增渗剂和离子导入对尼莫地平体外经皮渗透性的影响
作者：陈森 郑俊民 郝劲松 吴会斌
单位：沈阳药科大学药学系， 沈阳 110015
关键词：尼莫地平；药物透皮传递；离子导入
沈阳药科大学学报990205 摘 要 研究了增渗剂和离子导入技术对尼莫地平(NM)体外经皮渗透性的作用，渗透促进剂如3%和5%月桂氮卓酮及10%油酸的20%丙二醇溶液能增加药物的渗透性(P<0.05)，其增渗比分别为4.66、4.39及12.64.离子导入技术能够显著增加药物的渗透性(P<0.01)，渗透比为8.03.同时表明10%油酸和3%的月桂氮卓酮丙二醇溶液与离子导入并用，渗透比为15、85、8.92.
分类号 R94
Effect of Penetration Enhancers and Iontophoresis on Nimodipine in Vitro Permeation
, 百拇医药
Chen Sen,Zheng Junmin,Hao Jinsong,Wu Huibin
Department of Pharmacy,Shenyang Pharmaceutical University,Shenyang 110015
Abstract This study was intended to investigate the effect of an enhancer and iontophoresis on the in vitro permeability of nimodipine(NM) through full thickness mouse skin.It was shown that enhancers such as 3% azone,5% azone(AZ)and 10% oleic acid(OA)in combination with propylene glycol(PG) pretreatment increased the permeability of NM(P<0.01) with ER of 4.66，4.39 and 12.64 respectively.The use of iontophoresis was found to enhance the permation significantly(P<0.01) with ER of 8.03.It was also found that 10% OA/PG and 3% AZ/PG assisted by transdermal iontophoretic delivery of NM remarkably increased the permeation(P<0.01),with the ER of 15.85 and 8.92.
, 百拇医药
Key words nimodipine;transdermal drug delivery;iontophoresis
1 Introduction
It was shown in this study that iontophoresis had significant enhancement effects on transdermal delivery of many ionic charged drugs〔1〕.The permeability of neutral species could also be increased by iontophoresis due to volume flow via electroosmosis〔2〕.Chemical penetration enhancers could induce a temporary,reversible increase in skin permeability.It was also shown that there existed synergistic effects of iontophoresis and enhancers on the transport of some drugs〔3，4〕.
, 百拇医药
Nimodipine is a potent dihydropyridine calcium antagonist with preferential effect on cerebral vessels.However,this drug has a poor oral bioavailability(5%～13%) because of its high hepatic first pass effect,and it has a short elimination half-life(2 h)，therefore,it is a potential candidate for transdermal studies〔5〕.
The objective of this study is to investigate several enhancers,iontophoresis and the combined application for their ability to enhance the in vitro skin permeation of nimodipine.
, 百拇医药
2 Materials and methods
2.1 Chemicals
Nimodipine(Shandong Xinhua Pharmaceutical Factory,China);Borneol(purchased from a drug store);Poloxamer(Pharmaceutical factory of SPU,China);propylene glycol(PG)and Azone(AZ)were for pharmaceutical use;oleic acid(OA)and all other chemicals were of analytical grade.All solutions were prepared,using water for chromatography.
2.2 Skin and preparation of skin
, 百拇医药
2.2.1 Full-thickness skin
The mice(SD,20 g,male)were killed by cervical dislocation technique.The abdominal region was carefully shaved,using electric clipper(The American Oster Company).The skin form of this region was immediately exercised and the subcutaneous fatty tissue trimmed,then the skin was cleaned with normal saline(NS)and stored at -20℃ until use(less than 1 week).
2.2.2 Stripped skin preparation
, http://www.100md.com
The stratum corneum was removed by well developed stripping technique〔6〕.
2.3 Apparatus
Valia-Chien diffusion cell modified was employed,with an effective diffusion area of 1.43 cm2 and cell volume of 10 mL. During iontophoretic experiments,platinum electrodes were inserted in to the diffusion cells.The current required was generated from a minute pulsed direct current transdermal iontophoretic delivery setup designed by our laboratory,in which pulsed current with square waveform,frequency of 2 000 Hz,on/off ratio of 1/1 and current density of 0.49 mA/cm2 was used.
