WO2010092597A2

WO2010092597A2 - Liposomal citicoline injection

Info

Publication number: WO2010092597A2
Application number: PCT/IN2010/000074
Authority: WO
Inventors: Ambikanandan Rajnarayan Misra; Narendra Ishwarlal Gandhi; Mannalal Ramgopal Bajaj; Bharat Babulal Shah; Rajan Shantaram Samant; Ahmad Sattar Pinjari Jamil
Original assignee: Lyka Labs Limited
Priority date: 2009-02-11
Filing date: 2010-02-10
Publication date: 2010-08-19
Also published as: WO2010092597A3; CN102316852A; EP2395977A2; JP2012517421A

Abstract

Disclosed herein is a stable sustained release liposomal injection comprising Citicoline entrapped in the ammonium sulfate liposomes, useful to enhance brain uptake efficiency.

Description

"LIPOSOMAL CITICOLINE INJECTION"

TECHNICAL FIELD OF INVENTION:

The present invention relates to Liposomal injection preparations comprising Citicoline entrapped in liposomes which is useful to enhance brain uptake efficiency. More particularly the present invention relates to Liposomal injection comprising Citicoline entrapped in an ammonium sulfate Liposomes having, a high drug: lipid ratio. The said liposomes are prepared by a process that loads the drug by an active mechanism using a transmembrane ion gradient, preferably a transmembrane pH gradient.

BACKGROUND OF THE INVENTION:

Citicoline is a naturally occurring substance found in most life forms. It is an intermediate metabolite in major pathway for the synthesis of Phosphatidylcholine. Phosphatidylcholine is a phospholipid that is a major component of cell membranes. Phosphatidylcholine is necessary for the structure and function of all cells and is crucial for sustaining life.

Citicoline is synthesized in cells by the reaction of the nucleotide cytidine triphosphate or CTP with phosphocholine. The enzyme catalyzing the reaction is called CTP: phosphocholine cytidyltransferase. This reaction is the rate-limiting step in the synthesis of phosphatidyl choline.

Phosphocholine is synthesized from choline, and for the synthesis of phosphatidylcholine, Cm-choline reacts with diacylglyceride, catalyzed by the enzyme Citicoline: 1,2- diacylglycerol cholinephosphotransferase.

Citicoline raises level of acetylcholine which improves brain metabolism and overall energy and increases level of neurotransmitters, noradrenalin and dopamine. Citicoline is believed to protect nerve cells in neuronal ischemic condition and has been reported to accelerate recovery from stroke & traumatic brain Injury. It helps to enhance blood brain circulation and to remove excitatory amino acids responsible for neurodegeneration. It also accelerates functional reorganization in variety of neurological disorders such as cerebral ischemia, mental stroke, alzheimer's disease and Cognitive disorders of different causes. Citicoline is safe drug as shown by toxicological tests, has no significant systemic cholinergic effects and is well tolerated. In Rodents, oral administration of Citicoline increases blood plasma level of 'cytidine' and 'choline' whereas in Humans, blood plasma level of 'Uridine' increases instead of 'cytidine' after exogenous administration because of rapid degradation of Citicoline before systemic absorption, by liver and intestinal pathways. This rapid degradation is a challenge to the sustained delivery of Citicholine.

Citicoline is also a delivery form of choline and cytidine. Choline is a precursor of acetylcholine and betaine. Acetylcholine is a neurotransmitter whose deficiency in certain regions of the brain is belived to be an etiological factor in certain dementia syndromes, including Alzheimer's disease, Betaine is involved in the conversion of the amino acid homocysteine to the essential amino acid L-methionine. L-methionine is a protein amino acid. Cytidine, following conversion to cytidine triphosphate, participates in a few reactions, including the formation of citicoline and nucleic acids.

