Concentration Of FK506 Research Articles

FK506 in the treatment of children with nephrotic syndrome of different pathological types

To evaluate the efficacy of FK506 in the treatment of children with nephrotic syndrome of different underlying pathology. 12 patients were treated with FK506 with a dosage of 0.1 - 0.15 mg/kg/d while corticosteroid dose was tapered stepwise. This therapeutic course lasted 3 - 6 months during which the plasma concentration ofFK506 was monitored. 12 children with different pathological types nephrotic syndrome were treated with FK506, including 4 cases of MCN, 6 cases of MsPGN, and 1 case of MPGN and 1 case of FSGS. After 2-month duration, 8 patients got complete remission including 4 cases of MCN and 4 cases of MsPGN and 3 children including 1 case of MsPGN, 1 case of MPGN, and 1 case of FSGS got partial remission. Only 1 child with MsPGN was considered to be a treatment failure. The overall response rate was 91.67% with the plasma concentration of FK506 maintained at 5 approximately 12 ng/ml, and the response time was 10 - 38 days. After 1-month duration, all patients except one experienced a reduction in proteinuria to normal levels or a partial response (50% reduction in protein excretion), significant increase in serum albumin, decrease in serum cholesterol and triglyceride and disappearance of edema. 2 months later, in 11 patients, blood biochemical values had returned to normal levels. The drug was generally well-tolerated. 3 patients had anorexia, nausea, vomiting. 2 patients experienced transient elevated serum creatinine which was reversible after the adjustment of dosage. 3 patients had minor changes in urine NAG. Only 2 of all patients relapsed. FK506 is one of the effective immunosuppressants. In this study, FK506 in combination with a small doses of steroid while decreasing FK506 dosage plays a role in consolidating the curative effect and preventing relapse. In conclusion, FK506 may be effective in the treatment of nephrotic syndrome.

Implantation of FK506 drug delivery system into the anterior chamber for inhibiting corneal rejection in high risk rabbit corneal allograft

To study the effects of a biodegradable FK506 drug delivery system (DDS) on the inhibition of corneal rejection, to measure the concentration of FK506 in the aqueous humor and to study the relationship between intraocular concentration of FK506 and its immunosuppressive effects on corneal rejection. Corneal neo-vascularization was induced by 5 - 0 silk sutures in 68 New Zealand rabbits to establish a high risk corneal transplantation model. A unilateral 7 mm diameter central penetrating corneal transplantation was performed with 7.5 mm diameter grafts from health New Zealand rabbit donors. Grafted rabbits were randomized into five groups as follows: Group A: untreated control animals; Group B: DDS anterior chamber recipients without drug; Group C: 1 mg cyclosporine DDS anterior chamber recipients; Group D: 0.1% FK506-olive oil drop recipients; Group E: 0.5 mg FK506 DDS anterior chamber recipients. Grafts were examined with a slit lamp every other day and clinical conditions were scored for up to 28 weeks. The aqueous humor and cornea of Group D and Group E were collected, and the concentration of FK506 was determined. The expression of cytokines IL-2R alpha, MCP-1, Fas and FasL mRNA was detected with in situ hybridization method. The median survival time was (17.9 +/- 4.7) d in Group A (untreated corneal allograft), (20.0 +/- 3.7) d in Group B, (56.3 +/- 8.8) d in Group C, (78.1 +/- 7.2) d in Group D, and over 180 d in Group E. Statistically significant difference (F = 926.37, P = 0.0000) in the survival time of allograft has been found between Group E and other groups. The concentration of FK506 in aqueous humor was (15.7 +/- 2.6) ng/ml in Group E at one week and remained stable for at least 24 weeks, much higher than that in Group D (<0.3 ng/ml). The concentration of FK506 in cornea was also higher in Group E than that in Group D. The expression of cytokines IL-2R alpha and MCP-1 mRNA was detected, but the expression of Fas and FasL mRNA was not detected in Group A. FK506 DDS implanted in the anterior chamber can significantly prolong corneal allograft survival in high-risk corneal graft rejection model. This intraocular DDS may be a valuable adjunct for the suppression of immune graft rejection in high-risk corneal transplants.

