HK1137364A - Non-immunosuppressive cyclosporin for treatment of ullrich congenital muscular dystrophy - Google Patents

Description

Non-immunosuppressive cyclosporin for treatment of Ullrich congenital muscular dystrophy

Technical Field

The present invention relates to the use of non-immunosuppressive cyclosporin a derivatives for reducing mitochondrial dysfunction and decreasing the rate of apoptosis in muscle cells in patients diagnosed with Ullrich congenital muscular dystrophy or Bethlem myopathy.

Background

Genetic mutations in the collagen VI gene cause two major skeletal muscle diseases, Bethlem myopathy (BM, online human mendelian inheritance [ OMIM ]158810) and Ullrich congenital muscular dystrophy (UCMD, OMIM 254090).

BM is an autosomal phenotypic genetic disease characterized by slowly progressive debilitating weakness of the medial and proximal muscles, accompanied by flexion finger contractures (Bethlem and Wijngaarden. brain 1976; 99: 91-100; Merlini et al. neurousual Disord 1994; 4: 503-11). It is manifested as variability within the family and different clinical attack times, varying from prenatal to middle-aged. Prenatal episodes of the disease are characterized by decreased fetal movement; neonatal onset of disease is characterized by hypotonia or torticollis; onset of disease in young children is characterized by delayed motor milestones, muscle weakness and contractures; while adult onset is characterized by proximal weakness, achilles tendon and finger contracture. In some individuals over the age of 50, with outdoor activity requiring assistance, there are usually mild and slow progressive symptoms (Pepe et al biochem Biophys Res Commun 1999; 258: 802-07.De Visser et al, Muscle Nerve 1992; 15: 591-96). Cardiac function is usually normal.

UCMD is an autosomal recessive genetic disorder characterized by congenital muscle weakness with joint contracture and distal joint hyperrelaxation coexisting therewith (Ullrich. Z. Ges. neuron. Psychiator. 1930; 126: 171-201.Camacho Vanegas et al. Proc Natl Acad Sci USA 2001; 98: 7516-21.). Manifestations are usually hypotonia at birth, congenital hip dislocation, calcaneal eminence and temporary kyphosis. The moving milestones are delayed and most children are never able to walk independently. There is usually follicular hyperkeratosis in the extensor surfaces of the upper and lower extremities, and keloid and cigarette paper-like scars form. The medial axial muscle is severely affected, resulting in progressive scoliosis with spinal stiffness. Early and severe respiratory involvement may require artificial ventilation support in the first or second decade of life. Subjects affected by UCMD had normal intelligence and MRI showed normal brain development. Patients with recessive or neonatal heterozygous mutations usually have a severe and typical phenotype, although they may occasionally exhibit the symptoms of the lighter Bethlem-like disease (Pan et al. Am J Hum Genet 2003; 73: 355-69.Baker et al. Hum Mol Genet 2005; 14: 279-93.Demir et al. Am J Hum Genet 2002; 70: 1446-58.). In some patients with UCMD phenotype, no mutation in the collagen VI gene has occurred, indicating that genetic heterogeneity is still present even for this disease (Pan et al Am J Hum Genet 2003; 73: 355-69).

Recently, studies have shown that mice lacking collagen VI due to targeted inactivation of Col6a1 have latent mitochondrial defects caused by inappropriate opening of the Permeability Transition Pore (PTP), an inner membrane channel, which plays a role in several forms of cell death, and cyclosporin a desensitizes them(Irwin et al Nat Genet 2003; 35: 267-71.Bernardi et al FEBS J2006; 273: 2077-99.). This finding was further exploited in vivo and successful therapeutic intervention was achieved in a mouse model. Determining whether mitochondria are involved in the pathogenesis of genetic and clinical heterogeneous UCMD is a major challenge, which is a major obstacle to the use of established therapies in mouse models for therapeutic applications in humans. This challenge has now been overcome. Furthermore, the inventors of the present invention have been able to demonstrate that the presence of a latent mitochondrial defect in the cells of UCMD patients and the consequent high apoptosis rate can be caused by non-immunosuppressive cyclosporin A derivatives such as [ D-MeAla ]]³-[EtVal]⁴-CsA inhibition. Thus, the present invention provides novel methods for inhibiting apoptosis of muscle cells in patients with UCMD and the use of non-immunosuppressive derivatives of cyclosporine A, preferably [ D-MeAla ]]³-[EtVal]⁴-CsA, a novel method of treating this disease in these patients. There is currently no effective method for treating UCMD. Thus, there is a need for new methods of treatment as described herein. The inventors of the present invention have also found a similar mitochondrial defect sensitive to cyclosporin a in BM patients. Thus, the novel treatment methods for UCMD patients disclosed herein are also applicable to the treatment of BM patients.

Disclosure of Invention

The present invention results from the following findings: a key problem in UCMD is increased rate of skeletal muscle cell apoptosis. The inventors have found that an increased rate of apoptosis is a consequence of potential mitochondrial dysfunction that can be corrected by exposing cells to cyclosporin a. Importantly, the nonimmunosuppressive cyclosporin A derivative [ D-MeAla]³-[EtVal]⁴CsA is as effective as the parent compound in reducing mitochondrial dysfunction and inhibiting excessive apoptosis. Thus, the present invention relates to the use of a nonimmunosuppressive cyclosporin A (CsA) derivative of formula I, preferably a nonimmunosuppressive cyclosporin A derivative of formula II, and most preferably a nonimmunosuppressive cyclosporin A derivative of formula III [ D-MeAla ™ ]]³-[EtVal]⁴CsA reduces mitochondrial dysfunction and decline in UCMD patientsLow incidence of apoptosis in patients with UCMD. International patent application PCT/EP2004/009804 to NovartisAG (WO2005/021028) at pages 3-6 describes non-immunosuppressive cyclosporin A derivatives suitable for use in the present invention. U.S. Pat. No. 6,927,208 discloses [ D-MeAla ]]³-[EtVal]⁴CsA。

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), where Y cannot be 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 or 8' -hydroxy-MeBmt,

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

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

y' is MeLeu, gamma-hydroxy-MeLeu, Melle, MeVal, MeThr, MeAla, Meallel 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 cannot be MeLeu when R is Sar,

z is Val, Leu, MeVal or MeLeu,

q' is MeLeu, gamma-hydroxy-MeLeu or MeAla.

Formula III

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.

