Volker Schmitz

Functional interactions between P-glycoprotein and CYP3A in drug metabolism

The interaction between drug-metabolising enzymes and active transporters is an emerging concept in pharmacokinetics. In the gut mucosa, P-glycoprotein and cytochrome P450 (CYP)3A functionally interact in three ways: i) drugs are repeatedly taken up and pumped out of the enterocytes by P-glycoprotein, thus increasing the probability of drugs being metabolised; ii) P-glycoprotein keeps intracellular drug concentrations within the linear range of the metabolising capacity of CYP3A; and iii) P-glycoprotein transports drug metabolites formed in the mucosa back into the gut lumen. In comparison with the gut mucosa, in hepatocytes the spatial sequence of CYP3A and P-glycoprotein is reversed, resulting in different effects when the activity of one or both are changed. CYP3A and P-glycoprotein are both regulated by nuclear receptors such as the pregnane X receptor (PXR). There is significant genetic variability of CYP3A, P-glycoprotein and PXR and their expression and activity is dependent on coadministered drugs, herbs, food, age, hormonal status and disease. Future pharmacogenomic and pharmacokinetic studies will have to take all three components into account to allow for valid conclusions.

The influence of storage temperature during machine perfusion on preservation quality of marginal donor livers

BACKGROUND Although non-heart-beating donors have the potential to increase the number of available organs, the livers are used very seldom because of the risk of primary non-function. There is evidence that machine perfusion is able to improve the preservation of marginal organs, and therefore we evaluated in our study the influence of the perfusate temperature during oxygenated machine perfusion on the graft quality. METHODS Livers from male Wistar rats were harvested after 60-min warm ischemia induced by cardiac arrest. The portal vein was cannulated and the liver flushed with Lifor (Lifeblood Medical, Inc.) organ preservation solution for oxygenated machine perfusion (MP) at 4, 12 or 21 degrees C. Other livers were flushed with HTK and stored at 4 degrees C by conventional cold storage (4 degrees C-CS). Furthermore two groups with either warm ischemic damage only or without any ischemic damage serve as control groups. After 6h of either machine perfusion or cold storage all livers were normothermic reperfused with Krebs-Henseleit buffer, and functional as well as structural data were analyzed. RESULTS Contrary to livers stored by static cold storage, machine perfused livers showed independently of the perfusate temperature a significantly decreased enzyme release of hepatic transaminases (ALT) during isolated reperfusion. Increasing the machine perfusion temperature to 21 degrees C resulted in a marked reduction of portal venous resistance and an increased bile production. CONCLUSIONS Oxygenated machine perfusion improves viability of livers after prolonged warm ischemic damage. Elevated perfusion temperature of 21 degrees C reconstitutes the hepatic functional capacity better than perfusion at 4 or 12 degrees C.

Active drug transport of immunosuppressants: new insights for pharmacokinetics and pharmacodynamics.

Immunosuppressants have a narrow therapeutic index, and pharmacokinetic variability negatively affects long-term outcome of transplantation. Recently, it has become clear that active transport is a major determinant of the inter-and intraindividual variability of the pharmacokinetics and pharmacodynamics of immunosuppressants. Active transport plays a key role in (1) the poor correlation between oral doses and systemic exposure of cyclosporine, tacrolimus, sirolimus, and everolimus, (2) tissue distribution including distribution into lymphocytes, (3) hepatic and intestinal metabolism, (4) the pharmacokinetic variability of immunosuppressants after oral dosing, (5) drug-drug interactions, (6) disease-drug interactions, and (7) age, gender, and ethnicity-based differences in pharmacokinetics of immunosuppressants. Those new insights may significantly improve patient management and long-term outcome not only by reducing pharmacokinetic variability and avoidance of drug-drug interactions but also by identification of sensitive patient populations. They will also significantly impact preclinical and clinical development strategies of new immunosuppressants.

Urine metabolites reflect time-dependent effects of cyclosporine and sirolimus on rat kidney function.

