The present invention relates to pharmaceutical composition comprising L- Carnosine or pharmaceutically acceptable salts or derivatives thereof, and Glutathione, its precursors or pharmaceutically acceptable salts or derivatives thereof. The present invention also relates to method of treating disease or disorder in patient in need of such therapy, wherein treatment comprises administration of L-Carnosine or pharmaceutically acceptable salts or derivatives thereof, and Glutathione, its precursors or pharmaceutically acceptable salts or derivatives thereof, in same or different compositions to humans or animal.

BACKGROUND OF INVENTION

Oxidative stress, ion balance and pH balance are some the most critical factors involved in the major acute and chronic physiological imbalance. The depletion of powerful antioxidant renders cells particularly vulnerable to oxidative stress. The resulting damage is the key step in the onset and progression of many disease states.

Several endogenous and exogenous compound are known to protect against oxidative stress. These endogenous and exogenous compound are combined with other therapies like antibiotics, anti-cancer drugs to achieved combined desired therapy goals.

L-Carnosine is used as protective agent for many therapies. Glutathione is endogenous agent involved in anti-aging and other biochemical pathways providing shielding effect upon administration. While both these compounds are known to be administrated individual for their respective mechanism of action, individual administration has a limitation of affecting only their respective physiological involvement.

Therefore, there is an urgent need for the improved therapy with high anti oxidant, anti-aging protective effect for many disease or disorders including diabetic nephropathy and chronic kidney disease.

Till date, pharmaceutical composition comprising L-Carnosine or pharmaceutically acceptable salts or derivatives thereof, and Glutathione, its precursors or pharmaceutically acceptable salts or derivatives thereof are not known.

DESCRIPTION OF THE DRAWINGS

Figure 1 : Effect of combination of L- camosine and S-acetyl-L-glutathione on H2O2 scavenging activity.

Figure : 2 Effect of combination of L- carnosine and S-acetyl-L-glutathione on DPPH Scavenging activity.

Figure 3: Effect of 1-camosine and s-acetyl -1-glutathione on MDA levels in kidney tissues.

Figure 4: Effect of 1-camosine and s-acetyl-l-glutathione on catalase levels in kidney tissue.

Figure 5: Effect of 1-camosine and s-acetyl-l-glutathione on SOD levels in kidney tissues.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to pharmaceutical composition comprising L- Carnosine or pharmaceutically acceptable salts or derivatives thereof, and Glutathione, its precursors or pharmaceutically acceptable salts or derivatives thereof. The active ingredient as per present invention are used in therapeutically effective amount.

"Therapeutically effective amount" or "effective amount" refers to the amount of a pharmaceutically active agent when administered to a patient, is sufficient to affect such treatment for the disease. The therapeutically effective amount will vary depending on the disease and its severity, and the age, weight, and other conditions of the patient to be treated.

The term "pharmaceutical compositions" herein refers to any composition for administration to human or animal by any route and includes but are not limited to immediate release, delayed release, extended release and pulsed-release.

In an embodiment, the present invention relates to a pharmaceutical composition comprising a. therapeutically effective amount of L-Carnosine or pharmaceutically acceptable salts or derivatives thereof, and b. therapeutically effective amount of Glutathione, its precursors or pharmaceutically acceptable salts or derivatives thereof.

In an embodiment, the present invention relates to a pharmaceutical composition consisting of a. therapeutically effective amount of L-Carnosine or pharmaceutically acceptable salts or derivatives thereof, and b. therapeutically effective amount of Glutathione, its precursors or pharmaceutically acceptable salts or derivatives thereof c. one or more suitable inactive ingredients.

In an embodiment, the present invention relates to a pharmaceutical composition comprising c. therapeutically effective amount of L-Carnosine or pharmaceutically acceptable salts or derivatives thereof, and d. therapeutically effective amount of S-Acetyl Glutathione or pharmaceutically acceptable salts or derivatives thereof.

In an embodiment, the present invention relates to a pharmaceutical composition consisting of d. therapeutically effective amount of L-Carnosine or pharmaceutically acceptable salts or derivatives thereof, and e. therapeutically effective amount of S-Acetyl Glutathione or pharmaceutically acceptable salts or derivatives thereof f. one or more suitable inactive ingredients.

In a preferred embodiment, the present invention relates to a pharmaceutical comprising a. L-Carnosine or pharmaceutically acceptable salts or derivatives thereof in an amount equivalent to 50mg to 400mg of L-Carnosine, and b. S-Acetyl Glutathione or pharmaceutically acceptable salts or derivatives thereof in an amount equivalent to lOOmg to 600mg of

S-Acetyl Glutathione.

