Drug-induced liver injury associated with Complementary and Alternative Medicine: a review of adverse event reports in an Asian community from 2009 to 2014.

BACKGROUND: The use of Complementary and Alternative Medicine (CAM) has been increasing over the years. A recent review of adverse event reports (AERs) associated with CAM in Singapore found a notable number of AERs submitted. The objectives of this study are to analyse hepatotoxicity cases associated with CAM in Singapore based on spontaneous adverse event reporting to the Health Sciences Authority (HSA), and to highlight safety signals for specific herbal ingredients. METHODS: AERs associated with CAM and hepatotoxicity submitted to the Vigilance and Compliance Branch (VCB) of the HSA from 2009 to 2014 were compiled. The following information was extracted and analysed: Demographic information; time to onset; hospitalisation status; outcome; type of hepatotoxicity; ingredients of CAM, and the total daily doses (TDD); concurrent western medicines and health supplements; and reporter details. RESULTS: Fifty-seven reports were eligible for analysis. Thirty-five (61.4 %) cases involved Traditional Chinese Medicine (TCM). The Roussel Uclaf Causality Assessment Method was applied in 29 (82.9 %) of these cases, and the median score was 4 (range: 1-8). Chai Hu (Radix bupleuri) was suspected in 11 (31.4 %) cases. TDDs of most ingredients were within recommended doses of the Chinese Pharmacopoeia. CONCLUSIONS: Drug-induced liver injury is still poorly understood and more objective assessments are warranted. Reporting of adverse events should be strongly advocated to facilitate future analyses and the understanding of risk-benefit profiles of CAM.

Effect of mushroom diet on pharmacokinetics of gabapentin in healthy Chinese subjects.

AIMS: This study evaluated the pharmacokinetics of gabapentin in Chinese subjects who received a diet rich in shiitake mushrooms. Shiitake mushrooms have been shown to contain high amount of ergothioneine. In vitro studies have shown that OCTN1-mediated secretion of gabapentin is trans-stimulated by ergothioneine. This study also investigated the concentrations of ergothioneine in plasma at baseline and following mushroom consumption. METHODS: Ten healthy male subjects were recruited and received a diet containing no mushrooms (treatment A) or a high mushroom diet (treatment B; after at least a 7 day washout period) 1 day prior to administration of a single oral dose of gabapentin 600 mg. RESULTS: Ingestion of shiitake mushrooms produced significant increases in plasma ergothioneine concentrations that were sustained for more than 48 h. A statistically significant but modest increase in the renal clearance (CLR ) of gabapentin occurred after intake of the mushroom diet (91.1 ± 25.1 vs. 76.9 ± 20.6 ml min(-1) , P = 0.031). No significant changes in AUC(0,tlast ) of gabapentin were observed (P = 0.726). Creatinine clearance did not correlate with CLR of gabapentin at baseline (treatment A). After ingestion of the mushroom diet, creatinine clearance accounted for 65.3% of the variance in CLR of gabapentin. CONCLUSIONS: These data suggest that diet-drug pharmacokinetic interactions may occur during co-exposure to gabapentin and mushroom constituents. However, as it does not affect the AUC(0,tlast ) of gabapentin, it may not have clinically important consequences. Shiitake mushrooms can also be used as a source of ergothioneine for future clinical studies.

Functional analysis of novel variants in the organic cation/ergothioneine transporter 1 identified in Singapore populations.

The human organic cation/ergothioneine transporter 1 (hOCTN1, gene symbol SLC22A4) is responsible for the cellular uptake of substances, such as L-ergothioneine, which is an important antioxidant in mammalian cells. The common-function-altered variant L503F-hOCTN1 has been associated with susceptibility to Crohn's disease in certain populations. Previously, we identified eight novel nonsynonymous single-nucleotide polymorphisms (SNPs) in the SLC22A4 gene in the Chinese and Indian populations of Singapore. The present study evaluated the impact of these novel SNPs on hOCTN1 transport function in HEK-293 cells. Transport uptake assays with L-ergothioneine were used to assess the function of the variant transporters. Cell surface biotinylation and Western blot analysis were used to characterize cellular transporter expression. Comparative modeling was used to locate amino acid substitutions in the topology of hOCTN1 in order to account for altered transport function. Transporter activity was markedly impaired in four of the naturally occurring hOCTN1 variants (R63H, R83P, G482D, and I500N). Multiple glycosylated isoforms of hOCTN1 proteins were identified in the plasma membrane and in the whole cell. Either the total cellular or membrane expression of the functionally deficient transporter variants was lower than that of the wild-type hOCTN1. The underlying mechanism involves both impaired transporter-substrate binding affinity and turnover rate. Considered together, several naturally occurring SNPs in the SLC22A4 gene encode variant hOCTN1 transporters that may impact the cellular uptake of L-ergothioneine and other substrates, with the potential to influence the antioxidant capacity of human cells.

Quantification of L-ergothioneine in human plasma and erythrocytes by liquid chromatography-tandem mass spectrometry.

A sensitive analytical method has been developed and validated for the quantification of L-ergothioneine in human plasma and erythrocytes by liquid chromatography-tandem mass spectrometry. A commercially available isotope-labeled L-ergothioneine-d9 is used as the internal standard. A simple protein precipitation with acetonitrile is utilized for bio-sample preparation prior to analysis. Chromatographic separation of L-ergothioneine is conducted using gradient elution on Alltime C18 (150 mm × 2.1 mm, 5 µ). The run time is 6 min at a constant flow rate of 0.45 ml/min. The mass spectrometer is operated under a positive electrospray ionization condition with multiple reaction monitoring mode. The mass transitions of L-ergothioneine and L-ergothioneine-d9 are m/z 230 > 127 and m/z 239 > 127, respectively. Excellent linearity [coefficient of determination (r(2)) ≥ 0.9998] can be achieved for L-ergothioneine quantification at the ranges of 10 to 10,000 ng/ml, with the intra-day and inter-day precisions at 0.9-3.9% and 1.3-5.7%, respectively, and the accuracies for all quality control samples between 94.5 and 101.0%. This validated analytical method is suitable for pharmacokinetic monitoring of L-ergothioneine in human and erythrocytes. Based on the determination of bio-samples from five healthy subjects, the mean concentrations of L-ergothioneine in plasma and erythrocytes are 107.4 ± 20.5 ng/ml and 1285.0 ± 1363.0 ng/ml, respectively.

Functional analysis of pharmacogenetic variants of human organic cation/carnitine transporter 2 (hOCTN2) identified in Singaporean populations.

The human organic cation/carnitine transporter-2 (hOCTN2; SLC22A5) mediates the cellular influx of organic cations such as carnitine, which is essential for fatty acid oxidation. Primary carnitine deficiency has been associated with a wide range of hOCTN2 gene mutations. Six novel nonsynonymous single nucleotide polymorphisms in the hOCTN2 gene were identified recently in Chinese and Indian populations of Singapore. The present study evaluated the impact of these polymorphisms on hOCTN2 function and expression in HEK-293 cells. Transport function was markedly impaired in variants that encoded amino acid substitutions D122Y (<20% of wild-type control) and K302E (∼45% of wild-type) in the large extracellular loop and large intracellular loop of hOCTN2, respectively. The function of the other four variants was unimpaired (E109K, V175M, K191N and A214V). From biotinylation and immunofluorescence experiments, the expression of the D122Y and K302E-hOCTN2 variants at the plasma membrane of HEK-293 cells was decreased relative to the wild-type hOCTN2 but total cellular expression was unchanged. Transporter kinetic studies indicated a decrease in the V(max) for l-carnitine influx by K302E-hOCTN2 to 49% of wild-type control, while K(m) remained unchanged; kinetic evaluation of D122Y-hOCTN2 was not possible due to its low transport function. The K302E-hOCTN2 variant was also more susceptible than the wild-type transporter to inhibition by the drugs cimetidine, pyrilamine and verapamil. These findings indicate that impaired plasma membrane targeting of the D122Y and K302E-hOCTN2 variants that occur in Singaporean populations contributes to decreased carnitine influx.

Genetic variations of the SLC22A5 gene in the Chinese and Indian populations of Singapore.

Novel organic cation transporter 2 (OCTN2) is a multispecific, bidirectional, pH-dependent organic cation transporter. It can function as a carnitine co-transporter with higher affinity for carnitine than OCTN1 but also functions as a uniporter for other cations. Drugs such as verapamil, pyrilamine and beta-lactam antibiotics have been characterized as substrates of OCTN2 and/or inhibitors of carnitine transport. This study identified variants of the SLC22A5 gene in two distinct ethnic groups of the Singaporean population (n=192) by DNA sequencing. Twenty-eight genetic variants of SLC22A5, including 13 that were novel, were found: 14 were located in the coding exons, 10 in the introns, 1 in the promoter region, 2 in the 5'-untranslated region and 1 in the 3'-untranslated region. Among the novel nonsynonymous variants, Asp122Tyr was predicted to be functionally significant. Functional nonsynonymous variants detected include Ser467Cys and Arg254X; the latter resulted in a premature stop codon and is predicted to result in a truncated protein that is less than half the molecular mass of wild-type OCTN2. These data constitute fundamental information of value for future pharmacogenetic studies in Asian populations on drugs that are substrates of OCTN2.

Genetic variations of the SLC22A4 gene in Chinese and Indian populations of Singapore.

The novel organic cation transporter 1 (OCTN1) is a multispecific, bidirectional and pH-dependent organic cation transporter with low carnitine transport activity. It is a transporter of the physiological substance ergothioneine and mediates the transport of a variety of organic cations such as tetraethylammonium, pyrilamine and quinidine. This study identifies genetic variations of the SLC22A4 gene in two distinct ethnic groups of the Singaporean population (n=192) by DNA sequencing. Twenty four genetic variants of SLC22A4, including 14 found to be novel. 16 in the coding exons (10 nonsynonymous and 6 synonymous variations) and 8 in the introns. Among the novel nonsynonymous variations, Arg63His, Arg83Pro, Met344Lys and Ile500Asn were predicted to be functionally significant. These data should provide fundamental and useful information for pharmacogenetic studies on drugs that are substrates of OCTN1 in Asians.