In another embodiment, the invention relates to a nonimmunosuppressive cyclosporin A derivative of formula I, more preferably a nonimmunosuppressive cyclosporin A derivative of formula II, and most preferably a nonimmunosuppressive cyclosporin A derivative of formula III [ D-MeAla]³-[EtVal]⁴-use of CsA in the production of a medicament intended for the treatment of UCMD.

In another embodiment, the invention relates to a method of treating UCMD in a patient comprising administering to the patient an effective amount of a non-immunosuppressive of formula ICyclosporine A derivatives, 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-CsA. An effective amount of a nonimmunosuppressive cyclosporin a derivative is understood to produce a desired clinical response, such as amelioration, stabilization or slowing of the progression of the disease, when repeatedly administered to a patient with UCMD in a therapeutic regimen. When administered orally, an effective amount is between about 1mg/kg to about 100mg/kg, preferably from about 1mg/kg to about 20mg/kg, administered daily or thrice weekly. By intravenous route, the corresponding dosage specified 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 the treatment of UCMD comprising 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]⁴CsA, a pharmaceutically acceptable carrier and, optionally, excipients and diluents. The diluent is typically water. Excipients commonly added to parenteral formulations include isotonic agents, buffers or other agents to control pH, and preservatives. The composition may comprise other active ingredients such as antibiotics.

The invention further relates to a nonimmunosuppressive cyclosporin A derivative of formula I, more preferably a nonimmunosuppressive cyclosporin A derivative of formula II and most preferably a nonimmunosuppressive cyclosporin A derivative of formula III [ D-MeAla [)]³-[EtVal]⁴-use of CsA for reducing mitochondrial dysfunction and decreasing the rate of apoptosis in Bethlem myopathy patients. Furthermore, the present invention relates to a method of treating BM in a patient comprising administering to the patient 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]³-[EtVal]⁴-CsA. Furthermore, the invention relates to the use of a nonimmunosuppressive cyclosporin A derivative of formula I, II or III for the production of a medicament intended for use in therapyThe application in the medicine for treating BM. Finally, the present invention relates to a pharmaceutical composition for the treatment of BM comprising an effective amount of a non-immunosuppressive cyclosporin a derivative of formula I, II or III, a pharmaceutically acceptable carrier and optionally excipients and diluents.

Drawings

FIG. 1 shows mitochondrial membrane potential of myoblasts obtained from muscle biopsy from UCMD patients exposed to oligomycin in panels A and B, and apoptosis rate of myoblasts in UCMD patients and healthy individuals in panel C.

FIG. 2 is a typical experiment, test [ D-MeAla ]]³[EtVal]⁴-CsA pair isolated from Col6a (1)^-/-Effect of liver and muscle mitochondrial Calcium Retention Capacity (CRC) in mice. Wherein 10. mu.M Ca was added as indicated by the arrow²⁺Of (2) is performed. 5 hours after the last treatment, mitochondria examined in panels A1 and B1 were isolated from placebo-treated mice, and in panels AII and BII were from using [ D-MeAla ]]³-[EtVal]⁴-CsA treated mice. Lines a-d: is not added; line a '-d': adding [ D-MeAla ] to the culture medium]³-[EtVal]⁴-CsA (0.8. mu.M). Data are representative of 4 replicates.

FIG. 3 treatment with placebo or [ D-MeAla ]]³-[EtVal]⁴Col6a 1-/-mice liver and muscle after CsA treatment mitochondria were isolated and results summarized for CRC determination. The experiment was performed as described in figure 2. CRCmax directed to media addition of 0.8. mu.M [ D-MeAla ]]³-[EtVal]⁴CRC observed after CsA. FIG. A: each dot represents one mouse. Mice 24-27 with [ D-MeAla]³-[EtVal]⁴CsA treatment, mice 28-31 with placebo.

FIG. 4 oligomycin pairs isolated from placebo or [ D-MeAla ]]³-[EtVal]⁴-Col 6a 1-/-effect of mitochondrial TMRM fluorescence in mouse FDB fibers after CsA treatment. The TMRM detection was performed as described in example 4 (method). As described therein, 5. mu.M oligomycin (O) was addedOligo) or 4. mu.M carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Each line represents the fluorescence value of one fiber. The figure also shows the number of depolarized fibres after addition of oligomycin, relative to a randomly set threshold of 90% fluorescence.

FIG. 5 in the use of [ D-MeAla]³-[EtVal]⁴-incidence of apoptosis in CsA or placebo-treated Col6a 1-/-mouse diaphragm sections. Indicates that is derived from [ D-MeAla ]]³-[EtVal]⁴Per mm in diaphragm sections of each Col6a 1-/-mouse treated with CsA (group 1, mice 24-27) or placebo (group 2, mice 28-31)²The number of apoptotic nuclei. Bars represent the mean +/-SD of 20-30 sections per mouse. Data from untreated (basal) and CsA (cyclosporine a) treated animals were from historical samples.

FIG. 6 for [ D-MeAla ]]³-[EtVal]⁴-CsA or placebo-treated Col6a 1-/-assessment of fibers in the mouse diaphragm that undergo mitochondrial changes. Panel A shows the percentage of mitochondrial altered muscle fibers detected by electron microscopy in samples from individual animals. Graph B represents the average of the two treatment groups. Bars represent mean percent +/-SD. Significance was calculated using the Mann-Whitney test.

Detailed Description

To assess whether increased incidence of apoptosis and potential mitochondrial dysfunction occurred in UCMD patients, five UCMD patients were studied relative to healthy individuals. Patients 1 and 5 had UCMD for which gene validation had been obtained (Demir et al. variants in COL6A3cause segment and amplified phenol of Ullrich genetic variant. am J Hum Genet 2002; 70: 1446-58.Giusti et al. minor and sequential COL6A1 variants in Ullrich minor variant. Ann neuron 2005; 58: 400-10.). In patient 2, sequence analysis of exon 9 of COL6A1 indicated a 15 nucleotide deletion in heterozygosity (spanning nucleotides 35.374-35.388 of accession AJ011932, corresponding to a genomic clone comprising exons 1-20 of the COL6A1 gene; nucleotides 921-936 of accession NM-001848, corresponding to COL6A1 transcript). In patient 4, sequence analysis of exon 9 of COL6A1 indicated the presence of a G → A variation in heterozygosity (nucleotide 35.400 of accession No. AJ 011932; nucleotide 850 of accession No. NM-001848). The results of the genetic analysis of patient 3, which exhibited typical clinical and immunohistochemical characteristics of UCMD, were not provided.

