Title: A combination therapy for bone loss and/or muscle loss.

FIELD OF THE INVENTION

The present invention is in the field of medicine. More specifically, the invention is in the field of counteracting, treating or preventing a condition or disorder of bone loss and/or muscle loss, for example in frail elderly subjects. In particular, the invention relates to a combination therapy that can be employed to treat, counteract or prevent bone loss (such as osteopenia or osteoporosis) and/or muscle loss (such as sarcopenia) in subjects for example frail elderly subjects.

BACKGROUND TO THE INVENTION

Unintentional body weight loss caused by bone loss or muscle loss poses a significant health risk. Bone loss and muscle loss generally occur during prolonged periods of immobilization, during ageing, as a consequence of a wide variety of disorders, or is driven by combinations thereof. Bone loss increases bone fracture risk, whereas muscle loss adds to that risk by increasing the risk of falling.

It is generally known that the process of ageing affects physical abilities, and that it is associated with bone loss and muscle loss. Bone mass, and muscle mass and muscle strength, peak in early adulthood around an age of 30 years, and subsequently decline with age especially after the fifth decade (Curtis et al., J Cell Physiol., 230(11):2618-2625 (2015)). In subjects that are 50 years or older, muscle mass is lost at a rate of 1-2% per year and strength at a rate of 1.5-3% per year. In women, there is an accelerated period of bone loss p erim enopaus ally additional to the bone loss rates of approximately 1-2% annually (Curtis et al., J Cell Physiol., 230(ll):2618-2625 (2015)).

In the elderly population, bone tissue loss and muscle tissue loss lead to (physical) frailty, which is a disorder generally characterized by osteoporosis and/or sarcopenia. Frailty is a very common disorder in the elderly population and represents a major public health problem. Being able to counteract bone loss and to maintain muscle mass during aging will have a great impact on healthy aging and thus quality of life for elderly individuals, and will also reduce the financial burden for society that is associated with frailty.

There is thus a need for a therapy that counteracts bone loss and muscle loss, especially in the elderly population. A currently approved therapeutic agent for treating osteoporosis is zoledronate (also known as Aclasta®). Zoledronate belongs to the class of bisphosphonates, which counteract bone mass loss by inhibiting the bone-degrading effect of osteoclasts. Zoledronate only blocks further bone loss but does not lead to restoration of bone mass that is already lost at diagnosis. Follistatin is an agent that is inter alia being studied for its role in regulation of muscle growth, as an antagonist to myostatin.

It is an aim of the present invention to provide for a method of counteracting bone loss and/or muscle loss, especially in frail elderly subjects.

SUMMARY OF THE INVENTION

Unexpectedly, the inventors discovered that a combination therapy that comprises administration of both a follistatin and a bisphosphonate to a subject, advantageously counteracts bone loss and/or muscle loss in subjects as compared to a bisphosphonate monotherapy such as a zoledronate monotherapy. The combination therapy of the invention showed a statistically significant increase of trabecular bone mass in immobilized mice as compared to bisphosphonate monotherapy (Example 1, and Figure 1). Example 2, which tested the combination in a mouse model for ageing, corroborated this finding. It is known that trabecular bone loss is associated with increased risk of impact bone fractures, for instance in the upper femur, which consists of 25-50% trabecular bone depending on the region, in the vertebrae which comprise about 90% trabecular bone or in the wrist. Moreover, the inventors discovered that administration of a follistatin as disclosed herein advantageously counteracts muscle loss induced by immobilization even in the presence of a bisphosphonate (Example 1, Figure 4). Moreover, the Examples show that the combination therapy prevented loss of cortical bone mass, maintained bone strength and counteracted body weight loss. Beforehand, there was no reasonable expectation that a combination of a follistatin and a bisphosphonate would improve counteracting bone loss and/or muscle loss in a subject.

Therefore, the invention provides a follistatin in combination with a bisphosphonate for use in a method of counteracting, treating or preventing (a condition of, or a condition associated with) bone loss and/or muscle loss in a subject. In the same manner, the invention provides a follistatin for use in a method of counteracting, treating or preventing (a condition of, or a condition associated with) bone loss and/or muscle loss in a subject, wherein the subject is (also) administered a bisphosphonate. In the same manner, the invention provides a bisphosphonate for use in a method of counteracting, treating or preventing (a condition of, or a condition associated with) bone loss and/or muscle loss in a subject, wherein the subject is (also) administered a follistatin. In the same manner, the invention also provides a combination product comprising a follistatin and a bisphosphonate for use in a method of counteracting, treating or preventing (a condition of, or a condition associated with) bone loss and/or muscle loss in a subject.

In a preferred embodiment of a medical use of the invention, said method of counteracting, treating or preventing (a condition of, or a condition associated with) bone loss and/or muscle loss in a subject is a method of counteracting, treating or preventing (a condition of, or a condition associated with) bone loss and muscle loss in a subject. In preferred embodiments of a medical use of the invention, said condition of, or condition associated with, bone loss is a degenerative bone disease.

In embodiments of a medical use of the invention, said condition of, or condition associated with, muscle loss is a degenerative muscle disease.

In another preferred embodiment of a medical use of the invention, said condition of bone loss is osteopenia or osteoporosis.

In a further preferred embodiment of a medical use of the invention, the condition of muscle loss is sarcopenia.

In another preferred embodiment of a medical use of the invention, the subject is an elderly human.

In a further preferred embodiment of a medical use of the invention, the subject is a frail elderly human.

In another preferred embodiment of a medical use of the invention, the follistatin is a human follistatin.

In a further preferred embodiment of a medical use of the invention, the follistatin is a follistatin comprising the amino acid sequence of SEQ ID NO:1; or the amino acid sequence of SEQ ID NO:1 except that it comprises a modification in a heparin binding domain (e.g. a heparin binding domain as comprised in SEQ ID NO:1), wherein said modification results in inactivation of said heparin binding domain.

In a further preferred embodiment of a medical use of the invention, the follistatin is a protein comprising the amino acid sequence of SEQ ID NO:3.

In another preferred embodiment of a medical use of the invention, the follistatin comprises a C-terminal Fc domain, preferably a C- terminal mouse Fc domain or a C-terminal human (e.g. IgG) Fc domain. In another preferred embodiment of a medical use of the invention, the follistatin is a follistatin having an inactivated heparin binding domain; and wherein said follistatin optionally further comprises a C-terminal Fc domain, preferably a C-terminal mouse Fc domain or a C- terminal human (e.g. IgG) Fc domain.

In another preferred embodiment of a medical use of the invention, the follistatin is a protein that has at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO:1 or SEQ ID NO:3, and inhibits the activity of (human) bone formation inhibitor activin-A. Preferably, said protein comprises an inactivated heparin binding domain (e.g. wherein said inactivation is the result of an insertion, substitution and/or deletion mutation) or, in other words, does not comprise a heparin binding domain. Optionally, said protein may further comprise a C-terminal Fc domain, for example a C-terminal mouse Fc domain or a C- terminal human (e.g. IgG) Fc domain. Preferably, sequence identity is calculated over the whole length of the protein. If necessary, a minimum number of gaps is introduced to achieve optimal alignment. Sequence identity can e.g. be determined using EMBOSS Needle, e.g. at standard or default settings.

