FORM 2
THE PATENTS ACT 1970
(39 of 1970)
AND
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION:
"NOVEL INJECTABLE COMBINATION"
2. APPLICANT (S):
(a) NAME: BDR Pharmaceuticals International Pvt. Ltd.,
(b) NATIONALITY: Indian Company incorporated under the Indian
Companies Act, 1956
(c) ADDRESS: 407-408 Staarda chambers, New Marine Lines, Mumbai 400020,
Maharashtra, India.
3. PREAMBLE TO THE DESCRIPTION:
The following specification particularly describes the invention and the manner in which it is to be performed:

Technical field:
The present invention relates to stable injectable formulations comprising of a combination of a Cytidine derivative (I) and a pyrazolone derivative (II) of the following formulae, useful in the treatment of CNS disorders and ischemic strokes. The present invention also relates to process of preparation of the said formulation.

Background and prior art:
Formula (I) as mentioned above represents Cytidine derivatives which exhibit pharmacological action in the biosynthesis of phosphatidycholine (Ptd Cho) and shows beneficial effects in various CNS injuries and neurogenerative diseases. The neuroprotective action has resulted in its vide use in cerebral stroke treatment as well as in Alzheimer's disease. Rl represents amino group or substituted amino group. The substituents are alkyl, branched alkyl, ary] or heteroaryl group. Notable amongst the active Cytidine derivatives is a compound Citicoline (IA) wherein Rl is NH2. Citicoline is widely used in treatment of stroke and Cognitive Dysfunction in the elderly patients.
Citicoline (IA) also known as CDP-Choline or Cytidine 5'-diphosphocholine, is a form of the essential nutrient Choline. It exhibits clinical efficacy in elderly patients in treatment

of cognitive deficits, inefficient memory and early stage Alzheimer's disease. Citicoline is also been useful as a therapy in stroke patients. Produced endogenously, Citicoline serves as a Choline donor in the metabolic path ways for biosynthesis of acetylcholine and neuronal membrane phospholipids, chiefly phosphatidylcholine. Exogenous citicoline, as the sodium salt is useful for its cholinergic and neuroprotective action. Citicoline is available in tablet and injectable forms for therapeutic applications^ The recommended daily dosage is lgm /day.
Formula (II) represents Pyrazolone group compounds which exhibit antioxidant properties, wherein RI, R2 and R3 can be same or different including hydrogen or alkyl, branched alkyl, saturated or unsaturated alkyl, substituted alkyl such as hydroxyl, halo, nitro, amino, sulfonyl, straight chain or branched chain alkyl, substituted or un-substituted aryl, hetero alkyl or heteroaryl with one or more hetero items. Notable amongst the active pyrazolone compound is Edaravone (IIA) wherein Rl is methyl (CH3), R2 is hydrogen and R3 is phenyl (C6H5) group.
Edaravone (IIA) is chemically 3-methyl-l-phenyl-2-pyrazol-in-5-one. Being a free radial scavenger, it provides a potential treatment for Cerebral Ischemia and in regeneration of cells and tissues in cardiopulmonary arrest. It is administered as an intravenous injection. The recommended daily dosage is restricted to 60 mg/day.
Though Edaravone exhibits excellent antioxidant properties, its usage is restricted due to toxicity related issues associated with it. It is felt essential to augment beneficial effects of Edaravone without increasing the toxic effects, so that a formulation with enhanced efficacy without having additional side effects can be offered to the patients.
Therefore, the inventors of the current invention aimed to study the synergistic effects of Edaravone and Cytidine derivatives to achieve a greater efficacy, without increasing the daily recommended dosages of Edaravone. Further, the present invention also provides a stable injectable formulation comprising Edaravone and Cytidine derivatives which is stable under accelerated conditions.

