse, the obtained hybridomas may be intraperitoneally administered to the mammal such as, for example, mouse. The monoclonal antibodies produced by the culture in the abdominal cavities of a suitable mammal such as, for example, a mouse may then be obtained by collecting the fluid in the peritoneal cavity.
The monoclonal antibodies produced by culturing the obtained hybridomas in a suitable medium or in the abdominal cavities of a suitable mammal such as, for example, a mouse may be used directly or may be purified. Preferably, the monoclonal antibodies may be purified. The monoclonal antibodies may be purified by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the monoclonal
antibodies may be purified by ammonium sulphate precipitation, ion exchange chromatography or an anti-lgG antibody column.
If monoclonal antibodies are employed which have binding with HSD11 B1 (or its ligand) then such antibodies may be used in combination (or combined) with small molecule inhibitors of HSD11 B1.
In certain embodiments, the antibody of the present invention may be polyclonal. The polyclonal antibodies may be produced by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the polyclonal antibodies may be produced by immunising a mammal with an immunising antigen to induce the production of antibodies specific for the said immunising antigen. Suitable immunising agents are as defined above. Any suitable mammal may be used such as, for example, mice, rats or rabbits. The polyclonal antibodies produced by the immunised mammal may be obtained by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the polyclonal antibodies may be obtained by collecting the serum of the immunised mammal.
The polyclonal antibodies produced by immunising a mammal with an immunising antigen and collecting the serum of the immunised mammal may be used directly or may be purified. Preferably, the monoclonal antibodies may be purified. The polyclonal antibodies may be purified by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the polyclonal antibodies may be purified by ammonium sulphate precipitation, ion exchange chromatography or an anti-lgG antibody column.
The polyclonal antibodies may be screened for binding to HSD11 B1 using any suitable method. Suitable methods will be known to a person skilled in the art. For example, the polyclonal antibodies may be screened for binding to HSD11 B1 using enzyme immunoassay, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or surface plasmon resonance (SPR). Preferably, the polyclonal antibodies may be screened for binding to the HSD11 B1 using enzyme-linked immunosorbent assay (ELISA).
The antibody of the present invention may be any suitable isotype. Preferably, the antibody is of isotype IgG. For the avoidance of doubt, antibodies of the isotype IgG typically comprise four peptide chains, of which two are heavy chains and two are light chains, and have two fragment antigen-binding (Fab) regions. The Fab regions comprise
complementary determining regions (CDRs) which are the part of the antibody which bind to the antigen.
Whilst the inhibitor or inhibitors may comprise a whole antibody or whole antibodies, it may comprise an antibody fragment or a modified form thereof. Suitable examples of whole antibodies include, but are not limited to, monovalent or divalent antibodies. Suitable examples of antibody fragments include, but are not limited to, Fab, F(ab’)2, Fv, Fab/c having one Fab and complete Fc, and single chain Fv (scFv) having heavy (H) or light (L) chain Fvs connected by a suitable linker. Other antibody scaffold proteins may be employed, such as Nanobodies RTM (these constructs, marketed by Ablynx (Belgium), comprise synthetic single immunoglobulin variable heavy domain derived from a camelid (e.g. camel or llama) antibody), Domain Antibodies (marketed by Domantis (Belgium), comprising an affinity matured single immunoglobulin variable heavy domain or immunoglobulin variable light domain), UniBodies (marketed by Genmab, UniBodies are modified fully human lgG4 antibodies where the hinge region of the antibody has been eliminated), Trifunctional Antibodies (monoclonal antibodies with binding sites for two different antigens), Affibodies (marketed by Affibody, Affibodies are based on a 58-amino acid residue protein domain, a three helix bundle domain, derived from one of the IgG- binding domains of staphylococcal protein A), Anticalins (antibody mimetics synthesised from human lipocalins, which can also be formatted as dual targeting proteins, so-called Duocalins) or DARPins (Designed Ankyrin Repeat Proteins) (which are another example of antibody mimetic based on repeat proteins, such as ankyrin or leucine-rich repeat proteins, which are ubiquitous binding molecules).