In another preferred embodiment of a medical use of the invention, the follistatin is a protein comprising SEQ ID NO:2 or a variant protein thereof that has at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO:2 and inhibits the activity of (human) bone formation inhibitor activin-A. Preferably, said variant protein comprises an inactivated heparin binding domain (e.g. wherein said inactivation is the result of an insertion, substitution and/or deletion mutation) or, in other words, does not comprise a heparin binding domain. Preferably, said variant protein further comprises a C-terminal Fc domain, for example a C-terminal mouse Fc domain or a C-terminal human (e.g. IgG) Fc domain. Preferably, sequence identity is calculated over the whole length of the protein. If necessary, a minimum number of gaps is introduced to achieve optimal alignment. Sequence identity can e.g. be determined using EMBOSS Needle, e.g. at standard or default settings.

In another preferred embodiment of a medical use of the invention, said bisphosphonate is selected from the group formed by zoledronate, risedronate, alendronate, cimadronate, clodronate, tiludronate, etidronate, ibandronate, piridronate, pamidronate and functional analogues thereof, preferably zoledronate.

In another preferred embodiment of a medical use of the invention, in which said follistatin is for use in combination with said bisphosphonate, said follistatin is a protein comprising SEQ ID NO:2 and said bisphosphonate is a zoledronate.

In another aspect, the invention provides a kit comprising a follistatin and a bisphosphonate; wherein said follistatin is as disclosed in any one of the aspects and/or embodiments of a medical use of the invention; and wherein said bisphosphonate is as disclosed in any one of the aspects and/or embodiments of a medical use of the invention.

In another aspect, the invention provides a pharmaceutical composition comprising a follistatin and a bisphosphonate; wherein said follistatin is as disclosed in any one of the aspects and/or embodiments of a medical use of the invention; and wherein said bisphosphonate is as disclosed in any one of the aspects and/or embodiments of a medical use of the invention.

In a preferred embodiment of a kit of the invention or a pharmaceutical composition of the invention, said follistatin is a human follistatin.

In a preferred embodiment of a kit of the invention or a pharmaceutical composition of the invention, said follistatin is a follistatin comprising - the amino acid sequence of SEQ ID NO:1; or - the amino acid sequence of SEQ ID NO:1 except that it comprises a modification in a heparin binding domain (i.e. the heparin binding domain as comprised in SEQ ID NO:1), wherein said modification results in inactivation of said heparin binding domain.

In a preferred embodiment of a kit of the invention or a pharmaceutical composition of the invention, said follistatin is a protein comprising the amino acid sequence of SEQ ID NO:3.

In a preferred embodiment of a kit of the invention or a pharmaceutical composition of the invention, the follistatin comprises a C- terminal Fc domain, preferably a C-terminal mouse Fc domain or a C- terminal human (e.g. IgG) Fc domain.

In a preferred embodiment of a kit of the invention or a pharmaceutical composition of the invention, the follistatin is a follistatin having an inactivated heparin binding domain; and wherein said follistatin optionally further comprises a C-terminal Fc domain, preferably a C- terminal mouse Fc domain or a C-terminal human (e.g. IgG) Fc domain.

In a preferred embodiment of a kit of the invention or a pharmaceutical composition of the invention, the follistatin is a protein that has at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO:1 or SEQ ID NO:3, and inhibits the activity of (human) bone formation inhibitor activin-A. Preferably, said protein comprises an inactivated heparin binding domain (e.g. wherein said inactivation is the result of an insertion, substitution and/or deletion mutation) or, in other words, does not comprise a heparin binding domain. Optionally, said protein may further comprise a C-terminal Fc domain, for example a C-terminal mouse Fc domain or a C-terminal human (e.g. IgG) Fc domain. Preferably, sequence identity is calculated over the whole length of the protein. If necessary, a minimum number of gaps is introduced to achieve optimal alignment. Sequence identity can e.g. be determined using EMBOSS Needle, e.g. at standard or default settings. In a preferred embodiment of a kit of the invention or a pharmaceutical composition of the invention, the follistatin is a protein comprising SEQ ID NO:2 or a variant protein thereof that has at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO:2 and inhibits the activity of (human) bone formation inhibitor activin-A. Preferably, said variant protein comprises an inactivated heparin binding domain (e.g. wherein said inactivation is the result of an insertion, substitution and/or deletion mutation) or, in other words, does not comprise a heparin binding domain. Preferably, said variant protein further comprises a C-terminal Fc domain, for example a C-terminal mouse Fc domain or a C-terminal human (e.g. IgG) Fc domain. Preferably, sequence identity is calculated over the whole length of the protein. If necessary, a minimum number of gaps is introduced to achieve optimal alignment. Sequence identity can e.g. be determined using EMBOSS Needle, e.g. at standard or default settings.

In a preferred embodiment of a kit of the invention or a pharmaceutical composition of the invention, said bisphosphonate is selected from the group formed by zoledronate, risedronate, alendronate, cimadronate, clodronate, tiludronate, etidronate, ibandronate, piridronate, pamidronate and functional analogues thereof, preferably zoledronate.

In a preferred embodiment of a kit of the invention, said kit comprises a first container comprising said follistatin and a second container comprising said bisphosphonate. Preferably, said first container comprising said follistatin and said second container comprising said bisphosphonate are in the form of a (single) unit dose. An exemplary follistatin single unit dose range is 4-6000 mg, for example 400-1200 mg. An exemplary bisphosphonate single unit dose range is 0.4-120 mg, for example 4-12 mg.

In a preferred embodiment of a pharmaceutical composition of the invention, said composition is in the form of a single unit dose. An exemplary follistatin single unit dose range is 4-6000 mg, for example 400- 1200 mg. An exemplary bisphosphonate single unit dose range is 0.4-120 mg, for example 4-12 mg.

In a preferred embodiment of a kit or composition of the invention, the kit or composition is for use in a method of counteracting, treating or preventing (a condition of, or a condition associated with) bone loss and/or muscle loss in a subject, wherein said use is as disclosed in any one of the aspects and/or embodiments of a medical use of the invention.

The invention also provides a method of counteracting, treating or preventing (a condition of, or a condition associated with) bone loss and/or muscle loss in a subject, comprising the step of: - administering a therapeutically effective amount of a follistatin and a bisphosphonate to a subject; preferably wherein said follistatin is as disclosed in any one of the aspects and/or embodiments of a medical use, kit or composition of the invention; and preferably wherein said bisphosphonate is as disclosed in any one of the aspects and/or embodiments of a medical use, kit or composition of the invention.

In a preferred embodiment of a method of counteracting a condition of bone loss and/or muscle loss in a subject, said method is a method of counteracting a condition of bone loss and muscle loss in a subject.

In another preferred embodiment of said method, said condition of bone loss is osteopenia or osteoporosis.

In another preferred embodiment of said method, said condition of muscle loss is sarcopenia.

In another preferred embodiment of said method, said subject is an elderly human.

In another preferred embodiment of said method, said subject is a frail elderly human. In another preferred embodiment of said method, said follistatin is a human follistatin.

In another preferred embodiment of said method, said follistatin is a follistatin comprising

- the amino acid sequence of SEQ ID NO:1;

- the amino acid sequence of SEQ ID NO:1 except that it comprises a modification in a heparin binding domain, wherein said modification results in inactivation of said heparin binding domain; or

- the amino acid sequence of SEQ ID NO:3.

In another preferred embodiment of said method, said follistatin comprises a C-terminal Fc domain.

In another preferred embodiment of said method, said follistatin is a protein comprising SEQ ID NO:2.

In another preferred embodiment of said method, said bisphosphonate is selected from the group formed by zoledronate, risedronate, alendronate, cimadronate, clodronate, tiludronate, etidronate, ibandronate, piridronate, pamidronate and functional analogues thereof.

