Prediction of Transporter-Mediated Rosuvastatin Hepatic Uptake Clearance and Drug Interaction in Humans Using Proteomics-Informed REF Approach.

Suspended, plated, or sandwich-cultured human hepatocytes are routinely used for in vitro to in vivo extrapolation (IVIVE) of transporter-mediated hepatic clearance (CL) of drugs. However, these hepatocyte models have been reported to underpredict transporter-mediated in vivo hepatic uptake CL (CL uptake,in vivo ) of some drugs. Therefore, we determined whether transporter-expressing cells (TECs) can accurately predict the CL uptake,in vivo of drugs. To do so, we determined the uptake CL (CL int,uptake,cells ) of rosuvastatin (RSV) by TECs (organic anion transporting polypeptides/Na+-taurocholate cotransporting polypeptide) and then scaled it to that in vivo by relative expression factor (REF) (the ratio of transporter abundance in human livers and TEC) determined by liquid chromatography tandem mass spectrometry-based quantitative proteomics. Both the TEC and hepatocyte models did not meet our predefined success criteria of predicting within 2-fold the RSV CL uptake,in vivo value obtained from our positron emission tomography (PET) imaging. However, the TEC performed better than the hepatocyte models. Interestingly, using REF, TECs successfully predicted RSV CL int,uptake,hep obtained by the hepatocyte models, suggesting that the underprediction of RSV CL uptake,in vivo by TECs and hepatocytes is due to endogenous factor(s) not present in these in vitro models. Therefore, we determined whether inclusion of plasma (or albumin) in TEC uptake studies improved IVIVE of RSV CL uptake,in vivo It did, and our predictions were close to or just fell above our lower 2-fold acceptance boundary. Despite this success, additional studies are needed to improve transporter-mediated IVIVE of hepatic uptake CL of drugs. However, using REF and TEC, we successfully predicted the magnitude of PET-imaged inhibition of RSV CL uptake,in vivo by cyclosporine A. SIGNIFICANCE STATEMENT: We showed that the in vivo transporter-mediated hepatic uptake CL of rosuvastatin, determined by PET imaging, can be predicted (within 2-fold) from in vitro studies in transporter-expressing cells (TECs) (scaled using REF), but only when plasma proteins were included in the in vitro studies. This conclusion did not hold when plasma proteins were absent in the TEC or human hepatocyte studies. Thus, additional studies are needed to improve in vitro to in vivo extrapolation of transporter-mediated drug CL.

Transporter Expression in Noncancerous and Cancerous Liver Tissue from Donors with Hepatocellular Carcinoma and Chronic Hepatitis C Infection Quantified by LC-MS/MS Proteomics.

Protein expression of major hepatobiliary drug transporters (NTCP, OATPs, OCT1, BSEP, BCRP, MATE1, MRPs, and P-gp) in cancerous (C, n = 8) and adjacent noncancerous (NC, n = 33) liver tissues obtained from patients with chronic hepatitis C with hepatocellular carcinoma (HCV-HCC) were quantified by LC-MS/MS proteomics. Herein, we compare our results with our previous data from noninfected, noncirrhotic (control, n = 36) and HCV-cirrhotic (n = 30) livers. The amount of membrane protein yielded from NC and C HCV-HCC tissues decreased (31%, 67%) relative to control livers. In comparison with control livers, with the exception of NTCP, MRP2, and MATE1, transporter expression decreased in NC (38%-76%) and C (56%-96%) HCV-HCC tissues. In NC HCV-HCC tissues, NTCP expression increased (113%), MATE1 expression decreased (58%), and MRP2 expression was unchanged relative to control livers. In C HCV-HCC tissues, NTCP and MRP2 expression decreased (63%, 56%) and MATE1 expression was unchanged relative to control livers. Compared with HCV-cirrhotic livers, aside from NTCP, OCT1, BSEP, and MRP2, transporter expression decreased in NC (41%-71%) and C (54%-89%) HCV-HCC tissues. In NC HCV-HCC tissues, NTCP and MRP2 expression increased (362%, 142%), whereas OCT1 and BSEP expression was unchanged. In C HCV-HCC tissues, OCT1 and BSEP expression decreased (90%, 80%) relative to HCV-cirrhotic livers, whereas NTCP and MRP2 expression was unchanged. Expression of OATP2B1, BSEP, MRP2, and MRP3 decreased (56%-72%) in C HCV-HCC tissues in comparison with matched NC tissues (n = 8), but the expression of other transporters was unchanged. These data will be helpful in the future to predict transporter-mediated hepatocellular drug concentrations in patients with HCV-HCC.

Fampridine is a Substrate and Inhibitor of Human OCT2, but not of Human MATE1, or MATE2K.

