Title of Invention	A PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF TUBERCULOSIS
Abstract	The present invention pertains to a composition containig tocopherols as antimicrobial agents for treatment of bacterial diseases in general and tuberculosis in particular. Tocopherol may be used alone or in combination with one or more of antibiotics, or antimicrobial agents as active pharmaceutical lngredients and with one or more of other anti-oxidants in quantities which are theraperutically effective to generate either anti-microbial or anti-oxidant effect or both the effects; the antioxidant effect being reflected in lowered lipid peroxidation levels in blood of tuberculosis patients in mammals. A tocopherol based anti-tuberculosis single dose combination including tocopherol (or its esters such as tocophery acetate). ascorbic acid, Rifampicin, lsoniazid, Pyrazinamlde, and Enthambutal and method of its preparation are disclised. The six drug composition described showed synergistic mycobactericidal activity with reduction in oxidative stress, leading to reduction in lung cell damages. The present invention further demonstrates mycobactericidal activity to the antioxidants such as d-alpha tocopherol and tocopheryl acetate; and compositions based on tocopherol for treatment of bacterial diseases and methods of preparation the said compositions.

Title of Invention

A PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF TUBERCULOSIS

Abstract

The present invention pertains to a composition containig tocopherols as antimicrobial agents for treatment of bacterial diseases in general and tuberculosis in particular. Tocopherol may be used alone or in combination with one or more of antibiotics, or antimicrobial agents as active pharmaceutical lngredients and with one or more of other anti-oxidants in quantities which are theraperutically effective to generate either anti-microbial or anti-oxidant effect or both the effects; the antioxidant effect being reflected in lowered lipid peroxidation levels in blood of tuberculosis patients in mammals. A tocopherol based anti-tuberculosis single dose combination including tocopherol (or its esters such as tocophery acetate). ascorbic acid, Rifampicin, lsoniazid, Pyrazinamlde, and Enthambutal and method of its preparation are disclised. The six drug composition described showed synergistic mycobactericidal activity with reduction in oxidative stress, leading to reduction in lung cell damages. The present invention further demonstrates mycobactericidal activity to the antioxidants such as d-alpha tocopherol and tocopheryl acetate; and compositions based on tocopherol for treatment of bacterial diseases and methods of preparation the said compositions.

