HK1148665B - Non-immunosuppressive cyclosporin for the treatment of muscular dystrophy - Google Patents

Description

Non-immunosuppressive cyclosporin for the treatment of muscular dystrophy

The present invention relates to the use of non-immunosuppressive cyclosporin a derivatives for reducing induction of myofiber necrosis and muscle degeneration in a subject diagnosed with Limb Girdle Muscular Dystrophy (LGMD), in particular sarcoglycemia (sarcoglycanopathy).

Muscular Dystrophy (MD) includes multiple groups of genetic disorders that involve primarily striated muscle tissue, which lead to progressive muscle weakness, wasting, and in many cases, premature death. Many characteristic mutations causally related to human MD are associated with alterations in structural adhesion proteins that attach the basic contractile proteins to the basement membrane, thus providing rigidity to the skeletal muscle cell membrane (sarcolemma), or with proteins that directly stabilize or repair cell membranes, such as sarcoglycan (sarcoglycan) or dystrophin (dystrophin).

Genetic mutations in the α -, β -, γ -, and δ -sarcoglycan genes result in skeletal muscle diseases including the heterologous syndrome, LGMD and subgroups thereof, sarcoglycan disease. The syndrome or phenotype of sarcoglycan disease depends on which sarcoglycan gene is mutated and the type of genetic mutation (allelic variant), and shows four forms of specific dysfunction: LGMD type 2C, 2D, 2E and 2F (mutations in the γ -, α -, β -, and δ -myoglycan genes, respectively) account for 25% of all cases diagnosed as LGMD. Specifically, The different mutations specifically observed in The delta-sarcoscan gene resulted in severe dysfunction of LGMD2F type or LGMD2F (one line Mendelian Inheritance in Man [ OMIM ] #601287, genetic mutations [ OMIM ]601411.Emery et al, The Lancet, 2002, 359: 687-.

The different LGMD types are characterized by progressive wasting and weakness with atrophy, mainly involving muscles of the arms and legs proximal to the shoulders and hips, respectively. The disease phenotypes are similar to those of severe Duchenne (Duchenne) or Becker (Becker) muscular dystrophy syndrome. However, the latter two diseases involve different molecular mechanisms and genetic disorders. Prior to the advent of molecular diagnosis of LGMD, LGMD patients were often diagnosed with duchenne muscular dystrophy. The onset of the disease varies from mild to severe clinical types in infancy to adulthood. Up to 25% of patients show a severe form of disease, with severe lumbar lordosis, achilles tendon contractures, muscle hypertrophy, cardiomyopathy and cardiac conduction defects. Lower leg or tongue hypertrophy, muscle involvement selectivity and late cardiac complications are more or less specifically associated with each different clinical type (Daniere et al, IntJ. biochem Cell biol., 2007; 39: 1608-1624). Progressive weakness leads to restrictive lung disease and hypoventilation of the lungs requiring assisted ventilation. Generally, there is a difference in morbidity and mortality. The progression to death is very rapid in the early-onset. Death is often the outcome of respiratory complications.

To date, there is no specific treatment for patients with any one of the LGMD syndromes. Positive supportive therapies for preserving muscle function, such as orthopedics, surgery and physical therapy, maximize functional capacity and extend life expectancy. However, these measures do not prevent the eventual occurrence of myofibrosis and respiratory complications.

With the development of animals lacking specific genes involved in muscular dystrophy, the underlying molecular mechanisms of sarcoglycemia will soon be better understood. In recent years, it was shown that muscle cells of mice lacking delta-myoglycan (scgd-/-mice) have an increased tendency to calcium influx into the cells due to targeted inactivation of the delta-myoglycan gene (scgd). It is believed that loss of a component of the dystrophin-glycoprotein complex (DGC), such as dystrophin or sarcoglycan complex, results in a fundamental change in the physical properties of the muscle cell membrane, as well as an increase in the osmotic leakage of the muscle membrane. Activation of unregulated calcium influx channels due to membrane instability and fragility can cause dystrophic disorders in skeletal muscle, leading to muscle fibrosis and induction of muscle fiber necrosis.