, 百拇医药
2.4 Pretreatment procedure
Prior to the diffusion experiment,the full-thickness stripped skin was sandwiched between the two compartments with stratum corneum facing the donor compartment.The cell was immersed in a water bath at (32±1)℃ and stirred at a constant rate by external magnetics.The enhancer solution in the cell was added into the donor side,and the receptor side was full of NS.After 1h of pretreatment,all solutions were emptied,and washed with deionized water.The NS was regarded as a control.
, 百拇医药
2.5 Transdermal permeation
After the pretreatment,10 mL of nimodipine saturated solution(prepared by suspending an excess amount of drug crystals in 30% PG/water and stirring for 12 h)was introduced into the donor side,and 10.0 mL of 30% PG solution was added into the receptor side just as the receptor solution.The iontophoretic system was connected,during iontophoresis.The samples were collected from the receptor compartment and the fresh solvent totally replaced at predetermined intervals.All samples were filtered through a 0.45 μm filter,then analysed by the HPLC method.
, http://www.100md.com
2.6 HPLC assay
The Shimadzu HPLC system(LC-10A)and an ultraviolet visible spectrophotometric detector(SPD-10A)were used.The column was C18 spherisorb 4.6 mm ×250 mm.The UV detector was operated at the wavelength of 237 nm.
The mobile phase used for nimodipine assay consisted of a combination(64∶36) of CH3OH and H2O at a flow rate of 1.0 mL/min.The temperature in the oven was maintained at 35℃.
, 百拇医药
The recovery of determination was (100±2.64)% and the variations within a day and between days were both less than 5%.
2.7 Data analysis
The cumulative amounts(Q,ng/cm2) were plotted against time(t,h).From the slope of the linear portion of the permeation profile,steady state flux(Jss,ng/cm2.h) could be estimated,the permeation coefficient(Kp)was calculated as follows:Kp=Jss/ΔC.All the signs are the same as usual and the enhancement activity of the enhancers was expressed as follows:Enhancement ratio(ER)=KP(after treatment)/KP(before treatment)〔7〕.
, http://www.100md.com
Statistical analyses were made,using Student′s t-test.The level of significance was taken as P<0.05.
3 Results and discussion
Fig.1 Effect of etripping on the transdermal permeation of NM through mouse abdominal skin. Each data point
represents the mean±s of six determinations.
1—full-thickness skin;2—stripped skin.
, 百拇医药
Tab.1 Effect of enhancers on Jss and permeation coefficient(Kp) of NM during
passive transport Penetration
enhancer
Parameters
ER
Jss×10-3
/μg(cm2.s)-1
KP×105
, http://www.100md.com
/cm.s-1
Control
0.100±0.011
0.259±0.028
1.0
10%PO/H2O
0.087±0.008
0.225±0.020
0.9
10%EO/PG
0.116±0.021
, 百拇医药
0.298±0.053
1.2
3%AZ/PG
0.488±0.100
1.206±0.258
4.6
5%AZ/PG
0.441±0.084
1.136±0.216
4.4
10%OA/PG
1.270±0.167
, http://www.100md.com
3.274±0.430
12.6
Control,hydration with normal saline but without any enhancer The figare shows the permeation profile of NM throuh full-thickness skin and stripped skin.Poor skin permeablity of NM could be detected under the former condition.Stripping significantly increased the transport of the drug(P<0.01)，with the steady state flux 12 times higher than that of the control.The result demonstrated the remarkable barrier function of stratum corneum for NM.
