| Title of Invention | COMPOSITION AND METHODS OF NONIONIC SURFACTANT BASED VESICULAR FORMULATION FOR IMPROVED DELIVERY OF CYCLOSPORINE. |
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| Abstract | The present invention relates to a composition and methods of nonionic surfactant based vesicular formulation for improved delivery of cyclosporine. Particularly, the present invention relates to nonionic surfactant based vesicular formulation containing cyclosporine an immuno-suppressant widely used in organ transplantation for better patient compliance. Further more the present invention relates to prepare a pharmaceutically acceptable surfactant based vesicular formulation of cyclosporine with a high drug payload, which is essentially free from alcohol and cremophor (polyoxyethylated ester of castor oil) and essentially devoid of natural vegetable oil, which are prone to form flakes or jelly like structures on storage. The present invention further relates to a method for the preparation of nonionic surfactant based vesicular formulation of cyclosporine for improved absorption |
| Full Text | Composition and methods of nonionic surfactant based vesicular formulation for improved delivery of cyclosporine Field of invention: The present invention relates to a composition and methods of nonionic surfactant based vesicular formulation for improved delivery of cyclosporine. Particularly, the present invention relates to nonionic surfactant based vesicular formulation containing cyclosporine an immuno-suppressant widely used in organ transplantation for better patient compliance. Further more the present invention relates to prepare a pharmaceutically acceptable surfactant based vesicular formulation of cyclosporine with a high drug payload, which is essentially free from alcohol and cremophor (polyoxyethylated ester of castor oil) and essentially devoid of natural vegetable oil, which are prone to form flakes or jelly like structures on storage. The present invention further relates to a method for the preparation of nonionic surfactant based vesicular formulation of cyclosporine for improved absorption. Background of invention: Cyclosporine, a group of nonpolar cyclic oligopeptides with immunosuppressant activity widely used in organ transplantation, are known to be very poorly soluble in water and are also, known P-gp substrate, thus exhibit low therapeutic index with poor and variable bioavailability when administered orally. This reduced bioavailability is also attributed to involvement of P-gp (P glycoprotein) located in the intestine which efflux the CsA molecule back into GI lumen and thus decreases the blood concentration. It is also the case that blood/blood-serum cyclosporine levels achieved using available dosage systems exhibit extreme variation between peak and trough levels. That is, for each patient, effective cyclosporme levels in the blood vary widely between administrations of individual dosages. Because of poor and variable bioavailability daily dosages needed to achieve the desired blood levels need to be varied considerably in the existing dosage forms of cyclosporine and a concomitant monitoring of blood levels is essential. This adds an additional cost to the therapy. This variation in patient response has been found to be attributable to a significant extent to variation in the availability of naturally occurring surfactant components, e.g. bile acids and salts, within the gastro-intestinal tract of the subject treated, since cyclosporine is reported to have inhibitory effect on bile salt synthesis and moreover it reduces the efflux of bile from liver which is required for efficient absorption of cyclosporine. For the proposed niosomal / proniosomal formulations, the presence of such natural surfactants in sufficient quantity as an integral component along with other nonionic surfactants and at least one lipid may help to achieve satisfactory absorption of cyclosporine. Number of efforts have been made to develop cyclosporine formulations both i.v and orally. However oral formulation is preferred due to patient compliance. Intravenous administration of cyclosporine results in anaphylactic reactions but these side effects are not reported when it is administered orally. Oral formulations of cyclosporine, which have been developed, are currently marketed both as soft gelatin capsule and solution under the trademark of SANDIMMUNE and NEORALS. All the earlier approaches to enhance the bioavailability of cyclosporine were towards making the drug in emulsified form or substantially increasing the surface area by converting into micro-emulsion. The present invention overcomes the problems of existing formulations (described above) such as micelle formulations, emulsions, and microemulsions, liposomes, by providing unique triglyceride, oil and moreover cremophor free which are likely to be toxic or manifest instability problem in one or other way. Surprisingly, the present invention have found that compositions including a combination of a hydrophobic surfactant, nonionic surfactant, lipid and charge inducers which have capability of forming vesicles and to encapsulate therapeutically effective amounts of hydrophobic therapeutic agents like mainly cyclosporine A without recourse to the use of triglycerides, thereby avoiding the lipolysis dependence and other disadvantages of conventional formulations. Use of these formulations results in an enhanced rate and/or extent of absorption of the cyclosporine A and it can be extrapolated to other poorly soluble drugs. In one of our invention, it provides a pharmaceutical composition including a surfactant, cholesterol, charge inducer, bile salt and cyclosporine. The composition includes a nonionic surfactant, cholesterol, positive charge fatty acids and a ionic surfactant in amounts such that upon solvent evaporation in the presence or absence of inert excipients followed by hydration forms a clear free flowing granules which can be easily filled into capsule and further hydration it gives milky vesicular dispersion of therapeutic agent. It is a particular feature of the present invention that the carriers are substantially free of alcohol and cremophor, thereby providing surprising and important advantages over conventional formulations. Prior Art Cyclosporine A is basically an oligo undecapeptide, which is most successful agent in the treatment of organ rejection till date. Several formulation of this drug is known till date. Some of which are described below. Tarr & Yalkowsky (1989) has reported the intestinal absorption of cyclosporine measured in-situ in rats using an olive oil emulsion prepared by either stirring or homogenization. The apparent permeability of cyclosporine from the homogenized emulsion was about twice that of the emulsion prepared by stirring. The examination of absorption in different intestinal segment lengths suggested the presence of an "absorption window." The absorption of cyclosporine appeared to be concentration independent and, therefore, non-carrier mediated. The dependence of absorption upon the intestinal perfusion rate suggested that the stagnant aqueous layer is the rate limiting barrier in cyclosporine absorption. Abdallah and Mayersohn (1991) have reported tablet formulation prepared by direct compression and compared with the commercial oil solution of cyclosporine placed into soft gelatin capsules. No differences were observed in the pharmacokinetics of the intravenously administered CsA in the two formulations and proposed tablet formulation for CsA is equivalent in dogs to the commercial dosage form placed into soft gelatin capsules. (Sato et al, 1994) has reported cyclosporine derivative, dihydrocyclosporine D. This was evaluated with milk fat globule membrane (MFGM) as an emulsifier of lipophilic cyclopeptides. As compared with olive oil formulation, MFGM emulsion significantly enhanced the blood and lymphatic fluid concentrations of the cyclosporine derivative after intraduodenal dosing in rats. Thus, it was suggested that MFGM can be used as an intestinal absorption enhancer of cyclosporines. Benmoussa et al (1994) has reported effect of non-absorbable fat substitutes, sucrose polyester (SPE) and tricarballylate triester (TCTE)) on cyclosporin A (CsA) intestinal absorption. This study was conducted in rats using in-situ perfusion and gastric intubation techniques and confirmed whole blood CsA concentrations significantly decreased when administered with SPE and TCTE in comparison with olive oil (p Manconi et al (2002) has reported Tretinoin-loaded niosomes prepared from polyoxyethylene (4) lauryl ether, sorbitan esters and a commercial mixture of octyl/decyl polyglucosides, in the presence of cholesterol and dicetyl phosphate. Liposomes made of hydrogenated and non-hydrogenated phosphatidylcholine were also compared with reference. Release data showed that tretinoin delivery is mainly affected by the vesicular structure and that tretinoin delivery increased from MLVs to LUVs to SUVs. (Guinedi et al., 2005) have reported a possible approach to improve the low corneal penetration and bioavailability characteristics shown by conventional ophthalmic vehicles. Niosomes formed from Span 40 or Span 60 and cholesterol in the molar ratios of 7:4, 7:6 and 7:7 were prepared using reverse-phase evaporation and thin film hydration methods. The prepared systems were characterized for entrapment efficiency, size, shape and in-vitro