FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
THE PROVISIONAL SPECIFICATION
(See section 10)
POLY-TLR ANTAGONIST.
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 : POLY-TLR ANTAGONIST
The present invention provides poly-toll like receptor antagonist(TLR). The toll like receptors(TLR) are family of proteins. The innate immune system recognizes pathogens and initiates an effective and appropriate response through TLRs. TLRs are part of the larger IL-1R/TLR superfamily, which includes IL-IRs, IL-18Rs, and a group of orphan receptors. The family is defined by the presence of a cytoplasmic Toll-like—IL-1 resistance (TIR) domain, which is responsible for mediating downstream signaling. So far,
13 TLRs have been identified; TLRs 1-9 are common to mouse and human, whereas TLR10 is only functional in humans, and
TLRs 11, 12, and 13 have been found only in mice . Many but not all of these receptors have been assigned a role in the initial detection of, and response to, specific pathogen-associated
molecules (PAMs). In macrophages and neutrophils, this drives innate immune responses, such as inflammation and induction of microbicidal activity, whereas activation of TLRs expressed on dendritic cells leads to the initiation of adaptive immunity through induction of IL-12 and co-stimulatory molecules.
Though they are part of protective system of the bady, their over expression is associated with variety of diseases.
The diseases wherein one or more TLRs are over expressed or believed to be overexpressed include but not limited to following,
1.sepsis,
2.septicemia (including but not limited to endotoxemia),
endotoxemia resulting from gram negative bacteremia (with its accompanying symptoms of fever, generalized inflammation, disseminated intravascular coagulation, hypotension, acute renal failure, acute respiratory distress syndrome, adult respiratory distress syndrome (ARDS), hepatocellular destruction and/or cardiac failure)
3.various forms of septic shock (including but not limited to endotoxic shock).
4. localized or systemic inflammatory response to infection by different types of
organisms, including gram negative bacteria,
5. diseases related to translocation of gram negative bacteria or endotoxin from the gut.
6. systemic inflammatory response syndrome or SIRS (Bone, et al., Chest 1992; 101: 1644-55).
7. exacerbation of latent or active viral infections (e.g., infection with HIV,
cytomegaloviruses, herpes simplex, and influenza virus).

8. inborn or acquired predisposition to pulmonary bacterial infection.
9. Smoking
lO.Congestive heart failure with pulmonary edema. 11 .Chronic obstructive pulmonary disease
12. Bronchiectasis
13. Autoimmune diseases.
14. acute lung injuries due to smoke inhalation or heat exposure (e.g., thermal injury; inhalation of hot air or steam) ,Aspiration of gastric contents, Near-drowning ,inhalation of noxious substances
15. Trauma (e.g., chest trauma)
16. Cigarette smoking (emphysema; tendency towards bronchitis) 17.Intravenous substance abuse
18. Chronic exposure to polluted air
19.Seizure disorders (increased risk of aspiration, leading to, e.g., chemical injury by stomach acid)
20.Alcoholism
21. Intenstinal Ischemia and reperfusion
22. Renal failure/uremia 23.Hypotension and shock
24.Hepatic disease, including cirrhosis 25.Pancreatitis 26.Malnutrition
27. injuries
28. Viral pneumonias, including those caused by Myxovirus (e.g., influenza)
29. Intravascular infections (e.g., infective endocarditis) 30.crohn's disease

31. Ulcerative colitis
32.lupus
33. SLE
34. Autoimmune disease
35. Rheumatoid diseases
36. Burkitt lymphoma, follicular lymphoma,
37. multiple myeloma
38. Chronic hepatitis.
Some of these disorders are amenable to therapy while there is no definitive therapy for majority of the diseases.
Selected antibacterial, anti-inflammatory, and immunomodulating adjunctive therapies investigated in patients with severe sepsis and septic shock

Type of therapy Neutralisation of microbial toxins
Non-specific
anti-inflammatory
and
immunomodulating
drugs

