Title of Invention	LIPIDIC NANOPARTICLES BASED COMPOSITION AND METHOD OF FORMULATION AND USE THEREOF
Abstract	The oral solid dosage form of solid lipid nanoparticles (SLN). loaded with Amphotericin B having LD50 of atleast 200mg/kg in mice and process for making the same have been described. The method used for making present composion is a modified nanoprecipitaton process. It essentially requires dissolving Amphotericin B in organic solvent and dispersing Amphotericin B solution, lipid and emulsifler in an aqueous phase. The process of present invention is simple, cost effective and gives a stable product suitable for oral administration. Amphotericin B SLN compositions prepared by the process of the present invention may be administered orally to human beings for the treatment of fungal infections and second line treatment of leishmaniasis with substantially equivalent or greater efficacy and low drug toxicity as compared to the conventional parenteral composition containing Amphotericin B and sodium desoxycholate.

Title of Invention

LIPIDIC NANOPARTICLES BASED COMPOSITION AND METHOD OF FORMULATION AND USE THEREOF

Abstract

The oral solid dosage form of solid lipid nanoparticles (SLN). loaded with Amphotericin B having LD50 of atleast 200mg/kg in mice and process for making the same have been described. The method used for making present composion is a modified nanoprecipitaton process. It essentially requires dissolving Amphotericin B in organic solvent and dispersing Amphotericin B solution, lipid and emulsifler in an aqueous phase. The process of present invention is simple, cost effective and gives a stable product suitable for oral administration. Amphotericin B SLN compositions prepared by the process of the present invention may be administered orally to human beings for the treatment of fungal infections and second line treatment of leishmaniasis with substantially equivalent or greater efficacy and low drug toxicity as compared to the conventional parenteral composition containing Amphotericin B and sodium desoxycholate.

