WO2008082871A1

WO2008082871A1 - Formulations of levosimendan for parenteral administration

Info

Publication number: WO2008082871A1
Application number: PCT/US2007/087099
Authority: WO
Inventors: Jitendra Patel
Original assignee: Orion Corporation
Priority date: 2006-12-28
Filing date: 2007-12-12
Publication date: 2008-07-10
Also published as: JP2010514778A

Abstract

The present invention provides pharmaceutical compositions of levosimendan, in particular for intravenous administration, and methods of preparing said compositions. The compositions of the invention have increased solubility and stability without the use of solubility enhancing agents.

Description

FORMULATIONS OF LEVOSIMENDAN FOR PARENTERAL

ADMINISTRATION

RELATED APPLICATION This application claims priority to U.S. Application No. 60/882,311 , filed

December 28, 2006, incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a novel pharmaceutical composition of levosimendan, in particular for intravenous administration, and methods of preparing said composition. The composition of the invention has increased solubility and stability without the use of solubility enhancing agents.

BACKGROUND OF THE INVENTION

Levosimendan (Simdax®, Orion Pharma, Espoo, Finland) is an intravenous drug that is used for the treatment of congestive heart failure. Levosimendan interacts with cardiac troponin C (cTnC), stabilizing the calcium-induced conformational change in cTnC, and promoting the interaction of cTnC with its neighbors on the thin filament, and thus increasing contractility. The positive inotropic effect of levosimendan is achieved without increases in intracellular calcium and with little or no increase in myocardial oxygen consumption. Levosimendan also activates K-ATP channels, which are involved in the regulation of vascular tone. Due to the hyperpolarization resulting from the activation of K-ATP channels, levosimendan is able to relax pre-constricted vascular muscle. Among others, the use of levosimendan in the treatment of myocardial ischemia is described in U.S. Patent No. 5,512,572; its use in coronary graft vasospasm after coronary by-pass surgery is described in U.S. Patent No. 6,593,329; the use of levosimendan in pulmonary hypertension and renal failure are described in U.S. Patent Nos. 6,462,045 and WO 04/060375, respectively.

Administration of a drug by parenteral, e.g. intravenous, administration provides several advantages. These include immediate response, easy control of the therapeutic response and overcoming the oral administration because of inactivation of the drug or the unconscious state of the patient.

Levosimendan is synthesized as a crystalline powder with a pKa of about 6.2 and low solubility in water (0.04 mg/mL). Available formulations of levosimendan are ethanol concentrates and have been described, for example, in WO 01/19334. Before parenteral administration, the ethanol concentrate of levosimendan is diluted in an appropriate aqueous solution, for example 5% dextrose, half normal saline, Lactated Ringer's Injection, normal saline solution or other large volume parenteral diluent. Following dilution of the ethanol concentrate, the pH of the diluted drug solution may become as low as pH 3.0. It is known that levosimendan's solubility in an aqueous solution decreases as the solution's pH falls below the pK_a value. Due to its poor solubility at lower pH, levosimendan may start to precipitate after dilution.

To overcome this problem, present formulations of levosimendan also contain a solubility-enhancing agent, which increases the amount of levosimendan that can be maintained in a dissolved state after dilution in an aqueous solution. Surfactant and other solubilizing agents such as polysorbates, polyethylene glycol, polyoxamers or polyvinylpyrrolidone and fatty acids can be used for this purpose. However, some of these substances are not cleared readily by the kidneys and may interfere with the stability of levosmiendan or other co-administered drugs. In addition, certain regulatory agencies may not approve the use of solubility-enhancing agents in a product that is to be administered to patients. Accordingly, there is a need for and it is an object of the invention to provide pharmaceutical compositions of levosimendan, in particular for intravenous administration, which do not use a solubility enhancing agent and which maintain the active ingredient (i.e. levosimendan) in solution.

SUMMARY OF THE PRESENT INVENTION The present invention is directed to formulations of levosimendan for parenteral administration comprising the use of an agent that increases the pH of the aqueous solution, which prevents precipitation of the active ingredient.

In one aspect, the present invention provides a pharmaceutical composition comprising (a) a quantity of levosimendan or a pharmaceutically acceptable salt thereof; (b) a pharmaceutically acceptable diluent; and (c) a pharmaceutically acceptable alkalinizing agent or buffering agent. In the pharmaceutical composition, the quantity of levosimendan may be dissolved in an organic solvent. In one embodiment, the quantity of levosimendan and the organic solvent are provided as a drug concentrate. Preferably, the organic solvent is anhydrous ethanol. In another embodiment, the quantity of levosimendan may be in a freeze-dried form. Preferably, the pharmaceutical composition provides an alkalinizing agent or buffer in an amount sufficient to increase the pH of the pharmaceutical composition to about the pKa of said levosimendan. More preferably, the pKa is about 6.2. In another preferred embodiment, the diluent is selected from the group consisting of Ringers solution, Lactated Ringers solution and saline solution.

In another embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable organic acid. Preferably, the organic acid is selected from the group consisting of citric acid, lactic acid, tartaric acid or maleic acid. More preferably, the organic acid is citric acid.

