Title of Invention	A NOVEL PHARMACEUTICAL COMPOSITION OF TOPICAL METHOTREXATE GEL IN CHRONIC INFLAMMATORY CONDITION
Abstract	The present invention relates to a topical Novel drug delivery system (NDDS) wherein the therapeutic agent is photostabilized thereby avoiding the photosensitized reactions thereof. The disclosure provides lipid-containing compositions, including liposomes encapsulating methotrexate, an external ingredients such as hyaluronic acid or its salt and may comprise of atleast one of the flavonoids such as hesperitin, rutin, quercetin. kaemferol and pharmaceutical formulations thereof, as well as process for preparing the same. Thus described herein are novel pharmaceutical compositions for sustained delivery of methotrexate via a topical route. The invention is based on the discovery that topical methotrexate gel composition can be used for the treatment of chronic inflammatory disease, such as rheumatoid arthritis.

Title of Invention

A NOVEL PHARMACEUTICAL COMPOSITION OF TOPICAL METHOTREXATE GEL IN CHRONIC INFLAMMATORY CONDITION

Abstract

The present invention relates to a topical Novel drug delivery system (NDDS) wherein the therapeutic agent is photostabilized thereby avoiding the photosensitized reactions thereof. The disclosure provides lipid-containing compositions, including liposomes encapsulating methotrexate, an external ingredients such as hyaluronic acid or its salt and may comprise of atleast one of the flavonoids such as hesperitin, rutin, quercetin. kaemferol and pharmaceutical formulations thereof, as well as process for preparing the same. Thus described herein are novel pharmaceutical compositions for sustained delivery of methotrexate via a topical route. The invention is based on the discovery that topical methotrexate gel composition can be used for the treatment of chronic inflammatory disease, such as rheumatoid arthritis.

