FORM-2
THE PATENTS ACT 1970
(39 OF 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
(See section 10 and rule 13)
1. Mitogen activated protein kinase modulator.
2. CADILA PHARMACEUTICALS LIMITED. "Cadila Corporate Campus", Sarkhej-Dholka Road, Bhat, Ahmedabad-382210.
Gujarat, India
Nationality: An Indian Company.
3. The following specification describes the invention.

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Field of the invention:
The present invention relates to modulation of Mitogen-activated protein kinases (MAPK) using Mycobacterium w (Mw). Background of invention:
The mitogen-activated protein kinase (MAPK) cascade is a major signaling system that is shared by various types of cells. In mammalian cells, there are at least three MAPK subtypes; ie, extracellular signal-regulated kinase (ERK), p38 (Phosphorylated) MAPK and c- Jun amino-terminal kinase (JNK). They are serine / threonine kinases. They translocate on activation (phosphorylation) into nucleus. They phosphorylate / activate many different proteins including transcription factors including transcription factors that regulate expression of important cell-cycle and differentiation specific proteins.
These proteins mediate varieties of cellular responses and biological activities including morphogenisis, cell death, stress responses, cell proliferation, apoptosis, paraapoptosis, cell survival etc.
Activation of MAPK cascade is not restricted to immature cells, and this cascade is also activated in terminally differentiated cells such as neutrophils, suggesting that the MAPK cascade also plays an important role in some functions of terminally differentiated mature cells.

Activation of MAP kinase in two different cells can lead to similar or different cellular responses.
The mechanisms controlling the responses are poorly understood. It requires dual phosphorylation i.e. phosphorylation of both threonine and tyrosine. Phosphorylation of tyrosine occurs first.
The ERK cascade is activated in response to signals from receptor tyrosine kinases, hematopoietic growth factor receptors, or some heterotrimeric G-protein-coupled receptors and appears to mediate signals promoting cell proliferation or differentiation.
The p38 MAPK and JNK cascades are activated in response to heat shock, hyperosmolarity, UV irradiation, protein synthesis inhibi tors or inflammatory cytokines and appear to be involved in the cell responses to stresses.
Each MAPK subtype is activated by phosphorylation on threonine and tyrosine residues by an upstream dual-specificity kinase and phosphorylate substrates on serine or threonine adjacent to proline residues. Activation of the distinct MAPK subtype cascade is dependent on the types of cells and the stimuli used. The functional role of each MAPK subtype may be different according to the types of cells.
Cancer cells frequently produce their own growth factors which bind to the cell's surface to stimulate growth. Growth factors send signals from the cell's surface to the nucleus to switch on growth- related programs.
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This signal transduction from the cell surface to the nucleus is accomplished by a number of signal transduction pathways which are often composed of many proteins called protein kinases. Mitogen-activated protein kinase (MAPK) pathway is one of the signal transduction pathway.
Moreover, MAPK modulators are useful in management of melanomas (Clin Cancer Res. 2006 Apr 1: 2371s-2375s). MAPK modulators can have synergistic action with Paclitaxol (Mol Pharmacol. 2001 Aug;60(2):290-301. MAPK modulator are associated with programal cell death (J. Biol Chem. 2000 Dec 15;275(50):38953-6.
MAPK modulators works synergistically with biological therapy like antibacterial as well as chemotherapy. (Oncogene 2003:22,2034- 2044). MAPK modulator are useful in resensitivity resistant cells to chemotherapeutic agents. (Brit. J. Cancer 2001, 85: 1175-1184). MAPK modulator is assicated with chemotherapeutic effects of cancer chemotherapy like Taxol, Cisplatin (Oncogene 2001;20,147- 155; Onco gene 2000, 19; 5142-5152)
In addition p38 MAPK have been implicated in cancer, thrombin-induced platelet aggregation, immunodeficiency disorders, autoimmune diseases, cell death, allergies, osteoporosis and neurodegenerative disorders.
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Elevated levels of mitogen-activated protein kinase/extracellular regulatory kinase (MAPK/ERK) actively are frequently found in some cancer cells.
All three members of MAPK signaling pathway also participate to different extent in iNOS induction. This is done through its control over activation of an important iNOS transcription factor AP- 1.
US 6,994,981 describes modulators of paraptosis and related methods. Several other prior art patents are also based on MAPK inhibitors are EP1208748, WO 2004089929, WO2006117567.