In another embodiment, the invention provides a method of increasing the solubility of levosimendan in an aqueous solution comprising the step of combining levosimendan with an aqueous solution and an alkalinizing or buffering agent. The alkalinizing agent or buffering agent first may be combined with the aqueous solution before combining with said levosimendan. Preferably, the alkalinizing or buffering agent is selected form the group consisting of sodium bicarbonate and tromethamine. Preferably, the aqueous solution is 0.9% sodium chloride. Also preferably, the alkalinizing agent or buffering agent is a solution of 4% sodium bicarbonate. In another embodiment, the invention provides a composition suitable for intravenous injection comprising (a) a quantity of levosimendan or a pharmaceutically acceptable salt thereof; (b) a pharmaceutically acceptable organic solvent, (c) a stability enhancing amount of a pharmaceutically acceptable organic acid having a pKa in the range of from 2 to 4; and (d) a pharmaceutically acceptable aqueous diluent having a pH in the range of about 6.3 to about 9.5. Preferably, the organic solvent in anhydrous ethanol and the aqueous diluent is selected from the group consisting of normal saline and half normal saline. Also, preferably, the organic acid is selected from the group consisting of citric acid, lactic acid, tartaric acid or maleic acid. Even more preferably, the organic acid is citric acid. In another embodiment, the invention comprises a packaged pharmaceutical product comprising: (a) a quantity of levosimendan, (b) a pharmaceutically acceptable organic solvent, (c) a pharmaceutically acceptable organic acid, (d) a pharmaceutically acceptable diluent, and (e) a pharmaceutically acceptable alkalinizing agent. The pharmaceutical product may be packaged with parts (a), (b) and (c) as a single component. Alternatively, the pharmaceutical product may be packaged with parts (a) and (b) as a single component. In another alternative, the pharmaceutical product may be packaged with parts (b) and (c) as one component. In any of the aforementioned embodiments, the pharmaceutical product may be packaged with parts (d) and (e) as a single component. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph showing the effect of pH on levosimendan stability in 5% dextrose.

Figure 2 is a bar graph showing pH changes during a stability evaluation of levosimendan in 5% dextrose.

Figure 3 is a graph showing a pH stability profile for levosimendan in 0.01 M citrate and phosphate buffers.

Figure 4 is a graph showing the formation of degradation product OR- 1420 in 0.01 M citrate and phosphate buffers. Figure 5 is a bar graph showing pH changes during a stability study of levosimendan in 0.01 M citrate and phosphate buffers

Figure 6 is a graph showing the stability of levosimendan in various diluents.

Figure 7 is a graph showing the formation of levosimendan degradation product OR- 1420 in various diluents. Figure 8 is a bar graph showing the solubility of levosimendan in phosphate buffered half-normal saline solution.

Figure 9 is a bar graph showing the solubility of levosimendan in phosphate buffered normal saline solution.

Figure 10 is a graph showing the stability of levosimendan in phosphate buffered half-normal and normal saline solutions.

Figure 11 is a graph showing the formation of levosimendan degradation product OR- 1420 in phosphate buffered half-normal and normal saline solutions.

Figure 12 is a graph showing pH changes in levosimendan in phosphate buffered half-normal and normal saline solutions. Figure 13 is a graph showing the solubility of levosimendan in bicarbonate- buffered normal saline solution.

Figure 14 is a graph showing the stability of levosimendan in bicarbonate- buffered normal saline solution.

Figure 15 is a graph showing the formation of levosimendan degradation product OR- 1420 in bicarbonate -buffered normal saline solution.

Figure 16 is a graph showing the solubility of levosimendan in bicarbonate- buffered normal saline solution in the presence of varying concentrations of ethanol. Figure 17 is a graph showing the stability of levosimendan in bags containing bicarbonate buffered half-normal and normal saline solutions compared to SIMD AX®.

Figure 18 is a graph showing the formation of levosimendan degradation product OR- 1420 in bags containing levosimendan in either bicarbonate buffered half-normal or normal saline solution compared to SIMD AX®.

Figure 19 is a graph showing the formation of total degradation products in bags containing levosimendan in either bicarbonate buffered half-normal or normal saline solution compared to SIMD AX®. Figure 20 is a graph showing levosimendan stability post-dilution in bicarbonate-buffered normal saline bags using a citric acid drug concentrate compared to SIMD AX®.

Figure 21 is a graph showing the formation of levosimendan degradation product OR- 1420 post-dilution of levosimendan in bicarbonate-buffered normal saline bags using a citric acid drug concentrate compared to SIMDAX®.

DETAILED DESCRIPTION OF THE INVENTION Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting.

In one aspect, the invention provides a pharmaceutical composition, in particular for intravenous infusion, comprising levosimendan or a pharmaceutically acceptable salt thereof as the active ingredient; a pharmaceutically acceptable diluent and a pharmaceutically acceptable alkalinizing agent or buffer. The levosimendan or pharmaceutically acceptable salt thereof initially may be solubilized in a solvent for processing and the solvent then may be removed prior to forming the pharmaceutical composition. For example, the levosimendan may be solubilized in a solvent such as ethanol, t-butyl alcohol or water and then e.g. freeze-dried to remove the solvent. Alternatively, the pharmaceutical composition may comprise the pharmaceutically acceptable solvent (i.e. the solvent does not need to be removed.) In one embodiment, the levosimendan and solvent may be in the form of a drug concentrate. As used herein, "levosimendan" refers to the (-)-enantiomer of [4-(l,4,5,6- tetrahydro-4-methyl-6-oxo-3pyridazinyl)phenyl]hydrazono]propanedinitrile.

