RU2781401C2 - Use of myostatin antagonists, combination containing them and their use - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: group of inventions relates to the field of medicine and pharmaceutics; it can be used for increasing or maintaining muscle mass. A combination is proposed, including ActRII receptor inhibitor, which is bimagrumab, and a chemotherapeutic agent, which is everolimus, wherein bimagrumab and everolimus are in separate pharmaceutical compositions. The use of the specified combination is also proposed for increasing or maintaining muscle mass in a subject having an age-related condition.

EFFECT: additive effect of a combination is provided in terms of increasing muscle mass.

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Description

FIELD TO WHICH THE INVENTION RELATES

The present disclosure relates to the use of myostatin or activin antagonists and in particular activin type II receptor inhibitors (ActRII) in the treatment of cancer cachexia.

More specifically, the present invention relates to combinations containing (a) an activin type II receptor inhibitor (ActRII) and (b) a chemotherapeutic agent or a pharmaceutically acceptable salt thereof, for simultaneous, separate or sequential use, their uses or methods of treatment using them for treatment of cancer cachexia.

The present disclosure also relates to combinations and combination uses of a myostatin antagonist and an mTOR inhibitor. Such combined applications are used in cancer cachexia and age-related conditions.

BACKGROUND OF THE INVENTION

Cachexia affects most patients with advanced cancer and is associated with poor outcome, decreased treatment tolerance, response to therapy, quality of life, and survival. Loss of skeletal muscle mass appears to be the most significant event in cancer cachexia and cannot be fully restored with conventional nutritional support [Fearon et al 2011, Tan et al 2009]. It has recently been shown in mouse models of ectopic lung and colon cancer that direct inhibition of myostatin by a monoclonal antibody, as well as indirect inhibition using soluble ActRIIB-Fc, protects against muscle wasting and even increases survival [Benny Klimek et al 2010, Busquets et al 2012, Murphy et al 2011, Zhou et al 2010].

Several members of the transforming growth factor beta (TGF-β) superfamily, including myostatin, Activin A, and growth differentiation factor 11 (GDF-11), are known to negatively regulate skeletal muscle mass in animals and humans throughout the life cycle. The mechanism of myostatin signaling is complex due to the activation of several downstream signaling pathways [Elkina et al 2011]. Myostatin, Activin and GDF-11 bind to activin type II receptors (ActRII) and induce its binding to the activin type I receptor. Lack of myostatin in developing animals and humans results in a hypermuscular phenotype with increased number and size of muscle fibers [Lee and McPherron 2001, Schuelke et al 2004]. Similarly, inhibition of myostatin action in adult animals increases muscle mass, indicating that myostatin also inhibits adult skeletal muscle mass [Whittemore et al 2003, Lee et al 2005, Nakatani et al 2008]. In contrast, high levels of myostatin or Activin A have been reported to promote cachexia and subsequent muscle atrophy [Zimmers et al, 2002; Chen et al, 2014].

WO 07/067616 shows reduction in weight loss by administering a myostatin binding agent, such as myostatin binding peptide, to normal mice treated with 5-fluorouracil. However, this does not show that myostatin binding peptide reduces body weight loss or increases body weight in a mouse model bearing a tumor such as CT-26, either in the absence of treatment or when treated with anti-cancer agents, as shown in accordance with the present. disclosure.

Bimagrumab is a human monoclonal antibody designed to compete with ActRII with greater affinity than its natural ligands, myostatin and activin A. It induces skeletal muscle hypertrophy and protects against dexamethasone-induced atrophy in mice [Lach-Trifilieff et al., 2014] and has been shown to improve disease status in patients suffering from sporadic inclusion body myositis without causing serious side effects [Amato et al 2014]. Although pharmacological blockade of the ActRII pathway using a soluble receptor antagonist has been shown to protect against cancer-induced cachexia in mice [Busquets et al 2012, Zhou et al 2010], cachectic patients with advanced cancer are more likely to receive anticancer agents against their specific type cancer as a standard of care, and whether ActRII inhibition will remain effective when combined with anti-cancer agents remains to be seen.

In accordance with the present invention, the effect of a chimeric murine version of bimagrumab, which has been shown to retain the binding, selectivity, and efficacy profile of bimagrumab while reducing the risk of immunogenicity and allowing for long-term profiling studies in mice, was evaluated in a CT model of cachexia in mice with colon cancer. -26 to elucidate the interaction between bimagrumab and chemotherapy. In addition, intervention at the level of type II activin receptors using the Ab neutralizing bimagrumab is effective in protecting against cancer-induced cachexia, as previously reported through blockade of circulating ligands (anti-myostatin Ab or soluble ActRIIB-Fc).

Platinum-based drugs such as cisplatin are cytotoxic intercalators that prevent DNA replication in a very non-specific manner and are commonly used as first line therapy. Problematically, cisplatin has been shown to accelerate weight and muscle loss as a side effect. Thus, the authors first aimed to assess the potential of bimagumab in contrast to cisplatin-mediated effects on muscle atrophy. A follow-up study then evaluated the effect of more frequent doses of bimagrumab and everolimus, a less cytotoxic, new generation molecularly targeted drug that inhibits the mammalian target of rapamycin (mTOR), on cancer cachexia.

In addition, loss of muscle mass and concomitant loss of total body water (as part of the pathophysiology of cachexia) results in a smaller volume of distribution for chemotherapeutic agents [Parsons 2012]. This, in turn, causes a higher concentration (Cmax and AUC) of these cytotoxic agents, resulting in more adverse events and lower chemotherapy tolerance in cachectic patients than in patients with non-catechic cancer [Sjoblom 2015, Arrieta 2015]. The disclosure of this invention shows that it can lead to higher chemotherapy tolerance, more effective anti-cancer treatment, and better outcomes (including progression-free survival and overall survival) than treatment with anti-cancer therapy alone in patients with cancer cachexia.

There is currently no standard treatment for cancer cachexia.

Therefore, pharmacotherapeutic agents that can reduce or prevent weight loss or even increase body weight in the context of cancer, with or without treatment with chemotherapeutic agents and in particular mTOR inhibitors, are highly desirable.

In addition, the combination of a myostatin or activin antagonist and an mTOR inhibitor according to the present disclosure also has the potential to treat age-related conditions.

SUMMARY OF THE INVENTION

Thus, a first aspect of the present disclosure relates to a combination of an ActRII receptor inhibitor and a chemotherapeutic agent for the treatment of cancer cachexia.

Thus, another aspect of the present disclosure relates to methods or uses for the treatment of cancer cachexia of compositions containing a myostatin or activin antagonist, which may be a myostatin binding molecule or an ActRII binding molecule.

In accordance with the present disclosure, the effect of a chimeric murine version of bimagrumab, which has been shown to retain the binding, selectivity, and potency profile of bimagrumab, reducing the risk of immunogenicity and allowing for long-term profiling studies in mice, was evaluated in the CT- 26 to elucidate the interaction between bimagrumab and chemotherapy. In addition, intervention at the level of type II activin receptors using the Ab neutralizing bimagrumab is effective in protecting against cancer-induced cachexia, as previously reported through blockade of circulating ligands (anti-myostatin Ab or soluble ActRIIB-Fc).

Platinum-based drugs such as cisplatin are cytotoxic intercalators that prevent DNA replication in a very non-specific manner and are commonly used as first line therapy. Problematically, cisplatin has been shown to accelerate weight and muscle loss as a side effect. The initial goal was to evaluate the potential of bimagumab against cisplatin-mediated effects on muscle atrophy. A follow-up study then evaluated the effect of more frequent doses of bimagrumab and everolimus, a less cytotoxic, new generation molecularly targeted drug that inhibits the mammalian target of rapamycin (mTOR), on cancer cachexia.

