Title of Invention

A PROCESS OF PREPARING AMPHOTERICIN-B LIPOSOME FOR INJECTION

Abstract The delivery by liposome technology gives us the advantage to deliver toxic drugs to the without causing damage to the normal cells. Also, many drugs with low aqueous solubility and/or combination of both water-soluble and lipid soluble drugs can be formulated in the liposomes, increasing the bioavailability of the drug, reducing dosage and also reducing systemic toxicity. Amphotercin B is a amphiphilic drug and it is intercalated into a liposomal membrane. Amphotericin B is a antifungal used in the treatment of confirmed infections caused by various fungal species or visceral leishmaniasis, a parasitic infection. Additionally, Amp-B is used to treat patients who are refractory to or intolerant of conventional amphotericin B therapy. It is available both in the normal and liposomal injection form.
Full Text FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10 and rule 13]
Title: A process of preparing Amphotericin-B liposome for injection.
Applicant: Claris Lifesciences Limited,
Claris Corporate Headquarters, Nr.Parimal Crossing, Ellisbridge, Ahmedabad - 380 006, Gujarat, India.
The following specification particularly describes the invention and the manner in which it is to be performed.
DESCRIPTION: -
FIELD OF INVENTION
[0001] This invention relates to Liposomal Drug Delivery System for Amphotericin-B Drug.
BACKGROUND OF THE INVENTION
[0002] A. liposomal Amphotericin-B Intravenous Therapy
[0003] Liposomal drug delivery systems have been proposed for a variety of drugs, particularly those that are administered by parental routes. Liposome has potential to prove drug release mainly by two mechanism and that are by Endocytosis and deport formation. Amphotericin-B is mainly used as antifungal drug. The fermentation products of Streptomyces nodosus know as Amphotericin-B drug.
[0004] B. Potential Side Effects of liposomal Amphotericin-B Intravenous Therapy. The major acute reaction to intravenous Amphotericin B is fever and chills. Sometimes hypernea and respiratory stridor or modest hypotension may occur. It may develop hypoxia or hypotension. Azotemia may also occur due to use of Amphotericin B. The other symptoms are Headache, nausea, vomiting, malaise, weight loss and phlebitis at peripheral infusion sites etc.
[0005] The Amphotericin-B was intercalated into a liposomal membrane, it will decrease the dose of Amphotericin-B and by that way it will decrease the side effect of Amphotericin-B. Liposome in the body can worked mainly by two mechanisms and that is "endosytosis" and "deport" formation.
[0006] A composition of liposomal Amphotericin-B drug delivery complex comprising a narrower side effect and may yield a safer and more efficacious therapy. It is mainly used for the treatment of mucormycosis, cryptococcosis, fusariosis, alternariosis, trichosporonosis and penicilliosis marneffei. There exists a need for a liposomal Amphotericin-B drug delivery complex preparative method that results in a product with low cost, narrower side effect and distributed at a targeted sites as compared to existing compositions.
[0007] SUMMARY OF THE INVENTION
[0008] A very common object is to make a liposome Amphotericin-B complex, which decreases the side effect of Amphotericin-B.

[0009] Definitions
[0010] The term "Homogenization" as employed herein refers to a processes that is use to reduce the particle size of any type of solution.
[0011] The term "Hydration" as employed herein refers to a chemical reaction in which a hydroxyl group (OH") and a hydrogen cation (an acidic proton) are added to the two carbon atoms bonded together in the carbon-carbon double bond which makes up an alkene functional group. The reaction usually runs in a strong acidic, aqueous solution.
[0012] The term "Liposome" as employed herein refers to a membrane composed of a phospholipid and cholesterol bilayer (see on the right). Liposomes can be composed of naturally or synthetic derived of phospholipids with mixed lipid chains and Distearoyl Phosphatidyl Glycerol Sodium (DSPG).
[0013] The term "Lyophilization" as employed herein refers to a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase. The process consists of three separate, unique, and interdependent processes; freezing, primary drying (sublimation), and secondary drying (desorption),
[0014] The term "Membrane Extrusion" as employed herein refers to a membrane composed of a method of reducing the size of liposomes is to pass them through a membrane filter of defined pore size. The defined pore size can be achieved at much lower pressures than required for the french pressure cell. There are mainly two ways one is "Tortuous Path" and other is "Nucleation Track" to do membrane extrusion. The process which is used in this patent is "Nucleation Track" method.
[0015] The term "Spray drying" as used herein refers to Spray drying is a commonly used method of drying a liquid feed through a hot gas. Typically, this hot gas is air but sensitive materials such as pharmaceuticals, and solvents like ethanol require oxygen-free drying and Nitrogen gas is used instead, The liquid feed varies depending on the material being dried and is not limited to food or pharmaceutical products. This process of drying is a one step rapid process and eliminates additional processing.
[0016] DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention provides a process for the preparation of liposomal Amphotericin-B complex.

