FORM 2
THE PATENTS ACT 1970
(Act 39 of 1970)
&
THE PATENTS RULE, 2003
COMPLETE SPECIFICATION (SECTION 10 and Rule 13)
TITLE OF THE INVENTION "PHARMACEUTICAL COMPOSITIONS OF VITAMIN Kl"
Emcure Pharmaceuticals Limited, an Indian company, registered under the Indian Company's Act 1957 and having its
registered office at
Emcure House, T-184, M.I.D.C., Bhosari, Pune-411026, 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 pharmaceutical compositions comprising trans isomer of Vitamin Kl and processes for preparation thereof.
BACKGROUND OF THE INVENTION
Vitamin K is the generic name for 2-methyl-l,4-naphthoquinone derivatives having activity on blood-coagulation and electron transport systems. Vitamin K was first identified in 1929 by Danish scientist Henrik Dam during his investigations regarding the role of cholesterol in metabolism of chicken. These substances were found to be essential for synthesizing prothrombin in the liver, which is a precursor of the enzyme thrombin causing blood coagulation reaction and were also known to prevent release of calcium from the bones.
Amongst these derivatives, Vitamin Kl, also known as phylloquinone, phytonadione or phytomenadione is known to be the active ingredient which exhibits significant anticoagulant activity. Vitamin Kl, chemically identified as, 2-Methyl-3-[(2E,7R,llR)-3,7,ll,15-tetramethyl-2-hexadecenyl]-l,4-naphthalenedione has two geometrical isomers (cis / trans or Z / E isomers) as well as two asymmetric centres at C7 and CI 1, each generating a pair of two enantiomers. Thus, there are total eight diastereomers for the molecule, out of which, 2'trans-7R, 11R stereoisomer, having chemical structure as given below, is the only active isomer and hence, the desired diastereomer.

An injectable formulation of Vitamin Kl from Biovail Technologies, (brand name, Aquamephyton) was first approved in in 1960. Presently, there are two reference listed drugs in United States of America for Phytonadione injectable formulations. One of them is marketed by Hospira, (approved in 1983) under the brand name "Vitamin Kl" having equivalent base strength of lmg/ 0.5ml and 10 mg/ml) while the other is from International Medication (brand name - Phytonadione, approved prior to 1976); having equivalent base strength of lmg/ 0.5ml.

Vitamin Kl Injection (Phytonadione Injectable Emulsion, USP) is indicated for the following coagulation disorders which is due to formation of factors II, VII, IX and X when caused by vitamin K deficiency or interference with vitamin K activity.
a) anticoagulant-induced prothrombin deficiency caused by coumarin or indanedione derivatives;
b) prophylaxis and therapy of hemorrhagic disease of the newborn;
c) hypoprothrombinemia due to antibacterial therapy;
d) hypoprothrombinemia secondary to factors limiting absorption or synthesis of vitamin K, e.g., obstructive jaundice, biliary fistula, sprue, ulcerative colitis, celiac disease, intestinal resection, cystic fibrosis of the pancreas, and regional enteritis;
e) other drug-induced hypoprothrombinemia where it is definitely shown that the result is due to interference with vitamin K metabolism, e.g., salicylates.
The recommended dose of Vitamin K injection formulation in the adult patients is in the range of 2.5 to 25 mg or more (rarely up to 50 mg) to prevent the anticoagulant induced prothrombin deficiency as well as to treat hypoprothrombenemia.
The analytical method for injectable formulations of Vitamin Kl, as disclosed in US Pharmacopoeia (USP) comprises liquid chromatographic (LC) analysis using the stationary phase octadecyl silanes (ODS or CI8), chemically bonded to porous silica or ceramic particles, and a mixture of alcohol and water as mobile phase. For the API analysis, USP discloses LC method wherein the stationary phase comprises porous silica particles, and mobile phase is a mixture of n-hexane and n-amyl alcohol.
When the injectable formulation from Hospira was subjected to API assay method (USP), two peaks corresponding to trans and cis isomers of phytonadione were obtained respectively at RT 23.1 (86 A%) and 26.3 (14 A%). The same formulation, when analyzed by USP method for phytonadione injectable emulsion, the chromatogram comprised of a single peak at RT 17.0 (100A%).

