DESC:FILED OF THE INVENTION
The object of present invention is to provide a stable aqueous parenteral composition exhibiting enhanced bio-availability comprising 300000 to 600000 IU of Vitamin D3, substantially free from alcohol and oil.
BACKGROUND OF THE INVENTION
Vitamin D is a group of fat-soluble secosteroids responsible for increasing absorption of calcium, magnesium, and phosphate from intestine and kidney, and multiple other biological effects. In humans, the most important compounds in this group are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol).
Vitamin D3 controls the incorporation of calcium and phosphorus in the skeleton.
Vitamin D3 is synthesized by humans in the skin when it is exposed to ultraviolet-B (UVB) rays from sunlight. Foods may be fortified with vitamin D3.
Although the synthesis of Vitamin D3 occurs naturally in the skin with adequate sunlight exposure, Vitamin D3 is not active and needs to be converted to 25(OH) D3 in the liver. From the liver, 25(OH) D3 is transported to the kidney and hydroxylated by 25-hydroxyvitamin D31-hydroxylase to form the active hormone calcitriol. Said active form binds to vitamin D receptors (VDRs) that are present in the parathyroid gland, intestine, kidney, and bone to maintain parathyroid function and calcium and phosphorus homeostasis. VDR activation is essential for the proper formation and maintenance of normal bone. In the diseased kidney, the activation of Vitamin D is diminished, resulting in a rise of parathyroid hormone, subsequently leading to secondary parathyroidism and disturbances in the calcium and phosphorus homeostasis.
Inadequate Vitamin D supply often develops in individuals who are infrequently exposed to sunlight without protective sunscreens, have chronically inadequate intakes of Vitamin D, or suffer from conditions that reduce the intestinal absorption of fat soluble vitamins (such as Vitamin D). Inadequate Vitamin D supply can leads to numerous diseases and physical ailments. Rickets and osteomalacia are classic vitamin D deficiency diseases. In children, vitamin D deficiency causes rickets, which results in skeletal deformities. In adults, vitamin D deficiency can lead to osteomalacia, which results in muscular weakness in addition to weak bones.
Nutrients. 2014 Feb; 6(2): 729–775 discloses widespread prevalence of vitamin D deficiency in India. Factually, sun exposure is an untenable solution, for most individuals in India, towards attaining vitamin D sufficiency. Low calcium intake in conjunction with vitamin D deficiency makes matters worse. Therefore, the need for improvement in vitamin status of the Indian population is both important and urgent.
Vitamin D3 can be taken either orally or by intramuscular injection. Intramuscular injection is useful in severe Vitamin D deficiency disease with intestinal malabsorption. It also avoids repeated oral intake of Vitamin D.
Vitamin D3 is fat soluble vitamin. Therefore, first approach is to prepare oil based parenteral formulation for complete solubilization & stabilization of Vitamin D3. Vitamin D3 injection according to IP (Indian pharmacopoeia) & BP (British Pharmacopoeia) is oil based formulation available as 300000 and 600000 IU/ml of Vitamin D3. However, oil containing injectable formulation can cause pain, erythema, swelling nodule formation at the site of injection and delay release of active ingredient. The oil based preparation are also known to provide slow release of drug and it is found to be equivalent to oral Vitamin D3 up to 6 weeks (Indian J Endocrinol Metab. 2017 Jan-Feb;21(1):131-136).
To avoid the problems associated with oil based formulations, various attempts have been made in the art.
One of them is to prepare emulsion as disclosed in US 2832721, US 3032468, WO 2011063952 and WO 2017115316.
Another approach is to prepare parenteral formulation of containing alcohol as disclosed in US 20060183722. US ‘722 discloses pharmaceutical formulations of lipophilic therapeutic agents in which such agents are solubilized in largely aqueous vehicles along with alcohol, and processes for preparing and using the same. However, alcohol containing injection can cause pain on injection and tissue necrosis and is not popular.
It is well known that aqueous based injection is better tolerated than oil based preparation and alcohol based preparation. Aqueous based injection are also found to provide rapid rise in serum concentration as compared to oily preparation. Therefore, aqueous injectable formulations of vitamin D3 with high concentration have been strongly desired for the parenteral administration to reduce the side effects and provide quick recovery from Vitamin D3 deficiency.
The word “aqueous” as used herein defines the system made from, with, or by water (Merriam Webster) or the system which is of, like, or containing water (Collins Dictionary).
However, Vitamin D3 compounds are hydrophobic or lipophilic and are only sparingly or negligibly water-soluble. The poor water solubility of these active agents often results in major difficulties in formulation particularly when easily sterilizable and administrable homogeneous aqueous solutions are needed. Also the poor water solubility of these active agents raises concerns of stability upon storage and especially throughout the shelf-life of the product.
The lipophilicity of the vitamin D3 compounds makes it difficult to manufacture an efficacious formulation, particularly, an aqueous injectable formulation, which is preferred, for example, in renal dialysis patients.
Additionally, vitamin D compounds, such as Vitamin D3, are known to be oxygen-sensitive. They get oxidized when exposed to air, and thus, loose integrity. Further challenge which exists is that vitamin D is very light sensitive and is subject to oxidative degradation. The oxidation kinetics i.e., the rate of reaction increases with concentration; hence high dosage (more concentrated) vitamin D products have a greater propensity for oxidation compared with lower dosage (less concentrated) formulations.
One approach to circumvent this problem is to add an antioxidant directly to a formulation of the drug. However, certain antioxidants, such as ascorbic acid and sodium ascorbate, which are highly water soluble, and don’t provide stable formulation. Thus, there is a need for pharmaceutical injectable formulations of vitamin D compounds that overcome the limitations of the prior art compositions.
Thus, alcohol and oil free stabilized aqueous composition of Vitamin D3 at a high concentration have been strongly desired for the parenteral administration to overcome the limitations such as slow release and side effects associated with oil and alcohol based parenteral formulations.

