Claims:1. An alcohol and acid free, stable, pre-lyophilized pharmaceutical composition for parenteral administration, which is substantially free of impurities, comprising,
a) Levosimendan or a pharmaceutically acceptable salt thereof at least in an amount of 1 to 25mg/vial;
b) polyvinyl pyrrolidone in an effective amount of 100mg to 350mg in aqueous vehicle as a solubilizing agent;
c) Triethanolamine HCl, in an effective amount of 5 mg to 12 mg.as a pharmaceutically acceptable alkalizing agent, and
d) Sodium citrate in an effective amount of 100 to 300mg in aqueous vehicle as a buffering agent,

wherein, the composition is stable at a pH range of 6-8 and wherein, the aqueous vehicle comprises oxygen free water for injection having oxygen content less than 2 ppm.

2. The pre-lyophilized pharmaceutical composition according to claim 1, wherein, the ratio of the drug to polyvinyl pyrrolidone will be preferably in the range of 1:10 to 1:20, more preferably, 1:10 to 1:15.

3. The pre-lyophilized pharmaceutical composition according to claim 1, wherein, the ratio of drug to sodium citrate will be preferably in the range of 1:8 to 1:20; more preferably, 1:8 to 1:12.

4. The alcohol and acid free pre-lyophilized pharmaceutical composition for parenteral administration according to claim 1, which comprises,
a) Levosimendan or a pharmaceutically acceptable salt thereof at least in an amount of 12.5mg/vial;
b) 150 mg of Kollidone PF-12 in aqueous vehicle as a solubilizing agent;
c) 5.6 mg of Triethanolamine HCl, as a pharmaceutically acceptable alkalizing agent and
d) 125 mg of Sodium citrate in aqueous vehicle as a buffering agent;
wherein, the composition is stable at a pH range of 6 to 8 and wherein, the aqueous vehicle comprises oxygen free water for injection having oxygen content less than 2 ppm.

5. The pre-lyophilized pharmaceutical composition for parenteral administration according to claim 1, which comprises,
a) Levosimendan or a pharmaceutically acceptable salt thereof at least in an amount of 25mg/vial;
b) 300 mg of Kollidone PF-12 in aqueous vehicle as a solubilizing agent;
c) 11.2 mg of Triethanolamine HCl, as a pharmaceutically acceptable alkalizing agent, and
d) 250 mg of Sodium citrate in aqueous vehicle as a buffering agent;
wherein, the composition is stable at a pH range of 6 to 8 and wherein, the aqueous vehicle comprises
oxygen free water for injection having oxygen content less than 2 ppm.

6. The pre-lyophilized pharmaceutical composition for parenteral administration according to any one of the preceding claims, wherein, the oxygen free water for injection contains oxygen content less than 2ppm.

7. The pre-lyophilized pharmaceutical composition for parenteral administration according to any one of the preceding claims, wherein, the pharmaceutical composition is freeze dried to remove aqueous vehicle.

8. The pre-lyophilized pharmaceutical composition according to claim 7, wherein, the freeze drying is employed with the freeze dried cycle/run for duration of 46 hours in primary drying and 12 hours in secondary drying with a continuous up gradation of vacuum from 200mtorr to 10mtorr for the said period.

9. An alcohol and acid free stable freeze dried pharmaceutical composition, comprising;
a. Levosimendan or a pharmaceutically acceptable salt thereof at least in an amount of 1 to 25mg/vial;
b. polyvinyl pyrrolidone as a solubilizing agent in an effective amount of 100 to 350mg;
c. Triethanolamine HCl, as a pharmaceutically acceptable alkalizing agent in an effective amount of 5mg to 12mg, and
d. Sodium citrate as a buffering agent in an effective amount of 100mg to 300mg,
wherein, the freeze dried composition comprises water content to the tune of 2.2%w/w (Less than 3.0% w/w).

