The Evaluation And Mechanism Of Promoting Activity Of O-acylmenthol As Novel Percutaneous Absorption Enhancers

L-menthol(MT) may offer advantages over many enhancers because of its high enhancing activity,low toxicity and irritancy,high promoting activity both on lipophilic and hydrophilic drugs.However,its low melting point,pungent flavor and irritancy at high concentration restricts its application in clinical situations.In this study,l-menthol was selected as a lead compound to synthesize new types of O-acylmenthol derivatives as candidates for percutaneous absorption enhancers which presumably could overcome above defects of MT.To develop more effective compounds as penetration enhancers,two types of O-acylmenthol derivatives were synthesized,one type was synthesized by MT and saturated fatty acid,including acetic acid,propanoic acid,butyric acid,pentanoic acid,hexanoic acid, heptanoic acid,octanoic acid,nonylic acid,decanoic acid,dodecanic acid,tetradeconic acid, palmitic acid and stearic acid;the other type of O-acylmenthol was synthesized by MT and pharmaceutical excipient acids(lactic acid,cinnamic acid,salicylic acid and oleic acid), O-ethylmenthol(MET) was also synthesized as a reference penetration enhancer.The structures of final products were confirmed by ¹HNMR and MS.The physicochemical parameters of the O-acylmenthol derivatives such as solubilty parameter(SP),n-octanol/water partition coefficient(K_O/W) and polarizability(PLB) were calculated by MARVIN(?) or by the approaches of Hoftyzer/Van Krevelen.Their promoting activity on the percutaneous absorption of five penetrants having different physicochemical properties,5-fluorouracil(5-FU,base),isosorbide dinitrate(ISDN, neutrality),lidocaine(LD,base),ketoprofen(KP,acid),indomethacin(IM,acid),which were selected based on their lipophilicity represented by logK_O/W,were tested in vitro across full thickness rat skin with each of the evaluated drugs in isopropyl myristate(IPM) solution,the PBS buffer solutions were added in the receive side.In the case of the IPM as vehicle,we can conclude that for drugs of a similar size,the greater the difference in the SP values between drug and solvent,the larger the permeation coefficients(P) that can be obtained from the vehicle,interestingly,a reverse relationship between the melting point and flux was observed in IPM solution.The structure-activity relationships of O-acylmenthol derivatives synthesized by MT and saturated fatty acids as percutaneous absorption enhancers are summarized as follows:Tail chain length(C2-C18) has important effects on the enhancing activity,C6-C10 seems to be favorable for lipophilic drugs.A parabolic relationship between the carbon chain length of O-acylmenthol and skin permeation enhancement has been observed for hydrophilic drug(5-FU),2-isopropyl-5-methylcyclohexyl tetradecanoate(M-TET) with C14 alkyl chain is most effective.2-isopropyl-5-methylcyclohexyl 2-hydroxypanoate(M-LA) provided the highest increase of accumulation of 5-FU(3.74-fold) and LD(4.19-fold) in the receptor phase while 2-isopropyl-5-methylcyclohexyl cinnamate(M-CA) was ineffective for most of the drugs; Both 2-isopropyl-5-methylcyclohexyl 2-hydroxybenzoate(M-SA)and (E)-2-isopropyl-5-methylcyclohexyl octadec-9-enoate(M-OA) had better promoting effects on the drugs with low water-solubility.The four O-acylmenthol enhancers produced parabolic relationship between the lipophilicity(log K_O/W) of the model drugs(5-FU,ISDN,KP,IM) and their enhancement ratio of the permeation coefficient(ER_P),indicating that the lipophilicity of the penetrants has significant effect on the permeation results,r = 0.989,(P = 0.144) for M-LA,r = 0.965,(P = 0.216) for M-CA,r = 0.786,(P = 0.630) for M-SA,and r = 0.996,(P = 0.088) for M-OA.We selected alkylamine or alkanolamines(diethylamine,ethanolamine,diethanolamine, triethanolamine and N-(hydroxylethyl) piperidine) to react with diclofenac acid(DA) to prepare DA salts,differential scanning calorimetry(DSC) and fourier transform infrared spectroscopy(FT-IR) analyses have been used to identify the formation of DA salts.We investigated the effects of five organic amines upon the penetration of DA by O-acylmenthol in the solutions of IPM.A 4.51-fold