Citicoline is useful in the treatment of stroke and brain injury. It may be helpful in some with tardive dyskinesia, Parkinson's disease, Alzheimer's disease and other conditions characterized by impaired cognitive function, including memory loss. An indication may emerge for it to help visual acuity in those with amblyopia. Citicoline is more effective and have a number of advantages over other agents being developed for the reduction of infarct volume subsequent to an ischemic event. Being an endogenous compound, Citicoline is inherently safe. Citicoline has a very low toxicity and an extremely broad therapeutic index.

In numerous studies of Citicoline, favorable results have been obtained in cerebral ischemia and traumatic head injury. Its efficacy in these studies has been attributed to its apparent ability to increase phosphatidylcholine synthesis in the brain. In animal studies, it has been shown to enhance cell-membrane formation and repair, to restore intracellular enzyme function, to limit nerve damage and decrease oedema. The same mechanism, generally are said to account for favorable effects reported for it in the treatment of Parkinson's disease, Alzheimer's disease and a variety of cognitive disorders, including impaired memory associated with aging.

Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single membrane bilayer) or multilameller vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient toward the center of the bilayer while the hydrophilic "heads" orient towards the aqueous phase.

The original liposome preparation of Bangham et al. (J. MoI. Biol., 1965, 13:238-252) involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to "swell", and the resulting liposomes which consist of multilammellar vesicles (MLVs) are dispersed by mechanical means. This preparation provides the basis for the development of the small sonicated unilammellar vesicles described by Papahadjopoulos et al. (Biochim. Biophys, Acta., 1967, 135:624-638), and large unilamellar vesicles.

This class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in US Pat. No. 4,522,803 to Lenk, et al. and includes monophasic vesicles as described in US Pat. No. 4,588,578 to Fountain, et al. and frozen and thawed multilammellar vesicles (FATMLV)

In a liposome-drug delivery system, a bioactive agent such as a drug is entrapped in the liposome and then administered to the patient to be treated. For example, Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410; Paphadjopoulos et al., U.S. Pat. No. 4,235,871; Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578. Alternatively, if the bioactive agent is lipophilic, it may associate with the lipid bilayer. In general practice, remote loading has been used for loading of weakly acidic and alkaline drugs (Haran, G., et. al., 1993). The basic idea of the method is dependant on varying charge behavior and interaction of neutral and minimum charged molecules at different pH across the inside and outside of the lipid membrane. Hence, the drug molecules after acquiring the charge (on basis of varying pH inside the liposomes) will thereby be trapped in the liposomal interior, and will show low diffusion rate from liposomal bilayer.

From several studies, it has been proved that Citicholine encapsulation in liposomes improved the therapeutic benefits by preventing it's hepatic degradation. Citicholine bears +1 charge and also having 'amino' group in it's structure which can be explored for it's improved encapsulation into the liposomes by transmembrane pH gradient method.

In the present invention, the term "entrapment" shall be taken to include both the drug in the aqueous volume of the liposome as well as drug associated with the lipid bilayer.

Large multilameller vesicles (MLVs) (Gabizon et al., 1982, supra.), large unilamellar vesicles (LUVs) and small (sonicated) unilamellar vesicles (SUVs) (Gabizon et al., 1983, supra., Shinozawa et al., 1981, Acta. Med. Okayama, 35:395) have been utilized with lipid compositions incorporating variable amounts of positively charged and negatively charged lipids in addition to phosphatidylcholine (PC) and cholesterol.

Sterically stabilized stealth liposomes offer a benefit to liposomal membrane by bypassing hepatic metabolism due to highly hydrophilic nature which proved to be efficient for long circulation of liposomes.

Citicoline has strongly polar & is highly hydrophilic in nature. Hence, it does not readily cross Blood brain barrier (BBB). According to animal studies, only 0.5% and 2% of Citicoline brain uptake were observed following oral and intravenous routes respectively. Hence, drug incorporation in microspheres & Liposomes is expected to improve drug delivery to brain across BBB. Citicoline or its salt as an active ingredient is normally administered parenterally in the form of injectables or orally in the form of tablets, capsules sachets. Liposomal Citicoline Injection having higher brain uptake not yet reported in the prior art.