Change of the Blood Concentration of Tacrolimus after the Switch from Fluconazole to Voriconazole in Patients Receiving Allogeneic Hematopoietic Stem Cell Transplantation

The purpose of this study was to assess the impact by switching co-administered triazole antifungal agent from fluconazole (FCZ) to voriconazole (VCZ) on the blood concentration of tacrolimus (FK506) in patients receiving allogeneic hematopoietic stem cell transplantation. We performed a retrospective study presented as case reports. The blood concentration of FK506 was increased after the switch from FCZ to VCZ, resulting in increase of the concentration/dose (C/D) ratio of FK506. Thus, the mean C/D ratios of FK506 with oral administration was surprisingly increased over 4.5-fold after the switch. Therefore, it was necessary to reduce the FK506 dose when co-administered FCZ is switched to VCZ. We should be careful when interpreting the results of these case reports; however, in some patients, it is recommended that the dose of FK506 be reduced to one-fifth after the switch.

Relationship between MDR1 gene polymorphism and blood concentration of tacrolimus in renal transplant patients

To investigate the relationship between MDR1 exon 21 and exon 26 polymorphism and whole blood concentration of tacrolimus (FK506) in renal transplant patients. Blood samples were collected from 86 renal transplant patients who received FK506 peri-operationally. PCR-RELP was used to determine the MDR1 genotype. The patients were divided into 3 subgroups for every position: GG, GT, and TT in exon 21; and CC, CT, and TT in exon 26. Three, six, and twelve months after the transplantation ELISA was used to measure the whole blood concentration of FK506. The FK506 concentrations standardized by dosage and body weight (FK506 concentration per dose/kg) were compared among the 3 subgroups within the MDR1 exon 21 and exon 26 groups. Of the 86 patients 26 (30.2%), 35 (40.7%), and 25 (29.1%) were carriers of GG, GT, and TT in exon 21, and 26 (30.2%), 35 (40.7%), and 23 (26.8%) were carriers of CC, CT, and TT in exon 26. MDR1 exon 26 C3435T was in significant linkage disequilibrium with MDR1 exon 21 G2677T. Three, six, and twelve months after the transplantation a significant correlation between the whole blood FK506 concentration per dose/day and MDR1 exon 21 and exon 26 genotypes. For exon 21 there were significant differences among the 3 subgroups (all P < 0.01). The ratio for the patients with GG was remarkably lower than that of those with GT and TT, and the ratio with GT was also lower than the patients with TT (P < 0.05). For exon 26, there was also a significant difference among the 3 subgroups (all P < 0.01). The ratio for the patients with CC was remarkably lower than those of the patients with CT and TT, and the ratio of CT was also lower than that of TT (P < 0.05). The MDR1 gene polymorphism is correlated with the whole blood concentration of FK506. To obtain the similar blood concentration of FK506, the patients with GG and CC should take the drug at a higher dose than those with Ct and TT.

Co-administration of Ketoconazole and Tacrolimus Therapy: A Transplanted Rat Model

The aim of this work is to study the safety and the effect of addition of ketoconazole to experimental kidney transplanted rat treated with tacrolimus and predicts the percentage of tacrolimus dose reduction. The material of this work included 60 male Sprague Dawely rats subjected to renal allotransplantation. They were equally divided into five groups: Group I: served as control group, Group II: received FK506 3.2 mg/kg/bw, Group III: received FK506 2 mg/kg/bw, Group IV: received FK506 1 mg/kg/bw, Group V: received FK506 1 mg/kg/bw plus Ketoconazole 20 mg/kg/day. FK506 trough level and laboratory investigations were determined at 0, 3, 7, 10, 14, and 27 days post-transplantation. In all groups loss of body weight was observed at day 27 after treatment compared to that before transplantation. Serum creatinine significantly increased at day 27 compared to the basal level in groups treated with 1.0 and 3.2 mg FK506 (1.80 +/- 0.50 versus 0.39 +/- 0.06 P = 0.001) and (1.03 +/- 0.26 versus 0.50 +/- 0.07 P = 0.001) respectively, while for 2.0 mg or 1.0 mg plus keto groups, no significant differences in serum creatinine levels over time (0.56 +/- 0.22 versus 0.44 +/- 0.10 P = 0.106) and (0.55 +/- 0.30 versus 0.42 +/- 0.08 P=0.160) were observed. Concomitant administration of Ketoconazole and FK506 in transplanted rat model is safe and results in increase of blood trough level concentration of FK506 with 50% reduction of its dose.