Full Text	84 THE CAZF.TTE OF INDIA: EXTRAORDINARY [IPART II-SEC 3fii)] FORM 2 THE PATENTS ACT 1970 (39 of 1970) & The Patents Rules, 2003 COMPLETE SPECIFICATION (See section 10 and rule 13) 1. TITLE OF THE INVENTION: A NOVEL PHARMACEUTICAL COMPOSITION OF TOPICAL METHOTREXATE GEL IN CHRONIC INFLAMMATORY CONDITION 2. APPLICANT (SJ a) NAME: b) NATIONALITY: c) ADDRESS: 1. DR. (MRS.). ANURADHA MAJUMDAR INDIAN 31/202, KANAKIA'S "SANSKRUTT, Thakur complex. Kandrvali (E). Mumbai-400101. 2. MS. NORMA ANDRADES INDIAN Nalai Wadi Saloli. Vasai(W): 401201. Dist: Thane, State: Maharashtra. 3. PREAMBLE TO THE DESCRIPTION COMPLETE The following specification particularly describes the invention and the manner in which it is to be performed. Page l of 29 -8 AUG 2008 TITLE A novel pharmaceutical composition of topical methotrexate gel in chronic inflammatory condition. FIELD OF THE INVENTION This invention relates to the field of topical therapeutics in the form of novel drug delivery system with improved photostability and in particular to the treatment of chronic inflammatory conditions such as rheumatoid arthritis. PRIOR ART The following is a list of art which is considered to be pertinent for describing the state of the art in the field of the invention. Acknowledgement of these references herein will at times be made by indicating their number within brackets from the list below. 1 Chatterji, C. and Gallelli, F. Thermal and photolytic decomposition of methotrexate aqueous solutions. J. Pharm. Sci. 67: 526-31 (1978). 2 Chahidi, C. et al. Photosensitization by methotrexate photoproducts. Photochem. Photobiol. 38: 317-322 (1983). 3 Clarke, L. et al. Methotrexate associated lymphoprohferative disorder in a patient with rheumatoid arthritis presenting in the skin. J. Am. Acad. Dermatol. Case report, In Press (2006). 4 VanDooren-Greebe, R. et al. Methotrexate revisited: effects of long-term treatment in psoriasis. Br. J. Dermatol. 130, 204-210(1994). 5 Condit, P. The onset of action of amethopterin. Science. 134, 1421-1425 (1961). 6 Hwang, C. et al. Development and optimization of a methotrexate topical formulation. Drug Dev. Ind. Pharm. 21:1941-1952 (1995). 7 Asako, H. et al. Leukocyte adherence in rat mesenteric venules: effects of adhenosine and methotrexate. Gastroenterology. 104: 31-37 (1993). 8 Bonina, F. et al. In- vitro antioxidant and in-vivo photoprotective effect of three lyophilized extracts of Sedium telephium L. leaves. J. Pharm. Pharmacol. 52: 1279-1285 (2000). 9 Bonetti, A. et al. An extended-release formulation of methotrexate for subcutaneous administration. Cancer Chemother Pharmacol. 33: 303-306 (1994). 10 Black, H. Potential involment of free radical reaction in ultraviolet light mediated cutaneous damage. Photochem. Photobiol 46: 213-221 (1987). Page 2 of 29 11 Griffiths, H. et al. Molecular and cellular effects of ultraviolet light-induced Crit. Rev Clin. Lab. Sci. 35:189-237(1998). 12 Dhuval, C. et al. Inhibition of cutaneous inflammation by free radical savengers. Proc 17th IFSCC Congress, Yokohama, pp 453-461 (1992). 13 Morel, I. et al. Antioxidant and iron chelating activities of the flavonoids catechin, quercetin and diosmetin on iron-loaded rat hepatocyte cultures. Biochem, Pharmacol., 4: 13-19(1993). 14 Saija, S. et al. Flavonoids as antioxidant agents: importance of their interaction with biomembranes. Free. Rad. Bio. Med. 481-496(1995). 15 Van, A. et al. Structural aspects of antioxidant activity of flavonoids. Free Rad Bio Med. 20:331-342(1996). 16 Grecomoro, G. et al. Therapeutic synergism between hyaluronic acid and dexamethasone in the intra-articular treatment of osteoarthritis of the knee: a preliminary open study. Curr Med Res Opin. 13 :49-55 (1992). 17 Dahl, L. et al. Concentration and molecular weight of sodium hyaluronate in synovial fluid from patients with rheumatoid arthritis and other arthropathies. Ann Rheum Dis. 44: 817-822(1985). 18 Trommer, H. et al. The effects of hyaluronan and its fragments on lipid models exposed to UV irradiation. Int. J. Pharm. 254:223-234 (2003). 19 US patent Application publication no. WO2008030818 (A2). 20 US Patent no. 6485740. BACKGROUND OF THE INVENTION Background Primary joint inflammation is a necessary factor in the etiology of rheumatoid arthritis. Thus the right anti-inflammatory treatment at right time could be curative. The drugs used to treat rheumatoid arthritis are generally divided into two categories: non steroidal anti-inflammatory drugs (NSAIDS); corticosteroids (oral, intra-articular and parenteral routes) which provide rapid symptomatic relief but has no effect on the progression of joint damage; and disease modifying anti-rheumatic drugs (DMARDS) which reduce disease activity and slow down the progression of joint damage, thereby preserving function. Page 3 of 29 An estimated 2.1 million adults worldwide are affected by rheumatoid arthritis. Methotrexate (MTX) is largely prescribed as second line treatment for long term therapy in rheumatoid arthritis used alone or in combination with other DMARDS, including the newer biological agents. Problem MTX is known to undergo photodegradation under fluorescent light, a source of UV radiation, N-dealkylation to lose either a methyl or a pteridine aldehyde or derived carboxylic acid. The products sensitize the oxidation of histidine and tryptophan by singlet oxygen mechanism. Thus patients undergoing high-dose methotrexate show photo sensitization (1, 2). Long term use revealed localized eruption on nose spreading to involve cheeks, eyelids, and palate during the treatment duration. However discontinuation of the drug revealed clear skin (3). Although the drug efficacy is strong, systemic use of MTX provokes number of side effects (4) such as severe gastrointestinal problems, high first pass effect and hematological disturbances. Thus its use is limited only when the treatment proves advantageous over other therapeutic agents and its administration is judged to surpass the risk of side effects. To reduce these effects, clinical studies have been conducted with topical MTX (5, 6) suggesting an effective suppression of inflammation (7) which would also avoid some of the above mentioned side effects. A major problem in topical administration of MTX is that the drug has high molecular weight and high polarity and is mostly in the dissociated form at physiological pH, its capacity for passive diffusion is thus limited. Solution One of the possibilities for increasing the penetration of drugs through skin is the use of vesicular systems such as niosomes, liposomes etc. Also some of these vesicular systems such as liposomes are found to exert a photostabilization potential (8). Also the in vivo half-life of MTX is short, and repeated administrations are required for optimal efficacy. Hence developing a lipid-based drug-delivery system to provide an extended-release depot for methotrexate for subcutaneous administration was been studied (9) in cancer treatment. Page 4 of 29 Thus it was thought worthwhile to incorporate MTX into vesicular system liposomes which would not only provide photostability but also an extended MTX release over the entire application duration thereby providing a better efficacy. Topical drug delivery offers numerous potential advantages over systemic therapies, including avoidance of hepatic first-pass metabolism, improved patient compliance and ease of access to the absorbing membrane i.e. the skin and minimizing systemic toxicity of the drug due to direct administration of the drug to the pathologic site. In addition to these, pain associated with intra-articular injections could be avoided so also as quoted previously systemic side effects of MTX could be reduced thus patient compliance and safety are the major criteria in the selection of topical route of drug administration. In vivo and in vitro studies have shown that reactive oxygen species and free radicals are involved in both inflammatory response elicited by acute UV skin exposure (skin erythema) (10), and in photoaging and carcinogenic processes induced by chronic UV skin irradiation (11). UV radiations penetrates deeply into the skin, thus topical administration of enzymatic and non-enzymatic antioxidants provides an excellent means of enriching the endogenous cutaneous protection system and therefore is an effective strategy for protecting the skin against UV mediated oxidative damage (12). Thus addition of any of the flavonoid such as hesperitin, rutin, quercetin, kaemferol etc. exhibiting the antioxidant, iron chelating, free-radical scavenging activity and inhibition of propagation of lipid peroxidative chain reactions (13, 14, 15) could provide a stabilizing effect against the free radicals associated with photooxidative damage of MTX. Hyaluronic acid (HA) is the major constituent of the extracellular matrix commercially available in numerous molecular weight grades extending upto a maximum of 5000 kDa. It helps in linking proteoglycans into macromolecular aggregates, maintaining turgidity and hydration whilst ensuring solute flow. HA is used as a penetration enhancer due to its skin hydration properties hence also utilized for topical delivery (16). HA plays a major role in the pliable and resilient nature of articular cartilage and for maintenance of the viscoeiastic and lubricating properties of the synovial fluid (17). HA- based cosmetic formulations or sunscreens are capable of protecting the skin against UV irradiation due to free radical scavenging properties (18). Several of the HA's important physiochemical properties are molecular weight dependent and therefore discrete differences in iunction over the wide range of commercially available molecular weights enables HA to be used in a diverse set of applications. Page 5 of 29 Thus addition of hyaluronic acid or a salt thereof, possessing versatile properties such as biocompatibility, non-immunogenicity, biodegradability and viscoelasticity could provide a better MTX delivery in inflammatory condition. Many of the initial prior arts reveal topical application of MTX in psoriasis wherein some inventors also used liposomal encapsulated MTX. So also targeted delivery of MTX liposomes in arthritis, cancer and other diseases (19). However no such reports claim the improvement in photostability and topical application using such a combination of MTX gel in indication such as rheumatoid arthritis however, transdermal preparations of MTX containing organic amine have been reported in rheumatoid arthritis (20). Objective To develop a photostable, safe, effective and novel topical anti-arthritic formulation of Methotrexate targeted towards the treatment of chronic inflammatory condition such as rheumatoid arthritis. Invention The pharmaceutical composition of lipid based MTX gel is completely novel as topical formulation for chronic inflammatory condition such as rheumatoid arthritis, the composition providing advantage of improved photostability and sustained release of MTX therein. Advantageous effect Novel pharmaceutical composition of MTX gel is unique with respect to the processing step which would not only provide photostabilizing effect on MTX but also an altogether different route of drug delivery of liposomes encapsulating MTX in rheumatoid arthritis. Addition of the selected ingredients can provide a synergistic effect to develop a photostable and sustained released topical formulation of MTX which can be used in chronic inflammatory condition such as rheumatoid arthritis. Invention also provided a greater anti-inflammatory effect than the marketed best selling topical Voveran® Emulgel. SUMMARY OF THE INVENTION The present invention revealed a unique composition containing liposomal MTX with essential ingredients such as sodium hyaluronate and at least one of the flavonoids formulated as sustained release topical gel. Page 6 of 29 Two most important ingredients used in present investigation i.e. in developing the topical formulation are atleast one of the flavonoid and hyaluronic acid or a salt thereof. Both of them individually exhibited a vital role in achieving the therapeutic efficacy of MTX topical gel. Also, the present invention provided a process of preparing a novel pharmaceutical composition of MTX providing an advantage of improved photostability and sustained release of MTX therein. Further, the invention provides a novel method of treating a subject having an inflammatory condition, comprising administering to the subject a combination of an effective amount of MTX and at effective rate of MTX. In addition, the disclosure relates to a preferred, inflammatory condition to be treated in accordance with the invention is an autoimmune disorder, more preferably Rheumatoid Arthritis (RA). Thus the present invention is based on the finding that topical application of liposomal methotrexate led to a greater anti-inflammatory effect in an inflammatory arthritis animal model. Industrial Applicability Liposomes encapsulating MTX in gel is easy to scale up and addition of other ingredients such as hyaluronic acid or its salt and at least one flavonoid gave a very stable gel with appropriate release rate. Also the ingredients added served to be multipurpose providing a synergistic effect to achieve a therapeutic