Muscle regulation and ActRII receptors

Several members of the transforming growth factor beta (TGF-β) superfamily, including myostatin, Activin A, and growth differentiation factor 11 (GDF-11), negatively regulate skeletal muscle mass in animals and humans throughout the life cycle. Ligand signaling passes through type II activin receptors (both ActRIIA and B; and the Smad 2/3 pathway) to inhibit muscle protein synthesis and myocyte differentiation and proliferation. The absence of any of these ligands in developing animals and humans results in a hypermuscular phenotype with increased number and size of muscle fibers. Postpartum myostatin decline leads to skeletal muscle hypertrophy due to an increase in the size of existing muscle fibers (Lee et al 2005; Lee et al 2010; Trendelenburg et al 2012). Thus, the ability to modulate muscle growth by disrupting this signaling pathway at the receptor level is much more significant than previously assessed through direct anti-myostatin approaches.

A "myostatin antagonist", as used herein, refers to a molecule capable of antagonizing (e.g., reduce, inhibit, reduce, slow down) myostatin function, expression and/or signaling, (e.g., by blocking the binding of myostatin to the myostatin receptor, i.e., ActRIIB). Non-limiting examples of antagonists include myostatin binding molecules and ActRII (ActRII A ActRIIB, or ActRIIA/B) receptor binding molecules. In some embodiments of the disclosed methods, regimens, kits, processes, uses, and compositions, a myostatin antagonist is used.

By "myostatin-binding molecule" is meant any molecule capable of binding to the human myostatin antigen, either alone or in combination with other molecules. The binding reaction can be shown by standard methods (qualitative assays), including, for example, a binding assay, a competitive assay, or a bioassay to determine inhibition of myostatin binding to its receptor, or any type of binding assay, with reference to a negative control test that uses an antibody of unrelated specificity, but ideally of the same isotype, eg an anti-CD25 antibody. Non-limiting examples of myostatin binding molecules include small molecules, myostatin receptor decoys, and antibodies that bind to myostatin produced by B cells or hybridomas, and chimeric, CDR-grafted or human antibodies, or any fragment thereof, e.g., F(ab') ₂ and Fab fragments, as well as single chain or single domain antibodies. Preferably, the myostatin binding molecule antagonizes (eg, reduces, inhibits, reduces, slows down) the function, expression and/or signaling of myostatin. In some embodiments of the disclosed methods, schemes, kits, processes, uses, and compositions, a myostatin binding molecule is used.

By "ActRII receptor inhibitor" is meant any molecule capable of binding to the human ActRII receptor (ActRII A and/or ActRIIB), either alone or in conjunction with other molecules, and inhibit receptor signaling. The binding reaction can be shown by standard methods (qualitative assays), including, for example, a binding assay, a competitive assay, or a bioassay to determine inhibition of ActRII receptor binding to myostatin, or any type of binding assay, with reference to a negative control test that uses an antibody of unrelated specificity, but ideally of the same isotype, eg an anti-CD25 antibody. Non-limiting examples of ActRII receptor inhibitors include small molecule, myostatin decoys, and anti-ActRII receptor antibodies produced by B cells or hybridomas, and chimeric, CDR-grafted or human antibodies, or any fragment thereof, such as F(ab') ₂ and Fab fragments, as well as single-chain or single-domain antibodies. Preferably, the ActRII receptor-binding molecule antagonizes (eg, reduces, inhibits, decreases, slows down) the activating function, expression and/or signaling of myostatin. In some embodiments of the disclosed combinations, uses, methods, and compositions, an ActRII receptor inhibitor is used.

Bimagrumab

Bimagrumab, the pharmaceutically active compound used in accordance with the present invention, is a fully human monoclonal antibody (modified IgG1, 234-235-Ala-Ala, λ2) designed to competitively bind to the activin type II receptor (ActRII) with higher affinity, than its natural ligands that limit muscle growth, including myostatin and activin. Bimagrumab is cross-reactive with human and mouse ActRIIA and ActRIIB and is effective on human, macaque, mouse and rat skeletal muscle cells. Bimagrumab binds with extremely high affinity (KD 1.7 ± 0.3 pM) to human ActRIIB and with relatively low affinity to human ActRIIA (KD 434 ± 25 pM).

The present invention is based on the therapeutic principle that sufficient blocking of myostatin binding to its ActRII receptor (ActRIIB and/or ActRIIA) will significantly reduce the activity of myostatin and other ligands that inhibit skeletal muscle growth by acting on receptors while allowing some of these ligands to perform their other physiological functions through secondary receptors (Upton et al 2009). Other approaches to reduce myostatin activity, i.e. competitive soluble ActRII, causing soluble receptor precipitation can deplete a number of ActRII ligands with activity at other receptors, potentially creating a higher safety risk than using a receptor antagonist antibody such as bimagrumab.

Other approaches include the use of myostatin binding antibodies such as LY2495655 (Eli Lilly) which will then inhibit or reduce signaling through the ActRII receptor.

As a potent ActRII inhibitor, bimagumab blocks the effects of myostatin, activin A, GDF11, and possibly other ligands that work through this receptor.

The present invention thus provides, inter alia , a myostatin antagonist or an activin antagonist (such as Activin A, Activin B or Activin AB), preferably a myostatin binding molecule or antibody, and more preferably an inhibitor or more preferably an Anti-ActRII receptor antibody, most preferably bimagrumab, for use in the treatment of cancer cachexia.

Brief description of the drawings

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings, in which:

Fig. 1 : Effects of cisplatin and CDD866 on body weight (A, B, C), tumor volume (D), and weight (E) in CT-26 mouse colon cancer-induced cachexia, either alone or in combination. Values are expressed as means ± SEM (standard error of the mean) (n=10). The percentage change in body weight was calculated compared to the start of treatment on day 0; ^* : P < 0.05, ^** : P < 0.01 vs. non-tumor control; ^&& : P < 0.01 vs. CT-26 control; ⁺⁺ : P < 0.01 vs. non-tumor cisplatin; ^## : P < 0.01 vs. CT-26 cisplatin using Sidak's multiple comparison test after ANOVA.

Fig. 2 : Effects of cisplatin and CDD866 on muscle mass and time to disease progression in CT-26 mouse colon cancer-induced cachexia, either alone or in combination. Values are expressed as means ± SEM (n=10). Percent change in muscle mass, normalized to baseline body weight on day 0, was calculated compared to non-tumor controls (A, B, C); ^* : P < 0.05, ^** : P < 0.01 vs. non-tumor control; ^& : P < 0.05, ^&& : P < 0.01 vs. CT-26 control; ⁺⁺ : P < 0.01 vs. non-tumor cisplatin; ^xx : P < 0.01 vs. non-tumor CDD866; ^## : P < 0.01 vs CT-26 cisplatin; ^$ : P < 0.05, ^$$ : P < 0.01 vs. CT-26 CDD866 using Sidak's multiple comparison test after ANOVA. Time to disease progression, expressed as % of event, defined by interruption criteria (D); the average number of days elapsed before reaching the break criterion (E), expressed using a box and whisker chart with values from minimum to maximum (n=10); ^& : P < 0.05, ^&& : P < 0.01 vs. CT-26 control (Vehicle 1/Vehicle 2) using Dunn's multiple comparison test after ANOVA.

Fig. 3: Effects of everolimus and CDD866 on body weight (A, B, C), tumor volume (D), and weight (E) in CT-26 mouse colon cancer-induced cachexia, either alone or in combination. Values are expressed as means ± SEM (n=10). The percentage change in body weight was calculated compared to the start of treatment on day 0; ^* : P < 0.05, ^** : P < 0.01 vs. non-tumor control; ^&& : P < 0.01 vs. CT-26 control; ⁺⁺ : P < 0.01 vs non-tumor everolimus; ^## : P <0.01 vs. everolimus CT-26 using Sidak's multiple comparison test after ANOVA.

