Vijay Warty

Clinical Pharmacokinetics of Tacrolimus

SummaryTacrolimus, a novel macrocyclic lactone with potent immunosuppressive properties, is currently available as an intravenous formulation and as a capsule for oral use, although other formulations are under investigation.Tacrolimus concentrations in biological fluids have been measured using a number of methods, which are reviewed and compared in the present article. The development of a simple, specific and sensitive assay method for measuring concentrations of tacrolimus is limited by the low absorptivity of the drug, low plasma and blood concentrations, and the presence of metabolites and other drugs which may interfere with the determination of tacrolimus concentrations. Currently, most of the pharmacokinetic data available for tacrolimus are based on an enzyme-linked immunosorbent assay method, which does not distinguish tacrolimus from its metabolites.The rate of absorption of tacrolimus is variable with peak blood or plasma concentrations being reached in 0.5 to 6 hours; approximately 25% of the oral dose is bioavailable. Tacrolimus is extensively bound to red blood cells, with a mean blood to plasma ratio of about 15; albumin and α1-acid glycoprotein appear to primarily bind tacrolimus in plasma. Tacrolimus is completely metabolised prior to elimination. The mean disposition half-life is 12 hours and the total body clearance based on blood concentration is approximately 0.06 L/h/kg. The elimination of tacrolimus is decreased in the presence of liver impairment and in the presence of several drugs.Various factors that contribute to the large inter- and interindividual variability in the pharmacokinetics of tacrolimus are reviewed here. Because of this variability, the narrow therapeutic index of tacrolimus, and the potential for several drug interactions, monitoring of tacrolimus blood concentrations is useful for optimisation of therapy and dosage regimen design.

Baboon-to-human liver transplantation

Our ability to control both the cellular and humoral components of xenograft rejection in laboratory experiments, together with an organ shortage that has placed limits on clinical transplantation services, prompted us to undertake a liver transplantation from a baboon to a 35-year-old man with B virus-associated chronic active hepatitis and human immunodeficiency virus infection. Liver replacement was performed according to conventional surgical techniques. Immunosuppression was with the FK 506-prednisone-prostaglandin regimen used routinely for hepatic allotransplantation, to which a daily non-myelotoxic dose of cyclophosphamide was added. During 70 days of survival, there was little evidence of hepatic rejection by biochemical monitoring or histopathological examination. Products of hepatic synthesis, including clotting factors, became those of the baboon liver with no obvious adverse effects. Death followed a cerebral and subarachnoid haemorrhage that was caused by an angioinvasive aspergillus infection. However, the underlying cause of death was widespread biliary sludge that formed in the biliary tree despite a seemingly satisfactory choledochojejunostomy. During life and in necropsy samples, there was evidence of the chimerism that we believe is integral to the acceptance of both xenografts and allografts. Our experience has shown the feasibility of controlling the rejection of the baboon liver xenograft in a human recipient. The biliary stasis that was the beginning of lethal infectious complications may be correctable by modifications of surgical technique. In further trials, the error of over-immunosuppression should be avoidable.

Cardiovascular Depression Secondary to Ionic Hypocalcemia during Hepatic Transplantation in Humans

Cardiovascular function, serum ionized calcium (Ca+2), and serum citrate were measured intraoperatively in patients (n = 9) undergoing orthotopic hepatic homotransplantation. Serum citrate increased 20-fold (P < 0.0006) following transfusion of citrated blood products in the absence of a functional liver. Serum ionized calcium decreased (P < 0.003) with concomitant decreases in cardiac index (P < 0.005), stroke index (P < 0.004), and left ventricular stroke work index (P < 0.001). Hemodynamic depression and ionic hypocalcemia were reversed following the administration of CaCl2. In contrast to patients with normal hepatic function, who may tolerate large amounts of citrated blood, patients with end-stage liver disease demonstrate acute ionic hypocalcemia with concomitant hemodynamic depression when receiving citrated blood products during the course of hepatic transplantation.