PURPOSE: The renal clearance of fampridine (Fampyra®, or Ampyra®) significantly exceeds the glomerular filtration rate, suggesting active renal secretion is likely the major elimination pathway. The goal of this study was to identify the renal transporters that are involved in the renal active secretion, and elucidate the active renal secretion mechanism of fampridine. METHODS: The uptake of fampridine to HEK-293 cells overexpressing human OCT2, MATE1 or MATE2K was determined in the absence and presence of Cimetidine, the prototypical inhibitor of the transporters. The inhibition potential of fampridine on the renal transporters was evaluated by determining the uptake of TEA and Metformin, the probe substrates of the transporters of OCT2 and MATEs, respectively, in the absence or presence of fampridine. RESULTS: Significant time- and concentration-dependent uptake of fampridine by human OCT2 was observed. The Km and Vmax were determined as 51.0 ± 17.1 µM and 1107 ± 136 pmole/min/106 cells, respectively. Fampridine also inhibited OCT2 mediated uptake of Metformin with estimated IC50 of 66.8 µM. In contrast, there was not significant uptake of fampridine by human MATE1 or MATE2K, and fampridine did not inhibit MATE1 or MATE2K mediated uptake of TEA. CONCLUSION: The studies indicated fampridine is a substrate and inhibitor of OCT2, but not MATE1 or MATE2K. Results from the study suggested the active renal secretion of fampridine is mediated by human OCT2 but not MATE1 or MATE2K. To our knowledge, fampridine is the first reported substrate specific to OCT2 but not to MATE1 or MATE2K.

Novel small molecule inhibitors of TLR7 and TLR9: mechanism of action and efficacy in vivo.

The discovery that circulating nucleic acid-containing complexes in the serum of autoimmune lupus patients can stimulate B cells and plasmacytoid dendritic cells via Toll-like receptors 7 and 9 suggested that agents that block these receptors might be useful therapeutics. We identified two compounds, AT791 {3-[4-(6-(3-(dimethylamino)propoxy)benzo[d]oxazol-2-yl)phenoxy]-N,N-dimethylpropan-1-amine} and E6446 {6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole}, that inhibit Toll-like receptor (TLR)7 and 9 signaling in a variety of human and mouse cell types and inhibit DNA-TLR9 interaction in vitro. When administered to mice, these compounds suppress responses to challenge doses of cytidine-phosphate-guanidine (CpG)-containing DNA, which stimulates TLR9. When given chronically in spontaneous mouse lupus models, E6446 slowed development of circulating antinuclear antibodies and had a modest effect on anti-double-stranded DNA titers but showed no observable impact on proteinuria or mortality. We discovered that the ability of AT791 and E6446 to inhibit TLR7 and 9 signaling depends on two properties: weak interaction with nucleic acids and high accumulation in the intracellular acidic compartments where TLR7 and 9 reside. Binding of the compounds to DNA prevents DNA-TLR9 interaction in vitro and modulates signaling in vivo. Our data also confirm an earlier report that this same mechanism may explain inhibition of TLR7 and 9 signaling by hydroxychloroquine (Plaquenil; Sanofi-Aventis, Bridgewater, NJ), a drug commonly prescribed to treat lupus. Thus, very different structural classes of molecules can inhibit endosomal TLRs by essentially identical mechanisms of action, suggesting a general mechanism for targeting this group of TLRs.

Optimization of dose and route of administration of the P-glycoprotein inhibitor, valspodar (PSC-833) and the P-glycoprotein and breast cancer resistance protein dual-inhibitor, elacridar (GF120918) as dual infusion in rats.

Transporters can play a key role in the absorption, distribution, metabolism, and excretion of drugs. Understanding these contributions early in drug discovery allows for more accurate projection of the clinical pharmacokinetics. One method to assess the impact of transporters in vivo involves co-dosing specific inhibitors. The objective of the present study was to optimize the dose and route of administration of a P-glycoprotein (P-gp) inhibitor, valspodar (PSC833), and a dual P-gp/breast cancer resistance protein (BCRP) inhibitor, elacridar (GF120918), by assessing the transporters' impact on brain penetration and absorption. A dual-infusion strategy was implemented to allow for flexibility with dose formulation. The chemical inhibitor was dosed intravenously via the femoral artery, and a cassette of known substrates was infused via the jugular vein. Valspodar or elacridar was administered as 4.5-hour constant infusions over a range of doses. To assess the degree of inhibition, the resulting ratios of brain and plasma concentrations, Kp's, of the known substrates were compared to the vehicle control. These data demonstrated that doses greater than 0.9 mg/hr/kg valspodar and 8.9 mg/hr/kg elacridar were sufficient to inhibit P-gp- and BCRP-mediated efflux at the blood-brain barrier in rats without any tolerability issues. Confirmation of BBB restriction by efflux transporters in preclinical species allows for subsequent prediction in humans based upon the proteomic expression at rodent and human BBB. Overall, the approach can also be applied to inhibition of efflux at other tissues (gut absorption, liver clearance) or can be extended to other transporters of interest using alternate inhibitors.

Interindividual and Regional Variability in Drug Transporter Abundance at the Human Blood-Brain Barrier Measured by Quantitative Targeted Proteomics.