drug release. Multilamellar acetazolamide niosomes formulated with Span 60 and cholesterol in a 7:4 molar ratio were found to be the most effective and showed prolonged decrease in Intraocular pressure (IOP). After a lot of research carried out on cyclosporine absorption and bioavailability from several years, but still there is no complete and perfect formulation for cyclosporine in terms of bioavailability and safety terms in the market it is still remained as enigma, after some time the research as oriented towards to provesicular approach. (Ahn et al, 1995) has proposed the proliposomes and they are free flowing particles which are composed of drugs, phospholipids and a water soluble porous powder, and immediately form a liposomal dispersion upon hydration. The preparation can be stored sterilized in a dried state and, by controlling the size of the porous powder in proliposomes, relatively narrow range of reconstituted liposome size can be obtained. Because of these properties, proliposomes appear to be a potential alternative to liposomes in design and fabrication of liposomal dosage forms. They are prepared by the penetration of a methanol-chloroform solution of propranolol hydrochloride and lecithin into microporous sorbitol, with subsequent vacuum drying. They were characterized for surface morphology and flowability, and following the conversion to liposomes upon hydration, size distribution, drug content and in vitro drug release of the reconstituted liposomes were examined and said proliposomes can be potential candidates for the sustained drug delivery of propranolol when applied directly onto the mucosal membranes. Ritschel et al (1990) has proposed that rat as a suitable model for pharmacokinetic and bioavailability studies of cyclosporine A (CsA). Two peroral formulations in the form of microemulsions were compared with a commercially available P.O. solution (to be diluted for administration) and a solution for intravenous administration. Of the two microemulsions, one resulted in an extent of absolute and relative bioavailability significantly higher than that of the available P.O. solution. Biliary recycling was observed upon all routes of administration. If uncorrected for biliary recycling, both absolute and relative bioavailability is overestimated. Nicholas et al (1985) has reported procedure for the preparation of a dry, free-flowing granular product which, on addition of water, disperses/dissolves to form an isotonic liposomal suspension, suitable for administration either intravenously or by other routes. Various parameters have been investigated including the suitability of sorbitol and sodium chloride as carrier materials, the nature of the lipid(s) in the formulation, and the extent of lipid loading onto the carrier. These factors are shown to have a marked effect on both the dry product and the hydrated liposomes. Baillie et al (1988) reported Non-ionic surfactant vesicles, niosomes, as a delivery system for the anti-leishmanial drug, sodium stibogluconate. Hunter et al (1988) has reported vesicular systems (niosomes and liposomes) for delivery of sodium stibogluconate in experimental murine visceral leishmaniasis. Improved doxorubicin pharmacokinetics and tumoricidal activity has been reported by Uchegbu et al (1995; 1996) after a single intravenous dose of lOmg kg"1 doxorubicin loaded sorbitan monostearate (Span 60) and hexadecyl diglycerol ether) based niosomes in the mouse adenocarcinoma (MAC) tumour model. Fang et al, (2001) has established the data regarding proniosomal gel where the encapsulation efficiency and size of niosomal vesicles formed from proniosomes upon hydration were also characterized. The encapsulation (%) of estradiol in proniosomes with span surfactants showed a very high value of about 100%. Proniosomes with Span 40 and Span 60 increased the permeation of estradiol across skin. Alsarra et al (2005) has reported Proniosomes as a drug carrier for transdermal delivery of ketorolac. Conacher et al (2001) has reported the ability of non-ionic surfactant vesicles to induce systemic immune responses in mice following oral immunisation. This was studied using a standard antigen (bovine serum albumin), a synthetic measles peptide and an influenza sub-unit vaccine. The effectiveness of this formulation was significantly increased by incorporating bile salts (in particular deoxycholate) into the formulation. Mann et al, (2006) has reported the bilosomes as Protein antigens administered via the oral route are exposed to a hostile environment in the gastrointestinal tract, consisting of digestive enzymes and a range of pH (1-7.5). Using a delivery system can afford protection to entrapped components against degradation and permit delivery of antigen to the cells responsible for generating local and systemic immune responses. Oral preparations [U S Patent 5,980,939] such as soft and hard capsules containing cyclosporine, an oil component, a hydrophilic cosurfactant consisting of propylene carbonate or a mixture of propylene carbonate and polyoxyethylene-polyoxypropylene block copolymer, and a surfactant. A pharmaceutical composition [U S Patent 6,106,860] containing, cyclosporine, partial esters of fatty acids with a glycerol derivative has been described. Pharmaceutical compositions comprising a cyclosporine, a fatty acid triglyceride, a glycerol fatty acid partial ester or propylene glycol or sorbitol complete or partial ester has been described in U S Patent 5,759,997. A micro-emulsion concentrate [U S Patent 5,639,474; U S Patent 5,603,951] containing cyclosporine as an active ingredient, dimethylisosorbide as a cosurfactant, oil and a surfactant is suitable for the formulation of a soft capsule for oral administration. Dimethylisosorbide was used as a co-surfactant or a hydrophilic phase along with other ingredients to enhance the absorption of cyclosporine [European Patent App. Nos. 94110184.2, 95117171.9 and PCT/EP95/04187]. One of the most significant attempts to improve bioavailability of cyclosporine from its dosage form is described in U.S. Pat. No. 5,342,625. This art describes use of microemulsion pre-concentrate consisting of a three phase systems i.e. (1) a hydrophilic phase component (2) a lipophilic phase component and (3) a surfactant. Such composition has alcohol as an essential ingredient. Such composition upon dilution with water provides an oil-in-water microemulsion with an average particle size of less than 1000 ANG. Such an enhanced surface area results in increased bioavailability of cyclosporine when compared with conventional dosage forms. A comparison of bioavailability from micro-emulsion dosage form (U.S. Pat. No. 5,342,625) with the conventional ethanol-oil based dosage form earlier reported (U.S. Pat. No. 4,388,307) has been performed in healthy human volunteers with enhanced bioavailability 149.0% (± 48) compared to other compositions. Some of the later U.S. Patents (Nos. 5,866,159, 5,916,589, 5,962,014, 5,962,017. 6,007,840 and 6,024,978) also pertain to oil-in-water microemulsion compositions having particle size less than 2000 ANG. Oral compositions using monoglycerides, diglycerides and triglycerides along with castor oil derivatives is disclosed in U.S. Pat. No. 5,977,066. Sherman Bernard Charles (WO9848779) claim an emulsion preconcentrate comprising a cyclosporine dissolved in a solvent system comprising acetylated monoglycerides, and a surfactant. A homogeneous cyclosporine formulation (U S Patent 6,916,785) for oral administration in the form of emulsion containing cyclosporine A, triglycerides, polyethylene glycol, non-ionic surfactant and a viscosity reducing agent has been reported. An oil-in-water microemulsion has been described [U S Patent 5,929,030] containing cyclosporine wherein fully dissolved in the dispersed oil particles. The oil is fatty acid vegetable oil glycerides, and lecithin and another surfactant are included to form and stabilize the microemulsion in which the hydrophilic phase comprises propylene glycol. The U.S. Patent (No. 6,022,852) discloses the Cyclosporine A formulations using tocopherol polyethylene glycol 1000 succinate for improved absorption. Hong et al. (U.S. Pat. No. 6,028,067) disclose the use of lipophilic solvent chosen from an alkyl ester of polycarboxylic acid and a carboxylic acid ester of polyols, along with an oil; and a surfactant to form microemulsion preconcentrate of cyclosporine A. A formulation comprising a cyclosporine in admixture with at least one mono or triglyceride of a fatty acid is sufficient to dissolve the cyclosporine. The resulting solution can then easily be emulsified in water or an aqueous fluid (U.S. Pat. No. 4,990,337). Other galenic improvements in cyclosporine emulsion formulations include the use of tocopherol derivatives (EP 0724452), tocopheryl polyethylene glycol carboxylic acid ester (EP 0712631), dimethylisosorbide (EP 0711550, EP 0650721), alkylene polyether or polyester (WO 9423733), emulsion compositions (EP 0694308), anhydromannitol oleylether, lactoglyceride, citroglycerides (EP 656212), phosphatidyl ethanolamine (EP 0651995), as surfactants and stabilizers etc. A pharmaceutical composition has been described (U S Patent 5,998,365) in the form of a microemulsion pre-concentrate comprising a cyclosporine dissolved in a solvent system and hydrophobic component, such as tocol, tocopherols, tocotrienols, and derivatives, a hydrophilic component such as propylene