Parsons et al (j.biol.chem., 2007, 282: 10068-. Similar disease improvement following inhibition of calcineurin activity was not observed in mice lacking the mdx gene (dystrophin gene mutation) used as a model of becker muscular dystrophy, even though increased calcium influx was observed in both dystrophin models. Indeed, activation of the calcineurin transgene protects mdx mice (Chakkalakal et al, hum. mol. Genet., 2004, 13: 379-. Parsons et al identified calcineurin as a potential target for LGMD treatment. Based on their observations, Parsons et al suggest that inhibition of calcineurin activity may provide some benefit in selecting muscle disease types, such as limb-girdle muscular dystrophy, and therefore, cyclosporine (CsA) may be potentially beneficial.

The effector mechanisms leading to progressive myofibrosis induced by membrane permeability changes in LGMD have not been elucidated and are the subject of active research, which may provide the basis for the ultimate development of novel mechanism-based therapeutic strategies for patients suffering from such diseases. However, at this time, no effective method is available for treating patients with LGMD. Thus, there is a need for new treatment modalities such as those described herein.

It is an object of the present invention to provide a treatment for the induction and progression of myofibronecrosis and myodegeneration in patients with LGMD, and in particular with sarcoglycopathy, more in particular with LGMD type 2F. This treatment should protect the dystrophic skeletal muscles and also the cardiac and diaphragm muscles from necrosis and should delay the progression of the disease.

The present inventors have surprisingly found that the administration of non-immunosuppressive cyclosporin a (csa) derivatives which do not inhibit calcineurin to subjects suffering from LGMD, in particular from sarcoglycemia, more in particular from LGMD type 2F, is an effective treatment. They observed non-immunosuppressive cyclosporin A derivatives [ D-MeAla ]]³-[EtVal]⁴Administration of CsA reduces muscle pathology, and the degeneration and progression of the disease in subjects diagnosed with myofibronecrosis, particularly LGMD, more particularly LGMD type 2F, and normalizes the myofibroid area distribution by reducing the sensitivity of mitochondria to potential calcium overload.

The subject may be a human or a mammal such as, for example, a mouse, which shows a dominance of muscular dystrophy due to deletion or non-expression of genes responsible for the phenotype of the disease.

Thus, the present invention relates to non-immunosuppressive cyclosporin A derivatives of formula I, more preferably non-immunosuppressive cyclosporin A derivatives of formula II and most preferably non-immunosuppressive cyclosporin A derivatives of formula III [ D-MeAla]³-[EtVal]⁴-use of CsA for the manufacture of a medicament for preventing or reducing muscle degeneration in a subject suffering from LGMD, in particular a sarcoglycemia, more in particular LGMD type 2F. Non-immunosuppressive CsA derivatives suitable for use in the present invention are also described on pages 3-6 of International patent application WO2005/021028 to Novartis AG. [ D-MeAla ]]³-[EtVal]⁴-CsA is disclosed in International patent application WO 00/01715 by Wenger et al. [ D-MeAla ] of the formula III]³-[EtVal]⁴CsA has been attributed CAS registry number 254435-95-5.

The non-immunosuppressive CsA derivatives used in the present invention are cyclic undecapeptides described by the following formula:

formula I

Wherein

W is MeBmt, dihydro-MeBmt, 8' -hydroxy-MeBmt or O-acetyl-MeBmt,

x is alpha Abu, Val, Thr, Nva or O-methylthreonine (MeOTHr),

r is Pro, Sar, (D) -MeSer, (D) -MeAla or (D) -MeSer (O acetyl),

y is MeLeu, ThioMeLeu, gamma-hydroxy-MeLeu, MeIle, MeVal, MeThr, MeAla, MeaIle or MeaThr; N-EthylVal (EtVal), N-Ethyl Ile, N-Ethyl Thr, N-Ethyl Phe, N-Ethyl Tyr or N-Ethyl Thr (O acetyl), wherein Y is not MeLeu when R is Sar,

z is Val, Leu, MeVal or MeLeu,

q is MeLeu, gamma-hydroxy-MeLeu, MeAla or Pro,

T₁is (D) Ala or Lys,

T₂is MeLeu or gamma-hydroxy-MeLeu, and

T₃is MeLeu or MeAla.