[0018] Cholesterol
Cholesterol is a soft, waxy substance found in all parts of the body. This includes the nervous system, skin, muscle, liver, intestines, and heart. It is made by the body and also obtained from animal products in the diet.
[0019] Alpha tocopherol
Tocopherol, or vitamin E, is a fat-soluble vitamin in eight forms that is an important antioxidant. Vitamin E is often used in skin creams and lotions because it is claimed by the manufacturers to play a role in encouraging skin healing and reducing scarring after injuries such as burns.
[0020] Amphotericin-B
Amphotericin-B (Fungilin®, Fungizone®, Abelcet®, AmBisome®, Fungisome®, Amphocil®, Amphotec®) is a polyene antifungal drug, often used intravenously for systemic fungal infections. It was originally extracted from Streptomyces nodosus, a filamentous bacterium, in 1955 at the Squibb Institute for Medical Research from cultures of an undescribed streptomycete isolated from the soil collected in the Orinoco River region of Venezuela. Its name originates from the chemical's amphoteric properties. Two Amphotericin-A and Amphotericin-B are known, but only B is used clinically because it is significantly more active in vivo. Currently the drug is available as plain Amphotericin-B, as cholesteryl sulfate complex, as lipid complex, and as liposomal formulation. The latter formulations have been developed to improve tolerability for the patient but may show considerable pharmacokinetic characteristics compared to plain Amphotericin-B.
[0021] A process of preparing Amphotericin-B liposome for injection and use their of can be describe as following steps:
[0022] The first step is Preparation of organic mixture
[0023] As per the step [0022], a selected quantity of chloroform and methanol is mixed to gather, in a selected size of the bottle.
[0024] As per the step [0023], a selected quantity of chloroform was 175ml and a selected quantity of methanol is 175 ml.
[0025] As per the step [0024], the chloroform and methanol is ranged from 130 to 180ml.
[0026] As per the step [0023], the size of bottle is ranged from 500 to 1000 ml.
[0027] the bottle is sonicated in ultrasonicated bath for the selected time period.
[0028] As per the step [0027], the selected time is ranged from 10 minutes to 2 hours

[0029] The second step is Lipid solution preparation
[0030] The weighted quantity of lipids, alpha-tocopherol, drug as per the batch size, hydrogenated phosphatidylcholine (H$PC), DistearoyI Phosphatidyl Glycerol Sodium (DSPG), Cholesterol is dissolved in a separate conical flask with organic mixture and filter it from sterile 0.2 micron filter.
[0031] As per the step [0030], take Amphotericin-B in separate conical flask and then add organic mixture to make a drug suspension solution and then close it with glass stopper.
[0032] As per the step [0030], the DistearoyI Phosphatidyl Glycerol Sodium solution is kept at a selected temperature.
[0033] As per the step [0032], the selected temperature i$ ranged from 45 °C to 60 °C
[0034] The pH is adjusted between 1.0 to 3.0 with 2.5 N hydrochloric acid solution.
[0035] The solution is concentrated up to 1 to 30 % for the spray drying.
[0036] As per the step [0035], close the conical flask with glass stopper or aluminum foil and store it at a selected temperature and after this spray dry it.
[0037] As per the step [0036], the selected temperature is ranged from 10 °C to 35 °C.
[0038] The third step is preparation of Amphotericin-B - DistearoyI Phosphatidyl Glycerol Sodium Complex.
[0039] Amphotericin B -DSPG complex solution was heated at a selected temperature.
[0040] As per the step [0039], the selected temperature is ranged from 40 °C to 60 °C.
[0041] As per the step [0040], add the Distearoyl Phosphatidyl Glycerol Sodium solution in Amphotericin-B solution it became orange brown color at a selected temperature.
[0042] As per the step [0041], the selected temperature is ranged from 40 °C to 60 °C
[0043] As per the step [0042], keep the solution at 60 °C.
[0044] As per the step [0043], check the pH of the solution.
[0045] The fourth step is HSPC, cholesterol and alpha tocopherol
[0046] As per the step [0038] & [0045], Heat the Amphotericin-B-Distearoyl Phosphatidyl Glycerol Sodium complex at a selected temperature.
[0047] As per the step [0046], the selected temperature is ranged from 40 °C to 60 °C
6

