FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
Complete Specification
[See Sections 10 and rule 13]
Stable veterinary composition for enhanced phosphorous uptake
Applicant: (a) INTAS Pharmaceuticals Limited
(b) Company Registered under Indian Company ACT
(c) 2nd Floor, Chinubhai Centre,
Ashram Road, Ahmedabad 380009. Gujarat. India
The following specification particularly describes the invention and the manner in which it is to be performed:

Field of the invention
The present invention relates to stable veterinary phosphorous injection compositions and process for such preparation.
Background
Phosphorus is an important component of the animal body and is involved in a number of physiological functions. Ruminants and other domestic animals generally depend on dietary sources for its day to day requirements. Phosphorus in the animal body is present in organic as well as inorganic _forms. The inorganic phosphorus could be present intracellular as well as extracellular. The extra cellular inorganic phosphorous has been shown to play major role in cellular energy metabolism, especially in erythrocyte energy metabolism and shape change.
Phosphorous deficiency (hypophosphatemia) is common problem in animal, characterized by a number of non-specific -syndromes characterized by pica, unthriftiness and poor body conditions (Gherguria et al., 1994), arthritis (Mullins and Ramsay, 1959) lameness (Hoey et al., 1976), reproductive disorders such as anoestrous and increase in inter calving period (Roberts, 1971). Severe hypophosphatemia has also been found to be associated with bovine paraplegic syndrome (Gerloff and Swensen, 1996 and Rojas et al., 1997) and parturient haemoglobinuria in cows (Penny 1956 and .Mullins and Ramsay, 1959) and buffaloes (Nagpal et al., 1968 and Gautam et al., 1969).
The parturient haemoglobinuria due to hypophosphatemia characterized by acute hemolytic anemia and is recorded by sporadically with high mortality in untreated animals (Simesen and Kronfels, 1970). The study suggest that the basic biochemical defect in phosphorous deficiency is due to reduced uptake of glucose by erythrocytes leading ultimately to low cellular Adenosine triphosphate (ATP), reduced Nicotinamide adenine dinucleotide (NADH), 2,3-Diphosphoglycerate (2,3- DPG) and reduced glutathione. Consequent to depleted

erythrocyte ATP and oxidative stress, the globin part of hemoglobin is precipitated and aggregated as Heinz bodies.
The role of phosphorous in this syndrome is further strengthened by the observation that replenishment of inorganic phosphorous in- vivo or in- vitro. leads to correction of all metabolic defects and recovery. A number of therapies have been tried in ruminants for correcting hypophosphatemia, which includes replacement of phosphorous by administering inorganic phosphates intravenously and supportive therapy to correct consequent metabolic defects of hypophosphatemia.
Two major drugs used by field of veterinarians are sodium acid phosphate 60-80gm as 20% solution administered intravenously (Mullins and Ramsay, 1959 and Samad et al., 1979) and ascorbic acid as an antioxidant (Chugh and Mata, 1997). Other therapies found useful in phosphorous deficiency hemoglobinuria but not popular among the field veterinarians are epsilon amino caproic -acid and para amino benzoic acid, used as antifibrinolytic agents (Chugh et al., 1987), organic phosphorous, 4- dimethyl amino, 2- methyl phosphonoic acid (Bhikane et al., 1998), inosine (Goel et al., 1988) and botropase (Goel et al ., 1989). Another product comprising of Toldimfos sodium, which is a sodium salt of 4-dimethylamino-2-methyl-phenyl-phosphinous acid is indicated for the treatment and prophylaxis of disease which arise in connection with parturition, and the peri-partum period, development and nutritional disorders in young animals caused by disorders of calcium, magnesium and phosphorous metabolism.. However, according to a report submitted by committee for veterinary medicinal products for Toldimfos to European Agency for the Evaluation of Medicinal Products (EMEA), the precise mode of action of Toldimfos is unknown and questionable.
Blood journal, vol. 39, No. 4 (April), 1972 discloses in vivo effect of inosine, pyruvate and phosphate on oxygen-hemoglobin affinity in rhesus monkey. The

