WO2003063896A1

WO2003063896A1 - Process of preparing a pharmaceutical composition for immunity against tuberculosis in hiv positive individuals

Info

Publication number: WO2003063896A1
Application number: PCT/IB2003/000199
Authority: WO
Inventors: Bakulesh Mafatlal Khamar
Original assignee: Modi, Rajiv, Indravadan
Priority date: 2002-01-29
Filing date: 2003-01-25
Publication date: 2003-08-07

Abstract

Present invention relates to process of preparing a pharmaceutical composition for immunity against tuberculosis in HIV positive individuals. According to present invention, vaccine made from 'Mycobacterium w' (Mw) is found to be useful in providing prophylaxis against tuberculosis in HIV positive individuals.

Description

PROCESS OF PREPARING A PHARMACEUTICAL COMPOSITION FOR IMMUNITY AGAINST TUBERCULOSIS IN HIV POSITIVE INDIVIDUALS.

Tuberculosis is a major communicable disease worldwide. It is caused by mycobacterium tuberculosis. It is a major cause of morbidity and mortality worldwide which includes developing countries as well as developed countries. This is happening inspite of availability of effective chemotherapy.

The problem of tuberculosis has gained more attention recently due to spreading epidemic of tuberculosis worldwide. The immunity in HIV is compromised and it makes the individual more vulnerable to various infectious disease particularly tuberculosis. The decrease in immunity is more pronounced for cell mediated immunity than humoral immunity. The incidence of tuberculosis is much more in HIV positive individuals compared to normal subjects. It varies from 32% in Brazil to 64% in India in HIV +ve individuals. The increased risk of tuberculosis can also be judged by the fact that in normal individuals risk of tuberculosis is 5% in 5 years compared to 8% in in first year in HIV positive. Similarly if life time risk of developing tuberculosis is one in normal individuals than it is 113 in HIV positive individuals.

Thus there is a greater need to provide prophylaxis against tuberculosis in HIV positive individuals.

The immunity against tuberculosis is judged by a test called tuberculin test. It is performed by injecting antigens [purified protein derivative (PPD)] of mycobacterium tuberculosis. In persons having immunity against tuberculosis there develops reaction at site of injection, which is read at 48 to 72 hours after infection. The reaction which develops at injection site consists of a raised, red, and hard (indurate ) area in the skin. This is indicative of presence of cell mediated immunity against tuberculosis.,

The immunity as detected by this method is found in individuals who are given BCG vaccination or exposed to tuberculosis organisms. The only known vaccine in use for providing prophylaxis against tuberculosis is BCG. The BCG contains live microorganisms and so it can not be given to immunocompromised individuals like HIV positive individuals. The current recommendations are to provide prophylaxis to HIV positive individuals by chemotherapeutic agents like Isoniazid, Rifampicin etc. There is no accepted method for providing immunity against tuberculosis. Thus there is unmet requirement for providing immunity against tuberculosis in HIV positive individuals.

US patents 54724144, 5985287, 6160093, 6001361 describes use of mycobacterium vaccae or its various components effective for the purpose of providing immunity against tuberculosis in animals.

US patent 6210684 and WO 9406466 describes use of mycobacterium vaccae for treatment or prophylaxis of AIDS.

However when used in human who are HIV positive mycobacterium vaccae fails to provide immunity against tuberculosis even after 3 to 5 doses. (Johnson D et al. Vaccine 1999, 17(20-21): 2583-7: Marsha BJ et. al. Am J Med Sci 1997, 313(6):377-83,: Waddell RD, Clin Infect Dis 2000, 30 Suppl 3:s309-15)

Thus the need to provide immunity against tuberculosis in HIV positive individuals is not met.

The failure to elicit immune response with mycobacterium vaccae may be due to inability of depleting CD4 cells to function in a manner to improve cell mediated immunity against tuberculosis which is judged by tuberculin conversion.

Surprisingly according to present invention it is observed that it is possible to provide a pharmaceutical composition for immunity against tuberculosis in HIV positive individuals. The process of preparing pharmaceutical composition for this purpose involves use of mycobacterium 'w. 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 providing immunity against tuberculosis in HIV positive individuals as judged by tuberculin test.

