FORM 2
THE PATENTS ACT 1970
(39 of 1970)
AND
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule l3)
1. TITLE OF THE INVENTION:
"PARENTERAL FORMULATIONS OF LEVOSIMENDAN"
2. APPLICANT:
(a) NAME: GUFIC BIOSCIENCES LIMITED
(b)NATIONALITY: Indian Company incorporated under the Companies Act, 1956
(c) ADDRESS: Subhash Road-A, Vile Parle (East), Mumbai-400057, Maharashtra, India.
3. PREAMBLE TO THE DESCRIPTION:
The following specification particularly describes the invention and the manner in which it is to be formed.

Field of Invention:
The present invention relates to a novel formulation for water insoluble drug, Levosimendan, mainly meant for parenteral administration. More particularly, the invention provides an alcohol free formulation consisting of Levosimendan as the active ingredient that provides advantages such as increased solubility and stability of the molecule (water insoluble drug) and to the process of preparation thereof.
Background:
Levosimendan is a calcium sensitizer used in the management of acutely decompensated congestive heart failure (Source-Wikepedia).
Levosimendan increases the sensitivity of the heart to calcium, thus increasing cardiac contractility without a rise in intracellular calcium. Levosimendan exerts its positive inotropic effect by increasing calcium sensitivity of myocytes by binding to cardiac troponin C in a calcium-dependent manner. It also has a vasodilatory effect, by opening adenosine triphosphate (ATP) sensitive potassium channels in vascular smooth muscle to cause smooth muscle relaxation. The combined inotropic and vasodilatory actions result in an increased force of contraction, decreased preload and decreased after load. Moreover, by opening also the mitochondrial (ATP) sensitive potassium channels in cardiomyocytes, the drug exerts a cardioprotective effect. (Source-Wikepedia).
The hemodynamic effects of levosimendan in man are described in Sundberg, S. et al., Am. J. Cardiol., 1995; 75: 1061-1066. Pharmocokinetics of levosimendan in man after i.v. and oral dosing is described in Sandell, E.-P. et al., J. Cardiovasc. Pharmacol 26(Suppl.l), S57-S62, 1995. The use of levosimendan in the treatment of myocardial ischemia is described in WO 93/21921. Transdermal compositions of levosimendan are described in WO 98/01111.
In clinical trials levosimendan has been shown to reduce the risk of worsening CHF or death compared with dobutamine and placebo in patients with decompensated CHF. The drug is well tolerated, does not "appear to be proarrhythmic, has minimal potential for interactions with other drugs, and does not reduce short-or long-term (30-day) survival.

Thus, unlike some other agents administered to improve contractility in decompensated heart failure, IV levosimendan appears to offer therapeutic benefits without risk of arrhythmogenesis and/or uncertain impacts on survival, (source - The Internet Journal of Cardiology™ISSN:1528-834X-Dobutamine Kills Good Hearts! Levosimendan May Not-Satish Kumar MD)
It is apparent that the drug is mainly used in life saving cases with increased stability and no toxicity is the preferred norm.
Currently available formulations of Levosimendan internationally are in liquid vials.
Advantages of taking the drug by parenteral, e.g. intravenous administration are as following:
1. an almost immediate response.
2. the therapeutic response may be more readily controlled by administering the drug parenterally, and a drug can be administered parenterally to a patient when it cannot be administered orally because of the unconscious state of the patient, or because of inactivation or lack of absorption in the intestinal tract.
The manufacture of Levosimendan solutions, and particularly solutions suitable for intravenous use, involves a number of problems which are caused by the sensitivity of Levosimendan against chemical and physical influences. In solutions, Levosimendan is sensitive to chemical degradation which limits the shelf-life of solutions in water and may produce undesirable degradation products. Levosimendan is also poorly soluble in water and precipitates easily from aqueous solutions which are extremely dangerous. The solubility of Levosimendan decreases further strongly when the pH is lowered from neutral, so that low pH would in principle seem unfavorable.(Source-Pharmaceutical solutions of Levosimendan Patent 6730673 Issued on May 4, 2004)
Also, Levosimendan solutions contain anhydrous alcohol as major vehicle for the drug which requires use of specified non reactive packaging material (rubber stoppers).
There is therefore a need to develop an injectable formulation which overcomes all of above shortcomings and provides a more stable formulation which last throughout the shelf life of the product.