The invention also provides a use of a follistatin in combination with a bisphosphonate for the manufacture of a medicament for counteracting, treating or preventing (a condition of, or a condition associated with) bone loss and/or muscle loss in a subject; preferably wherein said follistatin and said bisphosphonate are as disclosed in any one of the aspects and/or embodiments of a medical use, kit or composition of the invention; and preferably wherein said use is as disclosed in any one of the aspects and/or embodiments of a medical use of the invention.

The invention also provides a use of a follistatin for the manufacture of a medicament for counteracting, treating or preventing (a condition of, or a condition associated with) bone loss and/or muscle loss in a subject; preferably wherein said follistatin is as disclosed in any one of the aspects and/or embodiments of a medical use, kit or composition of the invention; and preferably wherein said use is as disclosed in any one of the aspects and/or embodiments of a medical use of the invention; and wherein said follistatin is for use, or for administration, in combination with a bisphosphonate as disclosed herein.

The invention also provides a use of a bisphosphonate for the manufacture of a medicament for counteracting, treating or preventing (a condition of) bone loss and/or muscle loss in a subject; preferably wherein said bisphosphonate is as disclosed in any one of the aspects and/or embodiments of a medical use, kit or composition of the invention; and preferably wherein said use is as disclosed in any one of the aspects and/or embodiments of a medical use of the invention; and wherein said bisphosphonate is for use, or for administration, in combination with a follistatin.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “follistatin”, as used herein, includes reference to a protein that inhibits the activity of (human) bone formation inhibitor activin-A, which is also known as activin isoform BABA. The follistatin can be a human follistatin, for instance a follistatin that comprises the amino acid sequence of SEQ ID NO:1 or a follistatin that comprises the amino acid sequence of SEQ ID NO:1 except that 1-200 such as 1-100, 1-50 or 1-20, amino acid residues in said SEQ ID NO:1 are substituted, deleted and/or inserted. Alternatively, the follistatin is a protein comprising SEQ ID NO:1 or SEQ ID NO:3, or is a protein that has at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO:1 or SEQ ID NO:3, and inhibits the activity of (human) bone formation inhibitor activin- A. More preferably, the follistatin is a protein comprising SEQ ID NO:2 or an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO:2 and which inhibits the activity of (human) bone formation inhibitor activin-A. Preferably, sequence identity is calculated over the whole length of the protein. If necessary, a minimum number of gaps is introduced to achieve optimal alignment. Sequence identity can e.g. be determined using EMBOSS Needle, e.g. at standard or default settings. Standard tests are available to test for inhibition of activin-A activity (see e.g. Lodberg et al., FASEB J;33(5):6001-6010 (2019); PMID=30759349). The follistatin can thus be a human follistatin the amino acid sequence of which has not been genetically modified. Such a human follistatin can be recombinantly produced. Alternatively, the human follistatin can be genetically modified by insertion, substitution and/or deletion mutations, for instance to improve pharmacokinetic parameters of the molecule. Such a follistatin can also be recombinantly produced. The term “follistatin” also includes reference to follistatin derivatives. Such derivatives may have improved solubility, absorption and biological half-life properties. The derivatives may also provide for decreased toxicity of the follistatin, or eliminate or attenuate any undesirable side effect of the molecule, etc. If desired, a follistatin as described herein may be administered in the form of a pharmaceutically acceptable salt, hydrate or solvate to the subject. When reference is made to a follistatin herein, included in said definition is said follistatin in the form of its pharmaceutically acceptable salt, hydrate or solvate, etc.

The term “bisphosphonate”, as used herein, includes reference to any compound which is an analog of endogenous pyrophosphate whereby the central oxygen is replaced by carbon. Bisphosphonates include aminobisphosphonates. Bisphosphonates include, but are not limited to the following compounds: zoledronate (also referred to as zoledronic acid herein), risedronate, alendronate, cimadronate, clodronate, tiludronate, etidronate, ibandronate, piridronate or pamidronate, and functional analogues thereof. Preferably, the bisphosphonate is zoledronate (commercially also known as ACLASTA®). Preferably, a bisphosphonate as described herein is an inhibitor of osteoclast-mediated bone resorption.

The terms “zoledronate” and “zoledronic acid” are used interchangeably herein. These terms are meant to include the free acid itself, i.e., l-hydroxy-2-(imidazol-l-yl)ethane-l,l-diphosphonic acid, as well as any pharmaceutically acceptable salt, hydrate thereof, solvate thereof, etc. l-hydroxy-2-(imidazol-l-yl)ethane-l,l-diphosphonic acid and its pharmacologically acceptable salts, hydrates and solvates are well-known in the art.

The term “combination”, as used herein in relation to a combination therapy as described herein, includes reference to using a follistatin and a bisphosphonate as described herein in the same treatment. The follistatin and the bisphosphonate as described herein can be administered together at the same time (such as in the form of a single pharmaceutical composition), separately of each other at the same time (for instance in the form of separate pharmaceutical composition) or separately of each other staggered in time. Simultaneous, separate and sequential administration of a follistatin and a bisphosphonate in the same treatment schedule are expressly envisaged. As an example, the time between administration of said follistatin and said bisphosphonate can be at least one minute, at least fifteen minutes, at least sixty minutes, at least four hours, at least one day, at least one week or at least one month or at least one year, or anywhere in between such as between one minute and one year.

The terms “treating” or “treatment”, as used herein, include reference to the medical management of a subject, for instance with the intent to cure, ameliorate, stabilize, inhibit or prevent a disease, pathological condition or disorder. This term includes reference to active treatment, that is, treatment directed specifically towards the improvement of a disease, pathological condition or disorder as described herein, and may also include causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition or disorder as described herein. In addition, this term includes reference to preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting or counteracting the development of the associated disease, pathological condition or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; or (ii) inhibiting the disease, i.e. arresting its development.

The term “counteracting”, as used herein, includes reference to at least partially counteracting or inhibiting bone loss and/or muscle loss, for instance in the sense that bone loss and/or muscle loss as a cause of ageing, immobilization or exposure to reduced levels of gravity as compared to the gravity level on earth is at least partially counteracted as compared to the situation wherein no combination therapy as described herein is administered to a subject. In such a way, inter alia the risk of developing future bone fractures is decreased.

The term “condition”, as used herein, can be used interchangeably with the terms disease, disorder or illness, unless otherwise indicated. The term “condition” includes reference to a pathological condition. A condition of bone loss and/or muscle loss is considered a pathological condition because it inter alia increases risks of developing future bone fractures, especially in an elderly subject.

