Accumulation of nitidine chloride in rat heart and the underlying mechanism / 药学学报

Nitidine chloride (NC) is a compound with prominent anti-tumor activity. To determine potential cardiotoxicity of NC, this study was designed to investigate the distribution of NC in rat heart and the underlying mechanism. The animal studies were approved by Institutional Animal Care and Use Committee of Zhejiang University Medical Center (2015-380-01) and complied with the standards of animal welfare in China. At 0.25, 0.5 and 2 h after a single intravenous injection (iv) of 5 mg·kg-1 NC, the concentrations of NC in rat heart were 47.7, 71.1 and 63.2 μg·g-1 respectively, which were 576, 1 352 and 1 212 folds of that in plasma. This study also revealed that the NC concentration in heart was 458.5 μg·g-1 (7 336 folds of that in plasma) at 2 h after the last dose in rats, after daily iv administration of NC at 5 mg·kg-1·day-1 for successive 20 days. Further studies showed that the accumulations of NC in MDCK-hOCT1 and MDCK-hOCT3 cells were 16.1 and 4.99 folds higher than that of the mock cells, respectively. There is no significant difference between the accumulations of NC in MDCK cells transfected with hOCTN1, hOCTN2 or hPMAT and the mock cells. Additionally, quinidine, L-tetrahydropalmatine and Decynium 22, the inhibitors of OCTs, clearly reduced the accumulations of NC in primary cardiomyocytes and cardiac fibroblasts from neonatal rats. MTT assay showed that the LC50 of NC on cardiomyocytes and cardiac fibroblasts were 10.9 and 10.4 μmol·L-1, respectively. Moreover, treatment of the primary cardiomyocytes and cardiac fibroblasts with NC (1～15 μmol·L-1) for 48 h resulted in significantly increased LDH enzyme leakage. These results indicated that NC can be highly accumulated in the heart, and accumulation is mediated by OCT1 and OCT3, but not by OCTN1, OCTN2 and PMAT. The accumulated NC has potential cytotoxicity as shown in the results from primary cardiomyocytes and cardiac fibroblasts.

Polimorfismo Val/Met en gen OCT1 de respuesta a metformina en muestras de Lima y Puno. Enfoque farmacogenético de la diabetes / Val/Met polymorphism in metformin response gene OCT1 in Lima and Puno samples. Pharmacogenetic approach of diabetes

Introducción: La farmacogenética tiene utilidad clínica para evaluar los efectos de los fármacos según perfil genético y aporta a la medicina poblacional y personalizada. La diabetes mellitus tipo 2 (DMT2) es una enfermedad prevalente en el Perú y el mundo. Para su tratamiento existen varios fármacos, entre ellos la metformina. La respuesta individual puede estar influenciada por el polimorfismo Val/Met en el gen octT1 y las frecuencias varían según grupo étnico. Se ha relacionado al alelo Met con una menor respuesta a la metformina. Objetivo: Evaluar la distribución del polimorfismo Val/Met en el gen OCT1 en muestras de sangre de sujetos de Lima y Puno, e inferir su impacto en la farmacogenética de la DMT2. Diseño: Estudio descriptivo, transversal. Lugar: Facultades de Farmacia y Bioquímica, Medicina, Universidad Nacional Mayor Lima, Perú; Centro de Genética y Biología Molecular, Facultad de Medicina, Universidad de San Martín de Porres, Lima, Perú. Participantes: 56 individuos de Puno y 57 de Lima-ciudad. Intervenciones: Análisis del polimorfismo Val/Met en el gen OCT1 con la técnica PCR-RFLP. Principales medidas de resultados: Frecuencias genotípicas y alélicas. Resultados: Las frecuencias de los genotipos, en general,fueron: Val/Val=85,0% y Val/Met=15,0%. La frecuencia del alelo Val, en general, fue mayor al 93%; el alelo Met, asociado con una menor respuesta a metformina, se encontró presente en Amantaní (8,3%) y Lima (9,6%), y ausente en Taquile. Conclusiones: Para el alelo Val del gen OCT1, se ha encontrado la más alta frecuencia registrada en el mundo. Respecto al alelo Met, aunque es menos frecuente, existen diferencias entre las subpoblaciones peruanas evaluadas, y ese conocimiento puede ayudar en la farmacogenética y la toma de decisiones en el tratamiento con los antidiabéticos orales como la metformina.