Objects of the invention:
Therefore, the primary object of the current invention is to provide a formulation which does not require the use of alcohol, is stable than the liquid one by making the molecule available via a lyophilized powder injectable route.
The farther object of the invention is to provide a formulation with a pH in the range of 6-8 to make it more compliant for use.
Summary of the invention:
In accordance with the objectives, the present invention provides a pharmaceutical composition for parental administration comprising of Levosimendan or pharmaceutically acceptable salt thereof as the active ingredient along with a suitable solubilizing agent and an alkalizing or buffering agent and to the process of preparation thereof.
In a preferred aspect, the pharmaceutical composition is free of alcohol, the pH of the composition is maintained in the range of 6-8 using an alkalizing or buffering agent selected from sodium hydroxide, sodium bicarbonate, sodium carbonate, sodium phosphate, Disodium phosphate, sodium citrate, lactate, acetate and salts of a strong base, such as for example sodium hydroxide or potassium hydroxide with a weak acid e.g. carbonic acid, acetic acid or lactic acid.
The solubility of Levosimendan is increased by adding solubilizing agent selected from propylene glycol, surfactants, polymeric substances such as polysorbates, polyalkytene glycols, poloxamers or polyvinyl pyrrolidene etc.
In another preferred aspect, the pharmaceutical composition of Levosimendan, solubilizing agent and an alkalizing or buffering agent is freeze dried and is provided as a drug concentrate.
In another aspect, the present invention provides a method for of increasing solubility of levosimendan in an aqueous solution.
In yet another aspect, the present invention provides a pharmaceutical composition suitable for intravenous injection comprising of Levosimendan or pharmaceutically

acceptable salt thereof; a solubilizing agent; an alkalizing or buffering agent and a diluent selected form normal saline, half saline or dextrose.
Disclosure of the invention:
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects there of may be fully understood and appreciated.
As used herein 'Levosimendan' refers to a compound (usually a liquid) that dissolves in a soiid, liquid or gaseous solute, resulting in a solution. "As used here in, "diluent" refers to any liquid material used to dilute or carry an active ingredient to deliver the drug.
As used here in, the term "Pharmaceutically acceptable" refers to diluents, agents, excipients, stabilizers and the like, within the scope of sound medical judgment, which are suitable for use in contact with human tissues and lower animals without undue toxicity, irritation, allergic reaction and the like, in keeping with a reasonable benefit/risk ratio, and effective for their intended use.
As used herein, the term "Alkalinizing agent" refers to any compound that increases the pH of a solution to which it is added.
As used herein, the word "buffering agent" refers to an agent that adjusts / maintains / controls the pH of a solution. Generally, a buffering agent acts by driving an acidic or basic solution to a certain pH range and then maintaining the solution in that pH range. As used herein, the word "Solubilizing agent" refers to an agent that increases the solubility of Levosimendan in aqueous solution.
In an aspect, the present invention is directed to pharmaceutical composition/formulation for parenteral administration, comprising Levosimendan or pharmaceutically acceptable salt as the active ingredient with an increased pH of aqueous solution, so as to prevent the precipitation of active ingredient.
Levosimendan is a crystalline powder with a pKa of 6.2 and is poorly soluble in water (0.04mg/ml and precipitates easily from aqueous solutions. The solubility of Levosimendan decreases further strongly when the pH is lowered from neutral. However,

