WO2019180199A1

WO2019180199A1 - Stable aqueous composition of phosphocreatine

Info

Publication number: WO2019180199A1
Application number: PCT/EP2019/057230
Authority: WO
Inventors: Marcello DI SABATINO; Paola Maffei
Original assignee: Alfasigma S.P.A.
Priority date: 2018-03-22
Filing date: 2019-03-22
Publication date: 2019-09-26
Also published as: IT201800003896A1

Abstract

The present invention relates to a stable liquid pharmaceutical composition of phosphocreatine, or a pharmaceutically alkaline salt thereof useful in the treatment of cardiac and skeleton muscle protection in heart surgery, in cardiology area and in rehabilitation. The present invention also provides a process for the preparation of the liquid composition of phosphocreatine, or a pharmaceutically acceptable alkaline salt thereof.

Description

“STABLE AQUEOUS COMPOSITION OF PHOSPHOCREATINE”

Field of the Invention

Background of the Invention

Phosphocreatine (PC), or creatine phosphate (otherwise N-[imino- (phosphonoamino) methyl] -N-methyl-glycine, CAS number 67-07-2), plays a key role in the energetic mechanism of muscle contraction and it is represented by the chemical formula:

Phosphocreatine, or creatine phosphate, displays its activity in a wide spectrum of diseases and organs. It acts on striated musculature and exerts a stabilizing action on the cardiac sacrolemma, with a protective activity of that membrane, when used in cardioplegic solutions during cardiac surgery or infusion in myocardic ischemia. Endogenous phosphocreatine is produced in the liver and pancreas from arginine, glycine and methionine, it constitutes a form of energy reserve when it reacts with ADP (adenosine diphosphate) by releasing the phosphate group to produce ATP (adenosine triphosphate) and creatine and contributes to the maintenance of a constant level of ATP in cells, and in particular in muscle cells, wherein is plenty used for contraction.

Phosphocreatine plays an important role in the tissues and organs that have an energy requirement subjected to a rapid and temporary fluctuations, as in skeletal muscles, heart muscle, and even the brain. Since the amount of ATP in the heart is very low compared to the need, the cells of the myocardium continuously resynthesize it to maintain a normal vitality and contractile function.

An extensive clinical research has shown significant reduction in the availability of phosphocreatine occurring in a wide spectrum of both physiological and pathological conditions. The decrease in intracellular concentration of phosphocreatine results in a hypo-dynamic state of cardiac and skeletal muscle pathology.

The protective, preventive and curative action of exogenous phosphocreatine, both in cardiac surgery, and, in general, in cardiology, has therefore been investigated in numerous controlled clinical studies through the assessment of different clinical, hemodynamic and ultrastructural parameters.

Exogenous administration of phosphocreatine, e.g. by parenteral route, intravenously or intramuscularly, can improve cardiac performance in patients subjected to coronary angioplasty, as described by Ke-Wu D. et al. in Angiology 2015, 66(2) 163-168. Such beneficial effect has been applied to different pathological conditions such as heart failure, acute myocardial ischaemia, chronic ischaemic heart disease, cardiac surgery, hypotonia and hypotrophy of skeletal muscle and cerebral ischaemia, as more recently described by Strumia E. et al. in Adv Ther (2012) 29(2):99- 123, and by Gaddi AV, et al. Heart, Lung Circulation, (2017) 26 (10): 1026-1035. Phosphocreatine is used as cardioprotective adjuvant in cardioplegia, i.e. in the transitory cessation of contractile activity of the heart employed during cardiac surgery, or in the treatment of acute ischaemia of the myocardium as described by Balestrino M. et al in Amino Acids, 2016, 48: 1955-1967.

Cardioplegia is a method of myocardium protection for patients requiring cardiac surgery in which heart beat must be stopped. This method allows heart surgery to be performed with a diastolic (relaxed), non-beating heart, providing to the surgeons a blood-free operating field, while protecting the heart muscle from ischemic damage due to the interruption of blood flow in the coronary arteries. This is achieved by an induced global ischemia to the heart by cross-clamping the aorta, thereby preventing coronary perfusion, and the systemic blood circulation is transferred to the heart- lung machine (extracorporeal circulation). Although convenient for the cardiac surgery practice, global ischemia of the heart is harmful, as the ischemic duration increases, cellular and molecular changes become more severe causing damage to the cardiac muscle fibrocells up to lose their functionality. Hence, it is important during cardiac surgery to initiate cardioprotective procedures to minimize any ischemic injury as reported by Chambers D J. et al. in Pharmacol. Ther 127, 2010. 41-52.