Pharmacokinetics of Mycophenolic Acid after Mycophenolate Mofetil Administration in Liver Transplant Patients Treated with Tacrolimus

The pharmacokinetics of mycophenolic acid (MPA) was studied after oral administration of mycophenolate mofetil (MMF) in 8 liver transplant patients. The mean (± SD) maximum MPA plasma concentration of 10.6 (± 7.5) mg/ml was achieved within 0.5 to 5 hours. The mean (± SD) steady‐state area under the plasma concentration versus time curve (AUC012) was 40 (± 30.9) mg/ml/h. The mean (± SD) half‐life was 5.8 (± 3.8) hours. There was poor correlation between trough blood concentrations of tacrolimus (r = −0.004) or serum creatinine (r = 0.689) with MPA AUC, while the serum bilirubin concentrations correlated (r = 0.743) well with MPA AUC, suggesting impairment in MPA conjugation in patients with liver dysfunction. The mean (± SD) ratio of the AUC of mycophenolic acid glucuronide (MPAG) to MPA was 64 (± 84), which correlated significantly with serum creatinine (r = 0.72) but not with serum bilirubin concentrations (r = 0.309), indicating accumulation of MPAG in patients with renal dysfunction. In 7 primary liver transplant patients on the same dose of MMF, the trough plasma concentrations of MPA during the first week of therapy ranged from < 0.3 to 1.5 pg/ml. The MPA concentrations increased by several folds during the next few weeks, which correlates well with increases in serum albumin concentrations. Changes in albumin appear to partially contribute to the variations in the pharmacokinetics of MPA in liver transplant patients.

The effect of graft function on FK506 plasma levels, dosages, and renal function, with particular reference to the liver

Plasma FK506 was studied in 49 liver, 13 heart, 3 double-lung or heart-lung, and 21 kidney recipients. The levels were correlated with the drug doses used, kidney function, and liver function. In all verieties of recipients, there was an early rise in the FK506 plasma levels that occurred at the time of intravenous administration of the drug. At the same time or shortly after, there were increases in serum creatinine that were transitory except in liver recipients with continuing suboptimal graft function. The quality of hepatic function dominated all aspects of FK506 management in the liver recipients. Those who received well-functioning grafts could be given about the same drug doses as recipients of kidneys and the thoracic organs. Liver recipients with defective grafts had astronomical rises in plasma FK506, a high incidence of renal failure, and probably increased neurotoxicity. In kidney transplant recipients, the FK506 plasma levels and doses were essentially the same in patients with prompt versus delayed renal function. These studies have highlighted the necessity, first of close pharmacologic monitoring of patients who are given FK506 in the presence of abnormal liver function, and second, of using smaller intravenous induction doses than in past practice.