it is observed by the current inventors that it is possible to provide stable pharmaceutically acceptable composition/formulation for parenteral administration or intravenous infusion comprising of Levosimendan as the active, with pH in the range of 6-8 using a suitable alkalinizing or buffering agent. Such composition is found to be chemically and physically stable over an extended period of time and is suitable for pharmaceutical use.
Further, the solubility of Levosimendan can be increased using a suitable solubilizing agent, where, Levosimendan can be maintained in a dissolved state in the aqueous solution and thereby preventing crystallization or crystalline growth of Levosimendan.
Accordingly, in an embodiment, the present invention provides freeze dried pharmaceutical composition for parenteral administration, free of alcohol and an acid, as disclosed herein below; a) Ievosimendan or a pharmaceutically acceptable salt thereof,
(b) a pharmaceutically acceptable solubilizing agent, and
(c) a pharmaceutically acceptable alkalinizing agent or a buffering substance.
The pharmaceutically acceptable solubilizing agent can be selected from co solvents such as propylene glycol, surfactants, polymeric substances such as polysorbates, polyalkylene glycols, poloxamers or polyvinyl pyrrolidene etc. For intravenous administration, solubilizing agent is selected from polyvinyl pyrrolidene or ethanol or mixtures thereof, more preferably, polyvinyl pyrrolidene Kollidon PF12. The amount of solubilizing agent is in the range of 1-20%, more preferably 10% by weight of the composition.
The pharmaceutically acceptable alkalinizing agents for the purpose of present invention include sodium hydroxide, sodium bicarbonate, sodium carbonate, sodium phosphate, Disodium phosphate, sodium citrate, lactate, acetate and salts of a strong base, such as for example sodium hydroxide or potassium hydroxide with a weak acid e.g. carbonic acid, acetic acid or lactic acid.
As is known in the art, buffering agents have different properties, such as different solubility's, acidities and alkalinities. For purposes of this invention, a buffering agent also may function as an alkalinizing agent and is selected from Tris-buffers, or

triethanolamine. The alkalinizing or buffering agent is added in an amount necessary to adjust the pH to the desired range.
The pH adjuster, preferably, being 10% solution of Sodium Citrate in aqueous solution of 1 % Triethanolamine.
The resulting drug concentrate is freeze dried, after filtration from 0.22micron Durapore membrane filters.
The aqueous intravenous composition of the invention may also comprise a physiologically and pharmaceutically acceptable compound effective to render the aqueous intravenous composition isotonic, i.e. to have an osmotic pressure corresponding to that of a 0.9% solution of sodium chloride. Typical examples of such compounds are chloride salts such as NaCl and saccharides such as sorbitol, mannitol and dextrose/glucose. The preparation of isotonic solutions is well known for one skilled in the art. The diluents suitable for the purpose of present invention include 5% Dextrose, 5% Glucose, Ringers solution, Lactated Ringers solution, saline solution and half normal saline. If the diluted pharmaceutical composition is to be stored and infused at ambient temperature for a longer time period, e.g. for a period longer than about 10 hours, preferably, the diluent will be one other than 5% dextrose or 5% glucose.
In another embodiment, the present invention provides a method of increasing solubility of levosimendan in an aqueous solution comprising the step of combining levosimendan with an aqueous solution comprising a solubilising agent and an alkalinizing or buffering agent. The alkalinizing agent or buffering agent may be combined after combining levosimendan with a pharmaceutically acceptable solubilizing agent. Alkalinizing or buffering agent may be selected from the group consisting of Disodium phosphate and triethanolamine. Preferably the alkalinizing agent or buffering agent is a solution of 10% disodium phosphate.
In another embodiment, invention provides a method of increasing solubility of levosimendan in an aqueous solution comprising of adding solubilizing agent and an alkalinizing or buffering agent to the solution. The solubilizing agent is selected from pharmaceutically acceptable solubilizing agent, preferably Kollidone PF-12, and