In certain embodiments, the antibody may be optimised or may be humanised. Optimised’, and like terms as used herein, means that the amino acid sequence of the antibody is adapted, such as by mutation or modification including, for example, glycosylation, so as to be suitable for use in the patient to which it is to be administered. ‘Humanised’, and like terms as used herein, means that the amino acid sequence of the antibody is adapted, such as by mutation or modification including, for example, glycosylation, to reduce the composition of non-human amino acid sequences in the antibody.
The antibody, when humanised, may be partially humanised or may be substantially fully humanised. By‘partially humanised’ is mean that part of the amino acid sequence of the antibody has been adapted, such as by mutation or modification including, for example, glycosylation, to be the same as the amino acid sequence of the human antibody. The
antibody, when partially humanised, may be partially humanised in any region of the antibody. Preferably, the antibody, when partially humanised, may be partially humanised in one or more of the variable fragment antigen-binding (Fab) regions of the antibody. By ‘substantially fully humanised’ is meant that substantially all of the amino acid sequence of the antibody has been adapted, such as by mutation or modification including, for example, glycosylation, to be the same as the amino acid sequence of the human antibody. Preferably, the antibody is substantially fully humanised.
The optimised and/or humanised antibody and derivatives may be produced by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the optimised and/or humanised antibody and derivatives thereof may be produced using genetic engineering technology, chimaeric technologies, CDR grafting or veneering.
In certain embodiments, when the optimised and/or humanised antibody and derivatives thereof are produced using genetic engineering technology, a polynucleotide encoding the antibody may be isolated and cloned into an expression vector to obtain a recombinant plasmid, transforming a host organism with the obtained recombinant plasmids, culturing the transformants and causing expression of the polynucleotide encoding the antibody. When more than one polynucleotide is used, each polynucleotide may be cloned into the same or different expression vectors. Any suitable host organism may be transformed with the obtained recombinant plasmids. For example, the host organism may be prokaryotic, such as E.coli, bacilli including Bacillus subtilis and enterobacteriaceae including Salmonella typhimurium, or eukaryotic, such as yeast including Saccharamyces cerevisiae. It will be appreciated by a person skilled in the art that the step of cloning into an expression vector to produce a recombinant plasmid may be performed using standard methods in the same or different organism to the host organism. Preferably, the step of cloning into an expression vector to produce a recombinant plasmid may be performed using standard methods in E.coli.
The optimised and/or humanised antibody may bind to any suitable antigen. Preferably, the optimised and/or humanised antibody binds to HSD11 B1.
The antibody or antibody mixtures may be present in the composition at any suitable concentration. Preferably, the antibody may be present at a concentration of about 0.1 nanograms (ng) to 100 micrograms (mg), more preferably about 1 ng to 50 mg, most preferably about 10 mg to 50 mg.
In other embodiments, the inhibitor or inhibitors comprise a peptide or peptide mimetic thereof, or C-terminal amidated peptide thereof.
The terms“peptide” and“peptides” include compounds that have amino acid residues (H- Ca-[side chain]) but which may be joined by peptide (-CO-NH-) or non-peptide linkages.
Peptides may be synthesised by the Fmoc-polyamide mode of solid-phase peptide synthesis. Reagents for peptide synthesis are readily commercially available.
Purification of the peptides may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and (principally) reverse-phase high performance liquid chromatography. Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis.
The peptide may contain at least one D-amino acid residue, such as 1 , 2 or 3 or 4 or 5 or 6 or 7 or 8 D-amino acids. Typically, the composition of the invention may contain 0, 1 , 2 or 3 D-amino acids. The presence of D-amino acids in the composition of the invention may be useful in preventing degradation of the compound by proteases. Other methods for making peptides resistant to proteolytic degradation include blocking the N- and/or C- terminal amino acid residues. Thus, in some embodiments the N- and/or C-terminal amino acid residues are blocked. Suitable blocking methods include acetylation of the N- terminus or incorporating a pyroglutamate residue at the N-terminus.
