Christopher L. Marsh

First-pass metabolism of midazolam by the human intestine

The in vivo intestinal metabolism of the CYP3A probe midazolam to its principal metabolite, 1′‐hydroxymidazolam, was investigated during surgery in 10 liver transplant recipients. After removal of the diseased liver, five subjects received 2 mg midazolam intraduodenally, and the other five received 1 mg midazolam intravenously. Simultaneous arterial and hepatic portal venous blood samples were collected during the anhepatic phase; collection of arterial samples continued after reperfusion of the donor liver. Midazolam, 1′‐hydroxymidazolam, and 1′‐hydroxymidazolam glucuronide were measured in plasma. A mass balance approach that considered the net change in midazolam (intravenously) or midazolam and 1′‐hydroxymidazolam (intraduodenally) concentrations across the splanchnic vascular bed during the anhepatic phase was used to quantitate the intestinal extraction of midazolam after each route of administration. For the intraduodenal group, the mean fraction of the absorbed midazolam dose that was metabolized on transit through the intestinal mucosa was 0.43 ± 0.18. For the intravenous group, the mean fraction of midazolam extracted from arterial blood and metabolized during each passage through the splanchnic vascular bed was 0.08 ± 0.11. Although there was significant intersubject variability, the mean intravenous and intraduodenal extraction fractions were statistically different (p = 0.009). Collectively, these results show that the small intestine contributes significantly to the first‐pass oxidative metabolism of midazolam catalyzed by mucosal CYP3A4 and suggest that significant first‐pass metabolism may be a general phenomenon for all high‐turnover CYP3A4 substrates.

Quantitation of BK Virus Load in Serum for the Diagnosis of BK Virus–Associated Nephropathy in Renal Transplant Recipients

BK virus-associated nephropathy is an increasingly recognized cause of graft dysfunction among kidney transplant recipients, and definitive diagnosis requires renal biopsy. By using a newly developed, quantitative, real-time polymerase chain reaction (PCR) assay for BK virus DNA, a retrospective analysis was done of sequential serum samples (n=28) from 4 transplant recipients with histopathologically documented BK virus nephropathy and from samples (n=76) from 16 transplant recipient control patients. BK virus DNA was detected in serum samples from all 4 case patients versus 0 of 16 control patients (P< .0001, Fishers exact test) at a median of 32 weeks (range, 17-61 weeks) before the diagnosis of BK virus nephropathy. BK virus load decreased in 3 of 3 patients after the reduction of immunosuppression and/or nephrectomy. It is concluded that quantitative PCR for BK virus DNA in serum is useful both for identifying transplant recipients at risk for BK virus nephropathy and for monitoring the response to therapy.

EFFECT OF CYP3A5 POLYMORPHISM ON TACROLIMUS METABOLIC CLEARANCE IN VITRO

Previous investigations of solid organ transplant patients treated with tacrolimus showed that individuals carrying a CYP3A5*1 allele have lower dose-adjusted trough blood concentrations compared with homozygous CYP3A5*3 individuals. The objective of this investigation was to quantify the contribution of CYP3A5 to the hepatic and renal metabolic clearance of tacrolimus. Four primary tacrolimus metabolites, 13-O-desmethyl tacrolimus (13-DMT) (major), 15-O-desmethyl tacrolimus, 31-O-desmethyl tacrolimus (31-DMT), and 12-hydroxy tacrolimus (12-HT), were generated by human liver microsomes and heterologously expressed CYP3A4 and CYP3A5. The unbound tacrolimus concentration was low (4–15%) under all incubation conditions. For CYP3A4 and CYP3A5, Vmax was 8.0 and 17.0 nmol/min/nmol enzyme and Km,u was 0.21 and 0.21 μM, respectively. The intrinsic clearance of CYP3A5 was twice that of CYP3A4. The formation rates of 13-DMT, 31-DMT, and 12-HT were ≥1.7-fold higher, on average, in human liver microsomes with a CYP3A5*1/*3 genotype compared with those with a homozygous CYP3A5*3/*3 genotype. Tacrolimus disappearance clearances were 15.9 ± 9.8 ml/min/mg protein and 6.1 ± 3.6 ml/min/mg protein, respectively, for the two genotypes. In vitro to in vivo scaling using both liver microsomes and recombinant enzymes yielded higher predicted in vivo tacrolimus clearances for patients with a CYP3A5*1/*3 genotype compared with those with a CYP3A5*3/*3 genotype. In addition, formation of 13-DMT was 13.5-fold higher in human kidney microsomes with a CYP3A5*1/*3 genotype compared with those with a CYP3A5*3/*3 genotype. These data suggest that CYP3A5 contributes significantly to the metabolic clearance of tacrolimus in the liver and kidney.

