[2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl]ethanoate for use in lymphangioleiomyomatosis and other diseases

Abstract

The present invention relates to the use of the compound [2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl]ethanoate in a method of treatment or prophylaxis of vascular and respiratory disease, fibrotic disease and neurodegenerative disease, particularly interstitial pneumonia, tuberous sclerosis or lymphangioleiomyomatosis, collagen disease, interstitial lung disease, human kidney disease, nephritic syndrome, liver fibrosis or liver cirrhosis, Alzheimer's disease or Parkinson's disease.

Claims

1. A method of treatment of lymphangioleiomyomatosis comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising [2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl]ethanoate.

2. The method of claim 1, wherein the composition is formulated for intravenous administration or oral administration or inhalation.

3. The method of claim 1, wherein the composition further comprises N,N-dimethylimidodicarbonimidic diamide.

4. The method of claim 3, wherein the composition is formulated for intravenous administration or oral administration or inhalation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows ELISA and Western blotting on MEF cells using phospho-specific antibodies which detect both the human and the mouse forms of rpS6. In 3 independent experiments, performed on disease relevant assay system of MEF cells, the compound of the invention showed dose-dependent inhibition of the phosphorylation of the ribosomal protein S6.

(2) FIG. 2 shows ELISA and Western blotting on 621-101 cells. In 3 independent experiments, performed on disease relevant assay system of 621-101 cells, the compound of the invention showed dose-dependent inhibition of the phosphorylation of the ribosomal protein S6.

EXAMPLES

(3) The compound [2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl]ethanoate was tested for activity using the assays described in Examples.

(4) These assays are used to test the effect of a compound on the activity of the mTOR pathway. Phosphorylation of ribosomal protein rp60 is used as read-out for mTOR pathway activity. Deregulation/overactivation of the mTOR pathway has been shown to be the underlying mechanism of tuberous sclerosis and lymphangioleiomyomatosis. Thus, these assays mimic a disease causative mechanism.

(5) The tested compounds are commercially available.

Example 1 (FIG. 1)

(6) MEFs are mouse embryo fibroblasts derived from TSC2 knockout (MEF110) and TSC2+ (MEF157) mice. Tsc2 mutations are known to cause tuberous sclerosis and lymphangioleiomyomatosis in humans.

(7) All cells were maintained in DMEM tissue culture medium, with the addition of fetal calf serum to 10% to support cell proliferation, at 37° C. and 5% CO.sub.2.

(8) In MEFs, ribosomal protein S6, which is a component of the 40S ribosome, and phosphorylated at Ser236 by p70 S6 kinase can be assayed by ELISA and Western blotting, using phospho-specific antibodies which detect both the human and the mouse forms of rpS6. This assay is predicitve of compounds with protective or therapetic activities for respiratory and vascular diseases, fibrotic diseases, or neurodegenerative diseases.

(9) Cells were grown to 60-80% confluence in DMEM+10% serum. Medium was replaced with fresh serum free medium two hours before addition of test compound of the invention.

(10) Compound was added as a 1:1000 dilution of the stock solution in DMSO. DMSO was added as a vehicle control, and samples were incubated for a further 60 minutes.

(11) For ELISA, cells were harvested into 200 ul cell lysis buffer containing both protease and phosphatase inhibitors, to protect the phosphorylation status of rpS6. Lysates were stored at −80° C. prior to use. 50 ul of lysate per sample was used for the ELISA, which was carried out according to manufacturers' instructions. For Western blotting, cells were harvested into 200 ul cell lysis buffer containing both protease and phosphatase inhibitors and resolved by SDS-PAGE before transfer to PVDF membrane and incubation with anti-phospho-rpS6 and anti-total-rpS6 antibodies.

(12) The compound was well tolerated by the cells.

(13) ELISA data are normalised to the vehicle control, data from separate experiments are shown as individual bars. Western blots are included to verify that loss of rpS6 phosphorylation is not a consequence of loss of rpS6 protein.

(14) In 3 independent experiments, performed on disease relevant assay system of MEF cells, the compound of the invention showed dose-dependent inhibition of the phosphorylation of the ribosomal protein S6.

Example 2 (FIG. 2)

(15) 621-101 cells are human cells derived from an angiomyolipoma. They have verified TSC2 mutations and are the only disease-derived mutation-bearing cells used that are mimicking the disease Lymphangioleiomyomitosis.

(16) Cells were grown to 60-80% confluence in DMEM+10% serum. Medium was replaced with fresh serum free medium two hours before addition of test compound of the invention.

(17) Compound was added as a 1:1000 dilution of the stock solution in DMSO. DMSO was added as a vehicle control, and samples were incubated for a further 60 minutes.

(18) For ELISA, cells were harvested into 200 ul cell lysis buffer containing both protease and phosphatase inhibitors, to protect the phosphorylation status of rpS6. Lysates were stored at −80° C. prior to use. 50 ul of lysate per sample was used for the ELISA, which was carried out according to manufacturers' instructions. For Western blotting, cells were harvested into 200 ul cell lysis buffer containing both protease and phosphatase inhibitors and resolved by SDS-PAGE before transfer to PVDF membrane and incubation with anti-phospho-rpS6 and anti-total-rpS6 antibodies.

(19) The compound was well tolerated by the cells.

(20) In 3 independent experiments, performed on disease relevant assay system of 621-101 cells, the compound of the invention showed dose-dependent inhibition of the phosphorylation of the ribosomal protein S6