Full Text	1 Title of invention Synergistic formulation of antioxidants and antimycobacterial agents: A method of making the same. Technical field The present invention is a synergistic, novel single dose powdery combi-preparation and provides an improvement in drug compositions used for treatment of tuberculosis. The said improvement is achieved by addition of antioxidant vitamins with antituberculosis agents. . The present invention has general application also in the field of anti-microbial agents. It comprises use of tocopherols, or esters of tocopherols either alone, or in combination with other antioxidants and optionally with one or more chemotherapeutic agents for treatment of bacterial diseases in general. Background of invention: Mycobacterium tuberculosis bacilli (MTB) are the most common causative organism for tuberculosis, (TB) in human. Estimated around one quarter to one half of world's population is infected by these organisms and cause world wide death, of some 3 million people each year. Studies before the advent of chemotherapy showed that untreated tuberculosis is often fatal. One third of patients died within 1 year after diagnosis and one half within 5 years. Of the survivors after 5 years, 60% had undergone spontaneous remission whereas remaining 40% still continued to suffer and excrete tubercle bacilli. With the advent of chemotherapeutic agents effective against mycobacteria and the experience over past years from the use of various agents alone or in combination for treatment of tuberculosis in humans as well as animals has resulted in formulation of various treatment regimes. The aims and objectives sub served by various treatment regimes are: (1) To make a open case ( which shows presence tubercle bacilli in sputum) of pulmonary tuberculosis a closed case ( no tubercle bacilli are present in sputum), so as to stop further transmission and spread of disease to other members of society by way of droplet airborne spread, (2) To cure the affected individual of the disease, by using drugs in various combinations which either inhibit growth of mycobacteria or kill the mycobacteria, (3) To prevent emergence of multi drug resistant mycobacteria. This objective is achieved by using multiple drugs concurrently. These drugs are used either as separate dosage form or as a combination formulation in fixed dose combination preparation. WHO guidelines advocate "Directly observed therapy", wherein the drugs are given to the patient under supervision of 2 a health worker (DOT). This approach has further helped in reducing, multi drug resistant mycobacteria by reducing chances of interrupted drug therapy and noncompliance. The recommended regimes are, in large part, based on evidence from clinical trials and are rated on the basis of a system developed by the United States Public Health Service (USPHS) and the Infectious Diseases Society of America (IDSA). There are four recommended regimes for treating patients with tuberculosis caused by drug-susceptible organisms. Although these regimes are broadly applicable, there are modifications that should be made under specified circumstances, described subsequently. Each regime has an initial phase of 2 to 3 months followed by a choice of several options for the continuation phase lasting 4 to 7 months. The general approach in management of tuberculosis with first line antituberculosis agents is summarized as follows Two drug regime - Rifampin and Isoniazid Three drug regime - Rifampin, Isoniazid and Ethambutal Four drug regime - Rifampin, Isoniazid, Ethambutal, Pyrazinamide and /or streptomycin. Because of the relatively high proportion of adult hosts with tuberculosis caused by organisms that are resistant to Isoniazid, four drugs are necessary in the initial phase of 2 to 3-months where Rifampicin is always included to be maximally effective. Thus, in most circumstances, the treatment regime for patients with previously untreated tuberculosis should consist of a 2 to 3 months initial phase of four drugs, such as Isoniazid (INH), Rifampin (RIF), Pyrazinamide (PZA), and Ethambutal (EMB). To avoid development of drug resistance all these drugs should be administered concomitantly. Therefore there is need for development of single dose preparation containing all the active drugs. The initial phase should consist of INH, RIF, PZA and EMB given daily for 2 to 3 months. Examples of circumstances in which PZA may be withheld include severe liver disease, gout, and, perhaps, pregnancy. EMB should be included in the initial phase of regime until drug susceptibility is determined. The first-line antituberculosis medications should be administered concomitantly; split dosing should be avoided. Fixed-dose combination preparations have advantage of ease of administration over individual separate drug preparations and are shown to decrease the risk of acquired drug resistance and medication errors. Fixed-dose combination compositions can be used when treatment under DOT is given and are especially more useful when DOT is not possible. Most of the fixed dose drug combinations are Kits containing three or four individual 3 drug preparations packed together in a single foil. A range of dose of these first line antituberculosis agents administered are, Rifampicin 450 mg, INH 300 mg to 600mg, Ethambutal 600 mg to 800mg, and Pyrazinamide 1200mg to 1500mg. Second line drug regimes comprising of drugs other than first line drugs which are reserved for treatment of multi drug resistant cases i.e. those cases where the mycobacteria show resistance to the effect of one or more drugs used in first line treatment regime. The following drugs are reserved as second line antituberculosis agents such as amikacin, aminosalicylic acid, amoxicillin/clavulinic acid, azithromycin, capreomycin, ciprofloxacin, clarithromycin, clofazimine, cycloserine, ethionamide, kanamycin, norfloxacin, ofloxacin, rifabutin, streptomycin, thiacetazone. These second line drugs are not used as single agents but are always used as multiple agents given simultaneously with first line drugs to which mycobacteria are sensitive. Alt above first line and second line drugs are either mycobactericidal or mycobacteriostatic. At no time during therapy single drug is used. It is always a multiple drug therapy. One of the complications of tuberculosis is highly raised oxidative stress leading to increased lipid peroxidation levels which indicates extensive tissue cell damage due to free radicals. No drug used in the therapy of tuberculosis has antioxidant property and therefore these drugs can only get rid of mycobacteria but fail to control and reduce tissue damages caused by free radicals. This disadvantage of present regimen can be removed by quenching the free radicals by simultaneous use of antioxidant agents. Vitamin E, potent chain breaking antioxidant has 6-chromanol ring structure with a phytol side chain referred more generally as tocopherol and the other as tocotrienols having double bonds in the phytol side chain at 3', 7', and 11' positions. These tocopherols and tocotrienols are present in various natural substances like wheat germ oil, soybean oil, and rice bran oil. Naturally occurring 6-chromanol ring compounds are a group of four tocopherols i.e. alpha, beta, gamma & delta tocopherols and four tocotrienols i.e. alpha, beta, gamma & delta stereo isomers, all of which are lipid soluble. A major part of the nutritive or therapeutic value of these 6-chromanol ring compounds in humans appears to be related to its properties as an antioxidant. In acting as an antioxidant, these agents, presumably prevent oxidation of essential cellular constituents, or prevents the formation of toxic oxidation products such as peroxidation products formed from unsaturated fatty acids that have been detected in its absence. 4 6-chromanol ring derivatives are chain breaking antioxidants and are efficient peroxy radical scavenger, which protects polyunsaturated fatty acids in cell membranes from oxidation and thus prevent the cellular damage. These compounds also inhibit prostaglandin synthesis and activities of protein kinase C and phospholipase A2. These compounds are available in the form of capsules, emulsion, injections and powders. Ascorbic acid (Vitamin C), a water soluble antioxidant is also known to be useful in treatment of scurvy, idiopathic methaemoglobinimea, in high doses to prevent viral respiratory infection and in cancer patient. It acts synergistically as antioxidant with other antioxidant agents. It is a water soluble substance having antioxidant, antiseptic and preservative properties. It is available in market in tablets and injection forms. Ascorbic acid may also modulate the immune system via its role in the regulation of hormones associated with stress. Both these antioxidants are used as preservatives and additives in many pharmaceutical preparations to avoid oxidation of active ingredients in the preparation from atmospheric oxygen. In the context of present invention it is necessary to understand difference between the "Preservative" purpose served by the antioxidants and the "Therapeutically effective anti-oxidant" purpose served by the same antioxidants. The "Preservative" function is a very limited objective for which the amount of anti-oxidant required per dose is very small than when it is desired to serve therapeutically effective antioxidant" purpose which requires a very large dose of the same antioxidants per dose and the effect is visible in the patients by way of lowering lipid peroxidation levels in the blood. There is evidence that in many diseases of respiratory system like asthma, chronic obstructive pulmonary disease, lung cancer, there is increase in free radical generation and hence increased oxidative stress. Increased oxidative stress and raised levels of free radicals are known to damage and injure cell wall of bacterial as well human cells leading to cell death. Mycobacterium tuberculosis due to its specialized cell wall structure along with presence of abundant catalase enzyme which can destroy free radicals is protected from injury, due to raised free radical levels in host, whereas host cell membrane remains susceptible to such damages. All known antimycobacterial agents either kill or inhibit growth of mycobacteria. None of antimycobacterial agents have antioxidant properties. On the contrary one of the most commonly used antimycobacterial agent Isonicotinic acid hydrazide is shown to be pro oxidant. 5 Anti-oxidants have been incorporated in the compositions for treatment of tuberculosis as disclosed in US 6277836 B1 and WO 2002/087547 A1 in a small amount with a purpose of providing protection from oxidation during storage to the active components of the composition like Rifampicin. Its purpose is not to provide any therapeutic dose of antioxidant to the patient. In SU 1577795 A1, it is reported that the use of Isoniazid and Ethambutal combined with alpha tocopherol and sodium thiosulphate in the treatment of infiltrative tuberculosis increases the efficiency of the treatment and prevents side effects. However the total duration of treatment still remains 6-7 months. The advantage quoted is reduction of antimycobacteriai drugs used from 3 or more to 2. Further efficacy of tocopherol independent of the two antimycobacteriai drugs was not detected. The therapy in this patent, however, is not based primarily on synergy provided by antioxidant and antimicrobial property of tocopherol and there is no claim on a single dose composition. Today even with the advent of Rifampicin and a four drug regime the duration of treatment is minimum 6 months and intensive phase of treatment is for 2-3 months. There is still a need for better drug regime that can increase efficacy of treatment, prevent side effects, and reduce the duration of treatment more particularly the duration of intensive phase having an aim of converting a 'open case* of tuberculosis to "closed case" so that spread of infection to other healthy members of society can be prevented. Even 30 to 40 yrs after Rifampicin was introduced no new and safer antimycobacteriai drug of efficacy similar to or more than Rifampicin has been invented. Pulmonary tuberculosis has been shown to be associated with significantly raised levels of oxidative stress resulting in extensive tissue damage and therefore there is need for an agent which can reduce the oxidative stress and at the same time have an antibacterial activity. Summary of Invention: It was surprisingly found that efficacy of present antituberculosis agent based treatment improved when the same antituberculosis agents were combined with tocopherols or esters of tocopherol and other anti-oxidants used in such a dose that would result into lowering of blood lipid peroxidation levels. The antioxidant agent's dose used in this invention was far higher than the dose of anti-oxidants that would be used in the compositions to achieve the purpose of protecting the active ingredients of the composition from other ingredients of the same composition and oxidative effect of atmospheric oxygen during the storage. Further, the extent of effect achieved in this invention was surprisingly 6 beyond what would be expected from known effects of antioxidants used. The effect led to rapid reduction and ultimately to disappearance of tubercle bacilli from the sputum and the patients became a "closed case" in less than a month. This dose was far greater than the small dose of these antioxidants used in the compositions as preservatives and additives, meant for providing protection from oxidation in storage by oxygen and other chemicals present in the composition. As expected from the therapeutic dose of antioxidants used in this invention, the lipid peroxidation levels in the blood fell rapidly to desired level, which would give more vitality for the cells to fight the disease causing organisms. However, the speed with which the tubercle bacilli disappeared from the sputum making the patients "closed cases" was surprising and could not be explained just on the basis of increase in viability and increase in strength of the host cells on account of nutritional effects of tocopherols and esters of tocopherols. When investigated further, it was found that tocopherols and esters of tocopherols have anti-mycobacterial property, which worked synergistically in eliminating the disease organisms from the patients. It is obvious that since tocopherols are not metabolic intermediates, but is a chemical entity that has an anti-microbial activity i.e. in effect it shall not be specific to certain bacteria only, but anti-bacterial in general, it shall be useful not only in control of tuberculosis but to control other diseases due to other microorganisms too. Being itself an antioxidant vitamin, it shall have little or no side effects. Using this anti-mycobacterial property of tocopherols and esters of tocopherols, very effective anti-tuberculosis compositions were prepared by combining in a single unit dose preparation, anti-tuberculosis agents and tocopherol or esters of tocopherol in dosage effective as anti-oxidant for lowering lipid peroxidation levels in the blood and other antioxidants such as ascorbic acid. Although a six drug combination has been tested in this investigation, any obvious variation, such as use of tocopherols or ester of tocopherols alone or in combination with one or more chemotherapeutic agents active against tuberculosis and several other bacterial diseases are included in this invention. It is obvious that an anti-mycobacterial property is basically an anti-bacterial property which would be available against a range of other bacteria, pathogenic or otherwise. The present invention also focuses on a unique single dose composition comprising of all the active ingredients taken together along with required pharmaceutical additives and dispensed in a sachet for oral use. This composition has following advantages: 7 1) The contents of sachet can be dissolved in any beverage or water just before use and the palatable liquid preparation is consumed. Such consumption can be facilitated further by making the contents effervescent and with pleasing flavors and taste. 2) No disintegration time is required before the drug can be absorbed as required in other solid preparations. This means early and better therapeutic blood levels of active agents are achieved giving better therapeutic response. 3) As all the drugs are taken together no chances of missing the dose of one or other drug as can occur with separate individual drug preparation. 4) The composition of this invention differs from other antituberculosis fixed dose combinations in having addition of the antioxidant agents in therapeutically active concentration and not merely using antioxidants as therapeutically inactive substances as preservative or additives. 5) The present single dose preparation has shown synergistic effect by reducing the period required for making sputum free of mycobacteria to one month which is normally two to three months with all other treatment regimes currently in use. 6) The therapeutic use of antioxidants in present composition prevents the tissue damage due to increased oxidative stress associated with the disease. 7) The composition of this invention also differs from earlier compositions in that all the constituents of the combination treatment are combined in a single dose, and they are not required to be combined at the time of taking. This is strategically very important from the point of view of convenience. Detailed description of invention: Like many other diseases, one of the complications of the tuberculosis is exposure of the patient's cells to oxidative stress, it was thought that use of antioxidants at therapeutically effective level to reduce lipid peroxidation level in the blood shall relieve the patients of this stress and whether that shall help in increasing vitality of the cells leading to a relief to the patients from the effects of tuberculosis and help them in responding to the antituberculosis treatment more efficiently and with reduced side effects. Finding of the studies that tocopherols have anti-microbial activity of their own was totally unanticipated and surprising. Thus, the present invention relates on one hand, to the use of antioxidant, such as Vitamin E, tocopheryl acetate, all-rac-alpha tocopherol and /or RRR-alpha-tocopherol and pharmaceutical^ acceptable salts or derivatives thereof as agents having a mycobactericidal property, which is a new activity, useful in the treatment of tuberculosis as well as other diseases caused by bacteria, and on the other hand a novel single dose combination for use in the treatment of tuberculosis incorporating, some 8 antituberculosis agents along with antioxidant-antimycobacterial agent and other antioxidants, in a single dosage compound powder composition and a process for manufacture of the same. The combined formulation has synergistic, antimycobacterial action and at the same time causes reduction in the oxidative stress leading to decreased host cell injury and damage along with early conversion to "closed case* leading to reduction in duration of treatment of intensive phase. The present invention describes a new, and useful pharmaceutical composition, comprising of first line antituberculosis agents such as Rifampicin, Isonicotinic acid hydrazide, Ethambutal, and Pyrazinamide and antioxidant-antimycobacterial agents such as vitamin E, tocopherol acetate, all-rac-alpha tocopherol and /or RRR-alpha-tocopherol or pharmaceutically acceptable salts thereof and ascorbic acid, for use in the treatment of tuberculosis producing a synergistic effect in comparison with the use of above antituberculosis agents given together, without antioxidants. This is the combination which has actually been clinically experimented. However, its obvious variation will include vitamin E alone or with any other one or more antituberculosis agents. Although the present trend is of a therapy based on combination of four or more anti-tuberculosis agents , with the unexpected factor of anti-microbial effect of the therapeutic quantities of vitamin E with or without other anti-oxidants, the possibility of future change in the perspective of efficacy of single or more anti-tuberculosis agents given as a single dose combination along with therapeutically effective dose of vitamin E with or without other anti-oxidants in favor of use of less or even one chemotherapeutic agent are real and they are also included in this invention. The present invention also further describes a new therapeutically useful mycobactericidal activity to vitamin E, tocophery1 acetate, derivatives and pharmaceutically acceptable salts used in therapy such as all-rac-alpha tocopherol, RRR-alpha-tocopherol. The present invention further describes a process for preparing a single dosage compound powder composition containing these antimycobacterial substances and the antioxidants. Since the reduction in level of lipid peroxidation in the patients, accompanied by rapid reduction in tubercle bacilli in the sputum of the patients was so dramatic and surprising, that it did not account for a simple anti-oxidant effect of tocopherol administered. A culture of mycobacteria susceptible to first line antituberculosis agents as well as mycobacteria resistant to first line antituberculosis agents responsible for tuberculosis was made. Total inhibition of growth of mycobacteria of both the types was very surprising which indicated that their effect was also positively anti-microbial in nature. It is obvious extension of the observed fact that mycobacteria basically being bacteria, many other bacteria shall be susceptible to anti-microbial effect as observed. Since tocopherols are not metabolic Q analogues, their action is not likely to be of the type of antibiotics of any specific type against specific type of bacteria only, but a general anti-bacterial action at the concentration used. At that concentration, it has nutritional benefits to human beings, having a lipid peroxidation lowering effect, and a general bactericidal effect. This