The clinical use of the immunosuppressant calcineurin inhibitor cyclosporine is limited by its nephrotoxicity. This is enhanced when combined with the immunosuppressive mTOR inhibitor sirolimus. Nephrotoxicity of both drugs is not yet fully understood. The goal was to gain more detailed mechanistic insights into the time-dependent effects of cyclosporine and sirolimus on the rat kidney by using a comprehensive approach including metabolic profiling in urine ((1)H NMR spectroscopy), kidney histology, kidney function parameters in plasma, measurement of glomerular filtration rates, the oxidative stress marker 15-F(2t)-isoprostane in urine, and immunosuppressant concentrations in blood and kidney. Male Wistar rats were treated with vehicle (controls), cyclosporine (10/25 mg/kg/day), and/or sirolimus (1 mg/kg/day) by oral gavage once daily for 6 and 28 days. Twenty-eight day treatment led to a decrease of glomerular filtration rates (cyclosporine, -59%; sirolimus, -25%). These were further decreased when both drugs were combined (-86%). Histology revealed tubular damage after treatment with cyclosporine, which was enhanced when sirolimus was added. No other part of the kidney was affected. (1)H NMR spectroscopy analysis of urine (day 6) revealed time-dependent changes of 2-oxoglutarate, citrate, and succinate concentrations. In combination with increased urine isoprostane concentrations, these changes indicated oxidative stress. After 28 days of cyclosporine treatment, urine metabonomics shifted to patterns typical for proximal tubular damage with reduction of Krebs cycle intermediates and trimethylamine-N-oxide concentrations, whereas acetate, lactate, trimethylamine, and glucose concentrations increased. Again, sirolimus enhanced these negative effects. Our results indicate that cyclosporine and/or sirolimus induce damage of the renal tubular system. This is reflected by urine metabolite patterns, which seem to be more sensitive than currently used clinical kidney function markers such as creatinine concentrations in serum. Metabolic profiling in urine may provide the basis for the development of toxicodynamic monitoring strategies for immunosuppressant nephrotoxicity.

Toxicodynamic therapeutic drug monitoring of immunosuppressants: promises, reality, and challenges.

Although current immunosuppressive protocols have dramatically decreased acute rejection episodes, there has been less progress in terms of long-term graft survival after kidney transplantation over the last 2 decades. The key to reducing the damage to a transplanted organ as caused by chronic processes is early detection. Modern screening technologies in the fields of genetics, genomics, protein profiling (proteomics), and biochemical profiling (metabolomics) have opened new opportunities for the development of sensitive and specific diagnostic tools. Metabolic profiling appears to be a promising strategy because changes in the cell biochemistry are ultimately responsible for the histologic and pathophysiologic changes of the transplanted kidney and are most likely already detectable before histologic and pathophysiologic changes occur. Using truly no-targeted screening technologies as clinical diagnostic tools is not yet feasible, mostly because of the complexity of the data generated and the lack of algorithms to convert this information into clinically applicable information. A realistic and powerful targeted approach is the development of combinatorial biomarkers. These are biomarker patterns that typically consist of five or more individual parameters. Combined biomarker patterns confer significantly more information than a single measurement and, thus, can be expected to have better specificity and sensitivity. A series of studies in rats and healthy individuals evaluating the effects of immunosuppressants on urine metabolite patterns showed that immunosuppressant-induced changes of metabolite patterns in urine were associated with a combination of changes in glomerular filtration, changes in secretion/absorption by tubulus cells, and changes in kidney cell metabolism. These studies suggested that a combination of biomarkers that can be used for toxicodynamic therapeutic drug monitoring of immunosuppressants should include urine metabolites that constitute valid surrogate markers of these kidney functions.