OBJECT OF THE INVENTION

The object of present invention is to provide a stable aqueous parenteral composition comprising 300000 to 600000 IU of Vitamin D3 substantially free from alcohol and oil.
Another object of present invention is to provide a stable aqueous parenteral composition of Vitamin D3, which is less irritant at the site of administration.
Another object of the invention is to provide an aqueous parenteral composition of vitamin D3 with enhanced bio-availability.
Another object of the invention is to provide use of the aqueous parenteral composition of Vitamin D3 for treatment and prevention of vitamin D deficiency and insufficiency.
Another object of the invention is to provide a process for the preparation of an aqueous parenteral composition of Vitamin D3 as discussed in above objects.

SUMMARY OF THE INVENTION

It has been surprisingly found that an aqueous parenteral composition comprising Vitamin D3 at a high concentration prepared according to the present invention remains stable under room temperature.
In one aspect, the present invention provides an aqueous parenteral composition comprising 300000 to 600000 IU/ml of Vitamin D3, Glycofurol and a surfactant.

In another aspect, the present invention provides an aqueous parenteral composition comprising 300000 to 600000 IU/ml of Vitamin D3, Glycofurol at least 5% w/v, and a surfactant at least 5% w/v.

In another aspect, the present invention provides an aqueous parenteral composition comprising 300000 to 600000 IU/ml of Vitamin D3, Glycofurol at least 5% w/v, and a surfactant at least 5% w/v, wherein the ratio of Glycofurol + surfactant to Vitamin D3 is in the range of 16 to 50 and the ratio of surfactant to Vitamin D3 is greater than or equal to 8.

In another aspect, the present invention provides an aqueous parenteral composition comprising 300000 to 600000 IU/ml of Vitamin D3, Glycofurol at least 5% w/v, a surfactant at least 5% w/v, and water at least 50% to 90%, wherein the ratio of Glycofurol + surfactant to Vitamin D3 is in the range of 16 to 50 and the ratio of surfactant to Vitamin D3 is greater than or equal to 8.

In another aspect, the present invention provides an aqueous parenteral composition comprising 300000 to 600000 IU/ml of Vitamin D3, Glycofurol at least 5% w/v, a surfactant at least 5% w/v, water at least 20% to 90%, and optionally a chelating agent, an antioxidant and a buffer, wherein the ratio of Glycofurol + surfactant to Vitamin D3 is in the range of 16 to 50 and the ratio of surfactant to Vitamin D3 is greater than or equal to 8.

