Patent Application: US-48878109-A

Abstract:
curcumin has shown anti - inflammatory and anti - angiogenic properties that could be useful in treating various diseases such as those of rheumatology and oncology . however , curcumin is very poorly absorbed and has a very low bioavailability . this patent describes a method of increasing the delivery of curcumin by complexing it with cyclodextrins . cyclodextrins are well known in the food industry and have been used to carry other drugs to increase bioavailability . the new combination of cyclodextrins and curcumin has been tested in pre - clinical inflammation models where it has demonstrated efficacy superior to both the positive control and curcumin .

Description:
cyclodextrin may be purchased from a number of vendors including sigma - aldrich , inc . ( st . louis , mo ., usa ). in addition , other forms of amorphous cyclodextrin having different degrees of substitution or glucose residue number are available commercially . a method for the production of hydroxypropyl - p - cyclodextrin is disclosed in pitha et al ., u . s . pat . no . 4 , 727 , 064 which is incorporated herein by reference . to produce the formulations according to the invention , a pre - weighed amount of cyclodextrin , which is substantially pyrogen free is placed in a suitable depyrogenated sterile container . methods for depyrogenation of containers and closure components are well known to those skilled in the art and are fully described in the united states pharmacopeia 23 ( united states pharmacopeial convention , rockville , md . usa ). generally , depyrogenation is accomplished by exposing the objects to be depyrogenated to temperatures above 400 ° c . for a period of time sufficient to fully incinerate any organic matter . as measured in u . s . p . bacterial endotoxin units , the formulation will contain no more than 10 bacterial endotoxin units per gram of amorphous cyclodextrin . by substantially pyrogen free is meant that the hydroxypropyl - p - cyclodextrin contains less than 10 u . s . p . bacterial endotoxin units per gram using the u . s . p . method . preferably , the hydroxypropyl -( 3 - cyclodextrin will contain between 0 . 1 and 5 u . s . p . bacterial endotoxin units per mg , under conditions specified in the united states pharmacopeia 23 . make a 20 % hpβ - cyclodextrin solution , ph 11 - 12 : place 2 g hpβ - cyclodextrin ( sigma / aldrich # 332593 ) per 8 ml water in a stoppered graduated cylinder with at least a 50 % volume reserve ; shake vigorously to dissolve , then place in an ultrasonic bath briefly to clarify ; vortex to ensure homogeneous mixing . add a stir bar and 10 % naoh dropwise with stirring until ph is between 11 & amp ; 12 ; qs to 2 g of cyclodextrin / 10 ml water and re - mix by vortexing . use at room temperature . make a 15 mg / ml curcumin stock solution , ph 11 - 12 : suspend 15 mg of curcumin ( sigma c7727 ) per 0 . 9 ml of an ethanol / water ( 10 / 90 ) mix in a beaker ( with a stir bar ) that is large enough for a ph monitoring electrode . add 10 % naoh dropwise while stirring and using a pipette to wet any remaining powder with liquid until the drug is well suspended and suspension turns red / purple . insert ph electrode and continue adding naoh until ph is between 11 and 12 and suspended drug is dissolved ( clear purple ). then qs to 15 mg / 1 . 0 ml and stir for ˜ 5 more minutes to make sure drug is in solution . then add to cyclodextrin solution immediately ( see 0052 below ) as alkaline curcumin is unstable . make neutral 3 mg / ml cyclodextrin : curcumin ( cdc ) dosing solution : add 2 ml of alkaline curcumin stock per 8 ml of alkaline cyclodextrin in a beaker with stir bar ; curcumin should stay dissolved . then complex the curcumin with the cyclodextrin by slowly lowering the ph via dropwise addition of 0 . 