carbonate or polyethylene glycol. Several attempts have been made to improve the bioavailibility of cyclosporine. The oral dosage forms known in the market (i.e. those employing ethanol, olive oil as carrier medium in conjunction with Labrafil as surfactant (U.S. Pat. No. 4,388,307) are unpleasant tasting galenic forms. U.S. Pat. No. 4,388,307 also describes a drink solution containing cyclosporine in a base of Labrafil, Miglyol, Ethanol, Corn/olive oil. However, such preparation suffered from the draw back that it could be presented only as a liquid for dilution in drinking water/fluid before use; otherwise it is very difficult to give an accurate dose. Han Gua (Chinese Patent No. 94191895.5) explains the active compound of cyclosporine, fatty acid sugar ester and diluents carrier having good bioavailability. However, this compound suffers from the drawback that diluents degrades due to hygroscopicity of sugar ester and the stability is not of desired standards, ("Solid Surfactant Solution of active Ingredients in Sugar Ester" Pharmaceutical Research, Volume 6, No. 11, 1989, 958, and "Application of sucrose laurate a new pharmaceutical excipient, in Peroral formulation of Cyclosporine A "International Journal of Pharmaceutics, Vol. 92, 1993, 197). A stable solution of cyclosporine forming a polar lipid self-emulsifying drug delivery system ("PLSEDDS") typically consists of cyclosporine dissolved in a medium chain monoglyceride of fatty acids and a surfactant (U S Patent 5,858,401). Self- emulsifying oily formulation (SEOF) has been described in U S Patent 5,965,160 comprising an oil component and a surfactant, the SEOF being characterized in that the oil component comprises an oily carrier and a cationic lipid and optionally, a lipophilic oily fatty alcohol. An emulsified composition containing the following components; a lipid-soluble drug and a lipid; glycerol and water; a phospholipid and/or a water-soluble nonionic surfactant has been described in U S Patent 5,338,761. A selfemulsifiable formulation of cyclosporine (US 7056530) consisting of a medium chain triglyceride of caprylic acid and capric acid, and of labrasol, wherein labrasol also serves as a surfactant, which is combined with other selected surfactants, like cremophore RH 40 and/or polysorbate 80 and alcohol. Most of the patents reported use alcohol as an essential part of the formulation (US Patent 6,254,885, U S Patent 4,835,002, U S Patent 5,962,019, U S Patent 6,960,563, Russian Patent WO03017947) and the marketed formulation Sandimun (U.S. Pat. No. 4,388,307) and Neoral (U.S. Pat. No. 5,342, 625). These formulations suffer instability problem due to evaporation of low boiling solvent as alcohol. The U.S. Pat. No. 5,639,724 discloses pharmaceutical compositions comprising Cyclosporine, transesterification product of a natural vegetable oil with glycerol, which is exemplified in the specification as MAISINE (transesterification product of corn oil and glycerol), which is an essential component of the compositions. The cyclosporine must be mixed with a transesterfication product of a natural vegetable oil with glycerol. These compositions are not useful as drink solutions because of formation of jelly like lumps, since the transesterfication product is a jelly like substance at room temperature. Such compositions also preferably require the use of alcohol. This composition compares its bioavailability with that of older and inferior compositions based on U.S. Pat. No. 4,388,307 and does not compare bioavailability with a more recently marketed compositions (NEORAL) as defined in U.S. Pat. No. 5,342,625 ; U.S. Pat. No. 5,639,724 discloses the use of Labrafil as a preferred ingredient to be added to the composition of cyclosporine and MAISINE for a drink solution. However, this patent does not address the problem of flake like substances formed by the presence of MAISINE even though Labrafil has been added to the composition. Several other patents reported use of cremophor as essential component to solubilize Cyclosporine in the developed formulation (U S Patent 7,026,290; European Patent US7056530; Russian Patent RU2207870; U S Patent 6,346,511, World wide WO0222158) and the marketed formulation Sandimun (U.S. Pat. No. 4,388,307) and Neoral (U.S. Pat. No. 5,342, 625). These formulations suffer from severe toxicity imposed by cremophor, which is reported to be neurotoxic. However its toxicity is not to that extent when given orally. But the attempt has been made to avoid cremophor. All the earlier approaches to enhance the bioavailability of cyclosporine were towards making the drug in a emulsified form (U.S. Pat. No. 4,388,307) or substantially increasing the surface area by converting into microemulsion (U.S. Pat. No. 5,342,625, Japan Patent JP 2001122779). Other approaches used the preparation of liposomal formulation (U S Patent 5,688,525, U S Patent 5,683,714; which unilamellar liposomes comprising phosphatidylcholine, phosphatidylglycerol and a Cyclosporine, U S Patent 5,670,166; which comprises dissolving a combination of a neutral and negatively charged phospholipids and a Cyclosporinee in an organic solvent; drying the solution to form a solid phase, and hydrating the solid phase in an aqueous solution having a pH ranging from 7.5 to 9.5. [U S Patent 5,656,287]; comprising of phosphatidylcholine, cholesterol, dimyristoylphosphatidylglycerol and a cyclosporine. The lipids, which have been used in the preparation of liposomes are very costly and moreover they are very prone to oxidation. Freeze dried liposome mixture containing cyclosporine provides a freeze-dried liposome mixture having an amphipathic lipid and a cyclosporine or derivative thereof for use in possible liposome delivery of cyclosporine into cells [U.S. Pat. No. 4,963,362]. An improved liposomal cyclosporine therapeutic formulation [U S Patent 5,656,287], comprising phosphatidylcholine, cholesterol, dimyristoylphosphatidylglycerol and a cyclosporine. The formulations are useful as immunosuppressive agents and enhancers of antineoplastic agents in drug resistant cancer cells. A high dose pharmaceutical liposome aerosol composition comprising of a pharmaceutical compound such as cyclosporine A, and a phospholipid starting reservoir concentration are also reported [U S Patent 5,958,378]. A cyclosporine(s)-containing pharmaceutical preparation for oral application is [U S Patent 5,637,317] comprising cyclosporine, natural oil, 3-sn-phosphatidyl choline and phosphatidyl ethanol amine, and water, with the proviso that ionic and non-ionic surfactants are excluded. A process for preparing a liposomal cyclosporine therapeutic formulation, which comprises dissolving a combination of a neutral and negatively charged phospholipid and a cyclosporine in an organic solvent; drying the solution to form a solid phase, and hydrating the solid phase in an aqueous solution [U S Patent 5,688,525]. An improved liposomal cyclosporin therapeutic formulation, comprising phosphatidylcholine, phosphatidylglycerol and a cyclosporine is described in the literature [U S Patent 5,683,714; U S Patent 5,670,166]. The formulation includes unilamellar vesicles having reduced toxicity. A cyclosporine(s)-containing pharmaceutical preparation for oral application is disclosed [U S Patent 5,529,785] comprising cyclosporine, natural oil of natural origin, phosphatidyl choline and phosphatidyl ethanol amine, and water. Other approaches to deliver the cyclosporine comprises Cyclosporine entrapped microspheres or nanospheres in a biodegradable polymer poly (lactide) [U S Patent 5,641,745] such that the cyclosporine is substantially in an amorphous state. Nanoparticulate formulation in the range of 2000nm has also been used to improve the bioavailability [WO2006110802]. Robert Floc'h et al. (U.S. 5,827,822) disclosed aqueous suspension of amorphous cyclosporine nanoparticles wherein at least 50 percent of the drug is present as particles of less than about 1 micrometer. Cyclosporin A formulations are provided as amorphous nanoparticle dispersions [U S Patent 5,827,822] for physiologic absorption as concentrates comprising lower alkanols and a polyoxyalkylene surfactant. A controlled release formulation containing cyclosporine entrapped in a biodegradable polymer (poly-D, L-lactide or a blend of poly-D, L-lactide and poly-D, L-lactide-co-glycolide) to form microspheres or nanospheres [U S Patent 5,641,745]. A novel cyclosporine formulation has been described [US2006205639], which is a pro-nanodispersion at room temperature, featuring solid particles of a relatively large particle size (at least about 150 nm) and yet which is a nanodispersion at body temperature. A powder dosage form of cyclosporine possessing comparatively higher stability and to some extent bio-availability was also reported [Chinese Patent 9419189.5, EP 0702562]. The blood level arising out of such product has been compared with the standard formulations (U.S. Pat. No. 4,388,307) showed significant improvement in bioavailability. However, if compared with the micro-emulsion based formulations these formulations do not show any advantage as the drug is adsorbed on solid surface and needs an additional process of dissolution prior to become bioavailable. The effect of sucrose laurate-on the gastrointestinal absorption of cyclosporine is also described (Lerk-PC; Sucker-H, International Journal of Pharmaceutics; 1993; 92; 197-202). The evaluation of the dosage form containing sucrose Laurate was found to enhance the in vitro absorption of cyclosporine when normal epithelial tissue and Peyer's patch tissue of guinea pigs were used. Compared to the commercially available drinking solution, absorption was raised by a factor of 10. It was concluded that preliminary formulation experiments showed that a solid oral dosage form of cyclosporine could be made using sucrose laurate as an excipient. Several tablet formulations of cyclosporine [Abdallah-H Y; Mayersohn-M. Pharmaceutical Research; 1991, 8, 518-522] were prepared and comparatively examined with the commercial oil solution placed in a soft gelatin capsule in vitro and in dogs in a randomized crossover study. Cyclosporine may be rendered more soluble by the concomitant administration of alpha.