The term “bone loss”, as used herein, includes reference to a reduction in the amount, mass, volume and/or density of bone tissue within a specified bone such as the wrist, spine, hip and/or shoulder bone. The term includes reference to a reduction in the amount of bone mineral in bone tissue, which is also referred to as a reduction in bone mineral density (BMD) in bone tissue. Bone loss can inter aha be the consequence of aging, a period of immobilization or a period of exposure to a reduced level of gravity as compared to a level of gravity on earth. Bone loss leads to weak bones and causes disability, especially in the elderly population, for instance in the form of a condition referred to as frailty. The presence of bone loss can routinely be assessed by testing for bone mineral density within a specified bone, for instance at different points in time. The presence of bone loss is for instance assessable by X-ray measurements. Preferably, the bone loss is age-associated bone loss, i.e. bone loss that is the result of the process of ageing, such as in elderly subjects suffering from frailty as described herein. The term includes reference to osteopenia and osteoporosis. The term “osteopenia”, as used herein, is a disorder that is also known as “low bone mass” or “low bone density” and includes reference to a condition wherein bone mineral density is low. Osteopenia can for example be diagnosed using dual X-ray absorptiometry scanning, wherein a bone mineral density score is generated by comparing a test subject’s bone density to the bone density of a healthy (young) reference subject that is generally about 30 years old, e.g. wherein the reference is a mean bone mineral density value of Caucasian females aged 20-29 years old wherein the bone mineral density value is measured at the femoral neck or alternatively at the lumbar spine or total hip. Bone density scores between 1 and 2.5 standard deviations below the reference (e.g. measured at the femoral neck), indicate the presence of osteopenia. The term “osteoporosis”, as used herein, includes reference to a more severe form of osteopenia. This means that someone with osteopenia is more likely to fracture a bone than someone with a normal bone density but is less likely to fracture a bone than someone with osteoporosis. Osteopenia is also considered a midway point to osteoporosis. According to the World Health Organization (WHO) criteria, osteoporosis is defined as a bone mineral density (BMD) (e.g. measured at the femoral neck) that lies 2.5 standard deviation (SD) or more below the average value for a healthy (young) reference subject that is generally about 30 years old, e.g. wherein the reference is a mean bone mineral density value of healthy Caucasian females aged 20-29 years old wherein the bone mineral density value is measured at the femoral neck or alternatively at the lumbar spine or total hip. Preferably, a condition of bone loss as described herein is osteopenia or osteoporosis, more preferably osteopenia or osteoporosis (optionally in combination with muscle loss/wasting as described herein) as a symptom of (physical) frailty such as (physical) frailty in an elderly subject. In embodiments, the bone loss is bone weakness which can also be referred to as loss of bone strength.

The term “muscle loss” can be used interchangeably with the terms “muscle atrophy” and “muscle wasting”. The terms include reference to a loss of muscle mass and/or muscle strength such as a loss of skeletal muscle mass and/or skeletal muscle strength. Muscle loss leads to muscle weakness and causes disability, especially in the elderly population, for instance in the form of a disease also referred to as frailty. Muscle loss or muscle atrophy can inter alia be the consequence of ageing, a period of inactivity, a period of immobilization and/or a period of exposure to a reduced level of gravity as compared to a level of gravity on earth. Preferably, the muscle loss is age-associated muscle loss, i.e. muscle loss that is the result of the process of ageing, such as in elderly subjects suffering from frailty as described herein. A preferred example of muscle loss is sarcopenia. The term “sarcopenia”, as used herein, includes reference to a disorder of age-associated loss of skeletal muscle mass and function. Sarcopenia is common among adults of older age but can also occur earlier in life. One way of defining sarcopenia is a muscle mass that it at least two standard deviations below the mean muscle mass of a reference individual (for instance a healthy or normal reference individual that is about 30 or 35 years old). In embodiments, the muscle loss is muscle weakness which may also be referred to as loss of muscle strength.

The phrase “condition of, or condition associated with, bone loss”, as used herein, refers inter alia to a degenerative bone disease. Degenerative bone diseases are conditions that cause the deterioration of bones. When referring to the gradual reduction of bone mass, which makes the bones more brittle, degenerative bone disease includes reference to osteopenia and osteoporosis. Degenerative bone disease may also refer to degenerative joint disease (which is also referred to as osteoarthritis) or brittle bone disease. Preferably, a degenerative bone disease is a pathological condition of bone loss that occurs in an elderly subject such as a frail elderly subject. The phrase “condition of, or condition associated with, muscle loss”, as used herein, refers inter alia to a degenerative muscle disease. Degenerative muscle diseases, such as muscular dystrophies, are conditions that are characterized by weakness and/or wasting away of muscle tissue. Examples of degenerative muscle diseases are muscular dystrophies such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

The term “subject”, as used herein, includes reference to an animal, preferably a mammal, most preferably a human. Exemplary animals include, but are not limited to, mammals such as a mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon or rhesus monkey. The term “subject” can be used interchangeably with the term “patient”. Preferably, the subject has an age in the range of (i) 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 to (ii) 115. More preferably, the subject has an age in the range of 40 to 115, more preferably 50 to 115, and even more preferably 65 or 70 to 115. Preferably, the subject is an elderly human or belongs to the population of elderly humans. Even more preferably, the subject is a frail elderly human or belongs to the population of frail elderly humans. The subject can either be male or female, such as a perimenopausal or postmenopausal female. Preferably, the subject suffers from, is suspected of suffering from, or is at risk of suffering from a condition of bone loss and/or muscle loss as described herein. In preferred embodiments, the subject is a human, such as an elderly human which is, or which in the past 1-3 years has been, immobilized such as bedridden.

The term “frailty”, as used herein, includes reference to a common condition in the elderly population and is amongst others characterized by unintentional weight loss caused by bone loss and muscle loss. More specifically, frailty is also often characterized by diminished strength, endurance and reduced physiologic function which increases risk of adverse outcomes such as falls, disability, admission to hospital, or the need for longterm care. Preferably, in a medical use of the invention, frailty is associated with, or (at least partially) mediated by, bone loss and/or muscle loss as described herein.

The term “modification”, as used herein in relation to a modification in an amino acid sequence, includes reference to a substitution, insertion and/or deletion mutation in an amino acid sequence. One example of a modification is a substitution mutation in a heparin binding domain that rends said heparin binding domain inactive.

The term “heparin binding domain”, as used herein, includes reference to a protein domain on the surface of a protein that is generally rich in basic amino acid residues and which interacts with heparin through ion pairing, disulfide pairing and/or hydrogen bonding. One example of a heparin binding domain is a heparin binding domain that is comprised within the amino acid sequence ⁷⁵ KKCRMNKKNK ⁸⁴ of SEQ ID NO:1. One of many examples on how to inactivate such a heparin binding domain is by replacing said heparin binding domain with the amino acid sequence STCWDQTNN. The term “inactivation”, as used herein in relation to inactivation of a heparin binding domain, includes reference to at least partially or completely reducing binding interaction or binding affinity between heparin and a protein of interest such as a follistatin. Preferably, the binding interaction or binding affinity between heparin and a protein of interest such as a follistatin is reduced by at least 60%, more preferably reduced by at least 70% or 80%, even more preferably reduced by at least 90%, 95% or at least 99%. Most preferably, the binding interaction or binding affinity between heparin and a protein of interest such as a follistatin is completely abolished.

The term “therapeutically effective amount”, as used herein, refers to an amount of a therapeutic agent to treat, ameliorate, counteract, inhibit or prevent a desired disorder or condition, or to exhibit a detectable therapeutic or prophylactic effect. The precise effective amount needed for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. The therapeutically effective amount for a given situation can be determined by routine experimentation.

The term “pharmaceutical composition”, as used herein, refers to a composition that is made under conditions such that it is suitable for administration to mammals, preferably humans, e.g., it is made under GMP conditions. A pharmaceutical composition according to the invention may comprise pharmaceutically acceptable excipients, e.g., without limitation, stabilizers, bulking agents, buffers, carriers such as liquid (preferably aqueous) carriers, diluents, vehicles, solubilizers, and binders. The skilled person understands that the selection of appropriate excipients depends on the route of administration and the dosage form, as well as the active ingredient and other factors. A pharmaceutical composition according to the invention is preferably adapted for parenteral administration, such as intraperitoneal or intravenous administration.