As is known in the art, the word "solvent" generally refers to a compound (usually a liquid) that dissolves a solid, liquid or gaseous solute, resulting in a solution. "Organic solvents" are solvents that are organic compounds, meaning they contain carbon atoms. Preferably, the pharmaceutical composition of the invention comprises an organic solvent. Non-limiting examples of organic solvents useful in the invention include but are not limited to ethanol, dimethyl sulfoxide (DMSO), N- methyl pyrrolidone (NMP), propylene glycol, glycerol, t-butyl alcohol and polyethylene glycol.

As used herein, "diluent" refers to any liquid material used to dilute or carry an active ingredient. Non-limiting examples of diluents suitable for the 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.

As used herein, the term "pharmaceutically acceptable" refers to diluents, agents, excipients, stablizers 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. Non- limiting examples of alkalinizing agents include sodium hydroxide, sodium bicarbonate, sodium carbonate, sodium 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 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 is known in the art, buffering agents have different properties, such as different solubilities, acidities and alkalinities. For purposes of this invention, a buffering agent also may function as an alkalinizing agent provided that it increases the pH of the solution to which it is added. Preferably, the alkalinizing and/or buffering agent increases the pH of the composition to a value above the pK_a of levosimendan. Non- limiting examples of buffering agents suitable for the invention include sodium bicarbonate, sodium phosphate and sodium lactate. The representative examples of alkalinizing agents and buffers described herein are not intended to limit the scope of the invention, which contemplates any pharmaceutically acceptable alkalinizing agent or buffer that increases the pH of the pharmaceutical composition to a level that maintains the levosimendan in a dissolved state sufficient for its intended use. As used herein, the term "organic acid" refers to an organic compound that is an acid. Organic acids generally are weak acids characterized by a carboxyl (-COOH) group. Preferably, the organic acid is a 2-hydroxy alkanoic acid. Examples of such organic acids include but are not limited to citric acid, lactic acid, tartaric acid and maleic acid, the most preferred being citric acid. Generally, for an intravenous pharmaceutical solution (such as an aqueous solution) to be administrable to a patient, the active ingredient(s) of the composition needs to be dissolved sufficiently in the composition, i.e. be free from visible particles. Depending on the final desired concentration of levosimendan, the pH of the aqueous composition may vary such that the drug is sufficiently solubilized to achieve that concentration. For example, a levosimendan solution containing a final drug concentration of 0.05 mg/mL may require a pH of about 7.2 for the drug to be solubilized. For a solution containing a lower concentration of drug (e.g. 0.01 mg/mL), a lower pH (e.g. below 4.0) may solubilize the drug so as to achieve that concentration. Methods for determining the solubility and/or concentration of levosimendan in a pharmaceutical composition are well known to those of ordinary skill in the art. In a preferred embodiment, the alkalizing agent or buffer increases the pH of the composition to a value above the pK_a of levosimendan.

Methods for preparing the individual components of the invention are well known in the art. The synthesis of levosimendan is described, for example, in US Patent No. 5,569,657. Methods for preparing organic solvents, diluents and alkalinizing agents or buffering agents also are well known to those skilled in the art and can be found in standard scientific textbooks and other published scientific literature. The pharmaceutical compositions of the invention described herein, typically may be prepared by dissolving the levosimendan first in a solvent to form a levosimendan concentrate. A preferred solvent is dehydrated alcohol such as ethanol (USP). The resulting drug concentrate optionally may be freeze-dried (or treated in another manner) to remove the ethanol. Methods for freeze-drying are well-known to those of ordinary skill in the art. Optionally, the ethanol concentrate also may comprise an organic acid.

The organic acid may be combined with the solvent before being combined with the drug, or after the drug has been combined with the solvent. The organic acid also may be added to the drug first and then combined with the solvent. The amount of the organic acid preferably is in the range of 0.0005 to 2%, preferably 0.01 - 1%, by weight of the concentrate solution. In a more preferred embodiment, the organic acid is citric acid. Even more preferably, the amount of citric acid is 0.03 - 0.6% by weight. Thereafter, the freeze-dried drug or drug concentrate is diluted with a suitable diluent to which an alkalinizing agent or buffering agent has been added. Alternatively, the diluent may be a buffer that increases the pH of the solution to which it is added.. The diluent, in addition to serving as a means of diluting the composition to achieve a final concentration of drug, also dilutes the organic solvent prior to administration to a patient.