has given rise to prospects of single dose compositions for tuberculosis in presently standardized therapies of combination of chemotherapeutic agents. It has also opened door for possible use of tocopherols and esters of tocopherols as chemotherapeutic agents useful alone or in combination with other drugs for treatment of tuberculosis as well as other diseases caused by bacteria.. The present invention further describes a new method or approach in the treatment of tuberculosis over the prior art by addition and use of antioxidants for prevention of lung tissue damage caused by liberated free radicals during the disease process and the course of treatment. The pharmaceutical composition is intended for oral use. The following examples are included to demonstrate preferred embodiments of the present invention. For those of skill in the art, that the techniques disclosed in the examples which follow, represent techniques to function well in the practice of invention and thus can be considered to constitute preferred modes for its practice. However those of skill in the art should, in the light of present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of invention. Example"!: Newly diagnosed cases suffering from pulmonary tuberculosis were assessed for status of oxidative stress in comparison with the matching healthy volunteers by performing analysis of blood samples for lipid peroxidation levels using, Draper and Hadley's (1990), double heating method. Lipid peroxidation is used as marker of cell membrane damage. It was observed that the lipid peroxidation levels were raised in pulmonary tuberculosis patients significantly when compared with matching human healthy volunteers. This observation, suggest increase in free radical liberation and oxidative stress in patients of pulmonary tuberculosis. This observation provides a basis and rationale for an indication to use antioxidants. Example 2: About 100 newly diagnosed cases suffering from pulmonary tuberculosis demonstrating positive sputum smear, for acid fast bacilli (AFB), by Ziehl-Neelson staining 10 procedure, and positive x-ray, lung involvement were included in the study. They were randomly divided in two groups. Control group received first line four drugs regime, comprising of Rifampicin 450 mg, Isoniazid 300 mg, Pyrazinamide 1500mg, and Ethambutal 800mg, once daily by oral route for a period of two months. Study group received first line four drug regime as detailed above and two antioxidants d- alpha tocopherol in the dose of 400 IU , or tocopheryl acetate in the dose of 400mg and ascorbic acid in the dose of 500mg once daily by oral route for a period of two months. The outcome of clinical improvement was accessed by following parameters in every patient of both the groups and findings were compared and analyzed for statistical significance at the end of successful completion of treatment for two months. Sputum smear AFB staining test - In both the groups sputum of all the patients were analyzed for the presence of acid fast bacilli using standard Zeil-Nelson hot stain method at the end of 15 days, 30 days and 60 days. The reports were documented as positive for presence of bacilli and negative as absence of bacilli. The results of both the groups where compared at the end of 15 days, 30 days, and 60 days. At the end of 15 days treatment in study group 50 % patients showed sputum smear negative for acid fast bacilli as compared to 14 % in control group. A similar pattern was observed at the end of 30 days treatment, 86% versus 50% and 100% versus 86 % at the end of 60 days treatment in study group versus, control group respectively. The study group showed much earlier and augmented sputum smear negativity for acid fast bacilli, which is an evidence of a synergistic antimycobacterial effect, of the drug combination, used in study group. Lipid peroxidation levels (LPO) —Blood samples were collected from patients included in both the groups before the start of treatment but after confirmation of diagnosis of pulmonary tuberculosis and at the end successful completion of treatment of 60 days. The oxidative stress is indicated by lipid peroxidation was reported in terms of Malondialdehyde (MDA) nmol/gm of hemoglobin using Draper and Hadley (1990) double heating method. All the patients in study as well as control groups showed elevated LPO levels at the start of treatment. The LPO levels after 60 days showed significant reduction in study group patients receiving antioxidants along with four antituberculosis agents, indicating improvement in oxidative status. However in control group, patients showed a further rise in levels of LPO, indicating deterioration of oxidative status and confirming need for use of 11 antioxidants for improvement in oxidative status and prevention of cell membrane injury and damage. Radiological investigations - Chest X-ray of all the patients from both groups was done at the start of treatment and after the completion of treatment of 60 days. The findings were visually analyzed and compared for each patient before and after the treatment. The findings were reported as favorable & improved or unfavorable and deteriorated. All the patients from study group showed favorable response as compared to 80% in control group suggesting a lesser damage to lungs in study group. Example 3 In vitro Studies - Lowenstein and Jenson (LJ) medium is used as a selective medium to grow mycobacteria and test sensitivity, to antimycobacterial agents. A set of (LJ) media was prepared by incorporating double dilutions of d- alpha tocopherol, and another set incorporating double dilutions of tocopheryl acetate yet another set without addition of any drug was prepared. These slopes were inoculated with known freshly cultured virulent strain of mycobacterium tuberculosis. Growth was observed at the end of each week for six weeks after incubating the media at 37 degrees for the entire period. A control medium slant without any drug was inoculated with same freshly prepared culture strain of mycobacterium tuberculosis. At the end of 6 weeks no growth was observed on (LJ) slopes containing the antioxidants like d-alpha tocopherol or Tocopheryl acetate in a concentration of 100 microgram per ml whereas the control slant showed a carpet growth. The above experiment was repeated with known strains available in laboratory showing resistance to one drug i.e. Rifampicin or to two drugs i.e. .Rifampicin and Isoniazid and at the end of 6 weeks it was observed that both these strain resistant to either Rifampicin or Rifampicin and Isoniazid did not grow in (LJ) slopes containing antioxidants like d-alpha tocopherol or tocopheryl acetate in a concentration of 100 microgram per ml whereas the control slant showed a carpet growth. The results of in vitro studies have confirmed that both d-alpha tocopherol and tocopheryl acetate have mycobactericidal activity. The in vivo studies on patients of pulmonary tuberculosis also confirm these results based on the conspicuously enhanced sputum smear negativity for acid fast bacilli at the end of 15 days. 12 From all the examples cited above it is confirmed that there is more oxidative stress in patients of pulmonary tuberculosis and a need for use of antioxidants to reduce cell membrane injury to the host cells. The above cited examples further prove that the d- alpha tocopherol and tocopheryl acetate have mycobactericidal activity and therefore can be used along with other antimycobacterial agents The above cited examples further prove that such a use a combination of antimycobacterial agents and antioxidants referred in example is synergistic. Example 4: A known drawback of use of multiple drugs, is reduction in patient compliance i.e. failure to adhere to treatment protocol. This drawback is removed if multiple drugs are combined in single dosage composition. The inventors have further developed a process for preparation of pharmaceutical composition wherein two to four required primary line antituberculosis agents in the therapeutically desirable quanta can be combined along with therapeutically desirable quanta of the antioxidants like d-alpha tocopherol or tocopheryl acetate optionally with therapeutically desired quantum of ascorbic acid in a compound powder unit dose composition with the required additives, flavoring agents and suspending agents. The range of doses selected for individual agents per single unit dose preparation are as follows Rifampicin 3-{[(4-Methyl-1-piperazinyl) imino] methyl} rifamycin SV. 450mg powder Isonicotinic acid hydrazide (4-Pyridinecarboxylic acid hydrazide) 300 or 600mg powder Pyrazinamide (Pyrazine-2-carboxamide) 1200 or 1500mg powder Ethambutal d-N, N'-bis (1-hydromethylpropyl) ethylene diamine; 600 or 800mg powder d- alpha tocopherol acetate - 400 IU or 800 IU powder Or Tocopheryl acetate ~ 400 mg or 800 mg powder Ascorbic acid — 125, or 250, or 500mg powder Saccharin as sweetening agent as per taste Flavoring agents - lemon, orange, anise, liquorice, raspberry as per choice. Permissible fillers and adsorbents in sufficient quantity This particular mixing of aforesaid substances was done by using mixture machines used in pharmaceutical manufacturing practice and the contents were homogeneously mixed so that the end product is free flowing powder. The prepared free flowing compound powder is further weighed into individual unit dose packs based on the total weight of individual agents added together required in the single unit dosage fixed dose preparation and sealed in appropriate air tight, light impervious sachet for further use. 13 Claims We claim, 1. A single dosage composition of drugs comprising: a. Antituberculosis agents in therapeutically effective dosage in a drug regime which is selected from one or more of first line antituberculosis agents and second line antituberculosis agents; where a.1) first line antituberculosis agents consist of a single antimicrobial agent or a two drug combination regime containing Rifampicin or Rifabutin or Rifapentine and Isoniazid; a three drug combination regime as Rifampicin or Rifabutin or Rifapentine, Isoniazid and Ethambutol; a four drug combination regime as Rifampicin or Rifabutin or Rifapentine, Isoniazid, Ethambutol and Pyrazinamide ; a.2) second line antituberculosis agents consist of one or more of second line drugs including ethionamide, cycloserine, p-aminosalicylic acid, macrolides preferably azithromycin, clarithromycin, quinolones preferably ciprofloxacin, Levofloxacin, Moxifloxacin, Fleroxacin and sparfloxacin, b. One or more of drug or drugs with antioxidant and antimicrobial property, in therapeutically effective dosage, wherein b.1) the therapeutically effective dosage for antioxidant effect comprising improvement in therapeutic parameters related to oxidative stress, related to tissue damage including a dosage effective for achieving lowering of lipid peroxidation levels in blood in a treatment period of around 60 days or less, and b.2) the therapeutic effective dosage for antimicrobial property comprising improvement in therapeutic parameters related to infection from disease causing microorganisms including a dosage effective for achieving sputum negativity in a period of around 15 days; c. One or more of antioxidant or antioxidants in therapeutically effective dosage for antioxidant effect comprising improvement in therapeutic parameters related to oxidative stress, related to tissue damage including a dosage effective for achieving lowering of lipid peroxidation levels in the blood in a treatment period of 60 days or less; d. Optionally containing pharmaceutical^ acceptable excipients including binders, sweeteners, flavoring agents, fillers, adsorbents, diluents, coating agents, disintegrating agents, preservatives, lubricants, controlled release agents , dispersing and or suspending agents, emulsifying agents, and effervescent agents. 2. Composition of claim no. 1 wherein the range of use of the said antituberculosis agents individually are - Rifampicin used within a range of 60mg to 600mg , Rifabutin used within a range of 150 mg to 600 mg, Rifapentine used within the range of 300 to 900mg, Isoniazid used within a range of 60 mg to 1500mg, Ethambutol, used within a range of 60mg 14 to 2500mg , Pyrazinamide, used within a range of 100mg to 2000mg , Ethionamide used within a range of 100mg to 1000mg, cycloserine used within a range of 250mg to 750 mg, p-aminosalicylic acid used within a range of ( 8g to 12g), macrolide preferably azithromycin used within a range of 250 to 1500mg, clarithromycin, used within a range of 250mg to 1500mg, quinolones preferably ciprofloxacin, used within a range of 500 to 2000mg, ofloxacin, used within a range of 100 to 1200mg, fleroxacin used within a range of 150 mg to 1000mg, sparfloxin used within a range of 150mg to 1000mg. 3. Composition of claim no.1 wherein the said antioxidant or antioxidants with antimicrobial properties of sub claim b. of claim 1 is Vitamin E, comprising homologues of all natural as well as synthetic tocols and tocotrienols such as 5,7,8-Trimethy! tocol, 5,8- Dimethyl tocol, 7,8- Dimethyl tocol, 8-Methyl tocol, 5,7,8-Trimethyl tocotrienol, 5,8-Dimethyl tocotrienol, 7,8-Dimethyl tocotrienol, 8-Methyl tocotrienol and their derivatives including esters further including dl-a-Tocopheryl acetate, d.a-Tocopheryl acetate, dl-a-Tocopheryl acid succinate, d.a-Tocopheryl acid succinate, dl-a-Tocopheryl nicotinate, d,a-Tocopheryl nicotinate, characterized by derivatives of 6-chromanol ring with phytol side chain qualitatively exhibiting biological activity of natural alpha tocopherol. 4. Composition of claim no. 3 wherein Vitamin E is d alpha tocopherol or tocopheryl acetate used within the range of 200 IU to 2000 IU, more particularly at 400 IU to 800 IU per dose. 5. Composition of claim no. 1 wherein the said antioxidant or antioxidants of sub claim c of claim 1 are selected from one or more of a. Vitamin E, comprising homologues of all natural as well as synthetic tocols and tocotrienols including 5,7,8-Trimethyl toed, 5,8-Dimethyl tocol, 7,8- Dimethyl tocol, 8-Methyl tocol, 5,7,8-Trimethyl tocotrienol, 5,8-Dimethyl tocotrienol, 7,8- Dimethyl tocotrienol, 8-Methyl tocotrienol and their derivatives including esters further including dl-a-Tocopheryl acetate, d,a-Tocopheryl acetate, dl-a-Tocopheryl acid succinate, d,a-Tocopheryl add succinate, dl-a-Tocopheryl nicotinate, d.a-Tocopheryl nicotinate, characterized by derivatives of 6-chromanol ring with phytol side chain qualitatively exhibiting biological activity of natural alpha tocopherol used within a range of 200 IU to 2000 IU, more particularly within a range of 400 IU to 800 IU per dose; b. ascorbic acid or sodium ascorbate used within the range of 50 mg to 1000mg, more particularly within a range of 150 to 500 mg per dose. 6 Composition of claim 1 containing the said pharmaceutically acceptable excipients, which include sweetening agents, fillers, binders, diluents, coating agents, disintegrating agents, preservatives, lubricants, time delay agents, dispersing and or suspending agents, emulsifying agents, effervescent agents and adsorbents added, 15 when all the added excipients are put together, within pharmaceutical^ permissible range of 0.5 to 1 % of total weight of a single dose. 7. A single dose composition of claim 1 comprising: a. Anti tuberculosis agents in following quantities - i. Rifampicin powder 300 to 450 mg j. Isoniazid powder 300 to 600 mg k. Pyrazinamide powder 1200 to 1500 mg I. Ethambutal powder 600 to 800 mg b. Tocopheryl acetate 400 mg to 800 mg or d-alpha tocopherol 400IU to 800IU c. Ascorbic acid 125 to 500 mg d. Pharmaceutically acceptable excipients including sweetening agent saccharin added as per taste; flavoring agents as per choice including lemon, orange, anise, liquorice, raspberry; permissible fillers, adsorbents, diluents, coating agents, disintegrating agents, preservatives, lubricants, time delay agents, dispersing or suspending agents, emulsifying agents, and effervescent agents in sufficient quantity, all excipients put together added in the range of 0.5 to 1 % of total weight of a single dose. 8 An oral composition of claim no. 1 containing effervescent agents in amounts sufficient to produce effervescence when the composition is added to a suitable liquid; the said agents preferably comprising sodium bicarbonate and an addulant including tartaric or citric acid chosen in amount such that the effervescence generated effectively disperses the constituents of the composition in the said liquid and a neutral or slightly acidic pH is established after completion of effervescence. 9 Composition of claim 8 wherein sodium bicarbonate is preferably taken in the range of sum of equivalent weight of acidulants added, acidulant is taken equal to or in slight excess to the equivalence to the sodium bicarbonate taken and the said suitable liquid is one or more of water, milk, fruit juice, a beverage. 10 An oral dosage form essentially containing ingredients of claim 1 including one or more of: a. a satchet containing powder or granules of the ingredients, or b. a package containing a number of tablets collectively dispensing the recommended quantity of the ingredients of composition of claim 1 as a single dose per package, or c c. a package containing a number of capsules containing powder or granules of the ingredients collectively dispensing the recommended quantity of the ingredients of composition of claim 1 as a single dose per package, or d. a satchet containing a volume of liquid or a syrup as such or packaged in a convenient number of capsules collectively dispensing the recommended quantity of the ingredients of composition of claim 1 as a single dose per package, or e. the like.. 11 A method of use, alone or in combination with other Active Pharmaceutical Ingredients including antimicrobial agents, of Vitamin E, comprising homologues of all natural as well as synthetic tocols and tocotrienols including 5,7,8-Trimethyl tocol, 5,8-Dimethyl tocol, 7,8-Dimethyl tocol, 8-Methyl tocol, 5,7,8-Trimethyl tocotrienol, 5,8-Dimethyl tocotrienol, 7,8-Dimethyl tocotrienol, 8-Methyl tocotrienol and their derivatives including esters further including dl-a-Tocopheryl acetate, d,a-Tocopheryl acetate, dl-a-Tocopheryl acid succinate, d.a-Tocopheryl acid succinate, dl-a-Tocopheryl nicotinate, d,a-Tocopheryl nicotinate, characterized by derivatives of 6-chromanol ring with phytol side chain qualitatively exhibiting biological activity of natural alpha tocopherol including tocopherols or tocotrienols or tocopheryl acetate at dosages therapeutically effective for antimicrobial effect. 12 A Pharmaceutical Composition comprising incorporation, alone or in combination with other Active Pharmaceutical Ingredients including antimicrobial agents, of vitamin E, comprising homologues of all natural as well as synthetic tocols and tocotrienols including 5,7,8-Trimethyl tocol, 5,8-Dimethyl tocol, 7,8-Dimethyl tocol, 8-Methyl tocol, 5,7,8-Trimethyl tocotrienol, 5,8- Dimethyl tocotrienol, 7,8-Dimethyl tocotrienol, 8-Methyl tocotrienol and their derivatives including esters esters further including dl-a-Tocopheryl acetate, d,a-Tocopheryl acetate, dl-a-Tocopheryl acid succinate, d,a-Tocopheryl acid succinate, dki-Tocopheryl nicotinate, d,a-Tocopheryl nicotinate, characterized by derivatives of 6-chromanol ring with phytoi side chain qualitatively exhibiting biological activity of natural alpha tocopherol containing tocopherols or tocotrienols or tocopheryl acetate in therapeutic amounts effective as antimicrobial agents in general and as anti-mycobactericidal agents in particular. 13 A Pharmaceutical Composition of claim 12 containing one or more of a. tocopherol esters including one or more of tocopherol acetate, tocopheryl succinate or tocopheryl nicotinate used collectively in the range of 200 to 2000 mg, more particularly in the range of 400 to 800mg b. tocopherols or tocotrienols used collectively in the range of 200IU to 2000 IU, more particularly in the range of 400 IU to 800 IU. 14 A process of preparing Composition of claim no. 1, claim no. 7, claim 12 and claim no. 13 wherein: 17 15 a ingredients of the composition are taken as powder, or granules weighed and mixed, using mixer machines used normally in pharmaceutical manufacturing, to a homogeneous free flowing form, b. Weighing the powder or granules in quantities envisaged by one of the claims of claim no. 1, claim no. 7, claim no. 12 or claim no. 13, in a single unit dose c. Sealing the single dose in one or more forms of containers including sachets, bottles, which are preferably air tight and light impervious. A method of treating tuberculosis by using compositions of claim 1 or claim 7. 16 A method of treating microbial diseases using compositions of claim 1, claim 7, claim 12 or claim 13. ,0$-- •^ >^ £SM£L^W^ M&W) 18 Abstract The present invention pertains to a composition containing tocopherols as antimicrobial agents for treatment of bacterial diseases in general and tuberculosis in particular. Tocopherol may be used alone or in combination with one or more of antibiotics, or antimicrobial agents as active pharmaceutical Ingredients and with one or more of other anti-oxidants in quantities which are therapeutically effective to generate either antimicrobial or anti-oxidant effect or both the effects; the antioxidant effect being reflected in lowered lipid peroxidation levels in blood of tuberculosis patients in mammals. A tocopherol based anti-tuberculosis single dose combination including tocopherol (or its esters such as tocopheryl acetate), ascorbic acid, Rifampicin, Isoniazid, Pyrazinamide, and Ethambutal and method of its preparation are disclosed. The six drug composition described showed synergistic mycobactericidal activity with reduction in oxidative stress, leading to reduction in lung cell damages. The present invention further demonstrates mycobactericidal activity to the antioxidants such as d-alpha tocopherol and tocopheryl acetate; and compositions based on tocopherol for treatment of bacterial diseases and methods of preparation the said compositions.