Formula II

Wherein

W is MeBmt, dihydro-MeBmt, 8' -hydroxy-MeBmt;

x is α Abu, Val, Thr, Nva or O-methyl threonine (MeOTHr);

r is Pro, Sar, (D) -MeSer, (D) -MeAla or (D) -MeSer (O acetyl);

y is MeLeu, ThioMeLeu, gamma-hydroxy-MeLeu, MeIle, MeVal, MeThr, MeAla, MeaIle or MeaThr; n-ethyl Val (EtVal), N-ethyl Ile, N-ethyl Thr, N-ethyl Phe, N-ethyl Tyr, or N-ethyl Thr (O acetyl), wherein Y is not MeLeu when R is Sar;

z is Val, Leu, MeVal or MeLeu;

q is MeLeu, gamma-hydroxy-MeLeu, or MeAla;

T₁is (D) Ala;

T₂is MeLeu; and

T₃is MeLeu.

Formula III

Wherein

W is MeBmt;

x is α Abu;

r is (D) -MeAla;

y is N-ethyl Val (EtVal);

z is Val;

q is MeLeu;

T₁is (D) Ala;

T₂is MeLeu; and

T₃is a group of compounds which are MeLeu,

and wherein MeBmt is N-methyl- (4R) -4-but-2E-en-1-yl-4-methyl- (L) threonine, α Abu is L- α -aminobutyric acid, D-MeAla is N-methyl-D-alanine, EtVal is N-ethyl-L-valine, Val is L-valine, MeLeu is N-methyl-L-leucine, Ala is L-alanine, (D) Ala is D-alanine, and MeVal is N-methyl-L-valine. The conventional amino acid position numbering commonly used for reference of cyclosporin A is shown below the formula. The combination name is used for the derivatives of CsA,the combination name includes a first portion indicating the identity of and providing a site for a residue that is different from that in cyclosporin a, and a second portion labeled "CsA" that indicates that all other residues are identical to cyclosporin a. For example, [ MeIle]⁴-CsA is the same cyclosporin as cyclosporin A except that MeLeu in position 4 is replaced by MeIle (N-methyl-L-isoleucine).

In a further embodiment, the invention relates to a non-immunosuppressive cyclosporin A derivative of formula I, more preferably a non-immunosuppressive cyclosporin A derivative of formula II and most preferably a non-immunosuppressive cyclosporin A derivative of formula III [ D-MeAla]³-[EtVal]⁴-use of CsA for the treatment of LGMD, in particular sarcoidosis, more particularly LGMD type 2F.

In another embodiment, the invention relates to a method of preventing or reducing muscle degeneration in a subject suffering from LGMD, in particular a sarcoglycemia, more particularly LGMD type 2F, comprising administering to said subject an effective amount of a non-immunosuppressive cyclosporin a derivative of formula I, more preferably a non-immunosuppressive cyclosporin a derivative of formula II and most preferably a non-immunosuppressive cyclosporin a derivative of formula III [ D-MeAla a ]]³-[EtVal]⁴-CsA. An effective amount of non-immunosuppressive cyclosporin a is understood to be an amount that, when repeatedly administered to a subject suffering from LGMD, in particular from a sarcoglycemia, more particularly from LGMD type 2F, in the course of a treatment regimen, results in an improvement, stabilization or delay of progression of a targeted clinical response such as a disease. When administered orally, an effective amount will be from about 1mg/kg (body weight) to about 100mg/kg, preferably from about 1mg/kg to about 20mg/kg, administered daily or three times weekly (cube). By intravenous route, the corresponding effective dose indicated may be from about 1mg/kg to about 50mg/kg, preferably from about 1mg/kg to about 25 mg/kg.

Furthermore, the present invention relates to a pharmaceutical composition for preventing or reducing muscle degeneration in a subject suffering from LGMD, in particular from a sarcoglycemia, more in particular from LGMD type 2F, comprising an effective amount of a non-immunosuppressive cyclosporin a derivative of formula I, more preferably a non-immunosuppressive ring of formula IISporin A derivatives and most preferably non-immunosuppressive cyclosporin A derivatives of formula III [ D-MeAla]³-[EtVal]⁴-CsA, a pharmaceutically acceptable carrier, and optionally comprising excipients and diluents. The diluent is typically water. Excipients typically added to parenteral formulations include isotonic agents, buffers or other pH control agents, and preservatives. The composition may include other active ingredients such as antibiotics, glucocorticoids, corticosteroids such as, for example, prednisone.