In the present invention a novel approach is done to modulate with signal transduction through the MAPK pathway, wherein MAPK pathway is modulated interrupted by Mycobacterium w.
Summary Of the invention:
The object of the invention is to provide the modulator for the Mitogen activated protein kinase (MAPK) signal transduction pathway.
Detailed description of the invention:
In accordance with the present invention the MAPK modulators are prepared by the following process.
The invention includes the composition of 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
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present invention and scope of the present invention should not be limited
by them.
The process of preparing a Mycobacterium w :
A. Culturing of Mycobacterium w.
i) Preparation of culture 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, bactotrytone 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 preferably 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(indirect). Similarly the bioreactor is sterilized once more with Middle brook 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, b) Environmental parameter
i. Temperature: 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 pyrogen free isotonic fluid can be used as a pharmaceutical carrier, most preferably. The carrier is added in amount so as get to desired concentration of active in final form.
E. Quality control:
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i. The material is evaluated for purity, sterility,
ii. The organisms are checked for acid fastness after gram
staining,
iii. 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.
iv. Inactivation test: this is done by culturing the product
on L J medium to find out any living organism,
v. 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.
The Mycobacterium w so prepared was evaluated for its MAPK modulating activity.
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Following examples are illustrative of MAPK modulation by Mycobacterium w.
A. NFS 60 celline : NFS 60 celline represents murine leukemia.
Sample preparation using NFS 60 cells is as following :
Each cell palette is process as follows: Cell lysates - Cells are rinsed twice with Phosphate buffer saline (PBS), further making sure to remove any remaining PBS after the second rinse. Cells are solubilised at 1 x 107 cells/mL in Lysis Buffer (ImM EDTA, 0.5% Triton X-100, 5mM NaF, 6 M urea, 10 Mg/mL Leupeptin, 10 mg/mL Pepstatin, 100 mM PMSF, 3 u/mL Aprotinin in PBS, 2.5 mM sodium pyrophosphate, 1 mM activated sodium ortho-vanadate in PBS, pH 7.2 - 7.4. Vortex lysates briefly and allow to sit on ice for 15 minutes before use or store at -20° C in a manual defrost freezer. Before use, centrifuge at 2000 x g for 5 minutes and transfer the supernate into a clean test tube. For assaying, dilute lysates 6-fold with IC Diluent #8 (1 mM EDTA, 0.5% Triton X-100, 5 mM NAF in PBS, pH 7.2-7.4) and make further serial dilutions in IC Diluent #3 (ImM EDTA, 0.5% Triton X-100, 5mM NAF, 1 M urea in PBS, pH 7.2-7.4)
B. ELISA PLATE PREPARATION :
JNK Plate Preparation (Duo set kit Cat # DYC1387-2 R&D systems):
Capture antibody is diluted to a working concentration of 2.0 |ng/mL in PBS, without carrier protein. Immediately coat a 96 well micro plate with
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100 mL per well of the diluted Capture Antibody. Seal the plate and incubated overnight at room temperature.
Aspirate each well and washed with Wash Buffer, the process is repeated two times for a total 3 washes. Washed by filling each well with wash buffer (400 mL) using a squirt bottle, manifold dispenser or auto washer, Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
Plates are blocked by adding 300 mL of Block Buffer to each well. Incubate at room temperature 1 -2 hours. The aspiration/wash is repeated as in step 2. The plates are now ready for sample addition.
ERK Plate Preparation (Duo set kit Cat # DYC1018-2 R&D systems):
Capture antibody is diluted to a working concentration of 4 Hg/mL in PBS, without carrier protein. Immediately coat a 96 well micro plate with 100 mL per well of the diluted Capture Antibody. Seal the plate and incubated overnight at room temperature.
Aspirate each well and washed with Wash Buffer, the process is repeated two times for a total 3 washes. Washed by filling each well with wash buffer (400 mL) using a squirt bottle, manifold dispenser or auto washer. Complete removal of liquid at each step is essential for good
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performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
Plates are blocked by adding 300 mL of Block Buffer to each well. Incubate at room temperature 1 -2 hours. The aspiration/wash is repeated as in step 2. The plates are now ready for sample addition. p38 Plate Preparation (Duo set kit Cat # DYC1018-2 R&D systems)
Assay protocols were followed as per the manufacturer's instructions.