However, when the present inventors carried out the liquid chromatographic (LC) analysis of injectable formulation from Hospira using a chiral LC column having amylose tris (3,5-dimethylphenylcarbamate) coated on silica-gel as stationary phase, surprisingly it was observed that the formulation showed three significant peaks at RT 6.8 (45 A%), 8.8 (48.5 A%) and 16.5 (3 A%), along with two more impurity peaks.. Out of these, only the peak at RT 8.8, with area % of 48.5 corresponded to the desired active ingredient, 2-Methyl-3-[(2E,7R,11R)-3,7,1 l,15-tetramethyl-2-hexadecenyl]-l,4-naphthalene-dione (phytonadione).
Thus, it is evident that the API assay method from USP is capable of distinguishing the geometrical (also called cis / trans or Z/E) isomers, whereas the USP method for injectable emulsion does not exhibit separation for cis-trans isomers. However, none of these methods is capable of distinguishing diastereomers of Vitamin Kl. On the contrary, the LC method developed and used by present inventors is capable of separating geometrical isomers as well as the diastereomers of Vitamin Kl.
Current knowledge of stereochemistry demands responsible drug development. Most proteins, nucleic acids, polysaccharides, lipids and steroids are chiral. In other words, almost all of the main therapeutic targets are chiral and, therefore, show stereospecific interactions with endogenous and exogenous ligands. Stereoselectivity of interaction is a well-recognized criterion of specific drug-receptor interactions. Such stereoselectivity has been observed with all classes of receptors, with both physiological and non-physiological ligands.
Further, it is not always possible to determine the long term toxic effects of the minor quantities of unintended isomers in a product by conventional acute and chronic toxicity studies. This minor quantum of unintended isomers may not reveal their real character in the short period of the chronic toxicity study. It is particularly important to consider this fact for those compounds (drugs) which are likely to be administered for a lifetime.
Therefore, the regulatory authorities all over the world are keen on having pharmaceutical formulations comprising purest possible forms of the active ingredient, which is desirable for various reasons ranging from undesired effects of the associated impurities to excessive metabolic load on patients who consume these medicines. The same principle when applied to Vitamin K, it is desirable that the formulation should contain only the known active

diastereomer, i.e. 2'trans-7R, 11R stereoisomer and should be free from all other impurities which may be either toxic or inactive. Since the vitamin K group of formulations was identified way back in 1929, the initial formulations might have been the generic 2-methyl-1,4-naphthoquinone derivatives showing desired anti-coagulant properties, however, it is need of the time to develop new formulations comprising only the desired, active diastereomer of vitamin Kl in its pure form.
Resolution of individual chiral compounds is a daunting task. However, a number of advantages are reaped by using stereochemically pure drugs, including potentially reducing the total dose required, simplification of the dose-response relationship, a source of inter-object variability is removed, and toxicity from the inactive stereoisomer(s) is minimized or eliminated. Accordingly, the use of only the active isomer as therapeutic agent is likely to yield several benefits.
OBJECT OF THE INVENTION
It is therefore an object of the present invention to provide a pharmaceutical composition comprising primarily of the desired, active diastereomer of vitamin Kl in its pure form.
Another object of the present invention is to provide a simple formulation, suitable for parenteral administration comprising primarily of the active trans-isomer of vitamin Kl in its pure form.
Yet another object of the present invention is to provide a simple, commercially viable process for preparation of an injectable formulation of vitamin Kl.
SUMMARY OF THE INVENTION
The present invention relates to stable pharmaceutical compositions comprising a desired diastereomer of phytonadione. The formulation, specifically an injectable composition, contains the desired trans-isomer, in an amount of at least 50% of the total weight of active ingredient. In the preferred embodiment of the present invention, the desired trans isomer is at least about 75% of the total weight of the active ingredient in the formulation.