The invention will be further explained below with the aid of the following figures.

Figure 1(a) represents the baseline swelling of mitochondria of skeletal muscle from 6-week-old wild-type mice (Wt) (white bars) and scgd-/-mice (black bars), as measured by absorbance at 540 nm. Mitochondria from the metatarsal muscle group, quadriceps and tibialis anterior are pooled together. As shown by the lower absorbance measurements, mitochondria from the muscle of the scgd-/-mice were more swollen at baseline compared to mitochondria from Wt mice.

FIG.1(b) shows the change in mitochondrial swelling after 10 min treatment with calcium (Ca2+) or PEG-3350(PEG), measured as the difference in absorbance at 540nm between untreated and treated mitochondria from 6 week old wild type mice (Wt) (white bars) and scgd-/-mice (black bars). Mitochondria from the metatarsal muscle group, quadriceps and tibialis anterior are pooled together.

FIG.2(a) represents the administration of D- [ MeAla ] in scgd-/-mice]³-[EtVal]⁴-reduction of muscle pathology after CsA. Measurement from the vector (white bar) or D- [ MeAla]³-[EtVal]⁴Wild type (Wt) mice treated with CsA (black bar) or with vehicle (grey bar or penultimate bar in the group) or D- [ MeAla [)]³-[EtVal]⁴-CsA (spot bar or last bar of group) treated scgd-/-gastrocnemius (Gastroc.) of mice, quadriceps (Quad.), Tibialis Anterior (TA) and ratio of Muscle Weight (MW) in myocardium to Tibial Length (TL) (MW/TL). D- [ MeAla ] in scgd-/-mice]³-[EtVal]⁴CsA prevents an increase in muscle mass, which increaseIs associated with a disease.

FIG.2(b) is a representation of a peptide from D- [ MeAla]³-[EtVal]⁴Administration of D- [ MeAla ] in CsA-treated wild-type (Wt) mice and scgd-/-mice observed in representative hematoxylin and eosin stained sections of the quadriceps muscle of scgd-/-mice]³-[EtVal]⁴-reduction of muscle pathology after CsA. The corresponding vehicle control is also shown.

Figure 2(c) represents D- [ MeAla ala administration in scgd-/-mice as assessed by quantification of fibrotic area in trichrome stained sections from diaphragm (Diaph.), Tibialis Anterior (TA), gastrocnemius (gasroc.), quadriceps (Quad)]³-[EtVal]⁴-reduction of muscle pathology after CsA. For the vector from (white bar) or D- [ MeAla-]³-[EtVal]⁴Wild type (Wt) mice treated with CsA (black bar) and treated with vehicle (grey bar or penultimate bar in the group) or D- [ MeAla [ ]]³-[EtVal]⁴Sections of CsA (spot bar or last bar of the group) treated scgd-/-mice were evaluated. D- [ MeAla ]]³-[EtVal]⁴-CsA reduces fibrosis in scgd-/-mice.

FIG.3(a) represents the results from administration of D- [ MeAla ] as assessed by quantification of the area distribution of fibrosis in the tibialis anterior (muscle)]³-[EtVal]⁴-reduction of fibrotic area heterogeneity in muscle of scgd-/-mice post-CsA. The study involved the use of either the vector (white bars) or D- [ MeAla]³-[EtVal]⁴Wild type (Wt) mice treated with CsA (black bar) and treated with vehicle (grey bar or penultimate bar in the group) or D- [ MeAla [ ]]³-[EtVal]⁴CsA (spot bar or last bar of group) treated scgd-/-mice. ("<" means "less than" > "means" greater than "). D- [ MeAla ]]³-[EtVal]⁴CsA treatment normalized the area of fibrotic heterogeneity in scgd-/-mice.

