François A. Leblond

Emerging Evidence of the Impact of Kidney Disease on Drug Metabolism and Transport

Several lines of emerging evidence indicate that kidney disease differentially affects uptake and efflux transporters and metabolic enzymes in the liver and gastrointestinal (GI) tract, and uremic toxins have been implicated as the cause. In patients with kidney disease, even drugs that are eliminated by nonrenal transport and metabolism could lead to important unintended consequences if they are administered without dose adjustment for reduced renal function. This is particularly so in the case of drugs with narrow therapeutic windows and may translate into clinically significant variations in exposure and response.

Down-regulation of hepatic cytochrome P450 in chronic renal failure: role of uremic mediators

Chronic renal failure (CRF) is associated with a decrease in liver cytochrome P450 (P450). The mechanism remains poorly understood. The present study aimed to investigate the effects of the serum of rats with CRF on liver P450. Normal rat hepatocytes were incubated for 24 h with serum (concentration of 10%) from rats with CRF and from control animals in order to measure (1) total P450 level, (2) protein expression and mRNA levels of major P450 isoforms, and (3) some of their specific metabolic activities (N‐demethylation of erythromycin). Time‐course experiments (incubation time from 12 to 48 h) and dose‐response curves (concentration of serum ranging from 1 to 30%) have been conducted. In normal hepatocytes incubated for 24 h with serum (concentration of 10%) from rats with CRF, total P450 level, protein expression and mRNA levels of several P450 isoforms (CYP2C6, 2C11, 3A1 and 3A2) were decreased by more than 35% (P<0.001) compared to serum from control animals. The protein expression as well as the mRNA levels of CYP2D were similar in hepatocytes incubated with serum from either control or CRF rats. The N‐demethylation of erythromycin was decreased by more than 35% (P<0.001) in hepatocytes incubated with serum from rats with CRF. The inhibitory effect of serum from rats with CRF tended to peak at 48 h of incubation and was maximum at a concentration of 20%. In conclusion, uremic serum contains mediator(s) that down‐regulate the cytochrome P450 of normal hepatocytes secondary to reduced gene expression.

Studies on small (<350 μm) alginate‐poly‐L‐lysine microcapsules. III. Biocompatibility of smaller versus standard microcapsules

Microencapsulation of islets of Langerhans has been proposed as a means of preventing their immune destruction following transplantation. Microcapsules of diameters <350 microm made with an electrostatic pulse system present many advantages relative to standard microcapsules (700-1500 microm), including smaller total implant volume, better insulin kinetics, better cell oxygenation, and accessibility to new implantation sites. To evaluate their biocompatibility, 200, 1000, 1120, 1340, or 3000 of these smaller microcapsules (<350 microm) or 20 standard microcapsules (1247+/-120 microm) were implanted into rat epididymal fat pads, retrieved after 2 weeks, and evaluated histologically. The average pericapsular reaction increased with the number of small microcapsules implanted (p<0.05; 3000 vs. 200, 3000 vs. 1000, and 1000 vs. 200 microcapsules). At equal volume and alginate content, standard microcapsules caused a more intense fibrosis reaction than smaller microcapsules (p<0.05). In addition, 20 standard microcapsules elicited a stronger pericapsular reaction than 200 and 1000 smaller microcapsules (p<0.05) although the latter represented a 3.4-fold larger total implant surface exposed. We conclude that microcapsules of diameters <350 microm made with an electrostatic pulse system are more biocompatible than standard microcapsules.

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.

Current Understanding of Drug Disposition in Kidney Disease

Patients with chronic kidney disease (CKD) represent 13% of the American population. CKD has been shown to significantly alter drug disposition of nonrenally eliminated drugs. Indeed, modifications in the expression and function of intestinal and hepatic drug metabolism enzymes and uptake and efflux transporters have been reported. Uremic toxins, inflammatory cytokines, and parathyroid hormone have been implicated as causes. These changes can have an important clinical impact on drug disposition and lead to unintended toxicity if they are administered without dose adjustment in patients with impaired kidney function. This review summarizes recent preclinical and clinical studies and presents the current understanding of the effect of CKD on drug absorption, distribution, metabolism, and excretion.

Studies on smaller (approximately 315 microM) microcapsules: IV. Feasibility and safety of intrahepatic implantations of small alginate poly-L-lysine microcapsules.

The most successful transplantation site of nonencapsulated islets of Langerhans is the liver. Because usual alginate poly-L-lysine microcapsules were too large (700–1200 μm diameter) for intravascular implantations and were almost exclusively implanted intraperitoneally, the question of the preferred implantation site of microencapsulated islets has received little attention. The feasibility of implanting smaller (~315 μm) alginate poly-L-lysine microcapsules into the liver and the effect of such implantations on portal pressure and liver histology was evaluated in Wistar rats. A bolus of 10,000 microcapsules of 315 μm diameter was injected intraportally (group 1; n = 22). The portal pressure increased from 6.4 ± 1.8 mmHg to a maximum of 19 mmHg, returned to basal levels within 2 h, and remained normal after 2 months. In group 2 (n = 3), following the injection of 10,000 larger microcapsules (420 μm), the portal pressure increased to > 60 mmHg and two out of the three rats died within 3 h. When 5,000 microcapsules of 420-μm diameter were injected (group 3; n = 5), the portal pressure peaked to 30 ± 8 mmHg and remained elevated after 4 h (12 ± 3 mmHg), but returned to normal (8 ± 1 mmHg) after 2 weeks. Histological studies showed normal hepatic architecture without collagen deposition into portal tracts occupied by microcapsules. Conclusion: intrahepatic implantations of ~315-μm alginate poly-L-lysine microcapsules are feasible and safe. These results justify further investigation of this potential implantation site for microencapsulated islets.

