Preconcentrate of amphotericin b for preparing instantaneous safer nanoformulation

Abstract

The present invention relates to a pharmaceutical composition for intravenous infusion administration comprising Amphotericin B (AmB). In particular, the present invention relates to clear liquid preconcentrate composition comprising the antifungal active ingredient, a combination of lipid and monomerizing agent having weight ratio between 1:0.5 to 1:3 in a pharmaceutically acceptable vehicle, wherein preconcentrates spontaneously convert to a nanoparticulate suspension upon further dilution in aqueous medium, e.g., aqueous dextrose injection (5%), wherein not less than 90% of AmB is entrapped in the nanoparticles that ensures a safer and efficacious AmB formulations for intravenous administration. The present invention also relates to the process for preparation of said preconcentrate composition of Amphotericin B and to their use as medicaments.

Description

[0001]TITLE OF THE INVENTION PRECONCENTRATE OF AMPHOTERICIN B FOR PREPARING INSTANTANEOUS SAFER NANOFORMULATION FILED OF INVENTION The present invention relates to a pharmaceutical composition for intravenous infusion administration comprising Amphotericin B (AmB), a polyene antifungal agent as the active ingredient. In particular, the present invention relates to clear liquid preconcentrate composition comprising the antifungal active ingredient, a combination of lipids, and pharmaceutically acceptable vehicle, wherein preconcentrates spontaneously convert to a nanoparticulate suspension upon further dilution in aqueous medium, e.g., aqueous dextrose injection (5%), wherein not less than 90% of AmB is entrapped in the nanoparticles. The present invention also relates to the process for preparation of said preconcentrate composition of AmB and to their use as medicaments. BACKGROUND AND PRIOR ART OF THE INVENTION Amphotericin B (AmB) continues as the gold standard for the treatment of life threating systemic fungal infections and visceral leishmaniasis. The recent clinical indication of AmB for mucormycosis during the COVID-19 pandemic has intensified the importance of AmB nanoformulations. However, its usage has been marred by long therapeutic regimens and severe side effects. AmB injection side effects include severe nephrotoxicity and often hepatotoxicity. And hence, it is recommended that it should only be used to treat potentially life-threatening fungal infections and not to treat less serious fungal infections of the mouth, throat, or vagina in patients with a normal immune system. However, toxicity of the drugs can originate not only from biochemical intracellular adverse interactions or systemic allergic reactions linked to the specific chemical structure of the drug molecule, but also from its molecular aggregation state. The chemical structure of AmB as represented in Figure 1 comprises of a macrolide lactone ring containing 38 carbon atoms, which is α-glycosylated at C-19 hydroxyl position with a mycosamine sugar. The lactone ring contains a rigid, hydrophobic polyene chain made up of seven conjugated double bonds, a hydrophilic polyol chain made up of similar number (7) of hydroxyl groups and a six-membered ketal ring. Such aggregation is majorly ascribed to van der Waals forces (vdW) along the hydrophobic polyene chains of two AmB molecules, which may be stabilized by polar H-bonding interactions and coulombic forces. The toxicity of AmB drug is associated with its molecular aggregation states due to their self-assembling properties. AmB can exist as monomer or self-assemble to form aggregated and super-aggregated form. The serious / fatal AmB toxicity is linked to the aggregated state of AmB ascribed to interaction of the aggregated state with cholesterol in the host cell membrane. The monomeric state of AmB is a safe conformation which exhibits selective affinity to ergosterol in the pathogen cell membrane. Interaction of AmB molecules by apposition of their hydrophobic polyene chains results in the formation of the aggregated / dimer state. The aggregated state loses the specificity exhibited by the monomeric state and exhibits a high tendency to interact with cholesterol-rich membranes in the host cell, exhibiting severe toxicity. Therefore, monomer is safer state of AmB. Regarding super-aggregated form of AmB, it is considered as a monomer depot and releases the monomer slowly over time. Hence it exhibits safety like the monomer form. Among commercially available AmB nanoformulations, AmB is seen to exist in the super-aggregated state in liposomal formulations which exhibit high safety, while the micellar formulations wherein AmB exists in the aggregated state, manifest severe toxicity, further confirming the correlation of AmB state and toxicity. But liposomal formulations have some drawbacks such as high production costs, leakage and fusion of encapsulated drug, high possibilities of phospholipid oxidation and hydrolysis-like reaction that question on stability of said formulations. The first-generation formulation of AmB was a micellar dispersion of the drug in sodium deoxycholate, marketed under the brand name Fungizone®. However, the micelles on dilution in the body rapidly release free drug which self-associates to form toxic aggregated form, resulting in host cell binding and dose-dependent hepatotoxicity and nephrotoxicity on chronic use. It also involves tedious administration by IV infusion over a period of 4-6 hours for a duration of 4 weeks, which manifests various infusion related side effects like infection of the indwelling catheter, fever, chills and shaking due to RBC hemolysis, bone pain, thrombophlebitis and other surfactant related toxicities. (Ref: Wasan, K.M., et al., Highly effective oral amphotericin B formulation against murine visceral leishmaniasis. Journal of Infectious Diseases, 2009.200(3): p.357-360.) Significant decrease in AmB toxicity is demonstrated using a number of excipients. For instance Gruda et al demonstrated an inverse correlation of the acyl chain length of sucrose