FORM 2
THE PATENTS ACT, 1970 THE COMPLETE SPECIFICATION
(See section 10)
1. PROCESS FOR MANUFACTURING PHARMACEUTICAL COMPOSITION COMPRISES OF MYCOBACTERIUM W IN THE TREATMENT OF BRONCHIAL ASTHAMA ATTACK.
2. Dr. Bakulesh Mafatlal Khamar residing at 201, "Ashadha" Vasundhra Colony, Gulbai Tekra, Ellisbridge, Ahmedabad- 380006, Gujarat, India, Nationality Indian.
3. The following specification describes the nature of this invention.

PROCESS FOR MANUFACTURING PHARMACEUTICAL COMPOSITION COMPRISES OF MYCOBACTERIUM W IN THE TREATMENT OF ASTHAMA (OBSTRUCTIVE LUNG DISEASE)
.■^"'",»--* ■•■ * '
The invention relates to process for the preparation of formulations comprising a
microorganism Mycobacterium w for the management of bronchial Asthma
(obstructive lung disease).
Obstructive lung diseases are characterized by limitation of air flow. The limitation of airflow can be due to mechanical obstruction by tumor or edema or bronchial smooth muscle contraction. Mechanical obstruction needs one time management.
Other obstructive lung diseases are progressive and/or episodic in nature.
Bronchial asthma is a ill defined group of obstructive Sung disease. It is one of the most common disorder encountered in clinical medicine in both children and adults.Rather than being a single disease asthma is currently considered to be a group of different disorders ((complex syndrome with many clinical phenotypes) characterized by
1. Variable degree of airflow obstruction
2. air-way inflammation
3. broncho-hyperresponsiveness.
This syndrome arises as a result of interaction between multiple genetic and environmental factors, Some patients also exhibit acute hypersensitivity responses to common inhaled proteins, known as allergens.Very small amount of them is required to trigger mast cell degranulation. However large no. of patients do not have personal or family history of allergy and have negative skin tests.
Chronis inflammation is found to be underlying pathology in clinical syndrome of asthma. Histopathology of Jungs reveal hyperinflation, mucous'plugging in the airways, cluster of sloughed epithelial cells, and a crystalline precipitates of eosinophil derived proteins. Bronchial mucosa is are edematous, the no. of goblet cells is increased, the basement membrane is thickened, and the smooth muscle is hypertrophied.
Bronchial smooth muscle contraction as seen in bronchial asthma is a recurrent phenomenon. It occurs as an episodic or chronic ailment or as an episodic exacerbations of chronic disease
Various definitions are given to characterize this disorder
Asthma is defined as a eondition wherein there is a complete or partial reversibility of obstructive dysfunction after bronchodilators therapy.
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Asthma is also defined as chronic inflammatory disorder of the airways that occurs when individuals with genetic predisposition are exposed to appropriate trigger factors leading to disruption of airway epithelium, infiltration of inflammatory cells, thickning of basement membrane, as, well as smooth muscle spasm and hypertrophy.
Asthma is a disease characterized by an increased responsiveness of the trachea and bronchi to various stimuli resulting in airway obstruction that is reversible, either spontaneously or as a result of treatment.
Asthma is a chronic inflammatory pulmonary disorder that is characterized by reversible obstruction of the airways.
Asthma is a chronic obstructive disease characterized by tracheo-bronchial hyper-reactivity leading to paroxysmal airway narrowing, which may reverse spontaneously or as a result of treatment. It is charaterised clinically by wheezing, dyspnea, and cough. Allergic asthma is the most common form. Other precipitating factors include infection, exercise, occupational and environmental exposures, drugs, air pollution, and emotional factors.
Asthma is a chronic condition involving lungs in which narrowing of the passages from the lungs to the nose and mouth(airways) leads to diffuculty breathing. These changes commonly occur in response to changes in the environment including weather, allergens (such as dog or cat dander, mold, or dust), foods, or respiratory infections (colds).
Asthma is also defined as paroxysmal or chronic dyspnoea due to lung disorder.
Bronchial Asthma is also defined as a disease characterised by an increased responsiveness of the trachea and bronchi to various stimuli and manifested by wide spread narrowing of the airways that changes in severity either spontaneously or as a result of treatment.
Clinically, it is characterised by:
• Episodic or chronic wheezing, dyspnea, cough, and felling or tightness in the chest.