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 more fully understood and appreciated.
In accordance with the above objective, the present invention provides stable Injectable formulation comprising combination of Citicoline (IA) and Edaravone (Il-a) along with suitable injectable excipients useful in the treatment of CNS disorders and ischemic strokes. The route of administration of the Injection of the present invention is Intravenous drips.
The present invention encompasses the pharmaceutical salt, hydrate, solvate or polymorphic forms of the above two actives. Citicoline (IA) and Edaravone (Il-a) are present in the formulation in the range of ratio of 5:1 to 50:1, more preferably 25:1.5. Citicoline concentration can be adjusted in the range of 10 mg/ml to 100 mg/ml, preferably, 10 mg/ml to 50 mg/ml, more preferably 25 mg/ml concentration and Edaravone concentration can be adjusted in the range of 0.5 mg/ml to 10 mg/ml preferably 1.5 mg/ml
Total volume of the injection per dose is 20 ml containing 500 mg Citicoline and 30 mg Edaravone. The dosage is adjusted as per the therapeutically accepted quantity namely Citicoline 1 gm/day and Edaravone 60 mg/day. The dosage is generally in two divided doses, that is two injections per day.
In the preferred embodiment the present invention provides a stable Injectable formulation comprising combination of Citicoline (IA) and Edaravone (Il-a) with Cyclodextrin derivatives and suitable injectable excipients.
Cyclodextrin derivative used in the present invention can be either single component or a mixture of the commercially available derivatives selected from, Alpha Cyclodextrin, Beta-Cyclodextrin, modified derivatives of these Alfa or Beta Cyclodextrin in various proportions. The Cyclodextrin derivatives used in present invention is from 0.1% Mole

ratio to 20% Mole ratio with respect to the Edaravone employed. The solution preparation can be made either by using any injectable approved organic solvent such as aliphatic alcohols, dimethyl sulfoxide, polyethylene glycols or in absence of any such organic solvents.
The Injectable solutions can be additionally added with stabilizers such as chelating agents like EDTA and its salts, Citric Acid and its salts or Nitrilo acetic acid derivatives and its salts. Additionally the solution can be further stabilized by using antioxidants like Sodium Bisulphite, Sodium Metabisulphie, Cystiene Hydrochloride, Thiol acetic acid or Phenolic group derivatives. Also, approved stabilizers such as Butylated Hydroxy Anisole and its derivatives, Parabens and its salts can be employed in appropriate proportions.
The inventors of the present invention have carried out intensive investigations to study the effects to augment therapeutic efficacy of Edaravone while combining with other products having similar therapeutic effects and surprisingly found that antioxidant and neurogenerative action was greatly enhanced in animal models when Citicoline and Edaravone both were simultaneously administrated by injectable route. The antioxidant and neuroprotective action was found to be enhanced in comparison with the administration of either Citicoline or Edaravone alone. These findings prove that Citicoline and Edaravone combination exerts synergistic effect and because of this the antioxidant, neurogenerative and neuroprotective action is greatly enhanced. This enhanced activity helps in early recovery for patients suffering from CNS disorders and ischemic strokes. The combination of the present invention can be administered by infusion through intravenous route.
In another embodiment the present invention provides process for preparation of the said Injectable formulation as described herein below:
Water for Injection is taken in a glass flask, equipped with stirring arrangement, Nitrogen gas bubbling tube and addition funnel. The entire set-up is placed in thermostatically controlled water bath system.