Cardioplegic solutions are generally physiological saline solutions comprising high concentrations of potassium, which results in rapid depolarized cardiac arrest, and other preserving agents. The use of a cardioplegic solution has, in fact, the dual role of effectively arresting the heart and preventing possible ischemic and reperfusion damage to the heart tissue. Cardioplegic solutions are also used during cardiac removal from a donor and organ preservation during transport and waiting before subsequent replanting to a recipient. Robinson L. A. et al. report in J Thorac Cardiovasc Surg. 1984 87(2): 190-200 the use of phosphocreatine in St. Thomas’ Hospital cardioplegic solution (STH1) on a rat heart model for cardiopulmonary by-pass and ischemic arrest. The addition of phosphocreatine to cardioplegic solutions has been documented since 1987 as reported by Semenovsky ML et al., J Thorac Cardiovasc Surg. l987;94(5):762-769 wherein rapid recovery of hemodynamics after release of the aortic cross-clamp, a decreased frequency of fibrillation, and more frequent restoration of sinus rhythm were observed.

The administration of the phosphocreatine solution can occur both during cardioplegia, by addition thereof to the cardioplegic solution in coronary perfusion. Phosphocreatine is also used during the perioperative and post-operative period by parenteral administration, in different types of cardiac surgery, such as valve replacement, aorto-coronary bypass surgery, and the repair of congenital heart defects.

Phosphocreatine can be used in the treatment of acute and chronic heart failure and in the acute ischemic attack of the myocardium and in cardiac surgery.

In general, the administration of phosphocreatine may be indicated in the treatment of all diseases in which a decrease in its concentration is observed, i.e. all conditions caused by a decrease in available energy or an increase in energy demand.

The preparation of phosphocreatine, in particular its disodium salt tetrahydrate, is described by Ennor A.H. et al in Biochem J, 1948, 43 (2) 190-191.

Phosphocreatine can be industrially prepared by different processes, for example, according to EP 0150883, from dibenzyloxy-phosphoryl chloride and cyanamide, which react together to give the intermediate benzyloxyphosphoryl cyanamide, then converted to phosphocreatine.

Phosphocreatine is not stable in aqueous solution and it is commercialized in solid form, which is solubilized before the use with a suitable solvent. Xie Z et al reports in Iranian J Pharm Res 15(1), 119, 2016 that phosphocreatine is soluble in water and the instability in water into its related substances (creatine, creatinine, creatinine phosphare disodium salt) makes the major problem of this active ingredient.

EP 0199117 Al describes the use of liquid compositions of phosphocreatine disodium salt for treating heart ischemia and infarction, consisting of a parenteral composition at concentrations from about 0.5 to 2.5 M and a solution of phosphocreatine disodium salt from about 5 to 50 mM for infusion. This application does not mention the preparation of the solutions and their stability and therefore by the acknowledge of the instability of phosphocreatine it is deducible that the solid PC is solubilized just before the use. EP 0222257 A2 describes a pharmaceutical composition of phosphocreatine sodium salt for the preparation of aqueous solutions for parenteral administration consisting of a bottle containing an amount of 0.5-1 gram of phosphocreatine sodium salt and a bottle containing a sterile solution of distilled water to be diluted before use.

CN 101732263 describes a lyophilized composition containing phosphocreatine and at least an excipient selected from low molecular weight dextran, mannitol, sorbitol, glucose, sodium chloride to stabilize it and favour its dissolution before use.

The lyophilisation process requires high amount of excipients as sugar to stabilize PC, which are allowed for intramuscular or infusion administration. Moreover, a lyophilisation process is characterized to eliminate all the water and it is difficult to have a process able to provide reproducible batches of phosphocreatine sodium salt, which is the more soluble salt of PC.

Phosphocreatine disodium salt tetrahydrate is available on the market as a powder together with a solvent for parenteral administration as sterile preparation for injection, infusion or implant in human body or for cardioplegic perfusion, with the trademarks Neoton^® and Seldomalfa^®. The solutions of phosphocreatine disodium salt powder when reconstituted before use have concentration ranging from 20 to 150 mg/ml.