Drug delivery system using microspheres that contain tacrolimus in porcine small bowel transplantation

Rejection remains a major barrier to successful bowel transplantation, in spite of improved immunosuppressive techniques. Therefore, new, more effective, immunosuppressants, with fewer side effects, are needed. Biodegradable microspheres containing tacrolimus (FK506) were used in an experimental porcine small bowel transplantation. Twenty pigs underwent transplantation and were divided into four groups according to the immunosuppressive regimen. Group A (n = 5): no immunosuppression; group B (n = 6): 0.2 mg/kg per day of FK506; group C (n = 3): 1.0 mg/kg per day of FK506; group D (n = 6): 0.04 mg/kg per day of FK506 contained in biodegradable microspheres. Rejection was diagnosed macroscopically by endoscopic examination and histologically by biopsy specimen analysis. The mean survival time and standard deviation (SD) were 8.8+/-3.5, 11.0+/-1.4, 9.7+/-2.5 and 28.6+/-22.5 days for groups A, B, C, and D, respectively, with a statistically significant difference found between group D, on the one hand, and groups A, B and C, on the other. The mean trough blood concentration of FK506 was 10.5+/-2.2, 27.9+/-6.0 and 10.5+/-3.5 ng/ml in groups B, C and D, respectively. In groups A and B, all pigs died of rejection, without infection. In group C, all died of infection, without rejection. In contrast, none of the pigs in group D developed rejection or infection. Our results clearly show that the drug delivery system using biodegradable microspheres that contain FK506 is effective for controlling rejection with fewer side effects in the porcine small bowel transplantation.

Cyclosporine A, FK-506, 40-0-[2-hydroxyethyl]rapamycin and mycophenolate mofetil inhibit proliferation of human intrahepatic biliary epithelial cellsin vitro

To investigate the effect of cyclosporine A (CsA), FK-506, and mycophenolate mofetil (MMF) and 40-0-[2-hydroxyethyl]rapamycin (RAD) on proliferation of human intrahepatic biliary epithelial cells (BECs) in vitro. BECs were isolated from six human liver tissuespecimens with the immunomagnetic separation method and treated with different concentrations of CsA, FK-506, RAD, and MMF in vitro. Proliferation of the cells was measured by MTT assay at 24 and 48 h after treatment, respectively. One-way analysis of variance was used to analyze the results. Expression of CK 19 in BECs was monitored by flow cytometry and Western blot. Six lines of BECs were established. They survived for 4-18 wk in vitro. Flow cytometry analysis showed that these cells always expressed CK19. CsA, FK-506, RAD, and MMF inhibited proliferation of BECs in a dose-dependent manner. The lowest concentration of CsA, FK-506, RAD, and MMF to inhibit proliferation of BECs (P<0.05) was 500, 100, 0.25, and 100 mug/L, respectively. However, the expression of CK19 by BECs was not changed. CsA, FK-506, RAD, and MMF have an antiproliferative effect on human intrahepatic BECs in vitro, while RAD has the strongest growth-inhibitory effect. Their possible effects on liver regeneration and bile duct injury in transplant patients should be further investigated.