Transplantation of Mesenchymal Stem Cells Promotes Tissue Regeneration in a Glaucoma Model Through Laser-Induced Paracrine Factor Secretion and Progenitor Cell Recruitment†‡§

Among bone marrow cells, hematopoietic and mesenchymal components can contribute to repair damaged organs. Such cells are usually used in acute diseases but few options are available for the treatment of chronic disorders. In this study, we have used a laser‐induced model of open angle glaucoma (OAG) to evaluate the potential of bone marrow cell populations and the mechanisms involved in tissue repair. In addition, we investigated laser‐induced tissue remodeling as a method of targeting effector cells into damaged tissues. We demonstrate that among bone marrow cells, mesenchymal stem cells (MSC) induce trabecular meshwork regeneration. MSC injection into the ocular anterior chamber leads to far more efficient decrease in intraocular pressure (IOP) (p < .001) and healing than hematopoietic cells. This robust effect was attributable to paracrine factors from stressed MSC, as injection of conditioned medium from MSC exposed to low but not to normal oxygen levels resulted in an immediate decrease in IOP. Moreover, MSC and their secreted factors induced reactivation of a progenitor cell pool found in the ciliary body and increased cellular proliferation. Proliferating cells were observed within the chamber angle for at least 1 month. Laser‐induced remodeling was able to target MSC to damaged areas with ensuing specific increases in ocular progenitor cells. Thus, our results identify MSC and their secretum as crucial mediators of tissue repair in OAG through reactivation of local neural progenitors. In addition, laser treatment could represent an appealing strategy to promote MSC‐mediated progenitor cell recruitment and tissue repair in chronic diseases. STEM Cells 2013;31:1136–1148

Quantitative method for the evaluation of biomicrocapsule resistance to mechanical stress

A quantitative method has been developed for the evaluation of biomicrocapsule resistance to mechanical stress. Fluorescein isothiocyanate-labelled dextran (M.W. 2 x 10(6)) was microencapsulated in alginate-poly-L-lysine membranes. Microcapsules of 302.0 +/- 3.2 microns were mixed with 3 mm glass beads and continuously agitated for 0 to 144 h. The percentage of broken capsules was calculated by measuring the fluorescence in the supernatant and in the residual intact capsules after the latter were dissolved. The fluorescence method was validated by comparison with a manual method (handpicking under a stereomicroscope). The highest percentage of broken capsules was obtained with a ratio of 225 +/- 25 glass beads per 1000 microcapsules. The percentage of broken capsules increased linearly from 7.3% at 12 h to 48.3% at 72 h of continuous agitation. The applicability of the method was evaluated by studying microcapsules of potentially different levels of resistance. The results confirmed that capsule resistance is improved by increasing poly-L-lysine concentrations and incubation times. Microcapsules made with guluronic acid-rich alginate were stronger than those made with mannuronic acid-rich alginate. In conclusion, this is a simple, precise and sensitive method for the quantification of biomicrocapsule resistance to mechanical stress.

Interleukin-1β, interleukin-6, tumour necrosis factor-α and interferon-γ released by a viral infection and an aseptic inflammation reduce CYP1A1, 1A2 and 3A6 expression in rabbit hepatocytes

Abstract Inflammation reduces activity and expression of hepatic cytochrome P450 (P450) and therefore diminishes drug biotransformation. This study aimed to identify the serum mediators triggered by a viral infection and an aseptic inflammation that downregulate P450 isoforms. Incubation of hepatocytes with serum from rabbits with a turpentine-induced inflammation or humans with a viral infection decreased the amount of cytochrome 1A1 (CYP1A1), 1A2 and 3A6 mRNA and apoproteins. By serum fractionation and immuno-neutralization, we showed that in the aseptic inflammation, interleukin-6 and, to a lesser degree, interleukin-1β are involved in the downregulation of all three isoforms. In serum from humans with a viral infection, interleukin-1β, interleukin-6, interferon-γ and tumour necrosis factor-α contribute to the downregulation of P450 isoforms. CYP1A1 and 1A2 are regulated by serum mediators at the transcriptional level, while the expression of CYP3A6 appears to be under the control of pre- and posttranscriptional mechanisms.

Chemokine expression patterns in the systemic and genital tract compartments are associated with HIV-1 infection in women from Benin.

IntroductionUnderstanding the genital mucosal immunity and the factors involved in linking innate to adaptive immunity is crucial for the design of efficient preventive strategies against human immunodeficiency virus (HIV)-1.MethodsLevels of both genital mucosal and blood chemokines were compared between 58 HIV-1-uninfected and 50 HIV-1-infected female commercial sex workers (CSWs) as well as 53 HIV-1-uninfected non-CSW control women at low risk for exposure, recruited in Cotonou, Benin.ResultsHIV-1-infected CSWs had significantly higher blood and genital levels of monocyte chemotactic protein (MCP-3/CCL7) and monokine induced by gamma interferon (MIG/CXCL9) compared with those in both the HIV-1-uninfected CSW and non-CSW groups. In the HIV-1-infected group, levels of MCP-3 and MIG were significantly higher in the genital mucosa than in the blood. However, the blood levels of macrophage inflammatory protein (MIP-1a/CCL3) and MIP-1b/CCL4 were higher in HIV-1-uninfected CSWs compared with those in the other groups.ConclusionIncreased production of chemokines in the genital tract may favour the recruitment of HIV-1 target cells causing a mucosal environment that promotes viral replication and dissemination, whereas higher expression of β-chemokines at the systemic level is associated with protection from HIV-1 infection.