Phosphocreatine disodium salt tetrahydrate is available in composition 500 mg/ 4 ml solvent for intramuscular injection and 20 mg/ml, 100 mg/ml for infusion; it is used at concentration of 10 mmole/litre in cardioplegic solutions.

The industrial preparation of phosphocreatine in a solid, sterile, stable and ready- to-use form presents several problems due to the requirements of the Health Authorities, such as the use of dedicated equipment to avoid cross-contamination with other substances possibly prepared in the production plant.

In addition, phosphocreatine powder dissolution, before use, may lead to a not perfectly homogeneous solution, wherein phosphocreatine may be present in not completely solubilized particles, which may cause harm to the patient.

Therefore, there is a need of a pharmaceutical composition containing phosphocreatine disodium salt in solution composition, obtained in sterile form, ready for use, or diluted at the concentration required for the cardiologic diseases.

Summary of the Invention

The invention provides a pharmaceutical composition comprising phosphocreatine, or a pharmaceutically acceptable alkaline salt thereof, at a concentration from 300 to 700 mM and a pH value from 8.0 to 12.

The composition of the invention may be in unit dosage form ready for the use or to be diluted before the use.

In another aspect of the invention are isotonic phosphocreatine, or a pharmaceutically acceptable alkaline salt thereof, composition for parenteral use at a concentration from 50 to 250 mM at pH between 7.0 and 8.0. In another aspect of the invention are isotonic cardioplegic solutions comprising phosphocreatine or a pharmaceutically acceptable alkaline salt thereof, at concentration between 5 and 20 mM at pH between 7.0 and 8.0.

According to another aspect the invention is a process for the preparation of a liquid pharmaceutical composition comprising phosphocreatine, or a pharmaceutically acceptable alkaline salt thereof, in an aqueous, basic and stable solution ready for use as such or after dilution. The process comprises the steps of:

(a) dissolving an amount of phosphocreatine, or a pharmaceutically acceptable salt thereof, in distilled water up to obtain a final concentration from of 100 to 700 mM;

(b) adding an inorganic base, selected between sodium hydroxide and potassium hydroxide, up to a pH value from 8.0 to 12 and,

(c) filtering the solution through a 0.22 pm sterilizing filter,

(d) aliquoting the solution in bottles of desired volume.

The process to obtain a parenteral composition comprising phosphocreatine at concentration from 50 to 200 mM at pH value between 7.0 and 8.0 comprises the step of diluting the composition to a concentration from 100 to 700 mM with a phosphate solution at a concentration between 5 and 50 mM at pH between 4 and 5.

It is another object is a process for obtaining cardioplegic solutions by diluting the phosphocreatine, or a pharmaceutically acceptable alkaline salt thereof, composition with a cardioplegic solution. According to another aspect of the invention are isotonic compositions comprising phosphocreatine or a pharmaceutically acceptable alkaline salt thereof for use in cardiovascular disease, heart failure, acute myocardial ischemia, chronic ischemia, cardiac surgery, muscular hypotonia, hypotrophy of skeletal muscle and cerebral ischemia, organ and tissue preservation in hearth surgery, and in cardioplegia. In another aspect the invention is a kit comprising a vial comprising phosphocreatine or a pharmaceutically acceptable alkaline salt thereof, at a concentration from 300 to 700 mM and a pH value from 8.0 to 12, with and a vial containing a solvent in a volume to obtain a phosphocreatine concentration between 50 and 300 mM.

Detailed description of the invention

It is an object of the present invention a liquid pharmaceutical composition comprising phosphocreatine, or a pharmaceutically acceptable alkaline salt thereof at a concentration from 300 to 700 mM and a pH value from 8.0 to 12.

In another aspect, the composition of the invention has a phosphocreatine concentration from 300 to 600 mM and a pH value from 8.0 to 12.

The composition according to the invention may comprise a pharmaceutically acceptable salt selected from, sodium phosphate dibasic dihydrate (Na₂HP0₄-2H₂0), sodium bicarbonate (NaHC0₃), sodium acetate (CffXOONa) and sodium citrate (Na₃CeH₅0₇) at concentration from 10 to 250 mM.

The composition comprises phosphocreatine, or a pharmaceutically acceptable alkaline salt thereof at concentration from 300 to 700 mM and sodium phosphate dibasic dihydrate at a concentration from 15 to 150 mM, preferably between 15 and 30 mM at pH values between 8.0 and 12.