Drug delivery system using microspheres that contain tacrolimus in porcine small bowel transplantation

Abstract Rejection remains a major barrier to successful bowel transplantation, in spite of improved immunosuppressive techniques. Therefore, new, more effective, immunosuppressants, with fewer side effects, are needed. Biodegradable microspheres containing tacrolimus (FK506) were used in an experimental porcine small bowel transplantation. Twenty pigs underwent transplantation and were divided into four groups according to the immunosuppressive regimen. Group A (n= 5): no immunosuppression; group B (n= 6): 0.2 mg/kg per day of FK506; group C (n= 3): 1.0 mg/kg per day of FK506; group D (n= 6): 0.04 mg/kg per day of FK506 contained in biodegradable microspheres. Rejection was diagnosed macroscopically by endoscopic examination and histologically by biopsy specimen analysis. The mean survival time and standard deviation (SD) were 8.8±3.5, 11.0±1.4, 9.7±2.5 and 28.6 ±22.5 days for groups A, B, C, and D, respectively, with a statistically significant difference found between group D, on the one hand, and groups A, B and C, on the other. The mean trough blood concentration of FK506 was 10.5 ±2.2, 27.9 ±6.0 and 10.5 ±3.5 ng/ml in groups B, C and D, respectively. In groups A and B, all pigs died of rejection, without infection. In group C, all died of infection, without rejection. In contrast, none of the pigs in group D developed rejection or infection. Our results clearly show that the drug delivery system using biodegradable micro-spheres that contain FK506 is effective for controlling rejection with fewer side effects in the porcine small bowel transplantation.

Suppression of Tacrolimus on the expression of MMP-9 in rhHGF-stimulated ECV304 cell line

To investigate the stimulating effect of recombinant human hepatocyte growth factor (rhHGF) on the expression of matrix metalloproteinase-9 (MMP-9) in ECV304 cells and the suppression effect of FK506. The growth curve of ECV304 cells was drawn and the valid concentration of rhHGF and FK506 were determined. The expression levels of MMP-9 were analyzed by flow cytometry (FCM). (1) The optimal growth concentration of ECV304 cell was 1.0E+4/ml and logarithm growth time was 3-5 days. The valid concentration of rhHGF was 8 ng/ml and IC50 was 8.27 ng/ml. The valid concentration of FK506 was 50 ng/ml and IC50 was 51.63 ng/ml. (2) The expression level of MMP-9 was 39.74% in controls. Cell livability was 1.388169 +/-0.102033 (P<0.01) when 8 ng/ml rhHGF was added to culture medium, and the expression level of MMP-9 was 40.32%. Cell livability was 0.398764 +/-0.092476 (P<0.01) when 50 ng/ml FK506 was added to culture medium, and the expression level of MMP-9 was 14.61%. Cell livability was 0.767203 +/-0.02639 (P<0.01) when 8 ng/ml rhHGF was added 15 minutes after 50 ng/ml FK506 was added to culture medium, and the expression level of MMP-9 was 35.08%. rhHGF can stimulate the proliferation of ECV304 cells and increase the protein expression level of MMP-9. FK506 can suppress the proliferation of ECV304 cells and decrease the expression level of MMP-9. FK506 can inhibit the proliferation of ECV304 cells induced by rhHGF and decrease MMP-9 protein level.

Severe elevations of FK506 blood concentration due to diarrhea in renal transplant recipients.

The rate of metabolism in the intestine of oral administered FK506 decreases as FK506 passes on to the lower intestine. In transplant recipients with diarrhea given oral FK506, the main areas for absorption of FK506 shift to the lower intestine, where the ability to metabolize FK506 is weaker. Therefore it is considered likely that when FK506 is administered to recipients with diarrhea, the blood concentration of FK506 will be higher. Twenty recipients experiencing episodes of diarrhea were investigated to determine the trough level of FK506 and the time required for the FK506 trough level to return to the level that obtained before diarrhea. AUC0-4h and Cmax of FK506 were investigated in eight recipients. In cases with severe diarrhea, the daily fluctuations of FK506 blood concentration were also investigated. The FK506 trough level (p < 0.0001), AUC (p = 0.0173), and Cmax (p = 0.0173) were found to be significantly higher during episodes of diarrhea. In almost all cases, it took between 2 and 4 wk for the elevated FK506 trough level to return to its previous level following a bout of diarrhea. In the daily fluctuations of FK506 concentration, Tmax was prolonged. In some cases, the concentration was highest just before administration of FK506, when it should have been at trough level. Diarrhea caused significant elevations of trough level, AUC0-4h and Cmax of FK506, and the prolongation of Tmax in renal transplant recipients administered FK506.