These five patients represented a range of different severity of UCMD: all are congenital attacks; three people (patients 2, 3 and 5) were never able to stand and walk; one person (patient 4) can only stand on support; one person (patient 1) can walk. The reduction in collagen VI ranged from mild (patients 1 and 4), to significant (patients 2 and 3), to complete deficiency (patient 5). Mutations affected the COL6a1 gene in three cases (patients 2, 4 and 5) and the COL6A3 gene in one case (patient 1), which was not clear in one case (patient 3).

After patient consent was obtained and ethical committee approval, quadriceps muscle biopsies from five patients and one healthy volunteer were obtained. Apoptosis rates were assessed using the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. Frozen sections of seven microns thick were prepared from muscle biopsies and fixed in 50% acetone-50% methanol. TUNEL was performed using the apoltag in situ apoptosis detection kit (Chemicon). Samples were stained with peroxidase-diaminobenzidine, showing TUNEL-positive nuclei, and counterstained with Hoechst 33258(Sigma), labeling all nuclei. The total number of nuclei and the number of TUNEL-positive nuclei were determined in randomly selected fields of view using a Zeiss Axioplan microscope (40 x magnification) equipped with a digital camera. Data are presented as mean ± SD. Data were analyzed by unpaired Student t-test and values of P < 0.01 were considered significant. The results show that the frequency of apoptotic nuclei was much higher in all patient samples compared to healthy donors, with values increased approximately 10-fold for patients 1, 2 and 3 and over 200-fold for patients 4 and 5. Increased apoptosis corresponds to a significant decrease in collagen VI expression in muscle biopsy sections of all patients, as demonstrated by staining with selective antibodies against collagen VI.

Experiments were performed to test whether there is a causal relationship between collagen VI deficiency and apoptosis. Myoblast cultures were prepared from muscle biopsy sections of two unaffected controls and patients 1, 2, 3 and 4 by enzymatic and mechanical treatment and plated on Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum, penicillin, streptomycin and amphotericin b (sigma). To test for apoptosis, cells were fixed in 50% acetone-50% methanol and processed for TUNEL analysis using the Dead End fluorescent TUNEL system (Promega). All cores were visualized by staining with Hoechst 33258. Cultures from all patients showed a higher incidence of apoptosis compared to healthy donors. Coating or treatment with cyclosporin a on collagen VI completely normalized the incidence of apoptosis in patient samples. Muscle cell cultures from patients 2, 3 and 4 showed as expected a lower level to almost no collagen VI as previously shown in patients 1 and 5 (Demir et al 2002.giusti et al 2005).

To assess whether the anti-apoptotic effects of collagen VI and CsA were up-dated to mitochondria, mitochondrial function in living muscle cell cultures was investigated. As expected, the addition of oligomycin to cultures established from healthy donors did not cause mitochondrial depolarization, whereas the addition of the proton carrier carbonyl cyanide-p-trifluoromethoxyphenylhydrazone immediately caused mitochondrial depolarization. Mitochondrial depolarization occurred after addition of oligomycin instead to cells from all patients with UCMD. Obviously, with cyclosporin A or intracellular Ca²The chelating agent BAPTA-M, or coating the cells on collagen VI, completely restored the response to oligomycin to normal, indicating that the pathogenesis of UCMD is related to PTP. Mitochondrial membrane potential was assessed by accumulation of tetramethylrhodamine methyl ester (TMRM). It is noted that the addition of oligomycin to healthy respiratory cells is expected to cause hyperpolarization, where the mitochondrial membrane potential is maintained by the respiratory chain proton pump and the ATP synthesis is driven by a proton electrochemical gradient. Thus, mitochondrial depolarization induced by oligomycin in UCMD myoblast cultures is an abnormal response, suggesting that the membrane potential is not maintained by respiration, but rather by mitochondrial ATP synthesisThe enzyme works "in reverse" to pump protons from the matrix to the inter-membrane space, thereby consuming glycolytic ATP.

Muscle cell cultures established from healthy donors and patients 1, 2 and 3 were studied by electron microscopy. In the UCMD samples, the mitochondrial area/perimeter ratios in patients 1, 2, and 3 were significantly increased, 62.5, 75, and 50% higher (p < 0.05), respectively, than the healthy donors. This finding demonstrates that mitochondrial elongation is significantly less than in normal samples in UCMD muscle cells. It was also observed that the minor axis values increased (> 400nm) in 4-8% of mitochondria in patients compared to controls (< 300 nm). Taken together, these findings indicate the presence of an increased-scale mitochondrial fraction in UCMD cells. A small percentage of cells from UCMD patients (between 4 and 5%, 1% from healthy donors) also showed swollen mitochondria, with low density stroma, and a lack of cristae. Clearly, when UCMD cells were plated on collagen VI, the area/perimeter ratio and minor axis values became similar to healthy donors; while coating or treatment with cyclosporin a on collagen VI reduced the number of cells with swollen mitochondria to the values observed in cultures from healthy donors.

Treatment of cultures with the F1FO ATP synthase inhibitor oligomycin increased the percentage of cells with swollen mitochondria in the control to 4% and over 40% in UCMD patients, indicating the presence of latent mitochondrial dysfunction that can be selectively amplified by oligomycin, as demonstrated in the collagen VI deficient mouse model above. The effect of oligomycin can be prevented by treatment with cyclosporin a, restoring the percentage of cells with swollen mitochondria to values similar to the basal values of all cell cultures. Nonimmunosuppressive cyclosporin A derivatives [ D-MeAla]³-[EtVal]⁴CsA is as effective as cyclosporine a in preventing oligomycin-dependent mitochondrial depolarization of cells from UCMD patients and restores the incidence of apoptosis to the levels exhibited by cells from healthy donors. Experiments leading to these findings are discussed in example 1.