esters based surfactants and AmB toxicity in mice. A decrease in toxicity was observed with increase in chain length for instance, myristate (C14) < laurate (C12) < caprate (C10). (Ref: Gruda, I., Milette, D., Brother, M., Kobayashi, G.S., Medoff, G., Brajtburg, J., Structure-activity study of inhibition of amphotericin B (Fungizone) binding to sterols, toxicity to cells, and lethality to mice by esters of sucrose, Antimicrob. Agents Chemother.35 (1991) 24-8) Formulations comprising block copolymers including poly(ethylene glycol)-block- poly(ɛ-caprolactone-co-trimethylenecarbonate), Pluronic®, poly(ethylene oxide) block-poly(N-hexyl stearate l-aspartamide), poly(ethylene oxide)-block-poly(- benzyl-l-aspartate) or monoglycerides revealed reduced hemolysis and nephrotoxicity with improved efficacy of AmB. (Ref: Vandermeulen G, Rouxhet L, Arien A, Brewster ME, Preat V. Encapsulation of amphotericin B in poly (ethylene glycol)-block-poly (ɛ-caprolactone-co-trimethylenecarbonate) polymeric micelles. International journal of pharmaceutics.2006 Feb 17;309(1-2):234-40) High binding of amphiphilic polyglutamic acid (PGA) polymer and AmB ensured slow release of the monomeric AmB molecules from the PGA thereby limiting aggregation. (Ref: Dinh, T., Zia, Q., Zubair, S., Stapleton, P., Singh, R., Owais, M., Somavarapu, S., Novel biodegradable poly(gamma-glutamic acid)-amphotericin B complexes show promise as improved amphotericin B formulations, NANOMED- NANOTECHNOL. 13 (2017) 1773-83). Likewise, incorporating AmB inside the core of Angiopep-2 modified PE-PEG based polymeric micelles also contributed to monomeric AmB release in a sustained manner. (Ref: Shao K, Huang R, Li J, Han L, Ye L, Lou J, Jiang C. Angiopep-2 modified PE-PEG based polymeric micelles for amphotericin B delivery targeted to the brain. Journal of Controlled Release. 2010 Oct 1;147(1):118-26) Researchers prepared micelles using phenylboronic acid – attached polycarbonate / PEG diblock copolymer which enabled monomerization due to interaction of boronic acid with AmB by hydrogen bonding and ionic interactions and also demonstrated increased antifungal activity. (Ref: Wang, Y., Ke, X., Voo, Z.X., Yap, S.S., Yang, C., Gao, S., Liu, S., Venkataraman, S., Obuobi, S.A., Khara, J.S., Yang, Y.Y., Biodegradable functional polycarbonate micelles for controlled release of amphotericin B, Acta Biomater.46 (2016) 211-20) Oliveira and Benatti developed AmB vesicles by using quaternary ammonium surfactant. Monomerization of AmB was enabled by ionic interaction in quaternary ammonium surfactant vesicles (Ref: Oliveira, T.R., Benatti, C.R., Lamy, M.T., Structural characterization of the interaction of the polyene antibiotic Amphotericin B with DODAB bicelles and vesicles, Biochim Biophys Acta Biomembr.1808 (2011) 2629-37) The aggregated state of AmB is initiated by dimerization of two apposite hydrophobic polyene chains which progresses to form larger aggregates. This dimer creation can be inhibited by using long chain lipid excipients. Recently our group developed, AmB lipid polymer hybrid nanoparticles (LIPOMER), which consists of stearate lipids with a hydrophilic polymer Gantrez (GZ), which successfully developed super-aggregated AmB and solid lipid nanoparticles (SLN) containing only stearates, which successfully developed monomeric AmB, with improved antileshmaniasis activity. The strategy for super-aggregated AmB with LIPOMER was limiting the hydrophobic interactions by using lipids that could intercalate along the polyene chain and the hydrophilic polymer which could mask the polar regions of the macrolide ring, to further stabilize the super-aggregated state (Ref: Das, S., Devarajan, P.V., Enhancing safety and efficacy by altering the toxic aggregated state of Amphotericin B in lipidic nanoformulations, Mol. Pharm.17 (2020) 2186-95) Gupta Et al developed Sodium Alginate (SA) Cross-Linked Amphotericin B Encapsulated Glycol Chitosan Stearate (GCS) Nanoparticles with same objective of monomerization of AmB. The stearic acid chain in Nanoparticles inhibits self- association of AmB molecules due to strong interaction among SA polymer, GCS copolymer and AmB, thus prevents formation the aggregated state. (Ref: Gupta, P. K., Jaiswal, A. K., Asthana, S., Verma, A., Kumar, V., Shukla, P., Dwivedi, P., Dube, A., Mishra, P. R., Self-assembled ionically sodium alginate cross-linked amphotericin B encapsulated glycol chitosan stearate nanoparticles: applicability in better chemotherapy and non-toxic delivery in visceral leishmaniasis, Pharm. Res. 32 (2015) 1727−1740) Complex method of preparation, stability, and scalability for monomerization of AmB hamper translation of AmB nanocarriers. Todke and Devarajan developed super-aggregated AmB using amphiphilic polyoxyl-40-stearate excipient. Super-aggregated AmB slowly released the safe monomeric form without aggregate formation. (Ref: Todke PA, Devarajan PV. In- silico approach as a tool for selection of excipients for safer amphotericin B nanoformulations. Journal of Controlled Release.2022 Sep 1;349:756-64.) Till date whichever excipients are used for monomerization of AmB, they are not suitable (not acceptable for intravenous administration by FDA) for the treatment of Visceral leishmaniasis and systemic fungal infection by intravenous injection. Some of the researchers were working on chemical modification of AmB to make it less toxic (new derivatives of AmB). As disclosed in Indian patent application no.202217010289, the C16 ester derivatives of C2'epi-amphotericin B (C2'epiAmB) is synthesized by 11 step chemical synthesis route. The said approach is surely not economic as it involves synthesis of new active chemical moiety screening for safety and efficacy. Amphotericin B colloidal dispersion (ABCD) is a colloidal dispersion of a stable complex of amphotericin B with cholesteryl sulphate in a 1:1 proportion, forming uniform disk-shaped particles. ABCD is associated with less nephrotoxicity than conventional amphotericin B deoxycholate. (Ref:Patel, Robin (2000). Amphotericin B colloidal