Target (s) Endotoxin
Multiple
inflammatory and immune mediators

Agents
Anti-endotoxin antibodies,
anti-lipid A antibodies,
lipopolysaccharide analogues,
lipopolysaccharide removal
High dose corticosteroids, low dose corticosteroids, pentoxifylline, immunoglobulins, interferon gamma

Inhibition of specific Pro-inflammatory cytokines:
Thromboxane Dazoxiben, ketoconazole
Mediators

Tumour necrosis Factor
Interleukin-1 Phospholipid components: Phospholipase A2 Cyclo-oxygenase

Anti-tumour necrosis factor antibodies, soluble tumour necrosis factor receptors Interleukin-1 receptor antagonist
Phospholipase A2 inhibitor Ibuprofen

Platelet activating factor Platelet activating factor antagonists,
platelet activating factor
acetylhydrolase
Oxygen free radicals ^-acetylcysteine, selenium
Nitric oxide 7V-methyl-L-arginine
Bradykinin Bradykinin antagonist
Correction of Coagulation cascade Antithrombin III, tissue factor
coagulopathy pathway inhibitor, activated
protein C Inspite of all these morbidity associated with sepsis has not reduced.
Thus there is a need to provide better therapeutic options for such diseases.
Mycobacterium w is a non-pathogenic, cultivable, atypical mycobacterium, with
biochemical properties and fast growth characteristics resembling those belonging to
Runyons group IV class of Mycobacteria in its metabolic and growth properties but is not
indentical to those strains currently listed in this group. It is therefore thought that
(M.sub.w) is an entirely new strain. The species identity of M.sub.w has been defined by
polymerase chain reaction DNA sequence determination.
It has been found to share antigens with Mycobacterium leprae and Mycobacterium tuberculosis. It is found to provide prophylaxis against leprosy in humans by converting lepromin negative individuals to lepromin positivity. It is also found to provide prophylaxis against tuberculosis in animals. In leprosy it is also found to reduce duration of therapy for bacterial killing, clearance as well as clinical cure when used along with multi drug therapy.
Therapeutic agent which may be used in the present invention resembles Mw a nonpathogenic, cultivable, atypical mycobacterium, with biochemical properties and fast growth characteristics resembling those belonging to Runyons group IV class of Mycobacteria in its metabolic and growth properties but is not indentical to those strains currently listed in this group. It is therefore thought that (M.sub.w) is an entirely new strain.
The species identity of Mw has been defined by polymerase chain reaction DNA sequence determination and differentiated from thirty other species of mycobacteria. It however differs from those presently listed in this group in on respect or the other. By base sequence analysis of a polymorphic region of pattern analysis, it has been established that M.sub.w is a unique species distinct from many other known mycobacterial species examined which are: M. avium, M. intracellulare, M. scrofulaceum, M. kansasii, M. gastri, M. gordonae, M. shimoidei, M. malmoense, M. haemophilum, M. terrae, M. nonchromogenicum, M. triviale, M. marinum, M. flavescens, M. simian, M. szulgai, M. xenopi, M. asciaticum, M. aurum, M. smegmatis, M. vaccae, M. fortuitum subsp fortuitum, M. fortuitum subsp. Peregrinum, M. chelonae

subsp. Chelonae, M. chelonae subsp. Abscessus, M. genavense, M. tuberculosis, M. tuberculosis H.sub.37R.sub.v, M. paratuberculosis.
This has been described in various patents and publications.
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 109
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 0.1 ml
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 I. 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 lxlO10 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 lxlO10 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 lxlO10 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 contains
Ethanol Extract of lxlO10 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
I. Each dose of 0.1 ml of therapeutic agent contains
Liticase 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
J. Each dose of 0.1 ml of therapeutic agent contains Mycobacterium w (heat killed) 0.5x107

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
The bioreactor containing 9L distilled water is sterilized with live steam(indirect). 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. Temprature: 37±0.5°C ii. pH : 6.7 to 6.8 initially.
B. Harvesting and concentrating