Chronic renal dysfunction following liver transplantation

Abstract:  With most of the immunosuppressive protocols consisting of calcineurin inhibitors (CI), nephrotoxicity has become a major long‐term complication often compromising outcome. In a single‐center retrospective study, we reviewed 1173 liver transplantations to identify variables indicative for the occurrence of chronic renal dysfunction (CRD) (defined as ≥1 episode of serum creatinine increase ≥1.8 mg/dL ≥2 wk). Chronic renal dysfunction was found in 137 (11.7%) of all transplants [82 (7%) early (after 3–12 months), 55 (4.7%) late‐onset (>12 months)]. Compared to 5‐/10‐yr survival rates in non‐CRD transplants (84/74%) survival was significantly decreased in early (66/46%), but unchanged in late‐onset CRD (98/86%). Rates of alcoholic cirrhosis and prior renal dysfunction were significantly increased in patients with CRD. In a multivariate logistic regression analysis, only cyclosporine A (CyA) as immunosuppression remained an independent risk factor. No correlations to age, gender, rejection/retransplantation or diabetes were found. Surprisingly, renal function (creatinine) showed no difference between patients on CI monotherapy (FK/CyA) compared to those who had mycophenolate mofetil (MMF) added. In liver transplantation, early onset CRD significantly compromises survival. CyA‐based immunosuppression appears to have a stronger impact than FK. The fact that patients with long‐term severe chronic renal dysfunction failed to improve under MMF rescue therapy emphasizes the importance of new diagnostic strategies to earlier identify at‐risk patients.

In vivo Visualization of Early Microcirculatory Changes following Ischemia/Reperfusion Injury in Human Kidney Transplantation

To determine whether microcirculatory changes following ischemia/reperfusion (I/R) may serve as predictors for subsequent graft dysfunction, we used noninvasive orthogonal polarization spectral (OPS) imaging to directly visualize and quantify cortical kidney microcirculation. In a total of 13 combined kidney/pancreas recipients, following reperfusion (5/30 min) microcirculatory parameters such as capillary diameter, functional capillary density (FCD) and red-blood-cell velocity (VRBC) of the renal graft were analyzed. From these parameters, a heterogeneity index (HI) and volumetric capillary blood flow (vCBF) were calculated. In addition, the extent of graft injury was determined by daily analysis of serum creatinine, blood urea nitrogen, C-reactive protein and systemic leukocyte count for 7 days post-transplant. At early reperfusion, a heterogeneous perfusion pattern with oscillating flow and scattered microvascular thrombosis of peritubular capillaries, resembling a ‘no reflow’, was observed. FCD was constant throughout the entire reperfusion period, whereas HI, capillary diameters, VRBC and vCBF increased. The latter showed a significant positive correlation with creatinine changes between days 1 and 3. So far our finding of a positive correlation of early microvascular changes (vCBF) and clinical parameters (creatinine) indicate a possible therapeutic implication of OPS imaging to predict early I/R-induced renal graft dysfunction.

Surgical Complications and Long-Term Outcome of Different Biliary Reconstructions in Liver Transplantation for Primary Sclerosing Cholangitis—Choledochoduodenostomy versus Choledochojejunostomy

Choledochojejunostomy (CJS) is commonly used for biliary reconstruction in liver transplantation for primary sclerosing cholangitis (PSC). We alternatively performed choledochoduodenostomy (CDS) and side‐to‐side choledochodocholedochstomy in a large cohort of patients. Fifty‐one patients with PSC, transplanted between 1988 and 2000, were analyzed retrospectively. Biliary reconstruction was CDS in 25 (49%), CJS in 20 (39%) and CC in 6 transplantations (12%). Biliary leaks occurred in the early follow‐up (≤41 days) only in CDS patients (20%). However, in the late follow‐up (>4 months), stricturing of anastomosis was found once in CDS (4%) and CJS (5%). Later (>9 months), intrahepatic bile duct strictures were diagnosed in four CDS (16%), one CJS (5%) and one CC (17%) patient(s). In 48% of CDS (12/25), 60% of CJS (12/20) and 17% of CC (1/6) at least one incidence of cholangitis was observed. Overall, biliary complication rates were significantly higher in CDS (40%) than CJS (10%) and CC (17%); of those none in CC and 12% in CDS were anastomosis‐related. Graft/patient survival showed no significant differences among groups. Based on our results we consider CJS the standard method for biliary reconstruction in PSC; however, in selected cases where CJS is difficult to accomplish because of previous surgery or for retransplantation, CDS may present an alternative technique.

Association of immunosuppressant-induced protein changes in the rat kidney with changes in urine metabolite patterns: a proteo-metabonomic study.

The basic mechanisms underlying calcineurin inhibitor (CI) nephrotoxicity and its enhancement by sirolimus are still largely unknown. We investigated the effects of CIs alone and in combination with sirolimus on the renal proteome and correlated these effects with urine metabolite pattern changes. Thirty-six male Wistar rats were assigned to six treatment groups (n = 4/group for proteome analysis and n = 6/group for urine (1)H NMR metabolite pattern analysis): vehicle controls, sirolimus 1 mg/kg/day, cyclosporine 10 mg/kg/day, cyclosporine 10 mg/kg/day + sirolimus 1 mg/kg/day, tacrolimus 1 mg/kg/day, tacrolimus 1 mg/kg/day + sirolimus 1 mg/kg/day. After 28 days, 24 h-urine was collected for (1)H NMR-based metabolic analysis and kidneys were harvested for 2D-gel electrophoresis and histology. Cyclosporine affected the following groups of proteins: calcium homeostasis (regucalcin, calbindin), cytoskeleton (vimentin, caldesmon), response to hypoxia and mitochondrial function (prolyl 4-hydroxylase, proteasome, NADH dehydrogenase), and cell metabolism (kidney aminoacylase, pyruvate dehydrogenase, fructose-1,6-bis phosphate). Several of the changes in protein expression, confirmed by Western blot, were associated with and explained changes in metabolite concentrations in urine. Representative examples are an increase in kidney aminoacylase expression (decrease of hippurate concentrations in urine), up regulation of pyruvate dehydrogenase and fructose-1,6-bisphosphatase, (increased glucose metabolism), and down regulation of arginine/glycine-amidino transferase (most likely due to an increase in creatinine concentrations). Protein changes explained and qualified immunosuppressant-induced metabolite pattern changes in urine.

Metabolic Profiles in Urine Reflect Nephrotoxicity of Sirolimus and Cyclosporine following Rat Kidney Transplantation

Background: Cyclosporine and/or sirolimus impair recovery of renal transplants. This study examines the changes in urine metabolite profiles as surrogate markers of renal cell metabolism and function after cyclosporine and/or sirolimus treatment employing a rat kidney transplantation model. Methods: Using inbred Lewis rats, kidneys were transplanted into bilaterally nephrectomized recipients followed by treatment with either CsA (cyclosporine) 10, Rapa (sirolimus) 1, CsA10/Rapa1 or CsA25/Rapa1 mg/kg/day for 7 days. On day 7, urine was analyzed by 1H-NMR spectroscopy. Blood and kidney tissue drug concentrations, tissue high-energy compounds (including ATP, ADP) and oxidative stress markers (15-F2t-isoprostanes) in urine were measured by HPLC mass spectrometry. Results: Changes in urine metabolites followed the order Rapa1 < CsA10 < CsA10/Rapa1 < CsA25/Rapa1. Compared with controls, CsA25/Rapa1 showed the greatest changes (creatinine –36%, succinate –57%, citrate –89%, α-ketoglutarate –75%, creatine +498%, trimethylamine +210% and taurine +370%). 15-F2t-isoprostane concentrations in urine increased in the combined immunosuppressant-treated animals ([CsA25/Rapa1]: 795 ± 222, [CsA10/Rapa1]: 475 ± 233 pg/mg/creatinine) as compared with controls (165 ± 78 pg/mg creatinine). Rapa concentration in blood and tissues increased in the combined treatment (blood: 31 ± 8 ng/ml, tissue: 1.3 ± 0.4 ng/mg) as compared with monotherapy (blood: 14 ± 8 ng/ml, tissue: 0.35 ± 0.15 ng/mg). Drug blood concentrations correlated with isoprostane urine concentrations, which correlated negatively with citrate, α-ketoglutarate and creatinine concentrations in urine. Only CsA25/Rapa1 significantly reduced high-energy metabolite concentrations in transplant kidney tissue (ATP –55%, ADP –24%). Conclusion: Immunosuppressant drugs induce changes in urine metabolite patterns, suggesting that immunosuppressant-induced oxidative stress is an early event in the development of nephrotoxicity. Urine 15-F2t-isoprostane concentrations and metabolite profiles may be sensitive markers of immunosuppressant-induced nephrotoxicity.