Michael D. Perloff

Saint John's wort: An in vitro analysis of P-glycoprotein induction due to extended exposure

Chronic use of Saint Johns wort (SJW) has been shown to lower the bioavailability for a variety of co‐administered drugs including indinavir, cyclosporin, and digoxin. Decreases in intestinal absorption through induction of the multidrug resistance transporter, P‐glycoprotein (P‐gp), may explain decreased bioavailability. The present study characterized the response of P‐gp to chronic and acute exposure of SJW and hypericin (HYP, a presumed active moiety within SJW) in an in vitro system. Experiments were performed with 3 to 300u2003μgu2003ml−1 of methanol‐extracted SJW and 0.03 to 3u2003μM HYP, representing low to high estimates of intestinal concentrations. In induction experiments, LS‐180 intestinal carcinoma cells were exposed for 3 days to SJW, HYP, vehicle or a positive control (ritonavir). P‐gp was quantified using Western blot analysis. P‐gp expression was strongly induced by SJW (400% increase at 300u2003μgu2003ml−1) and by HYP (700% at 3u2003μM) in a dose‐dependent fashion. Cells chronically treated with SJW had decreased accumulation of rhodamine 123, a P‐gp substrate, that was reversed with acute verapamil, a P‐gp inhibitor. Fluorescence microscopy of intact cells validated these findings. In Caco‐2 cell monolayers, SJW and HYP caused moderate inhibition of P‐gp‐attributed transport at the maximum concentrations tested. SJW and HYP significantly induced P‐gp expression at low, clinically relevant concentrations. Similar effects occurring in vivo may explain the decreased bioavailability of P‐gp substrate drugs when co‐administered with SJW.

Differential modulation of P-glycoprotein expression and activity by non-nucleoside HIV-1 reverse transcriptase inhibitors in cell culture.

AbstractPurpose. This study investigated the effects of the non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTI) nevirapine (NVR), efavirenz (EFV), and delavirdine (DLV) on P-glycoprotein (P-gp) activity and expression to anticipate P-gp related drug-drug interactions associated with combination therapy.nMethods. NNRTIs were evaluated as P-gp substrates by measuring differential transport across Caco-2 cell monolayers. Inhibition of P-gp mediated rhodamine123 (Rh123) transport in Caco-2 cells was used to assess P-gp inhibition by NNRTIs. Induction of P-gp expression and activity in LS180V cells following 3-day exposure to NNRTIs was measured by western blot analysis and cellular Rh123 uptake, respectively.nResults. The NNRTIs showed no differential transport between the basolateral to apical and apical to basolateral direction. NNRTI transport in either direction was not affected by the P-gp inhibitor verapamil. DLV inhibited Rh123 transport, causing a reduction to 15% of control at 100 μM (IC50 = 30 μM). NVR caused a concentration-dependent induction of P-gp expression in LS180V cells resulting in a 3.5-fold increase in immunoreactive P-gp at 100 μM NVR. Induction attributable to EFV and DLV was quantitatively smaller. NVR significantly reduced cellular uptake of Rh123 into LS180V cells, indicating increased drug efflux due to induced P-gp activity; effects of EFV and DLV were smaller. Acute DLV treatment of LS180V cells previously induced with NVR or ritonavir did not reverse the decreased Rh123 cell accumulation.nConclusions. NNRTIs show differential effects on P-gp activity and expression in vitro. Clinical studies are required to elucidate the clinical importance of potential drug interactions.

Fexofenadine Transport in Caco‐2 Cells: Inhibition with Verapamil and Ritonavir

This study investigated fexofenadine (FXD) transport and the inhibition of FXD transport in Caco‐2 cell monolayer transwells, usingrhodamine 123 (RH123) transport as a positive control. FXD transport from the basolateral (B) to apical (A) compartment was fivefold higher than A to B transport. FXD transport was linear with respect to time (up to 270 min) and concentration (up to 300 μm). Similar results were seen with the positive control RH123. Ritonavir (100 μM) and verapamil (100 μm) reduced transport of FXD and RH123 by more than 80%, whereas transport was not inhibited by 100 m indomethacin or 2 mM probenecid. This suggests predominantly P‐glycoprotein (P‐gp)‐mediated transport as opposed to transport by multidrug resistance protein. In concentration‐response experiments, FXD transport was inhibited by verapamil and ritonavir with IC50 values of 6.5 μm and 5.4 μm, respectively. Results from this in vitro study demonstrate differential transport of FXD across Caco‐2 cell monolayers and inhibition of FXD transport by established P‐gp inhibitors. The findings support the use of FXD as an index or probe compound to reflect P‐gp activity in vivo.

Rapid assessment of P-glycoprotein inhibition and induction in vitro.