The peptide may be a peptide aptamer. Peptide aptamers typically consist of short, 5-20 amino acid residues long sequences that can bind to a specific target molecule.
There are a number of different approaches to the design and synthesis of peptide composition that do not contain amide bonds. In one approach, one or more amide bonds are replaced in an essentially isoteric manner by a variety of chemical functional groups.
Retro-inverso peptidomimetics, in which the peptide bonds are reversed, can be synthesised by methods known in the art. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Retro-inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are more resistant to proteolysis.
The peptide may be linear. Although, it may be advantageous to introduce a cyclic moiety
into a peptide-based framework. The cyclic moiety restricts the conformational space of the peptide structure and this may lead to an increased efficacy. An added advantage of this strategy is that the introduction of a cyclic moiety into a peptide may also result in the peptide having a diminished sensitivity to cellular peptidases.
In some embodiments of the invention the peptide may be joined to another moiety. Convenient moieties to which the peptide may be joined include polyethylene glycol (PEG) and peptide sequences, such as TAT and antennapedia which enhance delivery to cells.
PEGylation is a method well known to those skilled in the art wherein a (peptide or other compound) is modified such that one or more polyethylene glycol (PEG) molecules are covalently attached to the side chain of one or more amino acids. It is one of the most important molecule altering structural chemistry techniques (MASC). Other MASC techniques may be used; such techniques may improve the pharmacodynamic properties of a compound, for example extending its serum half-life in vivo. A PEG-peptide conjugate is formed by first activating the PEG moiety so that it will react with, and couple to, the compound of the invention. PEG moieties vary considerably in molecular weight and conformation, with the early moieties (monofunctional PEGs; mPEGs) being linear with molecular weights of 12kDa or less, and later moieties being of increased molecular weights. PEG2, a recent innovation in PEG technology, involves the coupling of a 30kDa (or less) mPEG to a lysine amino acid (although PEGylation can be extended to the addition of PEG to other amino acids) that is further reacted to form a branched structure that behaves like a linear mPEG of much greater molecular weight. Methods that may be used to covalently attach the PEG molecules to the peptides. The potential advantages of PEGylation of the compound of the invention include reduced renal clearance which, for some products, results in a more sustained adsorption after subcutaneous administration as well as restricted distribution, possibly leading to a more constant and sustained plasma concentrations and hence an increase in clinical effectiveness. Further potential advantages include reduced immunogenicity of the therapeutic compound, and lower toxicity.
In some embodiments, the inhibitor or inhibitors is/are pro-drugs of the peptide. A pro drug is a compound which is metabolised in vivo to produce the molecule, such as a protein. One of skill in the art will be familiar with the preparation of pro-drugs.
The peptide may be a peptide mimetic. A peptide mimetic is an organic compound having similar geometry and polarity to the molecules defined herein, and which has a
substantially similar function. A mimetic may be a molecule in which the NH groups of one or more peptide links are replaced by CH2 groups. A mimetic may be a molecule in which one or more amino acid residues is replaced by an aryl group, such as a napthyl group.
In other embodiments, an inhibitor or inhibitors comprise nucleic acid, such as single stranded DNA or RNA, which is capable of binding to and inhibiting HSD11 B1. It is envisaged that the same targets on HSD11 B1 are also suitable for targeting with peptides and peptide aptamers will also be suitable for targeting with RNA or modified RNA aptamers. Nucleic acids such as single stranded DNAs and RNAs may be provided that bind to and inhibit HSD11 B1 expression by binding to mRNA or DNA. Typically, the nucleic acids are single stranded and have from 100 to 5000 bases.