Full Text	FORM 2 (39cf1970) & The Fteterts Rules, 2003 PROVISIONAL/COMPLETE SPECIFICATION [See section 10 and rule 13] 1. TITLE OF THE INVENTION I Lipidic nanoparticles based composition and method of formulation \| and use thereof 2. APPLICANTS I. Applicant 1: Name: Vandana Bharat Patravale. ' Nationality: Indian Address: C-15, Divine Light Co-operative Housing Society, 137/139, M.V. Road, Andheri (E), Mumbai-400093 II. Applicant 2: Name: Pratikkumar Anilbhai Patel Nationality: Indian Address: 405-Tulsi Pushpa, Goddev Naka;'Bhayandar(E), Mumbai-401105 3. PREAMBLE TO THE DESCRIPTION: COMPLETE (The following specification particularly describes the invention and the manner in which it is to be performed) 4. DESCRIPTION (Description shall start from new page): 5. CLAIMS (not applicable for provisional specification. Claims should start with preamble-"l/We claim" on separate page) 6. DATE AND SIGNATURE (to be given at the end of last page of specification) 7. ABSTRACT OF THE INVENTION (to be given along with complete specification on a separate page) Lipidic nanoparticles based composition and method of formulation and use thereof Prior Arts US Patent 6699499 describes various amphiphilic materials and liposome formulations of amphiphilic class. US Patent 5444052 describes a formulation of Amphotericin B for the treatment of fungal infections by intravenous administration. Here AmB was used in a combination with various glycerol ethers. The patent also states that glycerol ether acts synergistically to reduce the minimum inhibitory concentration of amphotericin B. The combination is particularly effective against Cryptococcus and Candida species. US Patent 6770290 describes a novel composition and method for solubilizing amphotericin B in a small amount of organic solvent for use in formulation of liposomes. The phospholipids used in this process have been acidified with organic solvent. Further the solution of amphotericin B was added to the acidified phosphatidylycerol which results in a soluble complex. Finally the liposome composition incorporating the soluble complex was hydrated, the final pH of the hydrating aqueous buffer being carefully controlled. The Amphotericin B liposomes formed were reported to have markedly reduced.toxicity. The patent also describes the in-vitro and iv- vivo characterization of formed liposomes. US Patent 5194266 describes a formulation containing amphotericin B and cholesterol sulfate, in a molar ratio of between about 1:1 to 2:1. The formulation was freeze dried to get a dry particles for reconstitution, having particle size predominantly between about 50-250 run. Further the formulation was described to be significantly less toxic and more effective in treating fungal infection than prior amphotericin B formulations. US Patent 3965090 describes a new form of amphotericin B comprising of a complex of amphotericin B and succinic acid. This new form of amphotericin B was reported to be more active than amphotericin B and exhibited the advantage of being soluble in acidic or. alkaline solutions. US Patent 4054734 describes new forms of amphotericin B comprising of an amphotericin B, an acid anion, and calcium cation. These new forms of amphotericin B have been reported retain the activity of Amphotericin B and additively displayed the advantage of being soluble in acidic or alkaline solutions. Other References 1. Cohen, J., Antifungal Chemotherapy, Lancet, (J 982) II: 532-537. 2. Graybill, J.R. Craven, P.C., Antifungal Agencies and System Mycosis: Activity and Theraputic Use, Drugs, (1983) 25:41-62. 3. Graybill, Jr. R. et al., Treatment of Murine Cryptococcoses Liposomal Associated Amphotericin B., Journal of Infectious Disease (1983) 145: 748-752. 4. Gregoriadis, G. A. (Ed.), Liposome Technology, CRC Press: Boca Raton, CRC Press: Boca Raton, Florida (1983). 5. Lopez-Berestein, G., Treatment and Prophylaxis in Disseminated Infection Due to Candida Albicans, with Liposome Encapsulated Amphotericin B. Journal of Infectious Disease (1983)147:939-945. 6. Lopez-Berestein Treatment of Hepatosplenic Candidiasis with Liposomal-Amphotericin B, Journal of Clinical Oncology (1987) 5: 310-317. 7. Medoff, G. Kabayashi, G, Strategies and.Treatment of System Fungal Infections, N. Eng. J. Med., (1980) 302:145-155. 8. Mehta, R., Amphotericin B is Toxic to Fungal Cells but not to: Mammalian cells, Biochimica ET Biophysica Acta,(1982) 770:230-234. 9. Antileishmanial Activity of Amphotericin and Other Antifungal Agents Entrapped in Liposomes, J. Antimicrobial Chemotherapy (1981) 8:371-381. 10. Hiroshi Fukui,Tomohiro Koike, Akira Saheki, Satoru Sonoke and Junzo Seki., A novel delivery system for amphotericin B with lipid nano-sphere (LNS®)., International Journal of Pharmaceutics (2003) 265(1-2):37-45. 11. Wasan K. M., Brazeau G. A., Keyhani Al1 Role of liposome composition and temperature in distribution of amphotericin B in serum lipoproteins. Antimicrobial Agents Chemotherapy. (1993) 37:246-250. 12. Wasan K. M., Morton R. E., Rosenblum M. G., Lopez-Berestein G. Decreased toxicity of liposomal amphotericin B due to association of amphotericin B with high density lipoproteins: role of lipid transfer protein. Journal of Pharmaceutical Sciences (1994) 83(7):1006-1010. 13. Wasan, K. M. and Lopez-Berestein G., Modification of Amphotericin B's Therapeutic index by Increasing Its Association with Serum High-Density Lipoproteins. Annals of the New York Academy of Sciences, (1994) 730: 93-106. 14. David S. Perlin, Rosaria Santangelo, Padmaja Paderu, Guillaume Delmas, Zi-Wei Chen,Raphae, Mannino,and Leila Zarif. "Efficacy of Oral Cochleate-Amphotericin B in a Mouse Model of Systemic Candidiasis". Antimicrobial. Agents and Chemotherapy. (2004) 44 (9): 2356-2360. 15. Perlin D. S., Delmas G., Park S., Chen Z' W., Tan F., Kashiwazaki R. and Zarif, L.„ "Efficacy of Orally Delivered Cochleates Containing Amphotericin B in a Murine Model of Aspergillosis"., Antimicrobial Agents and Chemotherapy, (2002) 46(8):704-2707. 16. Steigmann F., Sait M. Z., Rajagopal K. G. and Villa F.,"Oral Amphotericin B in the Treatment of Ulcerative Intestinal Diseases'. Digestive: Diseases, (1970) 15(11):993-1002. 17. Kishor M. Wasan, Verica Risovic, Kristina Sachs-Barrable and Michael Boyd.," Potential Mechanisms by Which Peceol® Increases the Gastrointestinal Absorption of Amphotericin B". Drug Development and Industrial:Pharmacy, (2004).30(7):767-774. 18. Amarji, Basant; Ajazuddin; Raghuwanshi, Dharmendra; Vyas, S.P.; Kanaujia, Parijat.,Lipid Nano Spheres (LNSs) for Enhanced Oral Bioavailability of Amphotericin B: Development and Characterization, Journal of Biomedical Nanotechnology,(2007) 3(3):264-269. Field of the Invention The present invention relates to a process and composition of Solid Lipid Nanoparticles (SLNs) containing Amphotericin B for oral route of administration. In addition, the SLNs could also be used for parenteral, topical and mucosal route. Background of the Invention The rational for the design of novel formulation of the AmB is the reduction of its nephrotoxicity by lowering the serum concentrations of free drug and delivering the drug at therapeutic level at a site of action. Numerous strategies have been attempted for achieving this goal. The colloidal and particulate carrier systems,-'such as liposomes or nanoparticles, exhibit controlled drug release but are rapidly removed from the circulation by cells of the reticulo endothelial phagocyte system by Kupffer cells, enhancing the risk of hepatic disorders. This gives a very low serum half-life of AmB, and results in low bioavailability and an uneven distribution throughout the body. However, this phenomenon may be used for the target design of drug delivery system for indication such as. visceral leishmaniasis, where the parasite resides exclusively within phagocytes. The literature review shows that lipid-based formulations significantly reduced mortality risk by an estimated 28%, compared with conventional AmB. AmB lipid complex and liposomal AmB significantly reduced the risk of renal dysfunction and associated side-effects. Various formulation strategies have been tried, out to treat systemic fungal infections via parenteral administration of AmB. G.S. Kwon et.al. (1998) solubilized AmB in micelles of poly (ethylene oxide)-block-po!y ((3-benzyl-L-aspartate) (PEO-PBLA). The antifungal activity of the AmB-loaded PEO-block-PBLA micelles was found to be four to eight times higher than Fungizone® in terms of minimal inhibitory concentrations (MICs), while drug free PEO-PBLA micelle had no antifungal activity for concentrations up to 200 p,g/ ml. The underlying theory for the increase in antifungal activity of AmB-loaded PEO-PBLA micelles is yet unclear, but could be related to a stabilizing effect of the polymeric micelles against auto-oxidation of the AmB heptanes moiety or alternatively, an enhancement in membrane perturbation of fungal cells. Another approach to enhance drug performance and reduce toxicity is the conjugation of drug with polymeric carrier. Conjugation of an insoluble drug such as AmB to a water soluble biodegradable polymer [Arabinogalactan (AG)] was found to increase the water solubility of the drug, drug circulation time, and accumulation.in the diseased tissue, resulting in an improved therapeutic effect and reduced toxicity. J. Domb et al (1999) synthesized AmB-AG conjugates tested for efficacy against common pathogenic fungi and for toxicity in mice. The conjugate containing 20% by weight of AmB was found to be soluble in water (>100 mg/ml), was. as effective as the parent drug, and possessed a 20-fold-lower toxicity. In a direct comparison between AmB-AG conjugate