, 百拇医药
Tab.2 Enhancement ratio for NM during
iontophoresis and in combination with enhancer Penetration
enhancer(PE)
Enhancement ratio
E1
E2
E3
5%AZ/PO
4.65
1.11
, http://www.100md.com
8.92
10%OA/PG
12.58
1.97
15.85
E0=KP (iontophoresis)/Kp(passive)=8.03;
E1=KP with PE(combined with iontophoresis)/Kpwithout PE(passive);
E2=KP with PE(combined with iontophoresis)/Kpwithout PE(ionotophoresis)；
, 百拇医药
E3=KP with PE(combined with iontophoresis)/Kpwithout PE(passive) The effect of the enhancers on the in vitro permeation of NM through full thickness mouse skin is shown in Table 1.NM in 3% AZ/PG，5%AZ/PG and 10% OA/PG led to a remarkable increase in NM permeation with ER of 4.66,4.39 and 12.64.Azone would enhance the premeation of both hydrophobic and hydrophilic molecules〔8〕,even though more dramatic enhancements could be usuaully seen with hydrophilic drugs.Because nimodiping is a highly lipophilic drug,the effect of AZ on its transport was still significant but not strong,which is not difficult to understand.In the study of Naik,Pechtold,Potts et al.〔9〕, the mode of action of oleic acid in vivo in man was determined.Their results showed that OA inducing skin penetration enhancement resulted from a mechanism involving both SC lipid fluidization and phase separation,with the latter probably predominant.To elucidate the mechanism of enhancenent of skin permeation by OA, the estimated in vivo penteration profiles were analyzed based on a two-layer skin diffusion model with polar and nonpolar routes in the stratum corneum〔10〕.OA increased both the diffusivity and partitioning parameters in the nonpolar route.In our study,10% OA/PG took the most remarkable part in NM permeation,maybe because of the lipophilic property of the drug.It was shown that the permeation coefficient of transdermal iontophoretic delivery of NM was significantly greater(P<0.01) than passive transport though untreated skin with ER of 8.03.The enhancement of the transport of NM(a neutral ,nonpolar drug) during iontophoresis is perhaps a result of electrostatic repulsion and current-inducd solvent flow.In addition,Inado,Ghanem,Higuchi et al.〔11〕 demonstrated that the applied electric field may induce temporary， reversible pore in the skin,which,the so-called electroporation,can enhance the transport of the molecules.This phenomenon perhaphs was one of the reasons of the enhancenment effect of iontophoresis on NM transport.But as stated above, 3% AZ/PG and 10% of OA/PG could remarkably increase the steady state flux and permeation coefficient of NM versus control.The permeation coefficient of iontophoresis in combination with the enhancer was much greater than that of the enhancer pretreated passive diffusion(P<0.01).However,there wasn't evident change of flux between iontophoresis and iontophoresis in combination with AZ(P>0.05),We still can not account for the reason.The electrochemical reaction may be one of the reasons.
, 百拇医药
参考文献
1 Chien Y W,Banga A K.Iontophoretic(Transdermal) delivery of drugs:Overview of historical development.J Pharm Sci,1989，78(5)：535～354
2 Pkal M J.Transport mechanisms in iontophoresis I:A theoretical model for the effect of electroosmotic flow on flux enhancement in transdermal iontophoresis.Pharm Res,1990，7(2)：118～126
3 Ganga S,Ramarao P,Singh J,Effect of azone on the iontophoretic transdermal delivery of metoprolol tartrate through human epidermis in vitro.J Controll Release,1996，42：57～64
, 百拇医药
4 Bhatita K S,Gao S,Singh J.Effect of penetration enhancers and iontophoresis on the FT-IR spectroscopy and LHRH permeability through porcine skin.J Contr Rel, 1997，47：81～89
5 Langley M S, Sorokin E M.Nimodipine:A review of its pharmacodynamic and pharmacokinetic properties,and therapeutic potential in cerebrovascular diseases.Drugs,1989，37：669～699
6 Banga A K,Chien Y W.Characterization of in vitro transdermal iontophoretic delivery of insuln.Drug Dev Ind Pharm,1993，19(16)：2069～2087
, http://www.100md.com
7 Cornwell P A,Barry B W.Effect of penetration enhancer treatment on the statistical distribution of human skin permabilities.Int J Pharm,1995,117：101～112
8 Stoughton R B,Mc Clure W O.Azone:A new non-toxic enhancer of cutaneous penetraton.Drug Dev Ind Pharm,1983，9(4)：725～744
9 Naik A,Pechtold L A R M，Potts R O.Mechanism of oleic acid-induced skin penetration enhancement in vivo in humans.J Controll Release,1995，37：299～306
, http://www.100md.com
10 Yamashita F,Koyama Y,Kitano M.Analaysis of in vivo skin penetration enhancement by oleic acid based on a two-layer diffusion model with polar and nonpolar-routes in the stratum couneum.Int J Pharm,1995，117：173～179
11 Inado H,Ghanem A-H，Higuchi W I.Studies on the effects of applied voltage and duration on human epidermal membrane alteration/recovery and the resultant effects upon iontophoresis.Pharm Res,1994，11(5)：687～697
收稿日期:1998-09-21, 百拇医药
单位：沈阳药科大学药学系， 沈阳 110015
关键词：尼莫地平；药物透皮传递；离子导入
沈阳药科大学学报990205 摘 要 研究了增渗剂和离子导入技术对尼莫地平(NM)体外经皮渗透性的作用，渗透促进剂如3%和5%月桂氮卓酮及10%油酸的20%丙二醇溶液能增加药物的渗透性(P<0.05)，其增渗比分别为4.66、4.39及12.64.离子导入技术能够显著增加药物的渗透性(P<0.01)，渗透比为8.03.同时表明10%油酸和3%的月桂氮卓酮丙二醇溶液与离子导入并用，渗透比为15、85、8.92.
