Assessment of Statin Interactions With the Human NTCP Transporter Using a Novel Fluorescence Assay.

Sodium taurocholate cotransporting polypeptide (NTCP), which is highly expressed in the sinusoidal membrane of hepatocytes, maintains bile acid homeostasis and participates in the hepatic disposition of a variety of endogenous substances as well as xenobiotics. Manifested by the involvement of organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) in the hepatic uptake of statin drugs, sinusoidal membrane transporters play an important role in the pharmacokinetics and pharmacodynamics of these agents. It has been speculated that NTCP may function as an alternative pathway for statin hepatic uptake, complementary to OATP1B1 and OATP1B3. In the current study, we produced stable NTCP-expressing human embryonic kidney 293 (HEK293) cells and developed a fluorescence-based assay using flow cytometry for measuring NTCP transport with chenodeoxycholyl-(Nε-7-nitrobenz-2-oxa-1,3-diazole)-lysine (CDCA-NBD) as the substrate. NTCP-mediated CDCA-NBD transport was time-dependent and exhibited typical Michaelis-Menten kinetics, with a Km of 6.12 µM. Compounds known to interact with NTCP, including chenodeoxycholic acid and taurocholic acid, displayed concentration-dependent inhibition of NTCP-mediated CDCA-NBD transport. We report here a systematic evaluation of the interaction between statins and the NTCP transporter. Utilizing this system, several statins were either found to inhibit NTCP-dependent transport or act as substrates. We find a good correlation between the reported lipophilicity of statins and their ability to inhibit NTCP. The objective was to develop a higher-throughput system to evaluate potential inhibitors such as the statins. The in vitro assays using CDCA-NBD as fluorescent substrate are convenient, rapid, and have utility in screening drug candidates for potential drug-NTCP interactions.

Validation of interobserver agreement in lung cancer assessment: hematoxylin-eosin diagnostic reproducibility for non-small cell lung cancer: the 2004 World Health Organization classification and therapeutically relevant subsets.

CONTEXT: Precise subtype diagnosis of non-small cell lung carcinoma is increasingly relevant, based on the availability of subtype-specific therapies, such as bevacizumab and pemetrexed, and based on the subtype-specific prevalence of activating epidermal growth factor receptor mutations. OBJECTIVES: To establish a baseline measure of interobserver reproducibility for non-small cell lung carcinoma diagnoses with hematoxylin-eosin for the current 2004 World Health Organization classification, to estimate interobserver reproducibility for the therapeutically relevant squamous/nonsquamous subsets, and to examine characteristics that improve interobserver reproducibility. DESIGN: Primary, resected lung cancer specimens were converted to digital (virtual) slides. Based on a single hematoxylin-eosin virtual slide, pathologists were asked to assign a diagnosis using the 2004 World Health Organization classification. Kappa statistics were calculated for each pathologist-pair for each slide and were summarized by classification scheme, pulmonary pathology expertise, diagnostic confidence, and neoplastic grade. RESULTS: The 12 pulmonary pathology experts and the 12 community pathologists each independently diagnosed 48 to 96 single hematoxylin-eosin digital slides derived from 96 cases of non-small cell lung carcinoma resection. Overall agreement improved with simplification from the comprehensive 44 World Health Organization diagnoses (κ  =  0.25) to their 10 major header subtypes (κ  =  0.48) and improved again with simplification into the therapeutically relevant squamous/nonsquamous dichotomy (κ  =  0.55). Multivariate analysis showed that higher diagnostic agreement was associated with better differentiation, better slide quality, higher diagnostic confidence, similar years of pathology experience, and pulmonary pathology expertise. CONCLUSIONS: These data define the baseline diagnostic agreement for hematoxylin-eosin diagnosis of non-small cell lung carcinoma, allowing future studies to test for improved diagnostic agreement with reflex ancillary tests.