experiments suggest that the inosine, pyruvate and phosphate should be used administered for increasing 2, 3-DPG levels in erythrocytes. The journal article did not disclose formulation and the stability aspect of the preparations.
The Biochemistry Journal (1990). vol. 266, pg, No. 441-446, discloses incubation of erythrocytes in inosine, pyruvate and phosphate medium (1PP medium) results in the accumulation of more 5- phosphoribosyl 1-pyrophosphate (PRPP) which is required for purine nucleotides. On carrying out experiments, the disclosed 1PP medium shows precipitation during storage.
Thesis submitted by. Aneesh George Veigas from Bombay Veterinary College during 1998 discloses phosphorous preparation comprising buffered phosphorous (Na2HP04. 2H20), adenosine, inosine, sodium pyruvate, ascorbic acid. The disclosed preparation was based on single vial system and shows precipitation during storage.
Thesis submitted by Gonsalves Tina Louis from Bombay Veterinary College during October 2001 discloses composition comprising pentoxyfylline, sodium hydroxide, sodium acid phosphate and sodium pyruvate, where inventor tried to substitute inosine and adenine with pentoxyfylline which was based on single vial composition and shows precipitation during storage.
Phosphorous injection under development by Inta.s Pharmaceutical comprises adenine, inosine, sodium pyruvate and buffered phosphorous which was based on single vial preparation and shows precipitation / physical instability during storage.
Thus to overcome the stability problem associated with the above disclosed inventions and to have better phosphorous supplementation for treating hypophosphatemia, there is need to develop a stable phosphorous injection.

The present invention provides a stable veterinary phosphorous injection composition with better phosphorous supplementation and process for such preparation.
Objects of the invention
The main objective of the invention is to provide a stable veterinary phosphorous injection composition for treating hypophosphatemia.
Another object of this invention is to provide a stable veterinary phosphorous injection composition with better phosphorous supplementation.
Another object of the invention is to provide a process for preparing stable veterinary phosphorous injection composition.
Summary of the Invention
The present invention relates to a stable veterinary phosphorous injection composition with better phosphorous supplementation for treating hypophosphatemia.
Further this invention also relates to a process for preparing a stable veterinary phosphorous injection.
Detailed Description of the Invention
The present invention relates to stable veterinary phosphorous injection composition with better phosphorous supplementation for treating hypophosphatemia. To achieve stable veterinary phosphorous injection composition the present invention provides a two vial system wherein first viai comprises inosine or its pharmaceutically acceptable salts, sodium pyruvate and a pharmaceutically acceptable carrier and second vial comprise of phosphorous source and at least one pharmaceutically acceptable carriers.

The stable veterinary phosphorous injection composition comprises inosine or its pharmaceutically acceptable salts, wherein the concentration of Inosine is in the range of I to 50 mg/ml preferably in the range of 10 to 20 mg/ml.
The sodium pyruvate used in the composition of the present invention comprises in the concentration range of 1 to 50 mg/ml, preferably in the range of 10 to 20 mg/ml.
The phosphorous source used in the phosphorous injection comprises of one or combination of phosphate buffers comprising of sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate or the likes thereof. The phosphorous source used in the phosphorous injection comprises in the concentration range of 1 to 400 mg/ml.
The pharmaceutically acceptable carriers used in the phosphorous injection composition comprise of preservatives, water for injection or other pharmaceutically acceptable carriers which are conventionally used in injection compositions.
The preservatives used in the phosphorous injection comprise of sodium methyl paraben, sodium propyl paraben, benzyl alcohol or the likes thereof. The preservatives used in the phosphorous injection comprises in the concentration range of 0 to 5% wt of the composition.
The pH of the phosphorus injection is in the range of 4.0 to 8.0 which provides enhanced phosphorus uptake, preferably in the range of 6.5 to 7.5
Another embodiment • of this invention is directed towards a process for preparation of stable veterinary phosphorous injection. One of the preferred methods for preparation of the said stable veterinary phosphorous injection comprises of the following steps:

a) Process for preparation of composition in vial I (product vial) comprises
of:
j. Take water for injection (90% of total batch size) and sparge nitrogen for
30 min ii. Add and dissolve preservatives iii. Add and dissolve inosine or its salts iv. Add and dissolve sodium pyruvate v. Make up the volume to desired batch size vi. Filter bulk solution through 6.2 micron filter vii.- Fill the filtered bulk in clear glass vials viii. Sterilize filled vials.
b) Process for preparation of composition in vial II (buffer vial) comprises
of:
i. Take water for injection (90% of total batch size) and sparge nitrogen for
30 minutes. ii. Add and dissolve the required quantity of one or combination of
phosphate buffer iii. Make up the volume to desired batch size iv. Filter bulk solution through 0.2 micron filter v. Fill the filtered bulk in clear glass vials vi. Sterilize filled vials.
c) Reconstitution method comprises of the entire content of vial I ( product vial)
has to be withdrawn and mixed with vial 11 (buffer vial) aseptically and shaken
gently to mix both the solution properly before administration. This injectable
composition has a pH in the range in between 4.0 to 8.0; preferably in the range
of6.5 to 7.5.