分类号 R94
Effect of Penetration Enhancers and Iontophoresis on Nimodipine in Vitro Permeation
, 百拇医药
Chen Sen,Zheng Junmin,Hao Jinsong,Wu Huibin
Department of Pharmacy,Shenyang Pharmaceutical University,Shenyang 110015
Abstract This study was intended to investigate the effect of an enhancer and iontophoresis on the in vitro permeability of nimodipine(NM) through full thickness mouse skin.It was shown that enhancers such as 3% azone,5% azone(AZ)and 10% oleic acid(OA)in combination with propylene glycol(PG) pretreatment increased the permeability of NM(P<0.01) with ER of 4.66，4.39 and 12.64 respectively.The use of iontophoresis was found to enhance the permation significantly(P<0.01) with ER of 8.03.It was also found that 10% OA/PG and 3% AZ/PG assisted by transdermal iontophoretic delivery of NM remarkably increased the permeation(P<0.01),with the ER of 15.85 and 8.92.
, 百拇医药
Key words nimodipine;transdermal drug delivery;iontophoresis
1 Introduction
It was shown in this study that iontophoresis had significant enhancement effects on transdermal delivery of many ionic charged drugs〔1〕.The permeability of neutral species could also be increased by iontophoresis due to volume flow via electroosmosis〔2〕.Chemical penetration enhancers could induce a temporary,reversible increase in skin permeability.It was also shown that there existed synergistic effects of iontophoresis and enhancers on the transport of some drugs〔3，4〕.
, 百拇医药
Nimodipine is a potent dihydropyridine calcium antagonist with preferential effect on cerebral vessels.However,this drug has a poor oral bioavailability(5%～13%) because of its high hepatic first pass effect,and it has a short elimination half-life(2 h)，therefore,it is a potential candidate for transdermal studies〔5〕.
The objective of this study is to investigate several enhancers,iontophoresis and the combined application for their ability to enhance the in vitro skin permeation of nimodipine.
, 百拇医药
2 Materials and methods
2.1 Chemicals
Nimodipine(Shandong Xinhua Pharmaceutical Factory,China);Borneol(purchased from a drug store);Poloxamer(Pharmaceutical factory of SPU,China);propylene glycol(PG)and Azone(AZ)were for pharmaceutical use;oleic acid(OA)and all other chemicals were of analytical grade.All solutions were prepared,using water for chromatography.
2.2 Skin and preparation of skin
, 百拇医药
2.2.1 Full-thickness skin
The mice(SD,20 g,male)were killed by cervical dislocation technique.The abdominal region was carefully shaved,using electric clipper(The American Oster Company).The skin form of this region was immediately exercised and the subcutaneous fatty tissue trimmed,then the skin was cleaned with normal saline(NS)and stored at -20℃ until use(less than 1 week).
2.2.2 Stripped skin preparation
, http://www.100md.com
The stratum corneum was removed by well developed stripping technique〔6〕.
2.3 Apparatus
Valia-Chien diffusion cell modified was employed,with an effective diffusion area of 1.43 cm2 and cell volume of 10 mL. During iontophoretic experiments,platinum electrodes were inserted in to the diffusion cells.The current required was generated from a minute pulsed direct current transdermal iontophoretic delivery setup designed by our laboratory,in which pulsed current with square waveform,frequency of 2 000 Hz,on/off ratio of 1/1 and current density of 0.49 mA/cm2 was used.