concentration to alleviate the inflammatory condition. BRIEF DESCRIPTION OF THE FIGURES: A preferred embodiment will now be described with reference to the accompanying figures, in which: FIG 1. Depicts the graphical representation of the results of in vitro permeation studies of MTX in various formulations. FIG 2. Shows bar graphs of quantitative results of an in vitro test for phototoxicity evaluation using photosensitized peroxidation of linoleic acid model depicting extent of lipid peroxidation. FIG 3A, 3B, 3C and 3D. Shows pictorial representations of petri plates showing qualitative results of an in vitro test for phototoxicity evaluation using Candida yeast test model. Page 7 of 29 FIG 4. Shows skin photographs of guinea pigs which were used to evaluate the non-irritant potential of the novel MTX gel compared to the placebo gel. FIGS 5 A and 5B. Shows skin photographs of rats which were used to evaluate the acute dermal toxicity of the novel gel at the end of 14 days. FIG 6. Shows skin photographs of guinea pigs which were used to evaluate the non-photoirritant potential of the novel gel compared to the plain MTX gel and standard photoirritant drug psoralen containing Melanocyl* cream. FIG 7. Shows skin photographs of guinea pigs which were used to evaluate the non-photoallergic potential of the novel gel compared to the plain MTX gel and standard photoallergic drug psoralen containing Melanocyl® cream. FIGS 8A and 8B. Depicts the graphical representation of the results of an in vivo experiment showing the change in severity of arthritis as a function of time in complete Freund's adjuvant (CFA) induced arthritic animals in terms of percent inflammation in the CFA injected knee and percent inhibition of edema in the CFA injected knee; treated with either liposome encapsulated MTX gel; standard diclofenac containing Voveran® Emulgel and untreated arthritic animals (positive control). FIGS 9 A, 9B and 9C. Shows the histopathological changes of the dissected knee joints of the liposome encapsulated MTX in hyaluronate gel treated animals compared to untreated arthritic animals. DETAILED DESCRIPTION OF THE INVENTION Hereinafter, preparations examples of the above composition will be explained. Preparation example 1: liposome encapsulating MTX in hyaluronate gel (MTX lipogel). The novel MTX gel primarily consists of two components namely the essential ingredients and the optional ingredients- Essential Ingredients: 1. Liposomal encapsulated MTX in the range of 0.08% to 0.5% w/w used as a therapeutic agent more preferably in the range of 0.08% to 0.3 % w/w. 2. Hyaluronic acid or its salt form in the range of 0.5 to 8 % w/w used as a gelling agent and penetration enhancer more preferably in the range of 0.5 to 5 % w/w possesing a molecular weight of 100-500kDa more preferably within 100-300kDa. Page 8 of 29 3. At least one of the flavonoids such as hesperitin, rutin, quercetin, kaemferol etc. preferably quercetin in the range of 0.0005 to 0.01 % w/w used as an antioxidant and photoprotectant more preferably in the range of 0.0005 to 0.005 % w/w. The pharmaceutical composition of the invention may be formulated with other ingredients, which are formulated to a typical pharmaceutical gel, as necessary together with the essential ingredients. Other formulation ingredients, which can be added, may include a humectant (10 to 40 % w/w), chelating agent, antimicrobial agent/preservative and purified water. Further, other formulation ingredients, which can be added, are not limited to the above ingredients and any ingredients as mentioned above can be formulated within a range of not exerting a bad influence on the objects and effects of the invention, but are preferably added in a range of 0.01-5 % w/w, more preferably 0.01-3 % w/w of the total weight. A total amount was 100 % w/w by the addition of purified water and liposome encapsulating MTX in sodium hyaluronate gel was prepared. Although a preferable dosage of the pharmaceutical composition according to the invention is different according to ages, sexes, weights, symptoms and degrees of diseases, drug forms, and administration periods, it can be properly selected by a skilled in the art. However, considering a preferable effect, it is preferred that the pharmaceutical composition of the invention is administrated in an amount of 0.065-0.4 mg/kg per day. The application of the pharmaceutical formulation can be done as once a day. In addition, the dosage can be increased or decreased according to the ages, sexes, weights and degrees of diseases etc. Accordingly, the dosage does not limit a scope of the invention in any way. Preparation example 2: Plain MTX in hyaluronate gel. The plain MTX gel primarily consists of two components namely the essential ingredients and the optional ingredients. Essential Ingredients: 1. Plain MTX in the range 0.08% to 0.5% w/w used as a therapeutic agent more preferably in the range of 0.08% to 0.3 % w/w. 2. Hyaluronic acid or its salt form in the range of 0.5 to 8 % w/w used as a gelling agent and penetration enhancer more preferably in the range of 0.5 to 5 % w/w possesing a molecular weight of 100-500kDa more preferably within 100-300kDa. Other formulation ingredients, which can be added, may include a humectant (10 to 40 % w/w), chelating agent, antimicrobial agent/preservative and purified water. Page 9 of 29 Further, other formulation ingredients, which can be added, are not limited to the above ingredients and any ingredients as mentioned above can be formulated within a range of not exerting a bad influence on the objects and effects of the invention, but are preferably added in a range of 0.01-5 % w/w, more preferably 0.01-3 % w/w of the total weight. A total amount was 100 % w/w by the addition of purified water and plain MTX in hyaluronate gel was prepared. Preparation example 3: Plain MTX in carbopol gel. The plain MTX gel primarily consists of two components namely the essential ingredients and the optional ingredients. Essential Ingredient: 1. Plain MTX in the range 0.08% to 0.5% w/w used as a therapeutic agent more preferably in the range of 0.08% to 0.3 % w/w. 2. Carbopol in the range 0.5% to 8% used as a gelling agent more preferably in the range of0.5% to 5%w/w. Other formulation ingredients, which can be added, may include a humectant (10 to 40 % w/w), chelating agent, antimicrobial agent/preservative and purified water. Further, other formulation ingredients, which can be added, are not limited to the above ingredients and any ingredients as mentioned above can be formulated within a range of not exerting a bad influence on the objects and effects of the invention, but are preferably added in a range of 0.01-5 % w/w, more preferably 0.01-3 % w/w of the total weight. A total amount was 100 %w/w by the addition of purified water and plain MTX in carbopol gel.was prepared. Hereinafter the process for preparing the invention will be explained in a step by step manner. Preparation example 1: liposome encapsulating MTX in hyaluronate gel. Step 1. Liposomes encapsulating MTX containing appropriate quantity of lipids such as those obtained from egg lecithin or soya lecithins were used and formulated using reverse phase evaporation technique. Briefly the lipid solutions, the drug solution and organic solvent in appropriate quantity were taken into round bottom flask (RBF) and sonicated on a bath sonicator to form an w/o emulsion. Page 10 of 29 Later the organic solvent was slowly evaporated under low vacuum on a rotary evaporator simultaneously placing the RBF in a water bath maintained at constant temperature such as 35 to 50°C more preferably within 35 to 45°C. Finally the residual organic solvent was completely evaporated by increasing the pressure of the vacuum pump and the resultant concentrated liposomal dispersion obtained was diluted with the buffer and sonicated to a predetermined time. Step 2. This liposomal dispersion of MTX was further added to the accurately weighed quantity of hyaluronic acid or its salt and atleast one of the flavonoids like quercetin etc. followed by addition of rest all the ingredients and around 20-30 % w/w of purified water. Wherein the hyaluronic acid is auto-cross linked. Step 3. Later the container was kept overnight to achieve complete soaking of sodium hyaluronate and a proper gel consistency. Step 4. Finally the weight was adjusted to 100 %w/w using purified water. Preparation examples 2 and 3 were prepared in the same manner however the ingredients differ as described earlier. Hereinafter, the invention will be more specifically described with experimental examples, Experimental example 1: In vitro permeation studies of liposomes encapsulating MTX in hyaluronate gel compared to plain MTX in hyaluronic acid or its salt and carbopol gel. Method Permeation studies using freshly excised guinea pig skin were carried out to evaluate the permeability of various formulations containing MTX using phosphate buffer saline pH 7.4 as a receptor medium. Further at specified time intervals the sample aliquots were withdrawn and analysed spectrophotometrically for MTX permeated through skin as a function of time. Results Results revealed that plain MTX in carbopol gel showed a maximum of 58% permeability whereas plain MTX in hyaluronate showed a maximum of 64 % permeability. However the liposomes encapsulating MTX in hyaluronate showed 100% permeability at the end of 24 hours. Thus addition of hyaluronic acid or its salt was found to enhance the penetration thereby permeability of MTX. Page 11 of 29 Further formulating liposomes of MTX provided a greater improvement in permeability with a sustained cumulative release of MTX over the entire time period as shown in FIG 1. Experimental example 2: In vitro tests for evaluating the phototoxic potential of liposomes encapsulating MTX. 2-1. Photosensitized peroxidation of linoleic acid. Method A fatty acid, linoleic acid was irradiated with UV radiations in presence of the test solutions and lipid peroxidation of linoleic acid was measured using a thiobarbituric acid (TBA) assay to determine thiobarbituric acid reactive substances (TBARS) such as malondialdehyde (MDA). The test solutions consisted of plain MTX, liposomes encapsulating MTX and liposomes encapsulating MTX plus atleast one of the flavonoids. Results Invention revealed that plain MTX was found to produce 19.89 umoles/mL of MDA on UV exposure whereas the liposomes encapsulating MTX produce 11.7 pmoles/mL of MDA and further addition of atleast one of the flavonoids produced only 1.18 umoles/mL of MDA as shown in FIG 2. Thus results obtained justifies the use of atleast one of the flavonoids as an antioxidant and photoprotectant in the formulation to enhance the photo stabilizing effect on MTX liposomes. 2-2. Candida yeast test. Method Watman filter paper discs impregnated with test solutions were placed on Candida albicans (cell culture - 0.2 mL of 1 x 103 cells/mL) seeded sabouraud dextrose agar and irradiated with UV radiations for predetermined time. A similar set of experimental plates were kept in dark. Further, at the end of the exposure all the test plates were incubated at 25°C for 48 hours. Phototoxicity was indicated by a clear zone around the test material in the irradiated culture plates. Page 12 of 29 Results Plain MTX and psoralen containing Melanocyl cream being photolabile on exposure to UV radiations was found to produce toxic metabolites affecting the growth of C. albicans. However the dark control samples remained unaffected and a tremendous C. albicans growth was observed as shown in FIGS 3A and 3B. On formulating liposomes encapsulating MTX and so also liposomes encapsulating MTX gel did not affect the C. albicans growth on UV exposure as shown in FIGS 3C and 3D. Thus the techniques used in the experimental procedures to photostabilize MTX have been successful, which could be confirmed from further in vivo photoprotection studies. Experimental example 3: In vivo studies to evaluate the safety of dermal application of the novel liposomal encapsulated MTX gel composition. 3-1. Dermal irritation test. Method Female English short hair strain guinea pigs weighing 250-280 gm were procured from Hafflkine's Institute of Training and Research, Mumbai. Ethical clearance for the study was obtained from the Institutional Animal Ethics Committee of The Bombay College of Pharmacy. Fur on the posterior area of the dorsal region was removed with an animal hair clipper followed by treatment with depilator one day prior to the study. Further on experimental day (day 0) test material 0.5 gm (placebo and novel MTX gel on same animal within the dose range (0.08-0.3%) was applied topically to an area of approximately 1 sq cm of the shaven back of the animals and covered with a gauze patch for 5 hours. Further more, at the end of the exposure period the patch was removed and excess material was washed off with water. Later animals were evaluated for signs of erythema and edema till day 14 and the reaction was scored as per Draize's score. Results Both the groups tested showed no signs of erythema or edema thus proving the non-irritant potential of MTX gel as shown in FIG 4. 