In another aspect, the present invention provides an aqueous parenteral composition of Vitamin D3 as discussed in the above aspects, which is substantially free from alcohol and oil.

In another aspect, the present invention provides a process for the preparation of an aqueous parenteral composition of Vitamin D3 as discussed here in above aspects.

In another aspect, the present invention provides the use of an aqueous parenteral composition of Vitamin D3 as discussed in above objects for the treatment of vitamin D deficiency and insufficiency, and for the prevention or treatment of various medical conditions resulting from a deficient or insufficient vitamin D state.

The foregoing brief description has outlined, in general, the featured aspects of the invention and is to serve as an aid to better understanding the more complete detailed description which is to follow. In reference to such, there is to be a clear understanding that the present invention is not limited to the parenteral composition, detail of preparation, or application of use described herein.

Any other variation of preparation, use, or application should be considered apparent as an alternative embodiment of the present invention.

Also, it is understood that the phraseology and terminology used herein are for the purpose of description and should not be regarded as limiting. The use of “including”, “having” and “comprising” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items and equivalents thereof.

DESCRIPTION OF DRAWINGS:
Figure-1: shows in vivo comparative pharmacokinetic profile of Vitamin D3 (Cholecalciferol) aqueous injection as per the present invention and Vitamin D3 conventional injection for Vitamin D3.
Figure-2: shows in vivo comparative pharmacokinetic profile of Vitamin D3 (Cholecalciferol) aqueous injection as per the present invention and Vitamin D3 conventional injection for 25-hydroxy Vitamin D3 (Metabolite).

DETAILED DESCRIPTION

It should be noted that, as used herein, the term “vitamin D3” means cholecalciferol, (i.e. the pro-hormone form of vitamin D3) as opposed to the metabolically active form, calcitriol.
Vitamin D3 (Cholecalciferol) has a chemical name of (3ß,5Z,7E)-9,10-secocholesta-5,7,10(19)-trien-3-ol and can be represented by the following structural of formula (I):