Other non-limiting ways of preparing the compositions of the invention also are contemplated. For example, the levosimendan first may be dissolved in an organic solvent and then combined with an alkalinizing agent. Optionally, this preparation may be freeze-dried for storage. Thereafter, the levosimendan/organic solvent/alkalinizing agent combination may be diluted to an appropriate concentration with a diluent. In another example, a solid form of an alkalinizing agent may be added to solid levosimendan and the combined material dissolved in an organic solvent. This combination then can be diluted with an appropriate diluent. In yet another example, solid levosimendan may be combined with an alkalinizing agent dissolved in an organic solvent. This combination then may be combined with an appropriate diluent. In yet another example, the alkalinizing agent and diluent may be combined and then this mixture combined with a levosimendan concentrate. In another embodiment, the alkalinizing agent and diluent may be combined with the levosimendan concentrate simultaneously. As mentioned above, in all of the aforementioned preparation methods, optionally an organic acid may be combined with the organic solvent (prior to combining with the drug) or after the drug is combined with the organic solvent (i.e. the organic acid is combined with the drug concentrate.) Although not wishing to be bound to a specific mechanism of action, it is believed that the citric acid functions to stabilize the drug/organic solvent combination, particularly at room temperature, although the citric acid has no detrimental effect on the composition during storage at refrigerated temperatures. As indicated above, the alkalinizing agent may be provided either in the form of a solid or as a solution. Other components, such as additional stabilizers, either in solid or liquid form, also may be added, if desired.

The amount of each component will depend upon the solubility of the component and the drug concentration of levosimendan desired in the final pharmaceutical composition. If the alkalinizing agent is added in the form of a solution, the routine practitioner can readily adjust and/or vary other components to achieve the desired final concentration of drug in the pharmaceutical composition. In another aspect, the present invention provides a method to improve the stability and solubility of levosimendan using an alkalinizing agent or a buffer. In particular, the invention provides a method to improve the stability and solubility of levosimendan in an aqueous solution by combining the levosimendan with the aqueous solution and an alkalinizing agent or buffer. As noted above, the amount of aqueous solution needed will depend upon the final desired concentration of levosimendan in the pharmaceutical composition and the amount of alkalinizing agent needed will depend upon the desired degree of solubility of levosimendan. Those of ordinary skill in the art readily can titrate the amount the alkalinizing agent or buffer necessary to achieve the desired amount of levosimendan solubility. As a general guideline, the higher the pH of the pharmaceutical composition the more soluble the levosimendan.

Preferably, the alkalinizing agent or buffer is provided in an amount sufficient to maintain the levosimendan in a dissolved state. More preferably, the alkalinizing agent or buffer is provided in an amount sufficient to cause the pH of the resulting composition to be about or above the pKa of levosimendan. Furthermore, the ultimate pH value of the composition should not exceed the limit that can be tolerated safely by a patient upon its administration. In a preferred embodiment, the pH of the pharmaceutical composition is in the range of about pH 6.0 to about pH 9.0. In a more preferred embodiment, the pharmaceutical composition is in the range of about pH 6.2 to about pH 9.0. In another preferred embodiment, the pH is in the range of about 6.3 to about 9.0. In another preferred embodiment, the pH is in the range of about 6.3 to about 9.5. In a preferred embodiment, the present invention provides a composition comprising levosimendan and an alkalinized or buffered aqueous solution. Preferably, the levosimendan is an alcohol concentrate of the drug. Also preferably, the alkalinized aqueous solution is a saline solution comprising sodium bicarbonate. Typically, the levosimendan alcohol concentrate is contained in a separate container than the alkalinized saline solution. Immediately before parenteral administration, the levosimendan alcohol concentrate is mixed with the alkalinized saline solution so as to achieve the final desired therapeutic concentration of drug. To generate the parenteral solution, the alkalinized aqueous solution may be added to a container comprising the levosimendan concentrate. Alternatively, the levosimendan concentrate and the alkalinized aqueous solution may be enclosed in the same container, separated by a physical barrier that can be pierced by agitation or other simple manipulation to cause mixing of the two solutions. Typically, the levosimendan concentrate comprises about 95-100% anhydrous ethanol. In a preferred embodiment, the composition comprises levosimendan (2.5 mg/mL) and citric acid (1-2 mg/mL) dissolved in dehydrated ethyl alcohol. This concentrate then is diluted with normal saline to which has first been added an alkalinizing agent or buffering agent (preferably 4% sodium bicarbonate solution) in order to achieve a final levosimendan concentration of 0.05 mg/mL.

In another embodiment, the present invention provides a packaged pharmaceuticalproduct or a kit comprising (a) a quantity of levosimendan, (b) a pharmaceutically acceptable alkalinizing or buffering agent and (c) a pharmaceutically acceptable diluent. The levosimendan may be in a form that is freeze-dried from an organic solvent concentrate or provided as a drug concentrate (in an organic solvent). Optionally, the packaged pharmaceutical product may contain an organic acid. The levosimendan, organic solvent (if present) and organic acid (if present) may be packaged as a single component or as individual components. Alternatively, the levosimendan and organic solvent may be packaged as one component. Alternatively, the organic solvent may be packaged as one component and the levosimendan packaged as a separate component. The packaged pharmaceutical product or kit may further comprise instructions for combining the materials of the product or kit. It may optionally comprise a diluent for diluting the composition to a concentration that can be administered to a patient or the diluent may be supplied as a separate component. Any suitable combinations of packaging the pharmaceutical product are envisioned. Most preferably, the diluent is maintained as a separate component (as part of the packaged pharmaceutical kit or otherwise) and is used immediately prior to administration.