10. The alcohol and acid free stable freeze dried pharmaceutical composition according to claim 9, comprising,
a. Levosimendan or a pharmaceutically acceptable salt thereof at least in an amount of 12.5mg/vial;
b. 150mg of polyvinyl pyrrolidone as a solubilizing agent;
c. 5.6mg Triethanolamine HCl, as a pharmaceutically acceptable alkalizing agent, and
d. 125mg of Sodium citrate as a buffering agent,
wherein, the freeze dried composition comprises water content to the tune of 2.2%w/w (Less than 3.0% w/w).

11. The alcohol and acid free stable freeze dried pharmaceutical composition according to claim 9, comprising;
a) Levosimendan or a pharmaceutically acceptable salt thereof at least in an amount of 25mg/vial;
b) 300mg of polyvinyl pyrrolidone as a solubilizing agent;
c) 11.2mg Triethanolamine HCl, as a pharmaceutically acceptable alkalizing agent, and
d) 250mg of Sodium citrate as a buffering agent,
wherein, the freeze dried composition comprises water content to the tune of 2.2%w/w (Less than 3.0% w/w).

12. A method of increasing solubility of Levosimendan in an aqueous solution comprising, combining Levosimendan with aqueous solution of polyvinyl pyrrolidone as solubilizing agent in presence of sodium citrate as buffering agent and Triethanolamine HCl as an alkalizing agent.

13. A kit comprising Levosimendan composition for parental administration comprising,
a) freeze dried Levosimendan composition according to claim 9 and
b) diluent selected from 5% Dextrose solution and/ or 0.9% saline. , Description:Field of Invention:

The present invention relates to an improved formulation for water insoluble drug, Levosimendan, mainly meant for parenteral administration. More particularly, the invention relates to an alcohol free and acid 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 and prior art:

Levosimendan is a calcium sensitizer used in the management of acutely decompensated congestive heart failure (Source-Wikipedia).

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-Wikipedia).

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 intravenous (IV) and oral dosing is described in Sandell, E.-P. et al., J. Cardiovasc. Pharmacol., 26(Suppl.1), 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 CardiologyTMISSN: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 follows:
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 US6730673 Issued on May 4, 2004)

Also, Levosimendan solutions contain anhydrous alcohol as major vehicle for the drug which requires use of specified manufacturing facilities (Flame proof) and non reactive packaging material (rubber stoppers).

There is therefore a need to develop an alcohol and acid free 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 an improved formulation which does not require the use of alcohol and acid, is stable than the liquid one by making the molecule available via a lyophilized powder.
The further 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 an alcohol and acid free pharmaceutical composition for parental administration comprising Levosimendan or pharmaceutically acceptable salt thereof as the active ingredient along with a suitable solubilizing agent and an alkalizing or buffering agent and the process of preparation thereof.

In a preferred aspect, the pre-lyophilized pharmaceutical composition of levosimendan is free of alcohol and acid, the pH of the composition is maintained in the range of 6-8 using an alkalizing and buffering agent. The invention thus provides pre-lyophilized pharmaceutical compositions of Levosimendan which comprises polyvinylpyrollidone as solubilizing agent, Triethanolamine HCl as alkalizing agent and sodium citrate as a buffering agent in oxygen free aqueous vehicle. The composition is stable and free of acid and alcohol.

In another aspect, the freeze dried pharmaceutical composition of Levosimendan comprises polyvinyl pyrrolidene as solubilizing agent, Triethanolamine HCl as alkalizing agent and sodium citrate as a buffering agent. The freeze dried product thus obtained will have a water content of less than 3%w/w.

In another aspect, the present invention provides a method 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 Levosimendan or pharmaceutically acceptable salt thereof; a solubilizing agent, an alkalizing agent, a buffering agent and a diluent selected from 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 thereof may be fully understood and appreciated.

As used herein ‘Levosimendan’ refers to a compound (usually a liquid) that dissolves in a solid, 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 herein, 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 “Alkalizing 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.