Delayed Graft Function and Cast Nephropathy Associated with Tacrolimus Plus Rapamycin Use

Delayed graft function (DGF) occurs in 15 to 25% (range, 10 to 62%) of cadaveric kidney transplant recipients and up to 9% of living donor recipients. In addition to donor, recipient, and procedural factors, the choice of immunosuppression may influence the development of DGF. The impact of immunosuppression on DGF was studied. The frequency of DGF was evaluated in first cadaveric or living donor kidney allograft recipients (n = 144) transplanted at the University of Washington from November 1999 through September 1, 2001. Donor, recipient, and procedural factors, as well as biopsy results, were compared between patients who developed DGF and those who did not. DGF was more common in patients treated with rapamycin than without (25% versus 8.9%, P = 0.02) and positively correlated with rapamycin dose (P = 0.008). In those developing DGF, the duration of posttransplant dialysis increased with donor age (P = 0.003) but decreased with mycophenolate mofetil use (P = 0.01). All biopsies during episodes of DGF demonstrated changes of acute tubular injury. Of the patients with tubular injury, 12 treated with rapamycin and tacrolimus developed intratubular cast formation indistinguishable from myeloma cast nephropathy. Histologic, immunohistochemical, and ultrastructural studies indicated that these casts were composed at least in part of degenerating renal tubular epithelial cells. These findings suggest that rapamycin therapy exerts increased toxicity on tubular epithelial cells and/or retards healing, leading to an increased incidence of DGF. Additionally, rapamycin treatment combined with a calcineurin inhibitor may lead to extensive tubular cell injury and death and a unique form of cast nephropathy.

Effects of Rifampin on Tacrolimus Pharmacokinetics in Healthy Volunteers

Tacrolimus is a marketed immunosuppressant used in liver and kidney transplantation. It is subject to extensive metabolism by CYP3A4 and is a substrate for P‐glycoprotein‐mediated transport. A pharmacokinetic interaction with rifampin, an antituberculosis agent and potent inducer of CYP3A4 and P‐glycoprotein, and tacrolimus was evaluated in six healthy male volunteers. Tacrolimus was administered at doses of 0.1 mg/kg orally and 0.025 mg/kg/4 hours intravenously. The pharmacokinetics of tacrolimus were obtained from serial blood samples collected over 96 hours, after single oral and intravenous administration prior to and during an 18‐day concomitant rifampin dosing phase. Coadministration of rifampin significantly increased tacrolimus clearance (36.0 ± 8.1 ml/hr/kg vs. 52.8 ± 9.6 ml/hr/kg; p = 0.03) and decreased tacrolimus bioavailability (14.4% ± 5.7% vs. 7.0% ± 2.7%; p = 0.03). Rifampin appears to induce both intestinal and hepatic metabolism of tacrolimus, most likely through induction of CYP3A and P‐glycoprotein in the liver and small bowel.

The pharmacokinetics of a microemulsion formulation of cyclosporine in primary renal allograft recipients