Fig. 4 : Effects of everolimus and CDD866 on muscle mass and time to disease progression in CT-26 mouse colon cancer cachexia, either alone or in combination. Values are expressed as means ± SEM (n=10). Percent change in muscle mass, normalized to baseline body weight on day 0, was calculated compared to non-tumor controls (A, B, C); ^* : P < 0.05, ^** : P < 0.01 vs. non-tumor control; ^& : P < 0.05, ^&& : P < 0.01 vs. CT-26 control; ⁺⁺ : P < 0.01 vs non-tumor everolimus; ^xx : P < 0.01 vs. non-tumor CDD866; ^## : P <0.01 vs everolimus CT-26; ^$ : P < 0.05, ^$$ : P < 0.01 vs. CT-26 CDD866 using Sidak's multiple comparison test after ANOVA. Time to disease progression, expressed as % of event, defined by interruption criteria (D); the average number of days elapsed before reaching the break criterion (E), expressed using a box and whisker chart with values from minimum to maximum (n=10); ^& : P < 0.05, ^&& : P < 0.01 vs. CT-26 control (Vehicle 1/Vehicle 2) using Dunn's multiple comparison test after ANOVA.

Fig. 5: mTOR is overexpressed in the muscles of old rats compared to young rats.

1. mTOR is overactive in skeletal muscle of old rats compared to young rats

2. there is no corresponding down-regulation of mTOR after fasting in the skeletal muscles of old rats compared to young rats.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a combination comprising (a) an activin type II receptor inhibitor and (b) a chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, and uses thereof, for simultaneous, separate or sequential use in the treatment of cancer cachexia in a subject.

It also refers to a combination of (a) a myostatin or activin antagonist and (b) an mTOR inhibitor for the treatment of age-related conditions.

The combination may be fixed or non-fixed, preferably non-fixed.

The general terms used in this application are defined by the following meanings, unless expressly stated otherwise:

The terms "comprising" and "including" are used in this application in their open-ended and non-limiting sense, unless otherwise indicated.

Terms in the singular and like references in the context of the description of the invention (especially in the context of the following claims) should be construed as covering both the singular and the plural, unless otherwise indicated or clearly contradicted by the context. When the plural form is used for compounds, salts, and the like, it also means a single compound, salt, or the like.

The term "combination" or "pharmaceutical combination" means either a fixed combination in a single unit dosage form, or a non-fixed combination, or a kit, consisting of parts, for combined administration, where an activin type II receptor antagonist or blocker (ActRII) and a chemotherapeutic agent, or its pharmaceutically acceptable the salt may be administered independently at the same time or separately or sequentially at time intervals that allow the combination partners to exhibit a joint, eg additive or synergistic effect.

The term "fixed combination" means that the active ingredients or therapeutic agents are administered to the patient simultaneously in a single composition or dosage form.

The term "non-fixed combination" means that both the active ingredients and the therapeutic agents are administered to the patient in different compositions or dosage forms, either simultaneously, or in parallel, or sequentially without any particular time limitation, such administration providing therapeutically effective levels of three compounds in the body of a subject, for example, a mammal or a human in need thereof.

Preferably, the term "combination" or "pharmaceutical combination" is a non-fixed combination.

The term "pharmaceutical composition" is defined as referring to a mixture or solution containing at least one therapeutic agent for administration to a subject, such as a mammal or a human, for the treatment of a particular disease or condition from which the subject suffers.

The term "pharmaceutically acceptable" is defined as referring to those compounds, biological agents, materials, compositions, and/or dosage forms that are medically suitable for contact with the tissues of a subject, such as a mammal or a human, without undue toxicity, irritation, allergic reaction, or other complications, commensurate with a reasonable benefit/risk ratio.

The terms "co-administration" are defined herein as encompassing the administration of selected therapeutic agents to a single subject, eg, a mammal or a human, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.

The term "treatment" or "treatment" as used herein includes treatment that alleviates, lessens, or alleviates the severity of at least one symptom in a subject, or delays the progression of a disease, condition, and/or disorder. For example, treatment may be the reduction of one or more symptoms of the disorder, or the complete elimination of the disorder. In the meaning of the present invention, the term "treat" also means stopping, delaying the onset of development (ie, the period before the clinical manifestation of the disease) and/or reducing the risk of developing or worsening the disease.

The term "progression-free survival" as used herein includes the length of time during and after treatment for a disease, such as cancer, in which the patient lives with the disease but does not worsen. In a clinical trial, measuring progression-free survival is one way to see how well a new treatment is working. It is also called PFS.

The term "overall survival" in the context of this application includes the length of time either from the date of diagnosis or from the start of treatment for a disease, such as cancer, during which patients diagnosed with the disease are still alive. In a clinical trial, measuring overall survival is one way to see how well a new treatment is working. It is also called OS.

The term "pharmaceutically effective amount" or "therapeutically effective amount" of a combination of therapeutic agents is an amount sufficient to provide an observable improvement over baseline clinically observable signs and symptoms of the disease.

The term "synergistic effect" in the context of the present application refers to the action of two agents, such as, for example, (a) and (b), or their pharmaceutically acceptable salt, which gives an effect, for example, promoting and/or enhancing the immune response in the subject, which is greater than simply adding up the effects of each of the drugs administered separately. The synergistic effect can be calculated, for example, using suitable methods such as the sigmoid equation for calculating the maximum efficiency (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the Loewe additivity formula (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and the median effect formula (Chou, T. C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 ( 1984)). Each equation above can be applied to experimental data to construct a corresponding graph used to evaluate drug combination effects. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, the isobologram, and the combination index curve, respectively.

The term "subject" or "patient" as used herein includes animals that are capable of stimulating and/or enhancing an immune response and/or having an age-related condition. Examples of subjects include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and non-human transgenic animals. In a preferred embodiment, the subject is a human, such as a human who suffers from or is at risk of, or may potentially suffer from, cancer cachexia or an age-related condition.

The term "about" or "approximately" should mean within 10%, more preferably within 5% of a given value or range.

Hereinafter, the present invention is described in more detail and illustrated by examples.

The present invention is presented in the following aspects:

1. A combination comprising (a) an ActRII receptor inhibitor and b) a chemotherapeutic agent.

2. Combination according to aspect 1 for simultaneous, separate or sequential use.

3. The combination according to aspect 1 or 2, wherein a) the ActRII receptor inhibitor and b) the chemotherapeutic agent are in separate form.

4. The combination according to aspects 1-3, where a) is an antibody to ActRII receptor.

5. The combination according to aspects 1-4, wherein said anti-ActRII antibody is bimagrumab.

6. The combination according to aspects 1-5, where b) is an anticancer agent containing platinum.

7. Combination according to any of the above aspects for use as a drug.

8. A combination according to aspect 1-6 comprising (a) an ActRII receptor inhibitor and b) a chemotherapeutic agent for use in the treatment of cancer cachexia.

9. The combination for use according to aspects 1-6, wherein the treatment of cancer cachexia is to reduce body weight loss.

10. An ActRII receptor inhibitor for use in the treatment of cancer cachexia.

11. An ActRII receptor inhibitor for use according to aspect 11 wherein the cancer cachexia is caused by treatment with a chemotherapeutic agent.

12. An ActRII receptor inhibitor for use in accordance with any one of aspects 10-12, wherein the treatment of cancer cachexia is to reduce body weight loss.

13. An ActRII receptor inhibitor for use in accordance with aspects 10-12 to prolong the time to progression of cancer in a patient.

14. An ActRII receptor inhibitor for use according to aspects 10-12 for prolonging time to progression of cancer cachexia.

15. An ActRII receptor inhibitor for use according to aspects 10-12 for prolonging cancer survival.

16. An ActRII receptor inhibitor for use according to aspects 10-15, wherein the ActRII receptor inhibitor is an anti-ActRII receptor antibody.

17. An ActRII receptor inhibitor for use according to aspect 16 wherein the anti-ActRII receptor antibody is bimagrumab.

18. An ActRII receptor inhibitor for use according to aspects 11-17, wherein the chemotherapeutic agent is a platinum-containing anticancer agent.