The composition of the invention comprises phosphocreatine, or a pharmaceutically acceptable alkaline salt thereof at concentration from 300 to 450 mM and sodium phosphate dibasic dihydrate from 15 to 30 mM at pH values between 8.0 and 11.0. The composition of the invention comprises phosphocreatine, or a pharmaceutically acceptable alkaline salt thereof at concentration of 400 mM at pH values between 11 and -12 and sodium phosphate dibasic dihydrate 15 mM.

The composition of the invention comprising phosphocreatine at concentration from 300 to 700 M at a pH values between 8.0 and 12 may be used or diluted before the use to obtain the desired concentration.

The composition of phosphocreatine, or a pharmaceutically acceptable alkaline salt thereof, when used for medical use are considered acceptable when the assay is > 97.0% and the sum of the impurities or related substances are < 3%.

The composition of the invention is obtained in sterile form and it is stable at

5°C for a period of time of at least 12 months and at 25 °C for a period of time of at least 6 months.

The composition can be easily used by the medical worker by diluting it with a suitable solvent in order to obtain phosphocreatine solutions with desirable concentration to be ready to use in several pathological conditions.

The composition of the invention comprising phosphocreatine, or a pharmaceutically acceptable salt thereof, at a concentration between 50 and 300 mM at pH value between 7.0 and 9.0 ± 0.3 are isotonic.

In another aspect the invention provides a solution for cardioplegia comprising a concentration of phosphocreatine or a pharmaceutically acceptable salt from 5 to 20 mM.

In another aspect of the invention the cardiologic solution comprising the phosphocreatine composition is dissolved in St. Thomas’ Hospital solution at final concentration from 5 to 20 mM. The present invention provides a composition comprising phosphocreatine or a pharmaceutically acceptable salt thereof in liquid form, stable for at least six months at 25 °C and for at least one year at 5°C. In particular, the compositions with a phosphocreatine or a pharmaceutically acceptable salt at concentrations from 300 to 700 mM at pH 11 are stable for three months at 25 °C and for six months at 5 °C.

The compositions comprising phosphocreatine or a pharmaceutically acceptable salt thereof, at concentration from 300 to 700 mM and a phosphate salt at concentration from 5 to 50 mM at pH 11, are stable for at least 12 months at 5 °C and for at least 12 months at 25 °C.

The solutions maintain a grade of purity, higher than 97% in sterile concentrate solution and these solutions are ready to be diluted before parenteral use or for preparing cardioplegic solutions.

The composition for intramuscular administration may contain anaesthetic, such as lidocaine, and composition for cardioplegia may contain aminoacids and vitamins and salt, such as St. Thomas’ Hospital Solution.

The composition of this invention comprising phosphocreatine in concentrated form, may be also used for preparing composition in solid form, capsule and gel with pharmaceutically acceptable excipients and other adjuvants.

These compositions may comprise acidifying agents, alkalizing agents, anionic, cationic and non-ionic surfactants, anti-foaming agents, preservatives, antioxidants, biocompatible and biodegradable materials, buffering agents, complexing agents, emulsifying agents, emulsions stabilizing agents, solubilizing agents, solvents, tonicizing agents, thickening agents.

Acidifying agents are selected from, acetic acid, adipic acid, ascorbic acid, aspartic acid, citric acid monohydrate, gluconolactone, hydrochloric acid, lactic acid, maleic acid, malic acid, phosphoric acid, propionic acid, sulphuric acid, tartaric acid, sodium succinate.

Alkalising agents are selected from, ammonium sulphate, ammonium hydroxide, arginine, calcium hydroxide, diethanolamine, monoethanolamine, potassium hydroxide, sodium bicarbonate, sodium carbonate, sodium citrate dihydrate, sodium hydroxide and trometamine.

Anionic surfactants are selected from, sodium docusate and phospholipids.

Cationic surfactants are selected from, benzalkonium chloride, benzethonium chloride, phospholipids.

Non-ionic surfactants are selected from, alpha tocopherol, phospholipids, polyoxyethylene derivatives of castor oil, polysorbates (polyoxyethylene sorbitan esters of fatty acids), polyoxyethylene derivatives of stearic acid, sorbitan esters of fatty acids, tricapriline.

Anti-foaming agents are selected from, dimethicone and simeticone.