3-2. Acute dermal toxicity test. Page 13 of 29 Method Inbred female Sprague dawley rats weighing 180-220 gm were used for the study. Ethical clearance for the study was obtained from the Institutional Animal Ethics Committee of The Bombay College of Pharmacy. Fur on the posterior area of the dorsal region was removed with an animal hair clipper followed by treatment with depilator one day prior to the study. Further on experimental day (day 0) test material 0.5 gm (placebo and novel MTX gel in three test doses of MTX ranging from 0.05 to 5 %; test dose 3 was 10 % higher than the therapeutic dose selected for efficacy experiments) was applied topically to an area of approximately 10 % area of the total body surface of the shaven back of the animals and covered with a gauze patch for 24 hours. Further more, at the end of the exposure period the patch was removed and excess material was washed off with water. Later animals were evaluated for clinical signs of skin toxicity (erythema and edema reaction was scored as per Draize's score), gross behavioral changes and mortality till day 14. On day 15 further the animals were sacrificed and vital organs were excised and observed for gross morphological changes. Results Results revealed that placebo group, test dose 1 and test dose 2 did not show any signs of erythema and edema however, test dose 3 group produced a moderately irritant reaction as per the Draize's score as shown in FIGS 5 A and 5B. Various organs excised after sacrificing the animals were weighed and their organ to body revealed a statistical significant increase in liver, kidney and spleen only at test dose 3. Hence it can be concluded that test dose 1 and 2 are safe enough to produce efficacy without any signs of morbidity. Experimental example 4: In vivo studies to evaluate the photostability of novel liposomal encapsulated MTX gel composition. 4-1. Photoirritation test. Method Female Hartley strain albino guinea pigs weighing 210-250 gm were procured from Haffkine's Institute of Training and Research, Mumbai. Ethical clearance for the study Page 14 of 29 was obtained from the Institutional Animal Ethics Committee of The Bombay College of Pharmacy. Fur on the posterior area of the dorsal region was removed with an animal hair clipper followed by treatment with depilator one day prior to the study. Further on experimental day (day 0) test material 0.25 gm in duplicates was applied topically to an area of approximately 1 sq cm of the shaven back of the animals for 2 hours ±15 min. Test groups consisted of control skin surface, area applied with psoralen containing Melanocyl® cream serving as photoirritant positive control, plain MTX carbopol gel applied surface and novel liposomal encapsulated MTX in hyaluronate gel applied surface. All the test formulations were applied on same animal thus every animal served its own control. Further more, the animals were anesthetized partially wrapped with aluminium foil on single patch of each group serving as unirradiated control, and exposed to 15 J/cm2 of UV radiations for a specified time period serving as irradiated control. Later animals were examined for signs of erythema and edema at an interval of 24, 48 and 72 hours and the reaction was scored as per Draize's score. Result Results revealed that the irradiated surface as well as the unirradiated surface of the psoralen applied area showed a mild erythema reaction at 24 hours post drug application on the other hand at the end of 48 and 72 hours, the irradiated surface showed a more prominent well defined erythema reaction as compared to unirradiaed surface. Thus confirming an innate irritation potential and moderate photoirritation potential of psoralen containing Melanocyl cream. So also the plain MTX carbopol gel, showed a mild erythema reaction only at the irradiated surface at 48 and 72 hours post drug application as shown in FIG 6. This proves the photoirritation potential of plain MTX. As compared to psoralen (positive control) and plain MTX; control skin surface (negative control) and liposome encapsulating MTX in hyaluronate gel applied area was found to be absolutely normal without any signs of erythema on 24 hours, 48 hours as well as 72 hours post drug application and irradiation. Thus confirming the non-photoirrant nature ofMTXlipogel. 4-2. Dermal phototoxicity / Photoallergy test. Method Female Hartley strain albino guinea pigs weighing 210-250 gm were procured from Haffkine's Institute of Training and Research, Mumbai. Ethical clearance for the study Page 15 of 29 was obtained from the Institutional Animal Ethics Committee of The Bombay College of Pharmacy. Fur on the posterior area of the dorsal region was removed with an animal hair clipper followed by treatment with depilator one day prior to the study. Further on experimental day (day 0) test material 0.25 gm in duplicates was applied topically to an area of approximately 1 sq cm of the shaven back of the animals for 2 hours ± 15 min. Test groups consisted of control skin surface, area applied with psoralen containing Melanocyl® cream serving as photoirritant positive control, plain MTX carbopol gel applied surface and novel liposomal encapsulated MTX in hyaluronate gel applied surface within the dose range 0.08-0.3%. All the test formulations were applied on same animal thus every animal served its own control. Further more, the animals were anesthetized partially wrapped with aluminium foil on single patch of each group serving as unirradiated control, and exposed to 10 J/cm2 of UV radiations for a specified time period serving as irradiated control on day 0, 2, 4, 7, 9 and day 11 of the experimentation. Further the animals were again challenged with the same concentration of the test substance between days 20 to 24 under similar exposure conditions. Later animals were examined for signs of erythema and edema at an interval of 24, 48 and 72 hours and the reaction was scored as per Draize's score. Result Results revealed that the irradiated surface as well as the unirradiated surface of the psoralen applied area showed a mild erythema reaction at 24 hours post drug application on the other hand at the end of 48 hours, the irradiated surface showed a more prominent well defined erythema reaction as compared to unirradiated surface. Thus confirming an innate irritation potential and moderate photoallergic potential of psoralen containing Melanocyl® cream. So also the plain MTX carbopol gel, showed a mild erythema reaction only at the irradiated surface at 48 hours post drug application as shown in FIG 7. This proves the photoallergic potential of plain MTX. As compared to psoralen (positive control) and plain MTX; control skin surface (negative control) and liposome encapsulating MTX in hyaluronate gel applied area was found to be absolutely normal without any signs of erythema on 24 hours, 48 hours post drug application and irradiation. Thus confirming the non-photoallergic nature of MTX lipogel. Experimental example 5: In vivo studies to evaluate the efficacy of novel liposomes encapsulating MTX in hyaluronate gel. Page 16 of 29 Methods Inbred female Wistar rats weighing 160-190 gm were used for the study. Rats were maintained on a standardized pellet diet and supplied with tap water. Experiments were performed in accordance with the guidelines established by the Institutional Animal ethics committee of The Bombay College of Pharmacy, Mumbai, India. The rats were injected intra-articulariy into the appendages of the knee joint, a 0.2 mL suspension composed of complete Freund's adjuvant (CFA) with 5 mg/mL heat killed Mycobacterium tuberculosis, H37Ra, (Difco, Detroit, USA). Four groups each consisting of 6 animals each were studies. Group I served as negative control (vehicle) whereas group II served as standard treated with Voveran Emulgel containing diclofenac as therapeutic agent, group HI served as positive control with CFA injected knee but left untreated and group IV treated with liposome encapsulated MTX in hyaluronate gel within the dose range 0.08-0.3%. Treatment was initiated on day 2 after induction of arthritis and the formulations were applied topically once daily. The inflammatory intensity or the joint swelling was determined in accordance with the increase in the rat knee diameter, measured by dial type vernier caliper (Mitotoyo, Tokyo, Japan). Results Complete Freund's Adjuvant (CFA), induced arthritis model revealed that, Vehicle control group showed no signs of inflammation right from day 0. However rest all the animals were injected with 0.2 ml each of CFA and hence developed an inflammation in the injected knee upto a maximum of 40 % as shown in FIG 8A. Further the inflammation subsided gradually both due to treatement as well as the normal healing mechanism of the body. The extent of subsidation being higher with the treated animals However the secondary lesion revealed by inflammation in the non-injected knee was observed from day 5 and worsened without resolution similar to untreated arthritis induced group in standard treated animals. Liposome encapsulated MTX in hyaluronate gel treated animals showed absolutely no signs of secondary lesion in the contralateral non-injected knee compared to the standard marketed anti-inflammatory drug diclofenac which produced secondary lesions. Also the treated animals have shown a complete remission of the inflammation compared to diclofenac treated group in the injected knee. Experimental observations obtained at the end of the study showed a 91% inhibition of edema in the injected knee of the animals treated with standard marketed drug diclofenac. Page 17 of 29 However the liposome encapsulated MTX in hyaluronate gel treated animals have shown a significant inhibition in the edema over a period of 21 days and complete remission of arthritis as shown in FIG 8B. This significant reduction may be attributed to the increase permeation of liposome encapsulated MTX in hyaluronate gel as observed in in vitro permeation studies and thus more drug reaching the desired site. Histopathological findings revealed that articular and periarticular surfaces of positive control group IH showed moderate to marked chronic inflammatory changes in the form of thickening due to fibrous proliferation, mild leukocytic infiltration and mild to moderate chondroid degenerative changes as shown in FIG 9A. Samples from group IV revealed no abnormalities detected and hence assure the healing of arthritic lesions as shown in FIG 9B. However the standard diclofenac treated knee joints at the 21 day still showed a mild to moderate inflammation and slight cartilage damage, a decrease in invasive inflammatory pannus due to treatment as shown in FIG 9C. Reference example 1: Statistical analysis A paired t-test, which was used in the data analysis in the experimental examples I and 5. Significance was considered, based on p Page 18 of 29 We claim*. 1. A topical sustained release photostable gel formulation containing a novel pharmaceutical composition consisting of liposomal encapsulated methotrexate, hyaluronic acid or a salt thereof and atleast one of the flavonoids such as hesperitin, rutin, quercetin, kaemferol intended for use in chronic inflammatory disorders like rheumatoid arthritis. 2. The formulation of claim 1 wherein the therapeutic drug methotrexate encapsulated in liposomes is incorporated in the range of 0.08% to 0.3% w/w. 3. The formulation of claim 1, wherein the gel consists of auto-cross linked hyaluronic acid or its salt form in the range of 0.5 to 5 % w/w possesing a molecular weight within 100-300kDa serving as dual ingredient as a gelling agent as well as a penetration enhancer therein. 4. The formulation of claim 1 and 2 consisting of addition of atleast one of the flavonoids like hesperitin, rutin, quercetin, kaemferol in the range of 0.0005 to 0.005 % w/w as antioxidant and potent UV protectant thereby imparting a photostabilizing effect on methotrexate therein. 5. The process for preparing the novel gel with the unique composition as described in claim 1, 2, 3 and 4 comprises the following steps: i. Formulation of liposomes encapsulating MTX in the range of 0.08% to 0.3% w/w containing appropriate quantity of lipids such as those obtained from egg lecithin or soya lecithins using reverse phase evaporation technique, ii. Process includes addition of this liposomal dispersion of MTX to the accurately-weighed quantity of hyaluronic acid or its salt in the range of 0.5% to 5% w/w wherein the hyaluronic acid is auto-cross linked and atleast one of the flavonoids such as hesperitin, rutin, quercetin, kaemferol in the range of 0.0005% to 0.005% w/w. iii. Process includes the further addition of the ingredients such as suitable humectant from 10 to 40% w/w and chelating agent and antimicrobial/preservative from 0.01% to 3% and around 20-30 % w/w of purified water. iv. Keeping the container overnight to achieve complete soaking of hyahironate and a proper gel consistency. v. The weight adjusted to 100 %w/w using purified water. 6. The novel pharmaceutical composition of methotrexate gel prepared as per claim 5 could be intended for indications like chronic inflammatory disorders such as rheumatoid arthritis by topical application at the joint. Applicants: Dr. (Mrs.) Anuradha Majumdar Ms. Norma Andrades Dated this 8th day of August, 200 Page 29 of 29