Obviously, it can be obtained from patients with clinical symptoms of UCMDMitochondrial defects were found in primary cultures, whether the primary gene defect was in COL6a1 or COL6A3 genes, and the underlying mitochondrial abnormality did not predict clinical symptom severity. These findings suggest that mitochondria are involved in the pathogenesis of all UCMD cases, and that there are other genetic and/or environmental factors that play a role in an individual's susceptibility to muscle fiber death and regeneration. All mitochondrial abnormalities and consequent apoptosis can be cured by plating UCMD cells on collagen VI or exposing them to cyclosporine a. These findings demonstrate that, in principle, the pathogenic chain downstream of a genetic lesion can be blocked by a suitable drug, at least in the early stages. Cyclosporine a is known to cause immunosuppression, a major obstacle to long-term treatment of patients. The key observation of the present inventors is that the nonimmunosuppressive cyclosporin A derivative [ D-MeAla]³-[EtVal]⁴CsA is as effective as cyclosporin a in alleviating mitochondrial dysfunction and reducing the rate of apoptosis in UCMD cells. This finding demonstrates that the immunosuppressive activity of cyclosporine, independent of its cytoprotective effect, can provide new pharmacological treatments for patients affected by the dysregulation of collagen VI.

In accordance with these findings, the present invention relates to a nonimmunosuppressive cyclosporin A derivative of formula I, more preferably a nonimmunosuppressive cyclosporin A of formula I, and most preferably a nonimmunosuppressive cyclosporin A derivative of formula III [ D-MeAla]³-[EtVal]⁴-use of CsA for reducing mitochondrial dysfunction and apoptosis rate in cells of UCMD patients.

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-ethyl Val (EtVal), N-ethyl Ile, N-ethyl Thr, N-ethyl Phe, N-ethyl Tyr or N-ethyl Thr (O acetyl), where Y cannot be 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 or 8' -hydroxy-MeBmt,

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

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

y' is MeLeu, 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), where Y cannot be MeLeu when R is Sar,

z is Val, Leu, MeVal or MeLeu,

q' is MeLeu, gamma-hydroxy-MeLeu or MeAla.

Formula III

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.

Non-immunosuppressive cyclosporin a derivatives can be used to inhibit apoptosis in vitro in muscle cells prepared from biopsy from patients with UCMD. The finding that apoptosis is inhibited may be used as an indicator that treatment of patients with non-immunosuppressive cyclosporin A derivatives is effective in reducing the severity of the disease.

The invention also relates to a nonimmunosuppressive cyclosporin A derivative of formula I, more preferably a nonimmunosuppressive cyclosporin A derivative of formula II, and most preferably a nonimmunosuppressive cyclosporin A derivative of formula III [ D-MeAla]³-[EtVal]⁴-use of CsA for treating a patient with UCMD. The active compound, for example a nonimmunosuppressive cyclosporin A derivative, may be administered by a convenient route. Administration can be parenteral, for example in the form of injection solutions or suspensions, or in the form of injectable depot preparations. Preferably, the administration is oral, in the form of a drinkable solution or suspension, tablet or capsule. The examples describe compositions comprising a nonimmunosuppressive cyclosporin A derivative [ D-MeAla ]]³-[EtVal]⁴-pharmaceutical compositions for oral administration of CsA. As demonstrated in the examples, these pharmaceutical compositions generally comprise the preferred non-immunosuppressive cyclosporin A derivatives and one or more pharmaceutically acceptable carrier substances. Suitable pharmaceutical carriers are described, for example, in Remington's pharmaceutical Sciences, 17^thed. Mack Publishing Company, Easton, Pa (1990)The literature is described as reference text standard in the art. These compositions are sometimes concentrated and need to be mixed with a suitable diluent, such as water, prior to administration. Pharmaceutical compositions for parenteral administration will generally also include one or more excipients. Optional excipients include isotonic agents, buffers or other agents to control pH, and preservatives. These excipients may be added to maintain the composition and to obtain a preferred pH range (about 6.5-7.5) and osmolarity (about 300 mosm/L).

Other examples of cyclosporin formulations for oral administration can be found in U.S. patents 5,525,590 and 5,639,724 and U.S. patent application 2003/0104992. By oral administration, the indicated dose of the nonimmunosuppressive cyclosporin A derivative administered daily or thrice weekly may be from about 1mg/kg to about 100mg/kg, preferably from about 1mg/kg to about 20 mg/kg. By intravenous route, the corresponding prescribed 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 a nonimmunosuppressive cyclosporin A derivative is understood to produce a desired clinical response, such as amelioration, stabilization or slowing of the progression of the disease, upon repeated administration to a patient with UCMD during the course of treatment. These clinical responses can be evaluated, for example, by quantitative isometric muscle strength (QIS) testing. QIS can objectively assess muscle strength with the aid of pressure sensing and recording devices. Alternatively, normalization of the rate of apoptosis can be assessed in muscle biopsy sections by biochemical and immunohistochemical methods known to those skilled in the art. Finally, electromyography, which shows a muscle map rather than a nerve map, which can be quantified, can be used.

Initial phase I clinical study was performed to evaluate [ D-MeAla]³-[EtVal]⁴Safety of the CsA oral dose and determination of the pharmacokinetic properties and safety of the drug. Studies have shown that doses of microemulsion from 50 to 1600mg in water are well tolerated. Mild and transient side effects were observed, including nausea, vomiting, abdominal pain and mild headache. These side effects are not dose-related.

In determining the trial dosage for testing the efficacy of a non-immunosuppressive cyclosporin A derivative pharmaceutical composition of the present invention comprising formulae I, II or III, the clinician will consider a number of factors. The most important of these 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 patient's profile (including mechanical ventilation, clinical stage of disease, severity of symptoms), whether there are other medications in the patient, etc. The course of treatment requires repeated administration of the pharmaceutical compositions of the present invention. Typically, the dosage is administered about once a day in a sufficient amount. Because of the genetic nature of the disease, treatment may take a long time, possibly the life of the patient.

No effective pharmacological treatment of UCMD is currently known. Patients are supported against influenza and pneumococcal infections by vaccination, and any infection is aggressively treated with antibiotics. Thus, in addition to the non-immunosuppressive cyclosporin a derivative, the pharmaceutical composition of the invention may comprise one or more further active ingredients, such as, for example, one or more antibiotics. The cyclosporin a derivative and these other active ingredients may be administered together as part of the same pharmaceutical composition, or may be administered separately as part of a suitable dosage regimen designed to obtain the benefits of all the active ingredients. The appropriate dosage regimen, the dosage of each administration and the specific interval between dosages of each active ingredient will depend upon the particular combination of active agents employed, the condition of the patient being treated and other factors discussed in the preceding section. These other active ingredients will generally be administered in the same amounts as are known to be effective as therapeutic agents alone. FDA-approved doses of these active agents for human administration that have been approved by the FDA are available to the public.