Preservatives are selected from, acetone, sodium bisulphite, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, benzyl benzoate, betadex sodium sulphobutyl ether, boric acid, butylated hydroxyanisole, butyl paraben, calcium chloride, chlorhexidine, chlorobutanol, chlorocresol, cresol, edetic acid, ethanol, glycerine, methyl paraben, monothioglycerol, pentetic acid, phenol, phenoxyethanol, phenylmercuric nitrate, potassium metabisulphite, propyl gallate, propylene glycol, propyl paraben, sodium acetate, sodium benzoate, sodium lactate, sodium metabisulphite, sodium sulphite, tetrasodium edetate, thiomersal, zinc oxide.

Antioxidants are selected from sodium bisulphite, alpha tocopherol, alpha tocopherol hydrogen succinate, ascorbic acid, ascorbyl palmitate, butyl hydroxyanisole (BHA), butyl hydroxytoluene (BHT), citric acid monohydrate, cysteine hydrochloride, glutathione, histidine, gentisic acid, gentisic acid ethanolamine derivative, malic acid, methionine, mono thioglycerol, potassium metabisulphite, propionic acid, propyl gallate, sodium ascorbate, sodium ditionine, sodium formaldehyde sulfoxylate, sodium glutamate, sodium metabisulphite, sodium sulfite, sodium thioglycolate, sodium thiosulphate.

Biocompatible and biodegradable materials are selected from, aliphatic polyesters (polylactic acid esters, co-glycol polylactic acid esters and the like), phospholipids, and polydextrose.

Buffering agents are selected from, acetic acid, adipic acid, ammonium sulphate, arginine, asparagine, boric acid, citric acid monohydrate, glycine, histidine, benzetonium chloride, lysine acetate, lysine hydrochloride, maleic acid, malic acid, meglumine, methionine, monosodium glutamate, phosphoric acid, dibasic potassium phosphate, sodium acetate, sodium citrate dihydrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, trometamine.

Complexing agents are selected from, betadex sodium sulphobutyl ether, citric acid monohydrate, cyclodextrin, disodium calcium edetate, disodium edetate, edetic acid, calcium sodium versetamide, gelatin, gluconolactone, hydroxypropyl betadex, malic acid, pentetic acid, sodium citrate dihydrate, sodium dibasic phosphate, sodium monobasic phosphate, tartaric acid, tetrasodium edetate, trehalose.

Emulsifying agents are selected from, acacia, aluminium monostearate, sodium deoxycolate, diethanolamine, lecithin, medium chain triglycerides, methylcellulose, monoethanolamine, phospholipids, polyoxyethylene derivatives of castor oil, polysorbates (polyoxyethylene sorbitan fatty acid esters), poloxamers, polyoxyethylene derivatives of stearic acid, sodium citrate dihydrate, sodium lactate, sorbitan fatty acid esters, trometamine. Solubilising agents are selected from, acacia, benzalkonium chloride, benzyl benzoate, betadex sodium sulphobutyl ether, cyclodextrins, hydroxypropyl betadex, lecithin, methylpyrrolidone, phospholipids, polyoxyethylene derivatives of castor oil, polysorbates (polyoxyethylene sorbitan esters of fatty acids), povidone, sorbitan esters of fatty acids, tricapriline, triolein.

Solvents are selected from, almond oil, benzyl alcohol, benzyl benzoate, carbon dioxide, castor oil, maize oil, cottonseed oil, diethylene glycol monoethyl ether, dimethyl sulfoxide, dimethyllacetamide, ethanol, ethyl acetate, ethyl oleate, glycerine, glycofurol, triglycerides from medium chain fatty acids, methyl pyrrolidone, liquid paraffin, mono ethanolamine, peanut oil, polyethylene glycol, propylene glycol, safflower oil, sesame oil, soybean oil, poppy seed oil, sorbitol, tricapriline, triolein, water.

Tonicizing agents are selected frombetadex sodium sulphobutyl ether, glucose monohydrate (dextrose), glucose (dextrose anhydrous), glycerine, hydroxypropyl betadex, mannitol, potassium chloride, sodium chloride.

Thickening agents comprise for example ammonium sulphate, betadex sodium sulphobutyl ether, carboxymethyl cellulose sodium, dextrin, gelatin, glycerine, methylcellulose, sucrose, trehalose.