Full Text

1
Title of invention
Synergistic formulation of antioxidants and antimycobacterial agents: A method of making the same.
Technical field
The present invention is a synergistic, novel single dose powdery combi-preparation and provides an improvement in drug compositions used for treatment of tuberculosis. The said improvement is achieved by addition of antioxidant vitamins with antituberculosis agents. . The present invention has general application also in the field of anti-microbial agents. It comprises use of tocopherols, or esters of tocopherols either alone, or in combination with other antioxidants and optionally with one or more chemotherapeutic agents for treatment of bacterial diseases in general.
Background of invention:
Mycobacterium tuberculosis bacilli (MTB) are the most common causative organism for tuberculosis, (TB) in human. Estimated around one quarter to one half of world's population is infected by these organisms and cause world wide death, of some 3 million people each year. Studies before the advent of chemotherapy showed that untreated tuberculosis is often fatal. One third of patients died within 1 year after diagnosis and one half within 5 years. Of the survivors after 5 years, 60% had undergone spontaneous remission whereas remaining 40% still continued to suffer and excrete tubercle bacilli. With the advent of chemotherapeutic agents effective against mycobacteria and the experience over past years from the use of various agents alone or in combination for treatment of tuberculosis in humans as well as animals has resulted in formulation of various treatment regimes.
The aims and objectives sub served by various treatment regimes are: (1) To make a open case ( which shows presence tubercle bacilli in sputum) of pulmonary tuberculosis a closed case ( no tubercle bacilli are present in sputum), so as to stop further transmission and spread of disease to other members of society by way of droplet airborne spread, (2) To cure the affected individual of the disease, by using drugs in various combinations which either inhibit growth of mycobacteria or kill the mycobacteria, (3) To prevent emergence of multi drug resistant mycobacteria. This objective is achieved by using multiple drugs concurrently. These drugs are used either as separate dosage form or as a combination formulation in fixed dose combination preparation. WHO guidelines advocate "Directly observed therapy", wherein the drugs are given to the patient under supervision of