It is typically done at the end of 6 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.
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.
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. Example: Poly TLR antagonist:
Description:
TLR stimulation is tested by assessing NF-KP activation in HEK293 cells expressing a
given TLR. The antagonistic activity of sample is tested on human TLR: 2, 3, 4, 5, 7, 8
and 9.
This step is performed in triplicate.
Control ligands
hTLR2: HKLM (heat-killed Listeria monocytogenes) at and 2 xl07, 1 xl07,
2x106 and 1 x 106 cells/ml
hTLR3: Poly(I:C) at 20 and 10 ng/ml
hTLR4: E. coli K12 LPS at 2 and 1 ng/ml
hTLR5: S. typhimurium flagellin at 20 and 10 ng/ml
hTLR7: Loxoribine at 1 and 0.5 mM
hTLR8: ssRNA40 at 5 and 3 ug/mL
hTLR9: CpG ODN 2006 at 50 and 20 ng/ml
Samples: Mycobacterium W
General Procedure
The secreted alkaline phosphatase reporter is under the control of a promoter inducible by
the transcription factor NF-GB. TLR stimulation in the screening is tested by assessing
NF-LJB activation in the HEK293 cells expressing a given TLR. This reporter gene
allows the
monitoring of signaling through the TLR, based on the activation of NF-DB. In a 96-well
plate (200uL total volume) containing the appropriate cells (25,000-50,000 cells/well),
we add 20uL of heat killed Mw as well as the positive control ligands to the wells. The
media we add to the wells is designed for the detection of NF-DB induced SEAP
expression. After a 16-20hr incubation we read the OD at 650nm on a Beckman Coulter
AD 340C Absorbance Detector.

TLR POSITIVE CONTROL(A) POSITIVECONTROL+Mw(B) NEGATIVECONTROL B/A % Antagonism%
TLR3 1.881 1.445 0.131 76.8% 23.2%
TLR4 1.207 0.602 0.112 49.87% 50.13%
TLR5 2.227 1.134 0.104 50.92% 49.08%
TLR7 1.503 1.141 0.114 74.57% 25.43%
TLR8 0.591 0.431 0.149 72.92% 27.08%
TLR9 1.979 0.135 0.146 0.07% 99.93%

TLR2 2.21 2.17 0.140 98.19% 1.81%
The findings are suggestive of TLR antagonism when stimulated by a TLR agonist for TLR 3,4,5,7,8,9. The antagonism is not seen for TLR2 in this experiment.
Procedures:
Mice of 8-10 weeks are sacrificed and spleenocytes are isolated from spleen. The spleen cells are cultured with different concentrations of LPS and combination of LPS with Mycobacterium w heat killed cells. The Cells were cultured in RPMI 1640 media.

After 48 hrs the cells are harvested and checked for expression of different TLRs. The TLR expression is checked by amplifying the specific mRNA from the cell lysate (Cell-
cDNA II kit, Ambion) using TLR specific primers (R&D systems).
The amlifyed products are checked on 1.5% agarose gel using Ethidium Bromide staining.
Results:
In Vitro study 1: It has been observed that expression of tlr3,4,5,6 and 9 is reduced when cells are exposed to Mw+lps in comparison to lps alone. There is no effect seen on tlrl. (fig). Thus of Mw demonstrates antagonist activity to LPS induced induction of tlr3,4,5,6 and 9. It has no effect on LPS induced tlrl
In Vitro study 2: In vitro studies Mw when used along with lyphopolysaccharide (LPS) reduce (p) induced TNF/Aplha & also reduce IFN gamma secretion. The amount of inhibition seen is significant & is as good as basal level (complete inhibition).
Example: Demonstration of positive effect of antagonism.
Rabbits were prepared as for pyrogen testing and temperature was monitored. Rabbits were administered intravenous injection of e.coli. lysate to mimic endotoxin/lps induced pyrexia. Two hours later they were divided in to control arm and treatment arm. The treatment arm received injection of heat killed Mw. Animals in treated group demonstrated lowering of teprature while control animals continued to have increased temperature. The effect persisted till the end of experiment.

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