-cyclodextrin, either separately, but essentially simultaneously or, preferably, in admixture [U.S. Pat. No. 5,051,402]. Bile salts used in formulations as said in this invention. Ionic surfactants, including cationic, anionic and zwitterionic surfactants, are suitable hydrophilic surfactants for use in the present invention. Preferred anionic surfactants include fatty acid salts and bile salts. Specifically, preferred ionic surfactants include sodium oleate, sodium cholate, and sodium taurocholate. Description of the Related Art Pharmaceutical formulations may be administered through various routes of administration. For example, drugs may be administered orally and intravenously. The encapsulation of pharmaceuticals in bilosomes and proniosomes is useful in reducing toxicity and improving the therapeutic effectiveness of cyclosporine. Further, this can be applied to certain other drugs for example, insulin, heparin, cyclosporine and other drugs belonging to class IV of BCS classification etc., after encapsulation into proniosomes and bilosomes. Although they represent an improvement over the prior art, oral niosomal dispersion based formulations have been criticized because of their physical instability (e.g. aggregation, leakage, sedimentation) and potential destruction in gastric fluids. The use of niosomal formulations represents an alternative to liposomal formulations. The lipids used in the liposomal formulations are prone to oxidation and denaturation. The nonionic surfactants used in the preparation of niosomal formulations are more stable. The surfactant, e.g. a GRAS surfactant, is preferably approved by the FDA. The use of proniosomes represents an alternative to conventional niosomal formulations. Proniosomes are dry, free-flowing granular products, which, upon the addition of water, disperse to form a multilamellar bilosomal or niosomal suspension. The stability problems associated with conventional niosomes, including aggregation, susceptibility to hydrolysis and oxidation may be avoided by using proniosomes and bilosomes. Additionally the alternative nonionic surfactants (sorbitan fatty acid esters) are more stable compared to phospholipids, generally used in liposomal formulations. Further, the Proniosomal formulation is coated with enteric polymer overcomes the disadvantages of drug delivery systems known in the prior art. For example, the utility of previous systems for orally administering labile pharmacological substances has been limited by the need to use toxic amounts of delivery agents, the instability of the systems, the inability to protect the active ingredient, the inability to effectively deliver drugs that are poorly water soluble or labile, the inadequate shelf life of the systems, the failure of the drug delivery systems to promote absorption of the active agent and the difficulties inherent to manufacturing the systems. Among the various routes of drug administration, the oral route is preferred because of its ease of administration, safety and patient compliance. However, the therapeutic efficacy of many drugs in case of oral administration is reduced because many pharmaceuticals of class IV category under BCS exhibit limited absorption through intestinal membrane. The limited absorption and thus bioavailability may be due to extensive metabolism and/or efflux of the molecule (P-gp substrate) into GI lumen. Moreover, many drugs are labile or inactivated under the acidic conditions of the stomach. Contrary to using p-gp inhibitors, the problem of efflux can be addressed by increasing the concentration of solubilized drug at absorption site and thereby increasing the rate of flip flop of the molecule across inner and outer leaflet of the intestine membrane so that efflux pump can not keep a pace with it and concentration gradient is not established. Additionally employing bile salt as integral component of the formulation increases the absorption of the drug and further this can counteract CsA induced cholestasis. Bile acids are naturally occurring surfactants. They are a group of compounds with a common "backbone" structure based on cholanic acid found in all mammals and higher vertebrates. The number and orientation of hydroxyl groups substituted onto a steroidal nucleus largely determine the detergent properties of bile acids. Bile acids may be mono-, di- or tri-hydroxylated; they always contain a 3-.alpha, hydroxyl group, whereas the other hydroxyl groups, most commonly found at C.sub.6, C.sub.7 or C.sub. 12, may be positioned above (beta.) or below (alpha.) the plane of the molecule. Enteric coating materials have been applied to address the problem of instability in acidic conditions. Enteric coating materials are those that ensure that acid-labile drugs remain active in the stomach upon oral ingestion such that the active ingredient is released and absorbed in the intestine. Enteric coatings materials are well known in the pharmaceutical art and include alginates, alkali-soluble acrylic resins, hydroxy propyl methylcellulose phthalate, cellulose acetate phthalate, and the like. Although the use of proniosomes and bilosomes are independently known in the art, the delivery of cyclosporine using bile salt as an integral component in the niosomal formulation has not been disclosed. Surprisingly, when bile salt is incorporated as an integral component in the formulation, drug delivery is enhanced. In many embodiments of the present invent, this novel and unexpected enhancement, which results from the unique combination of a bile salt and other nonionic surfactants in the formulation, relates to increased drug absorption and bioavailability and is mostly useful to deliver proteins, peptides and antigens and antibodies as bile salt helps in enhanced absorption due to its enterohepatic circulation and receptors present in the small intestine. In many embodiments of the current invention, the combination of a coating and a proniosomal and bilosomal formulation overcomes the disadvantages of drug delivery systems known in the prior art. For example, the utility of previous systems for orally administering labile pharmacological substances has been limited by the need to use toxic amounts of delivery agents, the instability of the systems, the inability to protect the active ingredient, the inability to effectively deliver drugs that are poorly water soluble or labile, the inadequate shelf life of the systems, the failure of the drug delivery systems to promote absorption of the active agent and the difficulties inherent to manufacturing the systems. Our attempt has been to prepare a surfactant based vesicular formulation, which is essentially free from alcohol and cremophor (polyoxyethylated ester of castor oil) and essentially devoid of natural vegetable oil, which are prone to form flakes or jelly like structures on storage. Moreover it has been reported that endogenous bile salt being a necessary component for efficient absorption of cyclosporine. As shown in the results of Venkataramanan et al (1986) the absorption of cyclosporine was severely impaired in dogs when bile flow was diverted. Apart from this there is a profound inhibitory effect of cyclosporine on bile salt synthesis in vivo. In a study, a daily treatment with CsA for 1 week led to 50% reduction of total bile salt synthesis in rats, as determined by the washout technique applied to anesthetized animals (Chan and Shaffer, 1997). This relationship seems to be intriguing as bile is necessary for the efficient absorption of cyclosporine on one hand and inhibitory effects of cyclosporine on bile salt synthesis on the other. Therefore we have attempted to provide bile as a integral component of the formulation (surfactant vesicles) in a comparable concentration which exists physiologically in small intestine (10-15mM). The presence of sodium deoxycholate or taurodeoxycholate in the concentration of 15 mM increases the CsA aqueous solubility, a value; 400 fold larger than that of CsA in pure water. These findings confirm the major role of bile salts in CsA absorption as observed in clinical practice by the increased absorption of CsA from the oily formulation after restoration of biliary function by T-tube clamping in liver-transplanted patients (Mehta et al, 1988; Naoumov et al, 1989). Similarly, the absorption of CsA from the microemulsion formulation, Neoral, is not completely independent of bile flow. An inverse correlation was observed between the volume of externally drained bile and the bioavailability of CsA (Trull et al, 1995). However bile acids poorly mix with CsA and are not sufficient by themselves to improve CsA absorption significantly. A tight molecular association of the vesicular type seems necessary to ensure a large passage of CsA into the mucosa. Main aim of the present invention is to prepare a pharmaceutically acceptable surfactant based vesicular formulation of cyclosporine with a high drug payload, which is essentially 1. Free from alcohol and cremophor (polyoxyethylated ester of castor oil) 2. Devoid of natural vegetable oil, which are prone to form flakes or jelly like structures on storage. 