Establishment Of Emulsion-gel Of 3,5,4'-trimethoxy-trans-stilbene(BTM) And Pharmacokinetic Study On Local Application Of Rabbit Ankles

OBJECTIVESAs a naturally occurring trimethyl derivative of resveratrol (Res), 3,5,4'-trimethoxy-trans-stilbene (BTM) exhibited anti-inflammatory activitiy as Res which can prevent the occurrence and development of osteoarthritis (OA) and may become a new therapy choice for OA. Taking into account that OA was a kind of local organ diseases characterized by degeneration of articular cartilage, it's more effective and security for local administration to improve the concentration of therapeutic drugs in target tissue. Meanwhile, it was more lipophilia for BTM than Res to penetrate into the stratum corneum which was the biggest transdermal barrier, which indicated a possibility for transdermal application for BTM. Therefore, this subject proposed to establish the topical preparations of BTM on the basis of its physicochemical properties studies, and evaluate the feasibility of local application of OA through pecutaneous penetration in vitro and local pharmacokinetics which will provide experimental basis for BTM transdermal delivery system.METHODS AND RESULTS1. Physicochemical properties of BTMAs kind of light yellow or white crystalline powder and the melting point varing in a range of 56℃-57℃, BTM almost insoluble in water, slightly soluble in methanol, ethanol, and easily soluble in acetonitrile, ethyl acetate, chlorform and acetone. The solubility increased as gradient in 10%-40% ethanol saline and 10%-40% PEG-400 saline solution. BTM showed strong lipophilicity and the value of LogP was 4.36±0.12. BTM was unstable to light for its stilbene bond occuring cis-trans isomerization. The effect of lights on the isomerization was sunlight(5300-31300 lx)>strong light(4500 lx)>daylight lamp(231 lx) >red light(55 lx), which indicated the rate of isomerization increasing with the light intensity. The reaction reached its equilibrium in sunlight at 45 min with only 9% BTM of it's before irradiation. But during the exposure time of red light for 9 h and daylight lamp for 1-2 h, no isomerization of BTM was found, which indicated these two light sources can provide the safe operation of light environment for different experiments.2. Establishment of BTM emulsion-gel and percutaneous pentration studiesThrough screening formulation of BTM emulsion-gel with the appearance, size and distribution, stability as the evaluation index and studying the in vitro penetrion of BTM emulsion-gel by using modified Franz diffusion cell and piglet skin with enhancing the percutaneous permeation as the goal, the final formulation was obtained as follows: drug (BTM,2.0%), Medium chain triglycerides (MCT,10.0%), Polyoxyethylene hydrogenated castor oil (RH-40,4.0%), Geraniol (2.0%), Glycerol (8.0%), Water (25%),0.5% Carbomer 940 (50%)The emulsion was prepared by magnetic stirring and the process optimizated by orthogonal design was as follows:melting the oil phase (BTM, MCT, RH-40, Geraniol) at 60℃and adding the water phase (Glycerol, water, mixed in 60℃water bath) when maintaining the same temperature, then magnetic stirring at 800 rpm for 5 min, after cooling we obtained the emulsion.0.5% Carbomer 940 which was adjusted pH value to 7.4 then was added to emulsion to get BTM emulsion-gel which was milky white semi-solid and smooth uniform, with diameter at 250 nm-310 nm, PDI at 0.171-0.315, pH of about 7.0 and stable under 40℃constant temperature in the dark for 30 days.The effect of different dosages on the cumulative permeation amount(Cpa) of BTM in emulsion-gel was:4%≈2%>1%, and the effect of different penetration enhancers was:Geraniol>IPM>Oleic acid> Azone> Nerolidol> control. Compared with Geraniol used singly, combining enhancers (Propylene glycol+Geraniol, IPM+Geraniol) failed to significantly improved the Cpa of BTM(P> 0.05), while Isopropanol+Geraniol significantly decreased the Cpa(P<0.05). In the scope of screening, the Cpa of BTM emulsion-gel with 2% dosage and Geraniol as the enhancer reaches the largest. Its Cpa of 24 h was 6.07±1.17μg/cm2, with the penetration ratio of 3.31.3. Local pharmacokinetics study of BTM emulsion-gel36 rabbits (1.5-2.0 kg) first were randomly divided into two groups, and futher divided into six time-groups, then received respectively a transdermal administration of BTM emulsion-gel (equivalent to 5 mg/joint of BTM) and an oral administration of BTM suspension(30 mg/kg BTM), collected the heart blood at 0.5,1,3,6,9,12 h and then sacrificed (3 animals each time). Dissected the left and right ankle joint tissue of rabbits respectively and recorded these weight. Blood was centrifuged for supernatant and precipitated protein with methanol, joint tissue samples were extracted also with methanol and a HPLC analysis was applied to determine the concentrion of BTM. Results:all the plasma samples of local administration were below the detection limit(9.2 ng/mL) while oral group reached its Cmax of 334.6±42.8 ng/mL at 0.5 h and 17.9±17.0 ng/mL at 9 h, which indicated transdermal administration can decrease the system drug exposure amount and be more security. And the concentration of BTM of transdermal was lower than oral administration at 0.5 h as result of high plasma concentration, and no significant differences were found at 1,3 h between different administration routes(P >0.05), but higer at 6,9,12 h for transdermal than oral administration (P <0.05), which indicated the local administration can maintain the drug concentration longer on the site of therapy with sustained release effect.CONCLUSIONSCompared with oral adminstration, transdermal adminstration of BTM emulsion-gel can penetrate into the skin and reach the joint tissue with higher concentration and sustained release effect, and its release characteristics was suited for long-term treatment of chronic deseases as OA. The plasma concentration of transdermal administration was far lower than oral group which can reduce drug interactions and improve medication safety for OA patients. This paper provided experimental basis for the dosage form choice of BTM, and provided a method reference for the option of the route of administration and transdermal drug delivery system for OA therapy drugs. The pharmacodynamics of BTM for local administration of OA treatment and local stimulation were still needly studied further.