19. A combination comprising a) a myostatin antagonist and b) an mTOR inhibitor.

20. The combination according to aspect 19 wherein the myostatin antagonist is an ActRII receptor inhibitor

21. The combination according to aspect 20, wherein the ActRII receptor inhibitor is an anti-ActRII receptor antibody.

22. The combination according to aspect 21, wherein the anti-ActRII receptor antibody is bimagrumab.

23. The combination according to aspect 19-22, wherein the mTOR inhibitor is everolimus.

24. A combination according to aspects 19-23 for use as a drug.

25. A combination according to aspects 19-23 for use in the treatment of cancer cachexia.

26. The combination according to aspects 19-23, wherein the treatment of cancer cachexia is to prevent weight loss.

27. The combination according to aspects 19-23, wherein the treatment of cancer cachexia is weight maintenance.

28. The combination according to aspects 19-23, wherein the treatment for cancer cachexia is weight gain.

29. Combination in accordance with aspects 1-9 or for use in accordance with aspects 19-28, where the agents are in separate pharmaceutical compositions.

30. A combination according to any one of aspects 19-23 for use in the treatment of an age-related condition.

31. A combination of aspect 30 wherein the age-related condition is selected from the group consisting of sarcopenia, skin atrophy, muscle atrophy, brain atrophy, atherosclerosis, arteriosclerosis, pulmonary emphysema, osteoporosis, osteoarthritis, high blood pressure, erectile dysfunction, dementia, disease Huntington's disease, Alzheimer's disease, cataracts, age-related macular degeneration, prostate cancer, stroke, reduced life expectancy, impaired renal function and age-related hearing loss, age-related mobility impairment (eg, weakness), cognitive decline, senile dementia , memory impairment, tendon stiffness, cardiac dysfunction such as cardiac hypertrophy and systolic and diastolic dysfunction, age-related thymic involution, cancer, obesity and diabetes.

32. A myostatin antagonist for use in prolonging time to progression of cancer in a patient, wherein said patient is being treated with a chemotherapeutic agent.

33. A myostatin antagonist for use according to aspect 30, wherein said chemotherapeutic agent comprises platinum-containing anticancer drugs such as cisplatin or carboplatin, or an mTOR inhibitor such as everolimus.

34. A myostatin antagonist for use according to aspects 32-33, wherein said myostatin antagonist is an ActRII receptor inhibitor.

35. A myostatin antagonist for use according to aspect 34, wherein said ActRII receptor inhibitor is an anti-ActRII receptor antibody.

36. A myostatin antagonist for use according to aspect 35, wherein said anti-ActRII receptor antibody is bimagrumab.

37. A method of treating a subject with cancer cachexia, which comprises administering to said subject an ActRII receptor inhibitor in an amount that is effective against said cancer cachexia.

38. The method of aspect 37 wherein the cancer cachexia is induced by treatment with a chemotherapeutic agent.

39. The method of aspect 38 wherein the chemotherapeutic agent is an anti-cancer agent containing platinum.

40. The method of any one of aspects 37-39, wherein the treatment of cancer cachexia is to reduce body weight loss.

41. A method for prolonging time to cancer progression in a subject having cancer, which comprises administering to said subject an ActRII receptor inhibitor in an amount that is effective to prolong time to cancer progression.

42. A method for prolonging time to progression of cancer in a subject with cancer cachexia, which comprises administering to said subject an ActRII receptor inhibitor in an amount that is effective to prolong time to progression of cancer cachexia.

43. A method for prolonging cancer survival in a subject, comprising administering to said subject an ActRII receptor inhibitor in an amount that is effective to prolong cancer survival.

44. The method of aspects 37-44 wherein the ActRII receptor inhibitor is an anti-ActRII receptor antibody.

45. The method according to aspect 44, wherein the anti-ActRII receptor antibody is bimagrumab.

46. A method of treating a subject with cancer cachexia, which comprises administering to said subject a myostatin antagonist and an mTOR inhibitor.

47. A method of treating a subject with an age-related condition, which comprises administering to said subject a myostatin antagonist and an mTOR inhibitor.

48. The method of aspects 46-47 wherein the myostatin antagonist is an ActRII receptor inhibitor

49. The method of aspect 48, wherein the ActRII receptor inhibitor is an anti-ActRII receptor antibody.

50. The method of aspect 49 wherein the anti-ActRII receptor antibody is bimagrumab.

51. The method of aspects 46-50 wherein said mTOR inhibitor is everolimus.

52. The method of aspects 46-50, wherein the age-related condition is selected from the group consisting of sarcopenia, skin atrophy, muscle atrophy, brain atrophy, atherosclerosis, arteriosclerosis, pulmonary emphysema, osteoporosis, osteoarthritis, high blood pressure, erectile dysfunction, dementia , Huntington's disease, Alzheimer's disease, cataracts, age-related macular degeneration, prostate cancer, stroke, reduced life expectancy, impaired renal function and age-related hearing loss, age-related mobility impairment (eg, weakness), cognitive decline, senile dementia, memory impairment, tendon stiffness, cardiac dysfunction such as cardiac hypertrophy and systolic and diastolic dysfunction, thymic aging, cancer, obesity and diabetes.

53. A method of treatment in accordance with any of the above use or combination.

A preferred combination and uses thereof is bimagrumab and a platinum-containing anti-cancer agent such as cisplatin.

Another preferred combination and uses thereof is bimagrumab and an mTOR inhibitor such as everolimus.

Other aspects include:

A combination comprising a) an ActRII receptor inhibitor such as bimagrumab and b) a PI3K inhibitor.

A combination according to any of the above aspects, comprising an ActRII receptor inhibitor such as bimagrumab and b) a VEGF receptor inhibitor.

Another specific aspect is a combination comprising a) an ActRII receptor inhibitor such as bimagrumab and b) a chemotherapeutic agent for use in improving progression-free survival.

Another specific aspect is a combination comprising a) an ActRII receptor inhibitor such as bimagrumab and b) a chemotherapeutic agent for use in improving overall survival.

All aspects can be combined with each other within the scope of the present invention.

In the following aspects, the invention relates to pharmaceutical compositions, individually comprising an amount that is jointly therapeutically effective in the treatment of cancer cachexia, uses thereof, or methods of treating cancer cachexia using such pharmaceutical compositions, for prolonging time to progression of cancer/cancerous cachexia, for prolonging survival cancer, improving progression-free survival or overall survival, and for the treatment of an age-related condition, the combination partner (a) and the combination partner (b) administered simultaneously but separately or administered sequentially.

Bimagrumab

The preparation of bimagrumab has been described in WO2010/125003.

Bimagrumab includes an antigen-binding site containing at least one immunoglobulin heavy chain variable domain (V _H ), which includes in sequence the hypervariable regions of CDR1 SEQ ID No. 1, CDR2 SEQ ID No. 2, and CDR3 SEQ ID No. 3.

The use of antibodies having a 1, 2 or 3 residue substitution from any of the heavy chain CDR1, CDR2 and/or CDR3 sequences is also within the scope of the present invention.

Bimagrumab also includes an antigen-binding site containing at least one immunoglobulin light chain variable domain (V _L ) that contains in sequence the hypervariable regions of CDR1 SEQ ID No. 4, CDR2 SEQ ID No. 5 and CDR3 SEQ ID No. 6 or their CDR equivalents.

The use of antibodies having a 1, 2 or 3 residue substitution from any of the light chain CDR1, CDR2 and/or CDR3 sequences is also within the scope of the present invention.

Bimagrumab also includes a light chain of SEQ ID No. 7 or SEQ ID No. 8 and a heavy chain of SEQ ID No. 9.

In accordance with the present invention, the use of antibodies having 95% identity with the light chain and/or heavy chain is also included.

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Cachexia or wasting syndrome is weight loss, muscle atrophy, fatigue, weakness, and significant loss of appetite in a person who is not actively trying to reduce weight. The formal definition of cachexia is a loss of body weight (mass) that cannot be restored by nutrition: even if the affected patient eats more calories, lean body mass will be lost, indicating the presence of a primary pathology.

Cachexia is seen in patients with cancer, AIDS, chronic obstructive pulmonary disease, multiple sclerosis, heart failure, tuberculosis, familial amyloid polyneuropathy, gadolinium poisoning, mercury poisoning (acrodynia), and hormone deficiency.

