Patent Application: US-201415522763-A

Abstract:
the present invention relates to pharmaceutical compositions with mucoadhesive and sustained release characteristics containing the association of the extract or fractions of curcuma longa l . with curcuminoid content greater than 5 % and bidens pilosa extract , which has an important proliferative and anti - inflammatory effect , for the treatment of inflammatory diseases of the oral cavity and of the gastrointestinal tract , as , for example , mucositis induced by radiotherapy and / or chemotherapy and aphthous lesions . additionally , inflammatory skin lesions and vaginal mucositis . these are compositions obtained from a liquid base formulation containing an aqueous - based solvent system . the subject of the present invention is , therefore , to obtain pharmaceutical preparations , to develop their obtainment processes and to establish their uses . these preparations contain an association between the bidens pilosa extract and the curcuma longa extract at 1 : 10 to 10 : 1 proportions , incorporated into semi - solid , liquid or solid pharmaceutical forms containing the synthetic or adhesive bio component , diluents , carriers , binders , disintegrants , at the concentration ranges from 1 to 75 % by weight , of the active agents of vegetable origin .

Description:
the starting liquid formulations of the present invention constitute the base element for the preparation of different pharmaceutical or cosmetic forms , such as mouthwash , mouthwash , drops , syrups , elixirs , among others for internal or external use . in a first embodiment of the present invention , a liquid base formulation was prepared , and in the pharmaceutical forms derived therefrom , a block copolymer selected from poly ( oxyethylene ), poly ( oxypropylene ), poloxamer 407 , or poloxamer 188 , which was used because of its mucoadhesive and solubilizing properties . in another aspect , the present invention relates to the preparation of multiparticulate polymer systems containing curcumin at a concentration of 1 to 50 % by weight . such systems are comprised of at least one bioadhesive natural polymer . they are also composed of a polymeric or lipid component capable of releasing the active principle in a prolonged manner on the surface of the mucosa . solvents such as distilled water , propylene glycol and polyethylene glycol 400 were used as carriers and as solubilizers . antioxidants such as butylhydroxytoluene , sodium bisulfite and sodium metabisulfite were used to maximize the oxidative stability of the formulations in the usual amounts of 0 . 01 to 1 % v / v of the formulations . the following description relating to the preparation of the liquid base formulation of the present invention presents both the formulations which constitute the novel element of the present invention but also includes those steps which , by their unsatisfactory result , demonstrate the actual effectiveness of what has been successfully developed by the inventors of the present invention . initially , formulations containing distilled water or a mixture of water and propylene glycol ( 1 : 1 , v / v ) were prepared at room temperature with mechanical stirring ( 500 rpm ). the addition of the curcuminoids in these formulations was carried out under constant mechanical agitation and the solutions obtained in this way had a concentration of less than 2 . 5 mg / ml . the addition of poloxamer to this blend greatly increased the dissolution of the curcuminoids . incorporation of 15 % ( w / v ) of this polymer into a water : propylene glycol ( 1 : 1 ) mixture led to improved solubilization of the curcuminoids in the preparation . on the other hand , it was verified that the solutions obtained after the incorporation of 15 % ( w / v ) of poloxamer still had concentration of curcuminoids of less than 2 . 5 mg / ml , showing that the procedure was still unsatisfactory . by directly adding the poloxamer to propylene glycol , a coarse dispersion has occurred which can not be used for the medicinal purposes for which the preparation is intended . it is therefore concluded that as a first step in the process of preparing the liquid base formulation for the preparation of different pharmaceutical or cosmetic forms , such as mouthwash , mouthwash , drops , syrups , elixirs among others , for internal or external use , the basis of curcuminoids , is constituted of : 1 st stage — preparation of formulations containing distilled water or a mixture of distilled water and propylene glycol at 1 : 1 v / v at room temperature under mechanical agitation ( 500 rpm ); 2 nd stage — the curcuminoids is added under constant mechanical agitation , obtaining a solution with curcuminoids concentration of less than 2 . 5 mg / ml ; 3 rd stage — adding poloxamer to the water mixture with propylene glycol and curcuminoid ; the procedure of incorporating the poloxamer into the mixture included dividing its total mass into two equal parts , followed by adding them to water and with propylene glycol . after the dispersion of poloxamer , the fractions were mixed under mechanical agitation and the resulting formulation showed good physical appearance , but as discussed above , it was not able to dissolve entirely the curcuminoids ( see formulation pi01 , fig1 ). 