and AmBisome, the conjugate was superior in safety and efficacy against fungal infections and Leishmania amazonensis. and Leishmania donovani in mice. Methoxy poly ethylene Glycol (mPEG) has also been reported as a carrier for conjugation. Lipid Nano-spheres (LNS®) are small-particle lipid emulsion, and have been reported to exhibit lesser uptake by the liver than the conventional lipid emulsion. Therefore, low-dose therapeutic system with a lipid emulsion for AmB, a potent antifungal drug, was studied by Junzo Seki et. al. (2003). The formulated LNS-AmB was a homogeneous emulsion with the mean particle diameter ranging from 25 to 50 nm. Due to relatively less uptake by the liver, i.v administration of LNS loaded with the AmB yielded higher plasma concentrations of the drug than the Fungizone® when evaluated in mice, rats, dogs, and monkeys. In in vitro and in vivo studies, both LNS-AmB and Fungizone, demonstrated similar antifungal activity. Inspite of the higher plasma AmB concentrations achieved by LNS-AmB, in comparison with Fungizone®, LNS-AmB did not cause significant hemolysis, vomiting and toxicity in dog. Therefore, LNS-AmB, allowed the treatment of systemic fungal infections at low doses without the severe nephrotoxicity of Fungizone®. The topical drug delivery of AmB was reported by R. Cortesi et al. (2003) using two systems namely microemulsions and monoglyceride-water system. Both these system were studied for vaginal and skin mycosis. Results indicate that systems can be employed as alternative formulations for the topical delivery of AmB. The unique physicochemical properties (low solubility and high affinity for biological membranes) have made AmB as one of the. first drug of choice for formulation into liposomes. It was hypothesized that liposomes could provide a safer alternative to conventional deoxycholate-solubilized AmB by attenuating its serious renal toxicity while maintaining its fungicidal activity. In animal models and in initial clinical studies, the liposomes formulations appeared to increase the safety of AmB, while providing efficacy against systemic fungal infections. / Lawrence et al. shown that formulation of AmB using small unilamellar liposomes (SUVs) consisting of phospholipids and cholesterol also showed good tolerability in cancer patients with fungal infections. While neither of these early formulations progressed to commercial development, they served as a basis from which formulation optimization took place in divergent directions, leading to the development of the three different, yet effective lipid antifungal products currently available namely AMBISOME ®, ABELCET ® and AMPHOTEC®. C.R Middaugh et al have prepared the nanoparticle dispersion of AmB using oppositely charged polymers namely poly ethyleneimine (PEI) and dextran sulphate (DS), and zinc sulphate as stabilizers has been developed. The MIC of this formulation Was found to be 0.35ng per ml. Nanoparticles showed distinct advantages over liposomes, such as long shelf-life at room temperature, ability to withstand autoclaving for sterilization improved oral bioavailability and low production costs. The currently available marketed products of AmB are lipid based formulations-viz. AmBisome, Amphocil and Amphotec. All these form, and Arulation differ with respect to reticulo-endothelial clearance, volume of distribution (Vd), Cmaxea Under Curve (AUC) of AmB,. When compared with Fungizone® ( a conventional formulation), a liposomal AmB was found to have lower clearance, and consequently a very high Cmax and AUc which is of the major importance from clinical point of view. The limitation of AmB parenteral therapy is the need of hospitalization often making'the treatment expensive and inconvenient to the patients at large. So to overcome the cost of the therapy and for better patient compliance, many strategies have been developed to formulate the oral formulation of AmB such as oral rianosuspension of AmB Croft S.L et al(2003) and Villa F et al(1970). Various lipid and mixed-micelle formulations Eugene Choo et al(2003),Michael Boyd et al(2004, Oral Cochleate-AmB Leila Zarifet al(2000,2002),oral lipid based formulations wasan et. Al (2009), oral Amphotericin B in the management of cutaneous and mucosal candidiasis San Francisco et. Al (1964) Summary of the Invention The present invention features method for producing Solid lipid nanoparticles containing amphiphilic drugs. The objective of the present invention is to make solid lipid nanoparticles of amphophilic drugs like amphotericin B. Thus the present investigation was focused on developing an oral dried nanoparticle formulation, of AmB which will protect the drug in acidic media of Gl tract, prevent the photo degradation of drug, enhance the oral absorption of the drug and capable of targeting the drug to the liver, spleen, lungs etc. The aim was to tackle the aforementioned drawbacks at the same time exploiting advantages of lipid based nanocarriers namely improved absorption and decreased nephrotoxicity of the encapsulated moiety and no traces of organic solvents i.e. highly purified nanoparticles. Detailed Description of the Invention This invention is directed towards the development of dry solid lipid nanoparticulates compositions for the oral delivery of lipophilic substances, and the methods for preparing such compositions. The dry solid lipid mixtures in the present invention comprises of lipophilic substance, a solid lipid or mixture of solid lipids, one or more phospholipids and surfactant or mixture of surfactants. Advantageously, the dry lipids mixtures of the present invention may further comprise a cryoprotectant. Any of a wide variety of lipophilic substances can be utilized in these mixtures. Examples include lipophilic drugs, vitamins, and hormones. The other lipophilic substances include steroids, steroid antagonists, non-steroidal anti-inflammatory agents, antifungal agents, antibacterial agents, antiviral agents, anticancer agents, antihypertensives, antioxidants, anti-epileptic agents and antidepressants among many others. Additional examples of lipophilic drugs with very poor water solubility and low oral bioavailability which could benefit from oral dosage forms are the neurohormone melatonin, the antifungal agent amphotericin B, the anticancer drug etoposide, as well as tamoxifen and its analogs. More specific compounds include cannabinoids, as exemplified by dexahabinol, and vitamins, enzymes or coenzymes, as exemplified by COQ1o- The loading of the lipophilic substance in the final dry solid lipid mixture is in the range of about 10-40% of the total solid weight of the mixture. Examples of solid lipids suitable for the preparation of dry lipid mixtures of the present invention includes triglycerides composed of natural, even-numbered and unbranched fatty acids with chain lengths in the C10-C18 range, or microcrystalline glycerol triesters of saturated, even-numbered and unbranched fatty acids of natural origin such as tricaprin, trilaurin, trimyristin, tripalmitifi, and tristearin. The content of solid triglycerides in the final dry lipid mixture is in the range of about 10-15 % of the total solid weight of the mixture. The preferred phospholipids which constitute the third component of the dry lipid mixture in the present invention are natural phospholipids, such as: soybean lecithin, egg lecithin, phosphatidylglycerol, phosphatidylinositol, "'phosphatidylethariolamine, phosphatidic acid, sphingomyelin, diphosphatidylglycerol, phdsphatidylserine, phosphatidylcholine, cardiolipin, etc.; synthetic phospholipids, such as dimyristoylphosphatidylcholine, dimyristoyl-phosphatidyl glycerol, distearoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, etc.; and hydrogenated or 30 partially hydrogenated lecithins and phospholipids. The fourth component of the mixture is surfactant or mixture of surfactants. These substances stabilized the system by localising at the interphase of heterogeneous system. The examples of these include Tween 80, Span 80, Transciitol P, Solutol HS 15 etc.. The dry solid lipid mixtures may further contain a cryoprotectant material as known in the art, such as a sugar or an amino compound, to protect the formulation during freeze-drying or spray-drying processes. Preferred cryoprotectants include glucose, sucrose, lactose, maltose, and trehalose; polysaccharides, such as dextrose, dextrins, and cyclodextrins; and nonriatural polymers, such as polyvinylpyrrolidone (PVP). Other cryoprotectants may also be used, including amino acids, as disclosed in U.S. Pat. No. 5,472,706. Examples 1. AmB (50-100 mg) was dissolved in DMSO (0.6-0.8 g), lipid (0.2-0.5 g), surfactant (0.2-0.6 g) and Epikuron 200 (20-40 % of solid lipid) was mixed with AmB solution and heated at 50-60°C. Separately, double distilled (filtered through 0.45um nylon membrane) water was heated to 50-60°C. This was added to the molten lipid-surfactant mixture under stirring (3000-4500 rpm) for 5-10 min and stirring was continued further till it reaches the room . temperature to form SLN dispersion. The resultant SLN dispersion was subjected to probe sonication (Dakshine Probe Sonicator) for 5-10 min with one cycle of 15 sec ON and 15 sec OFF in ice bath. 2. DMSO from SLN dispersion was removed by dialysis. SLN dispersion, 50 mL was filled in dialysis bag having molecular weight cut off of 10,000-20,000 Da (Hi-Media, Mumbai) and sealed with medi-clip. It was then dialyzed for 6-8 hours in 5 lit purified water. The water was replaced with fresh purified water every 2-3 h. Complete removal of DMSO from, formulation was confirmed by Gas Chromatography (GC) by measuring DMSO content of SLN dispersion every hr. The particle size and P. I. of the SLN dispersion decreased significantly after dialysis. The particle size and P. I. of SLN were determined by photon correlation spectroscopy (PCS) using Beckman N4 Plus submicron particle size analyzer was found to be 200.1-250.87 ± 10.1 nm and P.I was 0.24. . 