The Role Of Organic Cation Transporters And CYPs In The Transportation And Toxicity Of Nitidine Chloride

Protoberberine alkaloids, such as berberine, palmatine, coptisine, epiberberine and jatrorrhizine, etc., were considered to have many pharmacological activities, including antibacterial, anti-inflammatory, analgesia, hypoglycemic effect. Nitidine chloride, a typical protoberberine alkaloid, is isolated from the root of Zanthoxylum nitidum, which can inhibit proliferation and induce cell apoptosis in several tumor types including lung cancer, oral squamous cell carcinoma and liver cancer. The toxicity of nitidine is one of the key factors to determin whether the compounds should be exclude from drug candidates. There are some reports on cytotoxicity in vitro as well as in vivo induced by nitidine, including the toxicity on primary cultured rat hepatocytes.The polyspecific organic cation transporters. (OCTs) of the SLC22 family, including three organic cation transporters (OCT1-OCT3), have been reported to play an important role in the transport of organic cations, such as weak bases and some neutral compounds. Since the protoberberine alkaloid is a kind of quaternary or tertiary ammonium alkaloid, it will mainly be a cation or neutral compouds at physiological pH (7.4). Based on the structures of protoberberine alkaloids, we deduced that OCTs may mediated the uptake of the alkaloids in the hepatocytes or nephrocytes. Thus, the aim of the present study was to study their interaction with OCTs and assess the role of OCTs in the transportation, hepatotoxicity or nephrotoxicity of nitidine in vitro or in vivo, and then to determine the contribution of CYPs-mediated metabolism in attenuating the nitidine-induced hepatocellular toxicity by the transgenic cells, human liver microsomal, human recombinase, and animal model. The results will give us more information about the possible mechanisms and relationship associated with hepatocellular or nephrotoxicity induced by nitidine in vitro or in vivo.1. Interaction of protoberberine alkaloids with human organic cation transporters 1 and 2.Protoberberine alkaloids, an important group of alkaloids with remarkable biological actives, is a kind of quaternary or tertiary ammonium alkaloid, it will mainly be a cation or neutral compouds at physiological pH. The aim of this study was to investigate the potential interaction between the protoberberines (berberine, coptisine, jateorhizine, nitidine, epiberberine, berberrubine, palmatine, (-)-tetrahydropalmatine, corydaline) with organic cation transporters 1 (OCT1) and 2 (OCT2) and whether the alkaloids were the substrates and/or inhibitors of OCT1/OCT2, and whether OCT1/OCT2 polymorphisms may affect the transport of berberine and coptisine in vitro. The results showed that all investigated alkaloids significantly inhibited the OCTs-mediated MPP+ uptake, moreover the uptake of berberine, coptisine, jateorhizine, nitidine, epiberberine and berberrubine in MDCK-hOCTl/MDCK-hOCT2 cells was significantly higher than that in mock cells. The hOCTl-and hOCT2-mediated uptake followed Michaelis-Menten kinetics. Additionally, berberine and coptisine uptake were decreased markedly in MDCK expressing OCT1-P341L and OCT2-A270S variants compared with that in the wild type cells, whereas the Clmt of berberine in MDCK expressing OCT1-P341L and OCT2-A270S were increased to 3.1- and 1.3-fold compared to that of the wild type cells as well as 3.6- and 2.8-fold for coptisine, respectively. Taken together, these in vitro studies showed that the investigated alkaloids significantly inhibit the OCT1- or OCT2-mediated drug transport, moreover, berberine, coptisine, jateorhizine, nitidine, epiberberine and berberrubine were the high affinity substrates of OCT1 and OCT2, moreover, the OCT1/2 inhibitors (quinidine and verapamil for OCT1, cimetidine and desipromine for OCT2) inhibit the OCT1-or OCT2-mediated the uptake of significantly berberine, coptisine, jateorhizine, nitidine, epiberberine and berberrubine in MDCK-hOCT1 or MDCK-hOCT2 cells, indicating the possibility of interaction between these alkaloids and other combination drugs.2. The contribution of human OCTs to the uptake and hepatocellular toxicity of nitidine chlorideNitidine chloride, a quaternary ammonium alkaloid, has many pharmacological effects, including anticancer effect, but its hepatocellular and nephrocellular toxicity also has been found. Since organic cation transporter 1 and 3 (OCT1 and OCT3) located in hepatocytes might mediate the uptake of nitidine, while human multidrug and toxin extrusion 1 (hMATEl) expressed in hepatocytes and nephrocytes might mediate the efflux of nitidine, the aim of the present study was to evaluate the role of human OCT1, OCT3 and hMATEl to the nitidine induced hepatocellular toxicity. The results revealed that the uptake of nitidine in MDCK-hOCT3/MDCK-hMATEl cells was much higher than that in mock cells, and the hOCT3-, hMATEl-mediated uptake followed Michaelis-Menten kinetics, the Km and Vmax values were 2.49±0.45 μmol/L and 25.4±1.1 pmol/mg protein/min for hOCT3, the Km and Vmax values were 0.897±0.085 μmol/L and 18.5±1.0 pmol/mg protein/min for hMATEl, respectively, indicating that nitidine was a substrate of hOCT3 and hMATE1, whereas the transport capacity of MATE1 (Clmt.20.6 mL/mg protein/min) might much lower than that of OCT1 (C/int: 311 mL/mg protein/min). The nitidine induced cytotoxicity on MDCK-hOCTl or MDCK-hOCT3 cells was obviously higher than that on mock cells or primary cultured rat hepatocytes. The IC50 values on MDCK-hOCTl and MDCK-hOCT3 cells were 0.163±0.027 μmol/L and 0.522±0.072 μmol/L, respectively, while the IC50 on mock cells and primary hepatocytes were 17.0±4.2 μmol/L and 2.83±0.53 μmol/L. The OCTl/3 inhibitors (quinidine and (+)/(-)-THP) significantly inhibited the uptake of nitidine in MDCK-hOCT1,MDCK-hOCT3 cells and rat primary hepatocytes, and subsequently OCT1/3 inhibitors, (+)/(-)-THP markedly attenuated the nitidine-induced cytotoxicity. Our results indicated that nitidine was a substrate of hOCT3 and hMATEl, both OCT1/OCT3 located in hepatocyte membrane mediate the uptake of nitidine, and subsequently cause hepatotoxicity, whereas OCTl/3 inhibitors could attenuate nitidine-induced toxicity.3. Role of CYP450 in the metabolism and attenuation hepatotoxicity of nitidine chlorideLiver possesses a wide range of xenobiotic metabolizing enzymes, which is a major contributor in the disposition of drugs. The above results revealed that the IC50 values of nitidine induced cytotoxicity on MDCK-hOCTl and MDCK-hOCT3 cells are much lower than that on primary cultured rat hepatocytes, we deduced hepatic CYPs might mediate the metabolism of NC and subsequently attenuate the cytotoxicity of NC. To confirm our deduction and which CYP isoforms were responsible for nitidine metabolism, we studied the metabolism of nitidine in human liver microsomes (HLM) and recombinant human CYP3A4, CYP2D6 and CYP2C8 (rhCYP3A4, rhCYP2D6 and rhCYP2C8). The results indicated that CYP3A4, CYP2C8, CYP2D6 and CYP2B6 might be involved in the metabolism of nitidine, and nitidine-induced toxicity on MDCK-hOCT1/hCYP3A4 cells was much lower than that on MDCK-hOCT1 cells. Additionally, CYP3A4 inhibitors, such as fluconazole and erythromycin, might inhibit the metabolism of NC and subsequently enhance the cytotoxicity of NC on MDCK-hOCT1/hCYP3A4. Our results indicated that CYPs, such as CYP3A4, in hepatocytes mediates the metabolism of nitidine and attenuates the nitidine-induced toxicity.4. The pharmacokinetics and tissue distribution study of nitidine chloride in ratsThe above results showed that the transportation and toxicity of nitidine mediated by OCTs in MDCK-hOCTs cells and rat primary hepatocytes in vitro, thereby, the aims of the present study were to investigate the pharmacokinetics of nitidine in rats after single oral administration or intravenous injection, and the tissue distribution of nitidine after single intravenous injection or 20-day repeated injection, and attempt to deduced preliminarily whether the toxicity induced by nitidine in vivo. A specific and accurate LC-MS/MS method has been developed and validated for determination of nitidine and successfully applied to evaluate the pharmacokinetics and tissue distribution of nitidine in rats. After single oral administration of 10 mg/kg, the plasma concentration of nitidine was low, whereas a much higher exposure was found when nitidine was intravenously injected to rats at 2 mg/kg. The absolute bioavailability (F) was estimated as 4.8%, indicating a very poor oral absorption of nitidine. The concentratin of nitidine in heart, liver, kidney, lung and small intestine was much higher than that in plasma at all detected time (0.25 h,0.5h and 2 h) after single injection of 5 mg/kg, moreover the concentration of nitidine in heart, liver, kidney of rats after successive injection of 5 mg/kg/day for 20 days was much higher than that of single dose. Based on the above results, it can be deduced that nitidine could induced toxicity in vivo when repected intravenous injection.5. A 20-day repeated-injection toxicity study of nitidine chloride in ratsThe studies have shown that OCT1/OCT3 located in hepatocyte membrane mediate the uptake of nitidine, and subsequently cause cytotoxicity, the absolute bioavailability of NC was very low after single oral administration of 10 mg/kg, and NC would accumulated in liver, kidney or heart after repeated injection to rats, indicating that the adverse effects of nitidine on rats, including some organ toxicity or animal overall toxicity, might be observed. The aim of this study was to evaluate the potential toxicity of nitidine in Sprague-Dawley rats after repeated intravenous injection at dose of 5 mg/kg/day for 20 days and attempt to illustrate the potential mechanism for NC induced-toxicity. Toxicity was assessed by mortality, general behavior, body weight, food consumption, serum chemistry, gross necropsy, organ weights and microscopic examination. Decreased food consumption and body weight was noted at 5 mg/kg/day in both genders compared to the vehicle control. The results revealed that urea nitrogen (BUN), lactate dehydrogenase (LDH) and kidney index were elevated, alkaline phosphatase (ALP) was decreased, furthermore, many rats in the treated group exhibited abnormal histopathological changes in kidney, including inflammation of the kidney, vacuolar degeneration of renal tubular cells. Whereas, there was no observed adverse effect in the control group. Furthermore, the nitidine induced cytotoxicity on MDCK-hOCT2 cells was obviously higher than that on mock cells. The OCT2 inhibitors (cimetidine and (+)/(-)-THP) significantly inhibited the uptake of nitidine in MDCK-hOCT2 cells, and subsequently markedly attenuated the nitidine-induced cytotoxicity, indicating that OCT2 located in nephrocyte membrane mediate the uptake of nitidine, and subsequently cause NC accumulation in kidney. Based on these results, we concluded that the accumulation of NC mediated by OCT2 may be responsible for cell damage observed in kidney at dose of 5 mg/kg/day administrated intravenously for 20 days. The data provide a foundation for the further research and evaluation of this promising anticancer agent.