BRIEF DESCRIPTION OF FIGURES:

Figure 1: Transmission Electron Microscope Image of Liposomes Figure 2: Comparative blood profile of Citicoline liposome and injection

OBJECT OF THE INVENTION:

Therefore, the main object of the present invention is to provide a stable Liposomal injection comprising Citicoline with improved drug delivery to brain across the Blood brain barrier which is useful in enhancing brain uptake efficiency and process for preparation thereof.

Another object of the present invention is to prepare ammonium sulfate liposomes by Thin Film hydration (TFH) followed with loading Citicoline in the said liposomes by pH gradient method.

SUMMARY OF THE INVENTION:

In accordance with the above objective, the present invention provides a stable Liposomal injection comprising Citicoline entrapped in ammonium sulfate liposomes, useful to enhance brain uptake efficiency.

In another aspect, the present invention provides multilamellar vesicles of ammonium sulfate prepared by Thin Film Hydration (TFH) technique. The liposome is composed of Hydrogenated soya phosphatidylcholine (HSPC), a negatively charged lipid Disteroyl phosphatidylglucerol (DSPG) and Cholesterol (CHOL) in the molar ratio of 7:1:2. Optionally, DSPE-mPEG 2000 is also added as 3 mole % (0.0015 mM) of the total lipid to impart stealth property to liposomes. In yet another aspect the process parameters like solvent system, speed of rotation, vacuum, solvent evaporation temperature, film formation time, hydration time, sonication cycle, annealing time, incubation time, concentration of hydration medium and some formulation variables such as ratio of lipids as HSPC, DSPG and CHOL, are optimized by keeping ammonium sulfate: total lipid ratio and hydration volume as constant for initial optimization experiments. The optimized conditions were used throughout further investigation. For conversion of MLVs to SUVs, probe sonicator was used at 5 cycle, 60 % A, 0.6 sec, 2 min., temp. 55 ± 3°C or optionally extruded through series of polycarbonate filters.

The pH gradient is created between internal and external compartment of ammonium sulfate liposome by exchanging exterior (unentrapped) ammonium sulfate against 10% sucrose solution using dialysis sac.

The dialysis process is carried out for 18 + hours. This novel technique is used to enhance the entrapment of Citicoline by developing the liposomal formulation with different internal and external pH (pH gradient). Finally loading of Citicoline is carried out by incubating the liposomal suspension for optimized time (1 hr. at temperature 55±3°C) in ammonium sulfate at molarity of (10OmM).

Citicoline encapsulated in ammonium sulfate Liposome after separation from free drug may be suspended in buffer and lyophilized after addition of cryoprotectant such as mannitol. This will lead to dry powder for reconstitution with water for injection before use.

DETAILED DESCRIPTION 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. The present invention describes a stable Liposomal injection comprising Citicoline entrapped in liposomes which is useful to enhance brain uptake efficiency. The said Liposomal injection is prepared by pH gradient method to achieve high drug loading.

In a preferred embodiment the present invention describes stable sustained release Liposomal injection comprising Citicoline entrapped in ammonium sulfate liposomes. The said ammonium sulfate liposomes is prepared by Thin Film hydration (TFH) technique followed with loading of Citicoline in the said liposomes by pH gradient method.

The ammonium sulfate liposomes of present invention is composed of lipids such as hydrogenated soya phosphatidylcholine (HSPC), Disteroyl phosphatidyl glycerol (DSPG) and cholesterol (CHOL) to form MLVs. The said lipids are present in molar ratio of 7:1:2.

In another embodiment, aπynonium sulfate liposomes of present invention is composed of lipids such as hydrogenated soya phosphatidylcholine (HSPC), Disteroyl phosphatidyl glycerol (DSPG), cholesterol (CHOL) and DSPE-mPEG 2000 to form MLVs. The said lipids are present in molar ratio of 7:1:2:0.0015.