3. The bile salt is incorporated as an integral component in the formulation drug delivery is enhanced. The novel and unexpected enhancement, results from the unique combination of a bile salt and other nonionic surfactants in the formulation, relates to increased drug absorption and bioavailability as bile salt helps in enhanced absorption due to its enterohepatic circulation and receptors present in the small intestine. 4. In the present invention the structured vehicle (nonionic surfactant vesicles) described herein is able to reduce P-gp mediated efflux, as the presentation of cyclosporine to P-gp transporters could have been modified as shown in the figure-4. 5. Moreover the cyclosporine is reported to inhibit bile synthesis in the liver (causes cholestatsis) and understanding its major role in the absorption of cyclosporine, bile salt has been incorporated as integral component in the formulation to achieve enhanced delivery. Objectives of the invention The main object of this invention is to provide a composition and methods of nonionic surfactant based vesicular formulation for improved delivery of cyclosporine. Yet another object of this invention is to provide bile salt independent cyclosporine formulation having bile salt as integral component. Yet another object of this invention is to provide a pharmaceutically acceptable surfactant based vesicular formulation of cyclosporine with a high drug payload, which is essentially free from alcohol and cremophor (polyoxyethylated ester of castor oil) and essentially devoid of natural vegetable oils. A further object of this invention is to provide a formulation for the use in organ transplantation for immunosuppression. Yet another objective of this invention is to provide niosomal/proniosomal formulation, which may be converted to well define niosomal dispersion when desired. This preparation is in contrast to widely reported formulations comprising microemulsions, liposomes, microparticles, nanoparticles and galenic forms and solutions. The other objective of this invention is based on the reduction of P-gp mediated efflux of CsA and thus increasing the blood concentration of CsA to have improved bioavailability. Summary of the invention: Accordingly the present invention provides a non-ionic surfactant based vesicular formulation for improved delivery of cyclosporine wherein the formulation comprising a pharmaceutically effective amount of cyclosporine, surfactant wherein at least one of the surfactant is non-ionic, lipid, bile salt, charge inducer. In an embodiment of the invention wherein the molar ratio of surfactant: lipid : charge inducer : bile salt: cyclosporine is in the range between 30-65 : 65-30 : 3-6 : 5-15 : 0.5 to 5 respectively. In another embodiment of the invention wherein the surfactant used is selected from a group consisting of sorbitan monolaurate (Span-20), sorbitan monopalmitate (Span-40), sorbitan monooleate (Span-80), sorbitan monostearate (Span- 60), sorbitan trioleate (Span-85), sorbitan tristearate (Span-65) In yet another embodiment of the invention wherein the bile salts used is selected from a group consisting of sodium cholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium taurodeoxy-cholate, sodium glycodeoxycholate, sodium ursodeoxycholate, sodium chenodeoxycholate, sodium taurochenodeoxycholate, sodium glyco chenodeoxycholate, sodium cholylsarcosinate, sodium N-methyl taurocholate. In a further embodiment of the invention wherein the ionic surfactants used is selected from a group consisting of Sodium caproate, Sodium caprylate, Sodium caprate, Sodium laurate, Sodium myristate, Sodium myristolate, Sodium palmitate, Sodium palmitoleate, Sodium oleate, Sodium ricinoleate, Sodium linoleate, Sodium linolenate, Sodium stearate, Sodium lauryl sulfate (dodecyl), Sodium tetradecyl sulfate, Sodium lauryl sarcosinate, Sodium dioctyl sulfosuccinate [sodium docusate.] In still another embodiment of the invention wherein the nonionic surfactant includes sorbitan fatty acid esters but not limited to sorbitan monostearate, sorbitan monopalmitate, sorbitan monooleate and polyethylene glycol derivatized fatty acids. In yet another embodiment of the invention wherein the lipid used is cholesterol. In an embodiment of the invention wherein the inducer used is selected from a group consisting of stearylamine , dicetyl phosphate. In still further embodiment of the invention wherein the entrapment efficiency of cyclosporine is more than 90%. In an embodiment of the invention wherein the formulation exhibit greater bioavailability of Cyclosporine as compared with Sandimmun Neoral as indicated by the higher AUC values of the subject formulations 72.8±27.9 [Figure 1-3 and Table -1]. In an embodiment of the invention wherein the relative bioavailability of Cyclosporine formulation AUCtest/AUCmarket is 1.73 (173%). In an embodiment of the invention wherein the in-vitro absorption of cyclosporine through everted gut sac technique, the degree of efflux is reduced 2-3 fold. In an embodiment of the invention wherein the formulation can be administered through oral, parenteral route. In an embodiment of the invention wherein the vesicular formulation is in the form selected from a group consisting of , bilosomes, proniosomes, enteric coated proniosomes. In an embodiment of the invention wherein the formulation is with a high drug payload essentially free from alcohol and cremophor (polyoxyethylated ester of castor oil) and essentially devoid of natural vegetable oils. In an embodiment of the invention wherein the formulation is for the use in organ transplantation for immunosuppression. Accordingly the present invention provides a process for preparation if formulation wherein the process steps comprising; (a) dissolving cyclosporine, surfactant, cholesterol, charged inducer in a molar ratio ranging between 0.5 to 5: 30 to 65: 65 to 30: charge inducer 3 to 6 respectively in a mixture of methanol and chloroform, (b) evaporating the solvent from the above mixture obtained in step (a) using rotary vacuum evaporator and drying in dessicator to obtain a film, (c) hydrating the film with plain aqueous solution containing bile in a molar ratio ranging between 5-15, at a temperature higher than 60°C. to obtain bilosomes, (d) alternatively, spraying the mixture obtained as mentioned in (a) on inert material followed by evaporation of solvent using rotary vacuum evaporator and drying in a dessicator to leave free flowing granules of proniosomes. In an embodiment of the invention wherein the inert material includes but not limited to sorbitol, lactose and maltodextrins The current invention relates to a drug delivery system comprising at least one pharmaceutically active agent, at least one nonionic surfactant (Sorbitan fatty acid esters), one lipid or phospholipid, one bile salt, one additive, one-charge inducers and a coating material. A particular advantage of the current invention is that it provides a simple and inexpensive system to facilitate the administration of medicaments. In many embodiments, this drug delivery system enhances the stability and bioavailability of pharmaceutically active agents. According to one aspect of this invention, the pharmaceutical formulation is administered through various routes including, but not limited to, oral, buccal, sublingual, nasal, topical, transdermal, vaginal, rectal, intravenous, intradermal, intramuscular, subcutaneous, and intraperitoneal. In one aspect of the invention, the method of preparing proniosomes is through freeze-drying. In another aspect, it can be administered in liquid dosage form as bilosomal system where it contains one but not more than two nonionic surfactant (sorbitan fatty acid esters), one bile salt and one charge inducers. In an embodiment, the freeze-drying method includes preparation of an isotropic mixture from non-aqueous solution of nonionic surfactant and lipid along with drug and aqueous solution of inert carrier along with appropriate concentration of bile salt. In another embodiment the nonionic surfactant includes sorbitan fatty acid esters but not limited to sorbitan monostearate, sorbitan monopalmitate, sorbitan monooleate and polyethylene glycol derivatized fatty acids. The inert material may include but not limited to sorbitol, lactose and maltodextrins. In further embodiment of the invention, the pharmaceutically active agent is very poorly water-soluble drug. In an embodiment of the present invention bile salts may be selected from sodium cholate, Sodium taurocholate, Sodium glycocholate, Sodium deoxycholate, Sodium taurodeoxycholate, Sodium glycodeoxycholate, Sodium ursodeoxycholate, Sodium chenodeoxycholate, Sodium taurochenodeoxycholate Sodium glyco chenodeoxycholate Sodium cholylsarcosinate Sodium N-methyl taurocholate. In an embodiment of the present invention surfactants used may be selected from Sorbitan monolaurate (Span-20), Sorbitan monopalmitate (Span-40), Sorbitan monooleate (Span-80), Sorbitan monostearate (Span-60), Sorbitan trioleate (Span-85), Sorbitan tristearate (Span-65) In an embodiment of the present invention the ionic surfactants used may be selected from Sodium caproate, Sodium caprylate, Sodium caprate, Sodium laurate, Sodium myristate, Sodium myristolate, Sodium palmitate, Sodium palmitoleate, Sodium oleate, Sodium ricinoleate, Sodium linoleate, Sodium linolenate, Sodium stearate, Sodium lauryl sulfate (dodecyl), Sodium tetradecyl sulfate, Sodium lauryl sarcosinate, Sodium dioctyl sulfosuccinate [sodium docusate (Cytec)] The following examples broadly illustrate the nature of this invention the manner in which it is to be performed without limiting the nature and scope of the invention Example -1 A mixture of cyclosporine A (10mg), span 40 (70mg), stearylamine (3mg) and cholesterol (27mg) were dissolved in a mixture of 2ml of methanol and 8ml of chloroform and whole quantity is taken into round bottom flask and subjected for solvent evaporation at 45° C by heating in steam jacketed vessel and then keeping for 24 hrs in dessicator. The evaporation is carried out in such a way that thin film has formed and it is further hydrated by vortexing with 10ml of phosphate buffer (pH 7.4) at 65°C to form vesicles. The entrapment efficiency was determined by dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency of cyclosporine was found to be 93% that comes to 0.93mg/ml of cyclosporine encapsulated in vesicles. Example-ll A mixture cyclosporine A (10mg), span 40 (70mg), stearylamine (3mg) and cholesterol (27mg) were dissolved in mixture of 2ml of methanol and 8ml of chloroform and whole quantity is taken into round bottom flask and subjected for solvent evaporation at 45°c by heating in steam jacketed vessel and then keeping for 24 hrs in dessicator. The evaporation is carried out such a way that thin film has formed and it is further hydrated by vortexing with 10ml of phosphate buffer (pH 7.4) containing 5mM of sodium desoxycholate at 65°c to form bilosomes. The entrapment efficiency was determined by dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency of cyclosporine was found to be 91% that comes to 0.91mg/ml of cyclosporine encapsulated in vesicles. Example-Ill A mixture cyclosporine A (10mg), span 40 (70mg), stearylamine (3mg) and cholesterol (27mg) were dissolved in mixture of 2ml of methanol and 8ml of chloroform and whole quantity is sprayed on sorbitol (4g) using solvent evaporation by controlling the flow rate at 0.5ml/min, and temperature 65° C. The evaporation is carried out till the granules becomes free flow which are further subjected to standard sieving by passing through sieve-20 and the formed granules are termed as proniosomes. The entrapment efficiency was determined by hydrating a quantity of proniosomal granules with Phosphate buffer (pH7.4) and further dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency of cyclosporine was found to be 90% that comes to 0.90mg/ml of cyclosporine encapsulated in vesicles. Example-IV A mixture cyclosporine A (10mg), span 40 (70mg), stearylamine (3mg) and cholesterol (27mg) were dissolved in mixture of 2ml of methanol and 8ml of chloroform and whole quantity is sprayed on sorbitol (4g) using solvent evaporation by controlling the flow rate at 0.5ml/min, and temperature 65° C. The evaporation is carried out till the granules becomes free flow which are further subjected to standard sieving by passing through sieve-20 and the formed granules are termed as proniosomes. The equivalent weight of granules (4g) were coated with cellulose acetate phthalate (80mg) priorly dissolved in acetone to form free flowing granules (enteric coated proniosomes) which are again subjected to sieving (sieve-20). The entrapment efficiency was determined by hydrating a quantity of proniosomal granules with Phosphate buffer (pH7.4) and further dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency was found to be more than 90%. Example-V A mixture cyclosporine A (10mg), span 60 (70mg), stearylamine (3mg) and cholesterol (27mg) were dissolved in mixture of 2ml of methanol and 8ml of chloroform and whole quantity is taken into round bottom flask and subjected for solvent evaporation at 45° C by heating in steam jacketed vessel and then keeping for 24 hrs in dessicator. The evaporation is carried out such a way that thin film has formed and it is further hydrated by vortexing with 10ml of phosphate buffer (pH 7.4) at 65°C to form vesicles. The entrapment efficiency was determined by dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency of cyclosporine was found to be 94% that comes to 0.94mg/ml of cyclosporine encapsulated in vesicles. Example-VI A mixture cyclosporine A (10mg), span 60 (70mg), stearylamine (3mg) and cholesterol (27mg) were dissolved in mixture of 2ml of methanol and 8ml of chloroform and whole quantity is taken into round bottom flask and subjected for solvent evaporation at 45°c by heating in steam jacketed vessel and then keeping for 24 hrs in dessicator. The evaporation is carried out such a way that thin film has formed and it is further hydrated by vortexing with 10ml of phosphate buffer (pH 7.4) containing 5mM of sodium desoxycholate at 65°c to form bilosomes. The entrapment efficiency was determined by dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency of cyclosporine was found to be 93% that comes to 0.93mg/ml of cyclosporine encapsulated in vesicles. Example-VII A mixture cyclosporine A (10mg), span 60 (70mg), stearylamine (3mg) and cholesterol (27mg) were dissolved in mixture of 2ml of methanol and 8ml of chloroform and whole quantity is sprayed on sorbitol (4g) using solvent evaporation by controlling the flow rate at 0.5ml/min, and temperature 65° C. The evaporation is carried out till the granules becomes free flow which are further subjected to standard sieving by passing through sieve-20 and the formed granules are termed as proniosomes. The entrapment efficiency was determined by hydrating a quantity of proniosomal granules with Phosphate buffer (pH7.4) and further dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency of cyclosporine was found to be 94% that comes to 0.94mg/ml of cyclosporine encapsulated in vesicles. Example-VIII A mixture cyclosporine A (10mg), span 60 (70mg), stearylamine (3mg) and cholesterol (27mg) were dissolved in mixture of 2ml of methanol and 8ml of chloroform and whole quantity is sprayed on sorbitol (4g) using solvent evaporation by controlling the flow rate at 0.5ml/min, and temperature 65° C. The evaporation is carried out till the granules becomes free flow which are further subjected to standard sieving by passing through sieve-20 and the formed granules are termed as proniosomes. The equivalent weight of granules (4g) were coated with cellulose acetate phthalate (80mg) priorly dissolved in acetone to form free flowing granules (enteric coated proniosomes) which are again subjected to sieving (sieve-20). The entrapment efficiency was determined by hydrating a quantity of proniosomal granules with Phosphate buffer (pH7.4) and further dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency was found to be more tan 90%. Example-IX A mixture cyclosporine A (10mg), span 60 (70mg), dicetyl phosphate (3mg) and cholesterol (27mg) were dissolved in mixture of 2ml of methanol and 8ml of chloroform and whole quantity is taken into round bottom flask and subjected for solvent evaporation at 45° C by heating in steam jacketed vessel and then keeping for 24 hrs in dessicator.. The evaporation is carried out such a way that thin film has formed and it is further hydrated by vortexing with 10ml of phosphate buffer (pH 7.4) at 65°C to form vesicles. The entrapment efficiency was determined by dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency of cyclosporine was found to be 92% that comes to 0.92 mg/ml of cyclosporine encapsulated in vesicles. Example-X A mixture cyclosporine A (10mg), span 60 (70mg), dicetyl phosphate (3mg) and cholesterol (27mg) were dissolved in mixture of 2ml of methanol and 8ml of chloroform and whole quantity is taken into round bottom flask and subjected for solvent evaporation at 45°c by heating in steam jacketed vessel and then keeping for 24 hrs in dessicator. The evaporation is carried out such a way that thin film has formed and it is further hydrated by vortexing with 10ml of phosphate buffer (pH 7.4) containing 5mM of sodium desoxycholate at 65°c to form bilosomes. The entrapment efficiency was determined by dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency of cyclosporine was found to be 90% that comes to 0.90mg/ml of cyclosporine encapsulated in vesicles. Example-XI A mixture cyclosporine A (10mg), span 60 (70mg), dicetyl phosphate (3mg) and cholesterol (27mg) were dissolved in mixture of 2ml of methanol and 8ml of chloroform and whole quantity is sprayed on sorbitol (4g) using solvent evaporation by controlling the flow rate at 0.5ml/min, and temperature 65° C. The evaporation is carried out till the granules becomes free flow which are further subjected to standard sieving by passing through sieve-20 and the formed granules are termed as proniosomes. The equivalent weight of granules (4g) were coated with cellulose acetate phthalate (80mg) priorly dissolved in acetone to form free flowing granules (enteric coated proniosomes) which are again subjected to sieving (sieve-20). The entrapment efficiency was determined by hydrating