3. Freeze drying of SLN dispersion as prepared by the above mentioned method was performed using Ref-Vac (Ref-Vac Consulting, India). Cryoprotectants with varying concentration were used for freeze drying (Table 1). The freeze-drying technology employed was freezing at -4G°C for 24 h, primary drying at 0°C for 5 h followed by 10°C for 2.5 h and 15°C for 2 h and secondary drying at 25°C for 2.5 h. The chamber pressure was maintained at 20 Pa and the temperature of the cold trap was maintained at -50°C during the entire process. Freeze-drying resulted in easily redispersible fine powder. The particle size of the SLN dispersion was determined by PCS prior to lyophilization and after the reconstitution of the lyophilized powders with double distilled water. The mean particle size of the optimized formulation prior to lyophilization was found to be 181.6 nm and that of the reconstituted dispersion was 235.6 nm. Table 1 Cryoprotectants Used For Freeze Drying Cryoprotectant (%w/w) Cryoprotectant (% w/w) Mannitol 10-100. Trehalose 10-100 Mannitol + Trehalose 5 + 5 ■ Mannitol + Trehalose 25 + 25 Arginine 5-10 Lysine 5-10 Lactose 250 Neusiline 1-5 Gelatin 1-10 Fructose 5-20 4. Spray Drying of SLN dispersion was performed using a laboratory scale spray dryer (JISL LSD-48, JISL, India). Lactose (5-10 % w/v) was used as anticaking agent. The solution was sprayed through a peristaltic pump with controlled nozzle into the main chamber (500 mrn x 215 mm). The feeding flow rate was controlled by the pump rotation speed. The aspirator rate was 44 % and the compressor air pressure was 2 kg/cm2. Inlet air temperature was varied between 80 °C and 120 °C whereas feed flow rate was varied between 5 and 15 % of total content. Process yield for these batches was calculated as a relationship between total solids content in the resulting powder and total solids content in the feed. The mean particle size prior to Spray Drying was found to be 181.6 nm and that of the reconstituted dispersion was 593.4 nm i.e. a significant change in mean particle size was obtained after Spray Drying. 5. Dried SLN, (100 mg) was dispersed in 250 mL of medium (each of pH 1.2, 4 and 7.4) in USP type II dissolution apparatus. At regular time intervals; 1 mL of the sample was withdrawn and replaced with fresh buffer. The aliquots were analyzed for drug content,. particle size and P. I. Similar study was performed with plain drug. It was observed that the degradation behavior of plain AmB (solubilized in DMSO: methanol, 1:9) was in accordance with the reports. However, encapsulation of AmB in SLN protected it to some extent. At pH 1.2, SLN had pronounced protective effect, whereas, at. the same pH the plain.drug exhibited a greater drug degradation. 6. Single dose acute toxicity study was.performed, to evaluate the safety of the developed formulation (Freeze dried SLN)- The acute oral toxicity of the formulations was determined as LD50 as per the Organization for Economic Co-operation and Development (OECD) 425 guidelines. The results indicated that animals treated at single dose levels with the developed formulation in all the groups exhibited normal activity and behavioral pattern without any signs of tremors, convulsions, salivations, diarrhea and lethargy. All the animals from different groups exhibited normal food intake and body weight gain during the entire study. The oral LD50 of developed formulation was found to be >2000mg/kg of body weight. 7. Adult male Wistar rats (200-250 g, 10-12 weeks) were used in the pharmacokinetic study Following an overnight fasting (12 to 16 h), the rats were divided into four groups and received a single dose of 200 mg/kg by oral route of the following formulations ■ AmB, 50 mg dissolved in 0.6 mL DMSO (reference oral solution) ■ 1.45 g (~ 50 mg of AmB) of GDL SLN dispersed in 4 mL double distilled water ■ 1.45 g (~ 50 mg of AmB) of Softisan SLN dispersed in 4 mL double distilled water The serum concentration of the formulation was developed SLN formulation was found to be the 124.93 ± 13.61 ng/mL. The half-life of the developed SLN was significantly greater than that of plain drug, indicating the controlled release of AmB from developed formulation. Overall pharmacokinetic results suggest the superiority of developed SLN. This is indeed justified by the in vitro development studies, where the developed SLN confirmed greater protection to the drug against pH degradation as compared to the plain drug. Claims We claim our invention as: 1. A method of making nanoparticles of a said therapeutic agent of bioactive molecules and composition thereof. 2. The said method of claim 1 comprises of: a. Dissolving the said therapeutic agent of bioactive molecules in a first liquid component. b. Adding lipids to a solution of claimed 2(a) containing the said therapeutic agent c. Heating the mixture obtained in claim 2(a) and 2(b) to get lipid melt. d. Add lipid melt as described in claimed 2(c) to a aqueous phase under mechanical shear to achieve a dispersion comprising a continuous phase and a dispersed phase in which the continuous phase is aqueous phase of the dispersion and the dispersed phase comprises lipid globules of the therapeutic agent dissolved in the first liquid component. e. First liquid of claim 2 (a) was removed from dispersion of claimed 1 (d) by a . suitable method. f. Drying the dispersion obtained in step 2 (e) by suitable processes after adding ' suitable excipients. 3. The method as claimed in claim 2(a), wherein the first liquid component is a polar solvent. 4. The polar solvent as described in claim 3 includes DMSO/methanol/DMF/acetone/IPA/THF/surfactants arid surfactants solutions. 5. The said therapeutic agent of bioactive molecules as described in claim 2(a) is drugs, vitamins, and hormones. 6. The said therapeutic agent of bioactive molecules as described in claim 5 is natural, synthetic and semisyntheic. antibiotics steroids, steroid antagonists, non-steroidal antiinflammatory agents, antifungal agents, antibacterial agents, antiviral agents, anticancer agents, anti-hypertensives, antioxidants, anti-epileptic agents and antidepressants among many others. Additional examples of lipophilic drugs with very poor water solubility and low oral bioavailability which could benefit. from oral dosage forms are the neurohormone melatonin, the antifungal agent amphotericin B, the anticancer drug etoposide, as well as tamoxifen and its analogs. More specific compounds include cannabinoids, as exemplified by dexanabinol, vitamins, enzymes or. coenzymes, as exemplified by CoQio. 7. The vitamins described in claim 5 are Vitamin A, Vitamin D, Vitamin E, and Vitamin K. 8. The method as claimed in claim 2, wherein the lipid used is a natural or synthetic or semi-synthetic lipids are solid lipid, liquid lipid, semi-solid lipid. 9. The formulation as claimed in. 1 and 2(b) wherein the nanoparticles comprise one of the lipids as phospholipids. , 10. The phospholipids as described in claim 9. belong to a group of phosphomonoglyceride, phosphatidic acid and sphingolipid. 11. The types of phospholipids as described in claim 10 in which the phospholipids is selected from phosphatidylcholine, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidylserine, diphosphatidylglycerol, .. phosphatidic acid dimyristoylphosphatidylcholine dimyristoylphosphatidylglycerol and mixtures thereof. 12. A formulation as claimed in Claim 2(b), in which the content of phospholipids in the final dry solid lipid mixture is in the range of about 2-40% of the total solid weight of the other lipids , more preferably about 5-30%, and still more preferably about 10-20% of total solid weight of other lipids. 13. The method as claimed in claim 2, wherein the aqueous phase is water, aqueous surfactant solution, preservatives solution. 14. The preservative as described in claim 13 are citric acid, BHT, BHA, methyl parabens and their salts, propyl parabens and their salts, t-buty! ammonium salts, Na-EDTA. 15. The surfactant solution as described in claim 13 are alkylbenzene sulfonates, polyethoxylated nonionics, fatty amine salts and quaternary ammoniums. 16. The dispersion as described in claim 2(d) is an emulsion, nanostructured lipid carrier or solid lipid nanoparticles. 17. The type of emulsion as described in claim 16 is a nanoemulsions, microemulsion, and macro emulsion. 18. The continuous phase as described in claim 2'(d) is an aqueous phase. 19. The dispersed phase comprises of lipid globules as described in claim 2 (d) are globules having a diameter between 200 and 400 nm. 20. The suitable method as described in claim 2(e) is an ultrafiltration, dialysis, and electrodialysis. 21. The Drying of dispersion by suitable method as described in claim 2(f) is a freeze drying, spray drying, flash evaporation and vacuum drying. . 22. The suitable excipients as described in claim 2(f) are cryoprotectants. 23. The nanoparticle system of claim 1, wherein said system is in the form of a solution, suspension, gel, paste, colloidal dispersion, tablet, capsule., or oral controlled release substance. 24. A method for treating fungal infection and leishmaniasis comprising administering a solid lipid nanoparticle using pharmaceutically acceptable carrier to a patient in need. 25. The method of claim 24, wherein administration is oral, parenteral, topical, mucosal sublingual, or buccal.