The MLVs so formed are converted to SUVs by sonicating the liposomal suspension using probe sonicator for 5 cycle, 60 % A, 0.6 sec, 2 min., Temp. 55 ± 3°C or optionally extruded through series of polycarbonate filters using extruder (MOC: SS-316) at high pressure (50 to 800 psi) of pure nitrogen gas.

MLVs of Ammonium sulfate liposomes are prepared by Thin Film Hydration technique followed by successful loading of Citicoline against the transmembrane pH gradient. Enhanced loading of 40% Citicoline into Ammonium sulfate liposomes is achieved by employing pH gradient method. The Citicoline liposomes so prepared are spherical in shape and have particle size ranging between 115.5nm to 124nm. The entrapment efficiency was found to be improved using pH gradient method compared to conventional loading methods. The stable liposomal injection of present invention comprising Citicoline entrapped in the ammonium sulfate liposome exhibits sustained release of the drug. The ratio of drug and lipid is 1:3 to 1:10. Drug encapsulated in Ammonium sulfate Liposome after separation from free drug is suspended in buffer and lyophilized after addition of cryoprotectant such as mannitol.

In another embodiment, the present invention provides process for preparing liposomal injection of Citicoline comprising steps of:

1) Dissolving Lipid such as HSPC, DSPG and CHOL, optionally DSPE-mPEG 2000 in a mixture of chloroform and methanol;

2) evaporating the solvent in the rotary flask evaporator to obtain thin dry film under vacuum;

3) hydrating the thin dry film of step 2 by using aqueous ammonium sulfate to obtain a MLVs liposomal suspension comprising ammonium sulphate entrapped in liposomes;

4) converting the MLVs of step 3 to SUVs either by sonicating the MLVs or optionally extruding through the series of polycarbonate filters;

5) allowing the suspension of step 4 to stand undisturbed for 60mins for anneling

6) establishing transmembrane ammonium sulfate gradient by dialysis exchange against 10% aqueous sucrose;

7) incubating the Ammonium sulfate SUVs with Citicoline in 0.01 M [4-(2- hydroxyethyl)-l-piperazine ethane sulphonic acid] (HEPES) pH 7.5 for predetermined time & temp;

8) separating free drug and liposome using Sephadex G-50 column pre-equilibrated with 0.15M sodium chloride;

9) sterilizing liposomes by filtering using PES filter to obtain the liposomal suspension comprising Citicoline entrapped in the liposomes; and

10) lyophilizing the Liposomal suspension of step 11 using Mannitol as a cryoprotectant or directly injecting the same intravenously to patients.

The dialysis was carried out for 18+ hours for sufficient exchange of exterior unentrapped ammonium sulfate against 10% sucrose solution to establish a transmembrane pH gradient.The mixture of chloroform and methanol used in the above process is present in the ratio of 3:1 v/v.

Characterization of liposomes:

Citicoline entrapped liposomes were evaluated for morphology, particle size, zeta potential, internal pH of liposome and percent drug entrapment. The photomicrograph obtained by Optical microscope and transmission electron microscopy (TEM) confirms the multilamellar nature of unsonicated liposome with spherical shape of liposomes respectively. The TEM photograph shows size of a particle in the range of 100-150nm (figure- 1). The average particle size of optimized batch before and after Citicoline loading was found to be 115.5nm and 124nm respectively. The zeta potential of optimized batch was recorded as -25.8mV. The internal pH of liposome prior to and following drug loading was found to be 5.52 and 7.33 respectively. The percent drug entrapment in optimized batch was found to be 39.88 %.

In- vitro drug diffusion study:

In-vitro drug diffusion study was performed using dialysis sac, in which drug release was observed in phosphate buffer saline (PBS) pH 7.4. The temperature of the external media was kept at 37±2°C for the simulation of the body condition. The volume of the external medium was 100ml. The plain drug and liposomal formulation was taken directly in diffusion cell and placed in beaker containing diffusion medium.