The process for the preparation of a liquid pharmaceutical composition comprising phosphocreatine, or a pharmaceutically acceptable alkaline salt thereof, in an aqueous, basic and stable solution ready for use as such or after dilution comprises the steps of:

(a) dissolving an amount of phosphocreatine, or a pharmaceutically acceptable salt thereof, in distilled water up to obtain a final concentration from 100 to 700 mM; (b) adding an inorganic base, selected between sodium hydroxide and potassium hydroxide, up to a pH value from 8.0 to 12,

(c) fdtering the solution through a 0.22 pm sterilizing fdter,

(d) aliquoting the solution in bottles of desired volume.

Parenteral composition comprising phosphocreatine at concentration between 50 and 200 mM at pH between 7.0 and 8.0, are obtained by diluting the composition at concentration of 300 and 700 mM with a phosphate solution at a concentration between 5 and 50 mM at pH between 4 and 5 under sterile conditions.

Cardioplegic solutions are prepared by diluting the phosphocreatine sodium salt, or a pharmaceutically acceptable alkaline salt thereof composition, with appropriate solutions, such as the St. Thomas’ Hospital cardioplegic solutions to a final concentration of phosphocreatine from 5 to 20 mM.

Optionally, one or more pharmaceutically acceptable excipients can be added to the solution, and the final solution is filtered through a sterilising filter at 0.22 pm or 0.1 pm. The solution is then aliquoted into vials, flasks, bottles, bags, sachets.

The composition of the invention comprising phosphocreatine, or a pharmaceutically acceptable alkaline salt thereof, is useful in the treatment of cardiovascular disease, heart failure, myocardial and acute myocardial ischemia, chronic ischemia, acute myocardial infarction, cardiac surgery, muscular hyphotonia, hypotrophy of skeletal muscle and cerebral ischemia, organ and tissue preservation in hearth surgery, and in cardioplegia.

The composition of the invention may be also administered in an amount of about 1 g/day to 10 g/day for about 10-20 days by parenteral administration, or of 0.5 g/day to 2 g/day by intramuscular route for 10-40 days. Phosphocreatine liquid composition can be administrated by injection with a daily dosage of 1 to 20 g divided into 1 -2 administration in a day.

Phosphocreatine sodium salt liquid composition may also be administrated intravenously, for example, by infusion of a daily dose from 1 to 10 g divided into 1-4 administrations. Infusions can last from 1 to 24 hours. Administration may start 1-3 days before cardiac surgery and continue for 1-5 days after surgery, depending on the severity of myocardial damage.

Phosphocreatine liquid composition can be administered before and after the cardiac surgery.

In another aspect, the invention provides a kit comprising a vial comprising phosphocreatine or a pharmaceutically acceptable alkaline salt thereof, at a concentration from 300 to 700 mM and a pH value from 8.0 to 12 and a vial containing a solvent in a volume to obtain a phosphocreatine concentration between 50 and 300 mM.

The following examples illustrate the embodiments of the composition object of the invention and a process for its preparation, without constituting any kind of limitation to its realization as it can be contemplated by the person skilled in the technical field of reference.

EXAMPLES

Example 1 - Preparation of Phosphocreatine Compositions A-B

Phosphocreatine Compositions A and B were prepared by dissolving 20 g and 4 g respectively of disodium phosphocreatine tetrahydrate (MW 327.15) in about 100 ml distilled water. The solutions were made up to volume with distilled water (V=200 ml) and the solutions were sterile-filtered through a 0.22 pm fdter.

Table 1: Phosphocreatine Compositions A-B

The phosphocreatine assays were measured according to the method described in Example 7.

Compositions A and B have a pH value corresponding to 8.2.

The solutions were divided into 15 ml aliquots in bottles and stored at 5 and 25 °C.

Example 2 - Preparation of Phosphocreatine Compositions C-H

Phosphocreatine compositions C-H were prepared by dissolving in water amounts of disodium phosphocreatine tetrahydrate, according to Table 2, in distilled water. pH values were measured and adjusted to a value of pH 9, pH 10 and pH 11, by the addition of 1 N NaOH. The final volumes were then adjusted with distilled water and the solutions were filtered under sterile conditions by the use of a 0.22 pm filter.

The phosphocreatine assays were measured according to the method described in Example 7. Table 2: Phosphocreatine Compositions C-H

The solutions were then divided in sterile bottles. Solutions C-H were stored at 5 and 25 °C.

5

Example 3 - Preparation of phosphocreatine compositions 1-0 with buffer

The phosphocreatine compositions with pH value higher than 8.2 in the presence of buffers, were prepared with different salts.