Full Text

FORM 2
(39cf1970)
&
The Fteterts Rules, 2003
PROVISIONAL/COMPLETE SPECIFICATION
[See section 10 and rule 13]
1. TITLE OF THE INVENTION I Lipidic nanoparticles based composition and method of formulation
| and use thereof
2. APPLICANTS
I. Applicant 1:
Name: Vandana Bharat Patravale.
' Nationality: Indian Address: C-15, Divine Light Co-operative Housing Society, 137/139, M.V. Road, Andheri (E), Mumbai-400093 II. Applicant 2:
Name: Pratikkumar Anilbhai Patel
Nationality: Indian
Address: 405-Tulsi Pushpa, Goddev Naka;'Bhayandar(E), Mumbai-401105
3. PREAMBLE TO THE DESCRIPTION:
COMPLETE (The following specification
particularly describes the invention and the
manner in which it is to be performed)
4. DESCRIPTION (Description shall start from new page):
5. CLAIMS (not applicable for provisional specification. Claims should start with preamble-"l/We claim" on separate page)
6. DATE AND SIGNATURE (to be given at the end of last page of specification)
7. ABSTRACT OF THE INVENTION (to be given along with complete specification on a separate page)

Lipidic nanoparticles based composition and method of formulation and use thereof
Prior Arts
US Patent 6699499 describes various amphiphilic materials and liposome formulations of
amphiphilic class.
US Patent 5444052 describes a formulation of Amphotericin B for the treatment of fungal
infections by intravenous administration. Here AmB was used in a combination with various
glycerol ethers. The patent also states that glycerol ether acts synergistically to reduce the
minimum inhibitory concentration of amphotericin B. The combination is particularly effective
against Cryptococcus and Candida species.
US Patent 6770290 describes a novel composition and method for solubilizing amphotericin B in
a small amount of organic solvent for use in formulation of liposomes. The phospholipids used in
this process have been acidified with organic solvent. Further the solution of amphotericin B was
added to the acidified phosphatidylycerol which results in a soluble complex. Finally the
liposome composition incorporating the soluble complex was hydrated, the final pH of the
hydrating aqueous buffer being carefully controlled. The Amphotericin B liposomes formed
were reported to have markedly reduced.toxicity. The patent also describes the in-vitro and iv-
vivo characterization of formed liposomes.
US Patent 5194266 describes a formulation containing amphotericin B and cholesterol sulfate,
in a molar ratio of between about 1:1 to 2:1. The formulation was freeze dried to get a dry
particles for reconstitution, having particle size predominantly between about 50-250 run.
Further the formulation was described to be significantly less toxic
and more effective in treating fungal infection than prior amphotericin B
formulations.
US Patent 3965090 describes a new form of amphotericin B comprising of a
complex of amphotericin B and succinic acid. This new form of amphotericin
B was reported to be more active than amphotericin B and exhibited the advantage of being
soluble in acidic or. alkaline solutions.
US Patent 4054734 describes new forms of amphotericin B comprising of an
amphotericin B, an acid anion, and calcium cation. These new forms of
amphotericin B have been reported retain the activity of Amphotericin B and additively
displayed the advantage of being soluble in acidic or alkaline solutions.

Other References
1. Cohen, J., Antifungal Chemotherapy, Lancet, (J 982) II: 532-537.
2. Graybill, J.R. Craven, P.C., Antifungal Agencies and System Mycosis: Activity and Theraputic Use, Drugs, (1983) 25:41-62.
3. Graybill, Jr. R. et al., Treatment of Murine Cryptococcoses Liposomal Associated Amphotericin B., Journal of Infectious Disease (1983) 145: 748-752.
4. Gregoriadis, G. A. (Ed.), Liposome Technology, CRC Press: Boca Raton, CRC Press: Boca Raton, Florida (1983).
5. Lopez-Berestein, G., Treatment and Prophylaxis in Disseminated Infection Due to Candida Albicans, with Liposome Encapsulated Amphotericin B. Journal of Infectious Disease (1983)147:939-945.
6. Lopez-Berestein Treatment of Hepatosplenic Candidiasis with Liposomal-Amphotericin B, Journal of Clinical Oncology (1987) 5: 310-317.
7. Medoff, G. Kabayashi, G, Strategies and.Treatment of System Fungal Infections, N. Eng. J. Med., (1980) 302:145-155.
8. Mehta, R., Amphotericin B is Toxic to Fungal Cells but not to: Mammalian cells, Biochimica ET Biophysica Acta,(1982) 770:230-234.
9. Antileishmanial Activity of Amphotericin and Other Antifungal Agents Entrapped in Liposomes, J. Antimicrobial Chemotherapy (1981) 8:371-381.
10. Hiroshi Fukui,Tomohiro Koike, Akira Saheki, Satoru Sonoke and Junzo Seki., A novel delivery system for amphotericin B with lipid nano-sphere (LNS®)., International Journal of Pharmaceutics (2003) 265(1-2):37-45.
11. Wasan K. M., Brazeau G. A., Keyhani Al1 Role of liposome composition and temperature in distribution of amphotericin B in serum lipoproteins. Antimicrobial Agents Chemotherapy. (1993) 37:246-250.
12. Wasan K. M., Morton R. E., Rosenblum M. G., Lopez-Berestein G. Decreased toxicity of liposomal amphotericin B due to association of amphotericin B with high density lipoproteins: role of lipid transfer protein. Journal of Pharmaceutical Sciences (1994) 83(7):1006-1010.
13. Wasan, K. M. and Lopez-Berestein G., Modification of Amphotericin B's Therapeutic index by Increasing Its Association with Serum High-Density Lipoproteins. Annals of the New York Academy of Sciences, (1994) 730: 93-106.
14. David S. Perlin, Rosaria Santangelo, Padmaja Paderu, Guillaume Delmas, Zi-Wei Chen,Raphae, Mannino,and Leila Zarif. "Efficacy of Oral Cochleate-Amphotericin B in a Mouse Model of Systemic Candidiasis". Antimicrobial. Agents and Chemotherapy. (2004) 44 (9): 2356-2360.
15. Perlin D. S., Delmas G., Park S., Chen Z' W., Tan F., Kashiwazaki R. and Zarif, L.„ "Efficacy of Orally Delivered Cochleates Containing Amphotericin B in a Murine Model of Aspergillosis"., Antimicrobial Agents and Chemotherapy, (2002) 46(8):704-2707.

