Use of a fluorescent substrate for the selective quantification of rat CYP3A in the liver and the intestine.

INTRODUCTION: Quantification of cytochrome P450 is a major issue in the development of new drugs. Different assays have been reported, but few are very selective for the 3A isoform or cytochrome P450. The benzyloxy-substituted lactone cyclooxygenase-2 inhibitor 3-[(3, 4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl) phenyl] furan-2(5H)-one has recently been used successfully to probe isoform 3A of cytochrome P450 in the liver. However, its selectivity for the rat isoform remains to be established as well as its applicability in other tissue, such as the intestine. The purpose of this study was to ascertain the specificity of this substrate for the rat 3A isoform of cytochrome P450 using Supersomes and its application in non-hepatic tissue (e.g., intestine). METHODS: Specificity of the 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl)phenyl] furan-2(5H)-one for the isoform 3A of rat cytochrome P450 was established by using either isoform-specific inhibitory antibody or microsomes expressing only one cytochrome P450 isoform. Activity was assayed in rat liver and intestinal microsomal protein preparations. RESULTS: Experiments with inhibitory antibodies revealed that in liver and intestinal microsomes, more than 90% of the substrate metabolism was inhibited by antibodies against isoform 3A. Selectivity of the substrate for rat 3A isoform was further determined by testing the metabolic activity of various Supersomes preparations. DISCUSSION: In conclusion, our results validate the usefulness of 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl)phenyl] furan-2(5H)-one as a simple and specific substrate to study the activity of the isoform 3A of cytochrome P450 in the rat liver and intestine.

Down-regulation of intestinal drug transporters in chronic renal failure in rats.

Chronic renal failure (CRF) is associated with an increased bioavailability of drugs by a poorly understood mechanism. One hypothesis is a reduction in the elimination of drugs by the intestine, i.e., drug elimination mediated by protein membrane transporters such as P-glycoprotein (Pgp) and multidrug-resistance-related protein (MRP) 2. The present study aimed to investigate the repercussions of CRF on intestinal transporters involved in drug absorption [organic anion-transportingpolypeptide (Oatp)] and those implicated in drug extrusion (Pgp and MRP2). Pgp, MRP2, MRP3, Oatp2, and Oatp3 protein expression and Pgp, MRP2, and Oatp3 mRNA expression were assessed in the intestine of CRF (induced by five-sixth nephrectomy) and control rats. Pgp and MRP2 activities were measured using the everted gut technique. Rat enterocytes and Caco-2 cells were incubated with sera from control and CRF rats to characterize the mechanism of transporters' down-regulation. Protein expression of Pgp, MRP2, and MRP3 were reduced by more than 40% (p < 0.01) in CRF rats, whereas Oatp2 and Oatp3 expression remained unchanged. There was no difference in the mRNA levels assessed by real-time polymerase chain reaction. Pgp and MRP2 activities were decreased by 30 and 25%, respectively, in CRF rats compared with control (p < 0.05). Uremic sera induced a reduction in protein expression and in activity of drug transporters compared with control sera. Our results demonstrate that CRF in rats is associated with a decrease in intestinal Pgp and MRP2 protein expression and function secondarily to serum uremic factors. This reduction could explain the increased bioavailability of drugs in CRF.

Role of parathyroid hormone in the downregulation of liver cytochrome P450 in chronic renal failure.

Chronic renal failure (CRF) is associated with a decrease in drug metabolism secondary to a decrease in liver cytochrome P450 (P450). The predominant theory to explain this decrease is the presence of factors in the blood of uremic patients. This study tested the hypothesis that parathyroid hormone (PTH) could be this factor. The objectives of this study were to determine (1) the role of PTH in the downregulation of hepatocyte P450 induced by rat uremic serum, (2) the role of PTH in the downregulation of liver P450 in rats with CRF, and (3) the effects of PTH on P450 in hepatocytes. For this purpose, (1) hepatocytes were incubated with serum from rat with CRF that was depleted with anti-PTH antibodies or with serum from parathyroidectomized (CRF-PTX) rat with CRF, (2) the effect of PTX on liver P450 was evaluated in rats with CRF, and (3) the effects of PTH on P450 in hepatocytes were determined. The depletion of PTH from CRF serum completely reversed the downregulating effect of CRF serum on P450 in hepatocytes. Addition of PTH (10(-9) M) to depleted CRF serum induced a decrease in P450 similar to nondepleted CRF serum. The serum of CRF-PTX rats had no effect on P450 in hepatocytes compared with CRF serum. Adding PTH to CRF-PTX serum induced a similar decrease in P450 as obtained with CRF serum. Finally, PTX prevented the decrease of liver P450 in rats with CRF. In summary, PTH is the major mediator implicated in the downregulation of liver P450 in rats with CRF.