4 th stage — adding to the mixture of water with propylene glycol , curcuminoid and poloxamer , with a co - solvent ; aiming to increase the amount of curcuminoids dissolved , another co - solvent ( polyethylene glycol 400 , peg 400 ) was added to the formulation ( formulation pi02 ). in this new formulation , the peg 400 concentration was 20 % ( v / v ), and it was incorporated at the expense of reducing the amount of water in the formulation ( from 50 % to 30 % of the total volume ). fig2 shows the improvement from the addition of this constituent . thus , the ternary water mixture : peg 400 : propylene glycol ( 30 : 20 : 50 , v / v / v ) containing poloxamer ( 15 %) could solubilize most of the mass of curcuminoids added ( 2 . 5 mg / ml ). however , visual inspection showed the presence of small amount of precipitated material ( fig2 , formulation pi02 ). to further increase the solubility of the curcuminoids in the formulation , another modification was made to the composition of the solvent system , which now contained a larger amount of peg 400 ( 30 %, v v ). this increase occurred at the cost of a further reduction in the amount of water in the preparation to 20 % ( v / v ) ( formulation pi03 ). this new change allowed total solubilization of the mass of curcuminoids added and the resulting solution contained 2 . 5 mg / ml of curcuminoids . the preferred solvent system in this base formulation is expressed below : ( 1 ) propylene glycol from 45 %- 55 %, plus preferably , 50 %; ( 2 ) polyethylene glycol 400 , 25 %- 35 %, plus preferably , 30 %; ( 3 ) purified water , 15 %- 25 %, plus preferably 20 %. the poloxamer polymeric surfactant was added at the concentration of 15 % ( w / v ), according to the procedure described above . the pi03 formulation represented the starting point for the development of solutions with a higher concentration of curcuminoids and for those containing curcuminoids and bidens pilosa extract , as described below . thus , the preferred , preferred base formulation of the present invention is composed of : solvent - propylene glycol 50 % v / v co - solvent - polyethylene glycol 400 30 % v / v surfactant - poloxamer 15 % p / v carrier - purified water 20 % v / v initially , it was observed that for the preparation of liquid compositions prepared from a synergistic mixture containing the combination of the liquid base formulation with curcuma longa extract and bidens pilosa extract , by adding higher concentrations of curcuminoids ( 7 . 5 mg / ml ) to the pi03 formulation , a large amount of precipitate is formed . this caused the alteration of the preparation technique of the composition of the present invention , as punctuated below . ( 1 ) a mixture of peg 400 was prepared with propylene glycol and this mixture was heated with the aid of a hotplate maintained at 65 ° c . ; ( 2 ) the poloxamer mass of the formulation was divided in equal parts and one of the parts was dispersed in the propylene glycol and heated peg - 400 mixture — stage i ; ( 3 ) the curcuminoids were then added to the mixture under mechanical stirring ( 500 rpm ) for 30 minutes , still under heating ( 65 ° c . ); ( 4 ) the other half of the poloxamer mass was added in the aqueous phase at room temperature — stage ii . ( 5 ) after complete dispersion , the solution of phase ii was poured into the mixture of stage i , containing propylene glycol and peg 400 . stage iii ( 6 ) the final mixture was then kept under stirring until the temperature reached 25 ° c . ( approximately 30 minutes ). by the application of this new procedure , it was possible to dissolve up to 25 mg / ml of curcuminoids in the preparation , which remained physically stable for prolonged time at room temperature and was denominated as formulation pi04 . the incorporation of the bidens pilosa extract into the pi04 formulation was carried out by replacing the entire distilled water mass and 20 % of the propylene glycol mass by the glycerinated vegetable extract .— stage iv . ( 1 ) propylene glycol , 30 % ( v / v ); ( 2 ) polyethylene glycol 400 , 30 % ( v / v ); ( 3 ) glycerinated extract of bidens pilosa , 40 % ( v / v ). in addition to these liquid components , the formulation contains the polymeric mucoadhesive agent ( poloxamer , 15 %, w / v ), as well as sodium bisulphite and butylhydroxytoluene in usual concentrations . the preparation method of the cbd formulation was the same as described above for the pi04 formulation . the active principles of the present invention have been successfully associated with the cbd formulation . no precipitation , cloudiness , lump formation , gelling or any other sign of incompatibility between the active ingredients were observed . in addition , the bioadhesive component was successfully incorporated in the presence of bidens pilosa extract constituents . the stability of these formulations was evidenced using an accelerated stability protocol , showing that the decay in the concentration of curcuminoids and total polyphenols was within the legally established limit . the remaining percentage of curcuminoids after 180 days of testing was 92 . 25 % and the percentage of total polyphenols was 96 . 20 % in relation to the initial content . preparation of a semi - solid formulation containing curcuminoids and bidens pilosa the development of the semi - solid formulation was carried out aiming the preparation of a cream gel - type base , containing in the oil phase , in amounts ranging from 10 to 14 . 