Liver X receptors and cholesterol homoeostasis: spotlight on the adrenal gland.

The LXRs (liver X receptors) (LXRalpha and LXRbeta) are nuclear hormone receptors that are activated by oxysterols, endogenous oxidative metabolites of cholesterol. These receptors regulate an integrated network of genes that control whole body cholesterol and lipid homoeostasis. A brief overview of the mechanism of this regulation by LXRs in the liver, macrophage and intestine will be outlined, followed by data from our recent work demonstrating that LXRalpha is crucial in maintaining adrenal cholesterol homoeostasis. In the adrenal gland, oxysterols are formed as intermediates in the conversion of cholesterol into steroid hormones and can act as endogenous activators of LXR. We have found using both gain- and loss-of-function models that LXR acts to maintain free cholesterol below toxic levels in the adrenal gland, through the co-ordinated regulation of genes involved in cholesterol efflux [ABCA1 (ATP-binding-cassette transporter A1)], storage (sterol-regulatory-element-binding protein-1c and apolipoprotein E) and metabolism to steroid hormones (steroidogenic acute regulatory protein). Furthermore, we show that under chronic dietary stress, the adrenal glands of LXR-null mice (and not wild-type mice) accumulate free cholesterol. These results support the role of LXR as a global regulator of cholesterol homoeostasis, where LXR provides a safety valve to limit free cholesterol in tissues experiencing high cholesterol flux.

Unmasking the dynamic interplay between efflux transporters and metabolic enzymes.

Drug efflux by intestinal P-glycoprotein (P-gp) is known to decrease the bioavailability of many CYP3A4 substrates. We have demonstrated that the interplay between P-gp and CYP3A4 at the apical intestinal membrane can increase the opportunity for drug metabolism by determining bidirectional extraction ratios across CYP3A4-transfected Caco-2 cells for two dual P-gp/CYP3A4 substrates, K77 (an experimental cysteine protease inhibitor) and sirolimus, as well as two negative control, CYP3A4 only substrates, midazolam and felodipine. Studies were carried out under control conditions, with a P-gp inhibitor (GG918) and with a dual inhibitor (cyclosporine). Measurement of intracellular concentration changes is an important component in calculating the extraction ratios. We hypothesize that the inverse orientation of P-gp and CYP3A4 in the liver will result in an opposite interactive effect in that organ. In vivo rat intestinal perfusion studies with K77 and rat liver perfusion studies with tacrolimus under control conditions and with inhibitors of CYP3A4 (troleandomycin), P-gp (GG918) and both CYP3A4/P-gp (cyclosporine) lend support to our hypotheses. These results serve as a template for predicting enzyme-transporter (both absorptive and efflux) interactions in the intestine and the liver.

Transporter-enzyme interactions: implications for predicting drug-drug interactions from in vitro data.

As discussed in earlier articles, predictions of in vivo drug-drug interactions from in vitro studies is a subject of high interest with obvious therapeutic as well as economic benefits. Up until now little attention has been given to the potential interplay between metabolic enzymes and transporters that could confound the in vivo-in vitro relationships. Drug efflux by intestinal P-glycoprotein (P-gp) is known to decrease the bioavailability of many CYP3A4 substrates. We have demonstrated that the interplay between P-gp and CYP3A4 at the apical intestinal membrane can increase the opportunity for drug metabolism by determining bidirectional extraction ratios across CYP3A4 transfected Caco-2 cells for two dual P-gp/CYP3A4 substrates, K77 (an experimental cysteine protease inhibitor) and sirolimus, as well as two negative control, CYP3A4 only substrates, midazolam and felodipine. Studies were carried out under control conditions, with a P-gp inhibitor (GG918) and with a dual inhibitor (cyclosporine). Measurement of intracellular concentration changes is an important component in calculating the extraction ratios. We hypothesize that the inverse orientation of P-gp and CYP3A4 in the liver will result in an opposite interactive effect in that organ. In vivo rat intestinal perfusion studies with K77 and rat liver perfusion studies with tacrolimus under control conditions and with inhibitors of CYP3A4 (troleandomycin), P-gp (GG918) and both CYP3A4/P-gp (cyclosporine) lend support to our hypotheses. These results serve as a template for predicting enzyme- transporter (both absorptive and efflux) interactions in the intestine and the liver.

Characterizing the expression of CYP3A4 and efflux transporters (P-gp, MRP1, and MRP2) in CYP3A4-transfected Caco-2 cells after induction with sodium butyrate and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate.