, 百拇医药
2.4 Pretreatment procedure
Prior to the diffusion experiment,the full-thickness stripped skin was sandwiched between the two compartments with stratum corneum facing the donor compartment.The cell was immersed in a water bath at (32±1)℃ and stirred at a constant rate by external magnetics.The enhancer solution in the cell was added into the donor side,and the receptor side was full of NS.After 1h of pretreatment,all solutions were emptied,and washed with deionized water.The NS was regarded as a control.
, 百拇医药
2.5 Transdermal permeation
After the pretreatment,10 mL of nimodipine saturated solution(prepared by suspending an excess amount of drug crystals in 30% PG/water and stirring for 12 h)was introduced into the donor side,and 10.0 mL of 30% PG solution was added into the receptor side just as the receptor solution.The iontophoretic system was connected,during iontophoresis.The samples were collected from the receptor compartment and the fresh solvent totally replaced at predetermined intervals.All samples were filtered through a 0.45 μm filter,then analysed by the HPLC method.
, http://www.100md.com
2.6 HPLC assay
The Shimadzu HPLC system(LC-10A)and an ultraviolet visible spectrophotometric detector(SPD-10A)were used.The column was C18 spherisorb 4.6 mm ×250 mm.The UV detector was operated at the wavelength of 237 nm.
The mobile phase used for nimodipine assay consisted of a combination(64∶36) of CH3OH and H2O at a flow rate of 1.0 mL/min.The temperature in the oven was maintained at 35℃.
, 百拇医药
The recovery of determination was (100±2.64)% and the variations within a day and between days were both less than 5%.
2.7 Data analysis
The cumulative amounts(Q,ng/cm2) were plotted against time(t,h).From the slope of the linear portion of the permeation profile,steady state flux(Jss,ng/cm2.h) could be estimated,the permeation coefficient(Kp)was calculated as follows:Kp=Jss/ΔC.All the signs are the same as usual and the enhancement activity of the enhancers was expressed as follows:Enhancement ratio(ER)=KP(after treatment)/KP(before treatment)〔7〕.
, http://www.100md.com
Statistical analyses were made,using Student′s t-test.The level of significance was taken as P<0.05.
3 Results and discussion
Fig.1 Effect of etripping on the transdermal permeation of NM through mouse abdominal skin. Each data point
represents the mean±s of six determinations.
1—full-thickness skin;2—stripped skin.
, 百拇医药
Tab.1 Effect of enhancers on Jss and permeation coefficient(Kp) of NM during
passive transport Penetration
enhancer
Parameters
ER
Jss×10-3
/μg(cm2.s)-1
KP×105
, http://www.100md.com
/cm.s-1
Control
0.100±0.011
0.259±0.028
1.0
10%PO/H2O
0.087±0.008
0.225±0.020
0.9
10%EO/PG
0.116±0.021
, 百拇医药
0.298±0.053
1.2
3%AZ/PG
0.488±0.100
1.206±0.258
4.6
5%AZ/PG
0.441±0.084
1.136±0.216
4.4
10%OA/PG
1.270±0.167
, http://www.100md.com
3.274±0.430
12.6
Control,hydration with normal saline but without any enhancer The figare shows the permeation profile of NM throuh full-thickness skin and stripped skin.Poor skin permeablity of NM could be detected under the former condition.Stripping significantly increased the transport of the drug(P<0.01)，with the steady state flux 12 times higher than that of the control.The result demonstrated the remarkable barrier function of stratum corneum for NM.