The plain drug took 8 hours for about 93.25% of drug release whereas only 89.54% drug released from the liposomal formulation after 24 hours. The release kinetics was studied by fitting different models of drug release kinetics. The data obtained indicate that Citicoline plain drug follows 'First Order Release (R² 0.978) whereas Citicoline liposomal formulation obeys 'Higuchi's Diffusion Controlled' model for drug release (R² 0.993).

In- vivo biodistribution study by radiolabelling:

In-vivo biodistribution study was carried out at Institute of Nuclear Medicine and Allied Sciences, New Delhi (INMAS) by radiolabelling technique. Radiolabelling of Citicoline injection and Citicoline liposome was done by 99mTc by previously reported direct labeling methods and parameters like stannous chloride concentration and incubation time was optimized to have maximum labeling efficiency. The optimum concentration of stannous chloride needed for maximum labeling efficiency was found to be 100 μg for Citicoline liposome and 150 μg for Citicoline injection. The incubation time required for high labeling efficiency was found to be 15 min for both Citicoline injection and Citicoline liposome while other parameters were kept constant. The quality control tests to assess strength of labeling and labeling efficiency were also carried out according to the method described earlier (Theobald A.E., 1990).

For bio distribution study, 0.1 ml of labeled Citicoline injection and Citicoline liposomes were administered into healthy balb/c mice via tail vein, and tissue distribution was studied at lhr, 2hr, 4hr, 6hr and 24 hr interval. The animals were sacrificed, blood sample was collected and radio activities in different organs like brain, liver spleen, lungs, stomach and kidney was recorded by Gamma Ray Spectrophotometer, expressed as % Injected Dose/gm of organ. The pharmacokinetic parameters like AUC, C_max, T_max, Ua were computed by Kinetica 4.4 PK/PD analysis software.

The result shows the radioactivity in whole blood, after 24 h of injection was found to be 0.85 % and 2.01 % for Citicoline and Citicoline liposomes respectively. The C_max for liposomal formulation in brain was 0.3 %ID/g while for Citicoline injection it was only 0.11%ID/g indicate higher penetration of liposomal formulation into brain compared to plain drug. The AUCo_-24 of Citicoline and Citicoline liposome were 38.38 h*% ID/g and 56.35 h*% ID/g, respectively. The ti_/2 of Citicoline and Citicoline liposomes was found to be 29.53h and 114.34 hr after iv injection, respectively.

From in-vivo biodistribution study, it is concluded that Citicoline liposomal formulation shows more area under the curve and hence stay for longer duration in body compare to Citicoline injection as shown in figure-2.

Stability data indicates the stability of liposomes when stored as lyophilized formulation prepared with cyroprotectants. The percentage drug retention of the rehydrated lyophilized liposomal formulation after storing at different temperatures & humidity upto period of 2 months showed comparatively higher drug retention (>99 %) on storing at refrigerated condition as compared to room temperature (>98%) confirming that lyophilization of liposomal formulation kept it stable for longer period at refrigerated condition.

EXAMPLES:

Example 1:

Preparation of Ammonium Sulfate Liposomes by TFH followed by Loading

Citicholine by pH Gradient Method.

Multilamellar vesicles comprising HSPC, DSPG and CHOL with entrapped ammonium sulfate were prepared by Thin Film hydration (TFH) technique (New R.R.C., 1990). Briefly, the lipids were dissolved in a mixture of chloroform and methanol (ratio 3:1 v/v) in a 250ml round bottom flask in different molar ratios as shown below:

The solvent was evaporated in the rotary flask evaporator under vacuum. The thin dry lipid film thus formed was hydrated using aqueous ammonium sulfate of different molarity (Such as 8OmM, 10OmM and 12OmM) as hydrating medium at 60 ± 3°C i.e. above phase transition temperature of lipid (Tg). The formed liposomal dispersion was sonicated (5 cycles, 60 % Amp, 0.6 sec, 2 min., Temp. 55± 3°C) in probe sonicator. The sonicated liposomes were then allowed to stand undisturbed for about 60 min, for annealing. Resultant Liposomes were subjected to centrifiigation at 3,000 rpm, 4⁰C for 10 minutes using Remi centrifuge to remove unhydrated lipid, if any. To establish a transmembrane ammonium sulfate gradient, dialysis exchange against aqueous 10% sucrose was carried out (Haran et.al. 1993). Briefly, A 4 cm long portion of the dialysis tubing was made into a dialysis sac by folding and tying up one end of the tubing with thread, taking care to ensure that there would be no leakage of the contents from the sac. The sac was then soaked overnight in 10% sucrose solution in distilled water.

The wet sac was gently opened and washed copiously with 10% sucrose solution. Then the sac was filled with ammonium sulfate liposomal preparation (2-4ml). The sac was once again examined for any leaks and then was suspended in a glass beaker containing 100 ml of 10% sucrose solution, which acted as a receptor compartment. The contents of the beaker were stirred using Teflon coated bar magnet (length = 2.5 cm, d = 0.5 cm) and the beaker was closed with the aluminium foil to prevent any evaporative losses during the experiment run. The dialysis was carried out for 18+ hours for sufficient exchange of exterior (unentrapped) ammonium sulfate against 10% sucrose solution to establish a transmembrane pH gradient.

After creation of gradient, liposomes were incubated with the drug solution (15 mg/ml) in 0.01M HEPES (pH-7.5) buffer for definite period of time at 55±3°C i.e. above Tg. For separation of free drug and liposome 'gel exclusion' chromatography was followed as reported in literatures (New R.R.C., 1990). Briefly, Sephadex G-50 column was prepared by soaking Sephadex G-50 into 0.15 M sodium chloride overnight. 0.15M sodium chloride was optimized to exchange with free Cϊticoline. Then column of 2cm was prepared by pouring sephadexG-50 slurry into a 2 ml syringe. The syringe was put into 10ml centrifuge tube and centrifuged at 1000 rpm for 10 min. to remove excess solvent in Remi cooling centrifuge. The column was pre-equilibrated with 0.15M sodium chloride by three consecutive passes and each time centrifuged to remove excess 0.15M sodium chloride. Then ImI liposomal suspension was applied at top of column and centrifuged at 1000 rpm for lOmin.The elute was collected which contains Citicoline liposome while the free drug was got entrapped into the column.

Liposomal suspension so formed was then characterized for vesicle size, zeta potential and percent drug entrapment (PDE). Example 2:

Multilamellar vesicles comprising HSPC, DSPG and CHOL with entrapped ammonium sulfate were prepared by Thin Film hydration (TFH) technique (New R.R.C., 1990). Briefly, the lipids were dissolved in a mixture of chloroform and methanol (ratio 3:1 v/v) in a 250ml round bottom flask in different molar ratios. The solvent was evaporated in the rotary flask evaporator under vacuum. The thin dry lipid film thus formed was hydrated using aqueous ammonium sulfate of different molarity (Such as 8OmM, 10OmM and 12OmM) as hydrating medium at 60 ± 3 ⁰C i.e. above phase transition temperature of lipid(Tg).

Liposomes were extruded through series of polycarbonate filters (pall incorporation) using Extruder (MOC: SS-316) at high pressure (50 to 800 psi) of pure nitrogen gas. Extrusion was performed above glass transition temperature of lipid i.e. 60±3°C. Allow the liposomes to pass through two filter holder placed one above other(Double stacked system).Liposomes were extruded till particle size was achieved below 100 nm with Particle distribution index (PDI) below 0.1. Cool the Liposomal suspension to room temperature and proceed for further step.