The therapeutically effective amount of levosimendan included in the pharmaceutical composition depends e.g. on the administration route of the composition, the treatment procedure and the condition to be treated. In general, the amount of levosimendan in the composition is within the range of about 0.001-5 mg/mL. The daily dosage of levosimendan in man is within the range of about 0.1-50 mg, preferably about 0.2-20 mg, depending on the administration route, age, body weight and condition of the patient. Preferred peak plasma levels of levosimendan in steady state for the treatment of congestive heart failure are within the range of from about 1 to about 300 ng/mL, more preferably from about 10 to about 150 ng/mL, and especially from about 20 to about 60 ng/ml. Levosimendan can be administered intravenously at an infusion rate in the range of about 0.005-100 micrograms/kilogram/minute (mcg/kg/min), typically 0.01 to 10 mcg/kg/min, more typically about 0.02 to 1 mcg/kg/min. For the treatment of heart failure, with continuous infusion the suitable rate is 0.05-0.4 mcg/kg/min of levosimendan.

Salts of levosimendan may be prepared by known methods. Pharmaceutically acceptable salts are useful as active medicaments; however, preferred salts are the salts with alkali or alkaline earth metals. In another aspect, the invention provides a method of increasing the solubility of levosimendan as the active ingredient after dilution in an aqueous solution comprising the step of increasing the pH value of the aqueous solution. The pH may be increased by combining the aqueous solution with an alkalinizing or buffering agent as described herein. Preferred alkalinizing agents are sodium bicarbonate or tomethamine. The preferred pH of the aqueous solution post-dilution is between about 6.3 to about 9.5.

Examples

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.

Materials: Levosimendan API and SIMD AX^® formulation were obtained from Orion Pharma (Espoo, Finland.) Dehydrated ethyl alcohol, United States Pharmacopeia (USP) was used to prepare levosimendan formulations. A commercially available 4% Sodium Bicarbonate Additive Solution (Neut®, Hospira, Lake Forest, IL, NDC 0409-06609-02) was used to increase the pH of the diluent. Large volume parenteral (LVP) diluents were commercially purchased from Hospira or provided by Abbott Japan. Equipment: The light obscuration particle count method employed a commercially available HIAC instrument that was capable of performing the test per USP requirements. HPLC was used for chemical analysis.

Procedure: Solubility experiments were conducted by mixing an excess of solid drug with solvent, filtering the contents and analyzing the filtrate. Stability studies were conducted by diluting the drug solution in the designated solvent, storing the samples at the requisite temperatures and performing the analysis by HPLC as a function of time. The pH measurements were done potentiometrically using a glass electrode. The drug product samples were visually examined for appearance (physical state, clarity, visible particulates) and color. The drug formulation samples were diluted in a LVP container for conducting compatibility experiments. The samples were analyzed for Levosimendan and degradation products by HPLC. The light obscuration particle count method was used to determine the number of particles >10 μm and >25 μm per container. The method is based on USP <788>. HPLC method was used for chemical analysis. The drug samples were injected directly or after dilution (mobile phase or ethanol) on to the HPLC column. Levosimendan and/or the degradation product reference standard were used for quantitation. A summary of the HPLC method parameters is provided in Table 1.

Table 1. HPLC Method Conditions

Detection UV at 380 and 450 nm

Example 1

A pH-stability study was conducted using 5% dextrose injection. The pH of the 5% dextrose injection (pH = 4.54) was adjusted down by using hydrochloric acid and adjusted up by the addition of increasing amounts of 4% sodium bicarbonate solution. Levosimendan dissolved in ethanol then was added to the 5% dextrose solutions such that the final concentration of levosimendan was about 10 mcg/mL and the final ethanol concentration was 2%. The stability of levosimendan in the pH- adjusted 5% dextrose was monitored at ambient temperature by HPLC. Figure 1 shows the loss of levosimendan as a function of time during the course of about two days. The data demonstrate an increased degradation rate when the pH of the diluent was higher than 3.22. The pH of the stability solutions also was monitored initially (no drug added), after drug addition, and at the end of the experiment. These data are shown in Figure 2 and they indicate a significant upward shift in pH for samples, particularly for those with an initial pH of 4.54 and 5.74. This was most likely due to limited buffer capacity of these solutions. This was addressed by conducting pH-stability studies in buffers (see Example 2 below).

Example 2 A pH-stability study was conducted at 20⁰C using citrate and phosphate buffers (0.01 M) adjusted to pH values ranging from 3.5 to 7.5. Levosimendan dissolved in ethanol was added to the buffer solutions such that the final concentration of drug was about 10 mcg/mL and the final ethanol concentration was 2%. The stability of levosimendan in the pH-adjusted buffers was monitored at 20⁰C by HPLC. Figure 3 shows the loss of levosimendan as a function of time during the course of more than three days. OR-1420 ((E)-2-cyano-2-[[4-(l,4,5,6-tetrahydro-4-methyl-6- oxo-3 pyridazinyl)phenyl]hydrazono]acetamide), the principal degradation product formed during these studies was also monitored by HPLC analysis. These data are shown in Figure 4. The pH of the stability solutions was monitored prior to drug addition (starting buffer), after drug addition at the start of the experiment, and at the end of the experiment (Figure 5).