8 m citric acid with stirring and ph monitoring until ph is between 6 & amp ; 7 . as ph drops the color will change to a clear orange / yellow . store drug in dark or amber vials . use immediately or store in the refrigerator ; the drug is still clear after overnight refrigeration . the drug can be dried in a speed vac and stored at room temperature . reconstitution can be achieved by mixing with water to the original concentration . efficacy : the efficacy of cdc was compared to native curcumin in a mouse paw edema model . purpose : to compare the oral efficacy of an aqueous curcumin suspension ( c ) with that of a curcumin cyclodextrin complex ( cdc ) in the mouse carrageenan paw inflammation model materials : curcumin ( c ) ( sigma c7727 , lot # 096k1092 ); hpβ - cyclodextrin ( cd ) ( sigma / aldrich 332593 , lot # 04806cj ); indomethacin ( sigma 18280 , lot # 098k1500 ); λcarrageenan ( fluka 22049 , lot # 1408463 ); ketamine ( bioniche pharma ndc 67457 - 001 - 10 ); xylazine ( lloyd labs , lot # la33806 ); calipers ( fisher 15 - 077 - 957 ); ethanol ( e ) ( decon labs # 2716 , lot # a01190801 l ). animals : 35 male cd - 1 mice , 20 - 30 g ( harlan ) experimental design / methods / groups : 1 . acclimatize the mice for 3 + days after receipt . weigh mice within 24 hrs of dosing to calculate dosing volume . remove food 2 hours before dosing ; restore food ˜ 2 hours after dosing . 2 . vehicle and drugs : vehicle : make 20 ml of 20 % cyclodextrin ( cd ) by dissolving 4 g cd / 20 ml sterile water . make 10 ml of ethanol / water ( 25 %/ 75 %) by mixing 2 . 5 ml of ethanol and 7 . 5 ml sterile water . make 5 ml of ethanol / water ( 5 %/ 95 %). separate cd into two 10 ml aliquots ; add 10 % naoh dropwise to one of them until the ph is 11 - 12 ; adjust the other to ph 6 - 8 if needed . for the vehicle group , make 5 ml of vehicle by mixing 1 ml of ethanol / water ( 25 %/ 75 %) with 4 ml of 20 % cd , ph 6 - 8 . store in fridge for up to 3 days . curcumin : make 5 ml of 3 mg / ml curcumin dosing suspension by suspending 15 mg of drug in 5 ml of ethanol / water ( 5 %/ 95 %); vortex and sonicate to make a fine suspension . check ability to push through an appropriately sized gavage needle ; if clumpy , homogenize and re - check . store in fridge overnight protected from light . cdc : make 5 ml of 15 mg / ml curcumin stock by suspending 75 mg in 5 ml of ethanol / water ( 25 %/ 75 %). then add 10 % naoh dropwise with stirring until ph reaches 11 - 12 and drug is dissolved ; stir 10 mins ( solution will be red / purple ). then make 10 ml of dosing solution by taking 2 ml of dissolved drug and adding slowly to 8 ml of ph 11 - 12 20 % cd solution with constant stirring ; mix for 10 mins . next complex the curcumin by slow dropwise addition of 0 . 8 m citric acid until ph is 6 - 7 and solution is clear yellow . store in fridge overnight protected from light . indomethacin : make 10 ml of dosing solution by suspending 5 mg in 10 ml of sterile water with stirring , then adding 0 . 1 n naco3 dropwise until solid disappears and ph is 7 - 8 . store in fridge for up to 1 week . 3 . carrageenan : make 2 ml of 1 % carrageenan by dissolving 20 mg in 2 ml of saline at 4 ° c . and then vortexing , warming and sonicating . test for injection resistance by 27g and 28 g needles . store in refrigerator overnight . 4 . groups : see group variables table below . dose mice p . o . 30 minutes before carrageenan injection . anesthetize with ketamine / xylazine ( 100 / 12 mg / kg , i . p .) 15 minutes before carrageenan injection . at t = 0 , inject left footpad of each mouse with 25 μl of 1 % carrageenan , using a 27 or 28 g needle . at 3 hours , measure thicknesses of each paw with calipers , holding mouse vertical and paw out with forceps . calculate edema by subtracting right paw thickness from left paw thickness . results : the paw was measured at 3 hours after oral gavage of curcumin ( 30 mg / kg ), indomethacin ( 5 mg / kg ), and cdc ( 30 mg / kg ) to mice . after 3 hours , the swelling was 0 . 59 mm for cdc , 0 . 66 mm for curcumin , 0 . 73 mm for indomethacin , and 0 . 78 mm for vehicle control . the difference between cdc and curcumin / indomethacin was statistically significant , with p & lt ; 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