This is a positive risk factor for death, meaning that if a patient has cachexia, the likelihood of death from the underlying disease increases dramatically. This may be a sign of various underlying disorders; when a patient has cachexia, the physician will usually consider the possibility of cancer, metabolic acidosis (from decreased protein synthesis and increased protein catabolism), certain infectious diseases (eg, tuberculosis, AIDS), chronic pancreatitis, and certain autoimmune disorders, or amphetamine addiction. Cachexia physically debilitates patients to a state of immobility due to loss of appetite, asthenia and anemia, and the response to standard treatment is usually poor. Cachexia includes sarcopenia as part of its pathology.

Cancer cachexia:

Cancer cachexia is a multifactorial syndrome defined by permanent loss of skeletal muscle mass (with or without loss of fat mass) that cannot be fully restored by conventional nutritional support and that leads to progressive functional impairment.

Chemotherapeutic agents:

Chemotherapeutic agents include platinum-containing anticancer drugs (eg, cisplatin, carboplatin), PI3K/mTOR inhibitor, everolimus, PI3K inhibitors, and VEGFR inhibitors.

More broadly referred to as "chemotherapy", compounds include alkylating agents (eg, cyclophosphamide, temozolomide), platinum-containing agents, antimetabolites (eg, 5-fluorouracil, methotrexate, hydroxyurea, cytarabine, gemcitabine), topoisomerase inhibitor (eg, doxorubicin, irinotecan) , a microtubule polymerizing/depolymerizing agent (eg, vinblastine, vincristine, paclitaxel, docetaxel), an endocrine agent (eg, bicalutamide, leuprorelin, tamoxifen, letrozole), and more recent molecularly targeted drugs (eg, kinase inhibitors, antibodies).

mTOR inhibitors:

In the context of the present application, the term "mTOR inhibitor" refers to a compound or ligand, or a pharmaceutically acceptable salt thereof, that inhibits mTOR kinase in a cell. In one embodiment, the mTOR inhibitor is an allosteric inhibitor. In one embodiment, the mTOR inhibitor is a catalytic inhibitor.

Allosteric mTOR inhibitors include the neutral tricyclic compound rapamycin (sirolimus), rapamycin-related compounds, which are compounds that have structural and functional similarities to rapamycin, including, for example, rapamycin derivatives, rapamycin analogs (also called rapalogues), and other macrolide compounds that inhibit mTOR activity.

Rapamycin is a known macrolide antibiotic produced by Streptomyces hygroscopicus having the structure shown in formula A.

(A)

(A)

See, for example, McAlpine, J. B., et al., J. Antibiotics (1991) 44: 688; Schreiber, S. L., et al., J. Am. Chem. soc. (1991) 113: 7433; US patent No. 3929992. There are various numbering systems proposed for rapamycin. To avoid confusion, when specific rapamycin analogs are named in this application, the names are given with reference to rapamycin using the formula A numbering system.

Rapamycin analogs useful in the present invention are, for example, O-substituted analogs in which the hydroxyl group on the cyclohexyl ring of rapamycin is substituted with an OR ₁ group in which R ₁ is hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl or aminoalkyl; for example, RAD001, also known as everolimus, as described in US 5665772 and WO94/09010, the contents of which are incorporated by reference. Other suitable analogs of rapamycin include compounds substituted at the 26- or 28-position. The rapamycin analog may be the epimer of the analog as defined above, in particular the epimer of the analog substituted at position 40, 28 or 26, and may optionally be further hydrogenated, for example as described in US 6015815, WO95/14023 and WO99/15530, the content of which is included by reference , e.g. ABT578, also known as zotarolimus, or the rapamycin analogue described in US 7091213, WO98/02441 and WO01/14387, the contents of which are incorporated by reference, e.g., AP23573, also known as ridaforolimus.

Examples of rapamycin analogs suitable for use in the present invention from US 5,665,772 include, but are not limited to, 40-O-benzyl-rapamycin, 40-O-(4'-hydroxymethyl)benzyl-rapamycin, 40-O-[4' -(1,2-dihydroxyethyl)]benzyl-rapamycin, 40-O-allyl-rapamycin, 40-O-[3'-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)- prop-2'-en-1'-yl]-rapamycin, (2'E,4'S)-40-O-(4',5'-dihydroxypent-2'-en-1'-yl)-rapamycin, 40 -O-(2-hydroxy)ethoxycarbonylmethyl-rapamycin, 40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin, 40-O-(6-hydroxy)hexyl-rapamycin , 40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-[(3S)-2,2-dimethyldioxolan-3-yl]methyl-rapamycin, 40-O-[(2S) -2,3-dihydroxyprop-1-yl]-rapamycin, 40-O-(2-acetoxy)ethyl-rapamycin, 40-O-(2-nicotinoyloxy)ethyl-rapamycin, 40-O-[2-(N- morpholino)acetoxy]ethyl-rapamycin, 40-O-(2-N-imidazolylacetoxy)ethyl-rapamycin, 40-O-[2-(N-methyl-N'-piperazinyl)acetoxy]ethyl-rapamycin, 39-O- desmethyl-39,40-O,O-ethylene-rapamycin, (26R)-26-dihydro-40-O-(2-hydroxy)ethyl-p apamycin, 40-O-(2-aminoethyl)-rapamycin, 40-O-(2-acetaminoethyl)-rapamycin, 40-O-(2-nicotinamidoethyl)-rapamycin, 40-O-(2-(N-methyl- imidazo-2'-ylcarbethoxamido)ethyl)-rapamycin, 40-O-(2-ethoxycarbonylaminoethyl)-rapamycin, 40-O-(2-tolylsulfonamidoethyl)-rapamycin and 40-O-[2-(4',5'- dicarboethoxy-1',2',3'-triazol-1'-yl)-ethyl]-rapamycin.

Other rapamycin analogs known to be useful in the present invention are those wherein the hydroxyl group on the cyclohexyl ring of rapamycin and/or the hydroxy group at position 28 is substituted with a hydroxyester group, such as the rapamycin analogs known from US Patent RE44768, such as temsirolimus.

Other rapamycin analogs useful in the present invention include compounds where the methoxy group at position 16 is substituted with another substituent, preferably (optionally hydroxy-substituted) alkynyloxy, benzyl, orthomethoxybenzyl, or chlorobenzyl, and/or where the methoxy group at position 39 is removed along with 39 carbon, thus the cyclohexyl ring of rapamycin becomes a cyclopentyl ring containing no methoxy group at position 39; for example, as described in WO95/16691 and WO96/41807, the contents of which are incorporated by reference. The analogs can be further modified such that the hydroxy group at the 40-position of rapamycin is alkylated and/or the 32-carbonyl is reduced.

Rapamycin analogs from WO95/16691 include, but are not limited to, 16-dimethoxy-16-(pent-2-ynyl)oxy-rapamycin, 16-dimethoxy-16-(but-2-ynyl)oxy-rapamycin, 16-dimethoxy -16-(propargyl)oxy-rapamycin, 16-demethoxy-16-(4-hydroxy-but-2-ynyl)oxy-rapamycin, 16-dimethoxy-16-benzyloxy-40-O-(2-hydroxyethyl)-rapamycin , 16-dimethoxy-16-benzyloxy-rapamycin, 16-demethoxy-16- ortho -methoxybenzyl-rapamycin, 16-demethoxy-40-O-(2-methoxyethyl)-16-pent-2-ynyl)oxy-rapamycin, 39 -demethoxy-40-deoxy-39-formyl-42-nor-rapamycin, 39-demethoxy-40-deoxy-39-hydroxymethyl-42-nor-rapamycin, 39-demethoxy-40-deoxy-39-carboxy-42-nor -rapamycin, 39-demethoxy-40-deoxy-39-(4-methyl-piperazin-1-yl)carbonyl-42-nor-rapamycin, 39-demethoxy-40-deoxy-39-(morpholin-4-yl)carbonyl -42-nor-rapamycin, 39-demethoxy-40-deoxy-39-[N-methyl,N-(2-pyridin-2-yl-ethyl)]carbamoyl-42-nor-rapamycin and 39-demethoxy-40- deoxy-39-(p-toluenesulfonylhydrazonomethyl)-42-nor-rapamycin.