alkalinizing agent or buffering agent being Tris-buffer, having concentration of at least 15% in 0.9% of aqueous solution of sodium chloride.
In another embodiment, invention provides a method of increasing solubility of levosimendan in an aqueous solution comprising the step of combining levosimendan with an aqueous solution comprising of solubilising agent with an alkalinizing or buffering agent. The alkalinizing1 agent or buffering agent may be added after combining levosimendan with a pharmaceutical^ acceptable solubilizing agent. Alkalinizing or buffering agent may be selected from the group consisting of Sodium Bicarbonate, preferably, a solution of 5% Sodium Bicarbonate.
Freeze-drying (also known as lyophilization or cryodesiccation) is a dehydration process typically used to preserve a perishable material or make the material more convenient for transport. Freeze-drying works by freezing the material and then reducing the surrounding pressure and adding enough heat to allow the frozen water in the material to sublime directly from the solid phase to the gas phase.
The freeze-drying process was developed as a commercial technique that enabled serum to be rendered chemically stable and viable without having to be refrigerated. Shortly thereafter, the freeze dry process was applied to penicillin and bone, and lyophilization became recognized as an important scientific technique for preservation of biologicals. Since that time, freeze drying has been used as a preservation or processing technique for a wide variety of products. Some of the applications include the processing of pharmaceuticals, diagnostic kits, restoration of historic/reclaimed boat hulls.
If a freeze-dried substance is sealed to prevent the re-absorption of moisture, the substance may be stored at room temperature without refrigeration, and be protected against spoilage for many years. Preservation is possible because the greatly reduced water content inhibits the action of microorganisms and enzymes that would normally spoil or degrade the substance.
Freeze-drying also causes less damage to the substance than other dehydration methods using higher temperatures. Freeze-drying does not usually cause shrinkage or toughening of the material being dried. In addition, flavors, smells and nutritional content generally remain unchanged, making the process popular for preserving food. However, water is

not the only chemical capable of sublimation, and the loss of other volatile compounds such as acetic acid (vinegar) and alcohols can yield undesirable results.
Freeze dried products can be rehydrated (reconstituted) much more quickly and easily because the process leaves microscopic pores. The pores are created by the ice crystals that sublimate, leaving gaps or pores in their place. This is especially important when it comes to pharmaceutical uses. Freeze-drying can also be used to increase the shelf life of some pharmaceuticals for many years.
The use of technology of lyophilization to develop a formulation of Levosimendan according to the present invention offers the following advantages;
1. Has a greater stability for the entire period of the Shelf life.
2. Does not involve usage of any alcoholic solvent and whose solution before lyophilization has a pH between 6.8 to 7.3, more harmonious with the human body.
3. Does not require use of Chlorbutyl or bromobutyl rubber closure with fluropolymer coating (specialized packaging material)
In yet another embodiment, the invention provides a process for preparation of pharmaceutical composition, in particular for intravenous infusion, comprising Levosimendan or a pharmaceutically acceptable salt thereof as the active ingredient, a pharmaceutically acceptable solubilizing aqueous vehicle, acceptable alkalizing or buffering agent. Accordingly, Levosimendan or pharmaceutically acceptable salt thereof initially may be solubilized in an aqueous solution containing solubilizing agent, preferably 10% Kollidone PF12 solution followed by addition of alkalizing or buffering agent, preferably 10% Sodium citrate in aqueous solution of 1% Triethanolamine and then freeze drying to remove aqueous vehicle.
The formulation of the current invention is meant for administration via the IV route, the selection of the above ingredients is done keeping in mind their compatibilities and stability during the rigorous process for lyophilization and also in terms of safety for use in patients as an injectable. Moreover, since Levosimendan has low stability in water (0.04 mg/ml), a combination of buffers is required to prepare a solution with the desired concentration ready for lyophilization at the desired pH.

Moreover, for intravenous pharmaceutical aqueous solution to be administrable to a patient, the active ingredient of the composition needs to be dissolved sufficiently in the composition i.e. be free from visible particles. To achieve this, freeze drying process is used advantageously to obtain clear yellow solution free from visible particles. Depending on final desired concentration of Levosimendan, the pH of aqueous composition is varied such that the drug is sufficiently solubilized to achieve that concentration. Freeze dried Levosimendan in solution containing final drug concentrate of 0.05 mg/ml, 0.025 mg/ml or 0.0125 mg/ml requires a pH of about 7.2 for the drug to be solubilized, which is achieved through use of alkalizing or buffering agent.
The pharmaceutical composition of the invention described herein is freeze dried composition, which may also be prepared by dissolving Levosimendan first in aqueous vehicle containing solubilizing agent and alkalinizing agent. The resulting drug concentrate is freeze dried. Developing freeze drying process for such a low soluble active ingredient needs special care and it is an art in its own way because of low concentration of solute (about 0.0125%) and maximum amount of aqueous vehicle e.g. water for injection. Thereafter, freeze dried drug may be diluted with suitable diluent. The final concentration of solution may be reduced to desired level; using 5% Dextrose infusion prior to administration to a patient.
The process of the present invention not only avoids use of anhydrous alcohol as medium of vehicle but also provides Levosimendan active as lyophilized powder which is a more stable form than liquid concentrated solution of the same. Also, the formulation comprising of Levosimendan active offers greater stability than the liquid formulation which sometimes tends to change color on storage during shelf life.
It is understood that the following Examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art.