dispersion. Expert Opinion on Pharmacotherapy, 1(3), 475–488. doi:10.1517 / 14656566.1.3.475) Hence, the key to development of safer and efficacious AmB safe formulations for intravenous administration lies in ensuring in formulating economic formulation of AmB in the monomeric state having capacity to form average particle size between 50 to 200nm, and more specifically absence of the aggregated state. Therefore, the objective of the present invention is to develop an economic and safe formulation of AmB for treatment of fungal infection. Wherein, the novel formulation disclosed in present invention is providing a monomeric state of AmB in blood stream, which helps in reduced hemolysis and often decreased nephrotoxicity with improved efficacy. Surprisingly, the inventors of the present invention has developed a preconcentrate composition of AmB in combination with at least one lipidic agent in solvent and said preconcentrates spontaneously convert to a nanoparticulate suspension having nanoparticles of lipid with AmB in the monomeric state, when further diluted in aqueous medium. The said nanoparticulate suspension of AmB formed is suitable for safe and effective injecting to a patient under treatment. Thus, the present invention achieves the objective to provide an easy to prepare, safe nanoformulation of AmB for intravenous infusion administration with reduced severe haemolytic, hyper sensitivity and nephrotoxicity side effects. OBJECTS OF THE INVENTION It is an objective of the present invention to develop a novel preconcentrate formulation of AmB that can be enable safe delivery of AmB to the patient. Still another objective of the present invention is to provide an innovative formulation of AmB to reduce the toxicity associated with AmB by combining the drug with a combination of lipids and solvent that limit formation of the unsafe aggregated state. One more objective of the present invention is to develop a preconcentrate formulation of AmB that can be easily made into nano formulation preferably a nanodispersion having average particle size between 50 nm to 200nm by diluting it with suitable aqueous medium before administration. Another objective of the present invention is to provide a nanoformulation of AmB which can be prepared by easy steps and avoids high-cost instrument use like required for preparation of emulsion or liposomal formulation and also freeze dryers. Another objective of the present disclosure is to improve the safety of AmB for fungal infection treatment and visceral leishmaniasis by providing AmB in safe monomeric state in blood stream. SUMMARY OF THE INVENTION The present invention provides a homogeneous pre-concentrate composition of drug amphotericin B for preparing instantaneous safer nano-formulation for intravenous infusion administration, said composition comprising: i) 0.132 to 1.33 % w / v Amphotericin B, ii) 0.025 to 6.66 % w / v of lipidic agent, iii) 0.132 to 3.99 % w / v of Monomerizing agent, iv) Solvent dimethylacetamide to quantity sufficient; Characterized by being able to form monomeric and super-aggregated nanodispersion of drug Amphotericin B with drug entrapment efficiency >90%, after contact with an aqueous solution, having particles of an average diameter between 50 nm-200 nm, measured by a brookhaven particle size analyzer. In preferred embodiment, the present invention provides a homogeneous pre- concentrate composition of drug amphotericin B, wherein composition comprising: i) 0.66 to 1.0 % w / v Amphotericin B, ii) 1.0 to 1.33 % w / v of lipidic agent, iii) 0.3 to 0.66 %w / v of Monomerizing agent which is cholesterol, iv) Solvent dimethylacetamide to quantity sufficient. Wherein, the lipidic agent are selected from Polyoxyl-32-stearate, Macrogol-32- glycerides, Polyoxyl-40 stearate, Polyoxyl-32-distearate, Polyoxyl-60 Hydrogenated Castor Oil, Polyoxyl-50-stearate, Polyoxyl-100-stearate, Polyoxyl- 150-stearate, Polyoxyl-150-distearate, campesterol, stigmasterol, β-sitosterol. The present invention provides that the homogeneous pre-concentrate composition of drug amphotericin B for preparing instantaneous safer nano- formulation for intravenous infusion administration is characterized by combination of lipidic agent and monomerizing agent and the ratio of lipidic agent and monomerizing agent is between 1:0.5 to 1:3 by weight and more preferably, ratio is between 1:1 to 1:2 by weight. Further, optionally the said homogeneous pre-concentrate composition of drug amphotericin B comprising of 0.001 to 2.0 % w / v of an antioxidant selected from Alpha-tocopherol, Ascorbyl palmitate, Monothioglycerol. DESCRIPTION OF DRAWINGS: Further aspects and advantages of the present invention will be readily understood from the following detailed description with reference to the accompanying figures of the drawings. The figures together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages but not limiting the scope of the invention. In the accompanying drawings: Figure 1: Illustrate chemical structure of AmB (Blue dotted box- polyol region, red dotted box- polyene region, green dotted box- mysosamine) Figure 2: Illustrate UV spectrum of different state of Amphotericin B. Figure 3: Illustrate UV spectrum of different state of Amphotericin B formulation F8 and F15. Figure 4: Illustrate UV spectrum of different state of Amphotericin B formulation F18 as per present invention. Figure 5: Illustrate a (A) Particle size distribution of Amphotericin B formulation F8, F15 and F18. Figure 6: Illustrates the following: (A) the dissolution profile of PS40-AmB LNP’s (F8), PS60-AmB LNP’s (F15) and PS60-AmB LNP’s with cholesterol (F18) in biorelevant intravenous medium Krebs-Ringer Buffer (KRB) at pH 7.4; (B) the UV spectra of PS60-AmB LNP’s (F15) at 15 minutes and at 6 hours, and (C) the UV spectra of PS60-AmB LNP’s with cholesterol (F18) at 15 minutes and 6 hours, showing a continuous increase in the monomeric form without the release of aggregate. Figure 7: Illustrates Erythrocyte toxicity of Formulation F8, F15 and F18 in comparison with prior art