[0048] Add hydrogenated phosphatidylcholine (HSPC) solution, cholesterol solution and alpha tocopherol solution.
[0049] As per the step [0048], adjust the pH at 4.50 by adding 2.5 N Sodium Hydroxide solution.
[0050] As per the step [0049], the pH is ranged from 3.0 to 4.50.
[0051] As per the step [0050], the color of the final solution is found to be orange brown.
[0052] The fifth step is spray drying
[0053] The spray drying was carried out at selected parameters like inlet temperature, outlet temperature, compressed air pressure, air flow and pump flow etc.
[0054] As per the step [0053], the selected inlet temperature is found to be 60 °C.
[0055] As per the step [0054], the selected inlet temperature is ranged from 50 °C to 90 °G
[0056] As per the step [0053], the selected outlet temperature is found to be 36.
[0057] As per the step [0056], the selected outlet temperature is ranged from 30 °C to 60 °C
[0058] As per the step [0053], the selected compressed air pressure is found to be 0.6 to 0.7.
[0059] As per the step [0058], the ranged of compressed air pressure is found to be 0.2 to 1.0.
[0060] As per the step [0053] to [0059], after spray dry, allow the assembly along with the spray-dried powder to cool down considerably in an air-conditioned environment.
[0061] As per the step [0060], there will be a drastic loss of dried powder due to its sticky nature, which may interfere during removal and collection of final product.
[0062] As per the step [0061], collect the dried powder in dry glass bottle and check the moisture content of this spray-dried powder.
[0063] The sixth step is Buffer preparation
[0064] Sucrose is dissolved in disodium succinate hexahydrate buffer is about 0.1% to 0.5%w/v in water for injection.

[0065] As per the step [0064], The pH of the solution is about 5.30 by using 2.5 N HO.
[0066] As per the step [0065], the pH is ranged from 5.0 to 6.0.
[0067] As per the step [0065], the solution was filter through 0.2 ^ filters.
[0068] The seventh step is Hydration of spray-dried mixture
[0069] Weight the required quantity of spray dried powder.
[0070] As per the step [0069], add required quantity of buffer and sonicated at selected temperature like 60 °C and for the selected time like 1 hour.
[0071] As per the step [0070], the selected temperature is ranged from 40 °C and 65 °C.
[0072] As per the step [0070], the selected time is ranged from 30 minutes to 2 hours.
[0073] As per the step [0072], after hydration check for pH and particle size.
[0074] As per the step [0073], the pH is ranged from 5.0 to 6.0
[0075] As per the step [0075], the particle size is less than 100 nm.
[0076] The eighth step is the particle size reduction by homogenization
[0077] The homogenizer is washed with water for injection for several times.
[0078] As per the step [0077], the homogenizer is taken in water bath and put it at selected 75 °C temperatures.
[0079] As per the step [0078], the selected temperature is ranged from 50 °C to 80 °C.
[0080] The solution is washed with 2 M sodium hydroxide solution and after that it is washed with water for injection till the pH come at 7.0 and after that it is washed with buffer solution.
[0081] As per the step [0080], start the homogenizer at selected pressure and at selected temperature.
[0082] As per the step [0081], the selected pressure is ranged from 10,000 to 15,000 psi.
[0083] As per the step [0081], the selected temperature is ranged from 65 °C to 70 °C
[0084] As per the step [0081], Pass the solution three times from homogenizer, check the particle size.

[0085] As per the step [0083], the particle size should between 80 to 90 nm.
[0086] As per the step [0085], pass the solution from membrane extrusion 1.0 micron 0.45 and 0.2 -micron filter.
[0087] As per the step [0086], the solution is checked for pH, particle size, assay and % entrapment.
[0088] As per the step [0087], the pH is ranged from 5.0 to 6.0.
[0089] As per the step [0087], the particle size is less than 100 run.
[0090] As per the step [0087], the assay is not less than 90%.
[0091] As per the step [0087], the % entrapment is not less than 90 %.
[0092] As per the step [0087], the solution is cool at room temperature and starts the filtration from 0.2- micron sterile filter.
[0093] As per the step [0092], Take final drug-loaded solution in 5-liter flask, and start the filtration from 0.2-micron sterile filter.
[0094] The ninth step is lyophilization
[0095] As per the step [0094], the lyophilization of the done by filling the solution in 10 to 20 ml glass vial and by freezing, primary and secondary drying criteria.
[0096] As per the step [0095], the solution is dried by frozen technique at selected temperature and for the selected time.
[0097] As per the step [0096], the selected temperature is -50 °C
[0098] As per the step [0096], the selected time is 5 hours.
[0099] As per the step [0095], the primary drying is carried out by maintaining the vacuum at 0.1 mb constantly and by changing temperature and time.
[0100] As per the step [0099], the temperature is ranged from -30 °C, -25 °C, -15 °C, -5 °C °C and 0 °C by changing the time 1 hour, lhour, 30 hours, 1 hour, 1 hour accordingly.
[0101] As per the step [0095], the secondary drying is carried out by maintaining the vacuum at 0.017 mb constantly and by changing temperature and time.
[0102] As per the step [0101], the temperature is ranged from 10 °C, 20 °C and 25 °C by changing the time 7 hours, 5 hours and 5 hours, accordingly.