Method of counteracting a condition of bone loss and/or muscle loss in a subject It was unexpectedly established that a combination therapy of a follistatin and a bisphosphonate can advantageously be employed in a method of counteracting a condition of bone loss and/or muscle loss in a subject.

Therefore, the invention provides a follistatin in combination with a bisphosphonate for use in a method of counteracting a condition of bone loss and/or muscle loss in a subject. In the same manner, the invention provides a use of a follistatin and bisphosphonate in the preparation of a medicament or kit for use in counteracting a condition of bone loss and/or muscle loss in a subject. In the same manner, the invention provides a method of counteracting bone loss and/or muscle loss in a subject, comprising the steps of: - administering a therapeutically effective amount of a follistatin as described herein and a bisphosphonate as described herein to a subject in need thereof. Preferably, the subject suffers from, is suspected of suffering from, or is at risk of suffering from a condition of bone loss and/or muscle loss as described herein.

Amongst others, it was surprisingly established that a combination therapy of the invention was especially effective in counteracting bone loss, especially trabecular bone loss. Preferably, the bone loss as described herein is trabecular bone loss. Trabecular bone, also called cancellous bone, is porous bone composed of trabeculated bone tissue. It can be found at the ends of long bones like the femur, where the bone is not solid but is composed of holes connected by thin rods and plates of bone tissue. In addition, trabecular bone is prominent in vertebrae of the spine. The form and structure of trabecular bone are organized to optimally resist loads imposed by functional activities, like jumping, running and squatting. It has previously been shown that once a human reaches adulthood, bone density steadily decreases with age, of which loss of trabecular bone mass is a contributor. Typical bone fracture sites in elderly individuals are sites that have lost substantial amounts of trabecular bone, such sites including the hip, spine, femur, wrist and shoulder. Therefore, in medical use of the invention, preferably said method of counteracting a condition of bone loss and/or muscle loss in a subject as described herein is a method for counteracting a condition of trabecular bone loss and/or muscle loss. Alternatively, a method of counteracting a condition of bone loss and/or muscle loss in a subject as described herein is a method for improved counteraction of (trabecular) bone loss and/or muscle loss as compared to bisphosphonate, preferably zoledronate, monotherapy.

Bone mass, and muscle mass and muscle strength, peak in early adulthood around an age of 30 years, and subsequently decline with age especially after the fifth decade (Curtis et al., J Cell Physiol., 230(11):2618- 2625 (2015)). In subjects that are 50 years or older, muscle mass is lost at a rate of 1-2% per year and strength at a rate of 1.5-3% per year. In women, there is an accelerated period of bone loss perimenopausally additional to the bone loss rates of approximately 1-2% annually (Curtis et al., J Cell Physiol., 230(11):2618-2625 (2015)).

Bone loss and muscle loss are especially a pathological condition in subjects with an age above 50. Bone loss and muscle loss inter alia incrementally increase risk of bone fracture and incrementally increase the risk of falling which further increases risk of bone fracture. At some point, bone loss develops into osteopenia and its more severe form osteoporosis, which are especially pronounced in the elderly population as one of the components of frailty. In the same manner, at some point, muscle loss develops into sarcopenia, which is also especially pronounced in the elderly population as one of the components of frailty, together with bone loss. The combination therapy of the invention is preferably employed for treating age-associated bone loss and/or age-associated muscle loss, for instance in the elderly population, preferably the frail elderly population.

In embodiments, sarcopenia is characterized by (i) low muscle strength optionally in combination with (ii) low muscle quantity or quality and/or (iii) low physical performance. Low muscle strength can e.g. be identified by using the EWGS0P2 sarcopenia cut-off points for low strength by chair stand and/or grip strength (Cruz-Jentoft et al., Age and Ageing, 48: 16-31 (2019), Table 3), more specifically a grip strength of <27 kg for men and a grip strength of <16 kg for women and/or a chair stand of >15 s for five rises for men is indicative of sarcopenia. Low muscle quantity can e.g. be identified by using the EWGSOP2 sarcopenia cut-off points for low muscle quantity (Cruz-Jentoft et al., Age and Ageing, 48: 16-31 (2019), Table 3), more specifically an appendicular skeletal muscle mass (ASM) of <20 kg for men and <15 kg for women and/or an ASM/height ² of <7.0 kg/m ² for men and <5.5 kg/m ² for women is indicative of sarcopenia. Low performance can e.g. be identified by using the EWGSOP2 sarcopenia cut-off points for low performance (Cruz-Jentoft et al., Age and Ageing, 48: 16-31 (2019), Table 3), more specifically a gait speed of <0.8 m/s for men and/or a Short Physical Performance Battery (SPPB) score of <8 point score for men and women and/or a Timed-Up and Go (TUG) of >20 s and/or a 400 m walk test that is not completed or >6 min for completion is indicative of sarcopenia.

The medical methods of the present invention are preferably directed to the treatment of a subject that is an elderly human, more preferably a frail elderly human.

Preferably, a subject that is a frail elderly human is a subject that suffers from, is suspected to suffer from, or at risk of suffering from (i) osteopenia or osteoporosis and/or (ii) sarcopenia.

One way of assessing frailty in an elderly subject is by using Fried’s phenotype method. Fried’s phenotype method involves classifying a subject as frail, pre-frail or non-frail based on five criteria that are weight loss, exhaustion, physical activity, walk time and grip strength. For each of the criteria, the subject can be classified as frail or not frail, for instance by using the following cut-offs: 1) Weight loss: more than 4.5 kg lost unintentionally in the last year; 2) Exhaustion: subject stating that he feels that everything he did was an effort or that they could not get going (from the CES-D Depression Scale) a moderate amount of the time or most of the time; 3) Physical activity (Minnesota Leisure Time Activity Questionnaire): energy expenditure <383 kcal per week for men and <270 kcal per week for women; 4) Walk time (4.6 meter walk): > 7 sec (men height < 173 cm, women height < 159 cm) or > 6 sec (men height > 173 cm, women height > 159 cm); 5) Grip strength (Jamar Dynamometer, Layfayette Instruments, USA) (average of three trials): < 29-32 kg for men (stratified by BMI classifications) and < 17-21 kg for women (stratified by BMI classifications). The subjects can then be classified as follows. Frail subjects score below the cut-offs for three or more criteria, pre-frail subjects score below the cut-offs for one or two criteria, and non-frail subjects did not score below the cut-offs for any criteria.

Follistatin in a combination therapy of the invention

In a combination therapy of the invention, a follistatin is to be administered in a therapeutically effective amount to a subject.