FIG.3(b) represents the results from administration of D- [ MeAla ] as assessed by quantification of the fiber area distribution in gastrocnemius]³-[EtVal]⁴Heterogeneous area of fibrosis in the muscle of scgd-/-mice after CsAAnd (4) reducing. The study involved the use of either the vector (white bars) or D- [ MeAla]³-[EtVal]⁴Wild type (Wt) mice treated with CsA (black bar) and treated with vehicle (grey bar or penultimate bar in the group) or D- [ MeAla [ ]]³-[EtVal]⁴CsA (spot bar or last bar of group) treated scgd-/-mice. ("<" means "less than" > "means" greater than "). D- [ MeAla ]]³-[EtVal]⁴CsA treatment normalized the area of fibrotic heterogeneity in scgd-/-mice.

FIG.3(c) represents the results from administration of D- [ MeAla ] as assessed by quantification of the area distribution of fibrosis in the quadriceps]³-[EtVal]⁴-reduction of fibrotic area heterogeneity in muscle of scgd-/-mice post-CsA. For vector (white bar) or D- [ MeAla-]³-[EtVal]⁴Wild type (Wt) mice treated with CsA (black bar) and treated with vehicle (grey bar or penultimate bar in the group) or D- [ MeAla [ ]]³-[EtVal]⁴CsA (spot bar or last bar of the group) treated scgd-/-mice were measured. ("<" means "less than" > "means" greater than "). D- [ MeAla ]]³-[EtVal]⁴CsA treatment normalized the area of fibrotic heterogeneity in scgd-/-mice.

If increased calcium concentrations act as an initiator for LGMD through myofiber necrosis, many downstream calcium-dependent effectors could potentially be considered pathogenic. For example, increased calcium can lead to myotube necrosis via the calcium-activated protease calpain (calpain), a critical signaling protein involved in skeletal muscle cell injury and regeneration after differentiation.

Parsons et al (2007) demonstrated that inhibition of calcium/calmodulin-activated serine/threonine protein phosphatase calcineurin (calceinin) activity by gene deletion in a murine model of LGMD reduced skeletal muscle and muscle fibrosis and inflammation, i.e., improved skeletal muscle pathology. This does not occur in the mouse model of Duchenne, where CsA-induced inhibition of calcineurin is detrimental to muscle pathology (Stupka et al, Acta neuropathohol, 2004, 107: 299-. This difference in the results of inhibition of calcineurin activity in different dystrophies characterized by elevated calcium concentrations in muscle cells is presumably due to their respective different gene deletions involved in and affecting different muscle cell membrane alterations and different signaling pathways.

Another major mechanism leading to cell necrosis is mitochondrial calcium overload, which secondarily increases the generation of Reactive Oxygen Species (ROS) and further promotes MPT (mitochondrial permeability transition). Increased submuscular calcium can also promote a local increase in Reactive Oxygen Species (ROS), resulting in larger defects in the cell membrane and additional calcium entry, further promoting cell necrosis and/or apoptosis.

Experiments were performed to experimentally detect the causal link existing between the absence of the scgd gene, the progressive degeneration of the muscle fibers and the necrosis and/or apoptosis of cells caused by mitochondrial dysfunction.

To assess whether calcium-induced mitochondrial dysfunction can initiate and drive progressive degeneration of muscle fibers associated with LGMD, the inventors compared mitochondria isolated from dystrophic skeletal muscle in scgd-/-mice and wild-type mice. Mitochondria isolated from the metatarsus muscle group, the quadriceps and the tibialis anterior by homogenization in sucrose-containing buffer (250mM sucrose, 10mM Tris (pH 7.4), 1mM EDTA) were suspended in isotonic buffer (120mM KCl, 10mM Tris (pH 7.4), 5mM KH) after washing centrifugation₂PO₄) And detected in the swelling assay. The assay includes isolated mitochondria and 200 μ M CaCl₂(swelling) or 5% (w/v) PEG-3350 (shrinkage). Swelling produced a decrease in absorbance at 540nm and shrinkage increased. The results are expressed as mean ± SEM (standard error of mean) (fig. 1(a) and 1 (b)). Student's t-test using both samples and values were considered significant only if p < 0.05.

Mitochondria isolated from skeletal muscle of scgd-/-mice were swollen at baseline compared to wild-type mice. They also tolerate additional swelling caused by exogenously applied calcium and do not show reversal of baseline swelling (fig. 1(a) and 1 (b)). Insensitivity of mitochondria in scgd-/-mice to additional calcium indicates that they are swollen and pathological, consistent with downstream pathological effects such as pseudo-hypertrophic responses in gastrocnemius and quadriceps at 6 weeks of age (this hypertrophy is associated with tissue inflammation), a dramatic decrease in muscle weight with age compared to wild-type mice, a characteristic increase in central nucleation of muscle fibers (indicating regeneration due to ongoing degeneration and extensive cycles of degeneration/regeneration), and destabilization of the sarcolemma damaged by calcium overload.