75 % w / w of the total formulation of the oily components , and containing the following components in the amounts preferential : ( 1 ) liquid vaseline ( 5 %, p / p ); ( 2 ) stearic acid ( 3 %, p / p ); ( 3 ) cetyl alcohol ( 0 . 4 %, p / p ); ( 4 ) cetostearyl alcohol ( 0 . 35 %, p / p ); ( 5 ) monoestearato de glicerila ( 3 %, p / p ); ( 6 ) isopropyl myristate ( 1 %, p / p ). poloxamer ( 15 %, w / w ) and propylene glycol ( 6 %, w / w ) were added to this phase . as preservatives methylparaben ( 0 . 1 %, w / w ), propylparaben ( 0 . 05 %, w / w ) and butylhydroxytoluene ( 0 . 05 %, w / w ) were used . all components of the oil phase were added to a jacketed vessel , and the temperature of the mixture was raised to 75 ° c . after the mixture reached the required temperature for the complete melting of the solids , curcuminoids were added to a final concentration of 25 mg / ml . after this addition , a new period of mechanical agitation ( 500 rpm ) was followed until the entire mass of curcuminoids was dissolved in the oily base . in a second jacketed vessel , the bidens pilosa glycolic extract ( 65 %, w / w ) was heated to a temperature of 75 ° c . the aqueous extract was then poured onto the oily phase under constant mechanical stirring ( 500 rpm ). after the mixture was complete the heating was switched off and stirring was continued until the resulting product reached room temperature ( about 25 ° c .). preparation of a solid formulation containing curcuminoids and bidens pilosa solid formulations , which may be tablets , tablets or capsules , and more preferably in tablet form , have been developed using mannitol as diluent and poloxamer as a mucoadhesive agent . the granulation liquid used was the bidens pilosa extract itself at the rate of 35 to 45 % w / v . the mass of each component of the blend is expressed as follows : ( 1 ) curcuminoids ( 25 - 200 mg per tablet ); mannitol ( 250 to 350 mg per tablet ); poloxamer ( 100 to 140 mg per tablet ); microcrystalline cellulose ( 200 to 400 mg ); sodium metabisulfite ( 5 to 15 mg ) and citric acid ( 20 to 40 mg . nine hundred and thirty grams of the above - described blends were inserted into the processing chamber of a fluidized bed apparatus for performing mixing and granulation operations . the mixing step of the powders was initiated by the injection of heated air ( 45 - 65 ° c .) at a flow rate of 15 to 25 m 3 per hour for a period of 10 to 15 minutes . after the mixing step , the granulation liquid was added thereto in three steps . between each of the granulation steps a drying step was performed ( maintenance of the fluidization conditions without addition of liquid ). all powders were sieved in stainless steel screens ( 250 micrometer aperture mesh ) prior to performing the blending operation . the granules were compressed in a rotary compression machine equipped with a table containing 8 sets of punches ( numbers 13 to 15 , depending on the formulation ). the hardness of the tablets was determined during the process . in vitro evaluations of mucoadhesive formulations containing curcuminoids , bd and their association . studies conducted by the inventors of the present invention have demonstrated that the association of the curcuma long fraction with curcuminoids content greater than 5 % to the glycerinated extract of bd showed an important hyperproliferative and anti - inflammatory effect superior to the effects of these substances when evaluated separately in animals exposed to 5 - fluoracil . a protective effect against 5 - fluoracil ( 5 - fu )- induced mucositis was observed to be superior to the protective effect of curcuminoids or bd evaluated separately , both prophylactically and therapeutically . the studies were conducted in male swiss mice , aged between 10 and 14 weeks , kept in an air - conditioned room under a constant temperature of 24 ° c . and ambient conditions of 12 : 12 hours in the light - dark cycle . the diet was classic , with standard commercial feed and water provided ad libitum . intestinal mucositis was induced intraperitoneally ( i . p .) with 5 - fu injection at the 4th , 5th and 6th days in a single dose of 200 mg / kg / day . the degree of the disease was assessed by the diarrhea score daily score of days 0 - 8 . each animal was examined twice daily . the severity of diarrhea was classified using the following scale : 0 , normal ( normal or absent stools ); 1 , light ( soft stool a little wet ); 2 , moderate ( faeces wet and with moderate perianal inflammation and alteration in coat color ) and 3 in severe faeces ( watery with severe perianal inflammation and alteration in coat color ). the mean values of diarrhea were used to assess the severity of diarrhea ( n = 5 per group ). a ) evaluation of the protective effect of curcuminoids ; b ) evaluation of the protective effect of bd ; c ) evaluation of the protective effect of the association of curcuminoids and bd . the treatments in the 3 studies were performed according to the scheme presented in fig3 . administration of the test substance for each group was performed orally ( gavage ) for 6 days , and doses of 5 - fu were administered intraperitoneally on the 4th to 6th day . the first study deals with the evaluation of a formulation containing only curcuminoids . treatment groups ( n = 5 / group ) were thus distributed : group i : 3 . 