As used herein, the term “water for injection” or “water” used for the preparing injectable preparations refers to “Oxygen free water for injection” which is used as a vehicle. More particularly, the presence of Oxygen in the water is less than 2ppm.

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 alkalizing 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.

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 solubility in water (0.04 mg/ml), a combination of buffer and alkalizing agents are required to prepare a solution with the desired concentration ready for lyophilization at the desired pH.

Further, 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 in the present invention 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 reconstituted solution containing final drug concentrate of 0.05 mg/ml, 0.025 mg/ml or 0.0125 mg/ml requires a pH of about 7.8 for the drug to be stabilized, which is achieved in the present invention through use of a combination of alkalizing and buffering agents.

The pharmaceutical composition of the invention described herein is freeze dried composition, which may be prepared by dissolving Levosimendan first in aqueous vehicle containing solubilizing agent, alkalizing agent and a buffering 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/acid 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 Levosimendan active offers greater stability than the liquid formulation which sometimes tends to change color on storage during shelf life.

Accordingly, in an embodiment, the present invention provides freed dried pharmaceutical composition for parenteral administration, free of alcohol and acid, as disclosed herein below:
(a) Levosimendan or a pharmaceutically acceptable salt thereof,
(b) a pharmaceutically acceptable solubilizing agent, and
(c) a pharmaceutically acceptable alkalizing agent and 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 pyrrolidone etc. The preferred solubilizing agent is polyvinyl pyrrolidone, (Kollidon PF12).

As is known in the art, buffering agents have different properties, such as different solubilities, acidities and alkalinities. For the purposes of this invention, a buffering agent also may function as an alkalizing agent, however, in a typical embodiment, the freeze dried pharmaceutical composition of Levosimendan comprises Triethanolamine HCl as alkalizing agent and sodium citrate as a buffering agent. The alkalizing or buffering agents are added in an amount necessary to adjust the pH to the desired range.

The resulting drug concentrate is freeze dried, after filtration from 0.22 micron Durapore membrane filters.

Accordingly, in a preferred embodiment, the invention provides an alcohol and acid free stable pre-lyophilized pharmaceutical composition for parenteral administration, which is substantially free of impurities, comprises,
a) Levosimendan or a pharmaceutically acceptable salt thereof at least in an amount of 1 to 25mg/vial;
b) an effective amount of Kollidone PF-12 in aqueous vehicle as a solubilizing agent;
c) an effective amount of Triethanolamine HCl, as a pharmaceutically acceptable alkalizing agent, and
d) an effective amount of Sodium citrate in aqueous vehicle as a buffering agent,
wherein, the composition is stable at a pH range of 6-8 and wherein, the aqueous vehicle comprises oxygen free water for injection (Oxygen content less than 2 ppm).

In our earlier Patent Application No. 11/MUM/2011, we have provided an alcohol and acid free, stable pre-lyophilized pharmaceutical composition for parenteral administration, comprising, Levosimendan; polyvinyl pyrrolidone (Kollidone PF-12) as solubilizing agent; Sodium citrate as buffering agent and Triethanolamine HCl as alkalizing agent in water for injection as aqueous vehicle. However, it has been observed some impurities in the composition during its shelf life. Therefore, the present inventors have developed an improved formulation, wherein oxygen free water was employed as an aqueous vehicle instead of plain water for injection so as to control the generation of impurities. The oxygen free water as referred herein above comprises oxygen content of less than 2ppm.

In yet another embodiment, the invention provides method for manufacturing stable freeze dried compositions of Levosimendan which method comprises;
a) providing pre-lyophilized aqueous solution of Levosimendan in Kollidon PF-12; sodium citrate and triethanolamine hydrochloride and
b) carrying out freeze drying of the aqueous solution for 46 hrs in primary drying followed by 12 hrs in secondary drying with a continuous upgradation of vacuum from 200mtorr to 10mtorr during the free dried period to obtain freeze dried product with a water content less than 3.0% w/w.