formulations. Figure 8: Illustrates Kidney (Cr:Creatinie, and BUN: Blood Urea Nitrogen) biochemistry after intravenous administration Formulation F8, F15 and F18 in comparison with prior art formulations. Figure 9: Illustrates liver (ALT: Alanine aminotransferase and AST: Aspartate aminotransferase) biochemistry after intravenous administration Formulation F8, F15 and F18 in comparison with prior art formulations. Figure 10: Illustrates Comparison of in Vitro Antileishmanial Efficacy and Safety of Formulation F8, F15 and F18 in comparison with prior art formulations. DETAILED DESCRIPTION OF THE INVENTION The following description of the present specification uses some terms that have the general meanings indicated below: The term “dispersible”, as used herein, refers to a liquid monophasic formulation which is readily dispersed in isotonic aqueous solution to form a two-phase nanosize dispersion. The term “preconcentrate” as used herein, refers broadly to a concentrated solution of active pharmaceutical agent for dilution with diluent before administration for patient treatment. The term “AmB”, as used herein, refers to the pharmaceutical drug Amphotericin B. The terminology and structural scientific terms used herein have the same meaning as that found in scientific literature. To the extent a term used, it should be given the broadest definition persons in the pertinent art have given that term, as reflected in printed publications and literature at the time of filing. The term “monomeric AmB” defines monomeric state of AmB which is a soluble safe conformation also known as deaggregated AmB which exhibits selective affinity to ergosterol in the pathogen cell membrane. The term “aggregated AmB” defines the interaction of AmB molecules by apposition of their hydrophobic polyene chains resulting in the formation of the aggregated / dimer state. The aggregated state loses the specificity exhibited by the monomeric state and exhibits high tendency to interact with cholesterol-rich membranes in the host cell, exhibiting severe toxicity. The term “super-aggregated AmB” defines the super-aggregated form of AmB is considered as a monomer depot and releases the monomer slowly over time. Hence it exhibits safety like the monomer form. The objectives and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure. The present invention relates to pharmaceutical compositions for intravenous infusion administration of Amphotericin B as an active pharmaceutical agent for treatment of fungal infections. More preferably, the present invention relates to single phase dispersible preconcentrates composition comprising the active ingredient Amphotericin B, at least one lipidic agent and solvent, wherein preconcentrates spontaneously convert to a nanoparticulate dispersion having average particle size between 50 nm to 500nm, more specifically between 50 to 200 nm, upon further dilution in aqueous medium, e.g., dextrose injection. The present invention also relates to the process for preparation of said preconcentrate composition of Amphotericin B and to their use as medicaments. In accordance to present invention, it provides pharmaceutical compositions of antifungal active pharmaceutical agent in the preconcentrate clear liquid composition which comprise Amphotericin B as active ingredient, at least one lipidic agent which consists of hydrophilic domain and sterols as excipients and dimethylacetamide as preferred solvent with or without any additional solvents suitable for pharmaceutical administration. In accordance to one of the embodiment of present invention, the preconcentrate clear liquid pharmaceutical composition of present invention forms a spontaneous nanodispersion in aqueous medium, more specifically when diluted with an aqueous 5% dextrose solution to provide a safe nanoparticulate super-aggregated or monomeric state of Amphotericin B suitable for intravenous administration. More specifically, in accordance to present invention, the dispersible preconcentrate composition for intravenous infusion administration of drug Amphotericin B, said composition comprising: 0.132 to 1.33% w / v Amphotericin B, 0.025 to 6.66 % w / v of the lipidic agents and solvent dimethylacetamide to quantity sufficient. In an embodiment, the said dispersible concentrate composition characterized by being able to form super-aggregated or monomeric nanodispersion of drug Amphotericin B, after contact with an aqueous solution, having particles of an average diameter between 50 nm-200 nm, measured by a Brookhaven particle size analyzer, wherein the ratio of Amphotericin B and the combination of two lipidic agents in the said formulation, the weight ratio of said two lipidic agent selected between 1:0.25 to 1:4, more preferably between 1:0.5 to 1:3. In an additional embodiment, the non-limiting examples of hydrophilic domain containing lipidic agents are selected from Polyoxyl-32-stearate, Macrogol-32- glycerides, Polyoxyl-40 stearate, Polyoxyl-32-distearate, Polyoxyl-60 Hydrogenated Castor Oil, Polyoxyl -50-stearate, Polyoxyl-100-stearate, Polyoxyl- 150-stearate, Polyoxyl-150-distearate and sterol containing excipients are cholesterol, campesterol, stigmasterol, β-sitosterol and mixtures thereof. Further, additional examples of lipidic agent are mixture of Monoglycerides and Polyoxyl stearate, Louroyl Polyoxyl 32-glycerides, Stearoyl-32-glycerides, mixture of Lauroyl Polyoxyl 32-glycerides and PEG-6000, mixture of PEG-6-stearate and PEG-32- stearate, mixture of Polyoxyl-6-stearate and Ethylene glycol stearates and Polyoxyl-32-stearates, soyabean oil, Glyceryl distearate and polyglyceroyl-3- dioleate, PEG-40 Hydrogenated Castor Oil, PEG-60 Hydrogenated Castor Oil, PEG-20 Stearate, mixture Sucrose Stearate and Sucrose Distearate, Glycol Distearate, Isocetyl Stearate, mixture Sorbitan Stearate and Sucrose Cocoate, mixture Glyceryl Stearate and PEG-100 Stearate, mixture Glyceryl Stearate and Polyglyceryl-6 Palmitate / Succinate and Cetearyl Alcohol, Polyoxyl Stearate, Macrogol Stearate; Polyoxyl 8 Stearate (PEG 8 