• Prolonged expiration and diffuse wheezing on physical examination.
• Limitation of airflow on pulmonary function testing, or positive bronchoprovocation challenge test.
• Complete or partial reversibility of obstructive dysfunction after bronchodilator therapy.
At the moment preferred first line therapy of such conditions is inhaled corticosteroids. If it is not adequate than bronchodilators like beta agonists like salmbutol, methylxanthines like theophylin anti cholinergics like ipratropium are added in form of inhaled or oral drug. Leukotriene antagonists may also
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be added which do not posses direct bronchodilators activity like glucocorticoids.
The management of an attack comprises of use of bronchodilators, Corticosteriodsand leucotrein Antagonists. They can be used orally, parenterally or in form of aerosols depending upon severity of disease and other factors. Corticosteriods and mast cell stabilizers like chromolyn sodium are also used to prevent the subsequent attack.
However it is not necessary that each episode needs to be treated with bronchodilators or each patient with chronic obstructive iung disease needs to be treated by bronchodilators.
Management of servere acute attack or acute exacerbation of chronic disease may need massive dose of parenteral glucocorticoids to control the attack.
Asthma is a chronic lung disease. It cannot be cured only controlled. In asthma airways are inflamed. That is, airway linings are swollen and red. Airways narrow and breathing becomes hard. This narrowing gets better (but not all the way in some patients), sometimes by itself, some times with treatment. Airways are super sensitive. They react to many things, such as cigarette somke, pollen, or cold air. Coughing, wheezing, tight chest, difficult breathing or an asthma episode may result following exposure to allergen.
The prevention of an attack comprises of eliminating 'trigger factors'. It includes measures to control house dust mite antigen, animal danders, avoidance of exposure to environmental factors including change place of work or residence, early treatment of upper respiratory tract and chest infections etc.
What is required in management of asthma is improvement in lung function
The present invention discloses such formulations and method of their manufacture and use.
Administration of pharmaceutical composition made as per present invention is found to result in reduction in severity of disease and frequency of asthmatic attacks. The dependent on drugs is decreased and quality of life improves.
Mycobacterium w is found to be useful in management of leprosy. It converts lepromin negative individuals to lepromin positive status. It also reduces the duration of therapy required for cure of multibacillary leprosy.
The pharmaceutical composition made as per present invention is found to be effective in management of asthma (obstructive lung disease)
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Summary of the invention
According to present invention, pharmaceutical composition made from 'Mycobacterium w' (Mw) is found to be useful in the management of asthma(obstructive lung disease).
Mycobacterium w used in the present invention 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 identical to those strains currently listed in this group. It is therefore thought that (Mw) 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 Mw 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 H37RV, M. paratuberculosis.
The object of the present invention is to provide a pharmaceutical composition containing 'Mycobacterium w' (Mw) with or without constituents obtained from Mw for the management of asthma (obstructive lung disease). Yet another object of the present invention is to provide a pharmaceutical composition containing 'Mycobacterium w' (Mw) with or without constituents
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obtained from Mw for prevention of attacks of asthma (obstructive lung
disease).
Yet another object of the present invention is to provide a pharmaceutical
composition containing 'Mycobacterium w' (Mw) with or without constituents
obtained from Mw for delaying attacks bronchial asthma (obstructive lung
disease).
Yet another object of the present invention is to provide a pharmaceutical composition containing 'Mycobacterium w' (Mw) with or without constituents obtained from Mw which reduces the requirement of drugs used to improve lung function in management of asthma(obstructive lung disease).
Yet another object of the present invention is to provide a pharmaceutical composition containing 'Mycobacterium w' (Mw) with or without constituents obtained from Mw which improves lung function in presence/absence of other drugs in asthma (obstructive lung disease).
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the invention the composition containing mycobacterium W , 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
Tween 80 0.1% w/v
Thiomerosal I. P. ... . 0.01% w/v
(As a Preservative)
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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 x109
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 1x1010 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 1x1010 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
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F. Each dose of 0.1 ml of therapeutic agent contains
Chloroform Extract of 1x1010 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 1x1010 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 1x1010 Mycobacterium w
Sodium Chloride l. 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 1x1010Mycobacterium 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 0.1ml
J. Each dose of 0.1 ml of therapeutic agent contains Mycobacterium w (heat killed) 0.5x107
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Extract of mycobacterium w obtained 1x103 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 0.1ml
Example 2. The Process of preparing a pharmaceutical composition
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, 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 preperabiy 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 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 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 pharmaceutically 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.