Full Text

84 THE CAZF.TTE OF INDIA: EXTRAORDINARY [IPART II-SEC 3fii)]
FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION:
A NOVEL PHARMACEUTICAL COMPOSITION OF TOPICAL METHOTREXATE GEL IN CHRONIC INFLAMMATORY CONDITION

2. APPLICANT (SJ
a) NAME:
b) NATIONALITY:
c) ADDRESS:

1. DR. (MRS.). ANURADHA MAJUMDAR INDIAN
31/202, KANAKIA'S "SANSKRUTT, Thakur complex. Kandrvali (E). Mumbai-400101.

2. MS. NORMA ANDRADES INDIAN
Nalai Wadi Saloli. Vasai(W): 401201. Dist: Thane, State: Maharashtra.

3. PREAMBLE TO THE DESCRIPTION
COMPLETE
The following specification particularly describes the invention and the manner in which it is to be performed.

Page l of 29

-8 AUG 2008

TITLE
A novel pharmaceutical composition of topical methotrexate gel in chronic inflammatory
condition.
FIELD OF THE INVENTION
This invention relates to the field of topical therapeutics in the form of novel drug delivery system with improved photostability and in particular to the treatment of chronic inflammatory conditions such as rheumatoid arthritis.
PRIOR ART
The following is a list of art which is considered to be pertinent for describing the state of the art in the field of the invention. Acknowledgement of these references herein will at times be made by indicating their number within brackets from the list below.
1 Chatterji, C. and Gallelli, F. Thermal and photolytic decomposition of methotrexate aqueous solutions. J. Pharm. Sci. 67: 526-31 (1978).
2 Chahidi, C. et al. Photosensitization by methotrexate photoproducts. Photochem. Photobiol. 38: 317-322 (1983).
3 Clarke, L. et al. Methotrexate associated lymphoprohferative disorder in a patient with rheumatoid arthritis presenting in the skin. J. Am. Acad. Dermatol. Case report, In Press
(2006).
4 VanDooren-Greebe, R. et al. Methotrexate revisited: effects of long-term treatment in psoriasis. Br. J. Dermatol. 130, 204-210(1994).
5 Condit, P. The onset of action of amethopterin. Science. 134, 1421-1425 (1961).
6 Hwang, C. et al. Development and optimization of a methotrexate topical formulation. Drug Dev. Ind. Pharm. 21:1941-1952 (1995).
7 Asako, H. et al. Leukocyte adherence in rat mesenteric venules: effects of adhenosine and methotrexate. Gastroenterology. 104: 31-37 (1993).
8 Bonina, F. et al. In- vitro antioxidant and in-vivo photoprotective effect of three lyophilized extracts of Sedium telephium L. leaves. J. Pharm. Pharmacol. 52: 1279-1285 (2000).
9 Bonetti, A. et al. An extended-release formulation of methotrexate for subcutaneous administration. Cancer Chemother Pharmacol. 33: 303-306 (1994).
10 Black, H. Potential involment of free radical reaction in ultraviolet light mediated
cutaneous damage. Photochem. Photobiol 46: 213-221 (1987).
Page 2 of 29

11 Griffiths, H. et al. Molecular and cellular effects of ultraviolet light-induced Crit. Rev Clin. Lab. Sci. 35:189-237(1998).
12 Dhuval, C. et al. Inhibition of cutaneous inflammation by free radical savengers. Proc 17th IFSCC Congress, Yokohama, pp 453-461 (1992).
13 Morel, I. et al. Antioxidant and iron chelating activities of the flavonoids catechin, quercetin and diosmetin on iron-loaded rat hepatocyte cultures. Biochem, Pharmacol., 4:
13-19(1993).
14 Saija, S. et al. Flavonoids as antioxidant agents: importance of their interaction with biomembranes. Free. Rad. Bio. Med. 481-496(1995).
15 Van, A. et al. Structural aspects of antioxidant activity of flavonoids. Free Rad Bio Med. 20:331-342(1996).
16 Grecomoro, G. et al. Therapeutic synergism between hyaluronic acid and
dexamethasone in the intra-articular treatment of osteoarthritis of the knee: a preliminary
open study. Curr Med Res Opin. 13 :49-55 (1992).
17 Dahl, L. et al. Concentration and molecular weight of sodium hyaluronate in synovial
fluid from patients with rheumatoid arthritis and other arthropathies. Ann Rheum Dis. 44:
817-822(1985).
18 Trommer, H. et al. The effects of hyaluronan and its fragments on lipid models exposed to UV irradiation. Int. J. Pharm. 254:223-234 (2003).
19 US patent Application publication no. WO2008030818 (A2).
20 US Patent no. 6485740.
BACKGROUND OF THE INVENTION
Background
Primary joint inflammation is a necessary factor in the etiology of rheumatoid arthritis. Thus the right anti-inflammatory treatment at right time could be curative.
The drugs used to treat rheumatoid arthritis are generally divided into two categories: non steroidal anti-inflammatory drugs (NSAIDS); corticosteroids (oral, intra-articular and parenteral routes) which provide rapid symptomatic relief but has no effect on the progression of joint damage; and disease modifying anti-rheumatic drugs (DMARDS) which reduce disease activity and slow down the progression of joint damage, thereby preserving function.
Page 3 of 29

An estimated 2.1 million adults worldwide are affected by rheumatoid arthritis. Methotrexate (MTX) is largely prescribed as second line treatment for long term therapy in rheumatoid arthritis used alone or in combination with other DMARDS, including the newer biological agents.
Problem
MTX is known to undergo photodegradation under fluorescent light, a source of UV radiation, N-dealkylation to lose either a methyl or a pteridine aldehyde or derived carboxylic acid. The products sensitize the oxidation of histidine and tryptophan by singlet oxygen mechanism. Thus patients undergoing high-dose methotrexate show photo sensitization (1, 2).
Long term use revealed localized eruption on nose spreading to involve cheeks, eyelids, and palate during the treatment duration. However discontinuation of the drug revealed clear skin (3).
Although the drug efficacy is strong, systemic use of MTX provokes number of side effects (4) such as severe gastrointestinal problems, high first pass effect and hematological disturbances. Thus its use is limited only when the treatment proves advantageous over other therapeutic agents and its administration is judged to surpass the risk of side effects.
To reduce these effects, clinical studies have been conducted with topical MTX (5, 6) suggesting an effective suppression of inflammation (7) which would also avoid some of the above mentioned side effects.
A major problem in topical administration of MTX is that the drug has high molecular weight and high polarity and is mostly in the dissociated form at physiological pH, its capacity for passive diffusion is thus limited.
Solution
One of the possibilities for increasing the penetration of drugs through skin is the use of vesicular systems such as niosomes, liposomes etc.
Also some of these vesicular systems such as liposomes are found to exert a photostabilization potential (8).
Also the in vivo half-life of MTX is short, and repeated administrations are required for optimal efficacy. Hence developing a lipid-based drug-delivery system to provide an extended-release depot for methotrexate for subcutaneous administration was been studied (9) in cancer treatment.
Page 4 of 29

Thus it was thought worthwhile to incorporate MTX into vesicular system liposomes which would not only provide photostability but also an extended MTX release over the entire application duration thereby providing a better efficacy.
Topical drug delivery offers numerous potential advantages over systemic therapies, including avoidance of hepatic first-pass metabolism, improved patient compliance and ease of access to the absorbing membrane i.e. the skin and minimizing systemic toxicity of the drug due to direct administration of the drug to the pathologic site.
In addition to these, pain associated with intra-articular injections could be avoided so also as quoted previously systemic side effects of MTX could be reduced thus patient compliance and safety are the major criteria in the selection of topical route of drug administration.
In vivo and in vitro studies have shown that reactive oxygen species and free radicals are involved in both inflammatory response elicited by acute UV skin exposure (skin erythema) (10), and in photoaging and carcinogenic processes induced by chronic UV skin irradiation (11).
UV radiations penetrates deeply into the skin, thus topical administration of enzymatic and non-enzymatic antioxidants provides an excellent means of enriching the endogenous cutaneous protection system and therefore is an effective strategy for protecting the skin against UV mediated oxidative damage (12).
Thus addition of any of the flavonoid such as hesperitin, rutin, quercetin, kaemferol etc. exhibiting the antioxidant, iron chelating, free-radical scavenging activity and inhibition of propagation of lipid peroxidative chain reactions (13, 14, 15) could provide a stabilizing effect against the free radicals associated with photooxidative damage of MTX.
Hyaluronic acid (HA) is the major constituent of the extracellular matrix commercially available in numerous molecular weight grades extending upto a maximum of 5000 kDa. It helps in linking proteoglycans into macromolecular aggregates, maintaining turgidity and hydration whilst ensuring solute flow.
HA is used as a penetration enhancer due to its skin hydration properties hence also utilized for topical delivery (16). HA plays a major role in the pliable and resilient nature of articular cartilage and for maintenance of the viscoeiastic and lubricating properties of the synovial fluid (17).
HA- based cosmetic formulations or sunscreens are capable of protecting the skin against UV irradiation due to free radical scavenging properties (18).
Several of the HA's important physiochemical properties are molecular weight dependent and therefore discrete differences in iunction over the wide range of commercially available molecular weights enables HA to be used in a diverse set of applications.
Page 5 of 29