Stearate), Sorbitan Stearate, Sorbitan Tristearate, Isopropyl Isostearate, Triglycerol Diisostearate, Ethylhexyl Hydroxystearate, PEG-660 and 12-Hydroxystearate, PEG-30 Dipolyhydroxystearate, Pentaerythrityl Tetraisostearate, PEG-25-Propylene Glycol Stearate, PEG-150 Pentaerythrityl Tetrastearate, mixture PEG-150 Pentaerythrityl Tetrastearate and PEG-6 Caprylic / Capric Glycerides, mixture Sorbitan Isostearate and Polyglyceryl-3 Polyricinoleate, Sorbitan Isostearate, Polyglyceryl-6 Distearate. In accordance with the present invention, the solvent dimethylacetamide optionally mixed with other suitable solvent selected from eg. N-Methyl-2-pyrrolidone and 2- pyrrolidone. In one embodiment, the composition further includes at least one antioxidant in concentration range between 0.001 to 2.0 % w / v. Any suitable antioxidant may be used. Non limiting examples of antioxidant include alpha-tocopherol, butylated hydroxyl anisole, ascorbic acid, sodium ascorbate, propyl gallate and vitamin E. Further, non-limiting examples of antioxidant includes Ascorbyl palmitate, Ascorbate, Bisulfite sodium, Butylated hydroxy anisole (BHA), Butylated hydroxy toluene (BHT), Cystein / cysteinate HCl, Dithionite sodium (Na hydrosulfite, Na sulfoxylate), Gentisic acid, Gentisic acid ethanolamine, Glutamate monosodium, Glutathione, Formaldehyde sulfoxylate sodium, Metabisulfite potassium, Metabisulfite sodium, Methionine, Monothioglycerol (Thioglycerol), Propyl gallate, Sulfite sodium, Alpha Tocopherol hydrogen succinate, Thioglycolate sodium, Tocopherol alpha, Sodium thioglycolate. The solution to problem addressed in present invention is achieved by developing a suitable combination of lipidic excipients and active drug for designing safer and effective and economic AmB formulation. The outcome of the present invention proposes the predicted approach as a highly promising and viable strategy to enable rapid development of safer AmB nanoformulations, more specifically combination of AmB and lipidic agents wherein AmB is retained in safe super-aggregated or monomeric state having average particle size in the range between 50 to 500 nm, more preferably between 50 to 200 nm. Further, in accordance to second embodiment, the present invention provides process to prepare preconcentrate formulation of Amphotericin B comprising step of mixing and dissolving active drug and excipients in solvent using sonication or stirrer to obtain a clear solution. The process for preparation of said formulation is simple and economic as it requires less high-tech equipment and less time. Further, the said formulation is available in preconcentrate form and therefore saves cost in manufacturing. Additional advantage of recent invention is that the formulation of Amphotericin B is provided in dispersible preconcentrate as monophasic clear liquid and the stability of single-phase formulation is taken as an advantage over multiphase formulations like liposomal formulations or suspensions. Additionally said preconcentrate may contain stabilizers, preservatives, tonicity builders and other excipients as non-limiting examples. Examples: The performance of present invention is more explained by following examples: Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting. EXAMPLE 1: Evaluation of formulation A. Drug content: AmB drug content were evaluated in triplicate. For drug content estimation, 1mL of preconcentrate dissolved in 5 mL of DMSO (dimethyl sulfoxide) by vortex mixing and sonication for 15 min, suitably diluted with DMSO and further dilution made in methanol to obtain AmB stock solution 5µg / mL, analyzed for AmB content by reverse phase HPLC method developed. Conclusion: AmB preconcentrates of AmB LNP’s revealed drug content of >98%. B. Determination of State of Drug Particles: State of AmB was monitored by UV-vis spectroscopy. The UV spectrum based on the maxima is an accepted tool to determine the state of AmB in AmB LNP’s. ^ The super-aggregated AmB state: the super-aggregated AmB exhibits a peak in the region 321-325 nm. (Refer Figure no.2) ^ Aggregated AmB state: peak observed between 330−346 nm indicated aggregated AmB state; (Refer Figure no.2) ^ Monomer AmB state: peak seen between 405-415 nm reflect AmB in the monomer form; (Refer Figure no.2) (Ref: Gaboriau F, Chéron M, Leroy L, Bolard J. Physico-chemical properties of the heat-induced ‘superaggregates’ of amphotericin B. Biophysical Chemistry.1997 May 21;66(1):1-2). Conclusion: The state of AmB in AmB LNP’s revealed safer super-aggregate and Monomeric form, illustrated in Figure no.3 and 4 respectively. C. Particle size and Morphology of AmB particles in Amphotericin B Lipidic Nanoparticles (AmB LNP’s) formulation of example: The particle size and polydispersity index (PDI) of the AmB LNP’s was measured after dispersing 100 µL in 10 mL aqueous 5 % dextrose solution using the NanoBrook 90 plus PALS particle size analyzer (Brookhaven Instruments, USA). The instrument utilizes a 35-mW red diode laser (λ = 659 nm) with a photodetector at 90° and a chamber set at 25°C to place the sample. Each sample was evaluated in triplicate. Conclusion: The AmB LNP’s showed average particle size 50-200 nm and PDI < 0.3 (Fig.5A), with good stability over 24 hours. D. Transmission electron microscopy (TEM): TEM analysis was performed using TECNAI 12 BT / FEI TEM at 120 kV. Briefly, a drop of AmB LNP’s was placed on a carbon grid (Ted Pella, Inc, Redding) and air dried followed by negative staining with 1% phosphotungstic acid. Conclusion: TEM image of AmB LNP’s revealed discrete particles with a spherical shape and average size between 50-200nm. The particle size by TEM was in agreement with the DLS measurements, illustrated in Figure no.5. E. Entrapment efficiency (EE%): To find out the drug entrapment in nano- suspended particles, the AmB LNP’s formulation prepared in examples were ultracentrifuged at 40,000 rpm for 30 min at 25oC. An aliquot (0.1 ml) of the supernatant, was withdrawn and diluted to 10 ml with methanol and the absorbance measured at λmax 405 nm. AmB concentration