Formula (I)
The preferred range of concentration of the vitamin D3 in the aqueous parenteral composition is in the range 300000 IU/ml to 600000 IU/ml (IU is an abbreviation of “International Unit”).
The aqueous composition of the present invention is transparent in appearance, and has an adequate viscosity like water desired for parenteral administration. The aqueous composition of the present invention is excellent in physical and chemical stabilities, stability in storage, and stability in circulation, and not changed over a period of time at room temperature.
The term “aqueous compositions” means liquid compositions containing water more than 50% by volume of the composition.
The term "substantially free" refers to compositions that have significantly reduced levels of the oil and alcohol. In one embodiment, oil and alcohol are not added to the composition, but may be otherwise present as a volatile impurity. In this context, the term "substantially free" means that the selected composition contains less than a functional amount of the oil and alcohol.
The term "stable" used herein refers to the stability of Vitamin D3 in a liquid composition, wherein the chemical assay of Vitamin D3 is 90% or more and the liquid composition looks clear on visual examination by naked eye when stored at or below 30°C for at least 12 months.
“Vitamin D insufficiency and/or deficiency” is generally defined as having serum 25-hydroxyvitamin D levels below 30 ng/ml (National Kidney Foundation guidelines, NKF, Am. J. Kidney Dis. 42:S1-S202 (2003), incorporated herein by reference).
It has been surprisingly found that an aqueous parenteral composition comprising Vitamin D3 at a high concentration remains stable under room temperature and the accelerated stability conditions.
The aqueous parenteral composition of Vitamin D3 of the present invention is advantageous with respect to currently available oily injectable composition of Vitamin D3 for their ease in administration and patient compliance.
It has surprisingly been found that an aqueous parenteral composition comprising 300000 to 600000 IU/ml of Vitamin D3, Glycofurol and a surfactant is stable under room temperature.
An aqueous parenteral composition comprising Vitamin D3 in amount of 300000 to 600000 IU facilitates the administration of high concentration of Vitamin D3 in a single dose by eliminating the side effects associated with oil and alcohol based Vitamin D3 containing parenteral formulations.
The term "about" can indicate a difference of 10 percent of the value specified. Numerical ranges as used herein are meant to include every number and subset of numbers enclosed within that range, whether particularly disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range.
The surfactant may be selected from anionic and/or non-ionic surfactant. The non-ionic surfactants include but are not limited to polyoxyethylenated (e.g. polysorbate 80 (Tween® 80)), polyoxyethylenated alkyl ethers; polyoxypropylated fatty alcohols such as polyoxypropylene-styrol ether; polyethylene glycol stearate such as Solutol®, polyoxyethylenated derivatives of castor oil (Cremophor®), polyglycerol esters, polyoxyethylenated fatty alcohols, polyoxyethylenated fatty acids, copolymers of ethylene oxide and propylene oxide such Poloxamer®, and the like. Preferable surfactant is Tween® 80, which is interchangeable with Solutol® and Poloxamer®.
The anionic surfactants include but are not limited to alkaline stearates, sodium, potassium or ammonium stearates; calcium stearate, triethanolamine stearate; sodium abietate; alkyl sulphates (e.g. sodium lauryl sulphate and sodium cetyl sulphate); sodium dodecylbenzenesulphonate, sodium dioctylsulphosuccinate; fatty acids, and the like.
The concentration of the surfactant in the parenteral aqueous composition preferably ranges from 5% w/v to 30% w/v.
In another embodiment, the present invention is to provide an aqueous parenteral composition comprising 300000 to 600000 IU/ml of Vitamin D3, Glycofurol, a surfactant and water.
The water as used for the purpose of the present invention is water for injection and the concentration of the water for injection in the parenteral aqueous composition ranges from 50% to 90% v/v, more preferably 65% to 90% v/v.
The concentration of the Glycofurol in the parenteral aqueous composition preferably ranges from 5% w/v to 50% w/v, preferably from 5% w/v to 40% w/v and more preferably from 5% w/v to 30% w/v.
In another embodiment, the present invention is to provide an aqueous parenteral composition comprising 300000 to 600000 IU/ml of Vitamin D3, Glycofurol at least 5% w/v, a surfactant at least 5% w/v, and water at least 50% to 90% v/v, wherein the ratio of Glycofurol + surfactant to Vitamin D3 is in the range of 16 to 50 and the ratio of surfactant to Vitamin D3 is greater than or equal to 8.
It has been observed that compositions comprising Vitamin D3, Glycofurol and surfactant when used in above given ratios provided clear and stable aqueous solution. However, if the concentration of Vitamin D3, Glycofurol and surfactant were not in above given ratios, such compositions does not provide clear and stable solution.
The chelating agent can be selected from disodium EDTA, edetic acid, citric acid, trisodium citrate and potassium citrate. The concentration of the chelating agent in the parenteral aqueous composition preferably ranges from 0.1 %w/v to 0.5%w/v, preferably 0.1 %w/v.
The antioxidant can be selected from ascorbic acid, ascorbyl palmitate, benzotriazol, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), citric acid, cysteine, cysteine hydrochloride, disodium calcium ethylenediaminetetraacetate, disodium ethylenediaminetetraacetate, dithiothreitol, DL-alpha-tocopherol, erythorbic acid, ethoxyquin, ethylenediaminetetraacetic acid salts, fumaric acid, glutathione, guaiac, homocysteine, isopropyl citrate, L-ascorbate stearate esters, monothioglycerol, nordihydroguaiaretic acid (NDGA), palmitic acid ascorbic acid, pentaerythrityl-tetrakis[3-(3,5-di-t-butyl-4-hydroxyphenyl)propionate]2-mercaptobenzimidazole, potassium dichloroisocyanurate, propyl gallate, rongalite (CH2OHSO2Na), sodium ascorbate, sodium bisulfite, sodium edetate, sodium erythorbate, sodium hydrogen sulfite, sodium metabisulfite, sodium pyrosulfite 1,3-butylene glycol, sodium sulfite, sodium thioglycolate, sodium thiosulfate, soybean lecithin, tert-butyl hydroquinone, thioglycerol, thiourea, TPGS (tocopherol polyethylene glycol succinate), vitamin E and derivatives thereof, a-thioglycerin or salts thereof, and mixture thereof, preferably, Sodium metabisulphite and Propyl gallate.
The concentration of antioxidant in the parenteral aqueous composition ranges from 0.2% w/v to 1 % w/v, preferably 0.5% w/v.
The buffering agent can be selected from amino acids such as arginine, alanine, histidine, glycine and lysine; citrate, glutamate, bicarbonate, tartrate, benzoate, lactate, gluconate, TRIS, acetate, meglumine, borate and phosphate buffer, preferably, phosphate buffer such as dibasic sodium phosphate and monobasic sodium phosphate.
The concentration of buffering agent in the parenteral aqueous composition ranges from 0.2%w/v to 1%w/v, preferably 0.6%w/v.
The compositions according to present invention may optionally further comprises one or more excipients suitable for parenteral formulation, for example non-specific stabilizer such as glycin.
In another embodiment, the present invention is to provide an aqueous parenteral composition comprising 300000 to 600000 IU/ml of Vitamin D3, Glycofurol at least 5% w/v, a surfactant at least 5% w/v, and water at least 50% to 90% v/v, wherein the ratio of Glycofurol + surfactant to Vitamin D3 is in the range of 16 to 50 and the ratio of surfactant to Vitamin D3 is greater than or equal to 8 and said composition is substantially free from alcohol and oil.
In another embodiment, an aqueous parenteral composition of the present invention can be prepared by suitable method for the parenteral preparation.