Dibasic sodium phosphate dihydrate (Na₂HP0₄-2H₂0, MW 177.99) sodium 0 acetate trihydrate (C₂H₃Na0₂· 3 H₂0, MW 136.08), and tribasic sodium citrate dihydrate (MW 294.1), were dissolved under stirring in distilled water in an amount according to Tables 3 and 4 and phosphocreatine tetrahydrate amounts were added respectively. The pH values were increased to pH 11 by the addition of 1 N NaOH solution. The solutions were finally brought to volume with distilled water and filtered 5 by a 0.22 pm sterilising filter.

The phosphocreatine assay was measured according to the method described in Example 7.

The solutions were then divided into 15 ml aliquots in bottles and stored at 5 and

25 °C temperatures.

0 Table 3: Phosphocreatine Compositions 1-0

Table 4 shows phosphocreatine solutions with different buffers

Table 4: Phosphocreatine Compositions P-S

*= sodium acetate trihydrate °= sodium citrate tribasic dihydrate

Example 4 - Preparation phosphocreatine solutions at pH 11 with phosphate buffer

Solutions T and U were prepared according to the amounts shown in Table 5. The phosphocreatine assay was measured according to the method described in

Example 7. Table 5: Phosphocreatine solutions T and U with phosphate buffer Na2HPQ₄

The solutions were then aliquoted in volume of 10 ml aliquots and stored at 5 and 25 °C.

Example 5 - Preparation dilution solution

0.39 g of sodium dihydrogen phosphate dihydrate (NaH₂P0₄ 2H₂0) were solubilised in 250 ml of distilled water by stirring until a complete dissolution. The solution has a pH value of 4.7.

Example 6 - Preparation ready-to-use sodium phosphocreatine composition

In a beaker a volume corresponding to 10 ml sodium phosphocreatine solution, prepared as described in Example 4 (Solution U) was added to 30 ml of solvent prepared as described in Example 5 and the solution was let under agitation for a few seconds, filtered under sterile conditions.

The final solution, with a volume of 40 ml, has a phosphocreatine concentration of 100 mM, a pH value of 7.1 and an osmolarity value of 290 mOsm/Kg. Example 7 - Determination of phosphocreatine assay solutions A-U

The determination of the phosphocreatine weight assay of the solutions A-U was obtained by chromatographic method. Measurements were made using an Agilent 1100 HPLC chromatograph equipped with a 100*4.6 mm Hypercarb Graphite column, with a particle size of 5 pm.

Phosphocreatine is eluted by the mobile phase of 0.1% (v/v) trifluoroacetic acid in water and 1% (v/v) acetonitrile, with an elution flow of 1.0 ml/min. The instrument was equipped with a UV detector with a wavelength of 200 nm. Phosphocreatine was eluted at temperature of 25 °C with a retention time (RT) of 3.3 minutes.

The quantitative determination of phosphocreatine was made with respect to a standard solution of sodium phosphocreatine, prepared by dissolving 50 mg of sodium phosphocreatine tetrahydrate in 100 ml of distilled water (0.05% w/v solution).

Solutions A-U were appropriately diluted to obtain a final concentration of 0.05% (w/v), filtered through a 0.22 pm nylon filter and then analysed in HPLC.

From the HPLC measurements the phosphocreatine assay was determined according to the calculation:

Ac * Pst * T% * FD * Vform g phosphocreatine

Ast * 100 * Vst * 1000 bottle

wherein:

Ac: area of sodium phosphocreatine in the sample solution;

Ast: mean area of sodium phosphocreatine in the reference solution;

Pst: average w.s. sodium phosphocreatine weight in the reference solutions, expressed in mg;

T%: sodium phosphocreatine tetrahydrate titre; Vst: Volume in ml of the comparison solution;

FD: Dilution factor

Vform: Volume in ml of the formulation.

Solutions A-U prepared according to the above examples were stored at 5 °C and 25 °C and analysed in HPLC using the method described above to determine the titre at 3 and 6 months, compared to TO.

Table 6 shows the assay values and the pH values of solutions A-U stored at

5°C. Table 6: Phosphocreatine assay values and pH values of solutions A-U stored at 5 °C

./. Con d Table 6

Solutions T and U are stable at 5 °C even at 18 months.

Table 7 shows the phosphocreatine assay and the pH values of the solutions A- U stored at 25 °C at 3 and 6 months compared to TO.