Thus addition of hyaluronic acid or a salt thereof, possessing versatile properties such as biocompatibility, non-immunogenicity, biodegradability and viscoelasticity could provide a better MTX delivery in inflammatory condition.
Many of the initial prior arts reveal topical application of MTX in psoriasis wherein some inventors also used liposomal encapsulated MTX. So also targeted delivery of MTX liposomes in arthritis, cancer and other diseases (19). However no such reports claim the improvement in photostability and topical application using such a combination of MTX gel in indication such as rheumatoid arthritis however, transdermal preparations of MTX containing organic amine have been reported in rheumatoid arthritis (20).
Objective
To develop a photostable, safe, effective and novel topical anti-arthritic formulation of Methotrexate targeted towards the treatment of chronic inflammatory condition such as rheumatoid arthritis.
Invention
The pharmaceutical composition of lipid based MTX gel is completely novel as topical formulation for chronic inflammatory condition such as rheumatoid arthritis, the composition providing advantage of improved photostability and sustained release of MTX therein.
Advantageous effect
Novel pharmaceutical composition of MTX gel is unique with respect to the processing step which would not only provide photostabilizing effect on MTX but also an altogether different route of drug delivery of liposomes encapsulating MTX in rheumatoid arthritis.
Addition of the selected ingredients can provide a synergistic effect to develop a photostable and sustained released topical formulation of MTX which can be used in chronic inflammatory condition such as rheumatoid arthritis.
Invention also provided a greater anti-inflammatory effect than the marketed best selling topical Voveran® Emulgel.
SUMMARY OF THE INVENTION
The present invention revealed a unique composition containing liposomal MTX with essential ingredients such as sodium hyaluronate and at least one of the flavonoids formulated as sustained release topical gel.
Page 6 of 29

Two most important ingredients used in present investigation i.e. in developing the topical formulation are atleast one of the flavonoid and hyaluronic acid or a salt thereof. Both of them individually exhibited a vital role in achieving the therapeutic efficacy of MTX topical gel.
Also, the present invention provided a process of preparing a novel pharmaceutical composition of MTX providing an advantage of improved photostability and sustained release of MTX therein.
Further, the invention provides a novel method of treating a subject having an inflammatory condition, comprising administering to the subject a combination of an effective amount of MTX and at effective rate of MTX.
In addition, the disclosure relates to a preferred, inflammatory condition to be treated in accordance with the invention is an autoimmune disorder, more preferably Rheumatoid Arthritis (RA).
Thus the present invention is based on the finding that topical application of liposomal methotrexate led to a greater anti-inflammatory effect in an inflammatory arthritis animal model.
Industrial Applicability
Liposomes encapsulating MTX in gel is easy to scale up and addition of other ingredients such as hyaluronic acid or its salt and at least one flavonoid gave a very stable gel with appropriate release rate.
Also the ingredients added served to be multipurpose providing a synergistic effect to achieve a therapeutic concentration to alleviate the inflammatory condition.
BRIEF DESCRIPTION OF THE FIGURES:
A preferred embodiment will now be described with reference to the accompanying figures, in which:
FIG 1. Depicts the graphical representation of the results of in vitro permeation studies of MTX in various formulations.
FIG 2. Shows bar graphs of quantitative results of an in vitro test for phototoxicity evaluation using photosensitized peroxidation of linoleic acid model depicting extent of lipid peroxidation.
FIG 3A, 3B, 3C and 3D. Shows pictorial representations of petri plates showing qualitative results of an in vitro test for phototoxicity evaluation using Candida yeast test model.
Page 7 of 29

FIG 4. Shows skin photographs of guinea pigs which were used to evaluate the non-irritant potential of the novel MTX gel compared to the placebo gel.
FIGS 5 A and 5B. Shows skin photographs of rats which were used to evaluate the acute dermal toxicity of the novel gel at the end of 14 days.
FIG 6. Shows skin photographs of guinea pigs which were used to evaluate the non-photoirritant potential of the novel gel compared to the plain MTX gel and standard photoirritant drug psoralen containing Melanocyl* cream.
FIG 7. Shows skin photographs of guinea pigs which were used to evaluate the non-photoallergic potential of the novel gel compared to the plain MTX gel and standard photoallergic drug psoralen containing Melanocyl® cream.
FIGS 8A and 8B. Depicts the graphical representation of the results of an in vivo experiment showing the change in severity of arthritis as a function of time in complete Freund's adjuvant (CFA) induced arthritic animals in terms of percent inflammation in the CFA injected knee and percent inhibition of edema in the CFA injected knee; treated with either liposome encapsulated MTX gel; standard diclofenac containing Voveran® Emulgel and untreated arthritic animals (positive control).
FIGS 9 A, 9B and 9C. Shows the histopathological changes of the dissected knee joints of the liposome encapsulated MTX in hyaluronate gel treated animals compared to untreated arthritic animals.
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, preparations examples of the above composition will be explained.
Preparation example 1: liposome encapsulating MTX in hyaluronate gel (MTX lipogel).
The novel MTX gel primarily consists of two components namely the essential ingredients and the optional ingredients-
Essential Ingredients:
1. Liposomal encapsulated MTX in the range of 0.08% to 0.5% w/w used as a therapeutic agent more preferably in the range of 0.08% to 0.3 % w/w.
2. Hyaluronic acid or its salt form in the range of 0.5 to 8 % w/w used as a gelling agent and penetration enhancer more preferably in the range of 0.5 to 5 % w/w possesing a molecular weight of 100-500kDa more preferably within 100-300kDa.
Page 8 of 29

3. At least one of the flavonoids such as hesperitin, rutin, quercetin, kaemferol etc. preferably quercetin in the range of 0.0005 to 0.01 % w/w used as an antioxidant and photoprotectant more preferably in the range of 0.0005 to 0.005 % w/w.
The pharmaceutical composition of the invention may be formulated with other ingredients, which are formulated to a typical pharmaceutical gel, as necessary together with the essential ingredients.
Other formulation ingredients, which can be added, may include a humectant (10 to 40 % w/w), chelating agent, antimicrobial agent/preservative and purified water.
Further, other formulation ingredients, which can be added, are not limited to the above ingredients and any ingredients as mentioned above can be formulated within a range of not exerting a bad influence on the objects and effects of the invention, but are preferably added in a range of 0.01-5 % w/w, more preferably 0.01-3 % w/w of the total weight.
A total amount was 100 % w/w by the addition of purified water and liposome encapsulating MTX in sodium hyaluronate gel was prepared.
Although a preferable dosage of the pharmaceutical composition according to the invention is different according to ages, sexes, weights, symptoms and degrees of diseases, drug forms, and administration periods, it can be properly selected by a skilled in the art. However, considering a preferable effect, it is preferred that the pharmaceutical composition of the invention is administrated in an amount of 0.065-0.4 mg/kg per day. The application of the pharmaceutical formulation can be done as once a day. In addition, the dosage can be increased or decreased according to the ages, sexes, weights and degrees of diseases etc. Accordingly, the dosage does not limit a scope of the invention in any way.
Preparation example 2: Plain MTX in hyaluronate gel.
The plain MTX gel primarily consists of two components namely the essential ingredients and the optional ingredients.
Essential Ingredients:
1. Plain MTX in the range 0.08% to 0.5% w/w used as a therapeutic agent more
preferably in the range of 0.08% to 0.3 % w/w.
2. Hyaluronic acid or its salt form in the range of 0.5 to 8 % w/w used as a gelling agent
and penetration enhancer more preferably in the range of 0.5 to 5 % w/w possesing a
molecular weight of 100-500kDa more preferably within 100-300kDa.
Other formulation ingredients, which can be added, may include a humectant (10 to 40 % w/w), chelating agent, antimicrobial agent/preservative and purified water.
Page 9 of 29