16. Steigmann F., Sait M. Z., Rajagopal K. G. and Villa F.,"Oral Amphotericin B in the Treatment of Ulcerative Intestinal Diseases'. Digestive: Diseases, (1970) 15(11):993-1002.
17. Kishor M. Wasan, Verica Risovic, Kristina Sachs-Barrable and Michael Boyd.," Potential Mechanisms by Which Peceol® Increases the Gastrointestinal Absorption of Amphotericin B". Drug Development and Industrial:Pharmacy, (2004).30(7):767-774.
18. Amarji, Basant; Ajazuddin; Raghuwanshi, Dharmendra; Vyas, S.P.; Kanaujia, Parijat.,Lipid Nano Spheres (LNSs) for Enhanced Oral Bioavailability of Amphotericin B: Development and Characterization, Journal of Biomedical Nanotechnology,(2007) 3(3):264-269.
Field of the Invention
The present invention relates to a process and composition of Solid Lipid Nanoparticles (SLNs) containing Amphotericin B for oral route of administration. In addition, the SLNs could also be used for parenteral, topical and mucosal route.
Background of the Invention
The rational for the design of novel formulation of the AmB is the reduction of its nephrotoxicity by lowering the serum concentrations of free drug and delivering the drug at therapeutic level at a site of action. Numerous strategies have been attempted for achieving this goal.
The colloidal and particulate carrier systems,-'such as liposomes or nanoparticles, exhibit controlled drug release but are rapidly removed from the circulation by cells of the reticulo endothelial phagocyte system by Kupffer cells, enhancing the risk of hepatic disorders. This gives a very low serum half-life of AmB, and results in low bioavailability and an uneven distribution throughout the body. However, this phenomenon may be used for the target design of drug delivery system for indication such as. visceral leishmaniasis, where the parasite resides exclusively within phagocytes. The literature review shows that lipid-based formulations significantly reduced mortality risk by an estimated 28%, compared with conventional AmB. AmB lipid complex and liposomal AmB significantly reduced the risk of renal dysfunction and associated side-effects. Various formulation strategies have been tried, out to treat systemic fungal infections via parenteral administration of AmB.
G.S. Kwon et.al. (1998) solubilized AmB in micelles of poly (ethylene oxide)-block-po!y ((3-benzyl-L-aspartate) (PEO-PBLA). The antifungal activity of the AmB-loaded PEO-block-PBLA micelles was found to be four to eight times higher than Fungizone® in terms of minimal inhibitory concentrations (MICs), while drug free PEO-PBLA micelle had no antifungal activity for concentrations up to 200 p,g/ ml. The underlying theory for the increase in antifungal activity of AmB-loaded PEO-PBLA micelles is yet unclear, but could be related to a stabilizing effect of the polymeric micelles against auto-oxidation of the AmB heptanes moiety or alternatively, an enhancement in membrane perturbation of fungal cells.
Another approach to enhance drug performance and reduce toxicity is the conjugation of drug with polymeric carrier. Conjugation of an insoluble drug such as AmB to a water soluble

biodegradable polymer [Arabinogalactan (AG)] was found to increase the water solubility of the drug, drug circulation time, and accumulation.in the diseased tissue, resulting in an improved therapeutic effect and reduced toxicity. J. Domb et al (1999) synthesized AmB-AG conjugates tested for efficacy against common pathogenic fungi and for toxicity in mice. The conjugate containing 20% by weight of AmB was found to be soluble in water (>100 mg/ml), was. as effective as the parent drug, and possessed a 20-fold-lower toxicity. In a direct comparison between AmB-AG conjugate and AmBisome, the conjugate was superior in safety and efficacy against fungal infections and Leishmania amazonensis. and Leishmania donovani in mice. Methoxy poly ethylene Glycol (mPEG) has also been reported as a carrier for conjugation. Lipid Nano-spheres (LNS®) are small-particle lipid emulsion, and have been reported to exhibit lesser uptake by the liver than the conventional lipid emulsion. Therefore, low-dose therapeutic system with a lipid emulsion for AmB, a potent antifungal drug, was studied by Junzo Seki et. al. (2003). The formulated LNS-AmB was a homogeneous emulsion with the mean particle diameter ranging from 25 to 50 nm. Due to relatively less uptake by the liver, i.v administration of LNS loaded with the AmB yielded higher plasma concentrations of the drug than the Fungizone® when evaluated in mice, rats, dogs, and monkeys. In in vitro and in vivo studies, both LNS-AmB and Fungizone, demonstrated similar antifungal activity. Inspite of the higher plasma AmB concentrations achieved by LNS-AmB, in comparison with Fungizone®, LNS-AmB did not cause significant hemolysis, vomiting and toxicity in dog. Therefore, LNS-AmB, allowed the treatment of systemic fungal infections at low doses without the severe nephrotoxicity of Fungizone®.
The topical drug delivery of AmB was reported by R. Cortesi et al. (2003) using two systems namely microemulsions and monoglyceride-water system. Both these system were studied for vaginal and skin mycosis. Results indicate that systems can be employed as alternative formulations for the topical delivery of AmB.
The unique physicochemical properties (low solubility and high affinity for biological membranes) have made AmB as one of the. first drug of choice for formulation into liposomes. It was hypothesized that liposomes could provide a safer alternative to conventional deoxycholate-solubilized AmB by attenuating its serious renal toxicity while maintaining its fungicidal activity.
In animal models and in initial clinical studies, the liposomes formulations appeared to increase the safety of AmB, while providing efficacy against systemic fungal infections. / Lawrence et al. shown that formulation of AmB using small unilamellar liposomes (SUVs) consisting of phospholipids and cholesterol also showed good tolerability in cancer patients with fungal infections. While neither of these early formulations progressed to commercial development, they served as a basis from which formulation optimization took place in divergent directions, leading to the development of the three different, yet effective lipid antifungal products currently available namely AMBISOME ®, ABELCET ® and AMPHOTEC®.
C.R Middaugh et al have prepared the nanoparticle dispersion of AmB using oppositely charged polymers namely poly ethyleneimine (PEI) and dextran sulphate (DS), and zinc sulphate as

stabilizers has been developed. The MIC of this formulation Was found to be 0.35ng per ml. Nanoparticles showed distinct advantages over liposomes, such as long shelf-life at room temperature, ability to withstand autoclaving for sterilization improved oral bioavailability and low production costs.
The currently available marketed products of AmB are lipid based formulations-viz. AmBisome, Amphocil and Amphotec. All these form, and Arulation differ with respect to reticulo-endothelial clearance, volume of distribution (Vd), Cmaxea Under Curve (AUC) of AmB,. When compared with Fungizone® ( a conventional formulation), a liposomal AmB was found to have lower clearance, and consequently a very high Cmax and AUc which is of the major importance from clinical point of view.
The limitation of AmB parenteral therapy is the need of hospitalization often making'the treatment expensive and inconvenient to the patients at large. So to overcome the cost of the therapy and for better patient compliance, many strategies have been developed to formulate the oral formulation of AmB such as oral rianosuspension of AmB Croft S.L et al(2003) and Villa F et al(1970). Various lipid and mixed-micelle formulations Eugene Choo et al(2003),Michael Boyd et al(2004, Oral Cochleate-AmB Leila Zarifet al(2000,2002),oral lipid based formulations wasan et. Al (2009), oral Amphotericin B in the management of cutaneous and mucosal candidiasis San Francisco et. Al (1964)
Summary of the Invention
The present invention features method for producing Solid lipid nanoparticles containing amphiphilic drugs. The objective of the present invention is to make solid lipid nanoparticles of amphophilic drugs like amphotericin B. Thus the present investigation was focused on developing an oral dried nanoparticle formulation, of AmB which will protect the drug in acidic media of Gl tract, prevent the photo degradation of drug, enhance the oral absorption of the drug and capable of targeting the drug to the liver, spleen, lungs etc. The aim was to tackle the aforementioned drawbacks at the same time exploiting advantages of lipid based nanocarriers namely improved absorption and decreased nephrotoxicity of the encapsulated moiety and no traces of organic solvents i.e. highly purified nanoparticles.