In a preferred embodiment, the present invention relates to an oral pharmaceutical composition comprising a. L-Carnosine in an amount between 50mg to 400mg, and b. S- Acetyl Glutathione in an amount between lOOmg to 600mg.

In a preferred embodiment, the present invention relates to an oral pharmaceutical composition comprising a. L-Carnosine in an amount of 200mg, and b. S- Acetyl Glutathione in an amount of 300mg. In a preferred embodiment, the present invention relates to an oral pharmaceutical composition comprising a. L-Carnosine in an amount of 200mg, and b. S- Acetyl Glutathione in an amount of lOOmg.

In an embodiment, the present invention relates to a method of treating disease or disorder in patient in need of such therapy, wherein treatment comprises administration of a. therapeutically effective amount of L-Carnosine or pharmaceutically acceptable salts or derivatives thereof, and b. therapeutically effective amount of Glutathione, its precursors or pharmaceutically acceptable salts or derivatives thereof, in same or different compositions to humans or animal.

In an embodiment, the present invention relates to a method of treating disease or disorder in patient in need of such therapy, wherein treatment comprises administration of a. therapeutically effective amount of L-Carnosine or pharmaceutically acceptable salts or derivatives thereof, and b. therapeutically effective amount of S-Acetyl Glutathione or pharmaceutically acceptable salts or derivatives thereof, in same or different compositions to humans or animal.

In an embodiment, the present invention relates to a method of treating Diabetic Nephropathy or Chronic Kidney Disease, wherein treatment comprises administration of a. therapeutically effective amount of L-Carnosine or pharmaceutically acceptable salts or derivatives thereof, and b. therapeutically effective amount of S-Acetyl Glutathione or pharmaceutically acceptable salts or derivatives thereof, in same or different compositions to humans or animal.

In an embodiment, the present invention relates to a method of treating Diabetic Nephropathy or Chronic Kidney Disease, wherein treatment comprises administration of an oral pharmaceutical composition comprising a. L-Carnosine in an amount between 50mg to 400mg, and b. S- Acetyl Glutathione in an amount between lOOmg to 600mg,

In one or more embodiments, a pharmaceutical composition as per present invention includes immediate release, delayed release, extended release or combination thereof.

In one or more embodiments, a pharmaceutical composition as per present invention includes for oral, intravenous or other routes of administration.

In one or more embodiments, a pharmaceutical composition as per present invention includes solid, liquid, semisolid or other dosage forms. L- CARNOSINE

Carnosine is an endogenous dipeptide, composed of b-alanine and L- histidine. Carnosine has pH-buffering, metal-ion chelation, and antioxidant capacity as well as the capacity to protect against formation of advanced glycation and lipoxidation end-products. Carnosine is found naturally mainly in the skeletal muscles, central nervous system, olfactory neurons and in the lens of the eye in some vertebrates, including humans.

CNDP1 gene is responsible for formation of camosinase into the serum. Carnosinase is responsible for formation of AGES (Advanced Glycation end products). The AGEs blocks the nephrons during the condition of diabetes. Low carnosinase secretion has been associated with low risk of developing diabetic nephropathy. Low levels of carnosinase secretion means less of this enzyme in the blood, which results in higher concentration of carnosine. It is known that carnosine prevents the formation of Advanced Glycation End products (AGEs) which cause kidney damage in diabetics.

In one or more embodiments, L-Carnosine or pharmaceutically acceptable salts or derivatives thereof is in the equivalent therapeutically effective amount of L-Carnosine. In a preferred embodiment, L-Carnosine or pharmaceutically acceptable salts or derivatives thereof is in an amount equivalent to 50mg to 400mg of L-Camosine.

GLUTATHIONE OR ITS PRECURSORS OR DERIVATIVES

Glutathione (g-L-glutamyl-L-cysteinylglycine, GSH), a water-soluble endogenous tripeptide, is the most abundant thiol present in mammalian cells. The liver has highest content of this tripeptide and is the main tissue participating in GSH biosynthesis. Glutathione helps remove the toxins and free radical scavenges from the body by the oxidation process. The kidneys are highly dependent on an adequate supply of glutathione (GSH) to maintain normal function. This is due, in part, to high rates of aerobic metabolism, particularly in the proximal tubules.

Reduced glutathione (GSH) is the preferred substrate for several enzymes in xenobiotic metabolism and antioxidant defence. It plays an important role in many cellular processes, such as cell differentiation, proliferation and apoptosis. GSH deficiency has been observed in aging and in a wide range of pathologies.