The invention also relates to a nonimmunosuppressive cyclosporin A derivative of formula I, more preferably a nonimmunosuppressive cyclosporin A derivative of formula II, and most preferably a nonimmunosuppressive cyclosporin A derivative of formula III [ D-MeAla]³-[EtVal]⁴-use of CsA for reducing mitochondrial dysfunction and decreasing the rate of apoptosis in cells of BM patients. Furthermore, the invention relates to a method for treating a patientA method of treatment of BM comprising administering to a patient an effective amount of a nonimmunosuppressive cyclosporin A derivative of formula I, more preferably a nonimmunosuppressive cyclosporin A derivative of formula II, and most preferably a nonimmunosuppressive cyclosporin A derivative of formula III [ D-MeAla ™]³-[EtVal]⁴-CsA. Furthermore, the invention relates to the use of a non-immunosuppressive cyclosporin a derivative of formula I, II or III for the manufacture of a medicament for the treatment of BM. Finally, the present invention relates to a pharmaceutical composition for the treatment of BM comprising an effective amount of a non-immunosuppressive cyclosporin a derivative of formula I, II or III, a pharmaceutically acceptable carrier and, optionally, excipients and diluents.

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

The invention is further illustrated by the following examples. The examples are provided for illustration to a person skilled in the art and are not intended to limit the scope of the invention as described in the claims. Accordingly, the present invention should not be construed as limited to the embodiments provided, but rather should be construed to include any and all variations which become apparent as the teachings herein are provided.

Examples

Example 1: cyclosporin A and nonimmunosuppressive cyclosporin A derivatives [ D-MeAla]³-[EtVal]⁴Effect of CsA on mitochondrial TMRM fluorescence (measure of mitochondrial membrane potential) and incidence of apoptosis in myoblast cultures from UCMD patients.

FIGS. 1A and B show mitochondrial membrane potential as a function of time in myoblasts from UCMD patients l (A) and 4 (B). Mitochondrial membrane potential was determined from the accumulation of tetramethylrhodamine methyl ester (TMRM). Myoblasts were seeded on 24mm round glass coverslips and grown in DMEM supplemented with 20% FCS for two days. Thus, the extent of mitochondria and cells loaded with potentiometric probes is affected by the activity of the plasma membrane multidrug resistance pump, which is inhibited by cyclosporine a. Thus treatment with cyclosporin A may cause an increase in mitochondrial fluorescence, which may be mistakedMisinterpretation is an increase in mitochondrial membrane potential. To avoid such artifacts and standardize loading conditions, experiments were performed in the presence of 1.6 μ M cyclosporin H, which inhibits the multidrug resistance pump but does not affect PTP. For mitochondrial membrane potential measurements, cells were rinsed once and then incubated for 30 minutes in serum-free DMEM supplemented with 1.6. mu.M cyclosporine H and loaded with 10nM TMRM. At the end of each experiment, mitochondria were completely depolarized by the addition of 4 μ M of the proton carrier carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP). The cell fluorescence images were obtained with an Olympusix71/IX51 inverted microscope equipped with a xenon light source (75W) for epi-illumination and a 12-bit digitally cooled CCD camera (Micromax, Princeton Instruments). For fluorescence detection, a 568+/-25nm band pass excitation and 585nm long pass emission filter setting was used. Images were collected with an exposure time of 100 milliseconds using a 40X, 1.3NA oil immersion objective (Nikon). Data were collected and analyzed using Cell R software (Olympus). A cluster consisting of several (10-30) mitochondria was identified as a target region, and a visual field containing no cells was used as a background. Successive digital images were obtained every 2 minutes and the mean fluorescence intensity was recorded and saved for later analysis. In the experiments shown in panels A and B, the cells on the slide were loaded into TMRM as discussed, at the positions indicated by the arrows, without further treatment (open symbols) or with 1.6. mu.M cyclosporin A (filled squares) or 1.6. mu.M [ D-MeAla ]]³-[EtVal]⁴After 30 min of CsA (filled triangles), 6. mu.M oligomycin (oligo) and 4. mu.M FCCP were added. C of FIG. 1: without further treatment (basal) or with 1.6. mu.M cyclosporin A or 1.6. mu.M [ D-MeAla ]]³-[EtVal]⁴After 2 hours of CsA treatment incubation, myoblasts from patients 1(P1) and 4(P4) were incubated on plastic dishes and the presence of TUNEL-positive nuclei was counted. Data are mean ± s.d of at least four independent experiments. (. x), P < 0.01 compared to the base condition. Apoptosis was assessed as described previously.

The results reported in figure 1 demonstrate that in preventing oligomycin-dependent mitochondrial depolarization in cells from UCMD patients and restoring the incidence of apoptosis to the levels shown by cells from healthy donorsImmunosuppressive cyclosporin A derivatives [ D-MeAla]³-[EtVal]⁴CsA is as effective as cyclosporin A.

Example 2: [ D-MeAla ]]³-[EtVal]⁴-synthesis of CsA

(translated from the doctor's paper entitled "De noveaux analogies decyosporin A corporate agent anti-VIH-1" by Jean Francois Guichou, Faculte des Sciences, University of Lausane, CH-1015 Lausane, Switzerland (2001)).

Example a:

synthesis of H-MeLeu-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt (Oac) -Abu-Sar-OMe:

to a solution of cyclosporin A (CsA) (8.3 mmol; 10g) in 100ml of acetic anhydride was added 4-Dimethylaminopyridine (DMAP) (41.5 mmol; 5.8 g). The solution was stirred at room temperature for 18 hours. The reaction mixture was then diluted with 600ml of ethyl acetate and washed twice with water and four times with saturated aqueous sodium bicarbonate solution. Passing the organic phase over anhydrous Na₂SO₄Drying, filtering and evaporating the solvent under reduced pressure. The yellow residue obtained is chromatographed on silica gel (eluent: 98: 2 dichloromethane/methanol) and recrystallized from ether. 9.5g MeBmt (OAc) -CsA were recovered as a white powder with a yield of 92%.