EXAMPLES
Prepare the organic mixture of chloroform and methanol at 0.5:5 ratios. Take 175 ml of chloroform and 175 ml of methanol in 500 to 1000 ml Dry glass bottle and sonicate them for 10 minutes in ultrasonicate bath. Then weight lipids, alpha-tocopherol drug as per batch size hydrogenated phosphatidylcholine (HSPC), DISTEAROYL PHOSPHATIDYL GLYCEROL SODIUM (DSPG), cholesterol and alpha-tocopherol, dissolve it in separate conical flask with organic mixture, and Biter it from sterile 0.2 micron filter, take Amphotericin B in separate conical flask, add organic mixture and make the drug suspension solution, close it with glass stopper, keep the DSPG solution at 45°C to 60°C and adjust pH between 1.0 to 3.0 with 2.5 N Hydrochloric acid solution should be clear. Heat the amphotericin B liposome suspension solution at 40°C to 60°C and add the DSPG clear solution drop in Amphotericin B solution. This solution gives clear orange brown color at 40°C to 60°C. The temperature should not go down. These solutions keep at 60°C and check the pH of solution. After preparation of Amphotericin B-DSPG complex add the HSPC solution, cholesterol solution and alpha tocopherol solution before adding this solution in Amphotericin B-DSPG complex solutions heat at 40°C to 60°C so amphotericin B-DSPG solution should not go down. Adjust pH 4.50 with using 2.5 N NaOH solution this solution is required clear and orange brown color solution. Take lipid solution in round bottom flask and attached it in rotary evaporator, keep the water bath temperature 60°C and evaporate the organic solvent, after the evaporation of organic solvent thin film form in round bottom flask, check the moisture content. The fresh buffer was prepared everyday. Prepared disodium succinate hexahydrate buffer. Dissolve sucrose and disodium succinate hexahydrate in Water For Injection. Adjust the pH to 5.30 using 2.5 N HC1. And filter the buffer solution through 0.2 micron filter. Add required volume of dissodium succinate hexahydrate buffer in thin film round bottom flask and sonicate at 60°C for 1 hour with sonication after hydration check the pH and particle size. Wash the assembly several times with water for injection before use. Homogenizer keep in water bath and temperature keep 75°C first wash with 2M NaOH solution after then Water for injection till pH decrease the 7.0 after then wash with buffer solution and start homogenization of solution at 10000 to
10

15000 psi at 65 to 70°C After three pass check the particle size if particle size got 80 to 90 run stop the homogenization and filter the solution from 0.45 micron filter and check the pH and particle size, total volume, assay and %entrapment. After then start the lyophilization of the solution, solutions fill up 10 ml in 20ml glass vial lyophilization technique.
EXAMPLE: 2
Prepare the organic mixture of chloroform and methanol at 0.5:5 ratios. Take 175 ml of chloroform and 175 ml of methanol in 500 to 1000 ml Dry glass bottle and sonicate them for 10 minutes in ultrasonicate bath. Then weight lipids, alpha-tocopherol drug as per batch size hydrogenated phosphatidylcholine (HSPC), DIMYRISTOYL PH06PHAT1DYLJGLYCEROL (DMPG Na), cholesterol and alpha-tocopherol, dissolve it in separate conical flask with organic mixture, and filter it from sterile 0.2 micron filter, take Amphotericin B in separate conical flask, add organic mixture and make the drug suspension solution, close it with glass stopper, koep the DMPG solution at 45 °C to 60°C and adjust pH between 1.0 to 3.0 with 2.5 N Hydrochloric acid solution should be clear. Heat the amphotericin B liposome suspension solution at 40°C to 60°C and add the DMPG clear solution drop in Amphotericin B solution. This solution gives clear orange brown color at 40°C to 60°C. The temperature should not go down. These solutions keep at 60°C and check the pH of solution. After preparation of Amphotericin B-DMPG complex add the HSPC solution, cholesterol solution and alpha tocopherol solution before adding this solution in Amphotericin B-DMPG complex solutions heat at 40°C to 60°C so amphotericin B-DMPG solution should not go down. Adjust pH 4.50 with using 2.5 N NaOH solution this solution is required clear and orange brown color solution. Take lipid solution in round bottom flask and attached it in rotary evaporator, keep the water bath temperature 60°C and evaporate the organic solvent, after the evaporation of organic solvent thin film form in round bottom flask, check the moisture content. The fresh buffer was prepared evervdav. Prepared disodium succinate hexahydrate buffer. Dissolve sucrose and disodium
succinate hexahydrate in Water For Injection. Adjust the pH to 5.30 using 2.5 N HC1.