Now these ERK, P38 & JNK plates are ready for culturing. Wherein NFS 60 cells are plated with Mw cells. Wherein NFS 60 cells are cultured with 4 different concentrations of Mw. 3mL of 6 x 10s NFS 60 cells/mL are plated with 3 mL of 4 different concentrations of Mw. The cells are mixed well and incubated at 37° C, 5% C02 for 24 Hrs in micro titer plates. (1)3 x 108 heat killed Mw cells/mL
(2) 1.5 x 108 heat killed Mw cells/mL
(3) 7 x 107 heat killed Mw cells/mL
(4) 3.5 x 107 heat killed Mw cells/mL.
lmL of sample from each of the above cell mixture is withdrawn at 1, 2, 4, 8 and 24 hrs. The samples are centrifuged at 4° C to separate cells from the media. The cells and the media samples are stored at -70° C till further analysis.
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NFS 60 cells when exposed to Mw cells at concentration of 3 x 106 inhibits the proliferation / growth. The NFS 60 cells at this concentration are in the physiologically arrested and do not show cell viability by standard MTT / MTS assay.
But at the same time point the cells are in the state where the do not take-up trypan blue staining very effectively suggesting the cells are physiologically freezed. All the concentration of Mw lower then this the cells are viable and convest the proliferation dye MTT/MTS. While the higher concentrations of the Mw in hibits the cell growth and does not show any dye conversion. Splenocytes :
Splenocytes were harvested from normal mice. To 107/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 phophorylated 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.
Immunisation with Mw : Effect of immunization with Mw on MAPK levels: Immunisation :
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Two groups of mice were immunized with PBS alone or Mw. The mice were sacrificed on 8 day.
The Mw group was further divided into single injection on day 1 and daily injection till day 7).
The spllenocytes are harvested for each group and cultured in vitro. Cells were analyzed for levels of p38, ERK and JNK levels.
b) Immunisation followed by re-stimulation :
The cells of mcie from each of 3 above groups were restimulated with PBS or Mw. The p38 gets down regulated with in vitro exposure to Mw in PBS control group.
E : Effort on MAPK:
i) Effect on JNK over time :
JNK levels in NFS 60 cell lines change over time in a pecerlica pattern. In cells not exposed to Mw the levels go up and down in relation to the levels seen at 1 hour. Mw exposure alters the level in comparison to unexposed NFS cells but follows the identical pattern over time. (Fig. 1). When JNK levels observed at various time points are plotted fro Mw concentration it does not follow any particular pattern. (Fig. 2).
ii) Effect on p38 overtime :
P38 level in NFS 60 cell not exposed to Mw decreases till 8 hours and then there is sudden significant increase Mw exposure upto 8 hours results in increase in p38 expression in relation to unexposed NFS cells. However at 24
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hours the values are significantly lower in Mw exposed cells compared to unexposed cells. (Fig. 3)
When p38 levels are evaluated at different Mw concentration it shows rising trend with increasing amount of Mw at 2 and 4 hrs. (Fig. 4)
iii) Effect on ERK overtime :
ERK levels in unexposed cells increase over time in relation to 1 hrs. Mw exposed NFS 60 cells show decrease in ERK levels in relation to unexposed NFS 60 cells at all time period. (Fig. 5).
ERK levels at 2 and 4 hrs are plotted at various concentration of Mw the plot show that ERK levels follow peculiar pattern it goes down at lower levels rises at intermediate levels and achieves lower level again at a higher concentration. (Fig. 6).
F : Effect on cytokine production :
MAPK modulation induced by Mw results in decrease in cytokine levels. The decrease is significantly lower compared to basal secretion. (Fig.
7).
G: Effect on Immunisation :
a)The p38 levels in mice spleenocytes drops with sigleshot and further down with 7 shots of Mw immunization while JNK levels drops with single shot but does not get further down regulated with 7 shots. (Fig. 8). While in Mw 1 shot and Mw 7 shot groups on restimulation with Mw in vitro the p38 levels get up regulated compared to the o nly in vitro stimulation.
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The JNK levels get down regulated with dose dependent manned when re-stimulated in vitro. These no significant effect is observed (Fig.9).
H : Effect of Mw on cellular responses :
Mw exposed cells stop proliferation at a lower do se. At a higher dose they remain viable as evaluated by Trypan blud staining but loose their physiological activity as revealed by tests like MTT /MTS assay. At a significant higher dose the cells start dying. The death is by apoptosis.
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