This study was a randomized, double-blind, 12-week comparison of the pharmacokinetics, safety, and tolerability of two cyclosporine (CsA) formulations, cyclosporine emulsion capsules and oral solution for microemulsion and cyclosporine, in the postoperative management of renal transplant patients. Of the 101 patients, aged 18 to 65, who entered the study, 89 were evaluable for pharmacokinetics. Initial dosage was 10 mg/kg per day, administered twice daily in two equal doses. Dosages were adjusted to achieve target CsA concentrations. The pharmacokinetic (PK) parameters (dose-normalized) of greatest interest were maximum blood concentration (C(max)/dose), time to reach maximum concentration (t(max), area under the blood concentration-vs.-time curve (AUC/dose), and trough blood concentrations (Co h/dose). The relative CsA bioavailabilty was found to be significantly enhanced with cyclosporine emulsion compared with cyclosporine with a 16% to 31% increase in AUC and a 32% to 42% increase in C(max). Intrapatient variability of PK parameters was significantly lower with cyclosporine emulsion than with cyclosporine for AUC, C(oh), t(max), and C(max) in many instances. This indicates a more consistent, rapid, and more complete total absorption of CsA. Despite higher CsA C(max) levels and AUCs with cyclosporine emulsion, safety and tolerability (detailed in a parallel report) were comparable to those of cyclosporine. The PK advantages of cyclosporine emulsion over cyclosporine are either independent of food conditions or possibly reflective of more consistent absorption of CsA with cyclosporine emulsion. The findings suggest that de novo use of cyclosporine emulsion may simplify and improve management of organ transplant recipients and that the PK advantages of cyclosporine emulsion may translate into clinical benefits.

Survival Benefit with Kidney Transplants from HLA-Incompatible Live Donors

BACKGROUND A report from a high-volume single center indicated a survival benefit of receiving a kidney transplant from an HLA-incompatible live donor as compared with remaining on the waiting list, whether or not a kidney from a deceased donor was received. The generalizability of that finding is unclear. METHODS In a 22-center study, we estimated the survival benefit for 1025 recipients of kidney transplants from HLA-incompatible live donors who were matched with controls who remained on the waiting list or received a transplant from a deceased donor (waiting-list-or-transplant control group) and controls who remained on the waiting list but did not receive a transplant (waiting-list-only control group). We analyzed the data with and without patients from the highest-volume center in the study. RESULTS Recipients of kidney transplants from incompatible live donors had a higher survival rate than either control group at 1 year (95.0%, vs. 94.0% for the waiting-list-or-transplant control group and 89.6% for the waiting-list-only control group), 3 years (91.7% vs. 83.6% and 72.7%, respectively), 5 years (86.0% vs. 74.4% and 59.2%), and 8 years (76.5% vs. 62.9% and 43.9%) (P<0.001 for all comparisons with the two control groups). The survival benefit was significant at 8 years across all levels of donor-specific antibody: 89.2% for recipients of kidney transplants from incompatible live donors who had a positive Luminex assay for anti-HLA antibody but a negative flow-cytometric cross-match versus 65.0% for the waiting-list-or-transplant control group and 47.1% for the waiting-list-only control group; 76.3% for recipients with a positive flow-cytometric cross-match but a negative cytotoxic cross-match versus 63.3% and 43.0% in the two control groups, respectively; and 71.0% for recipients with a positive cytotoxic cross-match versus 61.5% and 43.7%, respectively. The findings did not change when patients from the highest-volume center were excluded. CONCLUSIONS This multicenter study validated single-center evidence that patients who received kidney transplants from HLA-incompatible live donors had a substantial survival benefit as compared with patients who did not undergo transplantation and those who waited for transplants from deceased donors. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases.).

Association between abcb1 (multidrug resistance transporter) genotype and post-liver transplantation renal dysfunction in patients receiving calcineurin inhibitors

OBJECTIVE Renal dysfunction is a common and costly adverse outcome of long-term treatment with calcineurin inhibitors (CNIs). We conducted a retrospective, case-control study to test whether the risk of renal dysfunction in liver transplantation patients receiving CNIs is associated with the 2677G>T transversion in exon-21 of the gene (ABCB1) encoding P-glycoprotein. A total of 120 non-Hispanic white patients were evaluated. RESULTS The overall incidence of renal dysfunction by year 3 post-transplantation was 40%. The frequency of renal dysfunction was reduced among patients with an ABCB1 2677TT genotype, as compared to those with a 2677GG genotype. Subjects with a heterozygote genotype behaved phenotypically like the 2677GG group. Comparing those subjects with a 2677TT genotype to the combined group of subjects with a 2677GG, TG, AT, or AG genotype resulted in an odds ratio of 0.26 (0.09-0.77). When subjects were stratified by gender, the frequency of renal dysfunction was reduced among men with an ABCB1 2677TT genotype, relative to men with different genotypes. A similar odds ratio was obtained for women, but it did not achieve significance. When 18 subjects with an elevated SCr concentration just prior to surgery were excluded from the year 3 analysis, the association between the 2677TT genotype and chronic renal dysfunction in the remaining cohort was strengthened comparing genotype groups. CONCLUSIONS Based on these results, we conclude that homozygosity for the ABCB1 2677T (S893) allele is associated with reduced risk of chronic renal dysfunction among liver transplantation patients receiving an immunosuppressive regimen containing CNIs.

