The Relation Between The Intestinal Transport Characteristics Of Capsaicin And TRPV1or Multidrug Resistance Proteins

Background&ObjectiveCapsaicin (8-methyl-N-vanillyl-6-nonenamide) is a primary pungent and irritating principle present in chilies and red peppers. Mol Wt:305.42. It has been widely used not only as food additive, but also as a potent drug to treat neuropathic pain in clinical. The Food and Drug Administration (FDA) has approved the approval of Qutenza (capsaicin)8%patch to treat postherpetic neuralgia. In addition, there is some researches showed that capsaicin at sub-cytotoxic concentrations was capable of inhibiting the P-gp efflux activity to a comparable level as the established P-gp inhibitor, verapamil. It has demonstrated that capsaicin could reverse doxorubicin resistance in human multidrug resistant carcinoma KB-C2cells by inhibiting the P-gp efflux pump, and increased AB digoxin transport and inhibited BA digoxin transport across the Caco-2cells at low concentration. Also, capsaicin can alters both transcellular and paracellular pathways of intestinal cephalexin and cefazolin absorption. As hot chilli is consumed on a daily basis in many populations, it is necessary to understand the transport mechanism of capsaicin through intestine. However, the research and application of capsaicin mainly concentrated in percutaneous absorption, it is still unclear about the mechanism of capsaicin on intestinal absorption. Therefore, we studied the permeation of capsaicin across different intestinal segments to understand the relationship between capsaicin and transporters or channel on the membrane. This article selects transporters or channel is mainly considered based on below:The application of capsaicin as an analgesic to treat neuropathic pain is benefiting from its highly selective activating lig and for transient receptor potential vanilloid1(TRPV1). TRPV1is a detector for noxious heat (>43℃) and inflammatory pain. The permeation of some small metal ions acrossTRPVlis dependent on the activation of TRPV1. It has been found that TRPV1could be opened to increase the permeation of large cations by some chemical regulators such as capsaicin. Moreover, it has been proved that the TRPV1channel has at least two binding sites for CAP, extracellular and intracellular sides of the membrane. The binding of CAP to one site of the channel may interact the binding of CAP to another site. Consequently, we conjecture whether the action of TRPV1by capsaicin could also mediate the permeation of itself. The hypothesis can be judged by blocking the TRPV1with ruthenium red (a TRPV channel blocker) in the transport of capsaicin across different intestinal segments, including jejunum, ileum and colon. Besides, it has been reported that capsaicin is an inhibitor of P-gp. So far, it has been suggested that a P-gp inhibitor usually be also a P-gp substrate such as mibefradil. Additionally, most P-gp substrates are of relatively hydrophobic nature because they are able to access the hydrophobic binding site. Therefore, we suspected that capsaicin might be a P-gp substrate because of its hydrophobicity. We investigated the effect of P-gp on the transport of capsaicin across different intestinal regions in the study. Furthermore, the substrates of multidrug resistance proteins such as multidrug resistance-associated protein2(MRP2) and breast cancer resistance protein (BCRP) are often overlapped with those of p-gp, and no report has been published to evaluate the effect of MRP2or BCRP on the permeation of capsaicin. Therefore, we designed to use probenecid (MRP2inhibitor) and novobiocin (BCRP inhibitor) to study the probable impact of these transporters on the permeability of capsaicin across different intestinal segments.In the study, capsaicin with absence of the inhibitors was set as the control group. As the expression levels of TRPV1and multidrug resistance proteins along the intestine track appear to be difference, all of the experiments were performed across jejunal, ileac and colonic membranes. Additionally, Na-fluorescein (Na-Flu), an indicator of the paracellular pathway, was used to reveal the condition of the intestinal epithelial TJ barrier on different intestine. In all, in this study we evaluated the permeation differences of capsaicin across three intestinal segments. The contribution of TRPV1in the intestinal permeation of capsaicin was evaluated by co-using RR as a TRPV1blocker. Moreover, the roles of multidrug resistance proteins in the transporting of capsaicin were investigated by co-using verapamil, probenecid, and novobiocin, respectively. And in order to validate the correlation between in vitro and in vivo absorption of capsaicin, in situ closed loop method has been used to measure the plasma levels.Content&Results:First, we establish the HPLC method for the determination of capsaicin in the transdermal solution obtained from the intestinal transport study. There is no need to have Special Handling for the transdermal solution, it can be injected into HPLC after high speed centrifugation on15000r/m. Capsaicin was analyzed using a Ecosil C18(150mmx4.6mm,5μm) with a flow rate of1.2ml/min at30℃. The mobile phase consisted of methanol-water (70:30, v/v), detection wavelength were280nm in7min.A fluorospectro photometer method for determining the concenteation of R123and CF was established. The EX was set at485nm and Em was set at535nm for R123. Calibration Curve of R123was the follow:Y=1.0435X-1.0989(R2=0.9982), and the linearity was good in the range of15.625～500μg·L-1, with the approach recovery and within-day RSD of R123were98.9%and1.9%. The EX was set at490nm and Em was set at520nm for CF. Calibration Curve of CF was the follow: Y=0.639X+0.068(R2=0.9997), and the linearity was good in the range of7.8125～500μg·L-1, with the approach recovery and within-day RSD of CF were97.8%and2.7%.A HPLC-MS/MS detector method for determining the concenteation of capsaicin was established in rat plasma. Capsaicin was extracted from aliquots of plasma with acetone and ethyl acetate (15:85) as the solvent and chromatographic separation was performed using a ZorbaxEclipse Plus C18column. To quantify capsaicin and IS respectively, multiple reaction monitoring (MRM) transition of m/z306â†'137and m/z455â†'165was performed. The analysis time was2.5min in positive mode; the calibration curve was linear in the concentration