And filter the buffer solution through 0.2 micron filter. Add required volume of dissodium succinate hexahydrate buffer in thin film round bottom flask and sonicate at 60°C for 1 hour with sonication after hydration check the pH and particle size. Wash the assembly several times with water for injection before use. Homogenizer keep in water bath and temperature keep 75 °C first wash with 2M NaOH solution after then Water for injection till pH decrease the 7.0 after then wash with buffer solution and start homogenization of solution at 10000 to 15000 psi at 65 to 70°C. After three pass check the particle size if particle size got 80 to 90 run stop the homogenization and filter the solution from 0.45 micron filter and check the pH and particle size, total volume, assay and %entrapment. After then start the lyophilization of the solution, solutions fill up 10 ml in 20ml glass vial lyophilization technique.
EXAMPLE 3:
A process of Liposomal Drug Delivery System for Amphotericin-B Drug carried out in detail as follows:
1. The first step is Preparation of organic mixture
(a) As per the step 1, a selected quantity of chloroform and methanol is mixed to gather in a selected size of the bottle;
(b) As per the step 1 (a), a selected quantity of chloroform is 30% and a selected quantity of methanol is 80%;
(c) As per the step 1 (b), the chloroform and methanol is ranged from 0 to 100%;
(d) As per the step 1 (a), the bottle is sonicated in ultrasonicated bath for the selected time period;
(e) As per the step 1 (e), the selected time is ranged from 10 minutes to 2 hours.
2. The second step is Lipid solution preparation
(a) The weighted quantity of lipids, alpha-tocopherol, drug as per the batch size, hydrogenated phosphatidylcholine (HSPC), DISTEAROYL PHOSPHATIDYL GLYCEROL SODIUM (DSPG), Cholesterol is dissolved in a separate conical flask with organic mixture and filter it from sterile 0.2-micron filter;
(b) As per the step 2(a), take Amphotericin B in separate conical flask and then add organic mixture to make a drug suspension solution and then close it with glass stopper;

(c) As per the step 2 (b), the DISTEAROYL PHOSPHATIDYL GLYCEROL SODIUM solution is kept at a selected temperature;
(d) As per the step 2 (c), the selected temperature is ranged from 45 °C to 60 °C
(e) The pH is adjusted between 1.0 to 3.0 with 2.5 N hydrochloric acid solution;
(f) The solution is concentrated up to 1 to 30 % for the spray drying;
(g) As per the step 2 (f), the selected temperature is rang edfrom10°Cto35°C.
The third step is preparation of Amphotericin - B - DISTEAROYL PHOSPHATIDYL GLYCEROL SODIUM Complex
(a) As per the step 3, Amphotericin B suspension solution is heated at a selected temperature;
(b) As per the step 3 (a), the selected temperature is ranged from 40 °C to 60 °C
(c) As per the step 3 (a), add the DISTEAROYL PHOSPHATIDYL GLYCEROL SODIUM solution in Amphotericin B solution it became orange brown color at a selected temperature;
(d) As per the step 3 (c), the selected temperature is ranged from 40 °C to 60 °C;
(e) As per the step 3 (d), keep the solution at 60 °C
(!) As per the step 3 (e), check the pH of the solution.
The fourth step is adding HSPC cholesterol and alpha tocopherol in amphotericin B -DSPG complex
(a) Heat the Amphotericin B-DISTEAROYL PHOSPHATIDYL GLYCEROL SODIUM complex at a selected temperature.
(b) As per the step 4 (a), the selected temperature is ranged from 40 °C to 60 °C;
(c) Add HSPC solution, cholesterol solution and alpha tocopherol solution in amphotericn B -DSPG complex solution adjust the pH;
(d) As per the step 4 (c), adjust the pH at 4.50 by adding 2.5 N Sodium Hydroxide solution;
(e) As per the step 4 (d), the pH is ranged from 3.0 to 4.50;
(0 As per the step 4 (e), the color of the final solution is found to be orange brown.
The fifth step is spray drying
(a) The spray drying is carried out at selected parameters like inlet temperature, outlet temperature, compressed air pressure, air flow and pump flow etc;
(b) As per the step 5 (a), the selected inlet temperature is found to be 60 °C;
(c) As per the step 5 (b), the selected inlet temperature is ranged from 50 °C to 90 T;
(d) As per the step5 (a), the selected outlet temperature is found to be 36 °C;
(e) As per the step 5 (d), the selected outlet temperature is ranged from 30 °C to 60 T;
(f) As per the step 5 (a), the selected compressecl air pressure is found to be 0.6