The inventiveness further lies in the application of freeze dried cycle under specific condition, wherein the freeze dried cycle/run for the composition is carried out for a duration of 46 hours in primary drying and 12 hours in secondary drying with a continuous up gradation of vacuum from 200mtorr to 10mtorr during the freeze drying period, thereby obtaining the freeze dried product also with greater stability and control on degradation of impurities during the shelf-life and even after reconstitution. The freeze dried composition of Levosimendan, which is substantially free of impurities according to the above freeze dried cycle, shows water content to the tune of 2.2%w/w (Less than 3.0% w/w), which contributes better stability to the product.

In another embodiment, invention provides a method of increasing solubility of Levosimendan in an aqueous solution comprising the step of adding solubilizing agent and an alkalizing and buffering agent to the aqueous solution containing Levosimendan. The solubilising agent is pharmaceutically acceptable solubilising agent, preferably, polyvinyl pyrrolidone (Kollidone PF-12), the alkalizing agent being Triethanolamine HCl and Sodium citrate in aqueous vehicle as a buffering agent.

Thus the invention provides a method of increasing solubility of Levosimendan in an aqueous solution comprising the step of combining Levosimendan with an aqueous solution comprising solubilising agent with an alkalizing and/or buffering agent. The alkalizing agent or/ and buffering agent may be added after combining Levosimendan with a pharmaceutically acceptable solubilizing agent.

According to the invention, the pharmaceutical composition comprises Levosimendan or a pharmaceutically acceptable salt thereof in amount of 1 to 25 mg; polyvinyl pyrrolidone (Kollidone PF-12) in an amount of 100mg to 350mg; Sodium citrate in an amount of 100 to 300mg and Triethanolamine HCl in an amount of 5 mg to 12 mg. The composition is stable at a pH range of 6-8 and wherein, the aqueous vehicle comprises oxygen free water for injection.

The ratio of drug to sodium citrate will be preferably in the range of 1:8 to 1:20; more preferably, 1:8 to 1:12. Similarly, the ratio of the drug to polyvinyl pyrrolidone (Kollidone PF-12) will be preferably in the range of 1:10 to 1:20, more preferably, 1:10 to 1:15.

As an exemplary embodiment, the present invention provides two dosage forms of Levosimendan which comprises 12.5mg and 25mg dosages. Accordingly, the solubilizing agent, buffering and alkalizing agents are suitably adjusted to obtain clear solution or to avoid any particulate matter in the pharmaceutical composition prior to proceeding with freeze drying process.

According to one embodiment, the invention provides an alcohol and acid free pre-lyophilized pharmaceutical composition for parenteral administration, which comprises,
i. Levosimendan or a pharmaceutically acceptable salt thereof in an amount of 12.5mg/vial;
ii. 150 mg of polyvinyl pyrrolidone (Kollidone PF-12) in aqueous vehicle as a solubilizing agent;
iii. 5.6 mg of Triethanolamine HCl, as a pharmaceutically acceptable alkalizing agent, and
iv. 125 mg of Sodium citrate in aqueous vehicle as a buffering agent;
wherein, the composition is stable at a pH range of 6 to 8 and wherein, the aqueous vehicle comprises oxygen free water for injection.

According to another embodiment, the invention provides an alcohol and acid free pre-lyophilized pharmaceutical composition for parenteral administration, which comprises,
i. Levosimendan or a pharmaceutically acceptable salt thereof in an amount of 25mg/vial;
ii. 300 mg of polyvinyl pyrrolidone (Kollidone PF-12) in aqueous vehicle as a solubilizing agent;
iii. 11.2 mg of Triethanolamine HCl, as a pharmaceutically acceptable alkalizing agent, and
iv. 250 mg of Sodium citrate in aqueous vehicle as a buffering agent;
wherein, the composition is stable at a pH range of 6 to 8 and wherein, the aqueous vehicle comprises oxygen free water for injection.