THE PHARMACOKINETIC CHARACTERISTICS OF TACROLIMUS (FK506) IN THE CHINESE PATIENTS WITH THE FUNCTIONING KIDNEY ALLOGRAFT

P718 Objective: Tacrolimus (FK506) is a potent immunosuppressive agent that has been widely used in solid organ transplantation. However, its pharmacokinetic characteristics have not been well established in the ethnic Chinese patients. In this study, we examined the whole blood concentration of FK506 in the patients with the functioning kidney allografts. Materials and Methods: Sixty-four patients (46 men and 18 women) with the functioning kidney allografts were enrolled into this study. FK506 was given orally at 0.10-0.15 mg/kg bw for 30 days postoperatively and then 0.05-0.10mg/kg bw for the following 90 days in combination with mycophenolate mofetil and corticosteroids. Whole blood concentration was measured by using ELISA method. Multiple blood samples were collected just prior to and at 30 minutes, 1, 2, 3, 4, 6, 8, 10, and 12 hours after oral administration of FK506. We examined the pharmacokinetic parameters, individual concentration-time profiles of whole blood samples after single dose of FK506 in 21 patients and during chronic therapy in 64 patients. Results: The whole blood concentration of FK506 was maintained in 10-15ng/ml during the 30 postoperative days and 5-10ng/ml during 90 days. Conclusions: In this study, the lower dosage of FK506 reaches an optimal blood concentration in the Chinese patients with the functioning kidney allografts. The different pharmacokinetic characteristics of FK506 may suggest that the bioavailability of FK506 in the Chinese patients differs from that in the western ones. The steady state of FK506 whole blood concentration is reached after single dosing period, suggesting that the pharmacokinetic characteristics measured in the early postoperative period can be used for the therapeutic drug monitoring during the chronic therapy of the patients with the functioning kidney allografts. The larger interindividual variation between patients is observed, indicating that individual therapeutic drug monitoring of FK506 is mandatory.

Quantitative relationship between myocardial concentration of tacrolimus and QT prolongation in guinea pigs: pharmacokinetic/pharmacodynamic model incorporating a site of adverse effect.

Clinical cases have been reported of tacrolimus (FK506)-induced QT prolongation. We have previously demonstrated sustained QT prolongation by FK506 in guinea pigs. Herein, we aimed to conduct a pharmacokinetic/pharmacodynamic (PK/PD) analysis of FK506, using a model involving the myocardial compartment. The pharmacokinetics of FK506 and its effects on QTc intervals were investigated in guinea pigs. In the pharmacokinetic study, whole blood and ventricular FK506 concentrations were analyzed, using a 4-compartment model during and after intravenous infusion of FK506 (0.01 or 0.1 mg/hr/kg). Subsequently, the concentration-response relationship between ventricular FK506 concentration and change in QTc interval was analyzed, using the maximal effect (Emax) model. Pharmacokinetic profiles of FK506 showed a delayed distribution of FK506 into the ventricle. Furthermore, the observed QT prolongation paralleled the ventricular FK506 concentrations, with no lag-time between the two. The Emax model successfully described the relationship between changes in QTc interval and ventricular FK506 concentrations. In conclusion, the PK/PD model where the myocardial drug concentration of FK506 was linked with its adverse effect could describe, for the first time, the anti-clockwise hysteresis observed in the relationship between blood FK506 concentration and QTprolongation. Such a hysteresis pattern for QTprolongation might be caused, therefore, mainly by the delayed disposition of FK506 to ventricular myocytes.

Arteriolopathy in non-episode biopsies of renal transplant allograft.