To a solution of MeBmt (OAc) -Cs (7.5 mmol; 9.4g) in 60ml of dichloromethane was added trimethyloxonium tetrafluoroborate (22.5 mmol; 3.3 g). After 16 hours at room temperature, 35ml of a 0.26M solution of sodium methoxide in methanol was added. After 1 hour, 35ml of methanol and 35ml of 2N sulfuric acid are added and the reaction mixture is stirred for a further 15 minutes with saturated KHCO₃Neutralized to pH 6.0 (28ml) and extracted twice with ethyl acetate. The organic phase was washed twice with saturated NaCl and over anhydrous Na₂SO₄Dried and filtered. Then, the solvent was evaporated under reduced pressure. The residue is chromatographed on silica gel (eluent: 5: 1 ethyl acetate/methanol). 7.3g of H-MeLeu-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt (OAc) -Abu-Sar-OMe were obtained (yield:76％)。

HPLC tr＝268.23mn(98％)

ES/MS：m/z：1277.5[M+H⁺]，639.2[M+2H⁺]

example B:

synthesis of H-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt (OAc) -Abu-Sar-OMe:

to the direction of

DMAP (2.3 mmol; 334mg) and phenyl isothiocyanate (6.9 mmol; 0.75ml) were added to a 48ml tetrahydrofuran solution of H-MeLeu-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt (OAc) -Abu-Sar-OMe (4.6 mmol; 7 g). After 2 hours, the solvent was evaporated and the crude product was chromatographed on silica gel (eluent: 9: 1 tert-butyl methyl ether (MTBE)/ethyl acetate (1); 9: 1 MTBE/methanol (2)). 5.8g of Ph-NH-C (S) -MeLeu-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt (OAc) -Abu-Sar-OMe (90% yield) were obtained.

To a solution of the latter compound (4 mmol; 5.6g) in 290ml of dichloromethane was added 13.8ml of trifluoroacetic acid. After 1 hour of reaction, KHCO was used₃Neutralized and diluted with 500ml of dichloromethane. The organic phase was washed twice with saturated NaCl and over anhydrous Na₂SO₄Dried and filtered. Then, the solvent was evaporated under reduced pressure. The residue is chromatographed on silica gel (eluent: 9: 1 MTBE/ethyl acetate (1); 3: 1 MTBE/methanol (2)). 2.8g of H-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt (OAc) -Abu-Sar-OMe (61% yield) were obtained.

HPLC tr＝25.80mn(99％)

ES/MS：m/z：1050.5[M+H⁺]，547.7[M+2H⁺]

Example C:

Boc-D-MeAla-EtVal-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt(OAc)-Abu-NMe-CH₂-CH₂-synthesis of OH:

in the inert stateTo a solution of H-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt (OAc) -Abu-Sar-OMe (0.87 mmol; 1.00g), DIPEA (2.78 mmol; 0.48ml) and Boc-D-MeAla-EtVal-OH (0.96 mmol; 0.32g) in 15ml dichloromethane was added fluorine-N, N, N' -tetramethylformamidine hexafluorophosphate (TFFH) (0.96 mmol; 0.25g) under an atmosphere. After 15 minutes, the dichloromethane was evaporated and the residue was dissolved in ethyl acetate. With saturated NaHCO₃The organic phase was washed with the solution, 10% citric acid solution and saturated NaCl solution, then over anhydrous Na₂SO₄Dried and concentrated. Purification by chromatography on silica gel (eluent: 98: 2 ethyl acetate/methanol) gave 1.14g (90%) Boc-D-MeAla-EtVal-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt (OAc) -Abu-Sar-OMe.

The latter product (0.64 mmol; 0.93g) was dissolved in 45ml of anhydrous methanol and added with small amounts of sodium borohydride (25.5 mmol; 0.96g) every 15 minutes over 3 hours and 30 minutes. At 4 hours, the reaction mixture was cooled to 0 ℃, hydrolyzed by the addition of 10% citric acid and concentrated. The residue was dissolved in ethyl acetate. The organic phase was washed with 10% citric acid solution and saturated NaCl solution over anhydrous Na₂SO₄Dried and concentrated. After purification by chromatography on silica gel (eluent: 95: 5 ethyl acetate/methanol), 0.63g (81%) Boc-D-MeAla-EtVal-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt (OAc) -Abu-NMe-CH were obtained₂-CH₂-OH。

ES/MS：m/z：1434.9[M+H⁺]，717.9[M+2H⁺]

Example D:

synthesis of H-D-MeAla-EtVal-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt-Abu-OH:

to Boc-D-Me Ala-EtVal-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt (OAc) -Abu-NMe-CH₂-CH₂A solution of-OH (0.425 mmol; 610mg) in 42.5ml of methanone was added to methanesulfonic acid (3.18 mmol; 2.060ml), and the mixture was heated to 50 ℃ and maintained at this temperature. The progress of the reaction was monitored by HPLC and mass spectrometry. After 80 hours, the mixture isThe mixture was cooled to 0 ℃ and purified by addition of 1M NaHCO₃And is hydrolyzed. The methanol was removed and the residue was dissolved in ethyl acetate. First with 1M NaHCO₃The solution was then washed with saturated NaCl solution and the organic phase was passed over anhydrous Na₂SO₄Dried and concentrated. Product (557mg), H-D-MeAla-EtVal-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt (OAc) -Abu-O-CH₂-CH₂-NHMe for the next step without purification.

The product (0.42 mmol; 557mg) is dissolved in 20ml of methanol and combined under an inert atmosphere with a solution of sodium methoxide (1.26 mmol) in 1.26ml of methanol. After 18 hours at room temperature, the reaction mixture was cooled to 0 ℃ and 5ml of an aqueous solution of sodium hydroxide (4.2 mmol; 168mg) were added dropwise. After 21 hours at room temperature, the reaction was again cooled to 0 ℃ and quenched with 1M KHSO₄And (4) neutralizing. The methanol was removed and the residue was dissolved in ethyl acetate. The organic phase was washed with half-saturated NaCl solution and passed over anhydrous Na₂SO₄Dried and concentrated. The product (335 mg; 64%), H-D-MeAla-EtVal-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt-Abu-OH was used in the next step without purification.