In another embodiment, the present invention is to provide a process for the preparation of an aqueous parenteral composition of Vitamin D3, which comprises:
(a) dissolving Vitamin D3 in Glycofurol and surfactant,
(b) optionally adding buffering agent and antioxidant to the solution of step (a), and
(c) adjusting the final volume with water for injection.

In another embodiment, the present invention is to provide a process for the preparation of an aqueous parenteral composition of Vitamin D3, which comprises:
(a) dissolving Vitamin D3 in Glycofurol and surfactant,
(b) dissolving buffering agent and optionally antioxidant in water for injection,
(c) mixing the solution of step (a) and step (b),
(d) optionally adjusting the pH of the solution of step (c) by using alkaline solution, and
(f) adjusting the final volume with water for injection.

In another aspect, the present invention provides the use of an aqueous parenteral composition of Vitamin D3 as discussed in above objects for the treatment of vitamin D deficiency and insufficiency, and for the prevention or treatment of various medical conditions resulting from a deficient or insufficient vitamin D state.

The invention is now illustrated with non limiting examples.

Examples:
Comparative example-A and B: Composition A and B were prepared by using ratio of surfactant to Vitamin D3 is < 8.
Table-1
Ingredients Example-A
Example-B

(mg) % (mg) %
Vitamin D3 225 0.75 225 0.75
Propyl gallate 15 0.05 15 0.05
Glycofurol 3743 12.48 5999 19.99
Tween® 80 770 2.57 757 2.52
Glycine 300 1 300 1
Water for injection qs to 30 ml 83.15 qs to 30 ml 75.69

Process for the preparation:
(a) Vitamin D3 was dissolved in Glycofurol, Propyl gallate and Tween® 80,
(b) Glycine was dissolved in 20% of water for injection,
(c) mixed solution of step (a) and step (b), and
(d) adjusted the volume up to 30 ml by using water for injection.

It was found that the solution of comparative example A and B were obtained as hazy i.e. not clear when ratio of surfactant to Vitamin D3 was < 8.

Comparative example-C and D: Composition C and D were prepared without using glycofurol.
Table-2
Ingredients Example-C Example-D
(mg) % (mg) %
Vitamin D3 60 0.6 75 0.75
Propyl gallate 1 0.01 5 0.05
Tween® 80 2899 28.99 2520 25.20
Hydroxy propyl beta cyclodextrin 40 0.4 150 1.5
Glycine - - 150 1.5
Water for Injection qs to 10 ml 70 qs to 10 ml 71

Process for the preparation:
(a) Vitamin D3 was dissolved in Propyl gallate and Tween® 80,
(b) Glycine and/or Hydroxy propyl beta cyclodextrin were dissolved in 20% of water
for injection,
(c) mixed solution of step (a) and step (b), and
(d) adjusted the volume up to 10 ml by using water for injection.

It was found that Vitamin D3 was precipitated out immediately after the dilution with water in comparative example C and Vitamin D3 remained insoluble in comparative example D.