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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.lnactivation 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.
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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 of cell constituents results in improved efficacy of the product.
Example 3. Characteristics of constituents of Mycobacterium w by HPLC analysis.
The constituents of mycobacterium w. used for the purpose of invention when subjected to HPLC analysis gives a single peak at 11 minutes. No other significant peaks are found beyond. The peak is homogenous and devoid of any notch suggesting homogeneity of material obtained
HPLC analysis was done using a waters system high performance liquid chromatography apparatus
Column: Novapak c1860A, 4μm, 3.9 x 150mm.
The guard column: Novapak c 18
Column Temperature: 30° c
Flow rate: 2.5 ml/min
Injection volume: 25μL.
Mobile phase:
Solvent A: HPLC grade methanol.
Solvent B: HPLC grade methylene chloride Binary gradient: The HPLC gradient initially comprised 98%(v/v) methanol (solvent B).
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The gradient was increased linearly to 80%.
A and 20% B at one minute; 35% A and 65% B at 10 minutes, held for 5
seconds and then decreased over 10 seconds back to 98% A and 2% B.
Example: 4 The effect of pharmaceutical compositions and methods of*
use.
A symptomatic patient with severe form of asthma. Her breathlessness was
not controlled inspite of that she was on a maximal medical therapy for
asthma was given Mycobacterium w intradermally at the interval of one Week.
By four weeks patient became asymptomatic and number of drugs were
gradually discontinued. Patient remained asymptomatic inspite of that.
Thus Mycobacterium w is found to be useful in management of asthma in
making patient asymnptomatic when maximal medical therapy fails to achieve
this:
It is also useful in reducing the number of medicines patient is a taking.
Example: 5 The effect of pharmaceutical compositions and methods of use.
A group of patients who were getting exacerbation of disease periodically were given Mycobacterium w. It was observed that none of them had exacerbation of disease.
Thus mycobacterium w is found to be useful in eliminating/delaying exacerbation of the disease.
Example: 6 The effect of pharmaceutical compositions and methods of use.
In a group of patients diagnosed to have bronchial asthma were given conventional therapy in the form of bronchodilators and steroids. This resulted in improvement in lung function as determined by spirometry in terms of FEV1 and PEFR. The improvement with therapy was in the range of 15 to 20% from baseline, over a three month period of observation and it did not improve further. At the end of three months patients were administered mycobacterium w containing pharmaceutical compositions. It was administered as 0.1 ml at the interval of one week. Though these compositions are not known to have
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anti-inflammatory or broncho-dilator activity their administration resulted in
further improvement in lung function as determined by FEV1 and PEFR
values. This improvement was in the range of 15 to 20% over and above the
maximum values already achieved by conventional therapy.
The improvement in lung function was associated with subjective feeling of
well being and improvement, in quality of life. It also improved their
performance scale. It also resulted in improvement in amount of physical
exertion they can do without getting breathless.
Thus mycobacterium W is useful in improving lung function quality of life and
performance.
Example: 6 The effect of pharmaceutical compositions and methods of use.
In a group of patients having obstructive lung disease and who were controlled by conventional therapy were observed for a period of three months and then mycobacterium W containing compositions were added to the therapy and observed for another three months. Average requirement of antibiotics used to treat infections and associated exacebation of disease in the intial three months was 3.71. In the next three months when mycobacterium W was coadministered the requirement came down to 2 from 3.71. None of them needed any antibiotic in last month of combined therapy. Thus mycobacterium W is useful inreducing requirement of antibiotics. Example: 8 The effect of pharmaceutical compositions and methods of use.
In a group of patients having obstructive lung disease and who were controlled by conventional therapy but still requiring hospitalization from time to time for management of acute exacerbations were observed for a period of three months and then mycobacterium W containing compositions were added to the therapy and observed for another three months.The no. of exacerbations were found to be three per person in first part of the study. In the second part it came down to one per person.
Thus mycobacterium W is useful in reducing the no. of exacerbation of disease.