Features, integers, characteristics, compounds, molecules, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and figures), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
Detailed Description of the Invention
Embodiments of the invention are described below, by way of example only, with reference to the accompanying figures in which:
Figure 1 are graphs showing the effect of p38 inhibition on the regeneration of cardiotoxin- injured muscle as detailed in Example 1 (A) Relative mRNA expression of Pax7; ** p<0.05, cardiotoxin vs. cardiotoxin + Losmapimod 12mg/Kg (t-Test). (B) Number of Pax7 positive cells per mm2 in Tibialis anterior muscle sections; #p<0.01 .vehicle+vehicle vs. cardiotoxin+vehicle: *p<0.05, cardiotoxin+vehicle vs. cardiotoxin+ Losmapimod 12mg/Kg;
Figure 2 are graphs showing the average absolute numbers of Pax7 positive cells and the effect in MyoD, MyoG and fusion index after treatment with DMSO vehicle, 100 nM or
1 mM losmapimod with donors stratified as insulin resistant normal, insulin resistant sarcopenic, insulin sensitive normal and insulin sensitive sarcopenic, as detailed in Example 1. Left, stages in the development of MSC to myotubes, showing their phenotypic markers; green squares indicate the cell type assessed in the plots displayed to the right. Right, average absolute numbers of Pax7 +cells (including satellite stem cells, satellite cells and myoblasts), Pax7+ MyoD+ cells (myoblasts), MyoD+ cells (myoblasts and myocytes), MyoD single positive cells (myocytes) and fused myotubes (assessed in three different ways with MyoG, MHC and fusion index);
Figure 3 are graphs showing the effect of p38 inhibition on the development from MSC to fused myotubes after treatment (fold-change of Pax7 positive cells and the effect in MyoD, MyoG and fusion index after treatment with vehicle or 100 nM and 1 mM losmapimod, relative to their respective DMSO control at each timepoint for insulin sensitive normal and insulin sensitive sarcopenic). Top left, stages in the development of MSC to myotubes, showing their phenotypic markers; green squares indicate the cell type assessed in the plots displayed to the right. Top right, fold change in the absolute number of cells (normalised to the corresponding DMSO control at each timepoint) for Pax7 +cells (satellite stem cells, satellite cells and myoblasts), Pax7+ MyoD+ cells (myoblasts), MyoD+ cells (myoblasts and myocytes), MyoD single positive cells (myocytes) and fused myotubes (assessed by MHC and fusion index). Comparison of the increase in Pax7+ (satellite stem cells, satellite cells and myoblasts) and Pax7+ MyoD+ cells (myoblasts) relative to DMSO when insulin sensitive normal and insulin sensitive sarcopenic cells were treated with 1 uM Losmapimod; and
Figure 4 are graphs showing the effect of BEN2427 (ABT384) on a control, Sar c + DMSO, Sar c + inh 100nm, Sar c + inh 1 mM and Sar c + inh 10 mM.
Example 1
Tests for determining an increase proliferation of muscle satellite cells
PAX7 is essential for the maintenance and function of MSCs - in particular, PAX7-expressing muscle satellite cells are indispensable for adult skeletal muscle regeneration. Thus, PAX7 is widely used as a marker for MSCs.
The effect of p38 MAPK inhibition on the regeneration of cardiotoxin-injured muscle was examined in three groups of mice. C57BI/6J mice were treated with a single dose of cardiotoxin in both Tibilialis anterior (TA) muscles (intramuscular, 50 pi of 10 mM solution) and then administered with vehicle for Losmapimod for 21 days (p.o., 10 ml/kg, BID), starting one day after the injury. Losmapimod was dosed at 1 and 12 mg/kg per day and the expression of the MSC marker Pax7 determined by qPCR (Figure 1A) and immunofluorescence (Figure 1 B).
Total RNA was purified from the left TA samples by using the RNeasy Plus Universal Mini Kit. 2000 ng of RNA was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit. The expression of mouse MyoG and Pax7 (see above section entitled“Tests”) was evaluated with qPCR with the 7500 Real-Time PCR System (Applied Biosystems) using the relative standard curve method and TaqMan probe-based assays (Applied Biosystems). The data was analysed using the 7500 software v.2.0.5, Microsoft Excel 2013 and GraphPad Prism v.7.00. Outliers were detected using Grubb’s test and significant outliers were excluded. The results were normalized to the geometrical mean of the housekeeping genes Rps18 and Hprt (see Figure 1A).