a quantity of proniosomal granules with Phosphate buffer (pH7.4) and further dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency of cyclosporine was found to be 95% that comes to 0.95mg/ml of cyclosporine encapsulated in vesicles. Example-XII A mixture cyclosporine A (10mg), span 60 (70mg), dicetyl phosphate (3mg) and cholesterol (27mg) were dissolved in mixture of 2ml of methanol and 8ml of chloroform and whole quantity is sprayed on sorbitol (4g) using solvent evaporation by controlling the flow rate at 0.5ml/min, and temperature 65° C. The evaporation is carried out till the granules becomes free flow which are further subjected to standard sieving by passing through sieve-20 and the formed granules are termed as proniosomes. The entrapment efficiency was determined by hydrating a quantity of proniosomal granules with Phosphate buffer (pH7.4) and further dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency of cyclosporine was found to be 92% that comes to 0.92mg/ml of cyclosporine encapsulated in vesicles. Example-XIII A mixture cyclosporine A (10mg), span 60 (70mg), dicetyl phosphate (3mg) and cholesterol (27mg) were dissolved in mixture of 2ml of methanol and 8ml of chloroform and whole quantity is taken into round bottom flask and subjected for solvent evaporation at 45°c by heating in steam jacketed vessel and then keeping for 24 hrs in dessicator. The evaporation is carried out such a way that thin film has formed and it is further hydrated by vortexing with 10ml of phosphate buffer (pH 7.4) containing 10mM of sodium desoxycholate at 65°c to form bilosomes. The entrapment efficiency was determined by dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency of cyclosporine was found to be 94% that comes to 0.94mg/ml of cyclosporine encapsulated in vesicles. Example-XIV A mixture cyclosporine A (10mg), span 60 (70mg), dicetyl phosphate (3mg) and cholesterol (27mg) were dissolved in mixture of 2ml of methanol and 8ml of chloroform and whole quantity is sprayed on maltodextrin (4g) using solvent evaporation by controlling the flow rate at 0.5ml/min, and temperature 65° C. The evaporation is carried out till the granules becomes free flow which are further subjected to standard sieving by passing through sieve-20 and the formed granules are termed as proniosomes. The entrapment efficiency was determined by hydrating a quantity of proniosomal granules with Phosphate buffer (pH7.4) and further dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency of cyclosporine was found to be 93% that comes to 0.93mg/ml of cyclosporine encapsulated in vesicles. Example-XV A mixture cyclosporine A (10mg), span 60 (70mg), diacetyl phosphate (3mg) and cholesterol (27mg) were dissolved in mixture of 2ml of methanol and 8ml of chloroform and whole quantity is sprayed on maltodextrin (4g) using solvent evaporation by controlling the flow rate at 0.5ml/min, and temperature 65° C. The evaporation is carried out till the granules becomes free flow which are further subjected to standard sieving by passing through sieve-20 and the formed granules are termed as proniosomes. The equivalent weight of granules (4g) were coated with cellulose acetate phthalate (80mg) priorly dissolved in acetone to form free flowing granules (enteric coated proniosomes) which are again subjected to sieving (sieve-20). The entrapment efficiency was determined by hydrating a quantity of proniosomal granules with Phosphate buffer (pH7.4) and further dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency was found to be more than 87%. Example-XVI A mixture cyclosporine A (10mg), span 60 (70mg), diacetyl phosphate (3mg) and cholesterol (27mg) were dissolved in mixture of 2ml of methanol and 8ml of chloroform and whole quantity is sprayed on maltodextrin (4g) using solvent evaporation by controlling the flow rate at 0.5ml/min, and temperature 65° C. The evaporation is carried out till the granules becomes free flow which are further subjected to standard sieving by passing through sieve-20 and the formed granules are termed as proniosomes. The equivalent weight of granules (4g) were coated with hydroxyl propyl methyl cellulose phthalate (80mg) priorly dissolved in acetone to form free flowing granules (enteric coated proniosomes) which are again subjected to sieving (sieve-20). The entrapment efficiency was determined by hydrating a quantity of proniosomal granules with Phosphate buffer (pH7.4) and further dialyzing the formulation exhaustively against Phosphate buffer (pH 7.4) at 4° C for 24 hrs. The entrapment efficiency was found to be more than 87%. Example-XVII In vivo Bioavailability Studies for Formulations The bioavailability of Cyclosporine in formulations was studied by using the following protocol: Sprague Dawley rats weighing 250-350 gm were fed palletized standard food and water ad-libitum. One day prior to the experiment, silicone rubber cannulae were inserted into the right jugular and right femoral veins under light ether anesthesia. After overnight fast, CsA was administered by gavage. In addition to formulations prepared in the present invention the bioavailability of Cyclosporine in Sandimmune Neoral under analogous conditions was observed for comparison purposes. Following administration, 200 microliter blood samples were collected from the jugular vein in 0.5 ml polypropylene microfuge tubes containing 0.3 mg of lyophilized Na EDTA and vortexed immediately for 10 sec. The sampling times for animals subjected to oral formulations were 0, 0.5, 1, 2, 2.5, 3.5, 5, 7, 17, and 24 hr after administration. Cyclosporine in whole blood was estimated by High Performance liquid Chromatography equipped with column oven maintained at 65°C. Briefly, 250 ul of the blood was transferred to 2ml glass tube. To it 500ul of acetonitrile was added into the series of test tubes and kept aside for 45 minutes after vortexing at room temperature. The precipitated proteins were separated by centrifugation at 11,000xg. Appropriate volume of acetonitrile was aspirated and taken into fresh test tubes and subjected to drying. Finally it was reconstituted with 50ul of acetonitrile and injected into HPLC column (Hana et al., 2000). Various pharmacokinetic parameters were obtained from non-compartmental analysis. The peak concentration (Cmax) and the time at which the peak concentration occurred (Tmax) were estimated by inspection of the raw concentration-time profile for each rat. The area under the blood concentration-time curve (AUC) from time 0 through the last data point (AUC 0-t) was calculated according to the linear trapezoidal procedure. The residual area under the tail of the blood concentration-time curve (AUC t-infin.) was estimated as the ratio of the final observed concentration (C*) to the first-order rate constant associated with the terminal elimination phase of the concentration-time profile (.lambda z). The rate constant lambda z was determined by log-linear regression of the concentration-time data in the apparent terminal log-linear phase of the concentration-time profile (i.e., the final 3 to 5 data points, depending on the profile under analysis). The total AUC (AUC t-infin.) was taken as the sum of AUC 0-t and AUC t-infin. The relative bioavailability of the proposed formulations was calculated as the ratio of AUC of the test and AUC of the marketed formulation. The results of each formulation were compared with results of marketed preparation (sandimmun Neoral). The results demonstrate that, the proposed surfactant based vesicular formulation shows greater bioavailability of Cyclosporine is achieved as compared with Sandimmun Neoral as indicated by the higher AUC values of the subject formulations (Figure Removed) Example-XIX In-vitro absorption of cyclosporine through everted gut sac technique (depicting P-gp efflux phenomenon) The animals were fasted for 12-16 h and were sacrificed by cervical dislocation. A midline incision was made in the abdomen, and the small intestine was removed. After washing with normal saline, underlying muscularis was removed and the intestine was segregated into different segments. The duodenal segment was obtained by cutting 1 cm from the pylorus, the jejunum was obtained by cutting between the duodenum and the ileum, and the ileal segment was cut about 5 cm above the ileo-cecal junction. Each segment was reduced to 5 cm and washed before use. For estimation of permeation of various formulations bearing cyclosporine from the serosal to the mucosal side (secretion), the intestinal segments were used directly. The proximal end of each segment was legated with a glass receptor tube. The distal end was directly legated to create a closed compartment. A 1 g stainless steel weight was tied to the distal end to maintain the sac in a vertical position during the experiment. The segments so prepared were suspended in separate permeation assemblies consisting of 25mL test tubes, which contained 20mL solutions of the test substances in Krebs Ringer buffer (KRB). The segments were dipped in a manner that the whole of the outer wall of the intestine was exposed to the drug solution. The assemblies were maintained at 37°C in a water bath, and the donor compartment in each case was bubbled continuously with oxygen. Once the conditions of all three assemblies were equilibrated, 2mL of the blank KRB was filled into the sacs through the receptor tubes and the same was withdrawn at regular intervals using a hypodermic