HPLC tr＝26.27mn(86％)

ES/MS：m/z：1235.5[M+H⁺]，618.2[M+2H⁺]

Example E: [ D-MeAla ]]³-[EtVal]⁴-synthesis of CsA:

to a solution of (7-azaphentole-1-acyloxy) tripyrrolidinophosphonium hexafluorophosphate (PyAOP, 0.486 mmol; 254mg) in 3.2 l dichloromethane was added dropwise a solution of H-D-MeAla-EtVal-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeBmt-Abu-OH (0.162 mmol; 200mg) and sym collidine (1.78 mmol; 0.24ml) in 50ml dichloromethane under an inert atmosphere. After 72 hours, by adding 10% Na₂CO₃The solution hydrolyzes the reaction mixture. The dichloromethane was evaporated and the residue was dissolved in ethyl acetate. The organic phase was washed with 0.1N HCl solution followed by saturated NaCl solution over anhydrous Na₂SO₄Dried and concentrated. The crude product was purified on silica gel to give 110mg (59%) [ D-MeAla]³[EtVal]⁴-CsA。

HPLC tr＝30.54mn(100％)

ES/MS：m/z：1217.6[M+H⁺]，609.3[M+2H⁺]

Example 3: [ D-MeAla ]]³-[EtVal]⁴-oral formulations of CsA

Amounts are expressed in weight percent (w% w).

Example a:

[D-MeAla]³-[EtVal]⁴-CsA 10

glycogen (Glycofurol) 7535.95

Medium chain triglyceride (Miglycol) 81218

Polyoxyethylene ether (40) hydrogenated Castor oil 35.95

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

glycogen 7539.95

Medium chain triglyceride 81214

Polyoxyethylene ether (40) hydrogenated castor oil 36

Butyl Hydroxy Anisole (BHA) 0.05-0.1

Example D:

[D-MeAla]³-[EtVal]⁴-CsA 10

tetraethylene glycol 10

Caprylic/capric triglyceride 5

Polyoxyethylene ether (40) hydrogenated castor oil 74.9

Alpha-tocopherol 0.1

Example E:

[D-MeAla]³-[EtVal]⁴-CsA 10

ethanol 9

Propylene glycol 8

Polyoxyethylene ether (40) hydrogenated Castor oil 41

Glycerol monolinoleate 32

See british patent application 2,222,770 for each component of formulations a-D and the method of preparation.

Example 4: collagen VI knockout mice [ D-MeAla]³-[EtVal]⁴Therapeutic Effect of CsA

For a group of four collagen VI knock-out mice (Col6a 1)^-/-(ii) a 5-6 months old; sex balance) twice daily intraperitoneal administration for 5 days5mg/kg of [ D-MeAla ]]³-[EtVal]⁴-CsA. Four animals of the control group were similarly treated with the placebo formulation. After slaughtering the animals, blood samples were collected from each animal, then liver was isolated and muscle was isolated from hind legs, processed to prepare mitochondria for Calcium Retention (CRC) assay. Meanwhile, diaphragm and flexor brevis muscles were isolated and processed for histopathology and in situ mitochondrial function assessment (mitochondrial transmembrane potential). All in vivo experiments were approved by the appropriate institution and performed according to the guidelines of the institution.

Method

Preparation of mitochondria and CRC detection

As described in Fontaine et al 1998.j.biol. chem.273: 25734-: 335 and 345 mitochondria were prepared from liver and muscle homogenates by differential centrifugation. In Ca²⁺The CRC of mitochondrial preparations was assessed using a fluorescence spectrophotometer equipped with a magnetic stirrer and a temperature control device in the presence of the indicator Calcium Green-5N (molecular probes; excitation: 505 nm; emission: 535 nm). The medium contained 0.25M sucrose, 10mM Tris-MOPS, 5mM glutamic acid, 2.5mM malic acid, 1 (liver) or 10 (muscle) mM Pi-Tris, 20. mu.M Calcium Green-5N (pH 7.4).

Isolation and culture of skeletal muscle fibers and TMRM detection

Such as Irwin et al 2003.nat. Genet.35: 267-271. Coating intact muscle fibers on laminin (3. mu.g/cm)²) And cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. For this experiment, FDB fibers were placed in 1ml of Tyrode buffer and loaded with 20nM TMRM (molecular probes) as described by Irwin et al. Images of mitochondrial TMRM fluorescence were obtained using an Olympus IX71/IX51 inverted microscope.

TUNEL detection

After fixation in 4% paraformaldehyde and paraffin embedding, 7 μm sections of the muscle diaphragm were prepared. TUNEL was performed using the ApopTag in situ apoptosis detection kit (Intergen). The samples were stained with peroxidase/diaminobenzidine, and TUNEL positive nuclei were detected and counterstained with Hoechst 33258(Sigma), all nuclei labeled as described by Irwin et al. The total number of nuclei and the number of TUNEL positive nuclei in randomly selected fibers were determined using a Zeiss Axioplan microscope fitted with a Leica DC 500 camera.

Results

[D-Me Ala] ³ -[EtVal] ⁴ Ex vivo Effect of CsA on Permeability Transition Pore (PTP)

Monitoring mitochondrial in vitro Ca²⁺In the presence of Calcium Green-5N, was incubated with [ D-MeAla ] isolated from]³-[EtVal]⁴CsA or Col6a1 treated with placebo^-/-Competent mitochondria of the liver and hind leg muscles of mice. Then Ca was applied²⁺A series of pulses, each pulse added until a threshold concentration is reached that causes the PTP to open. This procedure allows a detailed analysis of the effect of PTP sensitizers and desensitizers (Fontaine et al 1998.). The results show that [ D-MeAla ] is used]³-[EtVal]⁴PTP vs Ca was present in both liver and muscle mitochondria in CsA-treated mice²⁺And Pi desensitization, showing Ca required for PTP opening²The threshold is increased (fig. 2). This effect is particularly pronounced for muscle mitochondria where CRC approaches the addition of [ D-MeAla directly to the medium]³-[EtVal]⁴Maximum achievable levels of CsA. See BII, lines d and d'. From four pairs of [ D-MeAla ]]³-[EtVal]⁴The analysis of mitochondria from CsA-treated (mice 24-27) and four placebo-treated mice (mice 28-31) gave a summary of the results shown in figure 3.

