FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION:

"STABLE AMPHOTERICIN B MICROEMULSION FOR PARENTERAL
USE"
2. APPLICANT:
(a) NAME: MAC CHEM PRODUCTS INDIA PVT. LTD.
(b) NATIONALITY: Indian Company incorporated under the Indian Companies
ACT, 1956
(c) ADDRESS: 304, Town Centre, Andheri-Kurla Road, Andheri (East),
Mumbai-400059, Maharashtra, India
3. PREAMBLE TO THE DESCRIPTION
The following specification describes the invention.


Technical field of invention:
The present invention relates to stable microemulsion composition comprising Amphotericin B alongwith suitable excipients for intravenous administration, which is capable of providing safe, stable and an efficient formulation obtained by simple and rapid process.
Background and prior art:
Amphotericin B (3-Amino-3, 6-dideoxy-beta-D-mannopyranosyl) oxy) - is a polyene antifungal drug, often used intravenously for systemic fungal infections, originally extracted from Streptomyces nodosus, a filamentous bacterium, in 1955. Two Amphotericins; Amphotericin A and Amphotericin B are known, but only Amphotericin B is used clinically, because it is significantly more active in vivo.
Currently available drugs are plain Amphotericin B, cholesteryl sulfate complex, lipid complex and liposomal formulation. The latter formulations have been developed to improve tolerability for the patient but may show considerably different pharmacokinetic characteristics compared to plain Amphotericin.
Amphotericin B (having brand names Fungilin, Fungizone, Abelcet, AmBisome, Fungisome, Amphocil, Amphotec) is a polyene antifungal drug, often used intravenously for systemic fungal infections. As with other polyene antifungal, Amphotericin B associates with ergosterol, a membrane chemical of fungi, forming a pore that leads to K+ leakage and fungal cell death. However, researchers have found evidence that pore formation is not necessarily linked to cell death. The actual mechanism of action may be more complex and multi-faceted.
Amphotericin B is believed to interact with membrane sterols (ergosterol) to produce an aggregate that forms a transmembrane channel. Intermolecular hydrogen bonding interactions among hydroxyl, carboxyl and amino groups stabilize the channel in its open form, destroying activity and allowing the cytoplasmic contents to leak out. Thus, water may substantially be an essential solvent in preparing all pharmaceutical formulations including anesthetic agents; it is very difficult to formulate lipid-soluble drugs such as Amphotericin B into pharmaceutical preparations containing water as the solvent. In order to solve such problem, lipid soluble drugs, which are generally poorly
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soluble in water, have been developed into such forms as emulsions, micro emulsions, micelles, etc. using a surfactant.
US Patent No: 7053061 describes the parenteral composition of oil-coated-Amphotericin B in structured emulsion form. Invention describes Amphotericin B composition with low toxicity in a structured emulsion of oil-in-water type, in this process Amphotericin B is used as such or is micronized before dispersing in oil phase which is selected from group of vegetable oils such as soyabean oil, sesame oil. Aqueous phase water tonicity modified agent such as glycerin, mannitol and dextrose dissolved in aqueous phase, and emulsifier such as natural phosphatides, dispersed in aqueous phase.
U.S. Patent No: 5364632 describes a pharmaceutical composition being an oil in water type emulsion which comprises an effective amount of a lypophilic drug wherein Amphotericin B was dissolved in methanol (0.8mg/ml) by bath sonication. Phospholipids E-80 was dissolved in chloroform. Both solutions were mixed and filtered through a combined filtering system comprising a fiber glass prefilter and 0.45u regenerated cellulose membrane filter (RC 5) (GF92) for removing pyogenes and aggregates. In Japanese Patent 4173736 discloses a product containing 0.005% to 0.5% Amphotericin B, 0.5% to 25% phospholipids, preferably egg lecithin has been described. The composition has an average particle size diameter of l00nm.