FLUCONAZOLE THERAPY IN TRANSPLANT RECIPIENTS RECEIVING FK506

Fungal infections remain an important cause of morbidity and mortality following solid organ and bone marrow transplantation (1–3). Use of amphotericin B, the antifungal agent of choice at present, is frequently associated with a high incidence of side effects. In organ transplant patients particularly, the nephrotoxicity of amphotericin may be amplified when combined with immunosuppressive agents such cyclosporine or FK506. Fluconazole, a new antifungal agent with low toxicity and excellent oral bioavailability has been shown to be effective in both the treatment and prophylaxis of fungal infections in immunocompromised patients (4, 5). However, fluconazole is known to inhibit the cytochrome P-450 enzyme system in humans and animals. Coadministration of fluconazole with cyclosporine, a drug that is primarily metabolized in the liver, results in increased cyclosporine blood concentrations in transplant patients (6). Since FK506 is also known to be primarily eliminated by hepatic metabolism, coadministration of fluconazole to patients on FK506 therapy is expected to increase FK506 concentrations in patients (7). In rats, fluconazole is known to increase FK506 blood concentrations (8). In this communication we report our observations on the interaction between FK506 and fluconazole in transplant patients. Twenty organ transplant patients under FK506 immunosuppression were evaluated while receiving a course of fluconazole therapy. The patient population included 11 patients who received livers (OLTX); 6 patients who received kidneys (KTX), 2 patients who received hearts (HTX) and one bone marrow patient. The immunosuppressive regimen included FK506 and low-dose steroids (20 mg/day). FK506 was given initially as a continuous i.v. infusion at 0.1 mg/kg/day, with conversion to the oral dose of 0.15 mg/kg every 12 hr with the return of normal bowel function. Subsequent dose adjustments were guided by the quality of the graft, the presence of any evidence of rejection, signs of toxicity, and trough FK506 plasma concentrations (normal range 0.5 to 2 ng/ml). Sixteen of the patients studied had documented fungal infection (7 urinary tract infections, 4 esophagitis, 2 pyelonephritis, 2 tracheobronchitis, and 1 cholangitis). Candida albicans was the most common pathogen isolated (14 patients), whereas Candida tropicalis and Cryptococcus neoformans were isolated in one patient each. Four additional patients were considered high-risk patients and were treated with prophylactic fluconazole. The median time from transplantation to the initiation of fluconazole therapy was 60 days (range 2–1680 days). In patients with documented fungal infection, fluconazole was given for a median of 14 days (range 7–100 days). Fourteen of the 16 patients were successfully treated and became culture-negative. Two failures were noted in two kidney transplant patients who required transplant nephrectomy for persistent fungal invasion of the graft. Four patients received prophylactic fluconazole for a median of 12 days (range 6–30 days), and all remained fungal culture negative. Fluconazole was used at a dose of 200 mg/day in 8 patients and 100 mg/day in 12 patients. FK506 plasma concentrations were measured by an ELISA (9). The intraday coefficient of variation for this assay ranged from 4.2 to 5.5% and the interday coefficient of variation ranged from 14.4 to 17% at concentrations measured in this study. Figure 1 shows the median plasma trough concentration of FK506 on days -1, 0, 1, 2, 3, 4, 5, and 7 after fluconazole therapy as a function of the fluconazole dose administered. On day 1, the median plasma trough concentration of FK506 increased 1.4-fold in patients receiving 100 mg/day of fluconazole and 3.1-fold in patients receiving 200 mg/day. Thereafter the FK506 plasma concentration decreased as the FK506 dose was reduced to prevent higher FK506 concentrations. Figure 2 shows the median dose of FK506 and the plasma concentration of FK506 on days -1, 0, 1, 2, 3, 4, 5, and 7 of fluconazole therapy. A median FK506 dose reduction of 56% (range 0% to 88%) was required in order to maintain the FK506 concentrations below 2 ng/ml. There was no change in the route of FK506 administration during the period studied. The initial increase in FK506 plasma concentration during fluconazole therapy was associated with acute renal dysfunction in 3 patients (hemodialysis required in one) and acute changes in the mental status in 2 patients. FIGURE 1 Median plasma trough concentration of FK506 in patients receiving 100 mg (continuous line) and 200 mg (interrupted line) of fluconazole. FIGURE 2 Median dose (mg/day) (continuous line) and plasma trough concentration (ng/ml) (interrupted line) of FK506 during fluconazole therapy. The pharmacokinetics of FK506 was evaluated in one patient on two occasions, once while on fluconazole therapy (100 mg oral dose, 1 hr prior to FK506 dosing of 3 mg) and again eight days after stopping fluconazole therapy. Figure 3 shows FK506 the plasma concentration vs. time profile over a dosing interval in this patient with and without fluconazole therapy. Discontinuation of fluconazole therapy resulted in a significant decrease in the area under the plasma concentration–versus–time curve of FK506 (from 13 ng/ml/hr to 5.4 ng/ml/hr). The half-life maximum plasma concentration and the trough plasma concentration of FK506 also decreased during this period. FIGURE 3 FK506 pharmacokinetic profile of one patient with (continuous line) and without (interrupted line) fluconazole therapy. FK506 is a new immunosuppressive drug with a proven beneficial effect over currently available agents (10–12). Its elimination through hepatic metabolism by cytochrome P-450 enzyme makes it susceptible to possible interactions with other drugs as that are known to induce or inhibit these enzymes, such as phenobarbital, phenytoin, rifampin, clotrimazole, ketoconazole, itraconazole, fluconazole, cimetidine, and erythromycin (7). In rats, FK506 blood concentrations are reported to increase in the presence of fluconazole, erythromycin, ketoconazole, and diltiazam (8). Erythromycin and methylprednisolone have been reported to increase FK506 plasma concentrations (7). Clotrimazole treatment also increases FK506 plasma concentrations in liver transplant patients (13). The data reported here show an increase in trough and maximal plasma concentration of FK506, along with an increase in the half-life of FK506 in the presence of fluconazole. This suggests that fluconazole inhibits FK506 metabolism. It is not possible to predict whether fluconazole decreases FK506 metabolism in the gut and therefore increases the extent of absorption or whether it affects only the hepatic metabolism of FK506. Additional studies are required to understand the extent to which this interaction takes place in the small bowel and/or the liver. The increases in the plasma concentration of FK506 were higher at a 200 mg/day fluconazole dose than at a 100 mg/day dose. This dose-related effect, also reported with CsA (6), may have important implications when fluconazole is used in the treatment of severe fungal infections. Recently, a new fluconazole dosing regimen (200–400 mg/day) has been suggested for the treatment of invasive candidiasis (14). FK506 concentrations should be carefully monitored when using these dosing regimens. Despite the similarities between fluconazole’s interaction with CsA and FK506, some differences were observed. In renal transplant recipients, fluconazole slowly increased cyclosporine trough concentrations over a two-week period, reaching a maximum of twice the baseline level (6). In our patients the highest concentration of FK506 was seen within three days after the introduction of fluconazole therapy. In summary, fluconazole therapy in patients under FK506 immunosuppression appears to be beneficial for both prophylaxis and treatment of fungal infections. Fluconazole doses of up to 200 mg/day can be safely and effectively administered to transplant patients on FK506 therapy provided that the FK506 dose is reduced by half. Use of higher doses of fluconazole would require careful monitoring of trough FK506 plasma concentrations and corresponding reduction in FK506 doses to avoid nephrotoxicity.