To establish a transmembrane ammonium sulfate gradient, dialysis exchange against aqueous 10% sucrose was carried out (Haran et.al. 1993). Briefly, A 4 cm long portion of the dialysis tubing was made into a dialysis sac by folding and tying up one end of the tubing with thread, taking care to ensure that there would be no leakage of the contents from the sac. The sac was then soaked overnight in 10% sucrose solution in distilled water.

The wet sac was gently opened and washed copiously with 10% sucrose solution. Then the sac was filled with ammonium sulfate liposomal preparation (2-4ml). The sac was once again examined for any leaks and then was suspended in a glass beaker containing 100 ml of 10% sucrose solution, which acted as a receptor compartment. The contents of the beaker were stirred using Teflon coated bar magnet (length = 2.5 cm, d = 0.5 cm) and the beaker was closed with the aluminium foil to prevent any evaporative losses during the experiment run. The dialysis was carried out for 18+ hours for sufficient exchange of exterior (unentrapped) ammonium sulfate against 10% sucrose solution to establish a transmembrane pH gradient. After creation of gradient, liposomes were incubated with the drug solution (15 mg/ml) in 0.01M HEPES (pH-7.5) buffer for definite period of time at 55±3°C i.e. above Tg.

Finally prepared liposomal suspension was sterilized by filtration method. Liposomes were passed through 0.2/0.45μm Polyethersulfone (PES) membrane. Filtered liposomes can be injected intravenously to patients.

Example 3:

Saturated Phosphatidylcholine, Phosphatidyl glycerol and Cholesterol in molar ratio of 7:1:2 were dissolved in Chloroform: Methanol (3:1 %v/v) and evaporated at 60 ⁰C to form a thin lipid film. Further film was hydrated by 100 mM aqueous Ammonium sulfate solution at 60 ⁰C for complete hydration of lipid film. Prepared Liposomes were extruded through series of Polycarbonate membrane with pressurized nitrogen gas. Extra- Liposomal Ammonium sulfate was removed via dialysis against 10% sucrose solution. Size reduced liposomes were incubated with drug solution, where drug:lipid was 1: 5 (Preliminary experiments), at 60 °C for 1.5 hrs. Free drug in prepared liposomes was removed using ion exchange chromatography technique.

Example 4:

Saturated Phosphatidylcholine, Phosphatidyl glycerol, DSPE-mPEG 2000, Cholesterol in molar ratio of 7:1: 0.0015:2 were dissolved in Chloroform: Methanol (3:1 %v/v) and evaporated at 60 ⁰C to form a thin lipid film. Further film was hydrated by 100 mM aqueous Ammonium sulfate solution at 60 ⁰C for complete hydration of lipid film. Prepared Liposomes were extruded through series of Polycarbonate membrane with pressurized nitrogen gas. Extra-Liposomal Ammonium sulfate was removed via dialysis against 10% sucrose solution. Size reduced liposomes were incubated with drug solution, where drug:lipid was varied from 1: 3.33 to 1:10 , at 60 ⁰C for 1.5 hrs. Free drug in prepared liposomes was removed using ion exchange chromatography technique.

Example 5:

Saturated Phosphatidylcholine, Phosphatidyl glycerol and Cholesterol in molar ratio of 5.25:1.75:3 were dissolved in Chloroform: Methanol (3:1 %v/v) and evaporated at 60 °C to form a thin lipid film. Further film was hydrated by 100 raM aqueous Ammonium sulfate solution at 60 ⁰C for complete hydration of lipid film. Prepared Liposomes were extruded through series of Polycarbonate membrane with pressurized nitrogen gas. Extra- Liposomal Ammonium sulfate was removed via dialysis against 10% sucrose solution. Size reduced liposomes were incubated with drug solution, where drug: lipid was 5, at 60 °C for 1.5 hrs. Free drug in prepared liposomes was removed using ion exchange chromatography technique.