2 a health worker (DOT). This approach has further helped in reducing, multi drug
resistant mycobacteria by reducing chances of interrupted drug therapy and
noncompliance.
The recommended regimes are, in large part, based on evidence from clinical trials and are rated on the basis of a system developed by the United States Public Health Service (USPHS) and the Infectious Diseases Society of America (IDSA). There are four recommended regimes for treating patients with tuberculosis caused by drug-susceptible organisms.
Although these regimes are broadly applicable, there are modifications that should be made under specified circumstances, described subsequently. Each regime has an initial phase of 2 to 3 months followed by a choice of several options for the continuation phase lasting 4 to 7 months. The general approach in management of tuberculosis with first line antituberculosis agents is summarized as follows
Two drug regime - Rifampin and Isoniazid
Three drug regime - Rifampin, Isoniazid and Ethambutal
Four drug regime - Rifampin, Isoniazid, Ethambutal, Pyrazinamide and /or streptomycin.
Because of the relatively high proportion of adult hosts with tuberculosis caused by organisms that are resistant to Isoniazid, four drugs are necessary in the initial phase of 2 to 3-months where Rifampicin is always included to be maximally effective. Thus, in most circumstances, the treatment regime for patients with previously untreated tuberculosis should consist of a 2 to 3 months initial phase of four drugs, such as Isoniazid (INH), Rifampin (RIF), Pyrazinamide (PZA), and Ethambutal (EMB). To avoid development of drug resistance all these drugs should be administered concomitantly. Therefore there is need for development of single dose preparation containing all the active drugs.
The initial phase should consist of INH, RIF, PZA and EMB given daily for 2 to 3 months. Examples of circumstances in which PZA may be withheld include severe liver disease, gout, and, perhaps, pregnancy. EMB should be included in the initial phase of regime until drug susceptibility is determined.
The first-line antituberculosis medications should be administered concomitantly; split dosing should be avoided. Fixed-dose combination preparations have advantage of ease of administration over individual separate drug preparations and are shown to decrease the risk of acquired drug resistance and medication errors. Fixed-dose combination compositions can be used when treatment under DOT is given and are especially more useful when DOT is not possible. Most of the fixed dose drug combinations are Kits containing three or four individual

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drug preparations packed together in a single foil. A range of dose of these first line antituberculosis agents administered are, Rifampicin 450 mg, INH 300 mg to 600mg, Ethambutal 600 mg to 800mg, and Pyrazinamide 1200mg to 1500mg.
Second line drug regimes comprising of drugs other than first line drugs which are reserved for treatment of multi drug resistant cases i.e. those cases where the mycobacteria show resistance to the effect of one or more drugs used in first line treatment regime. The following drugs are reserved as second line antituberculosis agents such as amikacin, aminosalicylic acid, amoxicillin/clavulinic acid, azithromycin, capreomycin, ciprofloxacin, clarithromycin, clofazimine, cycloserine, ethionamide, kanamycin, norfloxacin, ofloxacin, rifabutin, streptomycin, thiacetazone. These second line drugs are not used as single agents but are always used as multiple agents given simultaneously with first line drugs to which mycobacteria are sensitive.
Alt above first line and second line drugs are either mycobactericidal or mycobacteriostatic. At no time during therapy single drug is used. It is always a multiple drug therapy.
One of the complications of tuberculosis is highly raised oxidative stress leading to increased lipid peroxidation levels which indicates extensive tissue cell damage due to free radicals. No drug used in the therapy of tuberculosis has antioxidant property and therefore these drugs can only get rid of mycobacteria but fail to control and reduce tissue damages caused by free radicals. This disadvantage of present regimen can be removed by quenching the free radicals by simultaneous use of antioxidant agents.
Vitamin E, potent chain breaking antioxidant has 6-chromanol ring structure with a phytol side chain referred more generally as tocopherol and the other as tocotrienols having double bonds in the phytol side chain at 3', 7', and 11' positions. These tocopherols and tocotrienols are present in various natural substances like wheat germ oil, soybean oil, and rice bran oil. Naturally occurring 6-chromanol ring compounds are a group of four tocopherols i.e. alpha, beta, gamma & delta tocopherols and four tocotrienols i.e. alpha, beta, gamma & delta stereo isomers, all of which are lipid soluble.
A major part of the nutritive or therapeutic value of these 6-chromanol ring compounds in humans appears to be related to its properties as an antioxidant. In acting as an antioxidant, these agents, presumably prevent oxidation of essential cellular constituents, or prevents the formation of toxic oxidation products such as peroxidation products formed from unsaturated fatty acids that have been detected in its absence.

4 6-chromanol ring derivatives are chain breaking antioxidants and are efficient
peroxy radical scavenger, which protects polyunsaturated fatty acids in cell membranes
from oxidation and thus prevent the cellular damage. These compounds also inhibit
prostaglandin synthesis and activities of protein kinase C and phospholipase A2. These
compounds are available in the form of capsules, emulsion, injections and powders.
Ascorbic acid (Vitamin C), a water soluble antioxidant is also known to be useful in treatment of scurvy, idiopathic methaemoglobinimea, in high doses to prevent viral respiratory infection and in cancer patient. It acts synergistically as antioxidant with other antioxidant agents. It is a water soluble substance having antioxidant, antiseptic and preservative properties. It is available in market in tablets and injection forms. Ascorbic acid may also modulate the immune system via its role in the regulation of hormones associated with stress.
Both these antioxidants are used as preservatives and additives in many pharmaceutical preparations to avoid oxidation of active ingredients in the preparation from atmospheric oxygen.
In the context of present invention it is necessary to understand difference between the "Preservative" purpose served by the antioxidants and the "Therapeutically effective anti-oxidant" purpose served by the same antioxidants. The "Preservative" function is a very limited objective for which the amount of anti-oxidant required per dose is very small than when it is desired to serve therapeutically effective antioxidant" purpose which requires a very large dose of the same antioxidants per dose and the effect is visible in the patients by way of lowering lipid peroxidation levels in the blood.
There is evidence that in many diseases of respiratory system like asthma, chronic obstructive pulmonary disease, lung cancer, there is increase in free radical generation and hence increased oxidative stress. Increased oxidative stress and raised levels of free radicals are known to damage and injure cell wall of bacterial as well human cells leading to cell death. Mycobacterium tuberculosis due to its specialized cell wall structure along with presence of abundant catalase enzyme which can destroy free radicals is protected from injury, due to raised free radical levels in host, whereas host cell membrane remains susceptible to such damages.
All known antimycobacterial agents either kill or inhibit growth of mycobacteria. None of antimycobacterial agents have antioxidant properties. On the contrary one of the most commonly used antimycobacterial agent Isonicotinic acid hydrazide is shown to be pro oxidant.

5
Anti-oxidants have been incorporated in the compositions for treatment of tuberculosis as disclosed in US 6277836 B1 and WO 2002/087547 A1 in a small amount with a purpose of providing protection from oxidation during storage to the active components of the composition like Rifampicin. Its purpose is not to provide any therapeutic dose of antioxidant to the patient.
In SU 1577795 A1, it is reported that the use of Isoniazid and Ethambutal combined with alpha tocopherol and sodium thiosulphate in the treatment of infiltrative tuberculosis increases the efficiency of the treatment and prevents side effects. However the total duration of treatment still remains 6-7 months. The advantage quoted is reduction of antimycobacteriai drugs used from 3 or more to 2. Further efficacy of tocopherol independent of the two antimycobacteriai drugs was not detected. The therapy in this patent, however, is not based primarily on synergy provided by antioxidant and antimicrobial property of tocopherol and there is no claim on a single dose composition.
Today even with the advent of Rifampicin and a four drug regime the duration of treatment is minimum 6 months and intensive phase of treatment is for 2-3 months. There is still a need for better drug regime that can increase efficacy of treatment, prevent side effects, and reduce the duration of treatment more particularly the duration of intensive phase having an aim of converting a 'open case* of tuberculosis to "closed case" so that spread of infection to other healthy members of society can be prevented. Even 30 to 40 yrs after Rifampicin was introduced no new and safer antimycobacteriai drug of efficacy similar to or more than Rifampicin has been invented.
Pulmonary tuberculosis has been shown to be associated with significantly raised levels of oxidative stress resulting in extensive tissue damage and therefore there is need for an agent which can reduce the oxidative stress and at the same time have an antibacterial activity.
Summary of Invention:
It was surprisingly found that efficacy of present antituberculosis agent based treatment improved when the same antituberculosis agents were combined with tocopherols or esters of tocopherol and other anti-oxidants used in such a dose that would result into lowering of blood lipid peroxidation levels. The antioxidant agent's dose used in this invention was far higher than the dose of anti-oxidants that would be used in the compositions to achieve the purpose of protecting the active ingredients of the composition from other ingredients of the same composition and oxidative effect of atmospheric oxygen during the storage. Further, the extent of effect achieved in this invention was surprisingly