Detailed Description of the Invention
This invention is directed towards the development of dry solid lipid nanoparticulates compositions for the oral delivery of lipophilic substances, and the methods for preparing such compositions. The dry solid lipid mixtures in the present invention comprises of lipophilic substance, a solid lipid or mixture of solid lipids, one or more phospholipids and surfactant or mixture of surfactants. Advantageously, the dry lipids mixtures of the present invention may further comprise a cryoprotectant. Any of a wide variety of lipophilic substances can be utilized in these mixtures. Examples include lipophilic drugs, vitamins, and hormones. The other lipophilic substances include steroids, steroid antagonists, non-steroidal anti-inflammatory agents, antifungal agents, antibacterial agents, antiviral agents, anticancer agents, antihypertensives, antioxidants, anti-epileptic agents and antidepressants among many others. Additional examples of lipophilic drugs with very poor water solubility and low oral bioavailability which could benefit from oral dosage forms are the neurohormone melatonin, the antifungal agent amphotericin B, the anticancer drug etoposide, as well as tamoxifen and its analogs. More specific compounds include cannabinoids, as exemplified by dexahabinol, and vitamins, enzymes or coenzymes, as exemplified by COQ1o-
The loading of the lipophilic substance in the final dry solid lipid mixture is in the range of about 10-40% of the total solid weight of the mixture.
Examples of solid lipids suitable for the preparation of dry lipid mixtures of the present invention includes triglycerides composed of natural, even-numbered and unbranched fatty acids with chain lengths in the C10-C18 range, or microcrystalline glycerol triesters of saturated, even-numbered and unbranched fatty acids of natural origin such as tricaprin, trilaurin, trimyristin, tripalmitifi, and tristearin. The content of solid triglycerides in the final dry lipid mixture is in the range of about 10-15 % of the total solid weight of the mixture.
The preferred phospholipids which constitute the third component of the dry lipid mixture in the present invention are natural phospholipids, such as: soybean lecithin, egg lecithin, phosphatidylglycerol, phosphatidylinositol, "'phosphatidylethariolamine, phosphatidic acid, sphingomyelin, diphosphatidylglycerol, phdsphatidylserine, phosphatidylcholine, cardiolipin, etc.; synthetic phospholipids, such as dimyristoylphosphatidylcholine, dimyristoyl-phosphatidyl glycerol, distearoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, etc.; and hydrogenated or 30 partially hydrogenated lecithins and phospholipids.
The fourth component of the mixture is surfactant or mixture of surfactants. These substances stabilized the system by localising at the interphase of heterogeneous system. The examples of these include Tween 80, Span 80, Transciitol P, Solutol HS 15 etc..
The dry solid lipid mixtures may further contain a cryoprotectant material as known in the art, such as a sugar or an amino compound, to protect the formulation during freeze-drying or spray-drying processes. Preferred cryoprotectants include glucose, sucrose, lactose, maltose, and trehalose; polysaccharides, such as dextrose, dextrins, and cyclodextrins; and nonriatural

polymers, such as polyvinylpyrrolidone (PVP). Other cryoprotectants may also be used, including amino acids, as disclosed in U.S. Pat. No. 5,472,706.
Examples
1. AmB (50-100 mg) was dissolved in DMSO (0.6-0.8 g), lipid (0.2-0.5 g), surfactant (0.2-0.6
g) and Epikuron 200 (20-40 % of solid lipid) was mixed with AmB solution and heated at
50-60°C. Separately, double distilled (filtered through 0.45um nylon membrane) water was
heated to 50-60°C. This was added to the molten lipid-surfactant mixture under stirring
(3000-4500 rpm) for 5-10 min and stirring was continued further till it reaches the room
. temperature to form SLN dispersion.
The resultant SLN dispersion was subjected to probe sonication (Dakshine Probe Sonicator) for 5-10 min with one cycle of 15 sec ON and 15 sec OFF in ice bath.
2. DMSO from SLN dispersion was removed by dialysis. SLN dispersion, 50 mL was filled in
dialysis bag having molecular weight cut off of 10,000-20,000 Da (Hi-Media, Mumbai) and
sealed with medi-clip. It was then dialyzed for 6-8 hours in 5 lit purified water. The water
was replaced with fresh purified water every 2-3 h.
Complete removal of DMSO from, formulation was confirmed by Gas Chromatography (GC) by measuring DMSO content of SLN dispersion every hr. The particle size and P. I. of the SLN dispersion decreased significantly after dialysis.
The particle size and P. I. of SLN were determined by photon correlation spectroscopy (PCS) using Beckman N4 Plus submicron particle size analyzer was found to be 200.1-250.87 ± 10.1 nm and P.I was 0.24.
. 3. Freeze drying of SLN dispersion as prepared by the above mentioned method was performed using Ref-Vac (Ref-Vac Consulting, India). Cryoprotectants with varying concentration were used for freeze drying (Table 1). The freeze-drying technology employed was freezing at -4G°C for 24 h, primary drying at 0°C for 5 h followed by 10°C for 2.5 h and 15°C for 2 h and secondary drying at 25°C for 2.5 h. The chamber pressure was maintained at 20 Pa and the temperature of the cold trap was maintained at -50°C during the entire process.
Freeze-drying resulted in easily redispersible fine powder. The particle size of the SLN dispersion was determined by PCS prior to lyophilization and after the reconstitution of the lyophilized powders with double distilled water. The mean particle size of the optimized formulation prior to lyophilization was found to be 181.6 nm and that of the reconstituted dispersion was 235.6 nm.

Table 1 Cryoprotectants Used For Freeze Drying
Cryoprotectant (%w/w) Cryoprotectant (% w/w)
Mannitol 10-100. Trehalose 10-100
Mannitol + Trehalose 5 + 5 ■ Mannitol + Trehalose 25 + 25
Arginine 5-10 Lysine 5-10
Lactose 250 Neusiline 1-5
Gelatin 1-10 Fructose 5-20
4. Spray Drying of SLN dispersion was performed using a laboratory scale spray dryer (JISL
LSD-48, JISL, India). Lactose (5-10 % w/v) was used as anticaking agent. The solution was
sprayed through a peristaltic pump with controlled nozzle into the main chamber
(500 mrn x 215 mm). The feeding flow rate was controlled by the pump rotation speed. The
aspirator rate was 44 % and the compressor air pressure was 2 kg/cm2. Inlet air temperature
was varied between 80 °C and 120 °C whereas feed flow rate was varied between 5 and
15 % of total content. Process yield for these batches was calculated as a relationship
between total solids content in the resulting powder and total solids content in the feed.
The mean particle size prior to Spray Drying was found to be 181.6 nm and that of the reconstituted dispersion was 593.4 nm i.e. a significant change in mean particle size was obtained after Spray Drying.
5. Dried SLN, (100 mg) was dispersed in 250 mL of medium (each of pH 1.2, 4 and 7.4) in
USP type II dissolution apparatus. At regular time intervals; 1 mL of the sample was
withdrawn and replaced with fresh buffer. The aliquots were analyzed for drug content,.
particle size and P. I. Similar study was performed with plain drug.
It was observed that the degradation behavior of plain AmB (solubilized in DMSO: methanol, 1:9) was in accordance with the reports. However, encapsulation of AmB in SLN protected it to some extent. At pH 1.2, SLN had pronounced protective effect, whereas, at. the same pH the plain.drug exhibited a greater drug degradation.

