Preclinical species and human disposition of PF-04971729, a selective inhibitor of the sodium-dependent glucose cotransporter 2 and clinical candidate for the treatment of type 2 diabetes mellitus.

(1S,2S,3S,4R,5S)-5-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-1-hydroxymethyl-6,8-dioxabicyclo[3.2.1]octane-2,3,4-triol (PF-04971729), a potent and selective inhibitor of the sodium-dependent glucose cotransporter 2, is currently in phase 2 trials for the treatment of diabetes mellitus. This article describes the preclinical species and in vitro human disposition characteristics of PF-04971729 that were used in experiments performed to support the first-in-human study. Plasma clearance was low in rats (4.04 ml · min(-1) · kg(-1)) and dogs (1.64 ml · min(-1) · kg(-1)), resulting in half-lives of 4.10 and 7.63 h, respectively. Moderate to good bioavailability in rats (69%) and dogs (94%) was observed after oral dosing. The in vitro biotransformation profile of PF-04971729 in liver microsomes and cryopreserved hepatocytes from rat, dog, and human was qualitatively similar; prominent metabolic pathways included monohydroxylation, O-deethylation, and glucuronidation. No human-specific metabolites of PF-04971729 were detected in in vitro studies. Reaction phenotyping studies using recombinant enzymes indicated a role of CYP3A4/3A5, CYP2D6, and UGT1A9/2B7 in the metabolism of PF-04971729. No competitive or time-dependent inhibition of the major human cytochrome P450 enzymes was discerned with PF-04971729. Inhibitory effects against the organic cation transporter 2-mediated uptake of [(14)C]metformin by PF-04971729 also were very weak (IC(50) = ∼900 µM). Single-species allometric scaling of rat pharmacokinetics of PF-04971729 was used to predict human clearance, distribution volume, and oral bioavailability. Human pharmacokinetic predictions were consistent with the potential for a low daily dose. First-in-human studies after oral administration indicated that the human pharmacokinetics/dose predictions for PF-04971729 were in the range that is likely to yield a favorable pharmacodynamic response.

pH-sensitive interaction of HMG-CoA reductase inhibitors (statins) with organic anion transporting polypeptide 2B1.

The human organic anion transporting polypeptide 2B1 (OATP2B1, SLCO2B1) is ubiquitously expressed and may play an important role in the disposition of xenobiotics. The present study aimed to examine the role of OATP2B1 in the intestinal absorption and tissue uptake of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitors (statins). We first investigated the functional affinity of statins to the transporter as a function of extracellular pH, using OATP2B1-transfeced HEK293 cells. The results indicate that OATP2B1-mediated transport is significant for rosuvastatin, fluvastatin and atorvastatin, at neutral pH. However, OATP2B1 showed broader substrate specificity as well as enhanced transporter activity at acidic pH. Furthermore, uptake at acidic pH was diminished in the presence of proton ionophore, suggesting proton gradient as the driving force for OATP2B1 activity. Notably, passive transport rates are predominant or comparable to active transport rates for statins, except for rosuvastatin and fluvastatin. Second, we studied the effect of OATP modulators on statin uptake. At pH 6.0, OATP2B1-mediated transport of atorvastatin and cerivastatin was not inhibitable, while rosuvastatin transport was inhibited by E-3-S, rifamycin SV and cyclosporine with IC(50) values of 19.7 ± 3.3 µM, 0.53 ± 0.2 µM and 2.2 ± 0.4 µM, respectively. Rifamycin SV inhibited OATP2B1-mediated transport of E-3-S and rosuvastatin with similar IC(50) values at pH 6.0 and 7.4, suggesting that the inhibitor affinity is not pH-dependent. Finally, we noted that OATP2B1-mediated transport of E-3-S, but not rosuvastatin, is pH sensitive in intestinal epithelial (Caco-2) cells. However, uptake of E-3-S and rosuvastatin by Caco-2 cells was diminished in the presence of proton ionophore. The present results indicate that OATP2B1 may be involved in the tissue uptake of rosuvastatin and fluvastatin, while OATP2B1 may play a significant role in the intestinal absorption of several statins due to their transporter affinity at acidic pH.

Characterization of digoxin uptake in sandwich-cultured human hepatocytes.