syringe attached to a long needle, and replaced with fresh buffer. The samples were collected until 90 min, filtered and diluted with absolute ethanol then analyzed by HPLC. The efflux has been observed in the order of ileum >jejunum > duodenum [figure 4]. This behaviour parallels the concentration of P-gp transporters in GI tract in the order of ileum >jejunum > duodenum. The efflux has also been observed in the case of proposed formulations but the degree of efflux has been reduced 2-3 fold. (Figure Removed) Figure : 4 In-vitro efflux studies using normal oriented gut sac (from serosal to mucosal). * Values of apparent permeability coefficient have been represented as JO'7 cm/sec. Control (oily formulation : Depicts the phenomenon of p-gp efflux compared to oily formulation Form. -1 : SP 60 :Chol:SA (64.5:30.5:5m.r) containing 0 mM BS (sodium deoxycholate) Form. -2 : SP 60 :Chol:SA (64.5:30.5:5m.r) containing 10 mM BS (sodium deoxycholate) Advantages of the present invention Keeping in mind the fact of all the research, we have developed niosomal, bilosomal, proniosomal formulations of cyclosporine A so that it provides all the advantages such as it would be free from oil, triglycerides, and toxic excipients like cremophor varieties. As our invention involves the formulations superior in all the aspects particularly, the present invention also provides: a pharmaceutical composition in accordance with the invention, e.g. as herein described, claimed or exemplified, which is free or substantially free from ethanol and/or from any trans-esterification product of a vegetable oil (whether natural or hydrogenated) tri-glyceride and a polyalkylene polyol. Preferably the compositions in accordance with this aspect of the present invention are free or substantially free from any further solubilizer or co-solubilizer for the cyclosporine. Bioavailability levels achieved using existing oral cyclosporine dosage systems are also low and exhibit wide variation between individuals, individual patient types and even for single individuals at different times during the course of therapy. The reports in the literature indicate that currently available therapy employing the commercially available cyclosporine drink solution provides an average absolute bioavailability of 30% only, with marked variation between individual groups, e.g. between liver (relatively low bioavailability) and bone-marrow (relatively high bioavailability) transplant recipients. Reported variation in bioavailability between subjects has varied from anything between one or a few percent for some patients to as much as 90% or more for others. The absorption of CsA from the microemulsion formulation, Neoral, is not completely independent of bile flow and since the bile salt being a necessary component for efficient absorption of cyclosporine, this has been incorporated as integral component in the proposed formulation due to which improved bioavailability has been observed. In the present invention the structured vehicle (nonionic surfactant vesicles) described herein is able to reduce P-gp mediated efflux, as the presentation of cyclosporine to P-gp transporters could have been modified as shown in the figure-4. Moreover the cyclosporine is reported to inhibit bile synthesis in the liver (causes cholestatsis) and understanding its major role in the absorption of cyclosporine, bile salt has been incorporated as integral component in the formulation to achieve enhanced delivery. The formulation can be administered through oral, parenteral route with high guaranteed safety. We Claim: 1. A non-ionic surfactant based vesicular formulation for improved delivery of cyclosporine wherein the formulation comprising a pharmaceutically effective amount of cyclosporine, surfactant wherein at least one of the surfactant is non- ionic, lipid, bile salt, charge inducer wherein the molar ratio of surfactant: lipid : charge inducer : bile salt : cyclosporine is in the range between 30-65 : 65-30 : 3-6 : 5-15 : 0.5 to 5 respectively. 2. A formulation as claimed in claim 1 wherein the surfactants used is selected from a group consisting of sorbitan monolaurate (Span-20), sorbitan monopalmitate (Span- 40), sorbitan monooleate (Span-80), sorbitan monostearate (Span-60), sorbitan trioleate (Span-85), sorbitan tristearate (Span-65). 3. A formulation as claimed in claim 1 wherein the bile salts used is selected from a group consisting of sodium cholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium taurodeoxy-cholate, sodium glycodeoxycholate, sodium ursodeoxycholate, sodium chenodeoxycholate, sodium taurochenodeoxycholate, sodium glyco chenodeoxycholate, sodium cholylsarcosinate, sodium N-methyl taurocholate. 4. A formulation as claimed in claim 1 wherein the ionic surfactants used is selected from a group consisting of Sodium caproate, Sodium caprylate, Sodium caprate, Sodium laurate, Sodium myristate, Sodium myristolate, Sodium palmitate, Sodium palmitoleate, Sodium oleate, Sodium • ricinoleate, Sodium linoleate, Sodium linolenate, Sodium stearate, Sodium lauryl sulfate (dodecyl), Sodium tetradecyl sulfate, Sodium lauryl sarcosinate, Sodium dioctyl sulfosuccinate [sodium docusate.] 5. A formulation as claimed in claim 1 wherein the nonionic surfactant includes sorbitan fatty acid esters but not limited to sorbitan monostearate, sorbitan monopalmitate, sorbitan monooleate and polyethylene glycol derivatized fatty acids. 6. A formulation as claimed in claim 1 wherein the lipid used is cholesterol. 7. A formulation as claimed in claim 1 wherein the inducer used is selected from a group consisting of stearylamine , dicetyl phosphate. 8. A formulation as claimed in claim 1 wherein the entrapment efficiency of cyclosporine is more than 90%. 9. A formulation as claimed in claim 1 wherein the formulation exhibit greater bioavailability of Cyclosporine as compared with Sandimmun Neoral as indicated by the higher AUC values of the subject formulations 72.8±27.9 [Figure 1-3 and Table - 1]. 10. A formulation as claimed in claim 1 wherein the relative bioavailability of Cyclosporine formulation AUCtest/AUCmarket is 1.73 (173%). 11. A formulation as claimed in claim 1 wherein the in-vitro absorption of cyclosporine through everted gut sac technique, the degree of efflux is reduced 2-3 fold. 12. A formulation as claimed in claim 1 wherein the formulation can be administered through oral, parenteral route. 13. A formulation as claimed in claim 1 wherein the vesicular formulation is in the form selected from a group consisting of , bilosomes, proniosomes, enteric coated proniosomes. 14. A formulation as claimed in claim 1 wherein the formulation is with a high drug payload essentially free from alcohol and cremophor (polyoxyethylated ester of castor oil) and essentially devoid of natural vegetable oils. 15. A formulation as claimed in claim 1 wherein the formulation is for the use in organ transplantation for immunosuppression. 16. A process for preparation of formulation as claimed in claim 1 wherein the process steps comprising; (e) dissolving cyclosporine, surfactant, cholesterol, charged inducer in a molar ratio ranging between 0.5 to 5: 30 to 65: 65 to 30: charge inducer 3 to 6 respectively in a mixture of methanol and chloroform, (f) evaporating the solvent from the above mixture obtained in step (a) using rotary vacuum evaporator and drying in dessicator to obtain a film, (g) hydrating the film with plain aqueous solution containing bile in a molar ratio ranging between 5-15, at a temperature higher than 60°C. to obtain bilosomes, (h) alternatively, spraying the mixture obtained as mentioned in (a) on inert material followed by evaporation of solvent using rotary vacuum evaporator and drying in a dessicator to leave free flowing granules of proniosomes. 17. A formulation as claimed in claim 1 wherein the inert material includes but not limited to sorbitol, lactose and maltodextrins 18. A non-ionic surfactant based vesicular formulation for improved delivery of cyclosporine substantially as herein described with reference to the examples accompanying the specification. |
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842-del-2006-Claims-(03-07-2013).pdf
842-del-2006-Correspondence-Others-(03-07-2013).pdf
842-del-2006-correspondence-others-(09-01-2008).pdf
842-del-2006-correspondence-others.pdf
842-del-2006-description(complete).pdf
842-del-2006-description(provisional).pdf
842-del-2006-form-18-(09-01-2008).pdf
| Patent Number | 258311 | |||||||||||||||||||||||||||||||||
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| Indian Patent Application Number | 842/DEL/2006 | |||||||||||||||||||||||||||||||||
| PG Journal Number | 01/2014 | |||||||||||||||||||||||||||||||||
| Publication Date | 03-Jan-2014 | |||||||||||||||||||||||||||||||||
| Grant Date | 30-Dec-2013 | |||||||||||||||||||||||||||||||||
| Date of Filing | 28-Mar-2006 | |||||||||||||||||||||||||||||||||
| Name of Patentee | COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH | |||||||||||||||||||||||||||||||||
| Applicant Address | ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110001, INDIA. | |||||||||||||||||||||||||||||||||
Inventors:
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| PCT International Classification Number | A61K 9/127 | |||||||||||||||||||||||||||||||||
| PCT International Application Number | N/A | |||||||||||||||||||||||||||||||||
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