Further, other formulation ingredients, which can be added, are not limited to the above ingredients and any ingredients as mentioned above can be formulated within a range of not exerting a bad influence on the objects and effects of the invention, but are preferably added in a range of 0.01-5 % w/w, more preferably 0.01-3 % w/w of the total weight.
A total amount was 100 % w/w by the addition of purified water and plain MTX in hyaluronate gel was prepared.
Preparation example 3: Plain MTX in carbopol gel.
The plain MTX gel primarily consists of two components namely the essential ingredients and the optional ingredients.
Essential Ingredient:
1. Plain MTX in the range 0.08% to 0.5% w/w used as a therapeutic agent more preferably in the range of 0.08% to 0.3 % w/w.
2. Carbopol in the range 0.5% to 8% used as a gelling agent more preferably in the range of0.5% to 5%w/w.
Other formulation ingredients, which can be added, may include a humectant (10 to 40 % w/w), chelating agent, antimicrobial agent/preservative and purified water.
Further, other formulation ingredients, which can be added, are not limited to the above ingredients and any ingredients as mentioned above can be formulated within a range of not exerting a bad influence on the objects and effects of the invention, but are preferably added in a range of 0.01-5 % w/w, more preferably 0.01-3 % w/w of the total weight.
A total amount was 100 %w/w by the addition of purified water and plain MTX in carbopol gel.was prepared.
Hereinafter the process for preparing the invention will be explained in a step by step manner.
Preparation example 1: liposome encapsulating MTX in hyaluronate gel.
Step 1. Liposomes encapsulating MTX containing appropriate quantity of lipids such as those obtained from egg lecithin or soya lecithins were used and formulated using reverse phase evaporation technique.
Briefly the lipid solutions, the drug solution and organic solvent in appropriate quantity were taken into round bottom flask (RBF) and sonicated on a bath sonicator to form an w/o emulsion.
Page 10 of 29

Later the organic solvent was slowly evaporated under low vacuum on a rotary evaporator simultaneously placing the RBF in a water bath maintained at constant temperature such as 35 to 50°C more preferably within 35 to 45°C.
Finally the residual organic solvent was completely evaporated by increasing the pressure of the vacuum pump and the resultant concentrated liposomal dispersion obtained was diluted with the buffer and sonicated to a predetermined time.
Step 2. This liposomal dispersion of MTX was further added to the accurately weighed quantity of hyaluronic acid or its salt and atleast one of the flavonoids like quercetin etc. followed by addition of rest all the ingredients and around 20-30 % w/w of purified water. Wherein the hyaluronic acid is auto-cross linked.
Step 3. Later the container was kept overnight to achieve complete soaking of sodium hyaluronate and a proper gel consistency.
Step 4. Finally the weight was adjusted to 100 %w/w using purified water.
Preparation examples 2 and 3 were prepared in the same manner however the ingredients differ as described earlier.
Hereinafter, the invention will be more specifically described with experimental examples,
Experimental example 1: In vitro permeation studies of liposomes encapsulating MTX in hyaluronate gel compared to plain MTX in hyaluronic acid or its salt and carbopol gel.
Method
Permeation studies using freshly excised guinea pig skin were carried out to evaluate the permeability of various formulations containing MTX using phosphate buffer saline pH 7.4 as a receptor medium.
Further at specified time intervals the sample aliquots were withdrawn and analysed spectrophotometrically for MTX permeated through skin as a function of time.
Results
Results revealed that plain MTX in carbopol gel showed a maximum of 58% permeability whereas plain MTX in hyaluronate showed a maximum of 64 % permeability. However the liposomes encapsulating MTX in hyaluronate showed 100% permeability at the end of 24 hours.
Thus addition of hyaluronic acid or its salt was found to enhance the penetration thereby permeability of MTX.
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Further formulating liposomes of MTX provided a greater improvement in permeability with a sustained cumulative release of MTX over the entire time period as shown in FIG 1.
Experimental example 2: In vitro tests for evaluating the phototoxic potential of liposomes encapsulating MTX.
2-1. Photosensitized peroxidation of linoleic acid.
Method
A fatty acid, linoleic acid was irradiated with UV radiations in presence of the test solutions and lipid peroxidation of linoleic acid was measured using a thiobarbituric acid (TBA) assay to determine thiobarbituric acid reactive substances (TBARS) such as malondialdehyde (MDA).
The test solutions consisted of plain MTX, liposomes encapsulating MTX and liposomes encapsulating MTX plus atleast one of the flavonoids.
Results
Invention revealed that plain MTX was found to produce 19.89 umoles/mL of MDA on UV exposure whereas the liposomes encapsulating MTX produce 11.7 pmoles/mL of MDA and further addition of atleast one of the flavonoids produced only 1.18 umoles/mL of MDA as shown in FIG 2.
Thus results obtained justifies the use of atleast one of the flavonoids as an antioxidant and photoprotectant in the formulation to enhance the photo stabilizing effect on MTX liposomes.
2-2. Candida yeast test.
Method
Watman filter paper discs impregnated with test solutions were placed on Candida albicans (cell culture - 0.2 mL of 1 x 103 cells/mL) seeded sabouraud dextrose agar and irradiated with UV radiations for predetermined time.
A similar set of experimental plates were kept in dark.
Further, at the end of the exposure all the test plates were incubated at 25°C for 48 hours.
Phototoxicity was indicated by a clear zone around the test material in the irradiated culture plates.
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Results
Plain MTX and psoralen containing Melanocyl cream being photolabile on exposure to UV radiations was found to produce toxic metabolites affecting the growth of C. albicans. However the dark control samples remained unaffected and a tremendous C. albicans growth was observed as shown in FIGS 3A and 3B.
On formulating liposomes encapsulating MTX and so also liposomes encapsulating MTX gel did not affect the C. albicans growth on UV exposure as shown in FIGS 3C and 3D.
Thus the techniques used in the experimental procedures to photostabilize MTX have been successful, which could be confirmed from further in vivo photoprotection studies.
Experimental example 3: In vivo studies to evaluate the safety of dermal application of the novel liposomal encapsulated MTX gel composition.
3-1. Dermal irritation test.
Method
Female English short hair strain guinea pigs weighing 250-280 gm were procured from Hafflkine's Institute of Training and Research, Mumbai. Ethical clearance for the study was obtained from the Institutional Animal Ethics Committee of The Bombay College of Pharmacy. Fur on the posterior area of the dorsal region was removed with an animal hair clipper followed by treatment with depilator one day prior to the study.
Further on experimental day (day 0) test material 0.5 gm (placebo and novel MTX gel on same animal within the dose range (0.08-0.3%) was applied topically to an area of approximately 1 sq cm of the shaven back of the animals and covered with a gauze patch for 5 hours.
Further more, at the end of the exposure period the patch was removed and excess material was washed off with water.
Later animals were evaluated for signs of erythema and edema till day 14 and the reaction was scored as per Draize's score.
Results
Both the groups tested showed no signs of erythema or edema thus proving the non-irritant potential of MTX gel as shown in FIG 4.
3-2. Acute dermal toxicity test.
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Method
Inbred female Sprague dawley rats weighing 180-220 gm were used for the study. Ethical clearance for the study was obtained from the Institutional Animal Ethics Committee of The Bombay College of Pharmacy. Fur on the posterior area of the dorsal region was removed with an animal hair clipper followed by treatment with depilator one day prior
to the study.
Further on experimental day (day 0) test material 0.5 gm (placebo and novel MTX gel in three test doses of MTX ranging from 0.05 to 5 %; test dose 3 was 10 % higher than the therapeutic dose selected for efficacy experiments) was applied topically to an area of approximately 10 % area of the total body surface of the shaven back of the animals and covered with a gauze patch for 24 hours.
Further more, at the end of the exposure period the patch was removed and excess material was washed off with water.
Later animals were evaluated for clinical signs of skin toxicity (erythema and edema reaction was scored as per Draize's score), gross behavioral changes and mortality till day 14.
On day 15 further the animals were sacrificed and vital organs were excised and observed for gross morphological changes.
Results
Results revealed that placebo group, test dose 1 and test dose 2 did not show any signs of erythema and edema however, test dose 3 group produced a moderately irritant reaction as per the Draize's score as shown in FIGS 5 A and 5B.
Various organs excised after sacrificing the animals were weighed and their organ to body revealed a statistical significant increase in liver, kidney and spleen only at test dose
3.
Hence it can be concluded that test dose 1 and 2 are safe enough to produce efficacy without any signs of morbidity.
Experimental example 4: In vivo studies to evaluate the photostability of novel liposomal encapsulated MTX gel composition.
4-1. Photoirritation test.
Method
Female Hartley strain albino guinea pigs weighing 210-250 gm were procured from Haffkine's Institute of Training and Research, Mumbai. Ethical clearance for the study
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was obtained from the Institutional Animal Ethics Committee of The Bombay College of Pharmacy. Fur on the posterior area of the dorsal region was removed with an animal hair clipper followed by treatment with depilator one day prior to the study.
Further on experimental day (day 0) test material 0.25 gm in duplicates was applied topically to an area of approximately 1 sq cm of the shaven back of the animals for 2 hours ±15 min.
Test groups consisted of control skin surface, area applied with psoralen containing Melanocyl® cream serving as photoirritant positive control, plain MTX carbopol gel applied surface and novel liposomal encapsulated MTX in hyaluronate gel applied surface. All the test formulations were applied on same animal thus every animal served its own control.
Further more, the animals were anesthetized partially wrapped with aluminium foil on single patch of each group serving as unirradiated control, and exposed to 15 J/cm2 of UV radiations for a specified time period serving as irradiated control.
Later animals were examined for signs of erythema and edema at an interval of 24, 48 and 72 hours and the reaction was scored as per Draize's score.
Result
Results revealed that the irradiated surface as well as the unirradiated surface of the psoralen applied area showed a mild erythema reaction at 24 hours post drug application on the other hand at the end of 48 and 72 hours, the irradiated surface showed a more prominent well defined erythema reaction as compared to unirradiaed surface. Thus confirming an innate irritation potential and moderate photoirritation potential of psoralen containing Melanocyl cream.
So also the plain MTX carbopol gel, showed a mild erythema reaction only at the irradiated surface at 48 and 72 hours post drug application as shown in FIG 6. This proves the photoirritation potential of plain MTX.
As compared to psoralen (positive control) and plain MTX; control skin surface (negative control) and liposome encapsulating MTX in hyaluronate gel applied area was found to be absolutely normal without any signs of erythema on 24 hours, 48 hours as well as 72 hours post drug application and irradiation. Thus confirming the non-photoirrant nature ofMTXlipogel.
4-2. Dermal phototoxicity / Photoallergy test.
Method
Female Hartley strain albino guinea pigs weighing 210-250 gm were procured from Haffkine's Institute of Training and Research, Mumbai. Ethical clearance for the study
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was obtained from the Institutional Animal Ethics Committee of The Bombay College of Pharmacy. Fur on the posterior area of the dorsal region was removed with an animal hair clipper followed by treatment with depilator one day prior to the study.
Further on experimental day (day 0) test material 0.25 gm in duplicates was applied topically to an area of approximately 1 sq cm of the shaven back of the animals for 2 hours ± 15 min.
Test groups consisted of control skin surface, area applied with psoralen containing Melanocyl® cream serving as photoirritant positive control, plain MTX carbopol gel applied surface and novel liposomal encapsulated MTX in hyaluronate gel applied surface within the dose range 0.08-0.3%. All the test formulations were applied on same animal thus every animal served its own control.
Further more, the animals were anesthetized partially wrapped with aluminium foil on single patch of each group serving as unirradiated control, and exposed to 10 J/cm2 of UV radiations for a specified time period serving as irradiated control on day 0, 2, 4, 7, 9 and day 11 of the experimentation.
Further the animals were again challenged with the same concentration of the test substance between days 20 to 24 under similar exposure conditions. Later animals were examined for signs of erythema and edema at an interval of 24, 48 and 72 hours and the reaction was scored as per Draize's score.
Result
Results revealed that the irradiated surface as well as the unirradiated surface of the psoralen applied area showed a mild erythema reaction at 24 hours post drug application on the other hand at the end of 48 hours, the irradiated surface showed a more prominent well defined erythema reaction as compared to unirradiated surface. Thus confirming an innate irritation potential and moderate photoallergic potential of psoralen containing Melanocyl® cream.
So also the plain MTX carbopol gel, showed a mild erythema reaction only at the irradiated surface at 48 hours post drug application as shown in FIG 7. This proves the photoallergic potential of plain MTX.
As compared to psoralen (positive control) and plain MTX; control skin surface (negative control) and liposome encapsulating MTX in hyaluronate gel applied area was found to be absolutely normal without any signs of erythema on 24 hours, 48 hours post drug application and irradiation. Thus confirming the non-photoallergic nature of MTX lipogel.
Experimental example 5: In vivo studies to evaluate the efficacy of novel liposomes encapsulating MTX in hyaluronate gel.
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Methods
Inbred female Wistar rats weighing 160-190 gm were used for the study. Rats were maintained on a standardized pellet diet and supplied with tap water. Experiments were performed in accordance with the guidelines established by the Institutional Animal ethics committee of The Bombay College of Pharmacy, Mumbai, India. The rats were injected intra-articulariy into the appendages of the knee joint, a 0.2 mL suspension composed of complete Freund's adjuvant (CFA) with 5 mg/mL heat killed Mycobacterium tuberculosis, H37Ra, (Difco, Detroit, USA).
Four groups each consisting of 6 animals each were studies. Group I served as negative control (vehicle) whereas group II served as standard treated with Voveran Emulgel containing diclofenac as therapeutic agent, group HI served as positive control with CFA injected knee but left untreated and group IV treated with liposome encapsulated MTX in hyaluronate gel within the dose range 0.08-0.3%.
Treatment was initiated on day 2 after induction of arthritis and the formulations were applied topically once daily.
The inflammatory intensity or the joint swelling was determined in accordance with the increase in the rat knee diameter, measured by dial type vernier caliper (Mitotoyo, Tokyo, Japan).
Results
Complete Freund's Adjuvant (CFA), induced arthritis model revealed that, Vehicle control group showed no signs of inflammation right from day 0. However rest all the animals were injected with 0.2 ml each of CFA and hence developed an inflammation in the injected knee upto a maximum of 40 % as shown in FIG 8A.
Further the inflammation subsided gradually both due to treatement as well as the normal healing mechanism of the body. The extent of subsidation being higher with the treated animals
However the secondary lesion revealed by inflammation in the non-injected knee was observed from day 5 and worsened without resolution similar to untreated arthritis induced group in standard treated animals.
Liposome encapsulated MTX in hyaluronate gel treated animals showed absolutely no signs of secondary lesion in the contralateral non-injected knee compared to the standard marketed anti-inflammatory drug diclofenac which produced secondary lesions. Also the treated animals have shown a complete remission of the inflammation compared to diclofenac treated group in the injected knee.
Experimental observations obtained at the end of the study showed a 91% inhibition of edema in the injected knee of the animals treated with standard marketed drug diclofenac.
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However the liposome encapsulated MTX in hyaluronate gel treated animals have shown a significant inhibition in the edema over a period of 21 days and complete remission of arthritis as shown in FIG 8B.
This significant reduction may be attributed to the increase permeation of liposome encapsulated MTX in hyaluronate gel as observed in in vitro permeation studies and thus more drug reaching the desired site.
Histopathological findings revealed that articular and periarticular surfaces of positive control group IH showed moderate to marked chronic inflammatory changes in the form of thickening due to fibrous proliferation, mild leukocytic infiltration and mild to moderate chondroid degenerative changes as shown in FIG 9A. Samples from group IV revealed no abnormalities detected and hence assure the healing of arthritic lesions as shown in FIG 9B.
However the standard diclofenac treated knee joints at the 21 day still showed a mild to moderate inflammation and slight cartilage damage, a decrease in invasive inflammatory pannus due to treatment as shown in FIG 9C.
Reference example 1: Statistical analysis
A paired t-test, which was used in the data analysis in the experimental examples I and 5. Significance was considered, based on p Page 18 of 29