Single-center experience with primary orthotopic liver transplantation with FK 506 immunosuppression.

OBJECTIVE The efficacy for primary orthotopic liver transplantation of a new immunosuppressive agent, FK 506 (tacrolimus, Prograf, Fujisawa USA, Deerfield, IL), was determined. SUMMARY BACKGROUND DATA After 3 years of preclinical research, a clinical trial of FK 506 for orthotopic liver transplantation was begun in February 1989, first as a rescue therapy for patients with intractable rejection with conventional immunosuppression, then as a primary drug. METHODS Between August 1989 and December 1993, 1391 recipients (1188 adult and 203 pediatric) of primary liver allografts were treated with FK 506 from the outset. Results from these patients were analyzed and compared with those of 1212 historical control patients (971 adult and 241 pediatric) given cyclosporine-based immunosuppression. RESULTS Actuarial survival at 4 years was 86.2% with FK 506 versus 65.5% with cyclosporine in the pediatric patients (p < 0.0000) and 71.4% versus 65.5% in the adults (p < 0.0005). The need for retransplantation was reduced significantly for FK 506 patients. Four-year graft survival was 77.0% with FK 506 versus 48.4% with cyclosporine in the pediatric patients (p < 0.0000), and 61.9% with FK 506 versus 51.4% with cyclosporine in the adult recipients (p < 0.0000). Regression analysis revealed that reduction in mortality or graft loss from uncontrollable rejection, sepsis, technical failure, and recurrent original liver disease were responsible for the improved results with FK 506 therapy. CONCLUSIONS FK 506 is a potent and superior immunosuppressive agent for orthotopic liver transplantation.