Rapamycin analogs from WO96/41807 include, but are not limited to, 32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-40 -O-(2-hydroxyethyl)-rapamycin, 16-O-pent-2-ynyl-32-(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin, 32(S)-dihydro- 40-O-(2-methoxy)ethyl-rapamycin; and 32(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin.

Another suitable analog of rapamycin is umirolimus as described in US 2005/0101624, the content of which is incorporated by reference.

The mTOR (mammalian target of rapamycin) kinase exists as a multiprotein complex, described as the mTORC1 complex or mTORC2 complex, that senses nutrient and energy availability and integrates signals from growth and stress factors. The mTORC1 complex is sensitive to allosteric mTOR inhibitors such as rapamycin, it consists of mTOR, GβL, and mTOR regulatory associated proteins (raptor) and binds to the FKBP12 protein by peptidyl prolyl isomerase (FK506-binding protein 1A, 12 kDa). In contrast, the mTORC2 complex consists of mTOR, GβL, and rapamycin-insensitive mTOR co-proteins (rictor) and does not bind to the FKBP12 protein in vitro.

The mTORC1 complex has been shown to be involved in the translational control of the protein, acting as a growth factor and nutrient-sensitive apparatus for regulating growth and proliferation. mTORC1 regulates protein translation through two key downstream substrates: P70 S6 kinase, which in turn phosphorylates the P70 S6 ribosomal protein, and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1), which plays a key role in modulating eIF4E downregulated cap dependent translation. The mTORC1 complex regulates cell growth in response to cell energy and nutritional homeostasis, and mTORC1 deregulation is common to many different human cancers. The function of mTORC2 includes regulation of cellular survival by phosphorylation of Akt and modulation of actin cytoskeleton dynamics.

The mTORC1 complex is susceptible to allosteric mTOR inhibitors such as rapamycin and its derivatives, largely due to the mode of action of rapamycin, which involves intracellular complex formation with FKBP12 and binding to the FKBP12-rapamycin binding (FRB) domain of mTOR. This leads to a conformational change in mTORC1, which is thought to alter and impair interaction with its support protein raptor, which in turn prevents substrates such as P70 S6K1 from reaching mTOR and phosphorylating them. Rapamycin and rapalogues such as RAD001 have gained clinical relevance by inhibiting mTOR hyperactivation associated with both benign and malignant proliferative disorders.

RAD001, known as everolimus (Afinitor®), has a chemical name

(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-{(1R)-2-[(1S ,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa- 4-aza-tricyclo[30,3,1,04,9]hexatriaconta-16,24,26,28-tetraen-2,3,10,14,20-pentaone and the following chemical structure

.

.

Everolimus is an FDA (Food and Drug Administration) approved drug for the treatment of advanced kidney cancer and is being investigated in several other phase III clinical trials in oncology. Preclinical studies have shown that everolimus is able to inhibit the proliferation of a wide range of tumor cell lines both in vitro and in vivo, presumably through suppression of rapamycin-responsive mTORC1 function. Everolimus, as a derivative of rapamycin, is an allosteric mTOR inhibitor that is highly active in inhibiting part of the mTORC1 function, namely the P70 S6 kinase (P70 S6K) and the P70 S6 downstream substrate P70 S6. Allosteric mTOR inhibitors such as everolimus (and other rapamycin analogs) have little or no effect on inhibition of the mTORC2 pathway or associated activation of the Akt signal. The following examples of allosteric mTOR inhibitors include sirolimus (rapamycin, AY-22989), 40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin (also called temsirolimus or CCI-779), and ridaforolimus (AP-23573/ MK-8669). Other examples of allosteric mTOR inhibitors include zotarolimus (ABT578) and umirolimus.

Alternatively or additionally, catalytic ATP-competitive inhibitors of mTOR have been found to target the mTOR kinase domain directly and target both mTORC1 and mTORC2. They are also more complete inhibitors of mTORC1 than allosteric mTOR inhibitors such as rapamycin because they modulate rapamycin-resistant mTORC1 output signals such as 4EBP1-T37/46 phosphorylation and cap-dependent translation.

The combination of bimagrumab and mTOR inhibitors such as everolimus may be particularly effective for age-related muscle dysfunction, as bimagrumab increases muscle mass and everolimus improves muscle quality. Bimagrumab improves muscle mass by inhibiting the myostatin/activin pathway. Everolimus improves muscle function by inhibiting the mTOR pathway, which is overactive in aged muscle (unpublished internal data that the authors were able to add). mTOR inhibition may improve muscle function by enhancing mitochondrial function, reducing inflammation, and increasing autophagy. Improvement in muscle mass and function with the combination of bimagrumab and mTOR inhibitors such as everolimus is likely to be of therapeutic benefit in sarcopenia and heart failure. In addition, improved muscle mass and function may be of therapeutic benefit in diabetes mellitus by increasing muscle glucose uptake. Increased mTOR activity has also been demonstrated in muscle biopsies obtained from elderly healthy subjects (aged 60-84 years) compared to younger healthy subjects (aged 18-40 years) (Markofski M et al., Exp Geront, 2015)

EXAMPLES

Hereinafter, the present invention is described in more detail and specifically with reference to examples, which, however, are not intended to limit the present invention.

Materials and methods

materials

Bimagrumab is a human IgG1 Leu234Ala/Leu235Ala monoclonal antibody directed against ActRII. CDD866, a murinized version of bimagrumab where the human Fc region of the antibody has been replaced with a mouse Fc. CDD866 was produced in CHO cells from Novartis Pharma AG (Basel, Switzerland). Cisplatin (cis-diamindichloro-platinum(II)) was purchased from Sigma Aldrich (catalog number 479306). Everolimus was synthesized from Novartis Pharma AG.

Animal experiments

Adult male Balb/cJRj mice aged 11-12 weeks were purchased from Janvier Laboratories (Le Genest St Isle, France). The mice were allowed to acclimate for 7 days. Animals were kept in groups of 5 or fewer animals at 25° C. with a 12:12 light-dark hour cycle. They were fed a standard laboratory diet containing 18.2% protein and 3.0% fat with an energy content of 15.8 mJ/kg (NAFAG 3890, Kliba, Basel, Switzerland). Food and water were given ad libitum .

Mouse colon cancer cell line CT-26 was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotic-antimycotic solution at 37°C with 5% CO ₂ . CT-26 cells were harvested by treatment with ^Accutase® (PAA Laboratories GmbH, Pasching, Austria) and suspended in a solution containing 50% PBS and 50% phenol red free BD Matrigel™ matrix (catalog number 356237, BD Biosciences, Bedford, MA, USA). 0.1 ml of cell suspension containing 3×10 ⁵ cells was inoculated subcutaneously into the left flank of mice. When the tumors were palpable, mice having tumors of acceptable morphology and size were randomized into balanced groups in terms of mean and range of tumor size and body weight. Treatments started on the day of randomization.

A therapeutic impact study was performed to evaluate the effect of CDD866 either alone or in combination with anti-cancer agents. CDD866 was administered at 20 mg/kg subcutaneously once or twice a week in a volume of 5 ml/kg. Cisplatin was administered at a dose of 1 mg/kg intraperitoneally twice a week. Everolimus was administered at 5 mg/kg orally once a day. In the combination administration groups, treatment with cisplatin or everolimus was combined with subcutaneous administration of CDD866 once or twice a week, respectively. Body weights and tumor volume were measured 2-3 times per week. At the end of the experiment, the mice were euthanized with CO ₂ and the tumor, tibialis anterior, gastrocnemius-soleus-foot complex, quadriceps were harvested and weighed.