Comparative example-E: Composition E was prepared by using Glycofurol in amount less than 5% w/v.
Table-3
Ingredients Example-E
mg %
Vitamin D3 75 0.75
Glycofurol 400 4
Tween® 80 880 8.8
Propyl gallate 5 0.05
Sodium metabisulphite 50 0.5
Trisodium citrate 10 0.1
Na2HPO4 40 0.4
NaH2PO4.2H2O 20 0.2
Water for Injection qs to 10 ml 85.2

Process for the preparation:
(a) Vitamin D3 was in dissolved Glycofurol, Propyl gallate and Tween® 80,
(b) sodium meta-bisulphite, trisodium citrate, dibasic sodium phosphate and
monobasic sodium phosphate were dissolved in 20% of water for injection,
(c) mixed the solution of step (a) and step (b),
(d) adjusted the volume up to 10 ml by using water for injection,

It was found that the solution of comparative example E was hazy i.e. not clear.

Comparative example-F: Composition F was prepared by using ratio of Surfactant to Vitamin D3 is < 8.
Table-4
Ingredients Example-F
mg %
Vitamin D3 75 0.75
Glycofurol 1248.33 12.4833
Tween® 80 300 3
Propyl gallate 5 0.05
Sodium metabisulphite 50 0.5
Trisodium citrate 10 0.1
Na2HPO4 40 0.4
NaH2PO4.2H2O 20 0.2
Water for Injection qs to 10 ml 82.5

Comparative example F was prepared similarly to the comparative example E.
It was found that the solution of comparative example F was hazy i.e. not clear when ratio of Surfactant i.e. Tween® 80 to Vitamin D3 is < 8.

Comparative example-G: Composition G was prepared by without using Surfactant.
Table-5
Ingredient Example G
mg %
Vitamin D3 750 mg 1.5
Glycofurol 12.5 g 25
Water for Injection qs to 50 ml 73.5

Comparative example G was prepared by dissolving Vitamin D3 in Glycofurol. The final volume of the obtained solution was adjusted with water for injection up to 50 ml.

It was found that the solution of comparative example G was hazy i.e. not clear.

Comparative example-H: Composition H was prepared by using ratio of Surfactant + Glycofurol to Vitamin D3 is < 16

Table-6
Ingredient Example H
(mg) (%)
Vitamin D3 7.50 0.75
Gylcofurol 50.00 5.00
Tween® 80 60.00 6.00
Propyl gallate 0.50 0.05
Sod. metabisulphite 5.00 0.50
Trisodium citrate 1.00 0.10
Na2HPO4 4.00 0.40
NaH2PO4.2H2O 2.00 0.20
Water of injection (ml) q.s. to 1 ml 87

Comparative example H was prepared similarly to the comparative example E.
It was found that the solution of comparative example H was hazy i.e. not clear when ratio of Surfactant + Glycofurol to Vitamin D3 is < 16 even though Glycofurol is 5% and ratio of surfactant to Vitamin D3 is 8.

The results of the above comparative examples show that apart from the amount of glycofurol and/or surfactant, the ratio of Glycofurol + surfactant to Vitamin D3 and the ratio of Surfactant to Vitamin D3 are critical to obtain the clear and stable solution of Vitamin D3.

Examples of the present invention:
The parenteral compositions of Vitamin D3 of Example-1 to 3 were prepared in accordance to the present invention.

Example-1 to 3:
Table-7
Ingredient Example-1 Example-2 Example-3
(mg) (%) (mg) (%) (mg) (%)
Vitamin D3 7.5 0.75 15 1.5 75 0.75
Glycofurol 124.83 12.48 249.6 24.96 600 6
Tween® 80 88 8.8 176 17.6 880 8.8
Propyl gallate 0.5 0.05 1 0.1 5 0.05
Sodium metabisulphite 5 0.5 10 1.0 50 0.5
Trisodium citrate 1 0.1 1 0.1 10 0.1
Na2HPO4 4 0.4 4 0.4 40 0.4
NaH2PO4.2H2O 2 0.2 2 0.2 20 0.2
Water for injection (ml) qs to 1ml 76.7 qs to 1 ml 55.63 qs to 10 ml 83.2

Process for the preparation:
(a) Vitamin D3 was dissolved in Glycofurol, Propyl gallate and Tween® 80,
(b) sodium meta-bisulphite, trisodium citrate, dibasic sodium phosphate and
monobasic sodium phosphate were dissolved in 20% of water for injection,
(c) mixed solution of step (a) and step (b),
(d) pH was adjusted to 7.5 by using 1N NaOH solution, and
(e) adjusted the final volume up to 1 ml with water for injection.