The following exemplary immunohistochemical test methods were used to detect biological markers of interest of the present invention. For example, increased proliferation of muscle satellite cells and/or inhibition of differentiation of muscle satellite cells in a subject can be established by determining whether there is an increase in PAX7 expression in the muscle of the subject by using one of these methods. Suitable modifications of the methods described below, or other suitable methods known to those skilled in the art, may be applied to stain, isolate and/or detected/visualise markers of interest.
Monoclonal Mouse Anti-Human MF-20 (DSHB), which recognises all MHC (myosin heavy chain) isoforms, was used to stain for the myosin heavy chain, and the results compared with the test carried out using an isotype specific negative control (X0944, Dako). Goat anti-mouse IgG secondary antibodies (abeam 150113) labelled with Alexa flour 488 dye were used for visualization. For PAX7, a similar procedure was used using a rabbit polyclonal anti-human PAX7 monoclonal antibody and an anti-rabbit IgG labelled with Alexa 647. Using the abovementioned protocol, the content of PAX7 positive cells was assessed (Figure 1 B).
The results show that losmapimod promotes a dose-dependent increase in PAX7 expression, correlated with an increase in Pax7+ MSC content in the regenerating muscle. In conclusion, p38MAPK inhibition increases both the expression of pax7 and the MSC content in regenerating cardiotoxin injured muscle.
It is known that by the inhibition of glucocorticoid action through the blocking of HSD11 B1 , circulating cortisone is converted to active cortisol by the muscle HSD11 B1. It has been shown that Mouse HSD11B1 KO protected against cortisol induced sarcopenia (Morgan et al 2013). Increased skeletal muscle HSD11B1 mRNA is associated with lower muscle strength in ageing (Kilgour et al 2013) and (Hassan-Smith et al 2015) and increases with age and reduced grip strength (Hassan-Smith et al 2015).
In recent studies by the inventors (not shown), it has been established that HSD11 B1 inhibition also increases the number of pax7 expressing satellite cells derived from human muscle biopsies (both normal and sarcopenic) and that the inhibition does not have any effect on human satellite cells derived from a patient with insulin resistance/diabetes. This initial data suggests that HSD11 B1 inhibitors would appear to be suitable for treating sarcopenia and especially those individuals who are insulin sensitive.
Patient level in vitro assay for sarcopenia
The effect of BEN2427 (a HSD11 B1 inhibitor (ABT384)) was assessed at day 10 at 100nM, 1 mM and 10 mM. Table 1 below shows the results.
Table 1.
As shown in Table 1 above, there was a significant increase in %PAX7 at 100nM, which then plateaus with a mean increase is ~2x which is similar to effects of epigenetic modifiers. In a sarcopenia assay, as shown in Figure 4, BEN2427 increases pool of satellite cells in sarcopenic patients. Differentiation to myotube-forming cells was not found to affected.
In a sarcopenia assay (the results of which are detailed in Figure 2 and Figure 3), losmapimod increases the pool of satellite cells in sarcopenic myoblasts to more than in normal cells when compared to the corresponding DMSO controls at each timepoint. Differentiation to myotube-forming cells, assessed by MyoG, MHC and fusion index was found to be reduced during treatment and recovered from day 7 onwards, when treatment was removed. These results suggest that an intermittent dosing regime would be suitable for treating sarcopenia in insulin sensitive patients.
Example 2
Example Formulations and Treatments for Sarcopenia in Insulin Sensitive Patients
A number of example formulations are provided below along with suggested dosage regimes. It will be understood that these are for illustrative purposes and these would be optimized during further experimentation, which may include clinical trials. For simplicity, the formulations do not stipulate any non-active components (such as pharmaceutically acceptable carriers or excipients etc.)