We have been performing protocol biopsies since 1995 to predict the outcome of renal allograft. However, histopathological findings in renal allograft with stable function remain unclear. For this reason, we performed non-episode biopsy on long-surviving renal allograft and investigated the histopathological changes. Among the several diseases seen in non-episode biopsies, arteriolopathy, such as drug-induced nephropathy, is one of the most frequent diseases. However, it is unrelated to the dosage and the concentration of cyclosporine or tacrolimus. Consequently, we evaluated the clinicopathological findings of arteriolopathy in this study in order to clarify whether cyclosporine (CsA) or tacrolimus (FK506) is responsible for these findings. We defined non-episode biopsy as a case with a serum creatinine level less than 2.0 mg/dL and containing less than 500 mg/dL of urinary protein. Final results showed that 71 cases were identified as non-episode biopsy. We then evaluated the histopathological findings and the clinical characteristics of these cases. Thirty-two of the 71 non-episode biopsy specimens showed findings of arteriolopathy. The frequency and the severity of arteriolopathy are not concerned with dosage and concentration of CsA or FK506. The arteriolopathy seen in non-episode biopsy was related to the time of the biopsy and the kidney age. Arteriolopathy in nonepisode biopsy also had a relationship with hypertension, suggesting that it is important to strictly control blood pressure for graft survival.

Low-dose tacrolimus (FK 506)-based immunosuppressive protocol in living donor renal transplantation

In order to avoid the side effects of tacrolimus (FK506), a low-dose FK506-based regimen was started from 1 June 1991. The dose was adjusted to maintain the FK506 whole blood trough level at 15–20 ng/ml for 7 days postoperatively, at 10–15 ng/ml for 2 months, and under 10 ng/ml thereafter. The graft survival rates at 3 years and 5 years were 87.8 and 82.3 % (FK506) vs 86.8 and 86.8 % [cyclosporine (CyA)]. The incidence of acute rejection within the first 90 days was 31.6 % in the FK506 group which was lower than the 57.1 % of the CyA group (P = 0.0585). Grades of acute rejection episodes over IIA in the FK506 group were 20 %, which was lower than the 37 % in the CyA group. The mean oral dosages of FK506 were 0.061 and 0.04 mg/kg per day at 3 and 5 years, respectively. The incidence of new onset diabetes was 27.8 % in the FK506 group and 17.1 % in the CyA group. However, insulin therapy was withdrawn in all patients of the FK506 group within 5 months. The percentage of patients who required an antihypertensive agent was 28.6 % and 40 % in the FK506 group and 73.2 % and 88 % in the CyA group at 1 and 3 years, respectively (P < 0.05). Nephrotoxicity was seen in 20 % of the FK506 group and 14.3 % of the CyA group. Hypercholesterolemia was less frequent in the FK506 group than the CyA group. The FK506-based regimen described here is a protocol with the potential to reduce its adverse effects. The whole blood concentration of FK506 should be monitored and blood levels maintained in the range of 5–10 ng/ml after 90 postoperative days for optimal efficacy and minimal toxicity.

Relationship between in vivo FK506 clearance and in vitro 13-demethylation activity in living-related liver transplantation.

Although it is important to maintain an appropriate blood concentration of FK506 after liver transplantation, significant interindividual variability in the actual FK506 dosage has been observed, presumably due to the wide variability of cytochrome P450 3A4 activity in liver microsomes. A study was conducted in patients undergoing living-related liver transplantation and their donors to investigate the relationship between the in vitro FK506 demethylation activity in graft liver microsomes and the in vivo blood clearance of FK506. Liver biopsy tissue was obtained from 17 living donors to measure the in vitro formation rate of 13-demethyl derivative (M-I: the major metabolite of FK506). Erythromycin N-demethylation activity in vitro was also assessed in 11 cases. The FK506 blood clearance (CLss) was calculated from its constant infusion rate and steady-state blood concentration on day 4 after transplantation in 17 recipients. The FK506 infusion rate varied 4.6-fold from 8.3 to 38.4 ng/min/kg. The mean CLss of FK506 was 22.1+/-10.8 ml/min (10.1-45.2 ml/min). The M-I formation rate showed a wide variability, ranging from 0.098 to 0.571 nmol/min/mg protein. A significant correlation was observed between the in vitro estimated total metabolic ability of the graft for FK506 (M-I formation rate x graft weight) and the in vivo CLss of FK506 (r=0.770, P<0.001). Erythromycin N-demethylation (0.066-0.443 nmol/min/mg protein) showed a strong correlation with the M-I formation rate (r=0.891, P<0.01). The in vivo FK506 clearance can mainly reflect in vitro FK506 demethylation activity.