Observation: It was found that the solutions of the Example-1 to 3 prepared according to the present invention were remained transparent or clear during the storage.

Example-4 to 5:
Table-8
Ingredient Example-4 Example-5
(mg) (%) (mg) (%)
Vitamin D3 75 0.75 7.50 0.75
Glycofurol 1248.33 12.48 200.00 20.00
Tween® 80 500 5 40.00 4.00
Propyl gallate 5 0.05 0.50 0.05
Sodium metabisulphite 50 0.5 5.00 0.50
Trisodium citrate 10 0.1 1.00 0.10
Na2HPO4 40 0.4 4.00 0.40
NaH2PO4.2H2O 20 0.2 2.00 0.20
Water for injection (ml) qs to 10 ml 80.5 qs to 1 ml 74.00

Example-4 and 5 were prepared similarly to the example 1 to 3.

Observation: It was observed that the solution of Example-4 & 5 were initially clear but became hazy over the day during the storage because the ratio of surfactant to Vitamin D3 is <8 in both examples and amount of surfactant is < 5% in example-5.

Example-6to 8:
Table-9
Ingredient Example-6 Example-7 Example-8
mg % mg % mg %
Vitamin D3 225 0.75 225 0.75 225 0.75
Propyl gallate 15 0.05 15 0.05 15 0.05
Glycofurol 4896 16.32 3780 12.6 6039 20.13
Tween® 80 4499 14.99 4503 15.01 2634 8.78
Glycin 450 1.5 301 1 450 1.5
Water for injection (ml) qs to 30ml 66.4 qs to 30ml 70.6 qs to 30ml 68.8

Process for the preparation:
(a) Vitamin D3 was dissolved in Glycofurol, Propyl gallate and Tween® 80,
(b) Glycine was dissolved in 20% of water for injection,
(c) mixed solution of step (a) and step (b),
(d) pH was adjusted to 7.5 by using 1N NaOH, and
(e) adjusted the volume up to 30 ml by using water for injection.

Observation: It was found that the solutions of the Example-6 to 8 prepared according to the present invention were remained transparent or clear during the storage.

Example – 9: Testing of Physical and Chemical Stability
The parenteral composition of Example 1 was checked for chemical stability upon storage at room temperature (25°C/60% relative humidity) and at 40°C/75% relative humidity (accelerated storage stability condition). The assay of Vitamin D3 (% of label claim) and pH upon storage were determined at different time points and are tabulated below in Table 10:

Table 10: Stability study results for parenteral composition of example 1:
No. Parameter Initial 25°C/60% RH 40°C/75% RH
1M 3M 6M 12M 1M 3M 6M
1 Assay (%)
(Vitamin D3) 103.80
101.5 100.2 96.3 95.1 103.1 97.2 96.1
2 Physical observation Clear Clear Clear Clear Clear Clear Clear Clear
3 pH 7.5 7.32 7.39 7.42 7.40 7.3 7.46 7.56

The above data shows that the assay of Vitamin D3 was within 92%-110% upon storage. Thus, it can be concluded that the parenteral composition of Example 1 was found to be chemically stable when stored at room temperature (25°C/60% relative humidity) for 12 months and at 40°C/75% relative humidity (accelerated storage stability condition) for a prolonged period of at least 6 months.

As disclosed in table-10, when the parenteral composition of Example-1 was stored over a prolonged period of time (for at least 12 months) at room temperature (25°C/60% relative humidity) as well as at accelerated storage stability conditions such as at 40°C/75% relative humidity for 6 months, showed no signs of haziness i.e. the parenteral composition was physically stable.