Glutathione derivatives are modified glutathione which may act as a prodrug or have similar pharmacological properties as glutathione. S-Acetyl-glutathione is a GSH analog which is more stable in plasma, it is taken up directly by the cells and later converted to GSH. In one or more embodiments, S-Acetyl Glutathione or pharmaceutically acceptable salts or derivatives thereof is administered in the equivalent therapeutically effective amount of glutathione. In a preferred embodiment, S-Acetyl Glutathione or pharmaceutically acceptable salts or derivatives thereof is in an amount equivalent to lOOmg to 600mg of S-Acetyl Glutathione.

Glutathione presursors are compound involved in biochemical formation of glutathione in vivo. Such precursors includes but are not limited to glutamic acid or glycine or cysteine or derivatives thereof. Three essential amino acids glutamate, cysteine, and glycine combine to form the tripeptide glutathione. At the beginning, cysteine is joined to glutamate through the action of glutamate cysteine ligase to produce g-glutamylcysteine, which proceeds to link with glycine via glutathione synthase action.

N-Acetylcysteine is an exogenous antioxidant, which works as a free radical scavenger. It is also a glutathione precursor regarded as one of the most important intracellular antioxidants. It has been proven that supplementation with N- acetylcysteine activates antioxidant enzymes, prevents oxidative stress development, improves sensitivity towards insulin.

PHARMACEUTICAL COMPOSITION

The pharmaceutical compositions are the different type of medicinal preparation designed for the administration of targeted one or more drugs. The pharmaceutical compositions as per present invention includes immediate release, delayed release, extended release and pulsed-release. The pharmaceutical compositions can be prepared using uniform mixture of two or more drugs. In one or more embodiments, pharmaceutical composition can be prepared with one or more drugs in separate compartment within a single dosage form. The pharmaceutical compositions as per present invention can be administered by oral, intravenous or other routes of drug administration. The pharmaceutical compositions as per present invention can be solid, liquid, semisolid or any other dosage form. The pharmaceutical compositions as per present invention can be prepared as one or more drug in modified release and other drugs as immediate release in single dosage form.

The oral pharmaceutical dosage form are tablets, capsules, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, lozenges, dispersible powders and granules, medicated gums, chewing tablets, effervescent tablets, multi-particulate dosage forms and the likes. The multicompartment dosage form are bilayer tablets, capsule-in-capsule, tablet-in capsule and any other dosage form. The pharmaceutical compositions can be formulated by any techniques known to or appreciated by a person skilled in the art

In an embodiment, the oral pharmaceutical composition further includes optionally any one or a combination of one or more pharmaceutically acceptable excipients, such as but not limited to carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, antioxidants and solubility enhancers.

Having described the invention with reference to the different embodiments of the invention, other embodiments will become apparent to one skilled in the art from consideration of the specifications.

The innovation is further defined by reference to the following examples. It will be apparent to those skilled in the art that many modifications, both to the composition and treatment, may be practiced without departing from the scope of this invention. EXAMPLES

Example 1 - H202 scavenging assay. A solution of hydrogen peroxide was prepared in phosphate buffer. The concentration of hydrogen peroxide was determined by absorption at 230 nm using a spectrophotometer. Extract in distilled water was added to hydrogen peroxide and absorbance at 230 nm is determined after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. The ascorbic acid was used as standard in the assay. The test and standard solutions were prepared in millipore water. Different concentrations of the test and standard solutions were used in the assay.

The % scavenging of H2O2 was calculated using following formula. JC _ % Scavenging of H202 = — - - X100 iC where Ac is absorbance of Control; At is absorbance of test after reduction of blank reading from test reading.

Preparation of standard and test solution for H2O2 assay

The 40 mM solution was prepared in 50 mM phosphate buffer (pH - 7.4) Assay Procedure:

Results

The IC50 value of ascorbic acid was found to be 312.29±11.79 mM for 20 mM H202 solution.

The IC50 value of L-carnosine was found to be 1332.83±84.44 mM for 20 mM H202 solution. The IC50 value of S-acetyl 1-glutathione was found to be 309.95±6.27 mM for 20 mM H202 solution.

Effect of combination of L- carnosine and S-acetyl-L-glutathione on H2O2 scavenging activity

Data are shown as Mean± SEM (n=3).

Example 2 - DPPH Assay.

In order to evaluate the antioxidant potential through free radical scavenging by the test samples, the change in optical density of DPPH radicals was monitored. The sample was diluted with methanol and DPPH solution was added. After 30 min, the absorbance is measured at 517 nm. The ascorbic acid was used as standard in the assay. The test and standard solutions were prepared in millipore water. Different concentrations of the test and standard solutions were used in the assay. The DPPH solution (100 mM) was prepared in methanol.