According to another aspect of the invention, the above pharmaceutical composition is freeze dried to remove aqueous vehicle.

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.

According to the invention, the pharmaceutical composition is freeze dried under specific conditions to obtain stable freeze dried product characterized by water content of Less than 3.0% w/w. The specific conditions under which the composition is freeze dried comprises carrying out freeze dried cycle/run for duration of 46 hours in primary drying and 12 hours in secondary drying with a continuous up gradation of vacuum from 200mtorr to 10mtorr during the free dried period. The freeze dried cycle employed as above results in freeze dried product with greater stability and control on degradation impurities during the shelf-life and after reconstitution. The freeze dried pharmaceutical composition thus obtained shows water content to the tune of 2.2%w/w (Less than 3.0% w/w) which leads to better stability.

Accordingly, in another preferred embodiment, the invention provides an alcohol and acid free stable freeze dried pharmaceutical composition, which comprises;
a) Levosimendan or a pharmaceutically acceptable salt thereof at least in an amount of 1 to 25mg/vial;
b) an effective amount of polyvinyl pyrrolidone as a solubilizing agent;
c) an effective amount of Triethanolamine HCl, as a pharmaceutically acceptable alkalizing agent, and
d) an effective amount of Sodium citrate as a buffering agent,
wherein, the freeze dried composition comprises oxygen free water content to the tune of 2.2%w/w (Less than 3.0% w/w).

The freeze dried composition may be re-constituted using aqueous vehicle for intravenous administration. 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.

The results of stability studies of the products of example 4 & 5 indicates that the product is seen as stable at 25°C for an observed period of 6 months, with the total impurity content less than 1%.
In yet another embodiment, the invention provides a kit comprising levosimendan composition for parental administration which comprises,
a) freeze dried Levosimendan composition according to invention and
b) a diluent selected from 5% Dextrose solution and / or 0.9% saline.

The use of technology of lyophilization with specific cycle 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.
3. Does not require specialized manufacturing facilities (flame proof) and Chlorobutyl or bromobutyl rubber closure with fluropolymer coating (specialized packaging material).

The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

Examples
Example: 1
Ingredients Qty. per ml
Levosimendan 1.25mg 20ml vial used. 10ml filled volume.
Polyvinyl pyrrolidone (Kollidone PF 12) 15mg 10ml solution filled in each vial
Sodium citrate 12.5mg Grey bromo butyl plugs were used with full slotted
Triethanolamine 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 as stable at 2 - 8°C because at 25°C storage products shows impurity within limits which is at accelerated temperature. At 40°C impurity crosses limits.

For process validation, two trials were taken on similar line of Example 1 and results are summarized below in tabular form as Example 2 and Example 3.

Example: 2
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 Initial 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

Example: 3
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 upto 0°C at 150mbar vacuum in 33 hours, then at 75 mbar, raising temperature upto +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: 12.5mg Levosimendan per vial
Diluted the above with 5 ml of 5% Dextrose solution to get 2.5mg/ml.
Diluted this solution with 500ml of 5% Dextrose solution to obtain 25mcg/ml of Levosimendan

Reconstituted Solution stability study Report
Product : Levosimendan for injection 12.5mg/vial

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 injection. : Dissolved the contents of 1 vial in 5ml WFI

Storage condition : Kept at room temperature

Duration of solution stability Analysis
Date Wt.of content
in mg Diluted 1ml with 200ml 5% Dextrose Diluted 1ml with 200ml 0.9% Normal saline Diluted 1ml with 200ml water
Assay Related sub. Assay Related sub. Assay Related sub.
Limit
90 to 110% Max. single impurity
NMT 2.0% Total impurity
NMT 3.0% Limit
90 to 110% Max. single impurity
NMT 2.0% Total impurity
NMT 3.0% Limit
90 to 110% Max. single impurity
NMT 2.0% Total impurity
NMT 3.0%
Initial 20/10/10