as measured from a standard plot in the concentration range 1-6 µg / mL. (EE%) was calculated using the following equation. Entrapment efficiency (%) =(^୫^ ^୭^ୟ୪ –^୫^ ^^୮^୰୬ୟ^ୟ୬^) × 100 Conclusion: Formation of AmB LNP’s is based on the principle of in situ nanoprecipitation which is dictated by the Marangoni effect and is influenced by interfacial turbulence, and diffusion stranding of the solvent into the non-solvent phase. AmB preconcentrates revealed drug content of >98%. In situ approach enabled generation of AmB LNP’s with high entrapment efficiency of >98% with all the excipients. EXAMPLE 2: Preparation of clear homogeneous preconcentrate Amphotericin B formulation: Different formulations were prepared using different lipidic agent in combination with Amphotericin B. Process: Amphotericin B and excipient were dissolved in N,N-Dimethylacetamide (DMA) or other solvent using bath sonication for 5 min to obtain a clear preconcentrate. Wherein all formulations prepared in these inventions comprising of 0.085 % w / v of Alpha-tocopherol as antioxidant. Formulation no. 1 2 3 4 5 6 7 8 Formulation no. 9 10 11 12 13 14 15 16 Amphotericin B 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 % w / v 6 EXAMPLE 3: Evaluation of Preconcentrate formulations for intravenous infusion administration: Each Preconcentrate Amphotericin B formulation from 1 to 16 prepared in example 2 were mixed in aqueous 5% dextrose solution (9mL) resulted in instantaneous generation of Amphotericin B loaded Lipidic Nanoparticles (AmB LNP’s) and further analysis were conducted to check nanoparticle size, entrapment efficiency and state of AmB therein. Figure no.5 illustrated the DLS graph and TEM micrograph of PS40-AmB LNP’s (F8) and PS60-AmB LNP’s (F15). ^ Evaluation of Preconcentrate formulations containing Polyoxyl-40-stearate Formulatio 1 2 3 4 5 6 7 8 4 4. 0 2 Entrapemen t Efficiency 98 ± 99.88 ± 98.75 ± 99.25± 98.69± 92.0 99.56 98.67 044 23 17 095 045 ±166 ± 45 ± 25 2 er- egat ^ Evaluation of Preconcentrate formulations containing Polyoxyl-60 hydrogenated castor oil Formulation 9 10 11 12 13 14 15 16 no. 0 4 8 2 0 4 5 er e d Conclusion: The AmB LNP’s (10μg / mL) obtained by dilution of the preconcentrate with aqueous 5% dextrose solution were scanned in the absorbance range 300 to 450nm. Appropriate blanks were used as controls to negate effects due to the excipient. Methanol solution of AmB (10μg / mL) was used as monomeric AmB reference. Micellar AmB (Ampholyn®) and liposomal AmB (Lipholyn®) were also evaluated in a similar manner (Figure no.2). Figure no. 3 illustrates the UV scans of the formulation different states of AmB which were recorded within 10 min of preparation of the samples. Wherein (A) UV- vis spectra PS40-AmB LNP’s (F8) & PS60-AmB LNP’s (F15) and compared with marketed micellar AmB (Ampholyn®) (Reference) and marketed liposomal AmB / (Lipholyn®) (Reference) as shown in figure no.2. The UV spectrum scanned from range 300-450 nm; samples diluted with water to obtain an AmB concentration equivalent to 10 μg / mL;(Peak I- Super-aggregated state, II- Aggregated state, III- Monomer state). In a typical peak III, AmB UV spectrum monomeric state of AmB exhibits characteristic peaks between 350-420 nm. Peak II represents the aggregated form while peak I represents the super-aggregated form. In our study we also used micellar AmB as reference for the aggregated state and a liposomal AmB as reference for the super-aggregated state. The super-aggregated state is reported to display high-intensity peaks at 321-325 nm and low-intensity Peaks at 406–409, 383–385 nm and 360–363 nm. These structures are reported to demonstrate enhanced thermodynamic stability with safety and efficacy comparable to the monomeric form. Such a spectrum was seen only with PS40-AmB LNP’s (F8) and PS40-AmB LNP’s (F15) with a dominant peak at 322.5 nm. This confirmed that AmB was in super-aggregated form. Furthermore, the spectrum was comparable with liposomal AmB, which is established as the safest nanoformulation available as on date. In summary, the super aggregated state can be achieved if the following critical aspects are considered. Selection of lipidic molecules that can interact with the hydrophobic polyene chain of AmB as well as hinder the polar AmB-AmB interactions by interacting and complete wrapping around the mycosamine moiety and polyol chain of AmB. Such interaction was evident only with PS40 and PS60. EXAMPLE 4: Preparation of safe and stable dispersible preconcentrate Amphotericin B formulation as per present invention: ^ Preconcentrate formulations containing combination of lipidic agent and monomerizing agent. Different formulations were prepared using lipidic agent and monomerizing agent with Amphotericin B as shown in following table. The preparation method for formulation and their evaluation for intravenous infusion administration was done as same as that mentioned in example no.2. Further figure no.2A illustrated the Particle size distribution and Figure no.5 illustrated the DLS graph and TEM micrograph of PS60-AmB LNP’s with cholesterol (F18). Formulation no. 17 18 19 20 ^ Evaluation of Preconcentrate formulations combination of lipidic agent and monomerizing agent Formulation no. 17 18 19 20 Polydipsersity 0.253 ± DI) 0.010 0 0.246 ± Index (P .189 ± 0.017 0.25 ± 0.005 0.040 2 c EXAMPLE 5: Evaluation of stability of Preconcentrate formulations: The stability studies were also conducted on above formulations prepared in examples 3 and 4, when sample stored at temperature between 2 to 8 °C to monitor the change in state of aggregation therein. And results are tabulated as below: Stability Particle size PDI Drug content State of Period (nm) (%) AmB Stability Particle size PDI Drug content State of P i A B 6 Months 174.27±6.53 0.497±0.052 94.51±0.13 328 9 Months 122.81 ±7.35 0.286±0.024 89.86±0.20 330 TABLE N Stability Particle size PDI Drug content State of Period (nm) (%) AmB Con 8 °C, protected from air and light, for up to nine months. The results are tabulated in table no, 7 and 8. During three