6

beyond what would be expected from known effects of antioxidants used. The effect
led to rapid reduction and ultimately to disappearance of tubercle bacilli from the sputum
and the patients became a "closed case" in less than a month. This dose was far greater
than the small dose of these antioxidants used in the compositions as preservatives and
additives, meant for providing protection from oxidation in storage by oxygen and other
chemicals present in the composition.
As expected from the therapeutic dose of antioxidants used in this invention, the lipid peroxidation levels in the blood fell rapidly to desired level, which would give more vitality for the cells to fight the disease causing organisms. However, the speed with which the tubercle bacilli disappeared from the sputum making the patients "closed cases" was surprising and could not be explained just on the basis of increase in viability and increase in strength of the host cells on account of nutritional effects of tocopherols and esters of tocopherols.
When investigated further, it was found that tocopherols and esters of tocopherols have anti-mycobacterial property, which worked synergistically in eliminating the disease organisms from the patients. It is obvious that since tocopherols are not metabolic intermediates, but is a chemical entity that has an anti-microbial activity i.e. in effect it shall not be specific to certain bacteria only, but anti-bacterial in general, it shall be useful not only in control of tuberculosis but to control other diseases due to other microorganisms too. Being itself an antioxidant vitamin, it shall have little or no side effects.
Using this anti-mycobacterial property of tocopherols and esters of tocopherols, very effective anti-tuberculosis compositions were prepared by combining in a single unit dose preparation, anti-tuberculosis agents and tocopherol or esters of tocopherol in dosage effective as anti-oxidant for lowering lipid peroxidation levels in the blood and other antioxidants such as ascorbic acid. Although a six drug combination has been tested in this investigation, any obvious variation, such as use of tocopherols or ester of tocopherols alone or in combination with one or more chemotherapeutic agents active against tuberculosis and several other bacterial diseases are included in this invention. It is obvious that an anti-mycobacterial property is basically an anti-bacterial property which would be available against a range of other bacteria, pathogenic or otherwise.
The present invention also focuses on a unique single dose composition comprising of all the active ingredients taken together along with required pharmaceutical additives and dispensed in a sachet for oral use. This composition has following advantages:

7
1) The contents of sachet can be dissolved in any beverage or water just
before use and the palatable liquid preparation is consumed. Such consumption can be facilitated further by making the contents effervescent and with pleasing flavors and taste.
2) No disintegration time is required before the drug can be absorbed as required in other solid preparations. This means early and better therapeutic blood levels of active agents are achieved giving better therapeutic response.
3) As all the drugs are taken together no chances of missing the dose of one or other drug as can occur with separate individual drug preparation.

4) The composition of this invention differs from other antituberculosis fixed dose combinations in having addition of the antioxidant agents in therapeutically active concentration and not merely using antioxidants as therapeutically inactive substances as preservative or additives.
5) The present single dose preparation has shown synergistic effect by reducing the period required for making sputum free of mycobacteria to one month which is normally two to three months with all other treatment regimes currently in use.
6) The therapeutic use of antioxidants in present composition prevents the tissue
damage due to increased oxidative stress associated with the disease.
7) The composition of this invention also differs from earlier compositions in that all
the constituents of the combination treatment are combined in a single dose, and they are
not required to be combined at the time of taking. This is strategically very important from
the point of view of convenience.
Detailed description of invention:
Like many other diseases, one of the complications of the tuberculosis is exposure of the patient's cells to oxidative stress, it was thought that use of antioxidants at therapeutically effective level to reduce lipid peroxidation level in the blood shall relieve the patients of this stress and whether that shall help in increasing vitality of the cells leading to a relief to the patients from the effects of tuberculosis and help them in responding to the antituberculosis treatment more efficiently and with reduced side effects.
Finding of the studies that tocopherols have anti-microbial activity of their own was totally unanticipated and surprising. Thus, the present invention relates on one hand, to the use of antioxidant, such as Vitamin E, tocopheryl acetate, all-rac-alpha tocopherol and /or RRR-alpha-tocopherol and pharmaceutical^ acceptable salts or derivatives thereof as agents having a mycobactericidal property, which is a new activity, useful in the treatment of tuberculosis as well as other diseases caused by bacteria, and on the other hand a novel single dose combination for use in the treatment of tuberculosis incorporating, some

8
antituberculosis agents along with antioxidant-antimycobacterial agent and other
antioxidants, in a single dosage compound powder composition and a process for
manufacture of the same. The combined formulation has synergistic, antimycobacterial
action and at the same time causes reduction in the oxidative stress leading to decreased
host cell injury and damage along with early conversion to "closed case* leading to
reduction in duration of treatment of intensive phase.
The present invention describes a new, and useful pharmaceutical composition, comprising of first line antituberculosis agents such as Rifampicin, Isonicotinic acid hydrazide, Ethambutal, and Pyrazinamide and antioxidant-antimycobacterial agents such as vitamin E, tocopherol acetate, all-rac-alpha tocopherol and /or RRR-alpha-tocopherol or pharmaceutically acceptable salts thereof and ascorbic acid, for use in the treatment of tuberculosis producing a synergistic effect in comparison with the use of above antituberculosis agents given together, without antioxidants. This is the combination which has actually been clinically experimented. However, its obvious variation will include vitamin E alone or with any other one or more antituberculosis agents. Although the present trend is of a therapy based on combination of four or more anti-tuberculosis agents , with the unexpected factor of anti-microbial effect of the therapeutic quantities of vitamin E with or without other anti-oxidants, the possibility of future change in the perspective of efficacy of single or more anti-tuberculosis agents given as a single dose combination along with therapeutically effective dose of vitamin E with or without other anti-oxidants in favor of use of less or even one chemotherapeutic agent are real and they are also included in this invention.
The present invention also further describes a new therapeutically useful mycobactericidal activity to vitamin E, tocophery1 acetate, derivatives and pharmaceutically acceptable salts used in therapy such as all-rac-alpha tocopherol, RRR-alpha-tocopherol. The present invention further describes a process for preparing a single dosage compound powder composition containing these antimycobacterial substances and the antioxidants.
Since the reduction in level of lipid peroxidation in the patients, accompanied by rapid reduction in tubercle bacilli in the sputum of the patients was so dramatic and surprising, that it did not account for a simple anti-oxidant effect of tocopherol administered. A culture of mycobacteria susceptible to first line antituberculosis agents as well as mycobacteria resistant to first line antituberculosis agents responsible for tuberculosis was made. Total inhibition of growth of mycobacteria of both the types was very surprising which indicated that their effect was also positively anti-microbial in nature. It is obvious extension of the observed fact that mycobacteria basically being bacteria, many other bacteria shall be susceptible to anti-microbial effect as observed. Since tocopherols are not metabolic

Q
analogues, their action is not likely to be of the type of antibiotics of any specific type
against specific type of bacteria only, but a general anti-bacterial action at the concentration
used. At that concentration, it has nutritional benefits to human beings, having a lipid
peroxidation lowering effect, and a general bactericidal effect. This has given rise to
prospects of single dose compositions for tuberculosis in presently standardized therapies
of combination of chemotherapeutic agents. It has also opened door for possible use of
tocopherols and esters of tocopherols as chemotherapeutic agents useful alone or in
combination with other drugs for treatment of tuberculosis as well as other diseases caused
by bacteria..
The present invention further describes a new method or approach in the treatment of tuberculosis over the prior art by addition and use of antioxidants for prevention of lung tissue damage caused by liberated free radicals during the disease process and the course of treatment. The pharmaceutical composition is intended for oral use.
The following examples are included to demonstrate preferred embodiments of the present invention. For those of skill in the art, that the techniques disclosed in the examples which follow, represent techniques to function well in the practice of invention and thus can be considered to constitute preferred modes for its practice. However those of skill in the art should, in the light of present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of invention.
Example"!:
Newly diagnosed cases suffering from pulmonary tuberculosis were assessed for status of oxidative stress in comparison with the matching healthy volunteers by performing analysis of blood samples for lipid peroxidation levels using, Draper and Hadley's (1990), double heating method. Lipid peroxidation is used as marker of cell membrane damage. It was observed that the lipid peroxidation levels were raised in pulmonary tuberculosis patients significantly when compared with matching human healthy volunteers.
This observation, suggest increase in free radical liberation and oxidative stress in patients of pulmonary tuberculosis. This observation provides a basis and rationale for an indication to use antioxidants.
Example 2:
About 100 newly diagnosed cases suffering from pulmonary tuberculosis demonstrating positive sputum smear, for acid fast bacilli (AFB), by Ziehl-Neelson staining

10
procedure, and positive x-ray, lung involvement were included in the study. They
were randomly divided in two groups. Control group received first line four drugs regime,
comprising of Rifampicin 450 mg, Isoniazid 300 mg, Pyrazinamide 1500mg, and
Ethambutal 800mg, once daily by oral route for a period of two months.
Study group received first line four drug regime as detailed above and two antioxidants d- alpha tocopherol in the dose of 400 IU , or tocopheryl acetate in the dose of 400mg and ascorbic acid in the dose of 500mg once daily by oral route for a period of two months. The outcome of clinical improvement was accessed by following parameters in every patient of both the groups and findings were compared and analyzed for statistical significance at the end of successful completion of treatment for two months.
Sputum smear AFB staining test - In both the groups sputum of all the patients were analyzed for the presence of acid fast bacilli using standard Zeil-Nelson hot stain method at the end of 15 days, 30 days and 60 days. The reports were documented as positive for presence of bacilli and negative as absence of bacilli. The results of both the groups where compared at the end of 15 days, 30 days, and 60 days.
At the end of 15 days treatment in study group 50 % patients showed sputum smear negative for acid fast bacilli as compared to 14 % in control group. A similar pattern was observed at the end of 30 days treatment, 86% versus 50% and 100% versus 86 % at the end of 60 days treatment in study group versus, control group respectively. The study group showed much earlier and augmented sputum smear negativity for acid fast bacilli, which is an evidence of a synergistic antimycobacterial effect, of the drug combination, used in study group.
Lipid peroxidation levels (LPO) —Blood samples were collected from patients included in both the groups before the start of treatment but after confirmation of diagnosis of pulmonary tuberculosis and at the end successful completion of treatment of 60 days. The oxidative stress is indicated by lipid peroxidation was reported in terms of Malondialdehyde (MDA) nmol/gm of hemoglobin using Draper and Hadley (1990) double heating method.
All the patients in study as well as control groups showed elevated LPO levels at the start of treatment. The LPO levels after 60 days showed significant reduction in study group patients receiving antioxidants along with four antituberculosis agents, indicating improvement in oxidative status. However in control group, patients showed a further rise in levels of LPO, indicating deterioration of oxidative status and confirming need for use of