7 mg / kg curcuminoid + 5 - fu ; group ii : 7 . 5 mg / kg curcuminoid + 5 - fu ; group iii : 15 mg / kg curcuminoid + 5 - fu ; group iv : positive control ( 5 - fu ); group v : negative control ( water ). groups i , ii or iii received prophylactic treatment with curcuminoids containing formulation and on days 4 , 5 and 6 they received a 5 - fu injection 4 hours after treatment and were euthanized 24 hours after treatment . the duodenal portion of each animal was extracted and then the paraffin blocks ( 58 - 60 ° c .) were assembled to obtain microscopic images after staining using the hematoxylin and eosin technique . in addition , the proliferative effect of the mucosa was evaluated by immunohistochemistry with the cyclin d marker . in this study , the results showed that the animals treated with the curcuminoids formulation , in a dose - dependent manner , presented a lower weakness with or without diarrhea when compared to the group only exposed to 5 - fu . better results were observed with the dose of 7 . 5 mg / kg . the histopathological study ( fig4 ) showed that the exposure of animals to the protocol with 5 - fu induces the appearance of mucositis with loss of epithelial lining integrity , blistering , shortening of the crypts and shortening of the villi with the presence of large inflammatory infiltrate ( fig4 a ). on the other hand , treatment with the formulation of curcuminoids , especially the dose of 7 . 5 mg / kg preserved the integrity of the epithelium , the crypts and villi , as compared to the normal control , as seen in fig4 b and 4c . we also observed a tendency to increase villus size , suggesting a cellular proliferative stimulating effect . the second study was carried out with a formulation containing only glycerinated extract of bd in 3 ( three ) different doses ( 75 , 100 and 125 mg / kg ). the treatment groups ( n = 5 / group ) were thus distributed : group i : 75 mg / kg bd + 5 - fu ; group ii : 100 mg / kg bd + 5 - fu ; group iii : 125 mg / kg bd + 5 - fu ; group iv : positive control ( 5 - fu ); group v : negative control ( water ). the results obtained in this study demonstrated that the glycerinated extract of bd was effective to protect against the induction of mucositis by 5 - fu in a dose - dependent manner . the histopathological study seen in fig5 showed , as previously described , that exposure to 5 - fu induces a severe mucositis . on the other hand , treatment with glycerinated extract of bd , in particular the 125 mg / kg dose , preserved the integrity of the epithelium , crypts and villi when compared to normal control ( fig5 b and 5c ). the third study sought to evaluate a formulation with the association of curcuminoids ( 75 mg / kg ) and bd ( 125 mg / kg ) ( cbd ). treatment groups ( n = 5 / group ) were thus distributed : group i : cbd + 5 - fu ; group ii : cbd + 5 - fu ; group iii : cbd + 5 - fu ; group iv : positive control ( 5 - fu ); group v : negative control ( water ). in this study the results showed , in addition to the results reported above with the treatment of the substances alone , a drastic reduction of the inflammatory infiltrate with an increase in cell profiling , that is , the association promoted a protective effect superior to the isolated effect of curcuminoids and bd against mucositis . histopathological cut showed a reduction of local inflammation and with cellular hyperproliferation demonstrated by the increase of crypts and villi in the animals treated with the combination ( fig6 c ). from these animals , peripheral blood samples were collected by retro - orbital puncture . serum il - 1β levels were analyzed by elisa kits for il - 1β dosing according to the manufacturer &# 39 ; s instructions . the results demonstrated that the induction of mucositis in animals exposed to 5 - fu produced a significant increase of il - 1β in the control animals . however , the animals treated with the formulation containing the combination of curcuminoids ( 7 . 5 mg / kg ) and bd ( 125 mg / kg ) ( cbd ), levels of il - 1β were similar to those found in control animals . studies carried out with formulation containing only curcumin or containing only bd , also reduced il - 1β levels , but much less effectively . this suggested a superior effect of the association of assets . this result further suggests a reduction in apoptosis in the epithelial cells induced by the drug . in addition , in animals treated with the cbd formulation the severity of mucositis was lower when compared to the control group . the present invention is directed to the use of the formulation containing the combination of curcuminoids from curcuma longa and bidens pilosa for the treatment of mucosal diseases , for example mucositis of the gastrointestinal tract induced by radio and / or chemotherapy , based on formulation pharmaceutical product with mucoadhesive properties , controlled release that will provide an additional prophylactic and / or therapeutic alternative to the treatments used in the medical clinic . 1 . armstrong j a , mccaffrey r . the effects of mucositis on quality of life in patients with head and neck cancer . clin j oncol nurs . 2006 ; 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