months, no significant changes were observed in % drug content, particle size, or the physical state of AmB in both formulations. However, after three months, F8 and F15 start showing aggregation of AmB along with a reduction in drug content. Further, the Table no.9 represents the stability data for PS60-AmB LNP’s with cholesterol (F18). The AmB in formulation F18 remained stable when stored at 2– 8 °C, protected from air and light, for up to 12 months. Throughout this period, no significant changes were observed in terms of % drug content, particle size, or the physical state of AmB within the PS60-AmB LNP’s with cholesterol (F18). These formulations-maintained stability for up to twelve months. Figure no.4 illustrates the safe monomeric state of AmB in the nanoparticles with cholesterol which was confirmed from the dominant monomeric peaks seen between 350-420 nm in the UV-Visible spectrum. While monomeric AmB LNP’s formed due to combination of C18 fatty acid chain of lipid and cholesterol, which effectively interacts with the polyene and polyol to separate and stabilize AmB in nanoparticles, thus preventing the self-aggregation of AmB molecules. EXAMPLE 6: In vitro Release evaluation to monitor state of AmB in pH 7.4: Release of AmB in the monomeric / aggregated state was evaluated by direct addition method using USP-IV, in biorelevant parenteral medium (Krebs-Ringer Buffer pH 7.4 with albumin (4%) [KRB]). AmB state was determined by scanning dissolution medium in UV-visible region, and absorbance measured at 405nm to quantify AmB monomer, at different time points. Conclusion: The in vitro dissolution study revealed a comparable and safer release of monomeric AmB from AmB LNP’s in biorelevant intravenous dissolution media, specifically Krebs-Ringer Buffer (KRB) at pH 7.4. Moreover, the presence of super-aggregated AmB acted as a monomeric depot, enabling the continuous release of monomeric AmB. Figure no. 6 illustrates the following: (A) the dissolution profile of PS40-AmB LNP’s (F8), PS60-AmB LNP’s (F15) and PS60- AmB LNP’s with cholesterol (F18) in biorelevant intravenous medium Krebs- Ringer Buffer (KRB) at pH 7.4; (B) the UV spectra of PS60-AmB LNP’s (F15) at 15 minutes and at 6 hours, demonstrating a gradual increase in the monomeric form of AmB with a corresponding decrease in the super-aggregated state over time without the release of aggregated AmB; and (C) the UV spectra of PS60-AmB LNP’s with cholesterol (F18) at 15 minutes and 6 hours, showing a continuous increase in the monomeric form without the release of aggregated AmB. Example 7: Safety Evaluation of present invention formulation by Hemolysis of RBCs: Safety of AmB LNP’s was evaluated by the hemolysis assay. Anticoagulant treated human whole blood was centrifuged at 1008g for 15 min at 4°C. Erythrocytes were separated, washed and diluted with isotonic PBS (pH 7.4) to obtain a 5% (v / v) working erythrocyte suspension. Dispersions of PS40-AmB LNP’s (F8), PS60-AmB LNP’s (F15), PS60-AmB LNP’s (F18) with cholesterol and micellar AmB, and liposomal AmB diluted with aqueous 5% dextrose solution at concentrations of 25 to 500 μg / mL equivalent of AmB, were added to the erythrocyte suspension and incubated for 1hour at 37°C. Unlysed erythrocytes were removed by centrifugation at 1008g for 15 minutes (min), and absorbance (Abs) of released hemoglobin in the supernatant was measured at 540nm. Hemolysis of samples was compared with the positive control (deionized water) and negative control (PBS) and % hemolysis was calculated using the equation: Hemolysis (%) = (Abs sample – Abs PBS) / (Abs water – Abs PBS) × 100. Conclusion: The cholesterol sparing effect was evaluated by monitoring haemolysis of red blood cells (RBCs) ex-vivo using Liposomal AmB as the non-toxic reference and Micellar AmB as the toxic reference. Haemolysis of <15% with PS40-AmB LNP (F8), PS60-AmB LNP (F15) andliposomal AmB, compared with <5 % with PS60-AmB LNP with cholesterol (F18) confirmed high safety, with marginal superiority over liposomal AmB, illustrated in Figure no.7. Example 8: Safety Evaluation of present invention formulation in In-vivo toxicity study: Swiss albino mice were procured and housed at 22 ± 2˚C and relative humidity 60 ± 5% with 12 h light–dark cycle. The animals were divided into six groups. Each group was fed with standard commercial laboratory chow and supplied with purified water ad libitum. Dose for the test compound and standard drug was selected from the available literature. The animals were injected with (3mg / kg / day) of the different AmB formulations diluted in sterile 5% dextrose in the lateral vein tail. In each experiment, six mice were used. Mice were observed for death after injection and daily thereafter for 7 days. Blood samples (∼0.2 mL) were collected on 7th day post-treatment from retro-orbital plexus at 24 h after injection coagulating at 4 °C and then centrifuged for 10 min at 5000 rpm to collect the serum. Kidney (Cr: Creatinine, and BUN: Blood urea nitrogen) and liver (ALT: Alanine aminotransferase and AST: Aspartate aminotransferase) biochemical parameters were analyzed. Below table illustrates the mortality by treatment AmB LNP’s and comparison with control and marketed formulation. Group Treatment No. of animals Mortality 1 Negative control (No treatment 6 No mortality her evaluated through an in vivo toxicity study in mice, following OECD guidelines. The micellar AmB (Ampholyn®) when administered intravenously, revealed concentration-dependent hepatotoxicity and nephrotoxicity at doses exceeding 1 mg / kg body weight. In contrast, intravenous administration of AmB LNP’s formulated with cholesterol at a dose of 3 mg / kg body weight resulted in no mortality or treatment-related abnormalities, with outcomes comparable to those of marketed liposomal AmB (Lipholyn®). However, administration of AmB LNP’s (PS40) and (PS60) led to mortality after the 4th day of treatment. Furthermore, serum biochemical markers, including renal (creatinine, BUN) and liver (bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase) biomarkers