range of1.85-370ng/ml (R2=0.9997) ng/ml. The lowest limit of quantification (LLOQ) reachedl.85ng/ml. The recovery for the QC (3.7,37,370ng/ml) were77.34%,70.64%and78.02%, respectively.The permeability of capsaicin via different intestinal membranes was evaluated by an in vitro diffusion chamber system at different concentrations. Data in all experiments were presented as the mean±SD. Statistical differences were tested by Factorial design analysis of variance. Differences were considered to be significant when P<0.05.There were regional differences in the in vitro permeability of capsaicin(F=17.808, P=0.000). The Papp of capsaicin in colon was also significantly higher than that in jejunum or ileum in the M-S direction.The permeability of R123or CF via different intestinal membranes was evaluated by an in vitro diffusion chamber system, with or without the co-administration of capsaicin. Data in all experiments were presented as the mean±SD. Statistical differences were tested by Factorial design analysis of variance. Differences were considered to be significant when P<0.05.The mucosal-to-serosal (M-S) of R123was increased and its serosal-to-mucosal transport (S-M) transport was decreased with co-administration of capsaicin across jejunum transport. However, no action of capsaicin at test concentration was found on the transport of R123across ileum or colon.The mucosal-to-serosal (M-S) and serosal-to-mucosal transport (S-M) transport of CF were both increased with co-administration of capsaicin across jejunum transport. However, no action of capsaicin at test concentration was found on the transport of CF across ileum or colon.The contribution of TRPV1to the transport of capsaicin was performed by co-administrating RR as a blocker of the TRPV1. Data in all experiments were presented as the mean±SD. Statistical differences were tested by one-way ANOVA and independent-sample test. Differences were considered to be significant when P<0.05.The activity of LDH at2h after completing the permeation of capsaicin (200μM) through the intestine was detected by LDH kit. There is no significant effect on the release of LDH from jejunal, ileac or colonic membrane (t=-0.918, P=0.394; t=0.402, P=0.702; t=-2.002, P=0.092). It suggests that capsaicin may not cause intestinal membrane damage in the study.RR significantly reduced the M-S direction transport of capsaicin across colon region, while no effect on the transport of capsaicin across jejunum or ileum. However, there were no statistical differences in the M-S permeation of capsaicin among different intestinal regions in presence of ruthenium red. Additionally, there was no further decrease of the permeation of capsaicin across colon when the concentration of RR was increased to a higher concentration.The influence of transporter proteins in the intestinal bidirectional transportation of capsaicin, jejunum, ileum and colon were performed respectively. Data in all experiments were presented as the mean±SD. Statistical differences were tested by one-way ANOVA and independent-sample test. Differences were considered to be significant when P<0.05.Verapamil was applied as P-gp inhibitor, which showed it had no significant effect on M-S or S-M direction of three intestinal segments for the transport of capsaicin.There is no effect on the absorption of capsaicin across the different intestinal regions by co-using MRP2inhibitor probenecid. In addition, the efflux of capsaicin was not significantly reduced by probenecid.However, the BCRP inhibitor novobiocin significantly reduced the M-S transport of capsaicin across ileum and colon, while no effect on the M-S transport of capsaicin across jejunum. It did not affect the S-M transport of capsaicin in each intestinal region. Rat plasma concentrations and pharmacokinetic parameters of capsaicin were examined by in situ closed loop method. Data in all experiments were presented as the meaniSD. Statistical differences were tested by one-way ANOVA. Differences were considered to be significant when P<0.05.It was found that the largest plasma concentration (Cmax), area under curve (AUC) of capsaicin absorbed in colon were higher than it in jejunum or ileum. However, there is no statistics difference when analyzed by one-way ANOVA.Conclusion&DiscussionIn this primary study, we excitedly realized that it was the first time to find the permeability of capsaicin across the colonic mucosa in M-S direction is remarkably higher than that across jejunal or ileac mucosa. However, there is no statistics difference for the absorption of capsaicin by in situ across different intestinal regions. As the influencing factors are much complex in in situ, some indirect interaction may affect the absorption of capsaicin, which have not been studied clarity. The result suggests that there is still a further study for the correlation between in vitro and in situ method.It shows that capsaicin could affect the permeation of R123by inhibiting P-gp, and increased the permeation of CF by opening the paracellular pathway. However, these effects were only appeared through the jejunal membrane. This may because the regional distribution difference of P-gp and tight junction (TJ) along the intestine were both increased from jejunum to colon.Secondly, RR significantly reduced the M-S direction transport of capsaicin across colon region, while no effect on the transport of capsaicin across jejunum or ileum. However, there were no statistical differences in the M-S permeation of capsaicin among different intestinal regions in presence of ruthenium red. Additionally, there was no further decrease of the permeation of capsaicin across colon when the concentration of RR was increased to a higher concentration (20μM). It seems that RR alters the characteristic of capsaicin transport across the intestinal membranes. BCRP is also an efflux transporter belonging to multidrug resistance proteins, and the inhibition of BCRP by novobiocin could increase the permeability of its substrates. However, our data showed that the permeability of capsaicin was decreased dramatically in all the segments in presence of novobiocin, especially in ileal colonic mucosa. It seemed that the transport of capsaicin was not the substrate of BCRP. There should be another transporter, which could pump capsaicin from mucosal to serosal side, and the transporter could be inhibited by novobiocin.