For in vitro to in vivo extrapolation (IVIVE) of brain distribution of drugs that are transported at the human blood-brain barrier (BBB), it is important to quantify the interindividual and regional variability of drug transporter abundance at this barrier. Therefore, using quantitative targeted proteomics, we compared the abundance of adenosine triphosphate-binding cassette and solute carrier transporters in brain microvascular endothelial cells (BMECs) isolated from postmortem specimens of two matched brain regions, the occipital (Brodmann Area (BA)17) and parietal (BA39) lobe, from 30 adults. Of the quantifiable transporters, the abundance ranked: glucose transporter (GLUT)1 > breast cancer resistance protein > P-glycoprotein (P-gp) > equilibrative nucleoside transporter (ENT)1 > organic anion-transporting polypeptide (OATP)2B1. The abundance of multidrug resistance protein 1/2/3/4, OATP1A2, organic anion transporter (OAT)3, organic cation transporter (OCT)1/2, OCTN1/2, or ENT2 was below the limit of quantification. Transporter abundance per gram of tissue (scaled using GLUT1 abundance in BMEC vs. brain homogenate) in BA17 was 30-42% higher than BA39. The interindividual variability in transporter abundance (percentage of coefficient of variation (%CV)) was 35-57% (BA17) and 27-46% (BA39). These data can be used in proteomics-informed bottom-up IVIVE to predict human brain drug distribution.

A high-performance liquid chromatography-tandem mass spectrometry method for the clinical combination study of carboplatin and anti-tumor agent eribulin mesylate (E7389) in human plasma.

An LC/MS/MS method was developed to quantify carboplatin and eribulin mesylate (E7389) in human plasma and urine. For carboplatin, sample clean-up by protein precipitation and supernatant injection into a Waters Spherisorb((R)) S5 SCX column was used. Liquid-phase extraction and reverse-phase chromatography on a Polaris C18 column were used for eribulin. Quantitation involved LC/MS/MS with positive electrospray ionization. Accuracy, precision, linearity, range, specificity, recovery and stability were also evaluated. Both compounds were stable in human plasma (>or=80 days at -80 degrees C), at room temperature (>or=4h), following three freeze-thaw cycles and in 50/50 methanol/H(2)O (<4 degrees C for >or=252 days).

Design, synthesis, and pharmacological evaluation of fluorinated tetrahydrouridine derivatives as inhibitors of cytidine deaminase.

Several 2'-fluorinated tetrahydrouridine derivatives were synthesized as inhibitors of cytidine deaminase (CDA). (4R)-2'-Deoxy-2',2'-difluoro-3,4,5,6-tetrahydrouridine (7a) showed enhanced acid stability over tetrahydrouridine (THU) 5 at its N-glycosyl bond. As a result, compound 7a showed an improved oral pharmacokinetic profile with a higher and more reproducible plasma exposure in rhesus monkeys compared to 5. Co-administration of 7a with decitabine, a CDA substrate, boosted the plasma levels of decitabine in rhesus monkeys. These results demonstrate that compound 7a can serve as an acid-stable alternative to 5 as a pharmacoenhancer of drugs subject to CDA-mediated metabolism.

Pharmacokinetic characterization of a natural product-inspired novel MEK1 inhibitor E6201 in preclinical species.

PURPOSE: E6201 is a natural product-inspired novel inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1 (MEK1) and other kinases and is currently under development as an anticancer (parenteral administration) and antipsoriasis agent (topical application). In vitro and in vivo preclinical studies were performed to characterize the pharmacokinetics of E6201. Allometric scaling was applied to predict human pharmacokinetics of E6201. METHODS: In vitro metabolism studies for CYP induction and CYP inhibition were conducted using human hepatocytes and microsomes, respectively. Metabolic stability using microsomes and protein-binding studies using pooled plasma were performed for mice, rats, dogs, and human. Pharmacokinetics of E6201 and its isomeric metabolite, ER-813010, in mice, rats, and dogs was determined following single IV administration of E6201 at three dose levels. Bioanalysis was performed using LC/MS/MS. Pharmacokinetic parameters were determined using non-compartmental analysis, and allometric scaling with a two-compartment model was used to predict E6201 pharmacokinetics in humans. RESULTS: E6201 showed high plasma protein binding (>95%), and metabolic stability half-life ranged from 36 to 89 min across species. In vitro CYP inhibition (CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A) and CYP induction (CYP1A, 3A, 2C9, and 2C19) suggested no inhibitory or induction effect on the tested human CYPs up to 10 µM of E6201. Pharmacokinetics of E6201 in mice, rats, and dogs was characterized by mean clearance ranging from 3.45 to 10.92 L/h/kg, distribution volume ranging from 0.63 to 13.09 L/kg, and elimination half-life ranging from 0.4 to 1.6 h. ER-813010 was detected in all species with metabolite to parent exposure ratio (AUC(R)) ranging from 3.1 to 33.4% and exhibited fast elimination (<3 h). The allometry predicted high clearance and large volume of distribution of E6201 in humans and was in general in good agreement with the observed first human subject pharmacokinetics. CONCLUSIONS: E6201 exhibited high clearance, high to moderate distribution, and fast elimination in preclinical species. In vitro results suggested that E6201 has low risk of drug-drug interactions due to CYP inhibition and induction in humans. In the first-in-man study, E6201 exhibited high clearance, which was well predicted by allometric scaling.