Introduction: Pharmacogenetics can be used in clinical analysis to assess the efficiency of drugs according to the patient’s genetic profile, and it is becoming important for population genetics and precision medicine. The type 2 diabetes mellitus (T2DM) is highly prevalent all over the world, including Peru. Among the different drugs for T2DM, metformin is used the most and patient’s response to it can be influenced by the Val/Met polymorphism of the OCT1 (SLC22) gene, where Met is associated with a lower response. The frequencies of these polymorphisms vary according to ethnic origin. Objective: To evaluate the distribution of the Val/Met polymorphism in the OCT1 gene in samples of Lima and Puno, and to assess their impact on pharmacogenetics of T2DM. Design: Descriptive, cross-sectional study. Settings: Faculties of Pharmacy and Biochemistry, and Medicine, Universidad Nacional Mayor de San Marcos, Lima, Peru; Centro de Genética y Biología Molecular, Facultad de Medicina, Universidad de San Martín de Porres, Lima, Peru. Participants: DNA samples of 56 non-selected subjects from Puno and 57 from Lima regions. Interventions: Analysis of the Val/Met polymorphism in OCT1 gene using the PCR-RFLP technique. Main outcome measures: Phenotypic and allelic frequencies. Results: Genotype frequencies were Val/Val=85,0% and Val/Met=15,0%. The Val allele frequency was higher than 93%, the Met allele was associated with a lower response to metformin and was present in Amantaní (8.3%) and in Lima (9.6%), and absent in Taquile. Conclusions: We found the highest Val allele frequency in the world. Regarding the Met allele, less frequent, we found differences among the Peruvian subpopulations tested, and this knowledge can help in the pharmacogenetics and decision making about oral treatment of metformin against diabetes.

Molecular response to imatinib & its correlation with mRNA expression levels of imatinib influx & efflux transporters in patients with chronic myeloid leukaemia in chronic phase.

Background & objectives: Imatinib is the standard first-line treatment for chronic myeloid leukaemia (CML) patients. About 20 to 30 per cent patients develop resistance to imatinib and fail imatinib treatment. One of the mechanisms proposed is varying expression levels of the drug transporters. This study was aimed to determine the expression levels of imatinib transporter genes (OCT1, ABCB1, ABCG2) in CML patients and to correlate these levels with molecular response. Methods: Sixty three CML chronic phase patients who were on 400 mg/day imatinib for more than two years were considered for gene expression analysis study for OCT1, ABCB1 and ABCG2 genes. These were divided into responders and non-responders. The relative transcript expression levels of the three genes were compared between these two categories. The association between the expression values of these three genes was also determined. Results: No significant difference in the expression levels of OCT1, ABCB1 and ABCG2 was found between the two categories. The median transcript expression levels of OCT1, ABCB1 and ABCG2 genes in responders were 26.54, 10.78 and 0.64 versus 33.48, 7.09 and 0.53 in non-responders, respectively. A positive association was observed between the expression of the ABCB1 and ABCG2 transporter genes (r=0.407, P<0.05) while no association was observed between the expression of either of the ABC transporter genes with the OCT1 gene. Interpretation & conclusions: Our findings demonstrated that the mRNA expression levels of imatinib transporter genes were not correlated with molecular response in CML patients. further studies need to be done on a large sample of CML patients to confirm these findings.

Subtherapeutic Dose of Prazosincan Ameliorate Metformin Induced Lactic Acidosis in Renal Compromised Rats.

Incidence of lactic acidosis caused by metformin is rare but this can occur in renal compromised individuals because of metformin accumulation. The drug enters the liver through organic cationic transporter 1(OCT1) and reduces oxygen consumption in mitochondria resulting in reduced lactate clearance and lactic acidosis. In the present study, we investigated that inhibition of the transport of metformin in the liver could reduce the blood lactate levels. Eighteen healthy male albino rats were selected for the study. Group 1- control group included 6 rats, they were given normal saline for 10 days by i.p injection. Group 2- Twelve rats were induced nephrotoxicity by gentamicin at the doses of 40mg/kg given by i.p. route . Group 3- six rats from group 2 were given metformin according to human doses of 1000mg/day and group 4- included six rats from group 2 received metformin and prazosin at subtherapeutic dose i.e. according to 1mg/day human dose. Blood urea, serum creatinine and total urinary albumin were found to be significantly.

Análise da expressão e atividade das moléculas de influxo e de efluxo de drogas em células da leucemia mielóide crônica / Analysis of the expression and activity of drug influx and efflux molecules in chronic myeloid leukemia cells

"A leucemia mielóide crônica (LMC) é caracterizada pela presença da translocação t(9:22) que codifica a proteína quimérica oncogênica BCR-ABL. Imatinibe é uma droga alvo-específica que inibe a atividade tirosina quinase (TK) da proteína BCR-ABL. Entretanto, os níveis intracelulares do imatinibe podem ser alterados pelas proteínas transportadoras de efluxo de drogas: glicoproteína P (Pgp), proteína da resistência em câncer de mama (BCRP) e proteína relacionada à resistência à múltiplas drogas (MRP1), assim como a proteína transportadora de influxo de drogas (OCT1). O objetivo do presente estudo foi analisar a participação dessas proteínas, isoladamente ou em associação, na resistência ao imatinibe em linhagens celulares e células de pacientes com LMC. Para análise da atividade das proteínas transportadoras de efluxo, foi utilizado o fluorocromo rodamina-123 associado ao modulador ciclosporina-A (Rho+CSA) e o pheophorbide A associado ao fumitremorgin C (PhA+FTC), ambos através de citometria de fluxo. A análise dos RNAm dos genes ABCB1, ABCG2 e SLC22A1 que codificam as proteínas Pgp, BCRP e OCT1, respectivamente, foi realizada por PCR em tempo real. A inibição da TK BCR-ABL foi mensurada através dos níveis de fosforilação de CrkL (pCrkL), seu principal alvo de ativação. Observamos uma maior positividade para o ensaio Rho+CSA nas amostras que expressavam Pgp comparada com as que expressavam MRP1, sugerindo menor atividade dessa proteína em pacientes com LMC, ou ainda que tal ensaio possa ser menos específico para a atividade da MRP1. O ensaio PhA+FTC foi capaz de identificar a atividade da proteína BCRP em linhagens celulares e células de pacientes. Níveis reduzidos dos RNAm ABCB1 e SLC22A1, mas não do RNAm ABCG2, foram observados quando comparados com as amostras de indivíduos saudáveis. Não houve correlação entre os níveis da proteína Pgp e do RNAm ABCB1. A expressão de Pgp foi detectada na maioria das amostras de LMC, independente da fase da doença, e não foi associada com o prognóstico desfavorável. Variações nos níveis de expressão da Pgp foram observadas durante a evolução da LMC e relacionadas com o tratamento prévio. O imatinibe foi capaz de aumentar a expressão da Pgp, assim como os níveis do RNAm ABCB1 na linhagem K562-Lucena 1, Pgp+. Além disso, observamos uma maior redução de pCrkL e um maior percentual de morte celular nas células K562, Pgp-, quando comparadas à K562-Lucena 1, evidenciando um possível papel do imatinibe como substrato para a Pgp. Este fármaco também demonstrou ter potencial para funcionar como agente modulador da bomba de efluxo, uma vez que impediu o exporte de Rho das células K562-Lucena 1. Amostras de pacientes resistentes ao imatinibe exibiram altos níveis de atividade das proteínas transportadoras de efluxo de drogas (ensaio Rho+CSA). Nossos dados mostram que a atividade e/ou expressão dos transportadores de efluxo e influxo de drogas encontram-se alterados na maioria dos pacientes com LMC, porém não há correlação com a resposta ao imatinibe e o prognóstico na LMC. Entretanto, o conjunto dos resultados sugere um papel para a Pgp na resistência in vitro ao imatinibe"(AU)

"Chronic myeloid leukemia (CML) is characterized by the presence of the t(9:22) encoding the BCR-ABL chimeric oncogenic protein. Imatinib is a target specific drug that inhibits the activity of the tyrosine kinase (TK) protein BCR-ABL. However, imatinib intracellular concentration may be altered by transporter proteins. It was described that efflux proteins, P-glycoprotein (Pgp), breast cancer resistance protein (BCRP) and multidrug resistance related protein (MRP1), and the influx protein, the organic cation transporter protein (OCT1) may contribute for imatinib clinical resistance. The aim of this study was to evaluate the role of these proteins in imatinib resistance in cell lines and leukemic cells from CML patients. To analyze the activity of the efflux transporter proteins, fluorochrome rhodamine-123 associated with the modulator cyclosporin A (Rho+CSA) and pheophorbide A associated with fumitremorgin C (PhA+FTC) were used by flow cytometry. Analysis of ABCG1, ABCG2 and SLC22A1 genes, that encode the Pgp, BCRP and OCT1 proteins, respectively, was performed by real time PCR. The inhibition of BCR-ABL TK was measured by the levels of CrkL phosphorylation (pCrkL), its main target of activation. We observed a higher positivity for Rho+CSA assay in samples expressing Pgp, when compared with the ones expressing MRP1. These results suggest that patients with CML have lower activity of this protein, or this assay might be less specific to indicate the activity of MRP1. The PhA+FTC assay was able to identify BCRP activity in cell lines and cells from patients. Reduced levels of ABCB1 and SLC22A1, but not ABCG2 mRNA were observed when compared with samples from healthy individuals. There was no correlation between the levels of Pgp protein and ABCB1 mRNA. Pgp expression was detected in most samples of CML, regardless of disease stage and was not associated with poor prognosis. Changes in Pgp expression levels have been observed during the development of CML and were related to pretreatment. Imatinib was able to increase Pgp expression as well as ABCB1 mRNA levels in Pgp+ K562Lucena 1 cells. Moreover, we observed a greater pCrkL reduction and a higher percentage of cell death in Pgp- K562 cells compared to K562-Lucena 1, indicating a possible role of imatinib as a Pgp substrate. This drug has also been shown to have potential as an efflux pump modulating agent, once efflux of Rho was prevented in K562-Lucena 1. Imatinib resistant patient samples exhibited high levels of efflux transporter proteins activity (Rho+CSA assay). Our data show that the activity and / or expression of influx and efflux transporters of drugs are altered in most patients with CML, but no correlation with prognosis and response to imatinib in CML was observed. However, our results suggest a role for Pgp in imatinib resistance"(AU)

Genetic analysis of OCT1 gene polymorphisms in an Indian population.

BACKGROUND: Genetic variants of the organic cation transporter (OCT1) gene could influence interindividual variation in clinical response to metformin therapy. The genetic basis for the single-nucleotide polymorphism (SNP) of OCT1 gene has been established in other populations, but it remains to be elucidated in the Indian population. This study is focused on OCT1 gene variants rs2282143 (P341L, 1022C>T), rs628031 (M408V, 1222A>G) and rs622342 (1386C>A) frequency distributions in the South Indian Tamilian population. MATERIALS AND METHODS: A total of 112 unrelated healthy subjects of South Indian Tamilian origin, aged 18–60 years, of either sex were recruited for the study. Genotyping was determined using the quantitative real time-polymerase chain reaction and polymerase chain reaction followed by restriction fragment length polymorphism methods. RESULTS: Allele frequencies of rs2282143, rs628031and rs622342 polymorphisms were 8.9%, 80.3% and 24.5%, respectively. Interethnic differences in the genotype and allele frequencies of OCT1 gene polymorphism were observed when compared with other major populations. The SNPs rs2282143, T allele and rs628031, G allele were more common in Asians (5.5–16.8% and 76.2–81%) and African Americans (8.2% and 73.5%) than in Caucasians (0–2% and 57.4–60%). CONCLUSION: This is the first time the frequency of OCT1 gene polymorphism was determined in the Indian population, and is similar to the frequencies observed in African-Americans and other Asian populations but different from those in Caucasians. The data observed in this study would justify further pharmacogenetic studies to potentially evaluate the role of OCT1 gene polymorphism in the therapeutic efficacy of metformin.