Formulation 2A - AZD4017 - Oral Tablet for the Treatment of Sarcopenia in Insulin Sensitive Patients
Formulation 2B - ABT-384 - Oral Tablet for the Treatment of Sarcopenia in Insulin Sensitive Patients
Formulation 2C - INCB-13739 - Oral Tablet for the Treatment of Sarcopenia in Insulin Sensitive Patients
Formulation 2D - Bezafibrate - Oral Tablet for the Treatment of Sarcopenia in Insulin Sensitive Patients
Formulation 2E - Diflunisal - Oral Tablet for the Treatment of Sarcopenia in Insulin Sensitive Patients
Formulation 2F - BMS-823778 - Oral Inhaled Solution for the Treatment of Sarcopenia in Insulin Sensitive Patients
Sensitive Patients
Formulation 2H - Carbenoxolone - Oral Tablet for the Treatment of Sarcopenia in Insulin Sensitive Patients
Formulation 2K - ABT-384 - Oral Tablet for the Treatment of Sarcopenia in Insulin Sensitive Patients - Alternative Dosing
Formulation 2L - ABT-384 - Oral Tablet for the Treatment of Sarcopenia in Insulin Sensitive Patients - Alternative Dosing
The skilled addressee will of course understand that alternative HSD11 B1 inhibitors could also be employed in place of those outlined above. The therapeutically effective doses will of course depend on the activity and format of the chosen inhibitor.
The forgoing embodiments are not intended to limit the scope of the protection afforded by the claims, but rather to describe examples of how the invention may be put into practice.
Amino Acid Sequences
Amino acid sequence for the corticosteroid 11 -beta-dehydrogenase isozyme 1, protein (HSD11 B1) (SEQ ID No. 1):
Organism: Homo sapiens (Human) ("from http://www.uniprot.org/uniprot/P28845):
Claims
1. A composition comprising a HSD11 B1 inhibitor for use in the prevention, management and/or treatment of sarcopenia or a sarcopenic disease or disorder in an insulin sensitive individual.
2. The composition according to claim 1 , wherein the HSD11 B1 inhibitor is a selective inhibitor of HSD11 B1.
3. The composition according to either claim 1 or 2, wherein the HSD11 B1 inhibitor is selected from one or more of the following: AZD4017, ABT384, ABT305, INCB- 13739, BVT.3498, BVT 116429, bezafibrate; CRx-401 , diflunisal; BMS-823778; UE2343; and carbenoxolone.
4. The composition according to any preceding claim, wherein the composition is
administered using an intermittent dosing regime.
5. An inhibitor of HSD11 B1 for use in the prevention, management and/or treatment of sarcopenia or a sarcopenic disease or disorder in an insulin sensitive individual.
6. The inhibitor according to claim 5, wherein the HSD11 B1 inhibitor is selected from one or more of the following: AZD4017; ABT384; ABT305; INCB-13739; BVT.3498; BVT 116429; bezafibrate; CRx-401 ; diflunisal; BMS-823778; UE2343; and carbenoxolone.
7. The inhibitor according to claim 5 or 6, wherein the inhibitor is administered using an intermittent dosing regime.
8. A method of prevention, management and/or treatment of sarcopenia or a sarcopenic disease or disorder comprising the administration of a HSD11 B1 inhibitor in an individual who is insulin sensitive and in need of such prevention, management and/or treatment.
9. The method according to claim 8, wherein the HSD11 B1 inhibitor is selected from one or more of the following: AZD4017; ABT384; ABT305; INCB-13739; BVT.3498; BVT 116429; bezafibrate; CRx-401 ; diflunisal; BMS-823778; UE2343; and carbenoxolone.
10. The method according to claim 8 or 9, wherein the administration of a HSD11 B1 inhibitor is according to an intermittent dosing regime.
11. The method according to claim 10, wherein the intermittent dosing regime comprises dosing for 7 days followed by 21 days of no dosing repeated.
12. The method according any one of claims 8 to 11 , wherein the individual in need is identified by
a) measuring the level of cortisol in a biological sample taken from the individual and/or by analysing the DNA sequence, epigenetic regulation, transcript or protein levels, enzymatic activity or post-translational modification of their HSD11 B1 and/or NR3C1 genes; and
b) measuring the level of blood sugar in a biological sample taken from the individual and confirming that the individual is sensitive to insulin.
13. A method of identifying a human or non-human animal suitable for or in need of the prevention, management or treatment of sarcopenia or a sarcopenic disease or disorder by use or administration of an inhibitor of HSD11 B1 which method of identifying comprises:
(a) determining the baseline level of cortisol in the human or non-human animal;
(b) comparing the level determined in step (a) with a baseline of cortisol in a control population of humans or non-human animals which do not have sarcopenia or a sarcopenic disease or disorder;
(c) determining whether the and human or non-human animal is sensitive to insulin; and
(d) selecting for humans and non-human animals in which the baseline level determined in step (a) is greater than that seen in step (b) and which have been determined to be sensitive to insulin.
14. Use of an inhibitor of HSD11 B1 in the manufacture of a medicament for the prevention, management and/or treatment of sarcopenia or a sarcopenic disease or disorder in an insulin sensitive individual.
15. Use according to claim 14, wherein the HSD11 B1 inhibitor is selected from one or more of the following: AZD4017; ABT384; ABT305; INCB-13739; BVT.3498; BVT 116429; bezafibrate; CRx-401 ; diflunisal; BMS-823778; UE2343; and carbenoxolone.
16. A pharmaceutical composition, comprising a HSD11 B1 inhibitor and a pharmaceutically acceptable carrier, excipient, or diluent.
17. The pharmaceutical composition according to claim 16, wherein the HSD11 B1 inhibitor is selected from one or more of the following: AZD4017; ABT384; ABT305; INCB-13739; BVT.3498; BVT 116429; bezafibrate; CRx-401 ; diflunisal; BMS-823778; UE2343; and carbenoxolone.
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GBGB1821144.1A GB201821144D0 (en) | 2018-12-21 | 2018-12-21 | Compositions and uses thereof |
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Citations (3)
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WO2008006819A2 (en) * | 2006-07-14 | 2008-01-17 | Vib Vzw | Use of ppar-alpha agonists to treat skeletal muscle wasting disorders |
US20170014425A1 (en) * | 2015-07-13 | 2017-01-19 | Ajou University Industry-Academic Cooperation Foundation | Pharmaceutical composition for preventing or treatinghearing loss, comprising carbenoxolone or its pharmaceutically acceptable salts as effective compound |
WO2019186171A1 (en) * | 2018-03-28 | 2019-10-03 | Benevolentai Bio Limited | Treatment of sarcopenic diseases |
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2018
- 2018-12-21 GB GBGB1821144.1A patent/GB201821144D0/en not_active Ceased
-
2019
- 2019-12-23 WO PCT/GB2019/053682 patent/WO2020128529A1/en active Application Filing
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WO2008006819A2 (en) * | 2006-07-14 | 2008-01-17 | Vib Vzw | Use of ppar-alpha agonists to treat skeletal muscle wasting disorders |
US20170014425A1 (en) * | 2015-07-13 | 2017-01-19 | Ajou University Industry-Academic Cooperation Foundation | Pharmaceutical composition for preventing or treatinghearing loss, comprising carbenoxolone or its pharmaceutically acceptable salts as effective compound |
WO2019186171A1 (en) * | 2018-03-28 | 2019-10-03 | Benevolentai Bio Limited | Treatment of sarcopenic diseases |
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ALIXE H. M. KILGOUR ET AL: "Increased Skeletal Muscle 11[beta]HSD1 mRNA Is Associated with Lower Muscle Strength in Ageing", PLOS ONE, vol. 8, no. 12, 31 December 2013 (2013-12-31), pages e84057, XP055667695, DOI: 10.1371/journal.pone.0084057 * |
GERARD J. MAREK ET AL: "Efficacy and safety evaluation of HSD-1 inhibitor ABT-384 in Alzheimer's disease", ALZHEIMER'S & DEMENTIA: THE JOURNAL OF THE ALZHEIMER'SASSOCIATION, vol. 10, no. 5, 1 October 2014 (2014-10-01), US, pages S364 - S373, XP055598497, ISSN: 1552-5260, DOI: 10.1016/j.jalz.2013.09.010 * |
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