Experimental:
Several different trials were conducted and tested before narrowing down to the present formulation. Some of these trials are discussed in brief below to emphasize the inventiveness of the current invention.
The buffers used were selected from sodium citrate, sodium bicarbonate, monoethanolamine, tris-buffer and phosphate buffers.
Example: 1
Levosimendan (active) plus water was taken and citric acid 1 - 20% solution was added drop wise to the solution but the active remained insoluble. So no assay color done,
Example: 2
In Levosimendan plus water, 1 - 30% sodium bicarbonate solution was added drop wise
but the resulting solution changed color and assay of the active went down and out of
specifications when measured by spectrophotometer.
Assay: 67.75%
Example: 3
In Levosimendan plus water, 1 - 10% solution of monoethanolamine was added drop wise but the solution changed color and assay went out of specifications when measured by spectrophotometer. Assay: 54.38%
Example: 4
In Levosimendan plus water, I -50 times (weight of Levosimendan) powder of Disodium phosphate was added but the active was partly soluble on filtration, Golden yellow solution obtained showed -Assay: 52.55%
Example: 5
In Levosimendan plus water, 1-20% Tris buffer was added drop wise but the solution
changed colour and the went out of specification when measured by
spectrophotometer.

Assay: 69.50%
Likewise other pre-formulation trials were checked and it was observed that the products were either getting solubilized partly or assay was getting reduced to 60 to 80%.
Example 6:
A combination of Levosimendan plus Kollidone PF 12 as solubilizer was taken followed by addition of tri sodium citrate drop wise till pH 7.2 - 7.3. The %conc. of Tri sodium citrate solution was studied from 10% to 50% and finally the most optimum concentration was selected. This was followed by addition of the additive selected from Mannitol and the volume was made with water. The assay of the final solution was within specification when measured by spectrophotometer and HPLC. Assay: 69.10%
In another trial the product solution was prepared without Mannitol & checked as mentioned in above trial. Assay: 98.57%
The pH of above mentioned solutions was adjusted to 6.3 to 8.5, more preferably between 6.3 - 7.8 and this solution was then lyophilized.
The solution was filled individually in vials or filled in a sterile tray and subjected to a cycle of lyophilization to obtain unit lyophilized vials or lyophilized powder ready to be filled directly in vials. The lyophilized powder was diluted further with 5ml Dextrose solution (5%) and or saline.
This study was further Jinked up with solution stability studies at pre-formulation stage. Accordingly these examples were repeated quantitatively and testing was done using HPLC assay method and UV absorption spectra.
Example 7:

Ingredients Qty. per ml
Levosimendan 2.5mg 5ml clear glass vial USP I vial were used chlorobutyl stopper with fluorocoating were used.
Kollidone PF 12 10 mg

Anhydrous Citric acid 2mg

Absolute alcohol 1ml

Dissolved Kollidone PF12 & citric acid in alcohol. Added Levosimendan and stirred to dissolve. Filtered. Filled 5ml in each vial. The results are described in Table 1 herein below:
Tablel:

Period Assay PH Single impurity Total impurity Storage temp. Color
Initial 103.94 3.1 0.12 0.17 - Orange
Color
Clear solution
After 4 months 101.28 3.15 0.14 0.17 2-8°C

After 4.5 months 97.55 3.2 0.49 0.77 25°C

After 6 months 92.2 3A 0.50 1 0.79 25°C Dark orange
After 8 months 86.2 3.86 0.53 1.10 25°C Dark orange
After 9 months 86.31 3.91 3.32 3.32 25°C Dark orange
Conclusion: The product is stable for 6 months at 25°C and for 2 years at 2 - 8°C.
Example 8;
Same as Example 7 but in this trial alcohol was used aldehyde free after treatment with chemicals and distilled over sodium sulphate unhydrous.