In another embodiment, the present invention comprises a composition composed of the desired trans-isomer (phytonadione); and substantially free of other diastereomers, geometrical isomers and impurities.
In yet another embodiment, the present invention sets forth a simple composition for vitamin Kl suitable for parenteral administration; wherein the formulation when administered to a human subject exhibits pharmacokinetic properties such as Cmax and AUC, comparable with the marketed formulation but are achieved by a significant reduction in the therapeutic dose.
In still further embodiment, the present invention provides a 5 to 50 percent dose reduction of the active ingredient and yet achieves the desired pharmacokinetic profile required to observe the therapeutic effect.
In yet another embodiment, the present invention sets forth a safe and commercially viable process for preparation of an injectable composition for vitamin Kl that is sufficiently stable to provide an acceptable shelf life.
BRIEF DESCRIPTION OF THE DRAWINGS:
FIG. -1 is a chromatogram of the marketed injectable formulation, manufactured by Hospira, by using API assay method in United States Pharmacopoeia (USP).
FIG. -2 is a chromatogram of the marketed injectable formulation, manufactured by Hospira, by using USP method for phytonadione injectable emulsion.
FIG. - 3 is a chromatogram of the marketed injectable formulation, manufactured by Hospira, by using a chiral column.
FIG. - 4 is a chromatogram of the injectable formulation, developed according to an embodiment of the present invention, by using a chiral column.
FIG. - 5 is a linear plot of mean baseline corrected plasma concentrations versus time.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to a pharmaceutical composition comprising primarily of the desired, active diastereomer of vitamin Kl in its pure form. The formulation also contains suitable excipients which imparts to the novel formulation its desired properties.

Typically, the present invention relates to an injectable emulsion pharmaceutical compositions, suitable for parenteral administration, comprising desired diastereomer, 2-Methyl-3-[(2E,7R,11R)-3,7,1 l,15-tetramethyl-2-hexadecenyl]-l,4-naphthalene-dione (phytonadione).
As used herein the term "active ingredient" means all stereoisomers, including geometric isomers of vitamin Kl and their salts. The term "trans isomer" or "desired isomers" means a Vitamin Kl isomer having 2'trans-7R, 11R configuration and known chemically as 2-Methyl-3-[(2E,7R,llR)-3,7,ll,15-tetramethyl-2-hexadecenyl]-l,4-naphthalene-dione.
The term "emulsion" or "emulsion formulation" means a colloidal dispersion of two immiscible liquids in the form of droplets, whose diameter, in general, is less than 1 micron. An emulsion is denoted by the symbol O/W if the continuous phase is an aqueous solution and by W/O if the continuous phase is oil. Other examples of emulsions such as O/W/O include oil droplets contained within aqueous droplets dispersed in a continuous oil phase.
The term "oil" as used herein, means a general sense to identify hydrocarbon derivatives, carbohydrate derivatives, or similar organic compounds that are liquid at body temperatures, e.g., about 37 °C and are pharmacologically acceptable in injectable compositions. It includes glycerides or non-glycerides. These are generally non-polar compounds that are not immediately miscible with water.
The term "oil component" and "non-aqueous phase" are used interchangeably and refers to oil, or a combination of multiple oils and oil soluble pharmaceutically acceptable excipients.
As used herein, the term "AUCo-t" means the area under the plasma concentration versus time curve, from time 0 to the last measurable concentration as calculated by the linear trapezoidal method.
As used herein, the term "AUC0.jnf" means the area under the plasma concentration versus time curve from time 0 to infinity.
In one aspect, the present invention provides an injectable formulation of Vitamin Kl, wherein the desired trans isomer is the predominant isomer of the active agent. Typically, the desired trans isomer is present in an amount of at least 50 percent by weight of the active ingredient. In the preferred embodiment, the desired trans isomer constitutes about 70 to 80% by weight of the active ingredient and more preferably, the trans isomer is present in an amount of more