AbstractPurpose. Using rhodamine123 (RH123) cell exclusion, 17 clinically used compounds were screened for their inhibitory effect on P-glycoprotein (P-gp), which was compared with the drugs inhibitory activity against CYP3A4. The same assay was used to study induction of P-gp activity.nMethods. P-gp inhibition was assessed using RH123 accumulation into LS180V cells as well as Rh123 transport across Caco-2 monolayers. Inhibition of CYP3A4 was determined in human liver microsomes using triazolam-4-hydroxylation. Induction of P-gp expression and activity was measured using western blot analysis and RH123 accumulation into LS180V cells, respectively.nResults. The observed inhibition of RH123 cell exclusion ranged from little or no effect (digoxin, indinavir, fexofenadine) up to a nearly 10-fold increase in RH123 accumulation (ivermectin, terfenadine). No correlation between P-gp and CYP3A4 inhibition was observed. The rank order in P-gp inhibitory potency for terfenadine, verapamil, ritonavir, and indomethacin was identical in both LS180V and Caco-2 models. Ritonavir and St. Johns wort extract showed a concentration-dependent P-gp induction, with good correlation between western blot analysis and RH123 accumulation.nConclusions. The RH123 accumulation assay in LS180V cells can be used as a valuable screening tool to study both inhibition and induction of P-gp activity and expression. This assay has the potential to predict P-gp-mediated alterations in intestinal absorption of drugs.

Ritonavir and dexamethasone induce expression of CYP3A and p-glycoprotein in rats

1. The consequences of extended exposure to the human immunodeficiency viral protease inhibitor ritonavir (RIT) on the expression and function of CYP3A isoforms in the liver and in enteric mucosal cells, and on the expression of the efflux transport protein P-glycoprotein (P-gp) in enteric mucosa and in brain microvessel endothelial cells, were evaluated in rat. Dexamethasone (DEX), a known inducer of CYP3A and P-gp in rodents, served as a positive control. 2. Male CD-1 rats received RIT (20u2009mgu2009kg−1), DEX (80u2009mgu2009kg−1) or vehicle by oral/duodenal gavage once daily for 3 days. 3. Compared with vehicle control, CYP3A activity in liver microsomes (intrinsic clearance for triazolam hydroxylation in vitro) was increased by a factor of 2–4 by RIT, and by 10–14-fold by DEX. Similar increases were observed in expression of immunoactive CYP3A protein. Overall, maximum reaction velocity and immunoactive protein were highly intercorrelated (r2 = 0.89). Both RIT and DEX also increased function and expression of enteric CYP3A, although to a more modest extent (about 1.7-fold for RIT, about 3.3-fold for DEX). 4. Enteric P-gp expression was equally induced (by 2.8-fold) by both RIT and DEX. P-gp expressed in brain microvessel endothelial cells was increased by a factor of 1.3 by both compounds. 5. Thus, increased expression of CYP3A isoforms and of P-gp occurs with 3 days of exposure to RIT in rats. Qualitatively similar changes occur in human cell culture models and in clinical studies, and might contribute to drug interactions involving RIT (and other antiretroviral agents) in humans.

Induction of P‐glycoprotein expression and activity by ritonavir in bovine brain microvessel endothelial cells

Extended treatment with human immunodeficiency virus (HIV) protease inhibitors (HPIs) is standard in HIV/AIDS therapy. While these drugs have helped decrease the overall incidence of AIDS defining illnesses, the relative prevalence of HIV/AIDS dementia has increased. HPIs may cause induction of blood‐brain barrier (BBB) drug transporters (P‐glycoprotein; P‐gp) and thereby limit entry of HPIs into brain tissue, increasing the probability that the brain could become an HIV sanctuary site. Using bovine brain microvessel endothelial cells (BMEC) as an in‐vitro model of the BBB, the potential for the HIV protease inhibitor ritonavir to cause induction of P‐gp activity and expression was examined. BMEC were isolated from fresh cow brain by enzymatic digest and density centrifugation. Primary culture BMEC were co‐incubated with ritonavir or vehicle control for 120 h. Quantitative drug accumulation of rhodamine 123 (Rh123) and fluorescence microscopy were used as measures of P‐gp activity. P‐gp expression was assessed using quantitative Western blotting. Ritonavir decreased Rh123 cell accumulation and increased P‐gp immunoreactive protein in a concentration‐dependent manner. Fluorescent microscopy mirrored Rh123 quantitative studies. In BMEC pretreated with 30 μM ritonavir, Rh123 accumulation was decreased 40% and immunoreactive P‐gp protein increased 2‐fold. Collectively, a strong correlation between decreased Rh123 BMEC accumulation and increased P‐gp immunoreactive protein was observed (Spearman r2 = 0.77, P < 0.0001). Thus extended exposure of BMEC to ritonavir caused a concentration‐dependent increase in P‐gp activity and expression. Similar findings may occur at the clinical level with prolonged HIV protease inhibitor use, giving insight into the central nervous system as an HIV sanctuary site and eventual development of HIV dementia.