In U.S Patent No. 5389373 describes a process of preparing oil in water (o/w) emulsion of poorly soluble drug Amphotericin B. The process involves dissolving Amphotericin B in aqueous solution of high or low pH, adding the resulting solution or not more then l00ug/ml strength to a performed emulsion, adding to emulsion an amount of acid, base or buffer appropriate to neutralize and to adjust the pH of the product to a desire value. In U.S Patent No: 5534502 disclose that amphotericin B is de-crystallized using an acid and ethanol and then homogenously dispersed in a lipid, following which it is emulsified. In this process it is essential to dissolve Amphotericin B in ethanol; the most preferred quantity of ethanol is 400 to 600 ml/gm of amphotericin. The main weakness of this process is Amphotericin B is not stable in acid pH.
Korean Patent No: KR20030084051 describes the method of producing microemulsion containing Amphotericin B by solubilizing Amphotericin B using vitamin C. Thus intravenous injection including the microemulsion has enhanced stability and safety in human body. The said microemulsion contains 0.1 to 10 parts by weight of Amphotericm B, based on 100 parts by weight of an oil phase. Microemulsion is produce by dissolving
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vitamin C and Amphotericin B in an oil phase at 60 to 80° .C, and vitamin C is dissolved in water phase thereafter, the oil phase where Amphotericin B is dissolved is dispersed in the water phase where vitamin C is dissolved. The oil phase contains one or more organic solvent selected from ethyl oleate, propylene glycol, diethylamide and glycol. Further, Chinese Patent No: CN1850032 (which is a!so published as CN 100372524C) describes a nano particle preparation of amphotericin B (AmB) by using n-butyl poly cynoacrylate as carrier material of AmB. The animal test shows that it can permeate through blood-brain barrier, the measured medicine concentration in brain tissue is higher than that of AmB liposome preparation in clinical application and said nano preparation of antimycotic Amphotericin B is superior to Amphotericin B liposome preparation in therapeutic effect.
An International application number PCT/GB88/00486 (International publication no.WO 88/10116) discloses a process for preparing drug containing emulsion, comprising the steps of: (a) optionally dissolving the drug preferably a polyene antifungal compound such as Amphoteri'cfn B in a suftabfe co-sofvent, (b) mixing the drug or a solution thereof in the co-solvent with an aqueous preparation of a surfactant, (c) removing at least most of any co-solvent which is present, (d) mixing the product of step (c) with oil and, (e) forming an oil-in-water emulsion from the product of step(d). The drug is located in the surfactant layer of the resulting emulsion, which is stable and reduces the toxicity of drug. International Publication Number WO2003/039435 discloses the process of obtaining of compositions containing amphotericin B in its super aggregate form capable of treating infections caused by fungi, parasite and other agents susceptible to this antibiotic. Further, the stable formulation of Amphotericin B in its super- aggregate form in presence of sodium deoxycholate, freeze dried (lyophilized), comprises the formulation for injectable formulation for use in the treatment of enteral, parenteral or topical, with reduced toxicity in comparison to the conventional forms that transmit Amphotericin B. European patent EP0558750B1 discloses a process for stabilizing an amphiphilic drug, comprising a soluble complex of the amphiphilic drug and a phospholipid in an acidified organic solvent and, an organic solvent having a pH less than 4.5 and the phospholipid is a phosphatidylglycerol.
Novel fat emulsion invention, in European Patent EP0598116B1, comprises main ingredient a polyene antifungal antibiotic, a characteristic feature of the present invention resides in limiting a relative proportion of each ingredient of a polyene antifungal
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antibiotic, a simple lipid, phospholipid and water in composition comprising (a), (b), (c) in aqueous vehicle :-
(a) 0.005 %(w/v) of a polyene antifungal based on total weight.
(b) 0.5 to30 %(w/v) of a simple lipid based on total weight.
(c) 0.15 to 2 times (weight ratio)of phospholipid based on simple lipid Or a freeze dried preparation thereof.
Objective of the invention:
The object of the present invention is to provide novel and alternative microemulsion composition comprising Amphotericin B with oil phase in the oil-in-water preparation for intravenous use.
Summary of the invention:
Accordingly, in one aspect, the present inventioii provides an efficient, stable