Pharmacokinetic (PK) study:
In a controlled study, Vitamin D3 (Cholecalciferol) aqueous injection prepared according to the present invention (Example-1) was administered as intramuscular injection to 6 Male Wistar rats of one group and conventional Vitamin D3 (Cholecalciferol) injection (as per Indian Pharmacopoeia containing Vitamin D3 and ethyl oleate) was administered to 6 Male Wistar rats of second group with a dose of 62000 IU/kg body weight of Vitamin D3.
Plasma sample was collected from retro-orbital sinus node at 0 min, 30 min, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 24 hr, 48 hr, 72 hr, 96 hr, 120 hr and 144 hr. Vitamin D3 and its active metabolite (25-Hydroxy Vitamin D3) were measured and depicted in Table – 11, Figure 1 and 2.

Sampling point and plasma concentration of vitamin D3 and 25-Hydroxy Vitamin D3 are summarized in the following Table-11 below
Table - 11
Time (Hr) Vitamin D3 Conc. (ng/mL) 25-Hydroxy Vitamin D3 Conc. (ng/mL)
Vita-D3 Conventional Injection Vita – D3 Injection of the present invention Fold increase Vita D3 Conven. Injection Vita – D3 Injection of the present invention Fold increase
0 0 0 - 0 0 -
0.5 0 121.8783 - 0 0 -
1 12.842 147.05 11.45071 0 0 -
2 24.888 211.1315 8.483265 0 0 -
4 27.42625 343.785 12.53489 0 12.78 -
6 27.35367 450.1667 16.45727 11.72 18.32 1.56314
8 39.54317 593.126 14.99946 15.75 27.66 1.75619
24 50.89 670.144 13.16848 16.9 65.73 3.889349
48 67.82217 543.048 8.00694 17.4 107.68 6.188506
72 54.83833 310.0072 5.653111 13.47 86.43 6.416481
96 53.09433 239.5433 4.511655 13.14 77.08 5.866058
120 50.749 182.6273 3.598639 14.62 64.4 4.404925
144 50.89083 133.445 2.622181 15.15 47.07 3.106931

Table – 11 & Figure 1 and 2 demonstrate that Vitamin D3 aqueous injection of the present invention provides faster and higher level of Vitamin D3 and its active metabolite.

The plasma concentration of Vitamin D3 level achieved at 0.5 hrs. and maintained till end of study by an aqueous preparation of the present invention, which is not achieved by convention oily preparation.

Similarly, the plasma level of active metabolite of Vitamin D3 more than 25 ng/ml is achieved at 8 hrs and maintained till end of study by aqueous preparation of the present invention, which is not achieved by convention oily preparation.

Thus, the aqueous parenteral composition of the present invention comprising 300000 to 600000 IU/ml of Vitamin D3 provides enhanced bioavailability.
,CLAIMS:1. A stable aqueous parenteral composition comprising 300000 to 600000 IU/ml of Vitamin
D3, Glycofurol at least 5% w/v, and a surfactant at least 5% w/v.

2. The stable aqueous parenteral composition as claimed in claim 1, wherein the ratio of
Glycofurol + surfactant to Vitamin D3 is in the range of 16 to 50 and the ratio of
surfactant to Vitamin D3 is greater than or equal to 8.

3. The stable aqueous parenteral composition as claimed in claim 1, wherein the surfactant is
in the range of 5% w/v to 30% w/v.

4. The stable aqueous parenteral composition as claimed in claim 1, wherein Glycofurol is in
the range 5% w/v to 30% w/v.

5. The stable aqueous parenteral composition as claimed in any preceding claims, wherein the
surfactant is selected from polysorbate 80, Solutol® and Poloxamer®.

6. The stable aqueous parenteral composition as claimed in any preceding claims, wherein the
surfactant is polysorbate 80.

7. The stable aqueous parenteral composition as claimed in any preceding claims, wherein the
said composition further comprising a chelating agent, an antioxidant and a buffer.

8. The stable aqueous parenteral composition as claimed in any preceding claims is
substantially free from alcohol and oil.

9. A process for preparation of the stable aqueous parenteral composition as claimed in any
preceding claims comprising:
(a) dissolving Vitamin D3 in Glycofurol and surfactant,
(b) optionally adding buffering agent and antioxidant to the solution of step (a), and
(c) adjusting the final volume with water for injection.