The above findings strongly suggest that the calcium-dependent MPT process is the primary cause of the progressive degeneration of muscle fibers associated with LGMD, even though the underlying mitochondrial abnormality cannot predict the severity of clinical syndrome. In general, the pathogenic chain of events downstream of genetic lesions can be interrupted by appropriate drugs. For example, loss of myostatin (myostatin) activity can improve LGMD in scgd-/-mice by reducing fibrosis and inducing muscle regeneration. Inhibition of calcineurin activity has been reported to produce similar effects. Based on the new findings discussed above and those given in examples 1 and 2, modulation of the degeneration/regeneration cycle and reduction of skeletal muscle degeneration in a murine model of LGMD can be achieved by normalizing mitochondrial function. These findings of the inventors enable novel drug therapies for patients affected by LGMD that target mitochondrial function rather than calcineurin activity and do not result in immunosuppression.

Thus, the present invention relates to non-immunosuppressive CsA derivatives, most preferably D- [ MeAla ™]³-[EtVal]⁴-use of CsA for preventing or reducing muscle degeneration in a subject with LGMD. Non-immunosuppressive CsA derivatives may also be used to normalize mitochondrial function of mitochondria prepared from muscle biopsies of subjects with LGMD. It was found that tolerance to calcium overload in the mitochondria would be an indication that treatment of patients with non-immunosuppressive CsA derivatives would be effective in reducing the severity of the disease.

The active compound, i.e. the non-immunosuppressive cyclosporin a derivative, for use in the treatment of patients suffering from LGMD may be administered by any conventional route. It may be administered parenterally, for example, in the form of injectable solutions or suspensions, or in the form of injectable deposit formulations. Preferably, it will be administered orally in the form of a solution or suspension for drinking, a tablet or a capsule. Non-immunosuppressive cyclosporin A derivatives D- [ MeAla for oral administration]³-[EtVal]⁴Pharmaceutical compositions of-CsA are described in the examples. The pharmaceutical compositions typically comprise a selected non-immunosuppressive cyclosporin A derivative in combination with one or more pharmaceutically acceptable carrier substances. Suitable Pharmaceutical carriers are described, for example, in Remington's Pharmaceutical Sciences, 17 th edition, Mack Publishing Company, Easton, PA (1990), which is a standard reference in the art. Typically, these compositions are concentrated and need to be combined with an appropriate diluent, e.g., water, prior to administration. Pharmaceutical compositions for parenteral administration also typically include one or more excipients. Optional excipients include isotonic agents, buffers or other pH control agents, and preservatives. These excipients may be added for preservation of the composition, as well as for achieving a preferred pH range (about 6.5-7.5) and osmotic pressure (about 300 mosm/L).

Other examples of cyclosporin formulations for oral administration can be found in U.S. patent nos. 5,525,590 and 5,639,724, and U.S. patent application No. 2003/0104992. By the oral route, an effective dose of the non-immunosuppressive cyclosporin A derivative for daily or wednesday administration may be from about 1mg/kg (body weight) to about 100mg/kg, preferably from about 1mg/kg to about 20 mg/kg. By intravenous route, the corresponding effective dose may be from about 1mg/kg to about 50mg/kg, preferably from about 1mg/kg to about 25 mg/kg. An effective amount of non-immunosuppressive cyclosporin a is understood to be an amount that results in an improvement, stabilization or delay in progression of a targeted clinical response such as disease when repeatedly administered to a LGMD patient over the course of a treatment regimen. The clinical response can be assessed, for example, by quantitative isometric muscle strength (QIS) testing. QIS enables muscle strength to be assessed in an objective manner by means of a pressure transduction and recording device. Alternatively, the normality of the rate of apoptosis can be assessed in muscle biopsies by biochemical and immunohistochemical methods known to those skilled in the art. Finally, electromyography can be used, which shows a map of the muscle rather than the neurogenic one, which can be quantified.