Hemodynamics and low density lipoprotein metabolism. Rates of low density lipoprotein incorporation and degradation along medial and lateral walls of the rabbit aorto-iliac bifurcation.

We have investigated whether arterial wall low density lipoprotein (LDL) metabolism in areas of disturbed flow differs from the metabolism in adjacent regions of undisturbed flow. Using the rabbit aorto-iliac bifurcation as a model, we examined the rates of LDL incorporation and catabolism in vivo and correlated them to the arterial flow patterns in these regions. The trapped ligand method was used to quantitate the rates of LDL incorporation and degradation over a 20-hour period in three hemodynamic zones of the daughter iliac branch: 1) a region of flow separation where the shearing forces are elevated along the medial wall and reduced along the lateral wall, 2) a transition region where the flow patterns begin to approach the fully established situation, and 3) a unidirectional flow region with symmetric fluid shearing forces along the medial and lateral walls. Our results indicate an elevated rate of LDL incorporation into the lateral versus the medial wall in the proximal zone of flow separation (5.2 +/- 0.8 nl/mg/hr vs. 3.7 +/- 0.5 nl/mg/hr, p less than 0.01). A similar elevation in the degradation rate of the lateral over the medial wall of this most proximal zone was also observed (2.1 +/- 0.4 vs. 1.4 +/- 0.2, p less than 0.05). No such differences were observed regarding LDL incorporation and degradation in the transitional or unidirectional hemodynamic zones. These results suggest that modifications in arterial wall LDL incorporation and catabolism are induced by hemodynamic forces. The implications of these findings for the formation of the atherosclerotic lesion are discussed.

Reversible activation-inactivation of cholesterol 7α-hydroxylase possibly due to phosphorylation-dephosphorylation

Abstract The activity of microsomal cholesterol 7α-hydroxylase is shown to be increased in vitro by ATP, Mg 2+ , and a cytosolic protein fraction. There was a loss of enzyme activity in the presence of E. coli alkaline phosphatase which was proportional to the amount of phosphatase. Much of this loss was recovered upon addition of ATP, Mg 2+ , and a cytosolic protein fraction.

The acute effects of exercise intensity on HDL-C metabolism

To determine whether exercise intensity influences acute HDL-C responses, 12 male recreational runners (24.8 +/- 4 yr) who ran 15-30 miles.wk-1 exercised on a motor driven treadmill at 60% (L) and 75% (H) VO2max. A counterbalanced experimental design was utilized and energy expenditure was 800 Kcal. Fasting blood samples were obtained 24 h before exercise (24 PRE), immediately post-(IPE), 1 h post- (1 h PE), 6 h post- (6 h PE), and 24 h post- (24 h PE) exercise and analyzed for HDL-C and HDL2&3-C. In addition, postheparin plasma samples, obtained 24 h PRE, 6 h PE, and 24 h PE were analyzed for lipolytic activity--LPLA and HTGLA. An exercise trial by time interaction was observed for HDL-C (P < 0.01). Post-hoc analysis revealed no change in HDL-C following the L trial. However, an increase in HDL-C was observed 24 h PE (P < 0.01) following the H trial. The increase in HDL-C was attributed to an elevated HDL3-C (P < 0.01), with no change in HDL2-C. Analysis of plasma lipolytic activity revealed an increase in LPLA 24 h PE (P < 0.05) which may be responsible for the postexercise alterations in HDL-C. However, HTGLA decreased 6 h PE (P < 0.01) and 24 h PE (P < 0.05). We conclude that increases in HDL-C levels following endurance activity are influenced, in part, by the exercise intensity.