22/10/10 244.8

295.9 99.58%

0.68%

0.84% 101.80%

0.65%

0.83% 100.30%

0.61%

0.81%

After
4 hrs. 20/10/10

22/10/10 244.8

295.9 96.35%

1.36%

1.78% 101.40%

0.79%

1.01% 100.10%

0.76%

0.99%

After
24 hrs. 21/10/10

23/10/10 244.8

295.9 77.23%

5.69%

7.65% 100.80%

1.39%

1.83% 99.41%

1.36%

1.78%

Conclusion: Reconstituted infusion should be consumed within 4 to 6 hours if 5% Dextrose is used for infusion.

The formulation of example 3 provides water soluble Levosimendan as freeze dried formulation, stable for 24 months, when stored at 2 – 8°C.

Example: 4
Improved formula using water for injection having (Oxygen content Less than 2ppm) and process according to the present invention

Ingredients Qty. per ml
Levosimendan 1.25mg 20ml vial used. 10ml filled volume.
Polyvinyl pyrrolidone (Kollidone PF 12) 15mg 10ml solution filled in each vial
Sodium citrate 12.5mg full slotted Grey bromo butyl plugs were used
Triethanolamine HCl 0.56mg For freeze-drying
Water for injection
(Oxygen content
Less than 2ppm) To make 1ml
pH 7.7
Bulk Assay 99.5%

Preparation of freeze dried product:
The freeze dried cycle, run for the product of the above example is employed for duration of 46 hours in primary drying and 12 hours in secondary drying with a continuous upgradation of vacuum from 200mtorr to 10 mtorr during the freeze drying period.

The freeze dried pharmaceutical composition according to the above cycle shows water content to the tune of 2.2%w/w (Less than 3.0% w/w) which contributes to better stability of the freeze dried product.

Results of stability studies are shown below at different storage temperatures
No. Period Bulk Assay : 99.5 % pH of Bulk solution : 7.7
Assay Water
(Max.3.0%) pH
(6 to 8) Impurity
Single Impurities
Total Storage temp.
1 Initial 100.8 2.2 7.6 0.2 0.5 -
2 3 months 100.2 2.1 7.6 0.2 0.5 2 – 8°C
3 3 months 99.5 2.2 7.5 0.3 0.6 25°C
4 6 months 99.7 2.0 7.4 0.4 0.7 2 – 8°C
5 6 months 98.6 2.2 7.4 0.5 0.8 25°C

Conclusion: From above mentioned results, product can be seen stable at 25°C for an observed period of 6 months with the total impurity content less than 1%.

Example: 5
Improved formula using water for injection having (Oxygen content Less than 2ppm) and process according to the present invention
Label claim: 25mg Levosimendan per vial

Ingredients Qty. per ml
Levosimendan 2.50mg 20ml vial used. 10ml filled volume.
Polyvinyl pyrrolidone (Kollidone PF 12) 30mg 10ml solution filled in each vial
Sodium citrate 25.0mg Grey bromo butyl plugs were used with full slotted
Triethanolamine HCl 1.12mg For freeze-drying
Water for injection
(Oxygen content
Less than 2ppm) To make 1ml
pH 7.7
Bulk Assay 99.5%

Preparation of freeze dried product:

The freeze dried cycle, run for the product of the above example is employed for duration of 46 hours in primary drying and 12 hours in secondary drying with a continuous upgradation of vacuum from 200mtorr to 10mtorr during the freeze drying period.

The freeze dried pharmaceutical composition according to the above freeze dried cycle, shows water content to the tune of 2.2%w/w (Less than 3.0% w/w) which contributes to better stability of the product.

Results of stability studies indicates that the product is seen as stable at 25°C for an observed period of 6 months similar to the product of example 4, with the total impurity content less than 1%.