Preferably, the follistatin is a human follistatin. Follistatin is a protein that inhibits the activity of (human) bone formation inhibitor activin-A, the latter being also known as activin isoform BABA. The follistatin can be a human follistatin, for instance a follistatin that comprises the amino acid sequence of SEQ ID NO:1 or a follistatin that comprises the amino acid sequence of SEQ ID NO:1 except that 1-200 such as 1-100, 1-50, 1-20 or 1-10 amino acid residues in said SEQ ID NO:1 are substituted, deleted and/or inserted (while still inhibiting the activity of (human) bone formation inhibitor activin-A). Alternatively, the follistatin is a protein comprising SEQ ID NO:1 or SEQ ID NO:3, or is a protein that has at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO:1 or SEQ ID NO:3, and inhibits the activity of (human) bone formation inhibitor activin-A. Preferably, sequence identity is calculated over the whole length of the protein. If necessary, a minimum number of gaps is introduced to achieve optimal alignment. Sequence identity can e.g. be determined using EMBOSS Needle, e.g. at standard or default settings. Preferably, the follistatin is a protein comprising SEQ ID NO:2 or a protein comprising SEQ ID NO:2 except that 1-50, preferably 1- 20, more preferably 1-10 or 1-5, amino acid residues in said SEQ ID NO:2 are substituted, deleted and/or inserted (while still inhibiting the activity of (human) bone formation inhibitor activin-A). More preferably, the follistatin is a protein comprising SEQ ID NO:2 or an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO:2 and which inhibits the activity of (human) bone formation inhibitor activin-A. Preferably, sequence identity is calculated over the whole length of the protein. If necessary, a minimum number of gaps is introduced to achieve optimal alignment. Sequence identity can e.g. be determined using EMBOSS Needle, e.g. at standard or default settings. Standard tests are available to assess inhibition by a follistatin of activin-A activity (Lodberg et al., 2019; PMID=30759349). A follistatin of the invention can thus be a human follistatin of which the amino acid sequence is not genetically modified. Such a follistatin can be produced recombinantly by standard molecular cloning techniques. Alternatively, the human follistatin can be genetically modified by insertion, substitution and/or deletion of amino acid residues, for instance in order to improve pharmacokinetic properties of the molecule. An example of a pharmacokinetic property is the half-life, which may be increased by PEGylation or by fusing the follistatin to a second protein (e.g. an albuminbinding domain (ABD)) so as to reduce renal clearance. Another option to increase half-life is to formulate the follistatin in a carrier (such as a lipid vesicle) that allows for an increased blood circulation time. A carrier may also allow for tissue targeting. For instance, and preferably, the follistatin is a follistatin comprising the amino acid sequence of SEQ ID NO:1 except that it comprises a modification in a heparin binding domain, wherein said modification results in inactivation of said heparin binding domain. Preferably, said heparin binding domain is a heparin binding domain that is indicated by the amino acid sequence ⁷⁵ KKCRMNKKNK ⁸⁴ as comprised SEQ ID NO:1. As an example, such a heparin binding domain can be inactivated by replacing said sequence with the amino acid sequence STCWDQTNN. The latter sequence is also underlined in SEQ ID NO:2, which means that in SEQ ID NO:2 said heparin binding domain is inactivated. The skilled person can routinely devise and test other heparin binding domain-inactivating modifications.

Alternatively, but preferably in addition, the follistatin comprises a C-terminal Fc domain, more preferably a C-terminal mouse Fc domain or a C-terminal human (IgG) Fc domain. One example of such a mouse Fc domain is provided in SEQ ID NO:2, i.e. the second underlined amino acid region, which is located at the C-terminus of the protein.

Most preferably, the follistatin is a protein comprising the amino acid sequence of SEQ ID NO:2. A protein consisting of the amino acid sequence of SEQ ID NO:2 is also referred to herein as ARC205 or FST315- mFcAHBS. Such a protein is also described in WO2014/187807, the contents of which are incorporated herein by reference, especially the embodiments that describe the follistatin derivatives as such.

Therapeutic effective amounts and regimens for the administration of a follistatin according to the invention can be determined readily by those with ordinary skill in the art. Generally, a therapeutic effective amount of a follistatin can be provided by a single administration or by multiple administrations within the same treatment schedule to obtain the desired results. The follistatin dose may suitably be from about 0.1 to about 30 mg/kg bodyweight. A follistatin as described herein can be prepared in any appropriate pharmaceutical composition or formulation, but is preferably prepared in a pharmaceutical composition adapted for parenteral administration. Such a composition generally comprises a carrier such as for instance an aqueous or oily solution, dispersion, emulsion and/or suspension. Parenteral administration involves the injection or infusion into a body tissue or body fluid, whereby preferably a syringe, needle, or catheter is used. Preferably, the carrier is an aqueous solution, preferably distilled sterile water, saline, buffered saline, or another pharmaceutically acceptable excipient for injection. Examples of parenteral modes of administration are intravenous, intra-arterial, intra-peritoneal, intracap sular, subcutaneous and intramuscular administration, which are well known to the person skilled in the art. Alternatively, a follistatin as described herein can be employed in a dosage form such as a tablet or capsule for enteral (oral) administration. As a further example, the follistatin may be administered in the form of an implant material that comprises a slow-release matrix, capable of releasing follistatin over a period of time at a predetermined location. Treatment may be conducted over an extended period, such as with daily dosages in the range of about 0.01-30000 pg/kg body weight or about 0.1-3000 pg/kg body weight. Preferably, a follistatin as described herein is administered intraperitoneally, for instance biweekly at a dose of about 10 mg/kg.

A pharmaceutical composition comprising a follistatin can be obtained by mixing the follistatin with a pharmaceutically acceptable carrier or excipient, by means that are widely known in the art, such as by conventional mixing, granulating, dragee-making, dissolving, lyophilizing or similar processes.

In embodiments of a kit or pharmaceutical composition of the invention, the follistatin and the bisphosphonate are present in a single unit dose. An exemplary follistatin single unit dose range is 4-6000 mg, for example 400-1200 mg.

Bisphosphonates in a combination therapy of the invention

In a combination therapy of the invention, a bisphosphonate is to be administered in combination with a follistatin as described herein, in a therapeutically effective amount, to a subject.

A bisphosphonate is generally referred to as an analog of endogenous pyrophosphate whereby the central oxygen is replaced by carbon. Bisphosphonates include amongst others aminobisphosphonates. Bisphosphonates include, but are not limited to the following compounds: zoledronate (also referred to as zoledronic acid herein), risedronate, alendronate, cimadronate, clodronate, tiludronate, etidronate, ibandronate, piridronate, or p ami dr on ate and functional analogues thereof. If desired, a bisphosphonate may be administered in the form of a pharmaceutically acceptable salt, hydrate or solvate to the subject. When reference is made to a bisphosphonate, included in said definition is said bisphosphonate in the form of its pharmaceutically acceptable salt, hydrate or solvate, etc.. For instance, zoledronate can be administered in the form of a monohydrate.

Preferably, the bisphosphonate is zoledronate (commercially also known as AC LAS TA®).

Therapeutic effective amounts and regimens for the administration of a bisphosphonate according to the invention can be determined readily by those with ordinary skill in the art. Generally, the bisphosphonate dose can be provided with a single administration or with multiple administrations within the same treatment schedule to obtain the desired results. The dosage of a bisphosphonate may suitably be from about 0.1 mg to about 10 mg, preferably about 5 mg, as a single (intravenous) infusion in for instance a 100 ml aqueous solution. Alternatively, a bisphosphonate can be administered subcutaneously, for instance biweekly, in a dose of 0.01-1000 pg/kg body weight or about 0.1-100 pg/kg body weight.

A bisphosphonate as described herein can be prepared in any appropriate pharmaceutical composition or formulation, but is preferably prepared in a pharmaceutical composition adapted for parenteral administration. Such a composition generally comprises a carrier such as for instance an aqueous or oily solution, dispersion, emulsion and/or suspension. Parenteral administration involves the injection or infusion into a body tissue or body fluid, whereby preferably a syringe, needle, or catheter is used. Preferably, the carrier is an aqueous solution, preferably distilled sterile water, saline, buffered saline, or another pharmaceutically acceptable excipient for injection. Examples of parenteral modes of administration are intravenous, intra-arterial, intra-peritoneal, intracap sular, subcutaneous and intramuscular administration, which are well known to the person skilled in the art. Alternatively, a bisphosphonate as described herein can be employed in a dosage form such as a tablet or capsule for enteral (oral) administration. Treatment may be conducted over an extended period, such as with daily dosages in the range of about 0.01- 1000 pg/kg body weight or about 0.1-100 pg/kg body weight.