than 90 percent by weight of the active ingredient. The final formulation typically contains the desired trans isomer in the range of 10 mg / ml or 2 mg /ml of the formulation
Further, according to one embodiment of the present invention, the quantity of the trans isomer in the active ingredient is measured by using chiral columns. Typically, the column chromatography was performed using chiral column, Chiral Pak AD-H (250 X 4.6 mm, 5 mum), and using acetonitrile and 0.1 percent formic acid in water (960 :40 v/v) as mobile phase with a flow rate of 3 mL/min. As shown in figure 1 and figure 2, the USP method is not capable of separating the desired trans isomer from others.
In another aspect, the compositions of the invention are both chemically and physically stable and have the globule size in the sub-micron range. A "sub-micron size" refers to globule or droplet in the emulsion that has an average diameter of less than 1 micron as measured by conventional sizing techniques such as laser light scattering spectrometry. In certain embodiments, the emulsion contains droplets of the drug compositions that have an average diameter of less than 500, 450, 400, 350, 300 or 250 nm. Oil droplets of sub-micron size are desired for the safe passage of these droplets in, the capillary blood vessel in the circulation. In some defined aspects droplets are less than 5 microns in diameter. The compositions of the invention may need to be prepared in a sterilized formulation. An effective method of sterilization that is well-known in the art is filtration through a 0.2 micron sized filter membrane. Thus, in certain embodiments, the droplets of the emulsion compositions of the invention have an average diameter that is less than 0.2-micron (200 nm). Thus, in exemplary, but non-limiting embodiments, the emulsion droplets have an average diameter of less than about 150, 100, 75, 50, 25, 20, 15, or 10 nm.
In the preferred embodiment of the invention, the present formulation is presented as an emulsion suitable for parenteral administration. Typically, any emulsion is made up of an oil phase, an aqueous phase and emulsifiers. The oil phase of the emulsion comprises medium or long chain triglyceride which can be used either alone or in combination with one or more vegetable oils. Any injectable vegetable oil such as soybean oil, corn oil, safflower oil, sesame oil, olive oil, castor oil of a mixture thereof may be used for the oil phase. Preferably, polyehtoxylated fatty acid derivatives are preferred.

Similarly an aqueous phase is prepared by dissolving or mixing water soluble excipients in water. The aqueous phase may contain suitable tonicity adjusting agents such as dextrose, sodium chloride etc. suitable antimicrobial preservative such as benzyl alcohol, phenols, formaldehyde, quaternary ammonium compounds etc., suitable antioxidants such as ascorbic acid, tocopherols, butylated hydroxytoluene, butylated hydroxyl anisole etc. The composition may also contain suitable pH adjusting agents or buffers.
Further, the judicious selection of the excipients makes the formulation simple, convenient and easy to manufacture and helps to achieve the desired properties of the formulation such as efficacy, stability and desired pharmaceutical appearance.
Another embodiment of the present invention relates to a method for preparing an injectable formulation of Vitamin Kl. Typically, the formulations of the present invention are prepared as emulsions and the method comprises the steps of:
a) preparing the aqueous phase by dissolving water soluble excipients in water;
b) preparing an oil-phase;
c) mixing the oil phase and the aqueous phase in a homogenizer;
d) adjusting the pH;
e) filtering the resulting emulsion and filling in suitable vials.
Further, in another embodiment, when the present formulations were compared with the marketed injectable emulsion formulation, the present formulation showed improved pharmacokinetics properties. Typically, with the present formulation higher levels of maximum plasma concentration (Cmax) and Area under curve (AUCo-oo) were achieved. Therefore, in still another embodiment, a reduced dose of the formulation of the present invention is required to achieve the Cmax and AUCo-oo which are obtained by presently marketed formulation. Typically, a dose reduction of 5 to 50 percent is achieved by use of the present formulation. More specifically, by using the present formulation, a dose reduction of 10 to 30 % percent could be achieved. The mean Cmax (n=24) of the present formulation was about 145 ng/ml.
Thus, in another embodiment, the present invention relates to a reduced dose pharmaceutical composition comprising at least 50% of trans isomer of Vitamin Kl. The reduction in the dose

could be from 5 to 50 percent than the standard approved dosage of 2.5 to 25 mg or more. Typically, the composition could be provided in the form ampules or glass vials.
The principles, preferred embodiments, and modes of operation of the present invention have been described in the foregoing specification. The invention which is intended to be protected herein, however, is not to be construed limited to the particular forms disclosed, since these are to be regarded as illustrative rather than restrictive. Variations and changes may be made by those skilled in the art, without departing from the spirit of the invention.
The following examples further illustrate the invention but should not be construed as in any way limiting its scope. In particular, the processing conditions are merely exemplary and can be readily varied by one of ordinary skill in the art.
Examples:
Example 1, 2 & 3: Injectable formulations of Phytonadione
The formulations prepared as an emulsion are summarised in Table 1. The aqueous phase was prepared by dissolving water soluble excipients in water. The oil soluble excipients were dissolved in polyoxyethylated fatty acid derivative. The oil phase and the aqueous phase were mixed in a homogenizer. The pH was adjusted using hydrochloric acid and/or sodium hydroxide. The resulting emulsion was filtered and was filled in suitable vials.
Table :1 Parenteral Formulations of Phytonadione