When determined for testing a composition comprising a non-immunosuppressive cyclosporin A derivative, most preferably D- [ MeAla]³-[EtVal]⁴The trial dose of efficacy of the pharmaceutical composition of the invention of CsA will take into account numerous factors by the clinician. The basic factors are the toxicity and half-life of nonimmunosuppressive cyclosporin A derivatives. Other factors include the size of the patient, the age of the patient, the general condition of the patient (including mechanical ventilation, clinical stage of disease, severity of symptoms), the presence of other drugs in the patient, and the like. A course of treatment will require repeated administration of the pharmaceutical composition of the invention. Typically, a sufficient dose of drug is administered about once a day. Due to the genetic nature of the disease, treatment may need to last for a long period of time, possibly for the life of the patient.

No effective drug treatment of LGMD is currently known. Patients are supported by vaccination against influenza and pneumococcal infections, and any infection is actively treated with antibiotics. Thus, the pharmaceutical composition of the invention may comprise, in addition to the non-immunosuppressive cyclosporin a derivative, one or more other active ingredients such as, for example, one or more antibiotics. The non-immunosuppressive cyclosporin a derivative and said other active ingredient may be administered as part of the same pharmaceutical composition or may be administered separately as part of a suitable dosing regimen designed to obtain the benefits of all active ingredients. The appropriate dosage regimen, the amount of each dose administered, and the particular dosage interval between doses of each active agent will depend on the particular combination of active agents employed, the condition of the patient being treated, and other factors discussed in the preceding section. The other active ingredients will generally be administered in the same amount in which they are known to be effective as monotherapeutic agents. FDA-approved doses of such active agents that have been FDA-approved for human administration are publicly available.

All patents, patent applications, and publications cited herein are deemed to be incorporated by reference in their entirety.

The invention is further illustrated by the following examples. The examples are provided for illustrative purposes to those skilled in the art and are not intended to limit the scope of the invention as set forth in the claims. Thus, the present invention should not be construed as limited to the examples provided, but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.

Example (b):

example 1: stabilization of calcium-induced damage in the sarcolemma and reduction of muscular dystrophy progression and pathology

To stabilize calcium-induced damage to the sarcolemma and reduce disease progression through numerous cycles of degeneration/regeneration, scgd-/-mice were treated with D- [ MeAla]³-[EtVal]⁴-CsA therapy.

scgd-/-mice were administered a 50 mg/kg/day dose of D- [ MeAla subcutaneously]³-[EtVal]⁴CsA or vehicle (without active ingredient D- [ MeAla)]³-[EtVal]⁴-preparation of CsA) starting at 4 weeks of age and ending at 10 weeks of age. The different muscles cut, i.e., gastrocnemius, quadriceps, tibialis anterior and cardiac muscle, were weighed and the ratio of Muscle Weight (MW) to Tibial Length (TL) (MW/TL) was determined. The results in fig.2(a) and 2(c) are expressed as mean ± SEM (standard error of mean). One-way ANOVA was used to compare the means between 3 or more independent groups. Newman-Keuls post hoc test (post hoc test) was performed whenever multiple comparisons were performed using inst 3.0 (GraphPad software from Science inc.). Values were considered significant when p < 0.05.

In response to a plurality ofThe degeneration/regeneration cycle of (c), the scgd-/-mice initially appeared as hypertrophied skeletal muscle as seen in subjects with LGMD. Administration of D- [ MeAla ] in scgd-/-mice]³-[EtVal]⁴-CsA resulted in a reduction in myolesions observed as a reduction in pseudohypertrophic response in skeletal muscle in treated scgd-/-mice (fig. 2 (a)). In D- [ MeAla ] in comparison with vehicle-treated animals]³-[EtVal]⁴Reduction of skeletal muscle hypertrophy was about 1.3 fold in gastrocnemius (gasroc.), quadriceps (Quad), and Tibialis Anterior (TA) and about 1.2 fold in cardiac muscle (heart) in CsA treated scgd-/-mice.