We claim*.
1. A topical sustained release photostable gel formulation containing a novel
pharmaceutical composition consisting of liposomal encapsulated methotrexate,
hyaluronic acid or a salt thereof and atleast one of the flavonoids such as hesperitin, rutin,
quercetin, kaemferol intended for use in chronic inflammatory disorders like rheumatoid
arthritis.
2. The formulation of claim 1 wherein the therapeutic drug methotrexate encapsulated in liposomes is incorporated in the range of 0.08% to 0.3% w/w.
3. The formulation of claim 1, wherein the gel consists of auto-cross linked hyaluronic acid or its salt form in the range of 0.5 to 5 % w/w possesing a molecular weight within 100-300kDa serving as dual ingredient as a gelling agent as well as a penetration enhancer therein.
4. The formulation of claim 1 and 2 consisting of addition of atleast one of the flavonoids like hesperitin, rutin, quercetin, kaemferol in the range of 0.0005 to 0.005 % w/w as antioxidant and potent UV protectant thereby imparting a photostabilizing effect on methotrexate therein.
5. The process for preparing the novel gel with the unique composition as described in claim 1, 2, 3 and 4 comprises the following steps:
i. Formulation of liposomes encapsulating MTX in the range of 0.08% to 0.3% w/w containing appropriate quantity of lipids such as those obtained from egg lecithin or soya lecithins using reverse phase evaporation technique, ii. Process includes addition of this liposomal dispersion of MTX to the accurately-weighed quantity of hyaluronic acid or its salt in the range of 0.5% to 5% w/w wherein the hyaluronic acid is auto-cross linked and atleast one of the flavonoids such as hesperitin, rutin, quercetin, kaemferol in the range of 0.0005% to 0.005% w/w.
iii. Process includes the further addition of the ingredients such as suitable humectant from 10 to 40% w/w and chelating agent and antimicrobial/preservative from 0.01% to 3% and around 20-30 % w/w of purified water.
iv. Keeping the container overnight to achieve complete soaking of hyahironate and a proper gel consistency.
v. The weight adjusted to 100 %w/w using purified water.
6. The novel pharmaceutical composition of methotrexate gel prepared as per claim 5
could be intended for indications like chronic inflammatory disorders such as rheumatoid
arthritis by topical application at the joint.

Applicants: Dr. (Mrs.) Anuradha Majumdar Ms. Norma Andrades
Dated this 8th day of August, 200
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A NOVEL PHARMACEUTICAL COMPOSITION OF TOPICAL METHOTREXATE GEL IN CHRONIC INFLAMMATORY CONDITION

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