Preferably, a bisphosphonate as described herein is administered intravenously or subcutaneously, for instance as a single intravenous infusion, and preferably in a dose of about 0.1-10 mg, more preferably about 5 mg, for instance in the form of a 100 ml aqueous solution.

In embodiments of a kit or pharmaceutical composition of the invention, the follistatin and the bisphosphonate are present in a single unit dose. An exemplary follistatin single unit dose range is 4-6000 mg, for example 400-1200 mg. An exemplary bisphosphonate single unit dose range is 0.4-120 mg, for example 4-12 mg.

Preferably, subjects are appropriately hydrated prior to administration of a bisphosphonate, especially when bisphosphonate is for administration to an elderly subject (preferably > 65 years) and/or when subjects receive diuretic therapy. In addition, adequate calcium and vitamin D intake are recommended in association with administration of a bisphosphonate.

Preferably, a bisphosphonate is administered via a vented infusion line and given at a constant infusion rate. The infusion time is preferably not less than 15 minutes.

A bisphosphonate as described herein and a follistatin as described herein may be prepared in the same pharmaceutical composition. Alternatively, and preferably, a bisphosphonate as described herein and a follistatin as described herein are prepared in separate pharmaceutical compositions, optionally in the form of a kit-of-parts. Such separate pharmaceutical compositions are for simultaneous, concurrent or sequential administration. Preferably, said (different or the same) pharmaceutical composition(s) or said kit of parts is for use in a method of counteracting a condition of bone loss and/or muscle loss as described herein above.

For the purpose of clarity and a concise description, features are described herein as part of the same or separate aspects and preferred embodiments thereof, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.

The content of the documents referred to herein is incorporated by reference. FIGURE LEGENDS

Figure 1. Effects of combination therapy on trabecular bone MicroCT of trabecular bone measurements in femoral metaphyses. Abbreviations: BV/TV, trabecular bone volume fraction (trabecular bone volume divided by tissue volume); Tb.Th, trabecular thickness; Tb.N. trabecular number; Tb.Sp, trabecular spacing; SMI, structure model index; CD, connectivity density. * p<0.05 vs baseline, # p<0.05 vs Veh, f , p < 0.05 vs. BTX+Veh, $, p < 0.05 vs. BTX+BP.

Figure 2. Effects of combination therapy on cortical bone

DXA measurements of cortical bone measurements in femoral diaphysis. Abbreviations: BMC, bone mineral content; BMD, bone mineral density. * p<0.05 vs baseline, # p<0.05 vs Veh, f , p < 0.05 vs. BTX+Veh, $, p < 0.05 vs. BTX+BP.

Figure 3. Effects of combination therapy on bone strength

Force measurements at the diaphysis and femoral neck. Maximum force (Fmax) for both locations are indicated. * p<0.05 vs baseline, # p<0.05 vs Veh, t, p < 0.05 vs. BTX+Veh, $, p < 0.05 vs. BTX+BP.

Figure 4. Effects of combination therapy on muscle mass.

Muscle measurements. The muscle mass weights for the left and right femur muscles (rectus femoris) and tibiae (gastrocnemius) are indicated. The lower graph represents the average cross-sectional area (CSA) of the femoral muscle. * p<0.05 vs baseline, # p<0.05 vs Veh, f , p < 0.05 vs. BTX+Veh, $, p < 0.05 vs. BTX+BP.

Figure 5. Effects of combination therapy on body weight. Body weight development during 3-week treatment period. All animals received wet chow at day 8 for 3 days due to prior body weight loss. # p<0.05 vs Veh, f , p < 0.05 vs. BTX+Veh, $, p < 0.05 vs. BTX+BP.

Figure 6. Effects of combination therapy on trabecular bone in mouse model for ageing. jiCT of trabecular bone measurements in femoral metaphyses.

Abbreviations: BV/TV, trabecular bone volume fraction; Tb.Th, trabecular thickness; Tb.Sp, trabecular spacing; Tb.N. trabecular number; SMI, structure model index; Tb.Pf, trabecular patterning factor. Data are presented as mean ± SEM. * p<0.05 vs Veh.

Figure 7. Effects of combination therapy on cortical bone in mouse model for ageing. jiCT of cortical bone measurements in femoral diaphyses. Abbreviations: MOI, moment of inertia. Data are presented as mean ± SEM.

Figure 8. Effects of combination therapy on muscle mass and body weight in a mouse model for ageing.

Muscle, body weight and fat depot weights. Data are presented as mean ± SEM. * p<0.05 vs Veh.

SEQUENCES

SEQ ID NO : 1 : Follistatin FST315 without signal peptide

(a . a . 30-344 of UniProtKB - P19883 (Last modified : October 10 , 2002 - v2 ) )

1 GNCWLRQAKN GRCQVLYKTE LSKEECCSTG RLSTSWTEED VNDNTLFKWM I FNGGAPNCI 61 PCKETCENVD CGPGKKCRMN KKNKPRCVCA PDCSNITWKG PVCGLDGKTY RNECALLKAR 121 CKEQPELEVQ YQGRCKKTCR DVFCPGSSTC WDQTNNAYC VTCNRICPEP ASSEQYLCGN 181 DGVTYSSACH LRKATCLLGR SIGLAYEGKC IKAKSCEDIQ CTGGKKCLWD FKVGRGRCSL 241 CDELCPDSKS DEPVCASDNA TYASECAMKE AACSSGVLLE VKHSGSCNSI SEDTEEEEED 301 EDQDYSFPI S SILEW

SEQ ID NO : 2 : ARC205 (FST315-mFcAHBS)

1 GNCWLRQAKN GRCQVLYKTE LSKEECCSTG RLSTSWTEED VNDNTLFKWM I FNGGAPNCI 61 PCKETCENVD CGPGSTCWD QTNNPRCVCA PDCSNITWKG PVCGLDGKTY RNECALLKAR 121 CKEQPELEVQ YQGRCKKTCR DVFCPGSSTC WDQTNNAYC VTCNRICPEP ASSEQYLCGN 181 DGVTYSSACH LRKATCLLGR SIGLAYEGKC IKAKSCEDIQ CTGGKKCLWD FKVGRGRCSL 241 CDELCPDSKS DEPVCASDNA TYASECAMKE AACSSGVLLE VKHSGSCNSI SEDTEEEEED 301 EDQDYSFPI S SILEWPRGPT IKPCPPCKCP APNLLGGPSV FI FPPKIKDV LMI SLSPIVT 361 CVWDVSEDD PDVQI SWFVN NVEVHTAQTQ THREDYNSTL RWSALPIQH QDWMSGKEFK 421 CKVNNKDLPA PIERTI SKPK GSVRAPQVYV LPPPEEEMTK KQVTLTCMVT DFMPEDIYVE 481 WTNNGKTELN YKNTEPVLDS DGSYFMYSKL RVEKKNWVER NSYSCSWHE GLHNHHTTKS 541 FSRTPGK

SEQ ID NO : 3 : Follistatin FST315-AHBS

1 GNCWLRQAKN GRCQVLYKTE LSKEECCSTG RLSTSWTEED VNDNTLFKWM I FNGGAPNCI 61 PCKETCENVD CGPGSTCWD QTNNPRCVCA PDCSNITWKG PVCGLDGKTY RNECALLKAR