The data in Figures 3 and 4 demonstrate an increase in degradation rate of levosimendan when the pH of the buffer is sequentially increased from a value of 3.5 to 7.5. At pH 3.5 there was very little loss of levosimendan, while in the pH range 6.5 to 7.5 the degradation rates were very similar and about 3% loss was observed over a period of three days. The vast majority of the loss in levosimendan depicted in Figure 3 was accounted for by the appearance of the hydrolysis product OR- 1420 (shown in Figure 4.) The pH of the stability solutions was monitored prior to drug addition (starting buffer), after drug addition at the start of the experiment and at the end of the experiment. These data are shown in Figure 5.

A comparison of the data in Figure 3 with the results in Figure 1 indicate (note Y-scale differences) that the loss in potency was higher for studies conducted in 5% dextrose. The degradation amounts in 5% dextrose were roughly ten fold higher (Figure 1) at neutral pH at 48 hours when compared with the data in buffer systems (Figure 3). These data suggest that dextrose may be used as a diluent for shorter-term storage and infusion of the pharmaceutical composition.

Example 3 The stability of levosimendan was evaluated in water, normal saline and 5% dextrose solutions adjusted to a target pH of 7.5 (using either phosphate buffer

(0.01M) or sodium bicarbonate). Levosimendan was added to ethanol such that the final concentration was about 10 mcg/mL and the final concentration of ethanol was 2%. The stability of levosimendan in the pH-adjusted solutions was monitored at 2O⁰C by HPLC. The profiles in Figures 6 and 7 show that the degradation of levosimendan was significantly slower in solutions prepared using water and normal saline than in 5% dextrose. The buffer components (bicarbonate or phosphate) had no impact on the degradation rate. The rate of degradation in normal saline was very similar to that of water in both the bicarbonate and phosphate buffers. These studies demonstrate that water and normal saline are suitable larger volume parenteral (LVP) diluent for levosimendan at neutral pH.

Example 4

The solubility of levosimendan was determined at ambient temperature in 0.01M phosphate buffer to which had been added sodium chloride (NaCl) so that the resulting solution contained an equivalent of either 0.45% (half normal) or 0.9% (normal) salt concentration. The pH of these solutions was adjusted with dilute sodium hydroxide (NaOH) to initial pH values ranging from 7.0 to 9.0 prior to drug addition. The results of the solubility studies in half normal and normal saline are summarized in Figures 8 and 9, respectively. The initial pH (prior to drug addition) and the final pH (post drug equilibration) are shown on the abscissa in these Figures.

The results show that at pH of about 7.5 a solubility value of approximately 200 mcg/mL or about four times higher than the desired clinical drug concentration was achieved. Levosimendan solubility was slightly greater in half normal saline when compared to normal saline probably due the lower ionic strength of the half normal saline. The final pH for the buffers that were initially adjusted to 8.0, 8.5 and 9.0 was reduced to a value of about 7.5 due to the poorer buffering capacity of phosphate buffer in the pH 8-9 range. However, the data demonstrate the expected pH-solubility relationship and all the solubility values were substantially above the target 50 mcg/mL value.

Example 5

The stability of levosimendan was determined at 20⁰C in 0.01M phosphate buffer to which had been added NaCl so that the resulting solution yielded either 0.45% (half normal) or 0.9% (normal) salt concentration. The pH of these solutions was adjusted with dilute NaOH to initial values ranging from 7.0 to 9.0 prior to drug addition. The composition of these buffered salt solutions was identical to that used in the solubility experiments discussed above.

Levosimendan dissolved in ethanol was added to the buffered salt solutions such that the final concentration of levosimendan was about 10 mcg/mL and the final ethanol concentration was 2%. The stability of levosimendan in the pH-adjusted salt solutions was monitored at 20⁰C by HPLC over various time periods. The pH of the solutions also was monitored at the start and end of the stability study. The stability data in Figures 10 and 11 demonstrate that the degradation of levosimendan is very similar in normal and half-normal saline in the pH 7.0 to 9.0 range. The principal degradation product was OR- 1420 (Figure 11).

The target pH, the pH at the start of the kinetic study and the pH at the end of the study were monitored and are shown in Figure 12. The final pH for the buffers that had been adjusted initially to 8.5 and 9.0 was reduced to a value of about 8 due to the poorer buffering capacity of phosphate buffer in the pH 8.5-9.0 range.

Example 6

In one embodiment of the invention, the ethanol concentrate of levosimendan may be diluted in normal saline to which a solution of 4% sodium bicarbonate solution (Neut®) has been added to adjust the pH in the neutral range. This dilution protocol involves the addition of the contents of one Neut® vial (5 rnL) to one 500 rnL IV normal saline bag followed by the addition of 10 rnL of levosimendan (2.5 mg/mL) in ethanol. The final contents of the bag contain levosimendan at a concentration of 0.05 mg/mL and ethanol at 2% concentration. Therefore, the solubility and stability of levosimendan were evaluated in normal saline in the presence of different amounts of Neut®.