in the AmB LNP-treated mice were comparable to those in the control group and liposomal group, indicating no impairment of liver or kidney function, illustrated in Figure no.8 and 9. Importantly, these findings aligned with the established safety profile of gold-standard liposomal AmB (Lipholyn®). Example 9: In vitro Anti-leishmanial efficacy evaluation (IC50) Infected promastigotes J774A.1 macrophage cell lines were used to generate intracellular amastigote forms of the parasite. Varying dilutions of PS40-AmB LNP’s (F8), PS60-AmB LNP’s (F15), PS60-AmB LNP’s with cholesterol (F18), micellar AmB (Ampholyn®) and liposomal AmB (Lipholyn®) were added to amastigote (2.5 × 105 / well) infected J774A.1 cells seeded in a 96-well plate (5 × 104cells / well), followed by 72 h incubation at 37 °C in a CO2 incubator. At the end of incubation, the formulation containing medium in each well was replaced with 50 μL each of PBS and Steady-Glo reagent, followed by gentle mixing for 1−2 min. Luminescence of each well was measured. Parasitic growth inhibition which is directly proportional to Luciferase activity was measured as luminescence and expressed as relative luminescence unit (RLU). Parasitic growth inhibition was calculated as described: Percentage inhibition (RLUcontrol - RLUtreated / RLUcontrol ) × 100. Conclusion: Figure no.10 illustrates, studies on antileishmanial efficacy of AmB LNP’s which demonstrated significant activity against macrophage-internalized amastigotes of the Leishmania parasite. Notably, the PS60-AmB LNP’s with cholesterol (F18) exhibited significantly lower IC50 values compared to PS40- AmB LNP’s (F8), PS60-AmB LNP’s (F15), micellar AmB (Ampholyn®) and liposomal AmB (Lipholyn®). The PS60-AmB LNP’s with cholesterol (F18) showed an IC50 value that was two-fold lower against intracellular amastigotes compared to liposomal AmB, which is considered the gold standard for treating leishmaniasis. This was achieved without compromising safety, as indicated by comparable CC50values. These results highlight the potential of monomeric AmB LNP’s as highly promising nanoformulations, offering superior efficacy and safety. EXAMPLE 10: Preparation of clear homogeneous preconcentrate Amphotericin B formulations as per present invention: The following formulations were prepared as per present invention. Process: Amphotericin B and excipients were dissolved in DMA using bath sonication for 5 min to obtain a clear preconcentrate. Formulation no. 21 22 23* 24 25 26 27 28 Polyoxyl-40- 1.33 1.33 1.33 3.33 6.66 1.33 1.33 1.33 stearate (PS40) Liii ( n caes e o ane preconcenrae was no cear souon ence no anayze) Formulation no. 29 30 31 32 33 34 36 37 * Cholesterol % 0.66 0.66 0.6 0.66 0.66 0.66 0.66 0.66 w / v 6 i i p y ^ Evaluation of Preconcentrate formulations combination of lipidic agent and monomerizing agent Each Preconcentrate Amphotericin B formulation from 21 to 16 prepared in example 2 were mixed in aqueous 5% dextrose solution (9mL) resulted in instantaneous generation of AmB LNP’s and further analysis were conducted to check nanoparticle size, entrapment efficiency and state of AmB therein. Formulation no. 21 22 23 24 25 26 27 28 * 5.4 .01 gat (* indicates the obtained preconcentrate was not clear solution hence not analyzed) Formulation no. 29 30 31 32 33 34 36 37 Particle Size 104.14±3. 104.07±3 126.9±38. 125.59±3.118.72±2.84.77±9.387.32±3.6 53 .64 - 9 25 07 7 9 0.0 20 mer

Claims

CLAIMS We claim, 1. A homogeneous pre-concentrate composition of drug amphotericin B for preparing instantaneous safer nano-formulation for intravenous infusion administration, said composition comprising: i) 0.132 to 1.33 % w / v Amphotericin B, ii) 0.025 to 6.66 % w / v of lipidic agent, iii) 0.132 to 3.99 % w / v of Monomerizing agent, iv) Solvent dimethylacetamide to quantity sufficient; Characterized by being able to form monomeric and super-aggregated nanodispersion of drug Amphotericin B with drug entrapment efficiency >90%, after contact with an aqueous solution, having particles of an average diameter between 50 nm-200 nm, measured by a brookhaven particle size analyzer.

2. The homogeneous pre-concentrate composition of drug amphotericin B as claimed in claim 1, wherein composition comprising: i) 0.66 to 1.0 % w / v Amphotericin B, ii) 1.0 to 1.33 % w / v of lipidic agent, iii) 0.3 to 0.66 %w / v of Monomerizing agent, iv) Solvent dimethylacetamide to quantity sufficient.

3. The homogeneous pre-concentrate composition for intravenous infusion of drug Amphotericin B as claimed in claim 1, wherein lipidic agent are selected from Polyoxyl-32-stearate, Macrogol-32-glycerides, Polyoxyl-40 stearate, Polyoxyl-32-distearate, Polyoxyl-60 Hydrogenated Castor Oil, Polyoxyl-50- stearate, Polyoxyl-100-stearate, Polyoxyl-150-stearate, Polyoxyl-150- distearate, campesterol, stigmasterol, β-sitosterol.

4. The homogeneous pre-concentrate composition for intravenous infusion of drug Amphotericin B as claimed in claim 3, wherein lipidic agent are selected1from Polyoxyl -40-stearate, Polyoxyl-32-stearate, Macrogol-32-glycerides, Polyoxyl-60 Hydrogenated Castor Oil and mixture thereof.

5. The homogeneous pre-concentrate composition for intravenous infusion of drug Amphotericin B as claimed in claim 1, wherein monomerizing agent is selected from cholesterol.

6. The homogeneous pre-concentrate composition for intravenous infusion of drug Amphotericin B as claimed in claim 1, wherein the ratio of lipidic agent and monomerizing agent is between 1:0.5 to 1:3 by weight.

7. The homogeneous pre-concentrate composition for intravenous infusion of drug Amphotericin B as claimed in claim 6, wherein the ratio of lipidic agent and monomerizing agent is between 1:1 to 1:2 by weight.

8. The homogeneous pre-concentrate composition for intravenous infusion of drug Amphotericin B as claimed in claim 1, wherein composition comprising 0.001 to 2.0 % w / v of an antioxidant selected from Alpha-tocopherol, Ascorbyl palmitate, Monothioglycerol.2

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