A time to disease progression study was performed as a follow-up to assess whether or not the combination of CDD866 and cisplatin or everolimus slowed down the progression of cancer cachexia to an interruption criterion, which was defined as body weight loss reaching 20% or tumor volume exceeding 1500 mm ³ . The treatment regimen was the same as in the therapeutic intervention study. Body weight and tumor volume were measured 2-3 times a week for the first 2 weeks and then every day until the end of the experiment. Mice were euthanized with CO ₂ when body weight loss approached 20% or tumor volume exceeded 1500 mm ³ .

Protein analysis

Lysis buffer was added, consisting of extraction reagent (Phosphosafe; Novagen Inc., Madison, WI, USA) supplemented with 1% protease inhibitor cocktail (calbiochem# 539131) and 0.2% SDS. Precellys homogenates (FastPrep-Machine FP20) were separated by centrifugation for 20 minutes at 4°C (14000 rpm). Supernatants were collected and protein content was measured using a commercial protein detection kit (BCA Kit; Thermo Scientific). Samples were diluted in SDS-PAGE sample buffer and denatured for 10 minutes at 70°C. Equal amounts of protein were loaded onto a strip of 4 to 12% and 8% polyacrylamide gel (NuPAGE Bis-Tris gel, Invitrogen Corp., Carlsbad, CA, USA), separated by electrophoresis, and then transferred to nitrocellulose membranes. The membranes were blocked in TBS 0.1% Tween and 5% w/v. skimmed milk powder. Primary antibodies phospho-SMAD3 (Millipore #04 1042 diluted 1:1000) and α-Tubulin (Sigma T6199 diluted 1:5000) were incubated in TBS with 0.1% Tween 20 and 5% w/v. skimmed milk powder, and secondary antibodies in TBS with 0.1% Tween 20, 0.05% SDS and 5% skimmed milk. Immunoreactivity was determined using SuperSignal West Femto substrate with maximum sensitivity (Thermo Scientific) and exposed to film or determined using FusionSpectra. mTOR and IL-6 were quantified using the MesoScale Discovery assay kit using a MesoScale Discovery reader according to the manufacturer's instructions.

Statistical analysis

Values were expressed as mean ± SEM. Statistical analysis was performed using Sidak's multiple comparison tests after analysis of variance to compare treatment groups with control groups (non-tumor and tumor), anti-cancer treatment alone (cisplatin or everolimus) or CDD866 alone in a therapeutic intervention study, and Dunn's multiple comparison criteria. to study the time to disease progression. Differences were considered significant when the probability value was <0.05. Statistical analyzes were performed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA). Body weight was expressed as % change from day 0 as the start of treatment. Tumor volumes in cubic mm were calculated according to the formula (length×width ² )/2. Muscle mass was normalized to body weight on the day of cell inoculation (baseline body weight) and then expressed as % change from non-tumor control group.

Example 1: Bimagrumab Prevents Cisplatin-Induced Weight Loss

Extensive weight loss has become a key determinant of cancer mortality. Thus, the change in body weight during long-term treatment was monitored (Fig. 1A and B). Ten days after the start of treatment, tumor-bearing animals treated with cisplatin as monotherapy lost 20% of their initial body weight (Figure 1B and C). In contrast, vehicle-treated, tumor-bearing animals experienced a 10% decrease in body weight, while animals treated with CDD866 alone or in combination with cisplatin had a modest body weight loss of only 3 and 5%, respectively (Fig. 1B and C). In healthy control animals, cisplatin had no effect on body weight, and administration of CDD866 resulted in a marked increase in body weight in the absence and presence of cisplatin (Fig. 1A and C). These data show that cisplatin at an effective antitumor dose (see Fig. 1E) did induce weight loss in cachectic animals, and that CDD866 significantly reduced chemotherapy-induced wasting.

The main issues to be addressed in this study are potential drug-drug interactions that may reduce the effectiveness of chemotherapy and the effect of CDD866 on promoting tumor growth. At the initial stage of treatment, the average tumor volume was≥260 mm ³ (Fig. 1D). CDD866 did not accelerate tumor progression (FIGS. 1D and E) and did not impair the antitumor effect of cisplatin (FIGS. 1D and E). Thus, CDD866 is effective in reducing chemotherapy-induced weight loss in cancer cachexia without interfering with the antitumor effect of cisplatin.

Example 2: Bimagrumab Antagonizes Cisplatin-Induced Muscular Atrophy

Given the positive effect of CDD866 on body weight, the authors further determined the impact of various interventions on individual skeletal muscles. In the gastrocnemius muscle, cisplatin caused a 25% loss of muscle mass. Treatment with CDD866 generally reduced muscle mass loss by up to 13%, and this protective effect was maintained in the presence of cisplatin (12%) (FIG. 2B). A similar level of protection was observed in the quadriceps (Fig. 2C). The tibialis anterior benefited most from CDD866 treatment. In the tibialis anterior, cisplatin-treated animals experienced muscle atrophy of 34%, and co-administration of CDD866 significantly reduced muscle loss by up to 16% (FIG. 2A).

Example 3: Bimagrumab in Combination with Cisplatin Extends Time to Progression in Cancer Cachexia

Extensive tumor growth and subsequent weight loss are important predictors of mortality in cancer patients. Therefore, the authors wanted to evaluate whether the combination of CDD866 and cisplatin affects life expectancy. For ethical reasons, they refrained from classic survival studies. Instead, each mouse was subjected to individual euthanasia, either when a body weight loss of more than 20% of the initial body weight was confirmed, or when a tumor volume of 1500 mm ³ was reached, defined as the time to disease progression.

On average, animals treated with vehicle or cisplatin had to be sacrificed after 12 and 12 days, respectively (Fig. 2D and E). Animals treated with CDD866 were to be euthanized after 16 days, confirming previous evidence that treatment with CDD866 reduces body weight loss but does not promote tumor growth. Combination treatment with CDD866 and cisplatin was superior to any other intervention tested. Indeed, the combination treatment increased the time to disease progression to 21 days (FIG. 2E). The control was terminated after 39 days when 35% of the animals in the pooled group still had not reached one of the defined termination criteria (FIG. 2D).

Combination with cisplatin

Despite a significant inhibition of tumor growth, cisplatin accelerated weight loss in cachectic animals, probably due to the high toxicity of the anticancer agent. CDD866 completely prevented cisplatin-mediated weight loss, demonstrating that ActRII inhibition remains effective in the presence of cisplatin. Treatment with cisplatin alone and also in combination with CDD866 reduced CT-26 tumor mass to similar levels, highlighting that CDD866 did not adversely affect the anticancer effect of cisplatin.

Therefore, cisplatin treatment did not improve CT-26 tumor-induced skeletal muscle atrophy, but rather exacerbated skeletal muscle loss. In contrast, administration of CDD866 alone or in combination with cisplatin protected against loss of skeletal muscle mass compared to animals treated with cisplatin alone, further confirming that ActRII inhibition remains fully effective with cisplatin treatment. Thus, these results indicate that CDD866 in combination with cisplatin prevents muscle atrophy in cachectic animals compared to cisplatin treatment alone. Notably, CDD866 was only administered once per week, and the mice received only two injections throughout the study (except for survival studies). Since Activin release by cancerous tissues ¹⁷ can potentially compete with ActRII inhibition by CDD866, cancer cachexia may require a higher dosage or frequency of CDD866 administration to obtain more pronounced or maximal responses. Indeed, greater prevention of muscle wasting was seen with CDD866 alone in a pooled study with everolimus at a more frequent dosing regimen.

Cancer patients with low muscle mass are at increased risk of chemotherapy-associated toxic reactions and show an increase in overall mortality ¹⁸ . Therefore, CDD866 significantly slowed down the progression of the disease in large part by increasing muscle mass. The time to disease progression in cancer cachexia was further slowed by concomitant therapy with CDD866 and cisplatin, which both prevented muscle wasting and inhibited tumor growth.