Table 7: Phosphocreatine assay and pH values of compositions A-U stored at 25 °C

./. Con d Table 7

Solutions T and U are stable at 5 °C even after 18 months.

Example 8 - Industrial preparation of disodium phosphocreatine solution

A phosphocreatine solution (Solution A), prepared by dissolving 20 kg of disodium phosphocreatine tetrahydrate and 0.400 kg of dibasic sodium phosphate dihydrate with 133 kg of water for injectable preparations (WFI), was added with 1.9 L of 1N NaOH, and kept in agitation. The pH was increased to 11±0.3 by the addition of 1N NaOH, and finally an amount of water for injectable preparations was added to a final volume of 159.3 Kg of WFI water were added. The obtained solution was sterilized by filtration in an aseptic environment using EXPRESS^® SHF Hydrophilic PES 0.22 pm filters or equivalent. The filtered solution was then aliquoted in 10 ml volume sterile glass bottles and stored at 5 ± 0.3 °C. Each bottle contains 1.00 gram of disodium phosphocreatine hydrated base at pH 11.

This composition was stored at 5 °C and at 25 °C.

Example 9 - Preparation cardioplegic compositions

A volume corresponding to 40 ml of the solution prepared according to Example

8 was diluted 4 litres with St. Thomas’ Hospital (STH1) solution according to J Thorac Cardiovasc Surg. 1984 87(2): 190-200. The solution resulted to be isotonic with a pH value of 7.

Claims

1. A pharmaceutical composition comprising phosphocreatine, or a pharmaceutically acceptable alkaline salt thereof, at concentration from 300 to 700 mM and pH value from 8.0 to 12.

2. The composition according to claim 1, wherein the phosphocreatine concentration is from 300 mM to 600 mM and the pH value is from 8.0 to 12.

3. The composition according to claim 1, comprising a pharmaceutically acceptable salt, selected from sodium phosphate dibasic dihydrate, sodium bicarbonate, sodium acetate at concentration from 10 to 150 mM.

4. The composition according to claim 3, wherein the sodium phosphate dibasic dihydrate is at a concentration from 15 to 150 mM.

5. The composition according to claim 4, comprising phosphocreatine or a pharmaceutically acceptable alkaline salt thereof at concentration from 300 to 450 mM and dibasic sodium phosphate dehydrate at concentration from 15 to 30 mM, having pH value from 8.0 to 12.

6. The composition according to claim 5, comprising phosphocreatine at concentration of 400 mM and dibasic sodium phosphate dehydrate at concentration of 15 mM at a pH value from 11 to 12.

7. The composition according to claim 1 further comprising at least one anahestetic.

8. A parenteral composition comprising phosphocreatine or a pharmaceutically acceptable alkaline salt thereof at concentration from 50 to 200 mM, obtained by diluting the composition of claim 1.

9. A cardioplegic composition comprising phosphocreatine or a pharmaceutically acceptable alkaline salt thereof at concentration from 5 to 20 mM, obtained by diluting the composition of claim 1 with St. Thomas’ Hospital solutions.

10. A process for the preparation of a phosphocreatine composition according to claim 1, comprising the steps of:

a) dissolving phosphocreatine or a pharmaceutical acceptable salt thereof in distilled water up to a concentration from 50 to 700 mM;

b) adding an inorganic base, selected from sodium hydroxide and potassium hydroxide up to a pH value comprised between 8.0 and 12:

c) fdtering the solution on a 0.22 pm sterilizing filter

d) disposing the solution in sterile bottles.

11. A composition according to claims 1 for use in the treatment of in cardiovascular disease, heart failure, acute myocardial ischemia, chronic ischemia, cardiac surgery hypothermia, hypotrophy of skeletal muscle and cerebral ischemia, organ and tissue preservation in hearth surgery, and in cardioplegia.

12. The composition according to claim 11 administered in an amount of 1 g/day to 10 g/day for 10-20 days by parenteral route, or 0.5-2.0 g/day by intramuscular route for 10-40 days.

13. The composition according to claim 12 administered by infusion in a daily dosage from 1 to 20 g divided into 1-4 administrations.

14. The composition according to claim 11 administered before and after the cardiac surgery.

15. A kit comprising a vial containing the composition of claim 1 and a vial containing a solvent in a volume to obtain a phosphocreatine concentration between 50 and 200 mM.