The solubility of levosimendan in normal saline (no ethanol) in the presence of increasing amounts of Neut® is shown in Figure 13. The initial pH (prior to drug addition), the final pH (post drug equilibration) and the number of Neut® vials used per 500 mL saline volume are shown on the abscissa. The results show that the pH of normal saline is insensitive to the number of Neut® vials added to it and the pH after the addition is in the range of 7.6 to 7.7. This is due to the limited buffering capacity and the pKa of the bicarbonate ion. The results demonstrate that it is feasible to achieve levosimendan solubility of greater than 200 mcg/mL (or about four times higher than the desired clinical drug concentration) in the 7.2 to 7.6 pH range. The solubility data also supports the use of sodium bicarbonate as an additive to normal saline to increase levosimendan solubility. These solubility results are consistent with those obtained in phosphate buffered saline solutions.

Example 7 Levosimendan dissolved in ethanol was added to the bicarbonate buffered normal saline solutions such that the final concentration of levosimendan was about 10 mcg/mL and the final ethanol concentration was 2%. The quantity of bicarbonate was varied by adding one, two, four or eight vials of Neut® per 500 mL of saline. The stability of Levosimendan in these salt solutions was monitored at 20⁰C by HPLC. The stability data in Figures 14 and 15 demonstrate that changing the bicarbonate concentration eight fold does not significantly influence the degradation rate for levosimendan in normal saline. The principal degradation product was OR- 1420 (Figure 15). Furthermore, the degradation rate (and product distribution) was very similar to that observed in phosphate buffered saline solutions. Example 8

Since the bicarbonate buffered saline solutions of levosimendan intended for clinical administration by infusion may contain 2% ethanol (from the parent drug concentrate) the influence of ethanol on the solubility of levosimendan in bicarbonate buffered saline was studied. One vial (5 mL) of Neut® was added to 500 mL of normal saline. Anhydrous ethyl alcohol then was added in order to prepare solutions ranging from 0% to 8% ethanol. Solid drug was mixed with these solutions in order to determine the equilibrium solubility of levosimendan. The results of the study are shown in Figure 16. A least squares linear regression line is superimposed on the experimental data points. As Figure 16 shows, the solubility increases linearly with ethanol concentration. The pH of the solutions ranged from 7.2 to 7.3. The solubility and pH results are consistent with the previous experiments and the solubility is well above a preferred concentration of 50 mcg/mL.

Example 9 All the stability studies discussed above were conducted at a final levosimendan concentration of 10 mcg/mL in buffered normal and half normal saline in glass containers. In a preferred embodiment, the levosimendan drug concentrate is diluted to 50 mcg/mL (0.05 mg/mL) in IV bags using Neut® to buffer the saline solution. Therefore, stability studies were conducted at ambient and refrigerated temperature by diluting the drug concentration to a final concentration of 0.05 mg/mL in Neut® buffered normal saline and half normal saline. SIMD AX® diluted to a concentration of 0.05 mg/mL in a normal saline bag and held at ambient temperature was used as a control. Levosimendan and the degradation products were monitored by HPLC. The results are shown in Figures 17 and 18. All the samples in the bags were visually clear during the course of the studies. As expected the studies demonstrate a slower rate of degradation at refrigerated temperature when compared to ambient conditions. Also, no differences in degradation rates were observed in saline and half normal saline. The degradation rate for the formulation post dilution is higher than SIMD AX® because of higher pH (7.8 to 8.4) due to the presence of Neut® in the IV bags. The pH of the IV bag containing diluted SIMD AX® is much lower (pH=3.7) due to the citric acid in the SIMD AX® formulation.

The primary degradation route is hydrolysis, which results in the formation of OR- 1420 (Figure 18.) Total degradation products are shown in Figure 19. Although the degradation rate of the present invention is higher than SIMD AX®, the extent of degradation does not impair its use as a pharmaceutical composition.

Example 10

A preferred formulation of levosimendan (2.5 mg/mL) in dehydrated ethyl alcohol was evaluated in several preliminary compatibility studies by diluting the formulation to a concentration of 0.05 mg/mL. The primary purpose was to demonstrate suitability of use over a 24-48 hour period post-dilution in Neut® buffered saline (5 mL per 500 mL diluent) solutions in different types of IV containers. SIMD AX® was included in the studies as a reference formulation. In addition to chemical compatibility, the studies evaluated visual clarity, pH and sub- visual particulate by light obscuration (HIAC) techniques. The results in Table 2 demonstrate that using a variety of LVP containers sourced from US and Japanese suppliers the diluted drug formulations stored at ambient (RT) and refrigerated (2°C to 8°C) met USP sub-visual particulate requirements. The pH results in Table 3 demonstrate that the quantity of Neut® solution was adequate to maintain the pH above the pKa (6.2) of levosimendan for well over the target 48 hour period. All the diluted formulations were visually clear over a period of one week demonstrating the suitability of the dilution approach. The data in Table 4 demonstrate compatibility of the formulation with two PVC bag sizes in normal saline at ambient and refrigerated temperatures.