microemulsion formulation comprising Amphotericin B with pharmaceutical acceptable
excipients such as surfactant, co-surfactant and water for injection suitable for drug
delivery.
Another aspect of the present invention is to provide simple and rapid method of
preparing a microemulsion composition for intravenous injection.
The oily phase for microemulsion formulation is selected based on solubility studies of
drug in various oily phases, stability of oil and its toxicity.
In preferred aspect, the oily phase used is Long-chain monoglycerides such as Glyceryl
monooleate (1UPAC name: 2, 3 dihydroxypropyl (z)-octadec-genoate, syn: Peceol) in the
range of 12-15%, while the surfactant used is synthetic and non-ionic polyethylene glycol
15 hydroxystearate, which is a water soluble solubilizer that was developed for use in
parental formulations with lipophilic drugs and vitamins, in the range of 2-3%, whereas
the co-surfactant used is hydrophilic emulsifier such as polyethylene glycol fatty acid
ester (MYS-40 NIKKOL TM) in the range of 53-57%.
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Detailed Description:
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.
Generally microemulsion system consists of oil, water and appropriate emulsifier can form spontaneously and are therefore thermodynamically stable. In addition, the size of the droplets in such emulsion remains constant and ranges from 10-100nm. The present invention discloses the novel pharmaceutical composition in micro emulsion form, which comprises therapeutically effective amount of Amphotericin B with suitable excipients so as to make the formulation stable.
Therefore, according to a preferred embodiment, the composition of the present invention comprises therapeutically effective amount of Amphotericin B, a surfactant; co-surfactant and water, suitable for intravenous administration.
The microemulsion used in the present invention is characterised by comprising the oily phase, which is Long-chain monoglycerides such as Glyceryl monooleate (IUPAC name: 2, 3 dihydroxypropyl (z)-octadec-genoate, syn: Peceol), preferably in the range of 12-15%.
The microemulsion used in the present invention is characterised by comprising a surfactant such as Solutol HS 15 which is also known as polyethylene glycol 660 hydroxystearate (syn: polyethylene glycol 15 hydroxystearate), preferably in the range of 2-3%.
The microemulsion of the present invention is also characterised by comprising a co-surfactant, the hydrophilic emulsifier such as polyethylene glycol fatty acid ester (Mys-40 NIKKOL TM), preferably present in the range of 53-55%.
In another preferred embodiment, the invention provides a simple process for preparation of the said microemulsion, which comprises the following steps:
(a) adding accurately weighed amount of hydrophilic emulsifier and surfactant to a container;
(b) adding oily phase in above solution and mixing it for some time, making clear solution;
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(c) taking Amphotericin B in another beaker and adding HPLC grade DMF, sufficient to get clear solution;
(d) adding step (c) in step (b) and mixing it for some time under stirring to obtain a clear solution; *
(e) co-evaporating the oil and drug mixture and condensing the DMF volume;
(f) taking out the flask and adding filtrated water sufficient to obtain desired solution and
(g) stirring vigorously and distributing clear solution equivalent to 50 mg of Amphotericin B in lyophillized glass vials and lyophilizer.
The following non-limiting examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.
The Amphotericin B microemulsion of the present invention is presented in Table 1, the best formulations are shown in Table 2.
Example 1: Table 1:
Preparation of Amphotericin B microemulsion using varying concentration of excipients:-

Formula Amphotericin B Peceol
(%w/w) Solutol HS15 (%w/w) Mys-40
(%w/w) Water
1 50mg 12.0% 2.0% 53% 25ml
2 50mg 13.0% 2.2% 54% 25ml
3 50mg 14.0% 2.5% 54% 25ml
4 50mg 14.5% 2.8% 54% 25ml
5 50mg 15.0% 3.0% 55% 25ml
6 50mg 15.0% 2.2% 53% 25ml
7 50mg 13.8% 2.5% 54% 25ml
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Table 2:
Best Formulation:

Formula Amphotericin B Peceol
(%w/w) SolutolHS15
(%w/w) Mys-40
(%w/w) Water
1 50mg 14.5% 2.7% 55% 25ml
2 50mg 14.5% 2.5% 54% 25ml
3 50mg 14.0% 2.2% 53% 25ml

■ nd
Dated this 2nd day of September 2008


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Dr. P. Aruna Sree Agent for the Applicant