Digoxin is a drug that is commonly used to treat congestive heart failure. Because of digoxin's narrow therapeutic index, patients are susceptible to drug-drug interaction-mediated cardiotoxicity. Digoxin is primarily cleared renally; however, a significant component of clearance is due to multidrug resistance 1-mediated transport into bile. Digoxin is reported to be actively transported into human hepatocytes by the organic anion-transporting polypeptide 1B3 (OATP1B3); however, further characterization has not been fully described. The purpose of this study was to investigate the hepatic uptake mechanisms of [(3)H]digoxin using sandwich-cultured human hepatocytes (SCHH) and transporter-expressing cells. Digoxin uptake in SCHH involves both a saturable (carrier-mediated) process and a passive (nonsaturable) process. At low concentrations, the saturable component exhibited an apparent K(m) of 2.39 µM and a V(max) of 4.49 pmol/(min · mg protein). The calculated passive diffusion clearance was 1.25 µl/(min · mg protein). Uptake of [(3)H]digoxin in SCHH was not inhibited by a variety of substrates or inhibitors for OATP1B1, OATP1B3, OATP2B1, organic anion transporter 2, organic cation transporter 1, and monocarboxylate transporter 8. Cytochalasin B, which inhibits glucose transporters, did not significantly inhibit digoxin uptake, whereas the flavonoids quercetin and rutin inhibited uptake by ∼50%. Nonlabeled digoxin inhibited [(3)H]digoxin uptake by ∼50%. Studies with OATP-transfected human embryonic kidney cells or oocytes showed that digoxin is not a substrate of OATP1B1, OATP2B1, or OATP1B3. In conclusion, the data suggest that digoxin uptake in SCHH involves both saturable and passive processes. The saturable process is mediated by an as yet undetermined digoxin transporter(s).

Discovery of novel hepatoselective HMG-CoA reductase inhibitors for treating hypercholesterolemia: a bench-to-bedside case study on tissue selective drug distribution.

The design of drugs with selective tissue distribution can be an effective strategy for enhancing efficacy and safety, but understanding the translation of preclinical tissue distribution data to the clinic remains an important challenge. As part of a discovery program to identify next generation liver selective HMG-CoA reductase inhibitors we report the identification of (3R,5R)-7-(4-((3-fluorobenzyl)carbamoyl)-5-cyclopropyl-2-(4-fluorophenyl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoic acid (26) as a candidate for treating hypercholesterlemia. Clinical evaluation of 26 (PF-03491165), as well as the previously reported 2 (PF-03052334), provided an opportunity for a case study comparison of the preclinical and clinical pharmacokinetics as well as pharmacodynamics of tissue targeted HMG-CoA reductase inhibitors.

Physicochemical determinants of human renal clearance.

Kidney plays an important role in the elimination of drugs, especially with low or negligible hepatic clearance. An analysis of the interrelation of physicochemical properties and the human renal clearance for a data set of 391 drugs or compounds tested in humans is presented. The data set indicated that lipophilicity shows a negative relationship while polar descriptors show a positive relationship with renal clearance. Analysis of net secreted and net reabsorbed subsets revealed that hydrophilic ionized compounds are probable compounds to show net secretion and a possible drug-drug interaction due to their likely interaction with uptake transporters and inherent low passive reabsorption. The physicochemical space and renal clearance were also statistically analyzed by therapeutic area. In conclusion, ionization state, lipophilicity, and polar descriptors are found to be the physicochemical determinants of renal clearance. These fundamental properties can be valuable in early prediction of human renal clearance and can aid the chemist in structural modifications to optimize drug disposition.

Renal organic anion transporter-mediated drug-drug interaction between gemcabene and quinapril.

In humans and rats, a synergistic blood pressure reduction was observed when the fibrate gemcabene (CI-1027) was coadministered with the angiotensin-converting enzyme inhibitor quinapril. In a quinapril (3 mg/kg) pharmacokinetic rat study, there was a 40% decrease in urinary excretion and a 53% increase in plasma area under the curve from 0 to 24 h of the active metabolite quinaprilat when coadministered with gemcabene (30 mg/kg). This observation revealed a possible transporter-mediated drug-drug interaction (DDI) between gemcabene and quinapril. This led to a series of studies investigating the underlying clearance mechanisms associated with these compounds intended to elucidate renal transporter interactions between quinapril and gemcabene. In vitro transporter studies using human embryonic kidney 293 cells transfected with human or rat organic anion transporter 3 (hOAT3, rOat3) revealed that quinaprilat is a substrate in both species, with a K(m) value of 13.4 microM for hOAT3. Subsequent studies discovered that gemcabene inhibited quinaprilat uptake by hOAT3 and rOat3 at IC(50) values of 35 and 48 microM, respectively. Moreover, gemcabene acylglucuronide, the major metabolite of gemcabene glucuronidation, also inhibited hOAT3- and rOat3-mediated uptake of quinaprilat at IC(50) values of 197 and 133 microM, respectively. High plasma concentrations of gemcabene (>100 microM) achieved in humans and rats upon oral dosing corroborate with gemcabene inhibition of renal OAT3-mediated secretion of quinaprilat in vitro. This investigation established that a DDI between gemcabene and quinapril involving inhibition of renal transporters and subsequent elevation in plasma concentrations of quinaprilat is responsible for the apparent synergistic blood pressure reduction observed with these compounds.

