Patent Grant
8110204
This invention relates to the management of Human Immunodeficiency virus (HIV) Disease/Infection.
Human immunodeficiency virus (HIV) was first isolated in 1983. The causative agent for AIDS is known to be a virus of the retrovirus family called HIV (human immunodeficiency virus). Infection with HIV does not, however, immediately give rise to overt symptoms of AIDS. Three to six weeks following primary HIV infection more than 50% of individuals develop acute HIV syndrome, which is self limiting. Clinical findings seen during this period include fever, pharyngitis, lymphadenopathy, headache, arthralgia, myalgia, malaise, lethargy, nausea, vomiting, diarrhea, skin rash, mucocutaneous ulceration, meningitis, encephalitis, neuropathy etc. The only indication of exposure to the virus may be the presence of antibodies thereto in the blood of an infected subject who is then described as ‘HIV positive’. The infection may lie dormant; giving rise to no obvious symptoms, and the incubation period prior to development of AIDS may vary from several months to decades. Development of AIDS itself may be preceded by the AIDS-related complex (ARC), which is characterized by unexplained fever, weight loss, chronic cough or diarrhea. The development of AIDS and/or ARC is dependent on breakdown of immune system. The reasons for the variable period between infection with the virus and breakdown of the immune system in an infected individual are poorly understood. Factors at present unknown may trigger proliferation of the virus with consequential disruption of the immune system. The victims of the disease are then subject to various infections and malignancies, which, unchecked by the disabled immune system, lead to death. Thus HIV is characterized by the “acute HIV syndrome” followed by “asymptomatic stage” with clinical latency. Symptomatic stage sets in later with breakdown of immune system, which ultimately leads to the death of the individual infected with HIV.
Though the disease is caused by virus, the morbidity and mortality associated with disease is due to breakdown of immune system. The breakdown of immune system is characterized by decreased CD4+ T lymphocyte count. Because of this reason 1993 revised classification system for HIV infection is based on CD4+ T lymphocyte counts. HIV disease is empirically divided based on CD4+ count which is a measure of immunodeficiency.
Individuals with nonprogressive HIV disease are found to have steady CD4+ counts. They are also observed to have strong immune response against the virus.
There is evidence that in HIV infection, there is a dramatic loss of CD4+ T-cells, which results in very rapid development of overt symptoms of AIDS. Most AIDs defining opportunistic infections and true malignancies occur in advanced stage of disease where in CD4+ count is less the 200 cells/μL.
CD4+ Count and HIV
Though HIV is a viral infection, viral load can be determined by reasonable accuracy, CD4+ count (a measure of immune status) plays major role in management of HIV due to following reasons.
The method to treat HIV includes various therapeutic options. The options include management of symptoms and infections manifesting in HIV infected individuals at various stages of the disease. The antiretroviral drugs are used to keep the HIV infection (viral load) in control. They keep the viral load in control. The early antiretroviral drugs like azothymidine delayed progression of disease and had no significant effect on CD4+ count. Protease inhibitors like indinavir, ritonavir which are introduced recently do improve CD4+ count while reducing viral, load. All the drugs (antiretroviral) have their own side effects. The resistance to drugs is also noted. Thus there is need to provide alternate mechanism of treating HIV.
Since CD4+ count is important in maintaining immunity of individual and decreased CD4+ counts are associated with morbidity and mortality in HIV infection attempts are made to improve immunity for management of HIV. Various efforts have been done towards this end. This has resulted in introduction of immune modifying therapies with or without antiretroviral drugs. They comprise of antigens, cytokines organisms etc.
It has surprisingly been found during the course of research by us that formulations of ‘Mycobacterium w’ (Mw) with or without antigenic and/or immunomodulatory material derived from (Mw) is effective for management of Human Immunodeficiency Virus (HIV) disease/infection.
M. vaccae is apparently unique among known mycobacterial species in that heat-killed preparations retain its properties for the use as vaccine and immunotherapeutic. For example, M. bovis-BCG vaccines, used for vaccination against tuberculosis, employ live strains. Heat-killed M. bovis BCG and M. tuberculosis have no protective properties when employed in vaccines. A number of compounds have been isolated from a range of mycobacterial species, which have adjuvant properties. The effect of such adjuvants is essentially to stimulate a particular immune response mechanism against an antigen from another species.