PURPOSE: To examine the changes in expression levels of CYP3A4 and efflux transporters in CYP3A4-transfected Caco-2 (colon carcinoma) cells in the presence of the inducers sodium butyrate (NaB) and 12-O-tetradecanoylphorbol-13-acetate (TPA). To characterize the transport of [3H]-digoxin and the metabolism of midazolam in the cells under different inducing conditions. METHODS: CYP3A4-Caco-2 cells were seeded onto cell culture inserts and were grown for 13-14 days. Transport and metabolism studies were performed on cells induced with NaB and/or TPA for 24 h. The expression and localization of P-gp, MRP1, MRP2, and CYP3A4 were examined by Western blot and confocal microscopy. RESULTS: In the presence of both inducers, CYP3A4 protein levels were increased 40-fold over uninduced cells, MRP2 expression was decreased by 90%, and P-gp and MRP1 expression were unchanged. Midazolam 1-OH formation exhibited a rank order correlation with increased CYP3A4 protein, whereas [3H]-digoxin transport (a measure of P-gp activity) was unchanged with induction. P-gp and MRP2 were found on the apical membrane, whereas MRP1 was found perinuclear within the cell. CYP3A4 displayed a punctate pattern of expression consistent with endoplasmic reticulum localization and exhibited preferential polarization towards the apical side of the cell. CONCLUSIONS: The present study characterized CYP3A4-Caco-2 cell monolayers when induced for 24 h in the presence of both NaB and TPA. These conditions provide intact cells with significant CYP3A4 and P-gp expression suitable for the concurrent study of transport and metabolism.

The drug efflux-metabolism alliance: biochemical aspects.

The considerable overlap in the substrate selectivity and tissue localization of CYP3A and P-glycoprotein has led to the hypothesis that this transporter and enzyme pair act as a coordinated absorption barrier against xenobiotics. A historical perspective on the investigation of this interactive alliance is given, starting from the understanding of the role of intestinal metabolism in explaining cyclosporine clinical data. Several animal studies using mdr1a-/- knockout mice have demonstrated P-glycoprotein's importance in limiting drug absorption and decreasing bioavailability. Human clinical studies investigating the importance of intestinal CYP3A and P-glycoprotein through inhibition or induction of these proteins have provided further evidence of this interaction. Recent in vitro studies using CYP3A4-expressing Caco-2 cells are reported. These studies reveal that the role of P-glycoprotein in the intestine extends beyond simply limiting parent drug absorption but also includes increasing the access of drug to metabolism by CYP3A through repeated cycles of absorption and efflux.

Laser resurfacing for facial acne scars.

BACKGROUND: Most people have acne at some stage during their life, with about one per cent being left with permanent acne scars. Recent laser techniques are thought to be more effective than chemical peels and dermabrasion. OBJECTIVES: To assess the effects of laser resurfacing for treating facial acne scars. SEARCH STRATEGY: We searched MEDLINE (1966 to April 1999), EMBASE (1980 to April 1999), Science Citation Index (1981 to April 1999), the Cochrane Controlled Trials Register (April 1999), DARE (April 1999), INAHTA (April 1999), NHS HTA Internet site (April 1999). Dermatological Surgery (1995 to March 1999) and the British Journal of Dermatology (1995 to September 1999) were handsearched. We searched the reference lists of relevant articles and contacted experts and commercial laser manufacturers. SELECTION CRITERIA: Randomised controlled trials which compare different laser resurfacing techniques for treating patients with facial acne scars, or compare laser resurfacing with other resurfacing techniques or no treatment. DATA COLLECTION AND ANALYSIS: Two reviewers independently selected studies, assessed the quality of studies and extracted data. MAIN RESULTS: No randomised controlled trials where laser treatment was compared to either placebo or a different type of laser were found. Most of the 27 studies uncovered were poor quality case series with small numbers of acne-scarred patients. REVIEWER'S CONCLUSIONS: The lack of good quality evidence does not enable any conclusions to be drawn about the effectiveness of lasers for treating atrophic or ice-pick acne scars. Well designed randomised controlled comparisons of carbon dioxide versus Erbium:YAG laser are urgently needed.

Determination of p-aminosalicylic acid and its N-acetylated metabolite in human urine by capillary zone electrophoresis as a measure of in vivo N-acetyltransferase 1 activity.

A capillary zone electrophoresis method has been developed for the determination of p-aminosalicylic acid (PAS) and its metabolite, N-acetyl-p-aminosalicylic acid (N-acetyl-PAS), in urine. A linear relationship was observed between time-normalized peak area and the concentration of the parent and metabolite with correlation coefficients greater than 0.9990. The method could be applied to the determination of PAS and N-acetyl-PAS in human urine without any sample pretreatment. A good separation of the analytes is achieved in a run time of 12 min (15 min total, including capillary wash). Using PAS as a probe for N-acetyltransferase 1 activity, 20 healthy volunteers were phenotyped after oral administration of a 1 g dose. The preliminary results seem to indicate a bimodal distribution of N-acetyl-PAS/PAS molar ratios.