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We claim
1. The process of manufacturing a pharmaceutical compositopm 'for the management of asthma (obstructive lung disease) comprises of incorporating cells of mycobacterium w along with pharmaceutically acceptable carrier and optionally a preservative in a single formulation. '
2. The pharmaceutically acceptable carrier as claimed in claim "Ms added in a way so as to have more than or equal to 1x 105 mycobacterium w in a unitary dosage, more preferably equal to or more than 1x107 mycobacterium w in unitary dosage most preferably between 1x108 to 1x109 cells of mycobacterium w in a unitary dosage form.
3. The preservative as claimed in claim 1 is Thiomesol and is added to have final concentration of 0.01% w/v.
4. The process of manufacturing a pharmaceutical composition for the management of asthma ((obstructive lung disease) comprising the steps of incorporating disrupted cells of mycobacterium w along with pharmaceutically acceptable carrier and optionally a preservative.
5. Disruption of mycobacterium w as claimed in claim 4 is done by sonication or high pressure fractionometer.
6. The process of manufacturing a pharmaceutical composition useful for the management of asthma (obstructive lung disease) comprising the steps of incorporating solvent extraction of mycobacterium w along with pharmaceutically acceptable carrier and optionally a preservative.

7. Solvent extraction as claimed in claim 6 is done by using a solvent selected from chloroform, ethanol, methanol, acetone, phenol, isopropyl alcohol, acetic acid, urea, etc.
8. The process of manufacturing a pharmaceutical composition for the management of asthma (obstructive lung disease) comprising of incorporating enzymatic extraction of mycobacterium w along with pharmaceutically acceptable carrier and optionally a preservative .
9. The enzymes used for enzymatic extraction of cells of mycobacterium w is selected from lyticase and/or pronase.
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10. The process of manufacturing a pharmaceutical composition for the management of asthma (obstructive lung disease) comprising admixing product of claim 1 with product of claim 4 and/or claim 6 and/ or claim 8.
11. The process of manufacturing a pharmaceutical composition for the management of asthma (obstructive lung disease) comprise of adding adjuvant to product of claim 1, claim 4, claim 6, claim 8 or claim 10.

12. The adjuvant as claimed in claim 17 is selected from mineral oil, mineral oil and surfactant, Ribi adjuvant, Titer-max, syntax adjuvant formulation, aluminum salt adjuvant, nitrocellulose adsorbed antigen, immune stimulating complexes, Gebru adjuvant, super carrier, elvax 40w, L -tyrosine, monatanide (manide -oleate compound), Adju prime, Squalene, Sodium phthalyl lipopoly saccharide, calcium phosphate, saponin, melanoma antigen, muramyl dipeptide(MDP) and like.
13. A pharmaceutical composition prepared according to claim 1 to 3, claim 4 and 5, claim 6 and 7, claim 8 and 9, claim 10, claim 11 and 12 useful for the management of asthma (obstructive lung disease).
14. A pharmaceutical composition prepared according to claim 1 to 3, claim 4 and 5, claim 6 and 7, claim 8 and 9, claim 10, claim 11 and 12 when administered prevents attacks of asthma (obstructive lung disease).
15. A pharmaceutical composition prepared according to claim 1 to 3, claim 4 and 5, claim 6 and 7, claim 8 and 9, claim 10, claim 11 and 12 when administered delays attacks.of asthma (obstructive lung disease).
16. A pharmaceutical composition prepared according to claim 1 to 3, claim 4 and 5, claim 6 and 7, claim 8 and 9, claim 10, claim 11 and 12 when administered reduces the requirement of drugs used in management of asthma (obstructive lung disease).

17. A pharmaceutical composition prepared according to claim 1 to 3, claim 4 and 5, claim 6 and 7, claim 8 and 9, claim 10, claim 11 and 12 when administered improves lung function in presence/absence of other drugs.
18. Pharmaceutically acceptable carrier as claimed in claim 1, claim 4, claim 6, claim 8, claim 10, claim11 contains surfactant.
19. The surfactant as claimed in claim 19 is selected from Tween 80 or triton x100.
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20. The, concentration of surfactant as claimed in claim 19 and 20 is upto 0.4% preferably 0.1%.
21. Mycobacterium W as claimed in claim 1 to 20ch is a killed microrganism.
Dated this 28th, day February 2003

DR. BAKULESH KHAMAR
DIRECTOR - RESEARCH
FOR CADILA PHARMACEUTICALS LTD.
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