In U.S. Pat. No. 6,001,361 the invention is related to compounds and methods for the treatment of mycobacterial infections including Mycobacterium tuberculosis and Mycobacterium avium. The invention is further related to compounds that function as non-specific immune response amplifiers, and the use of such non-specific immune response amplifiers as adjuvants in vaccination or immunotherapy against infectious disease, and in certain treatments for immune disorders and cancer.
U.S. Pat. No. 4,716,038 discloses diagnosis of, vaccination against and treatment of autoimmune diseases of various types, including arthritic diseases, by administering mycobacteria, including M. vaccae.
U.S. Pat. No. 6,210,684 describes method for delaying the onset of AIDS using killed M. Vaccae. Onset of AIDS is related to decrease in CD4+ count is not disclosed in the patent. Published studies shows that killed M. Vaccae has no effect on CD4+ count in HIV positive individuals.
International Patent Publication WO 91/01751 discloses the use of antigenic and/or immunoregulatory material from M. vaccae as an immunoprophylactic to delay and/or prevent the onset of AIDS.
International Patent Publication WO 94/06466 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for therapy of HIV infection, with or without AIDS and with or without associated tuberculosis.
U.S. Pat. No. 6,001,361 discloses an invention that relates generally to the treatment by vaccination or immunotherapy of skin disorders such as psoriasis, atopic dermatis, allergic contact dermatitis, alopecia areata, and the skin cancers basal cell carcinoma, squamous cell carcinoma and melanoma. In particular, the invention is related to the use of compounds, which are present in or have been derived from Mycobacterium vaccae (M. vaccae) or from the culture filtrate of M. vaccae.
U.S. Pat. No. 5,599,545 discloses the use of mycobacteria, especially whole, inactivated M. vaccae, as an adjuvant for administration with antigens, which are not endogenous to M. vaccae. This publication theories that the beneficial effect as an adjuvant may be due to heat shock protein 65 (hsp 65).
International Patent Publication WO 92/08484 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for the treatment of uveitis.
International Patent Publication WO 93/16727 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for the treatment of mental diseases associated with an autoimmune reaction initiated by an infection.
International Patent Publication WO 95/26742 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for delaying or preventing the growth or spread of tumors.
Vacce is not associated with change in CD4+ count or viral load in HIV positive individuals. It does not provide any relief in HIV +ve individual.
However inspite of various patents issued in relation to mycobacterium vaccae, it fails to provide any significant change in CD4+ count as well as viral load in individuals who are HIV positive.
Similarly attempts have been made to improve CD4+ count using immunomodulator from various sources.
Remune is a HIV-1 specific immunogen in incomplete Freund's adjuvant. It includes inactivated HIV-1 from which gp 120 is depleted. It is found to be safe with immunogenic potential in persons infected with HIV. In initial studies it was found to improve CD4+-cell count in asymptomatic HIV cohort not taking antiretroviral agents. It is given intramuscularly as an injection into the triceps muscles. The recent study by Sukeepaisarncharoen w et at suggests that remune therapy is associated with stabilization of CD 4-cell counts. It is also suggests that it may be of value in participants with higher CD4+ T cell count.
One such immunomodulator consists of mixture of antigens of inactivated bacteria with antigens of influenza virus (poly antigenic immunomodulator). It has not been possible to achieve improvement in CD4+ count using it.
SB-73 is another immunomodulator made up of substance produced by pencillium P (PB-73 strain). In a small study it is found to improve CD4+ count in majority (10/14) of individuals, infected with HIV when given intramuscularly in a dose of 5 mgm.
Reticulose, a peptide-nucleic acid is another immunomodulator found to be useful in improving CD4+ count in HIV infected individuals when given subcutaneously.
It was given as two 1 ml subcutaneous (SC) injections per day for two weeks followed by 1 ml SC per day every other week for a total 60 days (30 days total treatment). It resulted in a significant improvement, in CD4+ count in absence of any other antiretroviral therapy.
Thymosin α1 is a 28-aminoacid peptide. It was evaluated for its efficacy to improve CD4+ along with interleukin-2. It was found to have no significant effect. Grenulocyte colong stimulating factor (Filgrastim) has also been evaluated to improve immune response in HIV without much success.
OKT3, a CD3 monoclonal antibody, has been successfully used in management of HIV along with antiretroviral and Interleukin-2 in three patients.
Of various cytokines used in management of HIV, Interleukin-2 (IL-2) is extensively studied and used. It is used as injection to be administered intravenously or subcutaneously. It is found to improve CD4+ count significantly when used alone or along with antiretroviral drugs. It is given as intermittent therapy the side effects seen are sometimes intolerable. They are seen only during period of active administration. The improvement seen in CD4+ count is found to be stable.
Other cytokines used in management of HIV includes IL-12 and IL-15.
U.S. Pat. No. 5,759,992 provides low molecular weight glycopeptide with a molecular weight of 919.2 dalton which is derived from supernatant of disrupted cells maintaining temperature of 4° C. through out the process. This is obtained from bacteria which includes E coli and Mycobacterium. It is found to improve CD4+ count in normal mice. It is not evaluated in HIV +ve animals.
U.S. Pat. No. 5,871,732 describes methods for preventing or treating AIDS, AIDS related complex and human immunodeficiency virus infection by anti-CD4+ antibody homologs to DNA sequences of encoding such homologs.
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 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.
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.
According to present invention, immunomodulator made from ‘Mycobacterium w’ (Mw) is found to be useful in the management of HIV infection. We have now found that the same therapeutic agent not only delays development of AIDS in patients infected by HIV, but also is capable of causing regression, or even removal, of overt symptoms of AIDS even in patients where the disease is far advanced. These effects have been found in patients suffering also from tuberculosis. These effects are also seen in patients who are suffering from HIV infection with or without AIDS and without associated tuberculosis. The immunomodulator as per present invention is also found useful in relieving symptoms of HIV.
Therapeutic agent which may be used in the present invention resembles Mw 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 an immunomodulator Mycobacterium w containing ‘Mycobacterium w’ (Mw) with or without obtained from Mw for the prophylaxis or therapy of AIDS or AIDS related complex, 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 immunomodulator derived from Mycobacterium w that are useful for the management of HIV infection.
Yet another object of the invention is to provide immunomodulator derived from Mycobacterium w to improve immune function of HIV +ve subjects.
Yet another object of present invention is to provide immunomodulator to improve CD4+ count in HIV infected individuals.
Yet another object of present invention is to provide immunomodulator effective in ameliorating symptoms associated with HIV infection.
Yet another object of present invention is to provide an immunomodulator effective in management of opportunistic infections of associated with HIV infection.
Yet another object to present invention is to provide an immunomodulator useful in improving immune function of HIV +ve subjects in presence or absence of antiretroviral drugs.
Yet another object of the invention is to provide methods for the treatment of HIV, which results in the amelioration of symptoms of symptomatic stage of the disease.
It is yet another object of the invention to provide for a method of treatment of HIV infection that results in improved CD4+ T cell count.
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.
Mycobacterium w., (heat killed)
Mycobacterium w (heat killed) 0.5 × 107
It is typically done at the end of 6th day after culturing under aseptic condition.
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 constituents of Mycobacterium w can be prepared for the purpose of invention by:
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 o I Mycobacterium w organisms or it can be added to the product containing Mycobacterium w.
Addition cell constituents results in improved efficacy of the product.
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, 4 μm, 3. 9×150 mm.
The guard column: Novapak c 18
Column Temperature: 30° C.
Flow rate: 2.5 ml/min
Injection volume: 25 μL.
Mobile phase:
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.
Mycobacterium w when used in healthy animals or humans is found to be safe well tolerated and has no effect on any organ system, biochemistry or hematology including various blood cells. It is found not to cause lymphocytosis and nor change ratio of CD4+:CD8 cells as seen with various other nonspecific immune stimulation.
The only effect seen is at injection site. It includes morphologically formation of erythema, induration ulceration and scar formation. Histologically the injection site is found to have infiltration of various kinds of lymptocytes, plasma cells, giant cells giving a histological picture of epitheloid cell granulomas.
11 subjects who were HIV +ve and getting recurrent attacks of fever, upper respiratory tract infection, and diarrhea were given Mycobacterium w (5×108) intradermally. All improved and showed no recurrence of symptoms after 2nd month of treatment.
a) When Immunomodulator alone is used:
In 17 HIV positive individuals who had symptoms attributed to HIV and seeking help for the same Mycobacterium w was used as a sole therapy. Mycobacterium w was administered intradermally over a deltoid region. The amount of Mycobacterium w injected was 5×108 in a single injection. At the time of inclusion in study Mycobacterium w was given as intradermal injection over both the deltoids making a total dose of 1×109 Mycobacterium w subsequently at the interval of a month a single intradermal injection was given over a deltoid region which included 5×108 organisms.
There was no mortality or morbidity seen during trial. All subjects tolerated the therapy well and completed the trial. Symptomatic relief was seen within two months of initiation of therapy.
All subjects were evaluated for their CD4+ count at the beginning of therapy and 5 months later. The mean pretreatment CD4+ count was 204.70 (range 430-6).
All subjects showed improvement in CD4+ count. At the end of 5 months mean change in CD4+ count was 163.17 (range 8-628). In seven (41.2%) individuals increase in CD4+ count was more than 80%. Improvement in CD4+ count was less than 40% in 3 individuals (17.6%) only.
The therapy was not associated with any side effects systemically. These were a local reaction seen at the site of injection. It was in the form of erythematous reaction which was associated with induration. It progressed to ulceration at the site of injection in few which healed spontaneously leaving behind a this scar.
None of the subjects participating in a trial received any antiretroviral therapy.
Summary of Results
Details of change in CD4+ count in each individuals. (Table 1)
The result is much better than what is achieved with use of interleukin-2 when used along with two antiretroviral drugs. It is also better than what is achieved with HAART (Highly Active AntiRetroviral Therapy) alone for the same period. It is also worth noting that all patients showed improvement of CD4+ count. Natural course of disease in absence of antiretroviral therapy is associated with decline in CD4+ count month by month. On average 12 cells are lost per month as per ziduvadine study for symptomatic individuals published in New Eng. J. Med. In a large cohort of 2664 HIV +ve asymptomatic persons the CD4+ count decline is 4.6 cells/month.
Irrespective of no. of CD4+ count at the beginning of therapy improvement in CD4+ count was seen in all individuals. The pretreatment CD4+ count ranged from 6 to 430. The regression analysis of improvement (
b) Two antiretroviral drugs (NRTI)+Immunomodulator
In an another set of subjects who were HIV +ve and had symptoms related to HIV Mycobacterium w was used along with two antiretroviral drugs (both NRTI). None of them had received any anti-retroviral prior to these. Mycobacterium w was administered intradermally over a deltoid region. The amount of Mycobacterium w injected was 5×108 in a single injection. At the time of inclusion in study Mycobacterium w was given as intradennal injection over both the deltoids making a total dose of 1×109 Mycobacterium w subsequently at the interval of a month a single intradermal injection was given over a deltoid region which included 5×108 organisms.
There was no mortality or morbidity seen during trial. All subjects tolerated the therapy well and completed the trial. Symptomatric relief was seen within two months of initiation of therapy.
All subjects were evaluated for their CD4+ count at the beginning of therapy and 5 months later. The mean pretreatment CD4+ count was 200.99 (286 TO 96). All subjects showed improvement in CD4+ count. At the end of 5 months mean change in CD4+ count was 137.37 (range 24-588)
The therapy was not associated with any side effects systemically. These were a local reaction seen at the site of injection. It was in the form of erythematous reaction which was associated with induration. It progressed to ulceration at the site of injection in few which healed spontaneously leaving behind a this scar.
None of the subjects participating in a trial received any antiretroviral therapy.
Summary of Results
Details of change in CD4+ count in each individuals. (Table 2)
All subjects were evaluated for their CD4+ count at the beginning of therapy and 5 months later. None of the subjects showed deterioration in CD4+ count. All irrespective of pretreatment CD4+ count (Range 96 to 286) showed improvement in CD4+ count. Natural course of disease suggest minimal or no change in CD4+ when two antiretroviral drugs are used as used in this study. Thus improvement seen in the steady is significantly much more and can not be attributed to antiretroviral therapy used in the study.