Ingredients w/w%

Example 1 Example 2 Example 3
Phytonadione 1-10 1-10 1-10
Polyoxyethylated fatty acid derivative 45-65 45-65 45-65
Dextrose hydrous 20-40 20-40 20-40
Benzyl alcohol 5-10 5-10 5-10
Ascorbic acid 0.001 to 0.1 - 0.001 to 0.1
Butylated Hydroxytoluene (BHT) - 0.001 to 0.1 0.001 to 0.1
Water For Injection q.s. to 1ml q.s. to 1ml q.s. to 1ml

Example 4: Stability Study
The formulations prepared in the above examples were analysed for various parameters such as pH, globule size, Zeta potential and total impurities. The results are summarised in the below Table 2. Table 2: Stability study results

Parameters Example 1 Example 2 Example 3
Description Yellow aqueous dispersion
pH 6.5 6.5 6.5
Globule Size Z avg (nm) 16 17 20
Zeta potential -15 -14 -15
Osmolarity 280-340 mOsm/L
Photo degradation product I NMT .5%
Photo degradation product II NMT 1.0%
Any unspecified impurity NMT 0.2%
Total impurities NMT 3.0%

Claims:
1. An injectable composition of Vitamin Kl for parenteral administration comprising Vitamin Kl and pharmaceutically acceptable excipients, wherein the amount of 2-methyl-3-[(2E,7R,llR)-3,7,ll,15-tetramethyl-2-hexadecenyl]-l,4-naphthalenedione (trans isomer) is at least 50 % by weight of active ingredient.
2. The pharmaceutical composition as claimed in claim 1, wherein 2-methyl-3-[(2E,7R,l 1R)-3,7,11,15-tetramethyl-2-hexadecenyl]-l,4-naphthalenedione comprises at least 75% of the total weight of active ingredient.
3. The pharmaceutical composition as claimed in claim 1, wherein 2-methyl-3-[(2E,7R,l 1R)-3,7,1 l,15-tetramethyl-2-hexadecenyl]-l,4-naphthalenedione comprises at least 90% of the total weight of active ingredient.
4. The pharmaceutical composition as claimed in claim 1, wherein the pharmaceutical acceptable excipients are selected from the group consisting of buffers, emulsifying agents, pH adjusters, antioxidants, antimicrobial preservatives, and mixtures thereof.
5. A reduced dose injectable composition of Vitamin Kl for parenteral administration comprising Vitamin Kl and pharmaceutically acceptable excipients, wherein the amount of 2-Methyl-3-[(2E,7R,llR)-3,7,ll,15-tetramethyl-2-hexadecenyl]-l,4-naphthalenedione (trans isomer) is at least 50 % by weight of active ingredient.
6. A reduced dose injectable composition of Vitamin Kl for parenteral administration comprising Vitamin Kl according to claim 5 wherein the dose reduction is in the range of 5 to 50% compare to standard dose of 2.5 to 25 mg.

7. A reduced dose injectable composition of Vitamin Kl for parenteral administration comprising Vitamin Kl according to claim 5, wherein the dose reduction is in the range of 10 to 30% compare to standard dose of 2.5 to 25 mg.
8. A method of treating coagulation disorder by using pharmaceutical composition of claim 1.
9. A process for preparing the pharmaceutical composition as claimed in claim 1, comprising the steps of:

(a) preparing the aqueous phase by dissolving water soluble excipients in water;
(b) preparing an oil-phase;
(c) mixing the oil phase and the aqueous phase in a homogenizer;
(d) adjusting the pH;
(e) filtering the resulting emulsion and filling in suitable vials.
10. The pharmaceutical composition as claimed in claim 1, wherein the quantification of trans
isomer is done by using liquid chromatography employing chiral columns.