Example 4: Bimagrumab and everolimus prevent cancer cachexia in an additive manner

In the next step, everolimus, a molecularly targeted agent against the mammalian target of rapamycin (mTOR), was chosen as the partner in the combination because mTOR is known to play a central role in cell growth and proliferation. In addition, the frequency of administration for CDD866 was increased to twice a week to provide a significant anti-cachectic effect when administered as a single agent, and the combination of everolimus and CDD866 was evaluated in non-neoplastic mice as well as in tumor cachectic mice.

In the non-tumor group, weight gain was not significantly altered by everolimus treatment. Conversely, weight gain increased significantly with CDD866 treatment, as expected (FIGS. 3A and C). Weight gain was slightly slower in the combination group (Fig. 3A and C), but still significantly different from the everolimus-only group, and was not significantly different from the CDD866-only group until the end of treatment on day 14. In the CT- 26, body weight was significantly reduced in the tumor control group at day 14 compared to the non-tumor control group (FIGS. 3B and C). Weight loss induced by CT-26 was completely prevented by everolimus, CDD866 and the combination of everolimus and CDD866. The effect of CDD866 on body weight was maintained in the presence of everolimus.

Everolimus retarded CT-26 tumor growth and the antitumor effect was maintained in the presence of CDD866 (FIG. 3D). CT-26 tumor mass was significantly reduced when treated with everolimus alone or in combination with CDD866. No significant effect of CDD866 treatment on CT-26 tumor weight was observed.

In the non-tumor group, mass of the tibialis anterior, gastrocnemius-soleus-foot and quadriceps muscle complex was unchanged by everolimus treatment and increased significantly by CDD866 treatment (Fig. 4A-C). The effect of CDD866 on muscle mass was maintained in the presence of everolimus. The CT-26 tumor induced a significant reduction in mass in the tibialis anterior, gastrocnemius-soleus-foot complex, and quadriceps compared to the non-tumor control group (Fig. 4A-C). Muscle loss induced by CT-26 was significantly reduced by treatment with everolimus or CDD866. Interestingly, the combination of everolimus and CDD866 appeared to reduce skeletal muscle weight loss in an additive manner, and the effect of combination treatment was significantly different from everolimus treatment alone.

Example 5: Bimagrumab in combination with everolimus prolongs time to progression in cancer cachexia

In addition to the positive effects of everolimus and CDD866 on CT-26-induced cachexia in the therapeutic intervention study, the effect of these treatments on cancer development and associated cachexia was assessed using the same criteria used in the cisplatin combination study. In the CT-26 control group, the median number of days to the cut-off criterion (time to disease progression) was 17.5 days after randomization and initiation of treatment (FIGS. 4D and E). Everolimus treatment significantly extended the time to disease progression to 23 days mainly due to its antitumor effect, while CDD866 showed only a slight trend to 21 days. The lack of significance of CDD866 in relation to time to disease progression is explained by the fact that although the treatment was very successful in preventing weight loss, it did not inhibit tumor growth, which was the second criterion for termination. Importantly, the combination of everolimus and CDD866 seemed to further slow the time to progression to 28.5 days, which was significant compared to the CT-26 control group.

Combination with everolimus

Since mTOR is known to play a key role in cell growth and proliferation, inhibition of mTOR by everolimus was expected to show a significant antitumor effect, both in the absence and presence of CDD866. This result clearly shows that the anti-cancer effect of everolimus does not adversely affect the inhibition of ActRII by CDD866. Along with the decrease in body weight caused by the CT-26 tumor, skeletal muscle mass was significantly reduced in the CT-26 control group. Treatment with everolimus or CDD866 alone significantly protected tumor mice from loss of skeletal muscle mass caused by CT-26 tumor. Interestingly, inhibition of ActRII by CDD866 not only remains effective in the presence of everolimus, but also showed a non-significant trend towards an additive effect on reversing skeletal muscle mass loss, despite the fact that mTOR is required for normal muscle growth. Similarly, in tumor-free mice, there was no effect on body weight when treated with everolimus, while CDD866 significantly increased body weight. The effect of CDD866 on body weight was maintained in the presence of everolimus, clearly demonstrating that mTOR inhibition did not alter the effect of CDD866 on body weight. Also, the anabolic muscle response observed with CDD866 treatment in tumor-free mice was significant and independent of mTOR inhibition at a dose apparently effective on the tumor.

Everolimus treatment alone extended the time to disease progression as a proxy for survival, and CDD866 also showed a trend of extension. Importantly, the combination of everolimus and CDD866 appears to further increase the time to disease progression. Each treatment worked as a complement to each other to provide a positive effect, with everolimus inhibiting tumor growth and CDD866 preventing cachexia. The trend towards an additive anti-cachectic effect seen with the combination of CDD866 and everolimus needs further investigation into how ActRII blockade and mTOR inhibition positively impact cachexic skeletal muscle.

mTORC1 has been reported to mean denervation-activated skeletal muscle atrophy, but the anti-atrophic effect of mTOR inhibition with rapamycin treatment has been inconclusive. mTOR activation has also been reported in other pathological conditions, such as aging, obesity, insulin resistance, and diabetes, where mTOR inhibition appears to be beneficial. In the present study, a significant increase in phosphorylation as well as total mTOR was observed in tumor-bearing mice. Therefore, it is possible that such aberrant mTOR activation in CT-26 colon cancer-induced cachexia also contributes to cell-induced cachexia, and therefore mTOR inhibition has shown an additional benefit in combination with ActRII blockade.

Example 6 Combination of mTOR inhibitor and myostatin antagonist in aging:

The combination of bimagrumab and mTOR inhibitors such as everolimus may be particularly effective for age-related muscle dysfunction, as bimagrumab increases muscle mass and everolimus improves muscle quality. Bimagrumab improves muscle mass by inhibiting the myostatin/activin pathway. Everolimus improves muscle function by inhibiting the mTOR pathway, which is overactive in aged muscle. mTOR inhibition may improve muscle function by enhancing mitochondrial function, reducing inflammation, and increasing autophagy. Improvement in muscle mass and function with the combination of bimagrumab and mTOR inhibitors such as everolimus is likely to be of therapeutic benefit in sarcopenia and heart failure. In addition, improved muscle mass and function may have a therapeutic benefit in diabetes mellitus by increasing muscle glucose uptake.

The data in Fig. 5 support the beneficial use of mTOR inhibitors and a myostatin/activin pathway antagonist (e.g., bimagrumab) in aging, and show that:

1. mTOR is overactive in skeletal muscle of old rats compared to young rats

2. mTOR is not properly regulated after fasting in the skeletal muscle of old rats compared to young rats.

LINKS

The entire contents of the following referenced documents, in particular their definitions and descriptions relating to them, are incorporated into this application by reference.

1. Fearon, K. et al. Definition and classification of cancer cachexia: an international consensus. Lancet Uncool. 12, 489-495 (2011).

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Claims (4)

1. A combination comprising a) an ActRII receptor inhibitor and b) a chemotherapeutic agent, wherein a) the ActRII receptor inhibitor and b) the chemotherapeutic agent are in separate pharmaceutical compositions, wherein the combination increases or maintains muscle mass, and wherein a) is bimagrumab and b) is everolimus. 2. Use of the combination of claim 1 to increase or maintain muscle mass in a subject having an age-related condition. 3. Use according to claim 2, wherein the age-related condition is selected from the group consisting of sarcopenia, skin atrophy, muscle atrophy, brain atrophy, atherosclerosis, arteriosclerosis, pulmonary emphysema, osteoporosis, osteoarthritis, high blood pressure, erectile dysfunction, dementia , Huntington's disease, Alzheimer's disease, cataracts, age-related macular degeneration, prostate cancer, stroke, reduced life expectancy, impaired kidney function and age-related hearing loss, mobility impairment (eg, weakness), cognitive decline , senile dementia, memory impairment, tendon stiffness, cardiac dysfunction such as cardiac hypertrophy and systolic and diastolic dysfunction, age-related thymic involution, cancer, obesity, and diabetes. 4. Use according to claim 3, wherein the age-related condition is sarcopenia or muscle atrophy.

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