increase in the accumulative amount of DA was observed by ion-pair formation with diethylamine for the improvement of lipophilicity,however,the alkanolamines with hydroxyl groups had negative effects on the transdermal delivery of diclofenac.M-TET was effective on the penetration of diclofenac diethylamine(D-DETA), also,it is exciting to note that combined use of diethylamine with M-TET produced a 9.74-fold increase in accumulation amount(Q) of diclofenac compared with DA in IPM.Tolterodine(TOL) was selected as a model drug to screen the most promising enhancer among O-acylmenthol derivatives which were applied in the dug in adhesive(DIA) patches. The in vitro permeation studies indicated that M-OA was the most promising enhancer both in IPM solution and DIA patch for transdermal delivery.The pharmacokinetic analysis of the TOL plasma concentration-time profiles after single-dose intravenous bolus administration was carried out by fitting the data to a two-compartment model(r = 0.993).The steady-state volume of distribution(V_d) and clearance(CL) of TOL were 10.93 L/kg and 0.51 L/h, respectively.The concentration of TOL was too low to be detected in rat plasma up to 6 h of sampling.The in vivo studies in rats were conducted in order to examine the ability of the TDDS of TOL to provide a steady-state plasma concentration of the drug as well to investigate the effects of enhancers on the skin reservoir and to predict the steady-state plasma concentrations (C_SS^P) of TOL from in vivo permeability data.Following topical administration of patches with M-OA,MT as enhancer or without enhancer,pharmacokinetic parameters were analyzed by non-compartment analysis.The pharmacokinetic parameters,including C_max,C_SS,AUC_0-24 and AUC_0-∞ of M-OA group were significantly larger than those of MT group and Control group (P<0.05);there were no significant difference between C_max,C_SS,AUC_0-24 values of Control and MT group(P>0.05),however,AUC_0-∞ of MT group was significantly different from that of Control group(P<0.05).The plasma level of TOL declined after the removal of the patches, and it is noteworthy that the TOL of the Control group was eliminated much faster than in the MT group,significant skin reservoir effects were observed,especially for the patches with MT and M-OA as enhancers,as the rate constants for terminal phase after removal of the patches were much smaller than the rate constant for the elimination phase after i.v bolus.The observed steady-state plasma concentrations(C_SS) of TOL after the application of patches without enhancer(0.89±0.11μg/mL),with MT(0.84±0.14μg/mL) and M-OA (1.47±0.08μg/mL) as enhancers in rats were comparable with the respective predicted C_SS^P (0.95,1.04 and 1.44μg/mL) obtained over the period 0 and 24 h from the in vitro permeation data.This indicates that in vitro experiments with rat skin may be used for further transdermal drug delivery studies with TOL.The correlation analysis using the SPSS(?) program indicated there was a good enhancing activity correlation between the Q₈ from IPM and the Q₈ from patches,especially between the Q₄₈ from patches in vitro and the AUC_0-48 in vivo when TOL was in the Control patch or using MT and M-OA as enhancers,the r is 0.941(P<0.05) and 0.993(p<0.05) respectively.To investigate the permeation mechanism of M-OA,morphological changes in the stratum corneum(SC) were observed by a scanning electron microscope(SEM),and the stretching vibration peak shifts of C-H and amide were estimated by attenuated total reflectance Fourier transform infared(ATR-FTIR).The wrinkles were increased and numerous concavities were observed on the skin surface,moreover,the intercellular space of the SC in the skin surface was enlarged after M-OA treatment.The ATR-FTIR results revealed that the stretching vibrations of v_SCH₂ andv_asCH₂ were shifted from 2849.5 cm^-1 to 2851.1 cm^-1 and from 2919.4 cm^-1 to 2920.9 cm^-1,respectively.Amideâ…¡stretching vibrations in the SC lipids were shifted from 1540.6 cm^-1 to 1539.1 cm^-1.There might be two reasons responsible for the penetration activity of M-OA,one is M-OA could extract the SC lipids,the other is to disorder the arrangement of lipids and increase the degree of freedom of C-H and amide.