to 0.7;
(g) As per the step 5 (f), the ranged of compressed air pressure is found to be 0.2 to 1.0;
(h) As per the step 5 (a) to 5 (g)„ after spray dry, allow the assembly along with the spray-dried powder to cool down considerably in an air-conditioned environment;
(i) As per the step 5 (h), there will be a drastic loss of dried powder due to itssticky nature, which may interfere during removal and collection of finalproduct;
(j) As per the step 5 (i), collect the dried powder in dry glass bottle and check the moisture content of this spray-dried powder.
6. The sixth step is Buffer preparation
(a) As per the step 6, Sucrose is dissolved in disodium succinate hexahydrate buffer is about 0.1 to 0.5%w/v in water for injection;
(b) As per the step 6 (a), The pH of the solution is about 5.30 by using 2.5 N HC1;
(c) As per the step 6 (b), the pH is ranged from 5.0 to 6.0;
(d) As per the step 6 (b), the solution is filtered through 0.2 \x filters.
7. The seven step is Hydration of spray dried mixture
(a) Weight the required quantity of spray dried powder;
(b) As per the step 7 (a), add required quantity of buffer and sonicated at selected temperature like 60 °C and for the selected time like 1 hour;
(c) As per the step 7 (b), the selected temperature is ranged from 40 °C and 65 °C;
(d) As per the step 7 (b), the selected time is ranged from 30 minutes to 2 hours;
(e) As per the step 7 (b), after hydration check for pH and particle size;
(f) As per the step 7 (e), the pH is ranged from 5.0 to 6.0;
(g) As per the step 7 (e), the particle size is less then 100 ran.
8. The eight step is the particle size reduction by homogenization
(a) As per the step 8, the homogenizer is washed with water for injection for several times;
(b) As per the step 8 (a), the homogenizer is taken in water bath and put it at selected 75 °C temperature;
(c) As per the step 8 (b), the selected temperature is ranged from 50 °C to 80
°C
(d) As per the step 8 (b), the solution is washed with 2 M sodium hydroxide
solution and after that it is washed with water for injection till the pH come at 7.0 and after that it is washed with buffer solution;
(e) As per the step 8 (d), start the homogenizer at selected pressure and at selected temperature;
(f) As per the step 8 (e), the selected pressure ranged from 10,000 to 15,000


psi.; (g) As per the step 8 (e), the selected temperature is ranged from 65 °C to 70
°C; (h) As per the step 8 (e), after three times passing the solution from
homogenizer, check the particle size; (i) As per the step 8 (h), the number of passes is ranged from 2 to 10; (j) As per the step 8 (i), the particle size should be between 80 to 90 nm; (k) As per the step 8 (j) pass the solution from membrane extrusion 1.0
micron 0.45 and 0.2 -micron filter; (1) As per the step 8 (i), the solution is checked for pH, particle size, assay
and % entrapment; (m) As per the step 8 (1), the pH is ranged from 5.0 to 6.0; (n) As per the step 8 (1), the particle size is less than 100 nm; (o) As per the step 8 (1), the assay is ranged from 90 to 110 %; (p) As per the step 8 (1), the % entrapment is not less than 90 %; (q) As per the step 8(p), Take final drug-loaded solution in 5-liter flask, and start
the filtration from 0.2-micron sterile filter.
9. The ninth step is lyophilization
(a) As per the step 9, the lyophilization of the done by filling the solution in 10 mi in to 20 ml gfass vial and by freezing, primary and secondary drying criteria;
(b) As per the step 9 (a), the solution is freeze dried at selected temperature and for the selected time;
(c) As per the step 9 (b), the selected temperature is -50 °C;
(d) As per the step 9 (c), the temperature is ranged from -40°C to -60°C;
(e) As per the step 9 (b), the selected time is 5 hours;
(f) As per the step 9 (e), the time is ranged frorn 2 hours to 10 hours;
(g) As per the step 9 (a), the primary drying is carried out by maintaining the vacuum at 0.1 mb constantly and by changing temperature and time;
(h) As per the step 9 (e), the temperature is ranged from -30 °C, -25 °C, -15 °C
-5 °C and 0 °C by changing the time 1 hour, Ihour, 30 hours, 1 hour, 1
hour accordingly; (i) As per the step 9 (a), the secondary drying is carried out by maintaining the
vacuum at 0.017 mb constantly and by changing temperature and time; Q) As per the step 9 (j), the temperature is ranged from 10 °C, 20 °C and 25 °C by changing the time
7 hours, 5 hours and 5 hours accordingly.