The % inhibition of DPPH was calculated using following formula.

Where Ac is absorbance of Control; At is absorbance of test. Preparation of standard and test solution for DPPH assay

Assay Procedure:

Results

The IC50 value of ascorbic acid was found to be 7.31±0.068 pg/ml for 100 mM DPPH solution. The IC50 value of L-carnosine was found to be 949.37±74.78 pg/ml for 100 mM DPPH solution.

The IC50 value of S-acetyl 1-glutathione was found to be 13.0±0.44 pg/ml for 100 mM DPPH solution.

Effect of combination of L- carnosine and S-acetyl-L-glutathione on DPPH Scavenging activity

Data are shown as Mean± SEM (n=3). Example - 3 In-vivo Studies - Assessment of renal oxidative stress parameters.

The kidney homogenate was used for estimation of renal oxidative stress parameters. Direct kidney homogenate was used for estimation of malonaldehyde (MDA) and reduced glutathione (GSH). Post nuclear supernatant was used for determination of catalase activity while post mitochondrial supernatant was used for determination of superoxide dismutase (SOD). MALONDI ALDEHYDE (MDA) ASSAY

The lipid peroxide level in animal tissues was measured according to method described by Ohkawa and co-workers (Ohkawa et ah, 1979).

Procedure

Preparation of assay standard and solutions

Stock was prepared by adding 17 mΐ of TMP (1,1,3,3-tetramethoxy propane) to 983 mΐ of distilled water to give 100 mM solution of TMP. Serial dilutions were done by diluting the stock with the distilled water to get 1, 3, 10, 30, 100 and 300 mM solutions of TMP. The 2-thiobarbituric acid solution was prepared by dissolving required quantity in 0.1 M phosphate buffer (pH- 7.4). Sodium dodecyl sulphate solution and glacial acetic acid solution was prepared in distilled water. Assay procedure of MDA assay

Calibration curve of standard was plotted and unknown concentrations of the MDA in samples were calculated. The results were expressed as amount of malondialdehyde formed as nmoles of MDA/ mg protein.

Results The diabetic control group showed significant increase in MDA levels when compared to normal control group. The treatment with 1-carnosine for 28 days at a dose of 36 mg/kg showed significant decrease in MDA when compared to diabetic control group. The animals treated with s-acetyl-l-glutathione at all selected dose levels showed significant decrease in MDA when compared with diabetic control group. Combination group also showed significant decrease in MDA when compared to diabetic control animals (Figure 3).

CATALASE ASSAY

Determination of catalase activity was carried by using post nuclear supernatant. Catalase activity was assayed according to the method of Luck (Luck, 1965).

Procedure Table 1 Determination of catalase activity

Calculations

Enzyme activity was measured using molar extinction coefficient of H2O2 (0.071) and was expressed as micromole of H2O2 decomposed/min/ mg protein)

Results

The diabetic control group showed significant decrease in catalase levels when compared to normal control group. The animals treated with s-acetyl-l-glutathione at all selected dose levels showed significant increase in catalase levels when compared with diabetic control group. Combination group also showed significant increase in catalase when compared to diabetic control animals (Figure 4).

SUPEROXIDE DISMUTASE (SOD)

Estimation of SOD was carried out by using post mitochondrial supernatant (PMS). SOD was estimated according to method described by Paoletti and Mocali (Paoletti et al., 1986). Procedure

Estimation of Superoxide dismutase

Calculation

One unit of enzyme activity is defined as the concentration of enzyme that gives half maximal inhibition of nitrobluetetrazolium (NBT) reduction. Result was expressed as U/ mg protein.

Statistical analysis

The differences among control and treatment groups were analysed using graph pad prism V5.0 for Windows. The significance level between control and experimental groups was assessed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. All data were expressed as Mean ± Standard Error of Mean (SEM); the P values less than 0.05 were considered to be significant.

Results

The diabetic control group showed significant decrease in superoxide dismutase (SOD) levels when compared to normal control group. The treatment with 1- carnosine for 28 days at a dose of 36 mg/kg showed significant increase in SOD levels when compared to diabetic control group. The animals treated with s-acetyl- 1-glutathione at all selected dose levels showed significant increase in SOD levels when compared with diabetic control group. Combination group also showed significant increase in SOD when compared to diabetic control animals (Figure 5). Example - 4 Representative tablet composition of L- Camosine and S-Acetyl Glutathione .