121 CKEQPELEVQ YQGRCKKTCR DVFCPGSSTC WDQTNNAYC VTCNRICPEP ASSEQYLCGN 181 DGVTYSSACH LRKATCLLGR SIGLAYEGKC IKAKSCEDIQ CTGGKKCLWD FKVGRGRCSL 241 CDELCPDSKS DEPVCASDNA TYASECAMKE AACSSGVLLE VKHSGSCNSI SEDTEEEEED 301 EDQDYSFPI S SILEW EXAMPLES

Example 1. Effects of combination therapy on botox-immobilized mice (model for muscle loss and bone loss)

Materials and methods

Female 16-week old wild type BALB/cJ mice were used for this experiment. To establish an immobilization model, the mice were injected i.m. with 2U I 100g body weight (BW) botulinum Toxin A (BTX; Botox, Allergan, Irvine, CA) to paralyze the right hind limb. A baseline group was included for reference at the start of the experiment and another group was included without the BTX treatment (Veh). Mice were treated with either 10 mg/kg FST315-mFcAHBS i.p. (ARC205; bi-weekly), 100 pg/kg s.c. zoledronate (BP; once at day 0) or the combination for 21 days (n=10-12). The vehicle (BTX + Veh) received the same injections but only with the solvents of both compounds. Then, the left femurs and the right rectus femoris and gastrocnemius were harvested and stored for analysis.

Body weight was monitored throughout the treatment period.

Bone analyses:

The distal femoral metaphysis and mid diaphysis were scanned with an isotropic resolution of 9 mm using a SkyScan 1076 system (Bruker microCT, Kontich, Belgium). The following settings were used: X-ray voltage and tube current were 40 kV and 0.25 mA, respectively. Beam hardening was reduced using a 1-mm aluminum filter, exposure time was 5.9 s, and a mean of 3 pictures was taken at each angle (0.9°) to generate final images. NRecon from Bruker microCT was used to reconstruct 3-dimensional images. Using different software packages from Bruker microCT (CtAn and Dataviewer), bone microarchitectural parameters were assessed in trabecular and cortical bone. For image processing, trabecular bone was manually selected, whereas cortical bone was automatically selected. A global threshold was applied for segmentation using threshold levels of 90 (lower) and 255 (higher) for trabecular bone measurements and levels of 130 (lower) and 255 (higher) for cortical bone measurements. The DXA was used to calculate cortical bone mineral density and content (PMID: 28377955). Bone strength, determined by a 3-point bending test (Lloyd Instruments, Fareham, United Kingdom) and a neck test (5566; Instron, Norwood, MA, USA) of the femur, was performed in a materials testing machine until fracture as previously described (PMID: 28377955).

Muscle analyses: upper and lower leg muscle groups were individually weighed.

Results

A combination therapy with a follistatin such as ARC205 and a bisphosphonate such as zoledronate strongly increases trabecular bone mass (Figure 1). Mice receiving the BTX injection and Veh treatment (BTX + Veh) lose trabecular bone mass (BV/TV; left top Figure 1), which is partially prevented by ARC205 but strongly stimulated above vehicle by BP. Surprisingly, the combination treatment further increases the BV/TV to approximately twice the bone volume of that of botox-treated mice receiving BTX + Veh. The combination treatment also led to a statistically significant increase of BV/TV in BTX-immobilized mice as compared to bisphosphonate monotherapy. The advantageous changes in BV/TV as a result of the combination therapy are best explained by a combination of an increase in trabecular number (Tb.N) and increase in connectivity density (CD).

A combination therapy with a follistatin such as ARC205 and a bisphosphonate such as zoledronate prevents cortical bone loss (Figure 2). The BTX treatment did not affect bone, marrow or tissue area. BTX did however, reduce cortical bone thickness, BMC and BMD in the botox treated Veh and ARC205 group. The combination treatments prevented loss of cortical bone mass.

A combination therapy with a follistatin such as ARC205 and a bisphosphonate such as zoledronate prevents loss of bone strength (Figure 3). Mice receiving BTX (Veh and ARC205 group) compromise on bone strength at the site of the femoral neck. This is counteracted by the combination treatment of the invention.

A combination therapy with a follistatin such as ARC205 and a bisphosphonate such as zoledronate prevents loss of muscle mass (Figure 4). Following BTX treatment of the right leg, there is permanent muscle mass damage in all groups. In the left leg, the combination treatment is able to counteract muscle loss resulting from right leg BTX-induced immobilization of the mice.

A combination therapy with a follistatin such as ARC205 and a bisphosphonate such as zoledronate counteracts body weight loss following BTX treatment (Figure 5). Following BTX, the Veh and BP group lose body weight over the 3-week period, which is completely counteracted by the combination therapy, which follows the weight development of the untreated Veh mice.

Example 2. Effects of combination therapy on prematurely ageing mice (model for rapid bone and muscle mass loss in ageing population).

Materials and methods Female 8-week old Erccld/- mice (Ahmad et al., PMID: 18541667), being heterozygous for a mutation in the DNA repair enzyme Erccl, displaying rapid bone and muscle mass loss and having short lifespan (30 weeks), were used for this experiment. In the same manner as in Example 1, the mice were treated with either 10 mg/kg FSTsis-mFcAHBS i.p. (ARC205; biweekly), 100 gg/kg s.c. zoledronate (ZOL; once at day 0) or the combination for 21 days (n=3 per group). The vehicle (Veh) group received the same injections but only with the solvents of both compounds. Then, the left femurs and the right rectus femoris and gastrocnemius muscle were harvested and stored for analysis.

Body weight was monitored throughout the treatment period.

Bone analyses: Bone analyses were performed as described in Example 1. Muscle analyses: upper and lower leg muscle groups were individually weighed.

Results

A combination therapy with a follistatin such as ARC205 and a bisphosphonate such as zoledronate strongly increases trabecular bone mass (Figure 6). Mice treated with ARC205 did not increase trabecular bone volume fraction (BV/TV) in these DNA enzyme repair deficient mice but the bisphosphonate did (Figure 6, top left graph). Surprisingly, the combination treatment with ARC205 and BP greatly further enhanced BV/TV, which was best explained by increased trabecular number (Tb.N), reduced trabecular spacing (Tb.Pf) and as a consequence increased connectivity as expressed by reduced trabecular patterning factor (Tb.Pf). There was a clear trend towards improved trabecular bone mass with the combination therapy as compared to the monotherapies. Although none of the treatments increased cortical bone mass, it appeared that the combination therapy with a follistatin such as ARC205 and a bisphosphonate such as zoledronate in this mouse model for ageing best preserved cortical bone area, cortical thickness and moment of inertia (MOI), a proxy for cortical bone strength (Figure 7).

A combination therapy with a follistatin such as ARC205 and a bisphosphonate such as zoledronate provides for muscle mass gain which drives body weight gains (Figure 8). Muscle mass loss, a hallmark in these mice, is preserved in the lower leg muscle, musculus gastrocnemius, of the groups that received the combination therapy during the 3-week period. In the femoral muscle (rectus femoris), even a weight gain was observed for the combination therapy group. ZOL had no (negative) effect on muscle mass for any of the muscles measured when administered in combination with a follistatin. In vehicle- and ZOL-treated animals, body weight was stable over the 3-week period. In contrast, the combination therapy increased body weight, most probably through muscle mass gain as fat mass of several depots (subcutaneous, gonadal and inguinal WAT) were all reduced in the mice that received the combination treatment