Comparison of the Abbott IMx Tacrolimus I and Tacrolimus II Assays

Tacrolimus (FK-506, Prograf, Fujisawa Pharmaceutical Co.), first approved in the United States for liver transplants, is now widely used in other organ transplants (1). Its immunosuppressive potency is 10- to 100-fold greater than cyclosporine A. Because subtherapeutic concentrations are associated with organ rejection and high concentrations are associated with nephrotoxicity, neurotoxicity, and opportunistic infections, therapeutic drug monitoring of FK-506 is well-accepted and is a common laboratory practice (2). Currently, there is no consensus on the therapeutic range for FK-506. Substantial amounts of the drug are present intracellularly, with a red blood cells-to-plasma ratio >4:1. In addition, clinical effects correlate better with whole blood concentrations than with serum or plasma concentrations (3). Therefore, routine laboratory assays determine concentrations on whole blood. The whole blood therapeutic range was originally thought to be 15–25 μg/L, but has subsequently been modified at most US centers to 5–20 μg/L (1). Several methods are available for monitoring the concentration of FK-506. These include bioassays, radioreceptor assays, HPLC, ELISA (Incstar Corp.), and microparticle enhancement immunoassay (MEIA;Abbott Diagnostics) (4 …

Low-dose tacrolimus (FK506)-based immunosuppressive protocol in living donor renal transplantation

Abstract In order to avoid the side effects of tacrolimus (FK506), a low-dose FK506-based regimen was started from 1 June 1991. The dose was adjusted to maintain the FK506 whole blood trough level at 15–20 ng/ml for 7 days postoperatively, at 10–15 ng/ml for 2 months, and under 10 ng/ml thereafter. The graft survival rates at 3 years and 5 years were 87.8 and 82.3 % (FK506) vs 86.8 and 86.8% [cy-closporine (CyA)]. The incidence of acute rejection within the first 90 days was 31.6% in the FK506 group which was lower than the 57.1% of the CyA group (P - 0.0585). Grades of acute rejection episodes over IIA in the FK506 group were 20 %, which was lower than the 37 % in the CyA group. The mean oral dosages of FK506 were 0.061 and 0.04 mg/kg per day at 3 and 5 years, respectively. The incidence of new onset diabetes was 27.8% in the FK506 group and 17.1 % in the CyA group. However, insulin therapy was withdrawn in all patients of the FK506 group within 5 months. The percentage of patients who required an antihypertensive agent was 28.6 % and 40 % in the FK506 group and 73.2% and 88 % in the CyA group at 1 and 3 years, respectively (P < 0.05). Nephrotoxicity was seen in 20% of the FK506 group and 14.3 % of the CyA group. Hypercholesterolemia was less frequent in the FK506 group than the CyA group. The FK506-based regimen described here is a protocol with the potential to reduce its adverse effects. The whole blood concentration of FK506 should be monitored and blood levels maintained in the range of 5–10 ng/ml after 90 postoperative days for optimal efficacy and minimal toxicity.

A calcineurin inhibitor, FK506, blocks voltage-gated calcium channel-dependent LTP in the hippocampus

The effects of FK506, an immunosuppressant and protein phosphatase 2B (calcineurin) inhibitor, on the voltage-gated calcium channel (VGCC)-dependent long-term potentiation (LTP) were investigated in the CA1 region of mice hippocampal slices. VGCC-dependent LTP was induced either by a brief application of a potassium channel blocker tetraethyleneanmonium (TEA), or by a strong tetanic stimulation under the blockade of NMDA-receptors. FK506 (1–50 μM) produced dose-dependent inhibition on TEA-induced LTP. Cyclosporin A (CysA 50 μM), another calcineurin inhibitor, showed a similar inhibitory effect on TEA-induced LTP. FK506 (10 μM) also blocked the strong tetanus-induced LTP, but had no effect on the post-tetanic potentiation. By using a subthreshold weak tetanic stimulation protocol, we also found that low concentration of FK506 (1 μM) produced neither inhibition nor potentiation on VGCC-dependent LTP. These results showed FK506 and CysA exerted inhibitory effects on VGCC-dependent LTP, and suggest that calcineurin is involved in the processes of this kind of synaptic plasticity.

