FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patent Rules, 2003
PROVISIONAL / COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. Title of the invention
"PREPARATION OF AMPHOTERICIN B LIPOSOMES BY SUPERCRITICAL FLUID TECHNOLOGY"

2. APPLICANT(s)
(a) NAME
(b) NATIONALITY
(c) ADDRESS

Dr. Misra Ambikanandan
Indian
Pharmacy Department, Faculty of Tech. & Engg., The Maharaja
Sayajirao University of Baroda, P.B.No 51,Kalabhavan, Vadodara-
390001, India

(a) NAME
(b) NATIONALITY
(c) ADDRESS

Mr. Parmar Nirav
Indian
Pharmacy Department, Faculty of Tech. & Engg., The Maharaja
Sayajirao University of Baroda, P.B.No 51,Kalabhavan, Vadodara-
390001, India

(a) NAME
(b) NATIONALITY
(c) ADDRESS

Mr. Naik Sachin
Indian
Pharmacy Department, Faculty of Tech. & Engg., The Maharaja
Sayajirao University of Baroda, P.B.No 51,Kalabhavan, Vadodara-
390001, India

(a) NAME
(b) NATIONALITY
(c) ADDRESS

Mr. Gaurang Patel
Indian
Pharmacy Department, Faculty of Tech. & Engg., The Maharaja
Sayajirao University of Baroda, P.B.No 51,Kalabhavan, Vadodara-
390001, India

3. PREAMBLE TO THE DESCRITION

4. DESCRIPTION (Description shall start from next page)
PROVISIONAL
The following specification describes invention

COMPLETE
The following specification particularly
describes the invention and the manner in
which it is to be performed

5. CLAIMS (not applicable for provisional specification. Claims should start with the preamble - "i/we claim" on separate page)
6. DATE AND SIGNATURE (to be given on the last page of specification)
7.ABSTRACT OF THE INVENTION (to be given along with complete specification on the separate page)

NOTE:
*Repeat boxes in case of more than one entry
* To be signed by the applicant(s) or the authorized registered patent agent
*Name of the applicant should be given in fuul, family name in beginning
*complete address of the applicant should be given starting with postal index no./code, state and country
'Strike out the column which is/are not applicable

PREPARATION OF AMPHOTERICIN B LIPOSOMES BY SUPERCRITICAL FLUID TECHNOLOGY
Liposome is a biphasic structure in which aqueous phase is enclosed by membrane composed of Phospholipid molecules. Hydrophilic drug can be entrapped in aqueous phase while hydrophobic drug in lipid phase.
Liposomes having multiples of bilayers are known as Multi lamellar vesicles (MLVs). Liposomes bearing single bilayer are known as unilamellar vesicles, it can be Small unilamellar vesicles (SUVs) or Large unilamellar vesicles (LUV). Newer concept is proliposomes.
Amphotericin B is a macrocyclic, polyene, antifungal antibiotic produced from a strain of Streptomyces nodosus. Amphotericin B has a molecular formula of C47H73NO17 and a molecular weight of 924.09. It acts by binding to the sterol component of a cell membrane leading to alterations in cell permeability and cell death. While Amphotericin B has a higher affinity for the ergosterol component of the fungal cell membrane, it can also bind to the cholesterol component of the mammalian cell leading to cytotoxicity.The liposomal preparation of Amphotericin B (LAB), has been shown to penetrate the cell wall of both extracellular and intracellular forms of susceptible fungi. LAB has shown in vitro activity comparable to Amphotericin B against the following organisms: Aspergillus species (A. fumigatus, A. flavus), Candida species (C. albicans, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis), Cryptococcus neoformans, and Blastomyces dermatitidis. However, standardized techniques for susceptibility testing of antifungal agents have not been established and results of such studies do not necessarily correlate with clinical outcome. LAB can overcome the side effects of Amphotericin B deoxycholate (IV Solution of Amp B) like Nephrotoxicity, Hepatotoxycity etc.
Liposomes have distinct advantage over conventional dosage forms because the particles act as drug containing reservoirs. Modification of particle composition or surface can adjust the affinity for target site and/or drug release rate, and slowing the drug release rate may reduce the toxicity of the drug. These drug carriers may play an increasingly important role in drug delivery.
There are some methods available for the preparation of Liposomes.
Thin film hydration method, Reverse phase evaporation, Double emulsification, pH
gradient method, Solvent dispersion (this method includes Ethanol injection method and
ether injection method.), Detergent removal method
All this methods give MLVs as a final product. To convert MLVs in LUVs/SUVs techniques
like sonication, extrusion etc are used.

Both kinds of the drugs, hydrophilic/hydrophobic, can be incorporatediin the Liposomes. Size can be adjusted by mean of sonication or extrusion. pH sensitive drug can be incorporated by pH gradient method. All of the above mentioned methods bear some problems:
• Use of sonication or extrusion can reduce entrapment of drug. There is no process which involves only single operation.
• Dry product is required for stability, which can not be obtained in any of the method so freeze drying or spray drying has to be carried out.
• Retention of organic solvents.
• Sterile product is not obtained by any method.
• Ultimately all this factors lead to high cost of product.
• Some methods are hard to scale up.
• All these methods are time consuming.
In summery there exists a need for a method which can result in Nano sized, dried, and sterile product. These requirements can be satisfied by a novel approach called "Supercritical Fluid Technology". The term Supercritical Fluid means a gas in the stage above to its critical temperature and pressure. Liposomes can be obtained by precipitation in the supercritical fluid. Among all the Supercritical Fluids Carbon Dioxide is most widely used due to its favorable critical parameters (Tc 31.1 °C, Pc 73.8 bar), low cost, low toxicity, and nonflammability and environmentally benign nature.
There are so many methods in "Supercritical Fluid Technology" for particle production:
• Rapid Expansion of Supercritical Solution (RESS): Precipitation from solution composed of SCF and solute.
• Antisolvent methods
Gas Antisolvent (GAS),
Supercritical Antisolvent (SAS),
Aerosol Solvent Extraction System (ASES)
Solution Enhanced Dispersion by SCF (SEDS)
All these methods use SC CO2 as an antisolvent along with organic solvent.
• Precipitation from Gas Saturated Solution (PGSS): Spraying of the SCF dissolved
molten solution through nozzle.

The method involves precipitations of lipids in dry powder form of liposomes from supercritical fluid and organic solvents. This dried form of liposome can be reconstituted in the aqueous media.
The product obtained is sterile dried Powder of Liposomes. Than can be reconstituted in aqueous media.or insitu liposome formation can be there for pulmonary route. Process Parameters:
• Flow Rate of SCF
• System Pressure
• Sample Spray Rate
• Temperature of different zones
• Concentration of solute Formulation Parameters:
• Choice of lipid
• Choice of organic solvents
• AMB: Lipid: Cholesterol

Signature:
1) Dr. Misra Ambikanandan
2) Mr. Nirav Parmar

3) Mr. Sachin Naik
4) Mr. Gaurang Patel