We claim:
1. A process of preparing Amphotericin-B liposome for injection comprising the following steps:
i) Preparing organic mixture of chloroform & methanol is prepared by mixing methanol and chloroform in a ratio of 1:1 followed by sonication for 10 minutes.
ii) Preparing lipid solution comprises of dissolution of hydrogenated phosphatidylcholine and Cholesterol in organic mixture of methanol and chloroform.
hi) Preparing Amphotericin - B - DISTEAROYL PHOSPHATIDYL GLYCEROL SODIUM Complex comprises addition of DSPG solution to Amphotericin B solution followed by heating at 60 °C.
iv) A solution of hydrogenated phosphatidylcholine, Cholesterol, and alpha-tocopherol is added to solution of Amphotericin B .- DISTEAROYL PHOSPHATIDYL GLYCEROL SODIUM Complex,
vii) Carrying out step of spray drying which comprises of drying of Liposomal solution at selected parameters that is; inlet temperature of 50 °C to 90 °C; outlet temperature is 30 °C to 60 °C; air pressure of 0.2 to 1.0
viii) Step of hydration of spray dried mixture carried out by hydration using disodium succinate hexahydrate buffer containing sucrose at temperature range from 40 °C to 60 °C. During hydration, sonication is applied to form small uni-lamellar vesicles.
ix) Step of homogenization comprises giving 3 pass of homogenization at 10000 to 15000 psi and 65 to 70 °C temperature.
x) Homogenized dispersion was extruded from membrane extruder filter to form small uni-lamellar vesicle dispersion.
xi) Carrying out the step of lyophilization which comprises of freezing the solution at - 50 °C for 5 hours, followed by primary drying by maintaining vacuum at 0.1 mb and secondary drying by maintaining vacuum at 0.017 mb.
2. Process as claimed in claim 1 wherein organic mixture of chloroform &
methanol is prepared by mixing a selected quantity of 30% chloroform and 80%
methanol which has been sonicated in ultrasonicated bath for the period 10
minutes to 2 hours.
3. Process as claimed in claim 1 wherein preparation lipid solution comprises-

a) dissolving separately the weighted quantity of lipids, alpha-tocopherol, drug as per the batch size, hvdrogenated phosphatidylcholine (HSPC), DISTEAROYL PHOSPHATIDYL GLYCEROL SODIUM (DSPG), Cholesterol with organic mixture and filter it from sterile 0.2-micron filter;
b) making separately drug suspension of Amphotericin B with organic mixture ;

c) keeping DISTEAROYL PHOSPHATIDYL GLYCEROL SODIUM solution is kept at a temperature 45 °C to 60 °C;
d) adjusting PH between 1.0 to 3.0 with 2.5 N hydrochloric acid solution;
e) concentrating the solution up to 1 to 30 % for the spray drying .
4. Process as claimed in claim 1 wherein preparation of Amphotericin - B -
DISTEAROYL PHOSPHATIDYL GLYCEROL SODIUM Complex comprises
(a) heating Amphotericin B suspension solution at a selected temperature of 40 °C to 60 °C;
(b) adding the DISTEAROYL PHOSPHATIDYL GLYCEROL SODIUM solution in Amphotericin B solution it became orange brown color at a selected tempera ture40 °C to60°C of;

c) keeping the solution at 60 °C;
d) checking the pH of the solution.
5. Process as claimed in claim 1 wherein step of adding HSPC, cholesterol and
alpha tocopherol in amphotericin B -DSPG complex comprises
a) heating the Amphotericin B-DISTEAROYL PHOSPHATIDYL GLYCEROL SODIUM complex at a selected temperature40 °C to 60 °C;
b) adding HSPC solution, cholesterol solution and alpha tocopherol solution in amphotericn B -DSPG complex solution;
c) adjusting the pH at 3.0 to 4.50 by adding 2.5 N Sodium Hydroxide solution;& observing till the final solution is found to be orange brown.
6. Process as claimed in claim 1 wherein step of spray drying comprises
selecting parameters like inlet temperature,outlet temperature, compressed air
pressure, air flow and pump flow etc.,
a) the selected inlet temperature is ranged from 50 °C to 90 T;

b) the selected outlet temperature is ranged from 30 °C to 60 T;
(c) the selected compressed air pressure is is ranged from 0.1 to 0.7;
7. Process as claimed in claim 1 wherein preparation of buffer comprises dissolving sucrose in disodium succinate hexahydrate buffer ,maintaining the pH of the solution ranged from 5.0 to 6.0. , filtering the solution to obtain spray dried powdered mixture.
8. Process as claimed in claim 1 wherein hydration of spray dried mixture are carried out by
a) taking the required quantity of spray dried powder;
b) adding required quantity of buffer and sonicated at selected temperature like
60 °C and for the selected time like 1 hour;
c)add required quantity of buffer and sonicated at selected temperature ranged from 40 °C and 65 °C and for the selected time 30 minutes to 2 hours like 1 hour; pH is ranged from 5.0 to 6.0 and particle size which is to be is less then 100 ran.
9. Process as claimed in claim 1 wherein step of homogenization comprises
carrying out the homogenization step at the homogenizer is taken in water bath
at selected . pressure is ranged from 10,000 to 15,000 psi., selected temperature is
ranged from 65 °C to 70 °C to obtain particle size between 80 to 90 nm, maintaining pH
5-6, filtration from 0.2- micron sterile filter.
10. Process as claimed in claim 1 wherein lyophilization comprises filling the
solution in and by freezing, primary and secondary drying criteria, by frozen
technique the primary drying is carried out by maintaining the vacuum at 0.1
mb,at selected temperature of -50 °C to 0 °C & the selected time is 1 hour to 5
hours. [0099] As per the step the secondary drying is carried out by maintaining
the vacuum at 0,017 mb constantly and by changing temperature and time, the
temperature is ranged from 10 °C, 20 °C and 25 °C by changing the time 7 hours to 5
hours.