By D- [ MeAla]³-[EtVal]⁴Reduction of lesions in CsA treated scgd-/-mice was also observed in histological sections from the quadriceps (fig. 2 (b)). In comparison with wild-type mice or scgd-/-mice treated with vehicle, in mice derived from D- [ MeAla]³-[EtVal]⁴Improvement of myofibrillar tissue and normalization of the fiber area distribution were noted in sections of CsA treated scgd-/-mice.

Evaluation of the percentage fibrosis by means of a biochemical assay, which quantifies the amount of D- [ MeAla ] with or without]³-[EtVal]⁴The hydroxyproline content in the diaphragm (Diaph.), Tibialis Anterior (TA), gastrocnemius (Gastroc.), and quadriceps (Quad.) muscles of CsA-treated wild-type and scgd-/-mice (Parsons et al, am.J.Pathol., 2006, 168: 1975-. By D- [ MeAla]³-[EtVal]⁴CsA treatment of scgd-/-mice reduced fibrosis in different muscles by approximately 1, 5% -3, 5% (fig. 2 (c)).

Example 2: by D- [ MeAla] ³ -[EtVal] ⁴ Small and large fibers in skeletal muscle of scgd-/-mice after CsA treatment Normalization of vitamin distribution and reduction of denaturation/regeneration cycles

By D- [ MeAla]³-[EtVal]⁴CsA treatment scgd-/-mice partially induced Tibialis Anterior (TA) (FIG. 3(a))Normalization of the fiber area distribution in gastrocnemius (Gastroc.) (FIG. 3(b) and quadriceps (Quad.) (FIG. 3(c)) showed a reduction in the degeneration/regeneration cycle, scgd-/-mice at a dose of 50 mg/kg/day of D- [ MeAla]³-[EtVal]⁴-CsA or vector is administered subcutaneously for 6 weeks. Histological analysis of fibers from three skeletal muscles of scgd-/-mice showed small diameter fibers (< 200 μm) relative to wild-type mice²) Indicates an increase in regenerated fibers, an increase in the number of ongoing degeneration/regeneration cycles, and disease progression. Administration of non-immunosuppressive D- [ MeAla ] to scgd-/-mice]³-[EtVal]⁴CsA slows down the regeneration rate of the fibers and partially normalizes the distribution of small and large fibers. By non-immunosuppressive D- [ MeAla compared to scgd-/-mice receiving vehicle only]³-[EtVal]⁴Fewer fibrils were observed in CsA treated scgd-/-mice.

Example 3: d- [ MeAla ]] ³ -[EtVal] ⁴ -oral dosage forms of CsA.

The amounts are expressed as% w/w.

Example a:

D-[MeAla]³-[EtVal]⁴-CsA 10

glycofurol 75(Glycofurol 75) 35.95

Medium chain triglyceride (Miglycol) 81218

Polyoxyethylene ether hydrogenated castor oil RH 4035.95

Alpha-tocopherol 0.1

Example B:

[D-MeAla]³-[EtVal]⁴-CsA 10

tetraethylene glycol 2

Captex 800 2

Nikkol HCO-40 85.9

Butylated Hydroxytoluene (BHT) 0.1

Example C:

[D-MeAla]³-[EtVal]⁴-CsA 10

tetrahydrofuran polyglycol ether 7539.95

Medium chain triglyceride 81214

Polyoxyethylene ether hydrogenated castor oil RH 4036

Butylated Hydroxyanisole (BHA) 0.05-0.1

Example D:

[D-MeAla]³-[EtVal]⁴-CsA 10

tetraethylene glycol 10

Caprylic/capric triglyceride (Myrinol) 5

Polyoxyethylene ether hydrogenated castor oil RH 4074.9

Alpha-tocopherol 0.1

Example E:

[D-MeAla]³-[EtVal]⁴-CsA 10

ethanol 9

Propylene glycol 8

Polyoxyethylene ether hydrogenated castor oil RH 4041

Glycerol monolinoleate 32

See british patent application No. 2,222,770 for individual components of formulations a-D and methods of preparation.

Claims (1)

1. A cyclic undecapeptide having the formula:

the application in the preparation of the medicine for treating limb girdle muscular dystrophy,

wherein W is MeBmt;

x is α Abu;

r is (D) -MeAla;

y is N-ethyl Val (EtVal); z is Val;

q is MeLeu;

T₁is (D) Ala;

T₂is MeLeu; and

T₃is MeLeu.