11
antioxidants for improvement in oxidative status and prevention of cell membrane injury and damage.
Radiological investigations - Chest X-ray of all the patients from both groups was done at the start of treatment and after the completion of treatment of 60 days. The findings were visually analyzed and compared for each patient before and after the treatment. The findings were reported as favorable & improved or unfavorable and deteriorated. All the patients from study group showed favorable response as compared to 80% in control group suggesting a lesser damage to lungs in study group.
Example 3
In vitro Studies - Lowenstein and Jenson (LJ) medium is used as a selective medium to grow mycobacteria and test sensitivity, to antimycobacterial agents. A set of (LJ) media was prepared by incorporating double dilutions of d- alpha tocopherol, and another set incorporating double dilutions of tocopheryl acetate yet another set without addition of any drug was prepared. These slopes were inoculated with known freshly cultured virulent strain of mycobacterium tuberculosis. Growth was observed at the end of each week for six weeks after incubating the media at 37 degrees for the entire period.
A control medium slant without any drug was inoculated with same freshly prepared culture strain of mycobacterium tuberculosis. At the end of 6 weeks no growth was observed on (LJ) slopes containing the antioxidants like d-alpha tocopherol or Tocopheryl acetate in a concentration of 100 microgram per ml whereas the control slant showed a carpet growth.
The above experiment was repeated with known strains available in laboratory showing resistance to one drug i.e. Rifampicin or to two drugs i.e. .Rifampicin and Isoniazid and at the end of 6 weeks it was observed that both these strain resistant to either Rifampicin or Rifampicin and Isoniazid did not grow in (LJ) slopes containing antioxidants like d-alpha tocopherol or tocopheryl acetate in a concentration of 100 microgram per ml whereas the control slant showed a carpet growth.
The results of in vitro studies have confirmed that both d-alpha tocopherol and tocopheryl acetate have mycobactericidal activity. The in vivo studies on patients of pulmonary tuberculosis also confirm these results based on the conspicuously enhanced sputum smear negativity for acid fast bacilli at the end of 15 days.

12
From all the examples cited above it is confirmed that there is more oxidative
stress in patients of pulmonary tuberculosis and a need for use of antioxidants to reduce
cell membrane injury to the host cells. The above cited examples further prove that the d-
alpha tocopherol and tocopheryl acetate have mycobactericidal activity and therefore can
be used along with other antimycobacterial agents The above cited examples further prove
that such a use a combination of antimycobacterial agents and antioxidants referred in
example is synergistic.
Example 4:
A known drawback of use of multiple drugs, is reduction in patient compliance i.e. failure to adhere to treatment protocol. This drawback is removed if multiple drugs are combined in single dosage composition. The inventors have further developed a process for preparation of pharmaceutical composition wherein two to four required primary line antituberculosis agents in the therapeutically desirable quanta can be combined along with therapeutically desirable quanta of the antioxidants like d-alpha tocopherol or tocopheryl acetate optionally with therapeutically desired quantum of ascorbic acid in a compound powder unit dose composition with the required additives, flavoring agents and suspending agents.
The range of doses selected for individual agents per single unit dose preparation are as follows
Rifampicin 3-{[(4-Methyl-1-piperazinyl) imino] methyl} rifamycin SV. 450mg powder
Isonicotinic acid hydrazide (4-Pyridinecarboxylic acid hydrazide) 300 or 600mg powder
Pyrazinamide (Pyrazine-2-carboxamide) 1200 or 1500mg powder
Ethambutal d-N, N'-bis (1-hydromethylpropyl) ethylene diamine; 600 or 800mg powder
d- alpha tocopherol acetate - 400 IU or 800 IU powder
Or Tocopheryl acetate ~ 400 mg or 800 mg powder
Ascorbic acid — 125, or 250, or 500mg powder
Saccharin as sweetening agent as per taste
Flavoring agents - lemon, orange, anise, liquorice, raspberry as per choice.
Permissible fillers and adsorbents in sufficient quantity
This particular mixing of aforesaid substances was done by using mixture machines used in pharmaceutical manufacturing practice and the contents were homogeneously mixed so that the end product is free flowing powder. The prepared free flowing compound powder is further weighed into individual unit dose packs based on the total weight of individual agents added together required in the single unit dosage fixed dose preparation and sealed in appropriate air tight, light impervious sachet for further use.

13
Claims
We claim,
1. A single dosage composition of drugs comprising:
a. Antituberculosis agents in therapeutically effective dosage in a drug regime
which is selected from one or more of first line antituberculosis agents and second
line antituberculosis agents; where
a.1) first line antituberculosis agents consist of a single antimicrobial agent or a two drug combination regime containing Rifampicin or Rifabutin or Rifapentine and Isoniazid; a three drug combination regime as Rifampicin or Rifabutin or Rifapentine, Isoniazid and Ethambutol; a four drug combination regime as Rifampicin or Rifabutin or Rifapentine, Isoniazid, Ethambutol and Pyrazinamide ; a.2) second line antituberculosis agents consist of one or more of second line drugs including ethionamide, cycloserine, p-aminosalicylic acid, macrolides preferably azithromycin, clarithromycin, quinolones preferably ciprofloxacin, Levofloxacin, Moxifloxacin, Fleroxacin and sparfloxacin,
b. One or more of drug or drugs with antioxidant and antimicrobial property, in
therapeutically effective dosage, wherein
b.1) the therapeutically effective dosage for antioxidant effect comprising improvement in therapeutic parameters related to oxidative stress, related to tissue damage including a dosage effective for achieving lowering of lipid peroxidation levels in blood in a treatment period of around 60 days or less, and b.2) the therapeutic effective dosage for antimicrobial property comprising improvement in therapeutic parameters related to infection from disease causing microorganisms including a dosage effective for achieving sputum negativity in a period of around 15 days;
c. One or more of antioxidant or antioxidants in therapeutically effective dosage
for antioxidant effect comprising improvement in therapeutic parameters related to
oxidative stress, related to tissue damage including a dosage effective for achieving
lowering of lipid peroxidation levels in the blood in a treatment period of 60 days or
less;
d. Optionally containing pharmaceutical^ acceptable excipients including
binders, sweeteners, flavoring agents, fillers, adsorbents, diluents, coating agents,
disintegrating agents, preservatives, lubricants, controlled release agents , dispersing
and or suspending agents, emulsifying agents, and effervescent agents.
2. Composition of claim no. 1 wherein the range of use of the said antituberculosis
agents individually are - Rifampicin used within a range of 60mg to 600mg , Rifabutin used within a range of 150 mg to 600 mg, Rifapentine used within the range of 300 to 900mg, Isoniazid used within a range of 60 mg to 1500mg, Ethambutol, used within a range of 60mg

14
to 2500mg , Pyrazinamide, used within a range of 100mg to 2000mg , Ethionamide used within a range of 100mg to 1000mg, cycloserine used within a range of 250mg to 750 mg, p-aminosalicylic acid used within a range of ( 8g to 12g), macrolide preferably azithromycin used within a range of 250 to 1500mg, clarithromycin, used within a range of 250mg to 1500mg, quinolones preferably ciprofloxacin, used within a range of 500 to 2000mg, ofloxacin, used within a range of 100 to 1200mg, fleroxacin used within a range of 150 mg to 1000mg, sparfloxin used within a range of 150mg to 1000mg.
3. Composition of claim no.1 wherein the said antioxidant or antioxidants with
antimicrobial properties of sub claim b. of claim 1 is Vitamin E, comprising homologues of all
natural as well as synthetic tocols and tocotrienols such as 5,7,8-Trimethy! tocol, 5,8-
Dimethyl tocol, 7,8- Dimethyl tocol, 8-Methyl tocol, 5,7,8-Trimethyl tocotrienol, 5,8-Dimethyl
tocotrienol, 7,8-Dimethyl tocotrienol, 8-Methyl tocotrienol and their derivatives including esters
further including dl-a-Tocopheryl acetate, d.a-Tocopheryl acetate, dl-a-Tocopheryl acid
succinate, d.a-Tocopheryl acid succinate, dl-a-Tocopheryl nicotinate, d,a-Tocopheryl
nicotinate, characterized by derivatives of 6-chromanol ring with phytol side chain qualitatively
exhibiting biological activity of natural alpha tocopherol.
4. Composition of claim no. 3 wherein Vitamin E is d alpha tocopherol or tocopheryl acetate used within the range of 200 IU to 2000 IU, more particularly at 400 IU to 800 IU per dose.
5. Composition of claim no. 1 wherein the said antioxidant or antioxidants of sub claim c of claim 1 are selected from one or more of
a. Vitamin E, comprising homologues of all natural as well as synthetic tocols
and tocotrienols including 5,7,8-Trimethyl toed, 5,8-Dimethyl tocol, 7,8- Dimethyl
tocol, 8-Methyl tocol, 5,7,8-Trimethyl tocotrienol, 5,8-Dimethyl tocotrienol, 7,8-
Dimethyl tocotrienol, 8-Methyl tocotrienol and their derivatives including esters further
including dl-a-Tocopheryl acetate, d,a-Tocopheryl acetate, dl-a-Tocopheryl acid
succinate, d,a-Tocopheryl add succinate, dl-a-Tocopheryl nicotinate, d.a-Tocopheryl
nicotinate, characterized by derivatives of 6-chromanol ring with phytol side chain
qualitatively exhibiting biological activity of natural alpha tocopherol used within a
range of 200 IU to 2000 IU, more particularly within a range of 400 IU to 800 IU per
dose;
b. ascorbic acid or sodium ascorbate used within the range of 50 mg to
1000mg, more particularly within a range of 150 to 500 mg per dose.
6 Composition of claim 1 containing the said pharmaceutically acceptable excipients,
which include sweetening agents, fillers, binders, diluents, coating agents, disintegrating agents, preservatives, lubricants, time delay agents, dispersing and or suspending agents, emulsifying agents, effervescent agents and adsorbents added,