, 百拇医药
Tab.2 Enhancement ratio for NM during
iontophoresis and in combination with enhancer Penetration
enhancer(PE)
Enhancement ratio
E1
E2
E3
5%AZ/PO
4.65
1.11
, http://www.100md.com
8.92
10%OA/PG
12.58
1.97
15.85
E0=KP (iontophoresis)/Kp(passive)=8.03;
E1=KP with PE(combined with iontophoresis)/Kpwithout PE(passive);
E2=KP with PE(combined with iontophoresis)/Kpwithout PE(ionotophoresis)；
, 百拇医药
E3=KP with PE(combined with iontophoresis)/Kpwithout PE(passive) The effect of the enhancers on the in vitro permeation of NM through full thickness mouse skin is shown in Table 1.NM in 3% AZ/PG，5%AZ/PG and 10% OA/PG led to a remarkable increase in NM permeation with ER of 4.66,4.39 and 12.64.Azone would enhance the premeation of both hydrophobic and hydrophilic molecules〔8〕,even though more dramatic enhancements could be usuaully seen with hydrophilic drugs.Because nimodiping is a highly lipophilic drug,the effect of AZ on its transport was still significant but not strong,which is not difficult to understand.In the study of Naik,Pechtold,Potts et al.〔9〕, the mode of action of oleic acid in vivo in man was determined.Their results showed that OA inducing skin penetration enhancement resulted from a mechanism involving both SC lipid fluidization and phase separation,with the latter probably predominant.To elucidate the mechanism of enhancenent of skin permeation by OA, the estimated in vivo penteration profiles were analyzed based on a two-layer skin diffusion model with polar and nonpolar routes in the stratum corneum〔10〕.OA increased both the diffusivity and partitioning parameters in the nonpolar route.In our study,10% OA/PG took the most remarkable part in NM permeation,maybe because of the lipophilic property of the drug.It was shown that the permeation coefficient of transdermal iontophoretic delivery of NM was significantly greater(P<0.01) than passive transport though untreated skin with ER of 8.03.The enhancement of the transport of NM(a neutral ,nonpolar drug) during iontophoresis is perhaps a result of electrostatic repulsion and current-inducd solvent flow.In addition,Inado,Ghanem,Higuchi et al.〔11〕 demonstrated that the applied electric field may induce temporary， reversible pore in the skin,which,the so-called electroporation,can enhance the transport of the molecules.This phenomenon perhaphs was one of the reasons of the enhancenment effect of iontophoresis on NM transport.But as stated above, 3% AZ/PG and 10% of OA/PG could remarkably increase the steady state flux and permeation coefficient of NM versus control.The permeation coefficient of iontophoresis in combination with the enhancer was much greater than that of the enhancer pretreated passive diffusion(P<0.01).However,there wasn't evident change of flux between iontophoresis and iontophoresis in combination with AZ(P>0.05),We still can not account for the reason.The electrochemical reaction may be one of the reasons.
, 百拇医药
参考文献
1 Chien Y W,Banga A K.Iontophoretic(Transdermal) delivery of drugs:Overview of historical development.J Pharm Sci,1989，78(5)：535～354
2 Pkal M J.Transport mechanisms in iontophoresis I:A theoretical model for the effect of electroosmotic flow on flux enhancement in transdermal iontophoresis.Pharm Res,1990，7(2)：118～126
3 Ganga S,Ramarao P,Singh J,Effect of azone on the iontophoretic transdermal delivery of metoprolol tartrate through human epidermis in vitro.J Controll Release,1996，42：57～64
, 百拇医药
4 Bhatita K S,Gao S,Singh J.Effect of penetration enhancers and iontophoresis on the FT-IR spectroscopy and LHRH permeability through porcine skin.J Contr Rel, 1997，47：81～89
5 Langley M S, Sorokin E M.Nimodipine:A review of its pharmacodynamic and pharmacokinetic properties,and therapeutic potential in cerebrovascular diseases.Drugs,1989，37：669～699
6 Banga A K,Chien Y W.Characterization of in vitro transdermal iontophoretic delivery of insuln.Drug Dev Ind Pharm,1993，19(16)：2069～2087
, http://www.100md.com
7 Cornwell P A,Barry B W.Effect of penetration enhancer treatment on the statistical distribution of human skin permabilities.Int J Pharm,1995,117：101～112
8 Stoughton R B,Mc Clure W O.Azone:A new non-toxic enhancer of cutaneous penetraton.Drug Dev Ind Pharm,1983，9(4)：725～744
9 Naik A,Pechtold L A R M，Potts R O.Mechanism of oleic acid-induced skin penetration enhancement in vivo in humans.J Controll Release,1995，37：299～306
, http://www.100md.com
10 Yamashita F,Koyama Y,Kitano M.Analaysis of in vivo skin penetration enhancement by oleic acid based on a two-layer diffusion model with polar and nonpolar-routes in the stratum couneum.Int J Pharm,1995，117：173～179
11 Inado H,Ghanem A-H，Higuchi W I.Studies on the effects of applied voltage and duration on human epidermal membrane alteration/recovery and the resultant effects upon iontophoresis.Pharm Res,1994，11(5)：687～697
收稿日期:1998-09-21, 百拇医药