[D-MeAla] ³ -[EtVal] ⁴ Effect of CsA on mitochondrial transmembrane potential in FDB fibers

Mitochondrial transmembrane potential is assessed by changes in mitochondrial fluorescence of tetramethylrhodamine methyl ester (TMRM), a fluorescent probe that accumulates in polarized mitochondria and is released when the transmembrane potential decreases. It has been shown that oligomycin, an inhibitor of mitochondrial F1F0-ATP synthase, can show Col6a1^-/-Mitochondrial dysfunction in muscle fibers. Treatment of Col6a1 to a treatment isolated from placebo^-/-The fibers of the mice had oligomycin added and the TMRM fluorescence in most of the fibers produced the expected decrease (FIG. 4A). In vivo use of [ D-MeAla]³-[EtVal]⁴CsA treatment significantly reduced the number of fibers on which mitochondrial depolarization was observed (fig. 4B). Table 1 summarizes the use of placebo or [ D-MeAla respectively]³-[EtVal]⁴Results for each individual mouse of CsA treatment.

Table 1: depolarized fibers after oligomycin addition

Group of	Mouse #	Number of depolarized fibers after addition of oligomycin	Total number of fibers
				2 (placebo)	28	3	6
	29	6	8
					30	4	8
	31	5	9
				1([D-MeAla]3-[EtVal]4-CsA)	24	4	12
	25	2	8
					26	4	10
	27	0	10

[D-MeAla]³-[EtVal]⁴Effect of CsA on diaphragm apoptosis

Will be from [ D-MeAla ]]³-[EtVal]⁴CsA (#24-27) or placebo (#28-31) -treated Co16a1^-/-The diaphragm of each mouse was sectioned. Twenty to thirty sections of each animal were examined by TUNEL detection. From mice treated with [ D-MeAla ] in comparison with placebo]³-[EtVal]⁴Diaphragm fibers of CsA-treated mice showed a significant reduction in apoptotic nuclei (fig. 5). With [ D-MeAla ]]³-[EtVal]⁴In vivo CsA treatment was as effective as CsA.

Ultrastructural evaluation

Will come from using [ D-MeAla]³-[EtVal]⁴CsA (#24-27) or placebo (#28-31) treated Col6a1^-/-The diaphragm muscle samples of the mice were gently elongated on dental wax supports to prevent contraction during fixation. For fixation, the samples were incubated in 0.1M phosphate buffer (pH7.4) containing 2.5% glutaraldehyde for 3 hours at 4 ℃, washed overnight in 0.15M phosphate buffer, postfixed in 1% osmium tetroxide solution in barbiturate, dehydrated and embedded in Epon 812 epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and observed under a Philips EM 400 transmission electron microscope operating at 100 kv. For the purpose of the statistical analysis, 300 muscle fibers from three different pieces of each sample tissue were studied (fig. 6). [ D-MeAla ] in comparison with control mice (#28-31)]³-[EtVal]⁴Altered numbers of fibers of mitochondria showing swelling or electrically dense inclusions in CsA-treated mice (#24-27) were significantly reduced.

For patients containing [ D-MeAla ]]³-[EtVal]⁴-a formulation of CsA or a placebo formulation of Col6a1 for oral treatment^-/-Mice were subjected to similar experiments. Observed from [ D-MeAla]³-[EtVal]⁴-CThe significant increase in CRC in the muscle mitochondria of the animals treated with sA indicates that cyclosporin causes PTP depolarization. In addition, TUNEL detection on diaphragm muscle sections is illustrated by [ D-MeAla ]]³-[EtVal]⁴In vivo treatment of CsA can cause a significant reduction in the rate of apoptosis.

Example 5: [ D-MeAla ]]³-[EtVal]⁴Preliminary clinical trials of CsA on UCMD and BM patients

Five patients were enrolled, four of them UCMD patients, and one of them BM patients. The patient had a mutation in COL6a1, COL6a2 or COL6A3 genes, respectively. Experiments were designed to test whether CsA treatment was effective in reducing mitochondrial dysfunction, as assessed by oligomycin causing mitochondrial depolarization in muscle fibers. Depolarization is detected as a change in TMRM fluorescence. The patient was treated with an oral dose of 5mg/kg CsA daily for thirty days. Muscle biopsies were taken before and immediately after treatment and processed for TMRM detection. The average results of five patients showed that oligomycin induced 90% muscle cell polarization before treatment, but only 37% after treatment. Thus, short-term treatment with CsA can substantially reduce mitochondrial dysfunction as measured by oligomycin-induced polarization.

Claims (13)

1. Use of a nonimmunosuppressive cyclosporin a derivative of formula I for reducing mitochondrial dysfunction and apoptosis rate in cells of patients with Ullrich congenital muscular dystrophy or Bethlem myopathy.

2. The use of claim 1, wherein the nonimmunosuppressive cyclosporin A derivative is a cyclosporin derivative of formula II.

3. The use of claim 1, whereinThe cyclosporin A derivative is non-immunosuppressive cyclosporin [ D-MeAla ] of formula III]³-[EtVal]⁴-CsA。

4. Use of a nonimmunosuppressive cyclosporin a derivative of formula I in the manufacture of a medicament intended for the treatment of Ullrich congenital muscular dystrophy or Bethlem myopathy.

5. The use of claim 4, wherein the nonimmunosuppressive cyclosporin A derivative is a cyclosporin derivative of formula II.

6. The use of claim 4, wherein the nonimmunosuppressive cyclosporin A derivative is the nonimmunosuppressive cyclosporin [ D-MeAla ] of formula III]³[EtVal]⁴-CsA。

7. A method of treating a patient with Ullrich congenital muscular dystrophy or Bethlem myopathy comprising administering to the patient an effective amount of a nonimmunosuppressive cyclosporin a derivative of formula I.

8. The method of claim 7, wherein the nonimmunosuppressive cyclosporin A derivative is a cyclosporin derivative of formula II.

9. The use of claim 7, wherein the nonimmunosuppressive cyclosporin A derivative is the nonimmunosuppressive cyclosporin [ D-MeAla ] of formula III]³[EtVal]⁴-CsA。

10. A pharmaceutical composition for treating Ullrich congenital muscular dystrophy or Bethlem myopathy comprising an effective amount of a nonimmunosuppressive cyclosporin a derivative of formula I, a pharmaceutically acceptable carrier and, optionally, excipients and diluents.

11. The pharmaceutical composition of claim 10, wherein the nonimmunosuppressive cyclosporin a derivative is a cyclosporin derivative of formula II.

12. The pharmaceutical composition of claim 10, wherein the nonimmunosuppressive cyclosporin A derivative is the nonimmunosuppressive cyclosporin [ D-MeAla ] of formula III]³[EtVal]⁴-CsA。

13. The pharmaceutical composition of any one of claims 10-12, further comprising an additional active ingredient.