Examples
The present invention has been described by way of example only, and it is to be recognized that modifications thereto which fall within the scope and spirit of the appended claims, and which would be obvious to a skilled person based upon the disclosure herein, are also considered to be included within the invention.
The above said invention of a stable veterinary phosphorous injection composition can be illustrated by but not limited to following examples.
Formula:
A. Product vial (20 ml):

Sr.No.'- Ingredients Range in mg/ml
1 Inosine 1-50
2 Sodium pyruvate 1-50
3 Benzyl alcohol IP 5-30
4 Water for injection IP q.s.
B. Buffer vial (30 mJ volume in 50 ml vial):

Sr. No. Ingredients Range in mg/ml
1 Sodium Dihydrogen Phosphate. Dihydrate 1 -100
2 Disodium hydrogen phosphate dodecahydrate 1 400
3 Water for injection IP q.s.
Entire content of Product vial has to be withdrawn and mixed with Buffer vial aseptically and shaken gently to mix both the solution properly before administration.
The pH of the injectable composition is in the range in between 4.0 to 8.0; preferably in the range of 6.5 to 7.5.

Stability Data A. Product Vial

Parameters initial 6Months at 40°C
& 75%RH 6Months at 25°C & 60%RH
Description Clear colour -less solution Clear colour - less solution Clear colour- less solution
pH 6.84 6.61 6.6
Assay of Inosine (%) 114.9 111.0 111.6
Assay of Sodium pyruvate (%) 127.4 114.9 120.3
Assay of Benzyl (%) alcohol 113.6 107.8 108.3
B. Buffer Vial

Parameters Initial 3Months at 40°C & 75%RH 3Months at 25°C & 60%RH
Description Clear colour -less solution Clear colour - less solution Clear colour - less solution
pH 7.4 7.21 7.20
Phosphorus content (%) 101.8 " 100.4 100.5
It can be observed from the stability data for the product vial (A) and buffer vial (B) as shown above, that the product vial and buffer vial are stable and do not show precipitation / physical instability. Further, pH of product vial as well as buffer vial does not deviate from the initial pH. Further, individual assays for inosine, sodium pyruvate, benzyl alcohol and phosphorus content does not show significant change, hence both the product vial and the buffer vial remains physically and chemically stable.

Claims:
!. A multiple vial system of a stable veterinary phosphorous injectable composition comprising of a phosphorous source, an inosine source and a pyruvate source; wherein at least a phosphorous source is in a separate vial.
2. The composition according to claim 1, wherein the said inosine source and pyruvate source are in single vial or separate vials.
3.- The composition according to claim 1, wherein the phosphorous source is a phosphate buffer which can be sodium dihydrogen phosphate, disodium.. hydrogen phosphate or mixtures thereof in the range of 1 to 400 mg/ml.
4. The composition according to claim 1, wherein the inosine source is inosine or its pharmaceutically acceptable salt in the range of 1 to 50 mg/ml.
5. The composition according to claim 1, wherein the pyruvate source comprises of sodium pyruvate and the likes thereof in the range of 1 to 50 mg/ml.
6. The composition according in claim 1, which further comprises of preservatives, water for injection and other pharmaceutically acceptable carriers.
7. The composition according in claim 6, wherein preservatives can selected from sodium methyl paraben, sodium propyl paraben, benzyl alcohol and mixtures thereof.

8. The composition according to any of the preceding claims, wherein the pH of the injectable composition is in the range of 4.0 to 8.0; more preferably in between 6.5 to 7.5.
9. A multiple vial system of a stable veterinary phosphorous injectable composition comprising of:
a) a vial having phosphorous source comprising:
Sodium dihydrogen phosphate dihydrate in a range of 1 - 100 mg/ml, Disodium hydrogen phosphate dodecahedra in a range of 1 - 400 mg/ml, quantity sufficient amount of pharmaceutically acceptable carrier; and
b) a product vial comprising:
Inosine or its pharmaceutically acceptable salt in a range of 1 - 50 mg/ml,
Sodium pyruvate in a range of 1 - 50 mg/ml,
quantity sufficient amount of preservative,
quantity sufficient amount of pharmaceutically acceptable carrier;
wherein the pH of the injectable composition is in the range of 4.0 to 8.0.
10. A multiple vial system of a stable veterinary phosphorous injectable
composition as herein described with foregoing description and examples.