N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine (CP-100,356) as a "chemical knock-out equivalent" to assess the impact of efflux transporters on oral drug absorption in the rat.

The utility of the diaminoquinazoline derivative CP-100,356 as an in vivo probe to selectively assess MDR1/BCRP-mediated drug efflux was examined in the rat. CP-100,356 was devoid of inhibition (IC(50) >50 microM) against major human P450 enzymes including P4503A4. In human MDR1-transfected MDCKII cells, CP-100,356 inhibited acetoxymethyl calcein (calcein-AM) uptake (IC(50) approximately 0.5 +/- 0.07 microM) and digoxin transport (IC(50) approximately 1.2 +/- 0.1 microM). Inhibition of prazosin transport (IC(50) approximately 1.5 +/- 0.3 microM) in human BCRP-transfected MDCKII cells by CP-100,356 confirmed the dual MDR1/BCRP inhibitory properties. CP-100,356 was a weak inhibitor of OATP1B1 (IC(50) approximately 66 +/- 1.1 microM) and was devoid of MRP2 inhibition (IC(50) >15 microM). In vivo inhibitory effects of CP-100,356 in rats were examined after coadministration with MDR1 substrate fexofenadine and dual MDR1/BCRP substrate prazosin. Coadministration with increasing doses of CP-100,356 resulted in dramatic increases in systemic exposure of fexofenadine (36- and 80-fold increase in C(max) and AUC at a CP-100,356 dose of 24 mg/kg). Significant differences in prazosin pharmacokinetics were also discernible in CP-100,356-pretreated rats as reflected from a 2.6-fold increase in AUC. Coadministration of CP-100,356 and P4503A substrate midazolam did not result in elevations in systemic exposure of midazolam in the rat. The in vivo methodology should have utility in drug discovery in selective and facile assessment of the role of MDR1 and BCRP efflux transporters in oral absorption of new drug candidates.

A comprehensive in vitro and in silico analysis of antibiotics that activate pregnane X receptor and induce CYP3A4 in liver and intestine.

We have investigated several in silico and in vitro methods to improve our ability to predict potential drug interactions of antibiotics. Our focus was to identify those antibiotics that activate pregnane X receptor (PXR) and induce CYP3A4 in human hepatocytes and intestinal cells. Human PXR activation was screened using reporter assays in HepG2 cells, kinetic measurements of PXR activation were made in DPX-2 cells, and induction of CYP3A4 expression and activity was verified by quantitative polymerase chain reaction, immunoblotting, and testosterone 6beta-hydroxylation in primary human hepatocytes and LS180 cells. We found that in HepG2 cells CYP3A4 transcription was activated strongly (> 10-fold) by rifampin and troleandomycin; moderately (> or = 7-fold) by dicloxacillin, tetracycline, clindamycin, griseofulvin, and (> or = 4-fold) erythromycin; and weakly (> 2.4-fold) by nafcillin, cefaclor, sulfisoxazole, and (> 2-fold) cefadroxil and penicillin V. Similar although not identical results were obtained in DPX-2 cells. CYP3A4 mRNA and protein expression were induced by these antibiotics to differing extents in both liver and intestinal cells. CYP3A4 activity was significantly increased by rifampin (9.7-fold), nafcillin and dicloxacillin (5.9-fold), and weakly induced (2-fold) by tetracycline, sufisoxazole, troleandomycin, and clindamycin. Multiple pharmacophore models and docking indicated a good fit for dicloxacillin and nafcillin in PXR. These results suggest that in vitro and in silico methods can help to prioritize and identify antibiotics that are most likely to reduce exposures of medications (such as oral contraceptive agents) which interact with enzymes and transporters regulated by PXR. In summary, nafcillin, dicloxacillin, cephradine, tetracycline, sulfixoxazole, erythromycin, clindamycin, and griseofulvin exhibit a clear propensity to induce CYP3A4 and warrant further clinical investigation.

Application of simple mathematical expressions to relate half-lives of drugs in mice to those in humans.

The use of rat half-life data to predict human half-lives for drugs and other xenobiotics has previously been shown to be of value. Since transgenic and knockout mice are increasingly used in early stages of preclinical drug development, we wondered whether the estimation of half-life values in mice might be used in the same way that has been recommended for rats. A dataset of mouse and human half-life values was assembled for 88 drugs. Three different regression models were then applied to the prediction of human half-life values from mouse values. The results showed that none of the models was superior to any other, and that all models, including the simplest linear regression model, could predict a human half-life value from a mouse half-life value such that there would be an 80% chance that the predicted value would be within three-fold (i.e. between 0.33 times and 3 times) of the actual human value.

Detection of cytokine protein expression in mouse lung homogenates using suspension bead array.