Period Assay pH Single impurity Total impurity Storage
temp. Color
Initial 100.5 3.10 0.01 0.01 - Orange color clear solution
After 1 month 94.31 3.42 0.4 0.42 25 °C Orange color clear solution
After 4 months 94.37 3.39 1.05 1.55 25°C Orange color clear solution
Results: Use of aldehyde free alcohol is not advisable as it shortens the stability duration. Example 9:

Ingredients Qty. per ml
Levosimendan 2.5mg
Disodium phosphate 2.5mg
0.5NNaOH q.s. to pH 8.89
Bulk Assay initial 101.58%
Bulk assay after 4 hrs. 100.67% (Solution at 2-8°C)
Bulk assay after 24 hrs. 93.76% (Solution at 2-8°C)
CONCLUSION : Degradation observed.

Example 10:

Ingredients Qty. per ml
Levosimendan 2.5mg
Trisodium citrate 5.0mg
0.5N NaOH q.s. to pH 10.90
Bulk Assay initial 98.63%
Bulk assay after 4 hrs. 99.10% (Solution at 2 - 8°C)
Bulk assay after 24 hrs. 98.70% (Solution at 2 - 8°C)
There is no change in Assay. So for freeze drying of products Example 9 and example 10 were repeated with Mannitol.
Example 11:

Ingredients Qty. per ml
Levosimendan 2.5mg
Disodium phosphate 5.0mg
Mannitol 20mg
0.5N NaOH q.s. to pH 8.17
Bulk Assay initial 68.91%
Example 12:
Conclusion: Trials assay went down with the use of
Mannitol. Example 13 (Current Invention):
Ingredients Qty. per ml
Levosimendan 2.5mg
Trisodium citrate 9.0mg
Mannitol 20mg
0.5N NaOH q.s. to pH 8.25
Bulk Assay initial 69.22%
Ingredients Qty. per ml
Levosimendan I.25mg 20ml vial used- 10ml filled volume.
KollidonePF 12 15mg 10ml solution filled in each vial
Sodium citrate 125mg Grey bromo butyl plugs were used with full slotted
Triethanot amine HCl 0.56mg For freeze-drying
Water for injection To make 1ml
pH 7.0
Bulk Assay 96.65%

After freeze drying testing was done.

Results of stability study at 25°C and 40°C are given below :

Period Assay PH Single
impurity Total impurity Storage temp.
Initial 96.77 7.39 0.72 1.05 -
After 4 months 95.37 7.42 1.64 2.38 25°C
After 4 months 90.8 7.50 2.67 3.79 40°C
Conclusion : From above results Product can be seen stable at 2 - 8°C because at 25°C Storage products shows impurity within limit which is accelerated with increase in temperature. At 40°C impurity crosses limits.
For process validation further two trials were taken on similar line of Example 13 and results are summarized in tabular form as follows as Example 14 & Example 15
Examplel4:

No. Period Bulk Assay: 103.02 pH of Bulk solution: 7.3
Assay Water
(Max.6%) PH
(6 to 8) Impurity Single Impurity Total Storage temp.
1 Initia! 100.81 4.83 7.44 0.72 1.05 -
2 3 months 100.08 4.85 7.31 - - 2-8°C
3 6 months 99.69 4.69 7.42 1.06 1.49 2-8°C
4 9 months 98.79 4.96 7.49 1.45 2.17 2-8°C
5 4 months 100.64 4.95 7.33 1.18 2.19 25°C
6 6 months 99.80 4.88 7.38 1.57 2.91 25°C
No. Period Bulk Assay: 101.03 pH of Bulk solution: 7.38
Assay Water (Max.6%) pH (6 to 8) Impurity Single Impurity Total Storage temp.
1 Initial 99.88 5.78 7.31 0.75 1.05 -
2 3 months 99.82 5.29 7.38 - - 2-8°C
3 6 months 99.12 5.64 7.29 1.11 1.42 2-8°C
4 9 months 98.16 5.36 7.42 1.22 1.87 2-8°C
5 3 months 100.72 5.88 7.38 1.53 2.17 25°C
6 6 months 99.25 5.82 7.36 1.53 2.68 25°C
Freeze drying process involved cooling of product to -30°C, raising temperature to +0°C at 150mbar vacuum in 33 hours, then at 75 mbar, raising temperature to +45°C in 12 hrs.