Documents:

414-MUM-2008-ABSTRACT(13-1-2011).pdf

414-MUM-2008-ABSTRACT(21-9-2011).pdf

414-MUM-2008-ABSTRACT(8-9-2010).pdf

414-MUM-2008-ABSTRACT(COMPLETE)-(28-2-2008).pdf

414-MUM-2008-ABSTRACT(GRANTED)-(18-10-2011).pdf

414-mum-2008-abstract.doc

414-mum-2008-abstract.pdf

414-MUM-2008-ASSIGNMENT(28-2-2008).pdf

414-MUM-2008-CANCELLED PAGES(21-9-2011).pdf

414-MUM-2008-CLAIMS(AMENDED)-(13-1-2011).pdf

414-MUM-2008-CLAIMS(AMENDED)-(21-9-2011).pdf

414-MUM-2008-CLAIMS(AMENDED)-(8-9-2010).pdf

414-MUM-2008-CLAIMS(COMPLETE)-(28-2-2008).pdf

414-MUM-2008-CLAIMS(GRANTED)-(18-10-2011).pdf

414-mum-2008-claims.doc

414-mum-2008-claims.pdf

414-mum-2008-correspondence(19-5-2008).pdf

414-mum-2008-correspondence(ipo)-(13-1-2010).pdf

414-MUM-2008-CORRESPONDENCE(IPO)-(18-10-2011).pdf

414-mum-2008-correspondence-received.pdf

414-mum-2008-description (complete).pdf

414-MUM-2008-DESCRIPTION(COMPLETE)-(28-2-2008).pdf

414-MUM-2008-DESCRIPTION(GRANTED)-(18-10-2011).pdf

414-mum-2008-form 18(19-5-2008).pdf

414-MUM-2008-FORM 2(COMPLETE)-(28-2-2008).pdf

414-MUM-2008-FORM 2(GRANTED)-(18-10-2011).pdf

414-MUM-2008-FORM 2(TITLE PAGE)-(13-1-2011).pdf

414-MUM-2008-FORM 2(TITLE PAGE)-(21-9-2011).pdf

414-MUM-2008-FORM 2(TITLE PAGE)-(8-9-2010).pdf

414-MUM-2008-FORM 2(TITLE PAGE)-(COMPLETE)-(28-2-2008).pdf

414-MUM-2008-FORM 2(TITLE PAGE)-(GRANTED)-(18-10-2011).pdf

414-mum-2008-form 3(28-2-2008).pdf

414-mum-2008-form 9(19-5-2008).pdf

414-mum-2008-form-1.pdf

414-mum-2008-form-2.doc

414-mum-2008-form-2.pdf

414-mum-2008-form-26.pdf

414-mum-2008-form-3.pdf

414-mum-2008-form-5.pdf

414-MUM-2008-MARKED COPY(8-9-2010).pdf

414-MUM-2008-POST-GRANT OPPOSITION(8-10-2012).pdf

414-MUM-2008-REPLY TO EXAMINATION REPORT(13-1-2011).pdf

414-MUM-2008-REPLY TO EXAMINATION REPORT(8-9-2010).pdf

414-MUM-2008-REPLY TO HEARING(21-9-2011).pdf

414-MUM-2008-SPECIFICATION(AMENDED)-(13-1-2011).pdf

414-MUM-2008-SPECIFICATION(AMENDED)-(21-9-2011).pdf

414-MUM-2008-SPECIFICATION(AMENDED)-(8-9-2010).pdf


Patent Number 249372
Indian Patent Application Number 414/MUM/2008
PG Journal Number 42/2011
Publication Date 21-Oct-2011
Grant Date 18-Oct-2011
Date of Filing 28-Feb-2008
Name of Patentee CLARIS LIFESCIENCES LIMITED
Applicant Address CLARIS CORPORATE HEADQUARTERS, NR.PARIMAL CROSSING ELLISBRIDGE, AHMEDABAD-380006,
Inventors:
# Inventor's Name Inventor's Address
1 MAJMUDAR CHETAN S. CLARIS CORPORATE HEADQUARTERS, NR.PARIMAL CROSSING ELLISBRIDGE, AHMEDABAD-380006.
2 BOSE MILINA CLARIS LIFESCIENCES LTD, CLARIS CORPORATE HEADQUARTERS, NR.PARIMAL CROSSING ELLISBRIDGE, AHMEDABAD-380006.
3 SHUTHAR MINESH CLARIS LIFESCIENCES LTD, CLARIS CORPORATE HEADQUARTERS, NR.PARIMAL CROSSING ELLISBRIDGE, AHMEDABAD-380006.
PCT International Classification Number A61K9/12; C07H17/08
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA