Organic Anion Transporting Polypeptide Gene Research Articles

Organic Anion-Transporting Polypeptide Genes Are Not Induced by the Pregnane X Receptor Activator Rifampin: Studies in Hepatocytes In Vitro and in Monkeys In Vivo.

Induction potentials of the pregnane X receptor (PXR) activator rifampin (RIF) on transporter genes [e.g., organic anion-transporting polypeptides (OATPs)] are still in its infancy or remain controversial in the field. The present investigations characterized changes in transporter gene expression by RIF in sandwich-cultured hepatocytes from multiple donors of human and cynomolgus monkey using real-time quantitative reverse transcription polymerase chain reaction method. Three-day treatment of RIF significantly induced CYP3A4 (∼60-fold induction), but not CYP1A2 and CYP2D6 genes. SLC51B was the most highly induced uptake transporter gene (>10-fold) in both human and monkey hepatocytes. A greater induction of CYP2C9 was observed in monkey hepatocytes than that in humans. ATP-binding cassette (ABC)B1 and ABCC2 were induced slightly above 2-fold in human and monkey hepatocytes and appeared to be dose-dependent. The induction of OATP and other transporter genes was generally less than 2-fold and considered not clinically relevant. SLCO2B1 was not detectable in monkey hepatocytes. To investigate in vivo OATP induction, RIF (18 mg/kg per day) was orally dosed to cynomolgus monkeys for 7 days. Pitavastatin and antipyrine were intravenously dosed before and after RIF treatment as exogenous probes of OATP and CYP activities, respectively. Plasma coproporphyrin-I (CP-I) and coproporphyrin-III (CP-III) were measured as OATP endogenous biomarkers. Although a significant increase of antipyrine clearance (CL) was observed after RIF treatment, the plasma exposures of pitavastatin, CP-I, and CP-III remained unchanged, suggesting that OATP function was not significantly altered. The results suggested that OATP transporters were not significantly induced by PXR ligand RIF. The data are consistent with current regulatory guidances that the in vitro characterization of transporter induction during drug development is not required. SIGNIFICANCE STATEMENT: Organic anion-transporting polypeptide (OATP) genes were not induced by rifampin in sandwich-cultured human and monkey hepatocytes OATP functions measured by OATP probe pitavastatin and endogenous marker coproporphyrins were not altered in monkeys in vivo by 7-day rifampin treatment. The data suggested that OATP transporters are unlikely induced by the pregnane X receptor ligand rifampin, which are consistent with current regulatory guidances that the in vitro characterization of OATP1B induction during drug development is not required.

Clinical Significance of Organic Anion Transporting Polypeptide Gene Expression in High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is considered the most deadly and frequently occurring type of ovarian cancer and is associated with various molecular compositions and growth patterns. Evaluating the mRNA expression pattern of the organic anion transporters (OATPs) encoded by SLCO genes may allow for improved stratification of HGSOC patients for targeted invention. The expression of SLCO mRNA and genes coding for putative functionally related ABC-efflux pumps, enzymes, pregnane-X-receptor, ESR1 and ESR2 (coding for estrogen receptors ERα and ERß) and HER-2 were assessed using RT-qPCR. The expression levels were assessed in a cohort of 135 HGSOC patients to elucidate the independent impact of the expression pattern on the overall survival (OS). For identification of putative regulatory networks, Graphical Gaussian Models were constructed from the expression data with a tuning parameter K varying between meaningful borders (Pils et al., 2012; Auer et al., 2015, 2017; Kurman and Shih Ie, 2016; Karam et al., 2017; Labidi-Galy et al., 2017; Salomon-Perzynski et al., 2017; Sukhbaatar et al., 2017). The final value used (K = 4) was determined by maximizing the proportion of explained variation of the corresponding LASSO Cox regression model for OS. The following two networks of directly correlated genes were identified: (i) SLCO2B1 with ABCC3 implicated in estrogen homeostasis; and (ii) two ABC-efflux pumps in the immune regulation (ABCB2/ABCB3) with ABCC3 and HER-2. Combining LASSO Cox regression and univariate Cox regression analyses, SLCO5A1 coding for OATP5A1, an estrogen metabolite transporter located in the cytoplasm and plasma membranes of ovarian cancer cells, was identified as significant and independent prognostic factor for OS (HR = 0.68, CI 0.49–0.93; p = 0.031). Furthermore, results indicated the benefits of patients with high expression by adding 5.1% to the 12.8% of the proportion of explained variation (PEV) for clinicopathological parameters known for prognostic significance (FIGO stage, age and residual tumor after debulking). Additionally, overlap with previously described signatures that indicated a more favorable prognosis for ovarian cancer patients was shown for SLCO5A1, the network ABCB2/ABCB3/ABCC4/HER2 as well as ESR1. Furthermore, expression of SLCO2A1 and PGDH, which are important for PGE2 degradation, was associated with the non-miliary peritoneal tumor spreading. In conclusion, the present findings suggested that SLCOs and the related molecules identified as potential biomarkers in HGSOC may be useful for the development of novel therapeutic strategies.

Heptamethine carbocyanine DZ-1 dye for near-infrared fluorescence imaging of hepatocellular carcinoma.

Near-infrared fluorescence (NIRF) dyes have recently emerged as promising tools for non-invasive imaging of different types of cancers. Here, we explored the potential utility of a NIRF DZ-1 dye, with dual imaging and tumour targeting functions, in hepatocellular carcinoma (HCC). We showed the preferential uptake of DZ-1 by HCC cells in vitro and in derived subcutaneous/orthotopic tumour xenografts, accompanied by a minimal effect on normal cells. DZ-1 simplified tumour growth profiling as well, since we were able to correlate NIRF signals with tumour volume and/or tumour-emitting luminescence in mice. Using both orthotopic tumour transplantation and cirrhosis models in parallel, we demonstrated the ability of DZ-1 to differentiate liver tumour from cirrhosis. DZ-1 showed superiority in HCC imaging over indocyanine green by demonstrating significantly enhanced tumour-targeting specificity. At the cellular level, DZ-1 was mainly retained in mitochondria and lysosomes. Additionally, DZ-1 fluorescence spectroscopy has been used for the intraoperative navigation of rabbit liver cancer, to determine surgical margins. We showed that tumor hypoxia and select organic anion-transporting polypeptide genes mediate NIRF dye uptake in HCC, which was supported by clinical evidence. All these findings represent the first evidence that DZ-1 is an effective molecular probe for tumour-specific imaging in HCC, and provide insights into the development of a new generation of imaging agents for intraoperative guidance of cancer surgery.

Near-infrared fluorescence heptamethine carbocyanine dyes mediate imaging and targeted drug delivery for human brain tumor

Brain tumors and brain metastases are among the deadliest malignancies of all human cancers, largely due to the cellular blood–brain and blood–tumor barriers that limit the delivery of imaging and therapeutic agents from the systemic circulation to tumors. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. Here we identified and synthesized a group of near-infrared fluorescence (NIRF) heptamethine carbocyanine dyes and derivative NIRF dye-drug conjugates for effective imaging and therapeutic targeting of brain tumors of either primary or metastatic origin in mice, which is mechanistically mediated by tumor hypoxia and organic anion-transporting polypeptide genes. We also demonstrate that these dyes, when conjugated to chemotherapeutic agents such as gemcitabine, significantly restricted the growth of both intracranial glioma xenografts and prostate tumor brain metastases and prolonged survival in mice. These results show promise in the application of NIRF dyes as novel theranostic agents for the detection and treatment of brain tumors.

Age- and sex-related differences of organic anion-transporting polypeptide gene expression in livers of rats

Organic anion-transporting polypeptides (Oatps) play important roles in transporting endogenous substances and xenobiotics into the liver and are implicated in drug-drug interactions. Many factors could influence their expression and result in alterations in drug disposition, efficacy and toxicity. This study was aimed to examine the development-, aging-, and sex-dependent Oatps expression in livers of rats. The livers from SD rats during development (−2, 1, 7, 14, 21, 28, 35, and 60 d) and aging (60, 180, 540 and/or 800 d) were collected and total RNAs were extracted, purified, and subjected to real-time PCR analysis. Total proteins were extracted for western-blot analysis. Results showed that Oatp1a1, Oatp1a4, Oatp1a5 and Oatp1b2 were all hardly detectable in fetal rat livers, low at birth, rapidly increased after weaning (21 d), and reached the peak at 60 d. The Oatps remained stable during the age between 60–180 d, and decreased at elderly (540 and/or 800 d). After birth, Oatp1a1, Oatp1a4, and Oatp1b2 were all highly expressed in liver, in contrast, Oatp1a5 expression was low. Oatp expressions are male-predominant in rat livers. In the livers of aged rats, the Oatp expression decreased and shared a consistent ontogeny pattern at the mRNA and protein level. In conclusion, this study showed that in rat liver, Oatp1a1, Oatp1a4, Oatp1a5 and Oatp1b2 gene expressions are influenced by age and gender, which could provide a basis of individual variation in drug transport, metabolism and toxicity in children, elderly and women.

Transport systems for opioid peptides in mammalian tissues

Transmembrane transport of endogenous as well as synthetic opioid peptides is a critical determinant of pharmacokinetics and biologic efficacy of these peptides. This transport process influences the distribution of opioid peptides across the blood-brain barrier and their elimination from the body. A multitude of transport systems that recognize opioid peptides as substrates have been characterized at the functional level, and these transport systems are expressed differentially at different sites in the body. Many of these transport systems have been identified at the molecular level. These include the H(+)-coupled peptide transporters PEPT1 and PEPT2, the adenosine triphosphate-dependent efflux transporters P-glycoprotein and multidrug resistance-related protein 2, and several members of the organic anion-transporting polypeptide gene family. There are however many additional transport systems that are known to transport opioid peptides but their molecular identities still remain unknown.

Identification and functional characterization of the promoter region of the human organic anion transporting polypeptide gene

The organic anion transporting polypeptide (OATP) of the basolateral hepatocyte membrane mediates multispecific uptake of anionic and other amphipathic substrates from sinusoidal blood plasma. To investigate the mechanisms controlling OATP expression, the 5'-flanking region of the human OATP gene was isolated from a P1-derived artificial chromosome genomic clone. Sequence analysis of the OATP promoter showed a number of consensus binding sites for both ubiquitous and liver-enriched transcription factors. Transfection of HepG2 cells with a series of 5'-deleted promoter-luciferase constructs identified the minimal promoter region within 91 base pairs relative to the transcription initiation site. A putative silencer element was localized in the -662/-440 region. The minimal promoter was also active in Chang liver, Madin-Darby canine kidney, and Chinese hamster ovary cells, indicating that basal promoter function is independent of liver-specific regulatory mechanisms. In transfected HepG2 cells, taurocholate (100 micromol/L) stimulated and triiodothyronine (1 micromol/L) inhibited OATP promoter activity, whereas hydrocortisone, dexamethasone, beta-estradiol, estrone-3-sulfate, and testosterone had no significant effect. Reverse-transcription polymerase chain reaction analysis showed an increase in OATP messenger RNA in the livers of four patients with chronic cholestatic liver disease compared with three noncholestatic controls. The up-regulation of OATP expression by taurocholate could serve to enhance the sinusoidal efflux of toxic intracellular compounds during chronic cholestasis. (Hepatology 1997 Oct;26(4):991-7)

Identification and functional characterization of the promoter region of the human organic anion transporting polypeptide gene.

The organic anion transporting polypeptide (OATP) of the basolateral hepatocyte membrane mediates multispecific uptake of anionic and other amphipathic substrates from sinusoidal blood plasma. To investigate the mechanisms controlling OATP expression, the 5'-flanking region of the human OATP gene was isolated from a P1-derived artificial chromosome genomic clone. Sequence analysis of the OATP promoter showed a number of consensus binding sites for both ubiquitous and liver-enriched transcription factors. Transfection of HepG2 cells with a series of 5'-deleted promoter-luciferase constructs identified the minimal promoter region within 91 base pairs relative to the transcription initiation site. A putative silencer element was localized in the -662/-440 region. The minimal promoter was also active in Chang liver, Madin-Darby canine kidney, and Chinese hamster ovary cells, indicating that basal promoter function is independent of liver-specific regulatory mechanisms. In transfected HepG2 cells, taurocholate (100 micromol/L) stimulated and triiodothyronine (1 micromol/L) inhibited OATP promoter activity, whereas hydrocortisone, dexamethasone, beta-estradiol, estrone-3-sulfate, and testosterone had no significant effect. Reverse-transcription polymerase chain reaction analysis showed an increase in OATP messenger RNA in the livers of four patients with chronic cholestatic liver disease compared with three noncholestatic controls. The up-regulation of OATP expression by taurocholate could serve to enhance the sinusoidal efflux of toxic intracellular compounds during chronic cholestasis.

Assignment of the human organic anion transporting polypeptide (OATP) gene to chromosome 12p12 by fluorescence in situ hybridization

Background/Aims: The organic anion transporting polypeptide (OATP) of human liver mediates the basolateral hepatocellular uptake of numerous cholephilic anions and steroidal compounds. The aim of this study was to clone the human OATP gene and to map its chromosomal localization by fluorescence in situ hybridization. Methods: A polymerase chain reaction-amplified fragment of the human OATP gene was used to isolate a genomic OATP clone from a P1-derived artificial chromosome human genomic library. Human metaphase chromosomes were hybridized with digoxigenin-labeled DNA from the genomic OATP clone and incubated in fluoresceinated antidigoxigenin antibodies for in situ detection of specific hybridization signals. Results: Sequence analysis revealed that the isolated P1-derived artificial chromosome clone contained a large portion of the human OATP gene. Fluorescence in situ hybridization of human chromosomes with the genomic OATP clone resulted in the specific labeling of the OATP gene on the short arm of chromosome 12 at band 12p12. Conclusions: Mapping of a genomic OATP clone to chromosome 12p12 represents a first step towards the molecular characterization of the human OATP gene. While no liver disease has so far been associated with cytogenetic abnormalities of the short arm of chromosome 12, the genomic OATP sequence provides the basis for studies on gene structure and on the tissue-specific regulation of OATP gene expression.