Role Of Organic Cation Transporters In Absorption And Excretion Processes Of Pramipexole

In recent years, with the development of molecular biology techniques, the molecular structure and function of the transporter protein in human tissues and cells have gradually been recognized. They play an important role in both influx and efflux for nutrients and drugs, with a high degree of substrate specificity. Organic cation transporters (OCTs) express in kidney, liver and small intestine, and mediate in vivo absorption and elimination of many endogenous amines and drugs. OCTs have a broad substrate/inhibitor overlap. Approximately50%of clinical drugs are organic cations under physiological conditions, including antiarrhythmic drugs, anti-Parkinson drugs, antihistamines,β-adrenergic receptor antagonist, etc. Pramipexole (PPX) is a cationic drug with a net positive charge at physiological pH, but its absorption and excretion processes mediated by OCTs in vivo remain unclear. Therefore, this study intended to research the role of OCTs in the pramipexole absorption across the intestinal and nasal mucosae as well as renal excretion. Moreover, this research explored the risk of drug-drug interaction (DDI) mediated by OCTs between pramipexole and cimetidine.Firstly, we developed and validated a robust and sensitive LC-MS/MS assay without matrix effect for accurate measurements of pramipexole in different kinds of biological matrices. Pramipexole and its stable isotope labeled internal standard (ds-pramipexole) were extracted from tissue samples and other matrices by protein precipitation (PPT) coupled with solid phase extraction (SPE) using weak cation exchange SPE cartridges and liquid-liquid extraction (LLE) using ethyl acetate, respectively. Matrix effects were studied using post-column infusion and post-extraction addition experiments by direct monitoring of typical phospholipids including glycerophosphocho lines (GPChos) and lysogrycerophosphocholines (Lyso-GPChos). Chromatographic separation was achieved on a Welch Ultimate(?) XB-CN column using isocratic elution with a run time of3.0min. The assay was linear (r>0.99) in the concentration range of0.05-100ng/mL. The intra-and inter-day precision was less than 10.6%, while the intra-and inter-day accuracy ranged from95.2to105.6%and from91.4to110.2%for plasma and tissue samples, respectively. The matrix effect at three QC concentrations was almost absent for PPX in plasma and tissues samples (-13.8%to-1.2%). The recovery of PPX at all QC levels in different matrices ranged from80.7%to90.3%. Spiked plasma and tissue samples were stable (RE<±15%) in all satability assay conditions. These results met the acceptance criteria, demonstrating that this method was reproducible and accurate.To investigate the role of OCTs in the absorption of pramipexole across intestinal membranes, the permeabilities of pramipexole across rat duodenum, jejunum and ileum in both absorption (AL-BP) and secretion (BP-AL) directions were evaluated by Ussing Chamber experiments. Then, transport inhibition effects of amantadine, guanidine, procainamide, TEA, quinine and verapamil on pramipexole transport were assessed. The cumulative permeation amounts of pramipexole across the duodenum, jejunum and ileum demonstrated linear kinetics over a concentration range of1-50μM in both transport directions. Interestingly, the cumulative permeation amounts in secretion direction were much higher than that in absorption direction. Furthermore, the cumulative permeation amounts of pramipexole were reduced by various cation transporter inhibitors including amantadine, guanidine, procainamide, TEA, quinine and verapamil in ileum. However, only part of them exhibited significant inhibition effects in duodenum and jejunum. Taken together, these findings implicated regional differences of organic cation transporters in rat intestine might be attributed to distinct efflux of pramipexole across rat intestinal membranes. In addition, we could not rule out the contributions of other efflux transporters reported in rat intestine, such as P-gp and Mrps. Due to the minor role of OCTs in PPX kitestinal absorption, DDI between PPX and other clinical drugs was not likely to occur in intestinal absorption stage.In contrast to the low expression level of OCTs in intestine, OCTs express in a relatively higher level in both nasal respiratory and olfactory mucosae. Since the targeting site of PPX is in the brain, PPX is required to pass through the intestinal mucosa after oral absorption into systemic circulation and cross the blood brain barrier (BBB) to achieve brain drug delivery. As a result, the delivery would be significantly decreased due to the existing barriers, which in turn limited the therapeutic effort of PPX. Thus, we tried to change the administration route to increase the delivery of PPX in the brain. Due to the specific connection between nose and brain, intranasal administration (IN) is considered as a promising alternative for oral or intravenous administration by bypassing the BBB to rapidly deliver therapeutic agents to the brain. Therefore,"brain-targeted" drug delivery of PPX following IN was tested in animal models in vivo. The plasma and brain tissue pharmacokinetics of PPX in mice following intravenous (Ⅳ), oral and IN administration were illustrated using ADAPT5software. Model fitting results showed that the pharmacokinetics characteristics of PPX followed first order kinetics after intravenous and oral administration. After IN administration, the absorption rate was fitted to the Michaelis-Menten combined with passive diffusion equation, and about20%of the drug was transported directly into the brain through the nose-brain pathways. Furthermore,tmax (from1h to30min), Cmax (from14.65±0.43ng/g to20.37±6.30ng/g) and brain targeting efficiency (from4.43to5.77) of PPX were all improved compared with the oral group. These findings indicated that IN administration may achieve faster drug delivery to the brain and enhance drug exposure in brain. Mouse brain slice model showed that a V-type trend of PPX concentration was observed in brain according to the order of brain slices within initial20min after IN administration, which was in good agreement with the features of IN administration. These results indicated that nasal drug delivery for PPX have more advantages than oral dosing, and nose-to-brain pathways play a vital role in the direct transport of PPX into the brain.On the basis of pharmacokinetic studies for PPX after IN administration, the mechanism underlying pramipexole transport in the nasal tissues was further investigated. Of particular interest was the evaluation of the potential involvement of carrier-mediated transport, via OCTs, in the penetration of PPX. Transport experiments were conducted across excised bovine olfactory and respiratory mucosae using Ussing Snapwell vertical diffusion chambers. Western blotting and immunohistochemistry were also performed to determine the expression and localization of OCT1and OCT2in the nasal mucosa. In the olfactory mucosa, PPX transport was found to be saturable and was reduced by a number of cationic transporter inhibitors in M-S direction. The mean flux results were fitted by a Michaelis-Menten type model giving Jmax of18.62±5.46ng cm-2min-1and a Km of44.66±22.65μM. Furthermore, prarmipexole transport was temperature-and membrane potential-dependent, but pH-and sodium-independent, hence likely to be partially mediated by organic cation transporters, including OCT1and OCT2. Conversely, carrier-mediated transport plays a minor role in absorption of pramipexole across the respiratory mucosa as a result of the absence of polarized transport and inhibition effects. The expression of OCT1and OCT2were observed as bands in both tissues via western blotting experiments. The immunohistochemistry results demonstrated that OCT1and OCT2were associated with a ciliated columnar epithelial layer and the nasal submucosal structures of the bovine explants, such as the nasal glands, blood vessels and lympha vessels. The submucosal localization of OCT1and OCT2may assist in the transport of PPX into or out of these regions, similar to that ascribed to OCTs in the kidneys.OCTs were also mediated the transport of PPX across the bovine nasal mucosa, which suggested that utilization of OCTs may provide the opportunity for improved systemic absorption and targeted CNS delivery of PPX. In addition, OCT2is also highly expressed in the kidney. It is a worthy concern whether OCTs are involvd in the disposition of PPX after absorption. Therefore, the present study examined the OCTs mediated mechanism of renal excretion of PPX. In vivo pharmacokinetics experiments, in vitro kidney slice model and hOCT2-HEK293transfected cell uptake studies were performed to investigate the mechanism of renal excretion for PPX. When PPX and cimetidine were co-administered intravenously in rats, the plasma concentrations of PPX were significantly increased in comparison with the control group. The AUC(0-12h)increased by2.45fold and plasma clearance (CLp) reduced by nearly2.30times. The cumulative urinary excretion of PPX during24h was significantly decreased, and renal clearance (CLr) was decreased by3-fold. These results indicated that PPX and cimetidine might share the same transporters in kidney. Furthermore, in vitro kidney slice experiments showed that guanidine (1mM), TEA (0.1-1mM) and cimetidine (0.5mM,1mM) significantly inhibited the uptake of PPX in dose-dependent manners. These findings suggested that the target transporter systems involved in renal excretion of PPX were related to OCTs. In addition, the results obtained from transfected cell line studies showed that cimetidine inhibited renal excretion of PPX by suppressing OCT2in the kidney at molecular level. A Cu, max/ICso value greater than0.1proposed by International Transporter Consortium would indicate an increased potential for a transporter based DDI. The Cu,max/IC50value of cimetidine for OCT2was0.87, which predicted that OCT2-mediated DDI is highly likely to occur between PPX and cimetidine in clinical treatments.