6. Single dose acute toxicity study was.performed, to evaluate the safety of the developed
formulation (Freeze dried SLN)- The acute oral toxicity of the formulations was determined
as LD50 as per the Organization for Economic Co-operation and Development (OECD) 425
guidelines.
The results indicated that animals treated at single dose levels with the developed formulation in all the groups exhibited normal activity and behavioral pattern without any signs of tremors, convulsions, salivations, diarrhea and lethargy. All the animals from different groups exhibited normal food intake and body weight gain during the entire study. The oral LD50 of developed formulation was found to be >2000mg/kg of body weight.
7. Adult male Wistar rats (200-250 g, 10-12 weeks) were used in the pharmacokinetic study
Following an overnight fasting (12 to 16 h), the rats were divided into four groups and
received a single dose of 200 mg/kg by oral route of the following formulations
■ AmB, 50 mg dissolved in 0.6 mL DMSO (reference oral solution)
■ 1.45 g (~ 50 mg of AmB) of GDL SLN dispersed in 4 mL double distilled water
■ 1.45 g (~ 50 mg of AmB) of Softisan SLN dispersed in 4 mL double distilled water
The serum concentration of the formulation was developed SLN formulation was found to be the 124.93 ± 13.61 ng/mL.
The half-life of the developed SLN was significantly greater than that of plain drug, indicating the controlled release of AmB from developed formulation. Overall pharmacokinetic results suggest the superiority of developed SLN. This is indeed justified by the in vitro development studies, where the developed SLN confirmed greater protection to the drug against pH degradation as compared to the plain drug.

Claims
We claim our invention as:
1. A method of making nanoparticles of a said therapeutic agent of bioactive molecules and composition thereof.
2. The said method of claim 1 comprises of:
a. Dissolving the said therapeutic agent of bioactive molecules in a first liquid
component.
b. Adding lipids to a solution of claimed 2(a) containing the said therapeutic agent
c. Heating the mixture obtained in claim 2(a) and 2(b) to get lipid melt.
d. Add lipid melt as described in claimed 2(c) to a aqueous phase under mechanical
shear to achieve a dispersion comprising a continuous phase and a dispersed
phase in which the continuous phase is aqueous phase of the dispersion and the
dispersed phase comprises lipid globules of the therapeutic agent dissolved in the
first liquid component.
e. First liquid of claim 2 (a) was removed from dispersion of claimed 1 (d) by a
. suitable method.
f. Drying the dispersion obtained in step 2 (e) by suitable processes after adding
' suitable excipients.
3. The method as claimed in claim 2(a), wherein the first liquid component is a polar solvent.
4. The polar solvent as described in claim 3 includes DMSO/methanol/DMF/acetone/IPA/THF/surfactants arid surfactants solutions.
5. The said therapeutic agent of bioactive molecules as described in claim 2(a) is drugs, vitamins, and hormones.
6. The said therapeutic agent of bioactive molecules as described in claim 5 is natural, synthetic and semisyntheic. antibiotics steroids, steroid antagonists, non-steroidal antiinflammatory agents, antifungal agents, antibacterial agents, antiviral agents, anticancer agents, anti-hypertensives, antioxidants, anti-epileptic agents and antidepressants among many others. Additional examples of lipophilic drugs with very poor water solubility and low oral bioavailability which could benefit. from oral dosage forms are the neurohormone melatonin, the antifungal agent amphotericin B, the anticancer drug etoposide, as well as tamoxifen and its analogs. More specific compounds include cannabinoids, as exemplified by dexanabinol, vitamins, enzymes or. coenzymes, as exemplified by CoQio.
7. The vitamins described in claim 5 are Vitamin A, Vitamin D, Vitamin E, and Vitamin K.
8. The method as claimed in claim 2, wherein the lipid used is a natural or synthetic or semi-synthetic lipids are solid lipid, liquid lipid, semi-solid lipid.

9. The formulation as claimed in. 1 and 2(b) wherein the nanoparticles comprise one of the lipids as phospholipids. ,
10. The phospholipids as described in claim 9. belong to a group of phosphomonoglyceride, phosphatidic acid and sphingolipid.
11. The types of phospholipids as described in claim 10 in which the phospholipids is selected from phosphatidylcholine, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidylserine, diphosphatidylglycerol, .. phosphatidic acid dimyristoylphosphatidylcholine dimyristoylphosphatidylglycerol and mixtures thereof.
12. A formulation as claimed in Claim 2(b), in which the content of phospholipids in the final dry solid lipid mixture is in the range of about 2-40% of the total solid weight of the other lipids , more preferably about 5-30%, and still more preferably about 10-20% of total solid weight of other lipids.
13. The method as claimed in claim 2, wherein the aqueous phase is water, aqueous surfactant solution, preservatives solution.
14. The preservative as described in claim 13 are citric acid, BHT, BHA, methyl parabens and their salts, propyl parabens and their salts, t-buty! ammonium salts, Na-EDTA.
15. The surfactant solution as described in claim 13 are alkylbenzene sulfonates, polyethoxylated nonionics, fatty amine salts and quaternary ammoniums.
16. The dispersion as described in claim 2(d) is an emulsion, nanostructured lipid carrier or solid lipid nanoparticles.
17. The type of emulsion as described in claim 16 is a nanoemulsions, microemulsion, and macro emulsion.
18. The continuous phase as described in claim 2'(d) is an aqueous phase.
19. The dispersed phase comprises of lipid globules as described in claim 2 (d) are globules having a diameter between 200 and 400 nm.
20. The suitable method as described in claim 2(e) is an ultrafiltration, dialysis, and electrodialysis.
21. The Drying of dispersion by suitable method as described in claim 2(f) is a freeze drying, spray drying, flash evaporation and vacuum drying. .
22. The suitable excipients as described in claim 2(f) are cryoprotectants.
23. The nanoparticle system of claim 1, wherein said system is in the form of a solution, suspension, gel, paste, colloidal dispersion, tablet, capsule., or oral controlled release substance.
24. A method for treating fungal infection and leishmaniasis comprising administering a solid lipid nanoparticle using pharmaceutically acceptable carrier to a patient in need.
25. The method of claim 24, wherein administration is oral, parenteral, topical, mucosal sublingual, or buccal.

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Patent Number

279598

Indian Patent Application Number

3329/MUM/2010

PG Journal Number

05/2017

Publication Date

03-Feb-2017

Grant Date

27-Jan-2017

Date of Filing

07-Dec-2010

Name of Patentee

VANDANA BHARAT PATRAVALE

Applicant Address

C-15, DIVINE LIGHT CO-OPERATIVE HOUSING SOCIETY, 137/139, M.V. ROAD, ANDHERI (E), MUMBAI-400 093

Inventors:

#	Inventor's Name	Inventor's Address
1	VANDANA BHARAT PATRAVALE	C-15, DIVINE LIGHT CO-OPERATIVE HOUSING SOCIETY, 137/139, M.V. ROAD, ANDHERI (E), MUMBAI-400 093
2	PRATIKKUMAR ANILBHAI PATEL	405-TULSI PUSHPA, GODDEV NAKA, BHAYANDAR (E), MUMBAI-401105

PCT International Classification Number

A61K47/48; A61K9/127

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA