FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
THE PROVISIONAL SPECIFICATION
(See section 10)
"P38 Inhibitor".
CADILA PHARMACEUTICALS LTD., "CADILA CORPORATE CAMPUS", SARKHEJ-DHOLKA ROAD, BHAT, AHMEDABAD, 382210, GUJARAT, INDIA, AN INDIAN COMPANY.
THE FOLLOWING SPECIFICATION DESCRIBES AND ASCERTAINS THE NATURE OF THIS INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED.

TITLE : P38 Inhibitor
FIELD OF THE INVENTION
This invention relates to a novel p38 inhibitors, processes for the preparation thereof, the use thereof in treating CSBP/p38 kinase mediated diseases and pharmaceutical compositions for use in such therapy.
BACKGROUND OF THE INVENTION
Mitogen-activated protein kinases (MAP) is a family of pro line-directed serine/threonine kinases that activate their substrates by dual phosphorylation. The kinases are activated by a variety of signals including nutritional and osmotic stress, UV light, growth factors, endotoxin and inflammatory cytokines.
One particularly interesting MAPK is p38, also known as cytokine suppressive anti
inflammatory drug binding protein (CSBP) and RK. The p38 kinases are responsible for
phosphorylating and activating transcription factors as well as other kinases, and are
themselves activated by physical chemical and radiation stress like
osmotic,anisomysin,UV etc, pro-inflammatory cytokines like II-1 and TNF and bacterial lipopolysaccharide.
More importantly, the products of the p38 phosphorylation have been shown to mediate the production of inflammatory cytokines, including TNF, IL-l, IL-6 and cyclooxygenase-2.
Each of these cytokines has been implicated in numerous disease states and conditions. For example, TNF alpha is a cytokine produced primarily by activated monocytes and macrophages. Its excessive or unregulated production has been implicated as playing a causative role in the pathogenesis of rheumatoid arthritis. TNF has also been implicated in viral infections, such as HIV, influenza virus, and herpes virus including herpes simplex virus type-1 (HSV-1), herpes simplex virus type-2 (HSV-2), cytomegalovirus (CMV), varicella-zoster virus (VZV), Epstein-Barr virus, human herpes virus-6 (HHV-6), human herpes virus-7 (HHV-7), human herpes virus-8 (HHV-8), pseudorabies and rhinotracheitis, among others.
Inhibition of TNF production has been shown to have broad application in the treatment of inflammation, inflammatory bowel disease, Alzheimer's disease, Crohn's disease, multiple sclerosis and asthma, chronic pulmonary inflammatory disease, silicosis, sarcoidosis, adult respiratory distress syndrome,cachexia,keloid,scar formation,ulcerative colitis etc.
In addition, MAPKs, such as p38, have been implicated in cancer, thrombin-induced platelet aggregation, immunodeficiency disorders, autoimmune diseases, cell death, allergies, osteoporosis and neurodegenerative disorders.

Inhibition of p38 kinase leads to a blockade on the production of both IL-1 and TNF. IL-1 and TNF stimulate the production of other proinflammatory cytokines such as IL-6 and IL-8 and have been implicated in acute and chronic inflammatory diseases and in postmenopausal osteoporosis.
Inhibitors of p38 have also been implicated in the area of pain management through inhibition of prostaglandin endoperoxide synthase-2 induction..
Following patents, patent applications describe p38 inhibitorsand its uses,

Patent/Publication No. Title
US7186737B2 Inhibitors of p38
US7169779B2 Inhibitors of p38
US7151010B2 Methods for assembling a stack package for high density integrated circuits
US6635644B2 Inhibitors of p38
US6632945B2 . Inhibitors of P38
US6608060B1 Inhibitors of p38
US6528508B2 Inhibitors of p38
US6509363B2 Heterocyclic inhibitors of p38
US6162613A Methods for designing inhibitors of serine/threonine-kinases and tyrosine kinases
US6147080A Inhibitors of p38
US6093742A Inhibitors of p38
US6800626B2 Inhibitors of p38
WO 1996021654A1 NOVEL COMPOUNDS
WO1999000357A1 INHIBITORS OF p38
WO1999058502A1 HETEROCYCLIC INHIBITORS OF P38
WO 1999064400 A1 INHIBITORS OF p3 8
WO2000017175A1 INHIBITORS OF p38
WO2000017204A1 INHIBITORS OF p38
US 6,949,560 B2 IMIDAZO-SUBSTITUTED COMPOUNDS AS P38 KINASE INHIBITORS
US 6852740 Pyrazole derivatives as p38 kinase inhibitors
US 6,982,270 Bl 3,4-DIHYDRO-(lH)QUINAZOLIN-2-ONE COMPOUNDS AS CSBP/P38 KINASE INHIBITORS
US 6630485 P 38 MAP KINASE INHIBITOR
US 7,189,400 B2 METHODS OF TREATMENT WITH ANTAGONISTS OF MU-1
US 7,115,557 B2 USE OF CERTAIN DRUGS FOR TREATING NERVE ROOT INJURY
US 7,078,431 B2 l,3-BIS-(SUBSTITUTED-PHENYL)-2-PROPEN-l-ONES AND THEIR USE TO TREAT VCAM-1 MEDIATED DISORDERS
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US 6759410 US 6696471 3,4-DIHYDRO-(lH)-QUINAZ0LIN-2ONES AND THEIR USE AS CSBP/P38 KINASE INHIBITORS AMINOPYRROLE COMPOUNDS .
US 6696443 PIPERIDINE/PIPERAZINE-TYPE INHIBITORS OF P38 KINASE
US 6649637 US 6638765 INHIBITION OF INTRACELLULAR REPLICATION BYPYRIDINYLIMIDAZOLESPLATFORM FOR THE DIFFERENTIATION OF CELLS
US 6509361 1,5-DIARYL SUBSTITUTED PYRAZOLES AS P38 KINASE INHIBITORS
US 6479507 P38 MAP KINASE INHIBITORS
US 6444696 PYRAZOLE DERIVATIVES P38 MAP KINASE INHIBITORS
US 6410540 INHIBITORS OF P38 ALPHA KINASE
US 6376527 PYRAZOLE DERIVATIVES P38 MAP KINASE INHIBITORS
US 6316466 PYRAZOLE DERIVATIVES P38 MAP KINASE INHIBITORSi
US 6316464 P38 MAP KINASE INHIBITORS
US 6096711 HSP 72 INDUCTION AND APPLICATIONS
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the invention the composition of a pharmaceutical composition the method of preparation, HPLC characteristic its safety and tolerability, methods of use and outcome of treatments are described in following examples. The following are illustrative examples of the present invention and scope of the present invention should not be limited by them.
Example" 1. The pharmaceutical compositions:
A. Each dose of 0.1 ml of therapeutic agent contains:
Mycobacterium w., (heat killed) 0.50 x 10
Sodium Chloride I. P 0.90% w/v
Tween80 0.1% w/v
Thiomerosal I. P. ... . 0.01% w/v
(As a Preservative)
Water for injection I. P. q. s. to 0.1ml
B. Each dose of 0.1 ml of therapeutic agent contains:
Mycobacterium w., (heat killed) 0.50 x 109
Sodium Chloride I. P 0.90% w/v
Triton x 100 0.1% w/v

Thiomerosal 1. P. ... . 0.01% w/v
(As a Preservative)
Water for injection I. P. q. s. to 0.1 ml
C. Each dose of 0.1 ml of therapeutic agent contains:
Mycobacterium w., (heat killed) 0.50 x 109
Sodium Chloride I. P 0.90% w/v
Thiomerosal I. P. ... . 0.01% w/v
(As a Preservative)
Water for injection I. P. q. s. to 0.1 ml
D. Each dose of 0.1 ml of therapeutic agent contains
Extract of Mycobacterium w after sonication from 1x10 Mycobacterium w
Sodium Chloride I. P 0.90% w/v
Thiomerosal I. P. ... . 0.01 % w/v
(As a Preservative)
Water for injection I. P. q. s. to 0.1 ml
E. Each dose of 0.1 ml of therapeutic agent contains
Methanol Extract of 1x10 Mycobacterium w
Sodium Chloride I. P 0.90% w/v
Thiomerosal I. P. ... . 0.01% w/v
(As a Preservative)
Water for injection I. P. q. s. to 0.1 ml
F. Each dose of 0.1 ml of therapeutic agent contains
Chloroform Extract of 1x10' Mycobacterium w
Sodium Chloride I. P 0.90% w/v
Thiomerosal I. P. ... . 0.01% w/v
(As a Preservative)
Water for injection I. P. q. s. to 0.1 ml
G. Each dose of 0.1 ml of therapeutic agent contains
Acetone Extract of 1x10 Mycobacterium w
Sodium Chloride I. P 0.90% w/v
Thiomerosal I. P. ... . 0.01% w/v
(As a Preservative)
Water for injection I. P. q. s. to 0.1 ml
H. Each dose of 0.1 ml of therapeutic agent contain
Ethanol Extract of lxlO10 Mycobacterium w

Sodium Chloride I. P 0.90% w/v
Thiomerosal 1. P. ... . 0.01% w/v
(As a Preservative)
Water for injection I. P. q. s. to 0.1 ml
I. Each dose of 0.1 ml of therapeutic agent contains
Liticase Extract of 1 x 10 Mycobacterium w
Sodium Chloride I. P 0.90% w/v
Thiomerosal I. P. ... . 0.01% w/v
(As a Preservative)
Water for injection I. P. q. s. to 0.1 ml
J. Each dose of 0.1 ml of therapeutic agent contains
Mycobacterium w (heat killed) 0.5x10
Extract of mycobacterium w obtained lxlO3 Mycobacterium w by disruption, solvent
extraction or enzymatic extraction.
Sodium Chloride I. P 0.90% w/v
Thiomerosal I. P. ... . 0.01% w/v
(As a Preservative)
Water for injection I. P. q. s. to 0.1 ml
Example 2. The Process of preparing a pharmaceutical composition
A. Culturing of Mycobacterium w.
i) Preparation of calture medium.
Mycobacterium w is cultured on solid medium like L J medium or liquid medium
like middle brook medium or sauton's liquid medium.
For better yield middle brook medium is enriched. It can be preferably enriched
by addition of glucose, bactotryptone, and BSA. They are used in ratio of 20:30:2
preferably.
The enrichment medium is added to middle brook medium. It is done preferably
in ratio of 15:1 to 25:1 more preperably in ratio of 20:1.
ii) Bioreactor operation
a) Preparation of vessel:
The inner contact parts of the vessel (Joints, mechanical seals, o-ring/gasket grooves, etc.) should be properly cleaned to avoid any contamination. Fill up the vessel with 0.1 N NaOH and leave as such for 24 H to remove pyrogenic materials and other contaminants. The vessel is then cleaned first with acidified water, then wit ordinary water. Finally, the vessel is rinsed with distilled water (3 times) before preparing medium.
b) Sterilization of bioreactor
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The bioreactor containing 9L distilled water is sterilized with live' steam(indirecl). Similarly the bioreactor is sterilized once more with Middlebrook medium. The other addition bottles, inlet/outlet air filters etc. are autoclaved (twice) at 121°C for 15 minutes. Before use, these are dried at 50 C oven.
c) Environmental parameter
i. Tempralure:37±0.5°C
ii. pH : 6.7 to 6.8 initially.
B. Harvesting and concentrating
It is typically done at the end of 6th day after culturing under aseptic condition. The concentration of cells (palletisation) is done by centrifugation.
C. Washing of cells
The pallet so obtained is washed minimum three times with normal saline. It can be washed with any other fluid which is preferably isotonic.
D. Adding pharmaceutical^ acceptable carrier.
Pyrogen free normal saline is added to pallet. Any other pyrogen free isotonic fluid can be used as a pharmaceutical carrier. The carrier is added in amount so as get to desired concentration of active in final form.
E. Adding preservative
To keep the product free from other contaminating bacteria for its self life preservative is added. Preferred preservative is thiomesol which is used in final concentration of 0.01 %
w/v.
F. Terminal Sterilization
Terminal sterilization can done by various physical methods like application of heat or ionizing radiation or sterile filtration.
Heat can be in the form of dry heat or moist heat. It can also be in the form of boiling or pasturisation.
Ionizing radiation can be ultraviolet or gamma rays or mircrowave or any other form of ionizing radiation.
It is preferable to autoclave the final product.
This can be done before after filling in a final packaging.
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G. Quality Control
i. The material is evaluated for purity, sterility.
ii.The organisms are checked for acid fastness after gram staining.
iii.Inactivation test : This is done by culturing the product on L J medium to find out any living organism.
iv.Pathogenicity and/or contamination with pathogen.
The cultured organisms are infected to Balb/c mice. None of the mice should die and all should remain healthy and gain weight. There should not be any macroscopic or microscopic lesions seen in liver, lung spleen or any other organs when animals are killed upto 8 weeks following treatment.
v.Biochemical Test:
The organism is subjected to following biochemical tests:
a) Urease
b) Tween 80 hydrolysis .
c) Niacin test
d) Nitrate reduction test
The organism gives negative results in urease, tween 80 hydrolysis and niacin test. It is positive by nitrate reduction test.
H. Preparation of constituents of Mycobacterium w.
The constituents of Mycobacterium w can be prepared for the purpose of invention by:
I. Cell disruption
II. Solvent extration
III. Enzymatic extraction.
The cell disruption can be done by way of sonication or use of high pressure fractionometer or by application of osmotic pressure ingredient.
The solvent extraction can be done by any organic solvent like chloroform, ethanol, methanol, acetone, phenol, isopropyl alcohol, acetic acid, urea, hexane etc.
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The enzymatic extraction can be done by enzymes which can digest cell wall/membranes. They are typically proteolytic in nature. Enzyme liticase and pronase are the preferred enzymes. For the purpose of invention cell constituents of Mycobacterium w can be used alone in place of mycobacterium w organisms or it can be added to the product containing mycobacterium w.
Addition cell constituents results in improved efficacy of the product.'
Surprisingly it is found that Mycobacterium W is also antagonist to many Toll like
receptors.
Following are some of the examples describing the invention.
In vitro studies :
Splenocytes were harvested from normal mice. To 10 / ml of splenocytes
Mycobacterium w was added for stimulation. After 48 hours cells were removed and lysed. Using Elisa kit for R&D systems quantitative measurements were made for phosphortlaled JNK ERK-1,2 and P38 levels.
The findings suggest that Mycobacterium w reduces P38 level to all most half while there is no effect on JNK, ERK-1,2 (Fig. 1).
In vivo studies :
In a similar experiments splenocytes were harvested from mice on day 8 after injection with placebo (phosphoted buffer saline) or Mycobacterium w and phosphorylated JNK, ERK-1,2 and P38 were measured. Finding suggests that there is a significant reduction in P38 level (Fig. 2).
Thus Mw behaves as P38 inhibitor.
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