BACKGROUND: The objective for this present study was to determine whether or not suspension bead array is a feasible method to detect changes in cytokine protein expression in mouse lung tissue homogenates. Here, we report on suspension bead array as a feasible method for detection of lipopolysaccharide (LPS)-evoked changes in cytokine protein expression in mouse lung tissue homogenates. MATERIALS AND METHODS: Mice were treated (0.2 ml, intraperitoneal, i.p.) with phosphate buffered saline (PBS) or LPS (0.25 mg/ml) and sacrificed at either 2- or 24-hours post treatment. Formalin-fixed and paraffin-embedded lung sections were evaluated by light microscopy. Flash frozen lung tissues were homogenized for measurement of various cytokine protein expression levels using suspension bead array, antibody array and ELISA. Comparison between groups was performed using a Wilcoxon signed rank test. RESULTS: Pulmonary perivascular edema and an accumulation of mixed cell infiltrates within blood and lymphatic vessels, as well as in the adjacent interstitium, were present at both 2- and 24-hours following LPS treatment. A minimal increase in the number of alveolar macrophages was also observed in the 24-hour LPS-treated mice only. The suspension bead array assay revealed statistically significant increases in mouse lung tissue homogenate levels of interleukin-6 (IL-6) and granulocyte/macrophage colony-stimulating factor (GM-CSF) proteins and a decrease in IL-2 protein at 24-hours post LPS-treatment only. Similar cytokine protein expression patterns were observed using antibody array. Significantly increased IL-6 protein expression levels were also detected using ELISA, which correlated with the suspension bead array data. CONCLUSION: The present study shows that suspension bead array is a feasible method to detect changes in cytokine protein expression in mouse lung tissue homogenates.

Acute coronary artery injury in dogs following administration of CI-1034, an endothelin A receptor antagonist.

The objective of this study was to characterize acute coronary artery injury evoked by the endothelin A receptor (ETAR) antagonist, CI-1034. Male dogs (n = 5) were intravenously administered CI-1034 at 120 mg/kg for 4 d. Control animals (n = 3) received vehicle. Macroscopically, drug-related hemorrhage was observed in the right coronary groove and atrium. Histologically, drugrelated coronary changes were characterized as medial hemorrhage and necrosis, with mixed inflammatory-cell infiltrates in the adventitia and media. Immunohistochemistry staining indicated increased expression of inducible nitric oxide synthase (iNOS), cleaved caspase-3, and S100A8/A9 (within in monocytes and neutrophils) proteins in coronary arteries of CI-1034-treated animals. However, there were similar expression levels of endothelial nitric oxide synthase (eNOS) among control and CI-1034-treated animals. Significant drug-related nitric oxide (NO) accumulation occurred on days 1 through 4 in serum. Increased interleukin (IL)-6 and fibrinogen in plasma and serum amyloid A (SAA) occurred on days 2 through 5 in CI-1034-treated animals. Increased levels of NO accumulation in serum; increased IL-6 and fibrinogen levels in plasma; increased SAA levels; and increased expressions of iNOS, cleaved caspase-3, and S100A8/A9 complex appear to be characteristic of CI-1034-induced acute vascular injury in dogs.

Comparative methods for multiplex analysis of cytokine protein expression in plasma of lipopolysaccharide-treated mice.

Changes in circulating cytokines might serve as predictors of compound-evoked inflammatory responses. CD-1 mice were treated with lipopolysaccharide (LPS; 0.2 ml of 0.25 mg/ml, intraperitoneal) for subsequent expression measurement of plasma cytokine protein expression at 24-h post-treatment using multiple antibody Western blot, and at both 2-h and 24-h post-treatment using antibody array and suspension bead array. Antibody array provided a semi-qualitative assessment and suggested significantly increased expression of GCSF at 2-h post-treatment and GCSF, IL-6, IL-12, MCP-1, MCP-5, RANTES and sTNFR1 at 24-h post-treatment. Densitometric analysis of multiple antibody Western blots provided a semi-quantitative assessment and indicated significantly increased expression of IL-6, IL-12, IL-17, GCSF, eotaxin, and MCP-2 at 24-h post-treatment. The suspension bead array yielded statistically significant cytokine protein expression increases for IL-6, IL-10, IFNgamma and TNFalpha at both 2-h and 24-h post-treatments, while significant expression at 24-h post-treatment only was noted for IL-1beta, IL-5, IL-12 and GM-CSF. Suspension bead array provided the greatest range of detection, revealing subtle increased expression of GM-CSF, IL-1beta, IL-5, IL-10, TNFalpha and IFNgamma at 24-h post-treatment, not detected by antibody array or multiple antibody Western blot. Suspension bead array proved to be the best method for detection of LPS-evoked changes in plasma cytokine levels.