Example 11

Levosimendan in dehydrated ethyl alcohol containing citric acid also was evaluated in several preliminary compatibility studies by diluting a drug concentrate to a concentration of 0.05 mg/mL levosimendan and 2% ethanol. Essentially, 2.5 mL of Neut® was added to a 250 mL PVC IV bag (Hospira) followed by the addition of 5 mL of drug concentrate (levosimendan (2.5 mg/mL) in ethanol with citric acid.). The drug concentrates containing 0, 1 and 2 mg/mL citric acid had been stored for six months in stability chambers at either 5°C or 25°C along with SIMD AX®. Formulations were evaluated in compatibility studies at ambient temperature for a period of three days.

All samples in the bags were visually clear during the course of the studies. As Figure 20 shows, the degradation rate of the new formulation post dilution was higher than SIMD AX® because of the higher pH (7.6 to 8.4) (caused by the addition of Neut®). The primary degradation route resulting in the formation of OR- 1420 is shown in Figure 21. The citric acid level in the formulations had no impact on this rate as evidenced from the parallel lines observed for the six experimental compositions. The pH of the IV bag containing diluted SIMDAX® was much lower (pH range 3.5 to 4.0) due to the citric acid in the final SIMDAX® composition. Table 2. HIAC Results for Formulation Compatibility in Normal Saline from US and Japan Bags and Bottles

Table 3. pH Results for Formulation Compatibility in Normal Saline from US and Japan Bags and Bottles

K*

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Table 4. HIAC Results for Formulation Compatibility in Normal Saline in 250 and 500 mL PVC Bags

Claims

WHAT IS CLAIMED IS

A pharmaceutical composition comprising: (a) a quantity of levosimendan or a pharmaceutically acceptable salt thereof;

(b) a pharmaceutically acceptable diluent; and

(c) a pharmaceutically acceptable alkalinizing agent or buffering agent.

2. The pharmaceutical composition of claim 1 wherein said quantity of levosimendan is dissolved in an organic solvent.

3. The pharmaceutical composition of claim 2 wherein said quantity of levosimendan and said organic solvent are provided as a drug concentrate.

4. The pharmaceutical composition of claim 2 wherein said organic solvent is anhydrous ethanol.

5. The pharmaceutical composition of claim 1 wherein said quantity of levosimendan is in a freeze-dried form.

6. The pharmaceutical composition of claim 1 wherein said alkalinizing agent or buffer is in an amount sufficient to increase the pH of said pharmaceutical composition to about the pKa of said levosimendan.

7. The pharmaceutical composition of claim 6 wherein said pKa of said levosimendan is about 6.2.

8. The pharmaceutical composition of claim 1 wherein said diluent is selected from the group consisting of Ringers solution, Lactated Ringers solution and saline solution.

9. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable organic acid.

10. The pharmaceutical composition of claim 9, wherein said organic acid is selected from the group consisting of citric acid, lactic acid, tartaric acid or maleic acid.

11. The pharmaceutical composition of claim 9, wherein said organic acid is citric acid.

12. A method of increasing the solubility of levosimendan in an aqueous solution comprising the step of combining said levosimendan with said aqueous solution and an alkalinizing or buffering agent.

13. The method of claim 12, wherein said alkalinizing agent or buffering agent first is combined with said aqueous solution before combining with said levosimendan.

14. The method of claim 12 wherein the said alkalinizing or buffering agent is selected form the group consisting of sodium bicarbonate and tromethamine.

15. The method of claim 12 wherein said aqueous solution is 0.9% sodium chloride.

16. The method of claim 12 wherein said alkalinizing agent or buffering agent is a solution of 4.0% sodium bicarbonate.

17. A composition suitable for intravenous injection comprising

(a) a quantity of levosimendan or a pharmaceutically acceptable salt thereof;

(b) a pharmaceutically acceptable organic solvent,

(c) a stability enhancing amount of a pharmaceutically acceptable organic acid having a pKa in the range of from 2 to 4; and

(d) an aqueous diluent having a pH in the range of about 6.3 to about 9.5.

18. The composition of claim 17, wherein the organic solvent in anhydrous ethanol.

19. The composition of claim 17, wherein the organic acid is selected from the group consisting of citric acid, lactic acid, tartaric acid or maleic acid.

20. The composition of claim 19, wherein the organic acid is citric acid.

21. The composition of claim 17, wherein the aqueous diluent is selected from the group consisting of normal saline and half normal saline.

22. A packaged pharmaceutical product comprising:

(a) a quantity of levosimendan,

(b) a pharmaceutically acceptable organic solvent,

(c) a pharmaceutically acceptable organic acid,

(d) a pharmaceutically acceptable diluent and (e) a pharmaceutically acceptable alkalinizing agent.

23. The pharmaceutical product of claim 22 wherein (a), (b) and (c) are packaged as a single component.

24. The pharmaceutical product of claim 22 wherein (a) and (b) are packaged as a single component.

25. The pharmaceutical product of claim 22 wherein (b) and (c) are packaged as one component.

26. The pharmaceutical product of claim 22, claim 23, claim 24 or claim 25 wherein (d) and (e) are packaged as a single component.