Unchanged cytochrome P450 3A (CYP3A) expression and metabolism of midazolam, triazolam, and dexamethasone in mdr(−/−) mouse liver microsomes

P-Glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) share common substrates and expression properties, but the relationship of mdrl deficiency to CYP3A-mediated metabolism and protein expression is not established. The in vitro kinetic parameters of CYP3A-mediated metabolism of midazolam (MDZ), triazolam (TRZ), and dexamethasone (DEX) were studied in liver microsomes from three mrdrla(-/-) mice, one mdrla/b(-/-) mouse, and mdrla/b(+/+) controls. The kinetic profiles of CYP3A-mediated MDZ 4-hydroxylation were not significantly different between mdrl-deficient animals and controls. Overall mean (+/- SEM, N = 8) values were: Vmax, 0.74+/-0.05 nmol/min/mg protein; Km, 28.2+/-2.7 microM; and estimated intrinsic clearance, 0.026+/-0.003 mL/min/mg protein. Likewise, rates of formation of alpha-OH- and 4-OH-TRZ (from 500 microM TRZ), and of DEX metabolites sensitive to ketoconazole inhibition, M1 and M5 (from 20 microM DEX), did not differ between mdrl-deficient and control animals. Immunoquantified microsomal CYP3A protein levels in mdrla(-/-), mdrla/b(-/-), and mdrla/b(+/+) mice were not different, with overall mean immunoreactive protein levels of 2.68+/-0.09 pmol/microg protein. Although CYP3A and P-gp share aspects of activity and expression, disruption of the mdrl genes does not affect CYP3A-mediated metabolism or protein expression in the mouse.

P‐glycoprotein Interactions of Nefazodone and Trazodone in Cell Culture

This study investigated the effects of nefazodone (NFZ) and trazodone (TZD) on P‐glycoprotein (P‐gp) activity and expression in cell culture. NFZ and TZD showed no differential transport between the basolateral to apical and apical to basolateral direction across Caco‐2 cell monolayers. Transport in either direction was not affected by verapamil. NFZ was a potent inhibitor (IC50 = 4.7 M) of rhodamine123 (Rh123)B to A transport across Caco‐2 cell monolayers, while TZD had minimal effect. Following 72‐hour exposure of LS180V cells to NFZ and TZD (10 M), a twofold increase in immunoreactive P‐gp was observed. Rh123 accumulation into these cells was reduced to 65% and 74% of control by NFZ and TZD (10 M), respectively. It was concluded that differential rates of transport of NFZ and TZD in Caco‐2 cells were not evident. However, NFZ is an inhibitor of P‐gp activity at clinically relevant in vivo concentrations and may have the potential to increase bioavailability of coadministered compounds that are substrates for transport. Concentrations of NFZ and TZD achieved in the intestine after chronic oral dosing may induce P‐gp expression and reduce absorption of coadministered drugs.

Identification of glycine-extended CCK peptides in endocrine cells and modulation of CCK amide and CCK gly content and secretion from endocrine tumor cells by an inhibitor of amidation

Immunoreactive glycine-extended CCK peptides are found in normal mouse cerebral cortex and are very abundant in some CCK expressing endocrine tumor cells in culture. The glycine-extended forms in mouse cortex and in cell lines mirror their respective amidated forms. Mouse cerebral cortex, mouse AtT20 and rat WE cells produce mainly CCK 8 amide and CCK 8 Gly. In contrast, mouse intestinal STC-1 cells produce CCK 22 and CCK 8 amide along with forms of CCK Gly which are slightly larger than their respective amidated forms. The CCK 8 Gly-like peptide from AtT20 cells, after desulfation, co-eluted on HPLC with unsulfated CCK 8 Gly. Addition of copper and ascorbate to culture medium of WE cells caused a small increase in secretion of amidated CCK, without changing cellular levels of this peptide. Treatment with the amidation inhibitor diethyldithiocarbamate greatly decreased cellular content and secretion of CCK amide while it increased cellular content and secretion of CCK Gly. These results provide further evidence that glycine-extended CCK peptides are the immediate precursors of amidated CCK peptides.

Reduced levels of substance P in the brains of Cpefat/Cpefat mice

This study examines the role of carboxypeptidase E (CPE) in processing pro tachykinin to form the final bioactive amidated undecapeptide, substance P (SP) in various rat brain regions. Cpe(fat)/Cpe(fat) mice brain tissue was analyzed for total SP forms (including intermediates), and final amidated SP was compared to Cpe+/Cpe+ and Cpe+/Cpe- controls. In all brain regions tested by radioimmunoassay, amidated fully processed SP was more than fivefold lower in Cpe(fat)/Cpe(fat) mice than in controls whereas total SP species levels were unchanged. This demonstrates that CPE is required for normal SP proteolytic processing. Substance P has numerous functions in the brain; therefore, SP deficiency due to the CPE mutation may contribute to the obese phenotype or even to other phenotypes not yet described in Cpe(fat)/Cpe(fat) mice.