15
when all the added excipients are put together, within pharmaceutical^ permissible range of 0.5 to 1 % of total weight of a single dose.
7. A single dose composition of claim 1 comprising:
a. Anti tuberculosis agents in following quantities -
i. Rifampicin powder 300 to 450 mg
j. Isoniazid powder 300 to 600 mg
k. Pyrazinamide powder 1200 to 1500 mg
I. Ethambutal powder 600 to 800 mg
b. Tocopheryl acetate 400 mg to 800 mg or d-alpha tocopherol 400IU to 800IU
c. Ascorbic acid 125 to 500 mg
d. Pharmaceutically acceptable excipients including sweetening agent saccharin
added as per taste; flavoring agents as per choice including lemon, orange, anise,
liquorice, raspberry; permissible fillers, adsorbents, diluents, coating agents,
disintegrating agents, preservatives, lubricants, time delay agents, dispersing or
suspending agents, emulsifying agents, and effervescent agents in sufficient quantity,
all excipients put together added in the range of 0.5 to 1 % of total weight of a single
dose.
8 An oral composition of claim no. 1 containing effervescent agents in amounts sufficient to produce effervescence when the composition is added to a suitable liquid; the said agents preferably comprising sodium bicarbonate and an addulant including tartaric or citric acid chosen in amount such that the effervescence generated effectively disperses the constituents of the composition in the said liquid and a neutral or slightly acidic pH is established after completion of effervescence.
9 Composition of claim 8 wherein sodium bicarbonate is preferably taken in the range of sum of equivalent weight of acidulants added, acidulant is taken equal to or in slight excess to the equivalence to the sodium bicarbonate taken and the said suitable liquid is one or more of water, milk, fruit juice, a beverage.
10 An oral dosage form essentially containing ingredients of claim 1 including one or more of:
a. a satchet containing powder or granules of the ingredients, or
b. a package containing a number of tablets collectively dispensing the
recommended quantity of the ingredients of composition of claim 1 as a
single dose per package, or

c
c. a package containing a number of capsules containing powder or granules
of the ingredients collectively dispensing the recommended quantity of the
ingredients of composition of claim 1 as a single dose per package, or
d. a satchet containing a volume of liquid or a syrup as such or packaged in a
convenient number of capsules collectively dispensing the recommended
quantity of the ingredients of composition of claim 1 as a single dose per
package, or
e. the like..
11 A method of use, alone or in combination with other Active Pharmaceutical Ingredients including antimicrobial agents, of Vitamin E, comprising homologues of all natural as well as synthetic tocols and tocotrienols including 5,7,8-Trimethyl tocol, 5,8-Dimethyl tocol, 7,8-Dimethyl tocol, 8-Methyl tocol, 5,7,8-Trimethyl tocotrienol, 5,8-Dimethyl tocotrienol, 7,8-Dimethyl tocotrienol, 8-Methyl tocotrienol and their derivatives including esters further including dl-a-Tocopheryl acetate, d,a-Tocopheryl acetate, dl-a-Tocopheryl acid succinate, d.a-Tocopheryl acid succinate, dl-a-Tocopheryl nicotinate, d,a-Tocopheryl nicotinate, characterized by derivatives of 6-chromanol ring with phytol side chain qualitatively exhibiting biological activity of natural alpha tocopherol including tocopherols or tocotrienols or tocopheryl acetate at dosages therapeutically effective for antimicrobial effect.
12 A Pharmaceutical Composition comprising incorporation, alone or in combination with other Active Pharmaceutical Ingredients including antimicrobial agents, of vitamin E, comprising homologues of all natural as well as synthetic tocols and tocotrienols including 5,7,8-Trimethyl tocol, 5,8-Dimethyl tocol, 7,8-Dimethyl tocol, 8-Methyl tocol, 5,7,8-Trimethyl tocotrienol, 5,8- Dimethyl tocotrienol, 7,8-Dimethyl tocotrienol, 8-Methyl tocotrienol and their derivatives including esters esters further including dl-a-Tocopheryl acetate, d,a-Tocopheryl acetate, dl-a-Tocopheryl acid succinate, d,a-Tocopheryl acid succinate, dki-Tocopheryl nicotinate, d,a-Tocopheryl nicotinate, characterized by derivatives of 6-chromanol ring with phytoi side chain qualitatively exhibiting biological activity of natural alpha tocopherol containing tocopherols or tocotrienols or tocopheryl acetate in therapeutic amounts effective as antimicrobial agents in general and as anti-mycobactericidal agents in particular.
13 A Pharmaceutical Composition of claim 12 containing one or more of
a. tocopherol esters including one or more of tocopherol acetate, tocopheryl
succinate or tocopheryl nicotinate used collectively in the range of 200 to
2000 mg, more particularly in the range of 400 to 800mg
b. tocopherols or tocotrienols used collectively in the range of 200IU to 2000
IU, more particularly in the range of 400 IU to 800 IU.
14 A process of preparing Composition of claim no. 1, claim no. 7, claim 12 and claim no.
13 wherein:

17

15

a ingredients of the composition are taken as powder, or granules weighed and mixed, using mixer machines used normally in pharmaceutical manufacturing, to a homogeneous free flowing form,
b. Weighing the powder or granules in quantities envisaged by one of the claims
of claim no. 1, claim no. 7, claim no. 12 or claim no. 13, in a single unit dose
c. Sealing the single dose in one or more forms of containers including sachets,
bottles, which are preferably air tight and light impervious.
A method of treating tuberculosis by using compositions of claim 1 or claim 7.

16 A method of treating microbial diseases using compositions of claim 1, claim 7, claim 12 or claim 13.

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18
Abstract
The present invention pertains to a composition containing tocopherols as antimicrobial agents for treatment of bacterial diseases in general and tuberculosis in particular. Tocopherol may be used alone or in combination with one or more of antibiotics, or antimicrobial agents as active pharmaceutical Ingredients and with one or more of other anti-oxidants in quantities which are therapeutically effective to generate either antimicrobial or anti-oxidant effect or both the effects; the antioxidant effect being reflected in lowered lipid peroxidation levels in blood of tuberculosis patients in mammals. A tocopherol based anti-tuberculosis single dose combination including tocopherol (or its esters such as tocopheryl acetate), ascorbic acid, Rifampicin, Isoniazid, Pyrazinamide, and Ethambutal and method of its preparation are disclosed. The six drug composition described showed synergistic mycobactericidal activity with reduction in oxidative stress, leading to reduction in lung cell damages. The present invention further demonstrates mycobactericidal activity to the antioxidants such as d-alpha tocopherol and tocopheryl acetate; and compositions based on tocopherol for treatment of bacterial diseases and methods of preparation the said compositions.

Documents:

390-MUMNP-2006-ABSTRACT(25-5-2009).pdf

390-mumnp-2006-abstract(granted)-(8-1-2010).pdf

390-mumnp-2006-abstract.pdf

390-MUMNP-2006-CANCELLED PAGES(25-5-2009).pdf

390-mumnp-2006-cancelled pages(26-8-2009).pdf

390-MUMNP-2006-CLAIMS(25-5-2009).pdf

390-mumnp-2006-claims(amanded)-(26-8-2009).pdf

390-MUMNP-2006-CLAIMS(AMENEDED)-(25-5-2009).pdf

390-mumnp-2006-claims(granted)-(8-1-2010).pdf

390-mumnp-2006-claims.pdf

390-mumnp-2006-correspondance-others.pdf

390-mumnp-2006-correspondance-received.pdf

390-MUMNP-2006-CORRESPONDENCE(25-5-2009).pdf

390-mumnp-2006-correspondence(26-8-2009).pdf

390-mumnp-2006-correspondence(ipo)-(9-2-2010).pdf

390-mumnp-2006-description (complete).pdf

390-MUMNP-2006-DESCRIPTION(COMPLETE)-(25-5-2009).pdf

390-mumnp-2006-description(granted)-(8-1-2010).pdf

390-mumnp-2006-form 18(5-6-2006).pdf

390-mumnp-2006-form 2(25-5-2009).pdf

390-mumnp-2006-form 2(granted)-(8-1-2010).pdf

390-MUMNP-2006-FORM 2(TITLE PAGE)-(25-5-2009).pdf

390-mumnp-2006-form 2(title page)-(granted)-(8-1-2010).pdf

390-mumnp-2006-form 5(5-6-2006).pdf

390-mumnp-2006-form-2.doc

390-mumnp-2006-from-1.pdf

390-mumnp-2006-from-2.pdf

390-mumnp-2006-from-3.pdf

390-mumnp-2006-from-5.pdf

390-mumnp-2006-wo international publication report(5-4-2006).pdf

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Patent Number

237837

Indian Patent Application Number

390/MUMNP/2006

PG Journal Number

3/2010

Publication Date

15-Jan-2010

Grant Date

08-Jan-2010

Date of Filing

05-Apr-2006

Name of Patentee

SHELGAONKAR, Meena

Applicant Address

"Manoram"242, Bhausaheb Survey Nagar, Ring Road, Nagpur 440 022

Inventors:

#	Inventor's Name	Inventor's Address
1	SHELGAONKAR, Meena	"Manoram"242, Bhausaheb Survey Nagar, Ring Road, Nagpur 440 022
2	SHELGAONKAR, Shriniwas	"Manoram"242, Bhausaheb Survey Nagar, Ring Road, Nagpur 440 022

PCT International Classification Number

N/A

PCT International Application Number

PCT/IN2004/000278

PCT International Filing date

2004-09-08

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	942/MUM/2003	2003-09-10	India