Summary of the invention

According to present invention, vaccine made from 'Mycobacterium w' (M_w) is found to be useful in providing prophylaxis against tuberculosis in HIV positive individuals. It is observed that administration of mycobacterium w containing vaccine is capable of converting tuberculin negative and hiv positive individuals into tuberculin positive status. These effects have been found in patients suffering from tuberculosis also. These effects are also seen in patients who are suffering from HIV infection with or without AIDS and with or without associated tuberculosis.

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 (M_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_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 H₃ R_V, M. paratuberculosis.

The object of the present invention is to provide a vaccine containing

'Mycobacterium w' (Mw) with or without constituents obtained from Mw for the prophylaxis against tuberculosis, to a subject - exposed to HIV infection or is HIV positive with or without overt symptoms of AIDS.

Yet another object of the invention is to provide a vaccine to convert tuberculin negative individuals who are HIV positive to tuberculin positive status.

Yet another object of the invention is to provide vaccine derived from

Mycobacterium w to improve tuberculin status of HIV +ve subjects.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention the composition of immunomodulator 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 δO 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

Extractof ^Mycobacterium. w after sonicationirom 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 F. Each dose of 0.1 ml of therapeutic agent contains Chloroform 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

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 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 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 0.1 ml

J. Each dose of 0.1 ml of therapeutic agent contains

Mycobacterium w (heat killed) 0.5x10⁷

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 0.1 ml

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 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^th 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. 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. 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 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). 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. Immunity against tuberculosis in HIV sero positive individuals

Ten HIV. positives (subjects) were enrolled in this study. All of them were tuberculin negative with a tuberculin reading of '0' m.m. and that was the reason of including them in study. All were administered intradermal mycobacterium w.. In all subjects, tuberculin test to determine tuberculin like delayed-type hypersensitivity reaction was repeated after ninety days.

Results of the study are shown in Fig In all 10 subjects repeat tuberculin test performed after 90 days revealed a reading of more than 5 m.m. In 8 of 10 subjects it was more than 10 m.m. Maximum reading seen was 17 m.m. and minimum was 6 m. m. The mean reading was 12.6 m.m.

In HIV positive individuals cut-off point for considering an individual tuberculin positive is 5 m.m. thus all the subjects got converted from tuberculin negative status to tuberculin positive status. Thus in all subjects immunity against mycobacterium tuberculosis as determined by tuberculin conversion from negative to positive was obtained after single intradermal injection.

The tuberculin negative status as seen in this study before enrollment is seen in spite of patients having active tuberculosis.

In HIV positive individuals immunity decreases with decrease in CD4 count.

This decreased cell mediated immunity results in change in tuberculin status also. Initialy tuberculin positive subjects become tuberculin negative with decrease in immunity.

In immunocompetent individuals tuberculosis can be diagnosed by positive tuberculin test in an individual who neither given BCG nor exposed to tuberculosis. Thus tuberculin negativity '0' m.m. reading inspite of active tuberculosis suggests difficult situation for tuberculin conversion.

The present invention provides tuberculin conversion and immunity against tuberculosis in highly vulnerable group and provides prophylaxis, a much desired effect.

Claims

We claim:

1. The process of manufacturing a pharmaceutical composition useful for management of cancer comprises of incorporating cells of mycobacterium w along with pharmaceutically acceptable carrier and optionally a preservative in a single formulation wherein cells of mycobacterium w are not alive.

2. The pharmaceutically acceptable carrier as claimed in claim 1 is 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 useful for management of cancer 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 management of cancer 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 useful for management of cancer 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.

10. The process of manufacturing a pharmaceutical composition useful for management of cancer 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 useful for management of cancer 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 when administered to a HIV positive individual in therapeutic dosage provide immunity against tuberculosis.

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 1 and

12 when administered to a HIV positive individual in therapeutic dosage provide immunity against tuberculosis, results in improvement in tuberculin status of individuals.

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 to a HIV positive individual in therapeutic dosage provide immunity against tuberculosis, results in tuberculin conversion from tuberculin negative to tuberculin positive.

16. Pharmaceutically acceptable carrier as claimed in claim 1 , claim 4, claim 6, claim 8, claim 10, claim11 contains surfactant.

17. The surfactant as claimed in claim 19 is selected from Tween 80 or triton x 100.

18. The concentration of surfactant as claimed in claim 19 and 20 is upto 0.4% preferably 0.1%.