| There is a physiological barrier composed of transporters and metabolic enzymes in the skin.P-glycoprotein(P-gp)is an adenosine triphosphate(ATP)binding cassette(ABC)transporter in epidermis and dermis that can transport a variety of substrates with different structures.Paeoniflorin(PF),a P-gp substrate with sedative,anti-inflammatory,analgesic,and antithrombotic effects,has been widely used in the topical treatment of skin diseases.Verapamil(VER)is a typical inhibitor of P-gp and many studies have reported that VER affected the intestinal transport and dynamic absorption of PF in rats via P-gp.However,the effects of VER in the transdermal absorption of PF via P-gp has rarely been reported.In addition,the effects of P-gp inhibitors on the disposition of different substrate-drugs in the skin has been described in some existing research reports;however,the effects of specific interactions between drugs and inhibitors on the transdermal permeation of drugs still need to be elucidated.It is therefore of great importance for the application of P-gp substrate-drugs in the treatment of non-intact skin diseases to investigate the effect of different concentrations of VER on the percutaneous absorption of PF via P-gp.In this paper,the effect of different concentrations of VER on the transdermal absorption of PF via P-gp was investigated,and its mechanisms of action were studied.The main results were as follows:First,the HPLC quantitative analysis methods of PF and VER were established and optimized.Both methods have good linearity range,special properties and high precision,and are both suitable for experimental quantitative analysis.PF and VER have good solubility(125.68 ± 1.50 mg/m L and 33.73 ± 1.21 mg/m L),high stability,do not interfere with each other in receiving fluids and are not easily metabolized by enzymes in the skin.Second,the effects of different concentrations of VER on the in vitro percutaneous permeability and retention of PF and the in vivo skin and muscle retention were investigated.The results showed that the in vitro permeability of PF across stripped rat skin was significantly increased at a VER concentration range of 6-18 mg/m L(p < 0.01).PF retention in viable skin and muscle in vivo was significantly different following the addition of different concentrations of VER.Compared with the control group,the retention of PF in the skin and muscle in vivo was significantly increased in the 6 mg/m L VER group but decreased in the 30mg/m L VER group(p < 0.05).In conclusion,both cumulative permeated amount and retention amount of PF exhibited a first rising and then falling trend with the rising concentration of added VER,and appeared to be maxima.In addition,the amount of PF accumulation in Hacat cells and Caco-2 cells in the presence of different concentrations of VER was investigated.The results showed that the addition of 6 μg/m L of VER significantly promoted the efflux of PF(p < 0.05),and the amount of PF accumulation varied with the concentration of VER,which indicated that P-gp was involve in the transdermal absorption process of PF and was affected by VER.Finally,the mechanism underlying the effect of VER on the percutaneous absorption of PF via P-gp was explored.By using P-gp substrate calcein-AM for cellular substrate accumulation study and molecular docking study,it was found that both PF and VER would obviously inhibit the transport activity of P-gp,and the inhibition effect of PF on P-gp was stronger than VER at the same concentration.The competitive binding of PF and VER to the nucleotide binding domain(NBD)of P-gp was one of the factors responsible for the differential percutaneous permeation of PF.In conclusion,different concentrations of VER acting on P-gp altered the transdermal permeability and retention of PF,thus providing a theoretical basis for guiding clinical medication for certain drugs that are substrates of P-gp in non-intact morbid skin. |
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