Asialoglycoprotein/asialoglycoprotein receptor (AGP-AGPr) interaction is an important mechanism for the uptake of FK506 by hepatocytes.

Hepatic tissue concentrations of FK506 have been correlated with early acute rejection following liver transplantation. Asialoglycoproteins (AGP) reputedly bind FK506 in blood. AGP are removed from the circulation by the liver via the AGP receptor (AGPr), which resides on hepatocytes. This study was undertaken to determine if the AGP-AGPr mechanism enhances the delivery of FK506 to hepatocytes. Human orosomucoid (OM) was used as a representative AGP. asialoOM (aOM) was prepared by desialation of OM. Fresh rat hepatocytes were isolated by collagenase digestion. Tritium labeled FK506 (FK) was used to identify and quantitate FK506. Quantitation of FK in serum and culture media was by direct counting. FK in animal tissues used a method developed in our laboratory for the purpose. AGPr on resting hepatocytes was demonstrated by flow cytometry using FITC-orosomucoid and FITC-BSA controls. AGPr were enhanced by 2 g glucose/L. Two serum FK-binding fractions, 44 kD and 15 kD, were identified by gel filtration. Exogenous OM avidly bound FK and displaced FK activity from the 15 kD fraction. Serum (1%) and the 44 kD fraction enhanced the uptake of FK by hepatocytes, while serum depleted of OM-aOM by affinity chromatography was only 72.5% as effective as control serum; aOM enhanced the uptake of FK by hepatocytes to a degree similar to that of control serum but OM did not significantly affect the uptake of FK. Cold FK506 blocked the uptake and was dose dependent; cold CsA had no effect. Affinity extraction of OM from serum to which FK had previously been added removed 28.4% of FK activity. Following i.v. infusion, the kidney had the highest and liver the lowest tissue concentration of FK at 1 hr and 3 hr. In contrast, after oral administration the liver had the highest concentrations of the other tissues tested. The AGP-AGPr mechanism plays a significant role in the delivery of FK506 to hepatocytes and is likely responsible for the differences in bioavailability observed after oral and i.v. administration. Factors governing the AGP-AGPr mechanism are germane to understanding both the efficacy and toxicity of FK506 and the development optimal therapeutic strategies.

Quality Assurance Procedure for Monitoring Tacrolimus (FK506) Concentrations in Whole Blood by IMx Assay

A Quality Assurance Program for the IMx assay for FK506 in whole blood samples was established to monitor the performance of the assay in clinical sites enrolled by Fujisawa USA, Inc. Forty investigative sites participating in the program were required to perform assay to establish intraassay variability, interassay variability, and performance on blinded samples. Only two of the sites were required to repeat part of the program. The intraassay and interassay results at the sites were in good agreement with the target values obtained at Fujisawa Research Laboratory. Most of the coefficients of variation (CV) were within +/- 15%, well within the acceptance range of +/- 30%. Only a few values were outside the acceptance window. For the blinded samples, the CVs were variable and depended on the concentration of FK506 in the sample. At lower blood FK506 concentrations (5-10 ng/ml), the mean CVs were often outside the acceptance window, and many individual values were not acceptable. At concentrations of 15-50 ng/ml, the CVs were generally acceptable. Thus individual sites can quickly learn to perform the FK506 IMx assay and achieve good within- and between-day results. The assay of lower blood concentrations of FK506 may show higher variability. Patients are usually monitored for clinical signs of rejection and toxicity in addition to blood FK506 concentrations.