Regression analysis (
c) HAART Therapy+Immunomodulator
In an another set of subjects who were HIV +ve and had symptoms related to HIV. Mycobacterium w was administered along with HAART therapy (three drugs). None of them had received any anti-retroviral prior to this. Mycobacterium w was administered intradermally over a deltoid region. The amount of Mycobacterium w injected was 5×108 in a single injection. At the time of inclusion in study Mycobacterium w was given us intradermal injection over both the deltoids making a total dose of 1×109 Mycobacterium w subsequently at the interval of a month a single intradermal injection was given over a deltoid region which included 5×108 organisms.
There was no mortality or morbidity seen during trial. All subjects tolerated the therapy well and completed the trial. Symptomatic relief was seen within two months of initiation of therapy.
All subjects were evaluated for their CD4+ count at the beginning of therapy and 5 months later. The mean pretreatment CD4+ count was 213.23 (range 536-40).
All subjects showed improvement in CD4+ count. At the end of 5 months mean change in CD4+ count was 258.79 (range 40-887).
The therapy was not associated with any side effects systemically. These were a local reaction seen at the site of injection. It was in the form of erythematous reaction which was associated with induration. It progressed to ulceration at the site of injection in few which healed spontaneously leaving behind a this scar.
None of the subjects participating in a trial received any antiretroviral therapy.
Summary of Results
All subjects were evaluated for change in CD4+ count at the beginning of therapy and end of therapy. All patients showed significant improvement in CD4+ count. The patients no. I to 15 had NNRTI as third drug. The patient No. 16 and 17 had protease inhibitor used as third drug. The improvement was significantly more than even reported in literature. The improvement in CD4+ count was significantly more when protease inhibitor is used compared to when NNRTI is used as part of HAART therapy. Regression analysis of results (
Effect of Immunomodulator in children is also evaluated. Immunomodulator was given as intradermal injection of 0.1 ml every month over a deltoid region for five months. Of the five children treated with Immunomodulator alone. All showed improvement as shown in Table 4.
Thus effect of immunomodulator is not restricted to age of HIV positive patients.
In another set of three subjects (HIV II positive), Immunomodulator alone was given as n therapy. It was given intradermally over a deltoid region. The amount of Immunomodulator injected was 0.1 ml in a single injection. At the time of inclusion in study Immunomodulator was given as intradermal injection over both the deltoids making a total dose of 0.2 ml subsequently at the interval of a month a single intradermal injection was given over a deltoid region.
All the three subjects showed improvement in CD4+ count as shown in Table 5.
Thus effect of immunomodulator is not found to be limited to HIV 1 only.
Seven HIV +ve subjects with tuberculosis cervical lymphadenopathy not responding to five anti tuberculosis drugs were given Immunomodulator intradermally. All had initial increase in size of cervical lymph node, which became erythematous. Within 3 weeks the size of lymph nodes decreased in size and over two months lymphodenopathy healed completely.
Mycobacterium w has been used in leprosy for faster clearance of M. Leprae from lesions and improved clinical out come making it possible to release the patients from therapy (MDT, multi drug therapy) at an earlier date. It has also been found to convert lepromin negative persons to lepromin positive and there by provide immunity against leprosy. According to present invention it is found useful in management of HIV. It is seen that HIV related symptoms disappear quickly when Mycobacterium w is administered. It is also found to improve immunity in the form of CD4+ count.
It does all this in absence of any anti retroviral therapy.
However when anti-retroviral are included along with Mycobacterium w in the form of HAART therapy, response is augmented.
The lack of systemic side effects as seen with all other therapies makes it even more suitable.
CD4+CD4+CD4+
| Number | Date | Country | Kind |
|---|---|---|---|
| 49MUM2001 | Jan 2001 | IN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB02/00097 | 1/17/2002 | WO | 00 | 1/20/2006 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO02/056906 | 7/25/2002 | WO | A |
| Number | Date | Country | |
|---|---|---|---|
| 20060193875 A1 | Aug 2006 | US |