Example 6:

Saturated Phosphatidylcholine, Phosphatidyl glycerol DSPE-mPEG 2000 and Cholesterol in molar ratio of 7:1:0.0015:2 were dissolved in Chloroform: Methanol (3:1 %v/v) and evaporated at 60 ⁰C to form a thin lipid film. Further, film was hydrated by Ammonium sulfate solution of 80 mM to 120 raM at 60⁰C for complete hydration of lipid film. Prepared Liposomes were extruded through series of Polycarbonate membrane with pressurized nitrogen gas. Extra-Liposomal Ammonium sulfate was removed via dialysis against 10% sucrose solution. Size reduced liposomes were incubated with drug solution, where drug:lipid was 5, at 60 ⁰C for 1.5 hrs. Free drug in prepared liposomes was removed using ion exchange chromatography technique.

Example 7:

Drug loaded Liposomes prepared in EXAMPLE 1 was filtered through Polyethersulfone membrane having pore size 0.2μm with 0.45 μm prefilter. Filtered Liposomes containing required dose were lyophilized using Mannitol as a cryoprotectant. 1.0 to 3.0% w/w cryoprotectant is used with respect to phospholipid concentration

Claims

We claim,

1. A stable sustained release liposomal injection comprising Citicoline entrapped in the ammonium sulfate liposomes, useful to enhance brain uptake efficiency.

2. The liposomal injection according to claim 1, wherein drug to lipid ratio is in the range of 1:3 to 1:10

3. The liposomal injection according to claim 1, wherein the Citicoline entrapped in the ammonium sulfate liposome is having particle size ranging between 115.5nm to 124nm.

4. The liposomal injection according to claim 1, wherein said ammonium sulfate liposomes is composed of lipids such as hydrogenated soya phosphatidylcholine (HSPC), Distearoyl phosphatidyl glycerol (DSPG) and cholesterol (CHOL).

5. The liposomal injection according to claim 4, wherein said lipids are present in molar ratio of 7: 1:2.

6. The liposomal injection according to claim 4, wherein the said ammonium sulfate liposomes further comprises of DSPE-mPEG 2000 in the ratio of 3 mole % of total lipid.

7. The liposomal injection according to claim 6, wherein said lipids are present in molar ratio of 7:1:2:0.0015.

8. The liposomal injection according to claim 1, wherein the said injection is prepared by pH gradient method to achieve high drug loading.

9. The liposomal injection according to claim 1, wherein said ammonium sulfate liposomes prepared by Thin Film Hydration technique followed by loading of Citicoline against the transmembrane pH gradient.

10. A process for the preparation of Liposomal injection comprising Citicoline, comprising the steps of; a. Dissolving Lipid such as HSPC, DSPG, and CHOL optionally, DSPE- mPEG 2000 in a mixture of chloroform and methanol; b. evaporating the solvent in the rotary flask evaporator to obtain thin dry film under vacuum; c. hydrating the thin dry film of step 2 by using aqueous ammonium sulfate to obtain a MLVs liposomal suspension ; d. converting the MLVs of step 3 to SUVs either by sonicating the MLVs or optionally extruding through the series of polycarbonate filters; e. allowing the suspension of step 4 to stand undisturbed for 60mins; f. establishing transmembrane ammonium sulfate gradient by dialysis exchange against 10% aqueous sucrose; g. incubating the Ammonium sulfate SUVs with Citicoline in 0.01 M HEPES pH 7.5 for predetermined time & temp; h. separating free drug and liposome using Sephadex G-50 column pre- equilibrated with 0.15M sodium chloride; i. sterilizing liposomes by filtering using PES filter to obtain the liposomal suspension comprising Citicoline entrapped in the liposomes; and j. lyophilizing the Liposomal suspension of step 11 using Mannitol as a cryoprotectant or directly injecting intravenously to patients.

11. The process according to claim 8, wherein MLVs are converted to SUVs by extruded through series of polycarbonate filters using extruder (MOC: SS-316) at high pressure of pure nitrogen gas.