Cyclodextrin derivative in desired proportion is added to the flask under stirring, and allowed to dissolve to obtain a clear solution. To this solution desired quantity of Edaravone is added and stirred for a while to obtain clear solution. Occasionally, to facilitate the easy dissolution of Edaravone, temperature of the water bath is increased if required, and after complete dissolution of Edravone, desired quantity of Citicoline Sodium, Stabiliser and anti-oxidant are added and the reaction mass is stirred under Nitrogen gas atmosphere. The pH of the solution is then checked and adjusted in the range of 3 to 6, most preferably 3.5 to 5.5. After adjusting the pH, the solution is further stirred for 20 to 30 minutes, and then filtered successively through 0.45 and 0.2 micron size Poly filter membranes. The bulk solution is then analyzed by HPLC and UV for Edaravone content and Citicoline Sodium content. The Solution is then aseptically filled in 20 ml Vials and sealed to obtain regular injection preparation.
The above experiment is repeated in similar manner, but without addition of the Cyclodextrin derivatives, for studying the stability of the injection solutions. The result of the same is depicted in Example 5 of the specification.
The vials containing Citicoline Sodium and Edaravone solution of different combinations of excipients are subjected for Accelerated and Long term Stability studies, following the methodology as per ICH Guidelines.
Parameters studied during stability studies are Assay Content of Edaravone and Citicoline Sodium, Color of the Solution, Clarity of the Solution and pH of the solution.
It is observed that, stable Injectable combination solution is achieved, when Edaravone and Citicoline Sodium are used in presence of Cyclodextrin derivatives and in presence of preservatives such as Cysteine Hydrochloride and Sodium metabisulphite in the pH range of3.5 to 6.5.
The study was done both ways using single combination injection containing Citicoline and Edaravone both in single injectable formulations in one set of study and simultaneously administering separate component formulations containing Edaravone and Citicoline injectables in another set of study. A kit was prepared for second set of study in

which two vials are packed having Edaravone injection and Citicoline injection and at the time of administration, both the injections are simultaneously injected through IV route.
The invention also provides methods of treating CNS disorders and ischemic strokes, which comprises administering 'an effective amount' of the 'formulation of the present invention' to the subject suffering from CNS disorders and ischemic strokes. The subject mentioned herein is human.
'An effective amount' according to present invention Citicoline in an amount of about 1 gm /day and Edaravone 60 mg / day.
The invention further discloses use of the 'composition of the present invention comprising combination of Citicoline and Edaravone' in preparing the medicament to treat CNS disorders and ischemic strokes.
The following non-limiting preparative examples are given by way of illustration only and not to be construed as limiting the spirit and scope of this invention.
Example 1:
Injectable formulation was prepared by using following method. Contents:
1) Citicoline Sodium : 5000 mg
2) Edaravone :300mg
3) Sodium Citrate : 300 mg
4) Absolute Alcohol : 50 ml
5) Sodium Edetate :10mg
6) Water for injection : To make volume 200 ml.
The pH of the solution was adjusted to 5 to 7 with addition of citric acid and the solution was filtered and filled in vials under sterile conditions. The volume per vial was adjusted to 20 ml per vial. This gave an injectable formulation containing Edaravone at 1.5 mg / ml and Citicoline at 25 mg/ml. Stability of this combination was found to be less. After a period of 10 days, the solution became yellow and slight turbid.

Example 2:
1) Water for Injection : 750.0 ml
2) Cystein HCI: 750 mg
3) Beta Cyclodextrin :10.0 gm
4) Edaravone : 1.51 gm
5) Sodium Bisulphite : 1 gm
6) Citicoline Sodium: 28.6 gm, equivalent to Citicoline 25 gm.
Process For Manufacturing:
In a Glass assembly, abovementioned components are mixed together, the final Volume of the solution is made-up to 1000 ml by adding appropriate amount of Water for Injection. The solution is stirred to dissolve and adjust the pH to 3.5 by using orthophosphoric acid. The solution is then successively filtered through 0.45 micron and 0.25 micron poly membranes and then aseptically filled in 30 ml capacity vials having 20 ml solution per vial. The solution of the vial contains Edaravone 1.5 mg per ml and Citicoline 25 mg per ml.
Example 3:
1) Water for Injection : 750.0 ml
2) Cystein HCI: 750 mg
3) HydroxyPropyl Beta Cyclodextrin : 10.0 gm
4) Edaravone : 1.51 gm
5) Sodium Bisulphite : 1 gm
6) Citicoline Sodium : 28.6 gm, equivalent to Citicoline 25 gm.
Process For Manufacturing:

In a Glass assembly, abovementioned components are mixed, the final volume of the solution is made-up to 1000 ml by adding appropriate amount of water for injection. The solution is stirred to dissolve and adjust the pH to 3.5 by using orthophosphoric acid. The solution is then successively filtered through 0.45 micron and 0.25 micron poly membranes and then aseptically filled in 30 ml capacity vials having 20 ml solution per vial. The solution of the vial contains Edaravone 1.5 mg per ml and Citicoline 25 mg per ml.
Example 4:
1) Water for Injection : 750.0 ml
2) Cystein HC1: 750 mg
3) HydroxyPropyl Beta Cyclodextrene :10.0 gm
4) Edaravone : 1.51 gm
5) Sodium Bisulphite : 1 gm
6) Citicoline Sodium: 28.6 gm, equivalent to Citicoline 25 gm.
7) Disodium EDTA : 200 mg
Process For Manufacturing:
In a Glass assembly, abovemnetioned components are mixed, the final volume of the solution is made-up to 1000 ml by adding appropriate amount of water for injection. The solution is stirred to dissolve and adjust the pH to 3.5 by using orthophosphoric acid. The solution is then successively filtered through 0.45 micron and 0.25 micron poly membranes and the solution is then aseptically filled in 30 ml capacity vials having 20 ml solution per vial. The solution of the vial contains Edaravone 1.5 mg per ml and Citicoline 25 mg per ml. The Stability of the Injectable solutions is found to be acceptable during a period of one year of study.
Example 5:
Summary of Stability Data for Injectable Combination Solution containing Citicoline 25 mg per ml and Edaravone 1.5 mg per ml concentration.

Sr.
Example Initial 1 2 3 6 Month 9 12
No. Assay Month Month Month Month Month
1 1 Eda 98.81 94.2 % Not There is * *
99.96%
Citi
99.48% % 98.89
% 98.2 % done further. deterioration in Edara.
2 2 Eda 99.61 99.2 % 99.10% 98.7 % 98.7 98.15
99.89%
Citi
99.68% % 99.49
% 98.5 % 98.42 98.41 98.12 97.94
3 3 Eda 99.81 99.62 99.60 % 99.7% 99.7 99.15
99.91%
Citi
99.72% %
99.54
% % 99.34
% 98.92 98.91 99.12 98.94
4 4 Eda 99.89 99.72 99.75 % 99.73 % 99.78 99.35
99.91%
Citi
99.72% %
99.54
% % 99.44
% 98.92 98.93 99.12 99.34
NOTES:
1) Edaravone Stability was found to be good in combination formulas 2, 3 and 4
2) Addition of EDTA helps in prevention formation of colouration to the injectable solution.
3) Formula 4 ( as given in example 4) is having highest stability

We claim,
1. A stable Injectable formulation comprising Citicoline and its pharmaceutically acceptable salt in combination with Edaravone and its pharmaceutically acceptable salt alongwith Cyclodextrin derivative and suitable injectable excipients.
2. The Injectable formulation according to claim 1, wherein Citicoline is Citicoline sodium.
3. The Injectable formulation according to claim 1, wherein Citicoline is present in an amount of 10 mg/ml to 100 mg/ml.
4. The Injectable formulation according to claim 1, wherein Edaravone is present in an amount of 0.5 mg/ml to 10 mg/ml.
5. The Injectable formulation according to claim 1, wherein Cyclodextrin derivative is selected from alpha-Cyclodextrin, beta-Cydodextrin or their derivatives.
6. The Injectable formulation according to claim 1, wherein suitable injectable excipients are selected from chelating agents and antioxidants.
7. Method of treating CNS disorders and ischemic strokes, which method comprises administering 'an effective amount' of the 'Injectable formulation' according to claim 1 to the subject suffering from said disorders.
8. The method according to claim 7, wherein said subject is human.
9. Use of Injectable formulation according to claim 1 in preparing the medicament intending to treat CNS disorders and ischemic strokes.