Quantifying the Risk of Incompatible Kidney Transplantation: A Multicenter Study

Incompatible live donor kidney transplantation (ILDKT) offers a survival advantage over dialysis to patients with anti‐HLA donor‐specific antibody (DSA). Program‐specific reports (PSRs) fail to account for ILDKT, placing this practice at regulatory risk. We collected DSA data, categorized as positive Luminex, negative flow crossmatch (PLNF) (n = 185), positive flow, negative cytotoxic crossmatch (PFNC) (n = 536) or positive cytotoxic crossmatch (PCC) (n = 304), from 22 centers. We tested associations between DSA, graft loss and mortality after adjusting for PSR model factors, using 9669 compatible patients as a comparison. PLNF patients had similar graft loss; however, PFNC (adjusted hazard ratio [aHR] = 1.64, 95% confidence interval [CI]: 1.15–2.23, p = 0.007) and PCC (aHR = 5.01, 95% CI: 3.71–6.77, p < 0.001) were associated with increased graft loss in the first year. PLNF patients had similar mortality; however, PFNC (aHR = 2.04; 95% CI: 1.28–3.26; p = 0.003) and PCC (aHR = 4.59; 95% CI: 2.98–7.07; p < 0.001) were associated with increased mortality. We simulated Centers for Medicare & Medicaid Services flagging to examine ILDKTs effect on the risk of being flagged. Compared to equal‐quality centers performing no ILDKT, centers performing 5%, 10% or 20% PFNC had a 1.19‐, 1.33‐ and 1.73‐fold higher odds of being flagged. Centers performing 5%, 10% or 20% PCC had a 2.22‐, 4.09‐ and 10.72‐fold higher odds. Failure to account for ILDKTs increased risk places centers providing this life‐saving treatment in jeopardy of regulatory intervention.

Cyclosporine-associated thrombotic microangiopathy/hemolytic uremic syndrome following kidney and kidney-pancreas transplantation

Cyclosporine-associated thrombotic microangiopathy (CsA-TMA) is characterized by anemia, acute renal failure, and renal TMA. We report a case-control study of 13 patients (seven kidney-alone transplant recipients and six kidney-pancreas transplant recipients) who developed TMA (12 CsA, 1 FK506). Once CsA-TMA was identified, CsA or FK506 was discontinued and isradipine, aspirin, and pentoxifylline were started. Cyclosporine was reinstituted in all patients once serum creatinine reached the previous baseline value. Patients developing further decreases in renal function on rechallenge with CsA were converted to FK506 (n = 3). Rechallenge with CsA was successful in nine of the 13 patients (69%), with three (23%) converted to FK506 for a total salvage rate of 92%. The creatinine clearance at 6 months, 1 year, and 2 years following transplantation was 73.2 +/- 25.7 mL/min, 54.7 +/- 18.8 mL/min, and 57.0 +/- 32.0 mL/min, respectively, for patients successfully rechallenged with CsA compared with 67 +/- 17 mg/min, 71.8 +/- 21.2 mL/min, and 69 +/- 19 mg/min, respectively, for controls (P = NS). The average creatine clearance for patients converted to FK506 was 44.7 +/- 31.2 mL/min at 6 months following transplantation (n = 3) and 27.0 +/- 11.3 mL/min at 1 year. In this case-controlled retrospective series of renal transplant patients with documented CsA-TMA, the triple-drug combination of isradipine, aspirin, and pentoxifylline allowed for the successful reinstitution of CsA or conversion to FK506 in the setting of TMA, and resulted in increased transplant survival compared with previous reports.