Characterization of chronic constriction of the saphenous nerve, a model of neuropathic pain in mice showing rapid molecular and electrophysiological changes.

Neuropathic pain is one of the most inextricable problems encountered in clinics, because few facts are known about its etiology. Nerve injury often leads to allodynia and hyperalgesia, which are symptoms of neuropathic pain. The aim of this study was to understand some molecular and electrophysiological mechanisms of neuropathic pain after chronic constriction of the saphenous nerve (CCS) in mice. After surgery, CCS mice displayed significant allodynia and hyperalgesia, which were sensitive to acute systemic injection of morphine (4 mg/kg), gabapentin (50 mg/kg), amitriptyline (10 mg/kg), and the cannabinoid agonist WIN 55,212-2 (5 mg/kg). These behavioral changes were accompanied after surgery by an increase of c-Fos expression and by an overexpression of mu-opioid and cannabinoid CB1 and CB2 receptors in the spinal cord and the dorsal hind paw skin. In combination with the skin-nerve preparation, this model showed a decrease in functional receptive fields downstream to the injury and the apparition of A-fiber ectopic discharges. In conclusion, CCS injury induced behavioral, molecular, and electrophysiological rearrangements that might help us in better understanding the peripheral mechanisms of neuropathic pain. This model takes advantage of the possible use in the future of genetically modified mice and of an exclusively sensory nerve for a comprehensive study of peripheral mechanisms of neuropathic pain.

Effects of serum from patients with chronic renal failure on rat hepatic cytochrome P450.

1. In humans, chronic renal failure (CRF) is associated with decreased hepatic drug metabolism, particularly that mediated by the cytochrome P450 (P450). The mechanisms remain poorly understood. The present study aimed to investigate the effects of the serum of patients with CRF on liver P450, and to evaluate whether renal replacement therapies (dialysis or transplantation) impede the inhibition of CRF serum on P450. 2. Rat hepatocytes were incubated for 24 h with serum from patients with severe CRF and from controls to measure (1) P450 level, (2) protein expression and mRNA levels of P450 isoforms and (3) metabolic activities of CYP3A and CYP1A. Similar experiments were performed with serum of patients once on chronic hemodialysis and after kidney transplantation. 3. In rat hepatocytes incubated for 24 h with serum from patients with CRF, P450 level and protein expression, as well as mRNA levels of P450 isoforms (CYP1A2, 2C6, 2C11, 2D1/2D2, 3A2 and 4A1/4A3), were decreased by more than 45% (P<0.001) compared to control serum, while the levels of CYP2E1 were not modified. CYP3A and CYP1A activities were decreased by 51 and 59% (P<0.001), respectively. The inhibitory effect of serum obtained from patients before first dialysis was similar after 1 or 6 months on chronic hemodialysis but was lost after successful kidney transplantation. In CRF serum, the fraction containing proteins between 10 and 15 kDa decreases P450. 4. Human uremic serum contains mediator(s) that decreases rat hepatic P450 activity and expression secondary to reduced gene expression. The inhibitory effect of serum persists even after initiation of dialysis, but disappears after normalization of renal function following kidney transplantation.

Inflammatory response to peritoneal implantation of alginate-poly-L-lysine microcapsules.

A thorough understanding of the mechanisms involved in the host reaction to alginate-poly-L-lysine microcapsules (HRM) is important to design methods for the evaluation, selection, and development of biocompatible biomaterials and microcapsules or treatments to control this reaction. The objective of this study was to identify those immune cells and cytokines involved in the pathogenesis of the HRM. The total and differential cell counts were evaluated, and the mRNA expression of TNF-alpha, IL-1beta, IL-6 and TGF-beta1 was measured in peritoneal washings at 3, 17, 48, 96 and 168 h after saline or microcapsule injections. Neutrophil number and IL-1beta and IL-6 m-RNA expression presented an early transient increase, with no differences between saline and microcapsule injections, suggesting a reaction to the procedure. Macrophages, lymphocytes and TNF-alpha were significantly more activated over a longer period of time, after microcapsule implantation than saline injection. They are likely involved in transforming the reaction into a chronic inflammatory process. TGF-beta1 and IL-1beta presented a late (day 7) significant increase after microcapsule but not saline injections. They are likely involved in transforming the reaction into a fibrogenic process. These results suggest that macrophages, lymphocytes, TNF-alpha, IL-1beta and TGF-beta1 play a role in the pathogenesis of the HRM.