The freeze dried product so obtained dissolved easily in 2ml water for injection, to form clear golden yellow solution.
The solution stability of product was checked after dilution with 5% Dextrose injection and results are summarized below-
Label claim: \2.5mg Levosimendan per vial
Diluted with 5 ml 5% Dextrose solution to get 2.5mg/ml
Diluted this solution to 500ml of 5% Dextrose solution to obtain 25mcg/ml of
Levosimendan
Reconstituted Solution stability study Report
Product : Levosimendan for injection 12,5mg/vial
B.No. : 031/10, Mfg. date: 12/10/10
Label claim : Each vial contains: Levosimendan 12.5mg
Standard Description : Yellow coloured powder/cake in 20ml tubular clear glass vial USP
Type-I with grey bromo butyl rubber stopper and flip-off seal cap
Av. Weight of content : 287.0mg/vial
Reconstitution of inj. : Dissolve content of 1 vial in 5ml WFI
Storage condition : Kept at room temperature.


Initial 20/10/ 10 244. 8 99.58 % 101.80 % 100. 30%
22/10/ 10 295. 9 0.68% 0.84% 0.65% 0.83% 0.61% 0.81%

After 20/10/ 244. 96.35 101.40 100.
4 hrs. 10 8 % % 10%
22/10/ 10 295. 9 1.36% 1.78% 0.79% 1.01% 0.76% 0.99%

After 21/10/ 244. 7123 100.80 99.4
24 10 8 % % 1%
hrs. 23/10/ 10 295. 9 5.69% 7.65% 1.39% 1.83% 1.36% 1.78%
Conclusion : Reconstituted infusion should be consumed within 4 to 6 hours if 5% Dextrose in used for infusion.
In summary, the present invention provides water soluble Levosimendan as freeze dried stable formulation, stable for 24 months, when stored at 2 - 8°C.

We claim,
1. An alcohol and acid free pharmaceutical composition for parental administration, comprising; i. levosimendan or a pharmaceutically acceptable salt thereof as an active
ingredient in an amount of 12.5mg/vial; ii. 10% solution of Kollidone PF-12 in aqueous solution as a solubilizing agent;
and iii. pharmaceutically acceptable alkalinizing or buffering agent; wherein said composition is stable at a pH in the range of 6-8.
2. The pharmaceutical composition according to claim 1, wherein said composition is freeze dried.
3. The pharmaceutical composition according to claim 1, wherein said alkalinizing or buffering agent is selected from sodium hydroxide, sodium phosphate, Disodium phosphate, sodium citrate, lactate, acetate and salts of a strong base, such as for example sodium hydroxide or potassium hydroxide with a weak acid e.g. carbonic acid, acetic acid or lactic acid, tris buffer, triethanolamine.
4. The pharmaceutical composition according to claim 3, wherein said alkalinizing agent or buffering agent is 10% solution of sodium citrate in aqueous solution of 1% triethanolamine.
5. A method of increasing solubility of levosimendan in an aqueous solution, comprising combining levosimendan with aqueous solution of 10% solution of Kollidone PF-12 as solubilizing agent and 10% disodium phosphate as alkalinizing or buffering agent.
6. A method of increasing solubility of levosimendan in an aqueous solution comprising, adding 10% solution of Kollidone PF-12 as solubilizing agent and Tris-buffer, having concentration of at least 15% in 0.9% of aqueous solution of

sodium chloride as an alkalinizing or buffering agent to the aqueous solution of levosimendan.
7. A method of increasing solubility of levosimendan in an aqueous solution, comprising combining levosimendan with aqueous solution of 10% solution of Kollidone PF-12 as solubilizing agent and 5% Sodium Bicarbonate as alkalinizing or buffering agent.
8. A kit comprising composition for parental administration comprising
i. freeze dried levosimendan composition according to claim I and ii. diluent selected from 5% Dextrose solution and/ or 0.9% saline.