Facile fabrication of a novel disposable pencil graphite electrode for simultaneous determination of promising immunosuppressant drugs mycophenolate mofetil and tacrolimus in human biological fluids.

An innovative electrochemical sensor was proposed for simultaneous determination of mycophenolate mofetil (Mph) and tacrolimus (TAC) for the first time. A novel sensor based on electro-polymerization of multi-walled carbon nanotubes (MWCNTs) and a novel Cu-1N-allyl-2-(2,5-dimethoxyphenyl)-4,5-diphenyl-1H-imidazole metal organic framework (Cu-ADPPI MOF) on disposable pencil graphite electrode (dPGE). Many techniques were used to characterize the electrochemical activity and surface structure of the fabricated sensor. The proposed sensor exhibited good catalytic performance towards Mph and TAC oxidation due to the synergistic effect. Under optimal conditions, the proposed sensor has achieved a linear range of 0.85-155 × 10-8 M and 1.1-170.0 × 10-8 M with LODs of 0.28 × 10-8 M and 0.36 × 10-8 M for Mph and TAC, respectively. The designated sensor showed good reproducibility, repeatability, stability, and selectivity for the determination of Mph and TAC. Moreover, the simultaneous determination of Mph and TAC in different human biological fluids was carried out with acceptable results. As a result, the proposed sensor opens a new venue for the use of electro-polymerized MOFs in combination with other conductive materials such as MWCNTs for electrochemical sensing of different analytes with the desired sensitivity and selectivity. Graphical abstract Construction of disposable graphite electrode, based on electro-deposition of multilayer films of multi-walled carbon nanotubes and a new generation of Cu-MOFs, for simultaneous analysis of tacrolimus and mycophenolate mofetil for the first time.

Measurement and compartmental modeling of the effect of CYP3A5 gene variation on systemic and intrarenal tacrolimus disposition.

We evaluated the hypothesis that cytochrome P450 3A5 (CYP3A5) expression can affect intrarenal tacrolimus accumulation. Tacrolimus was administered orally to 24 healthy volunteers who were selected on the basis of their CYP3A5 genotype. As compared with CYP3A5 nonexpressors, expressors had a 1.6-fold higher oral tacrolimus clearance and 2.0- to 2.7-fold higher metabolite/parent area under the curve (AUC) ratios for 31-desmethyl tacrolimus (31-DMT), 12-hydroxy tacrolimus, and 13-desmethyl tacrolimus (13-DMT). In addition, the apparent urinary tacrolimus clearance was 36% lower in CYP3A5 expressors as compared with nonexpressors. To explore the mechanism behind this observation, we developed a semiphysiological model of renal tacrolimus disposition and predicted that tacrolimus exposure in the renal epithelium of CYP3A5 expressors is 53% of that for CYP3A5 nonexpressors, when normalized to blood AUC. These data suggest that, at steady state, intrarenal accumulation of tacrolimus and its primary metabolites will depend on the CYP3A5 genotype of the liver and kidneys. This may contribute to interpatient differences in the risk of tacrolimus-induced nephrotoxicity.

Pharmacokinetics of tacrolimus during pregnancy.

BACKGROUND: Information on the pharmacokinetics of tacrolimus during pregnancy is limited to case reports despite the increasing number of pregnant women being prescribed tacrolimus for immunosuppression. METHODS: Blood, plasma, and urine samples were collected over 1 steady-state dosing interval from women treated with oral tacrolimus during early to late pregnancy (n = 10) and postpartum (n = 5). Total and unbound tacrolimus as well as metabolite concentrations in blood and plasma were assayed by a validated liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) method. A mixed-effect linear model was used for comparison across gestational age and using postpartum as the reference group. RESULTS: The mean oral clearance (CL/F) based on whole-blood tacrolimus concentration was 39% higher during mid-pregnancy and late pregnancy compared with postpartum (47.4 ± 12.6 vs. 34.2 ± 14.8 L/h, P < 0.03). Tacrolimus-free fraction increased by 91% in plasma (f(P)) and by 100% in blood (f(B)) during pregnancy (P = 0.0007 and 0.002, respectively). Increased fP was inversely associated with serum albumin concentration (r = -0.7, P = 0.003), which decreased by 27% during pregnancy. Pregnancy-related changes in f(P) and f(B) contributed significantly to the observed gestational increase in tacrolimus whole-blood CL/F (r² = 0.36 and 0.47, respectively, P < 0.01). In addition, tacrolimus whole-blood CL/F was inversely correlated with both hematocrit and red blood cell counts, suggesting that binding of tacrolimus to erythrocytes restricts its availability for metabolism. Treating physicians increased tacrolimus dosages in study participants during pregnancy by an average of 45% to maintain tacrolimus whole-blood trough concentrations in the therapeutic range. This led to striking increases in unbound tacrolimus trough concentrations and unbound area under the concentration-time curve, by 112% and 173%, respectively, during pregnancy (P = 0.02 and 0.03, respectively). CONCLUSIONS: Tacrolimus pharmacokinetics are altered during pregnancy. Dose adjustment to maintain whole-blood tacrolimus concentration in the usual therapeutic range during pregnancy increases circulating free drug concentrations, which may impact clinical outcomes.

Absorption, distribution, and excretion of 14C-labeled tacrolimus (FK506) after a single or repeated ocular instillation in rabbits.

PURPOSE: The aim of this study was to investigate the absorption, distribution, and excretion of radioactivity in male rabbits after a single or repeated instillation of (14)C-labeled tacrolimus (FK506) ophthalmic suspension or an intravenous (i.v.) administration of (14)C-FK506. METHODS: The 0.3% (14)C-FK506 suspension was administered in single and repeated (three times, 5-min intervals) instillation studies, and 1 mg/kg of (14)C-FK506 was administered in the i.v. dose study. RESULTS: Results for single and repeated instillation studies were similar. In eyeball microautoradiograms, 15 min after dosing, the level of radioactivity in the cornea was the highest, followed by conjunctiva. After 1 h, little specific distribution was detected in the corneal epithelium, stroma, or Descemet's membrane. At 24 h, the level of radioactivity in the cornea decreased. Whole-body autoradiograms showed that the radioactivity was distributed to the digestive tract through the nasal meatus and esophagus and then was excreted into the feces. In the i.v. dose study, the distribution of radioactivity in whole-body autoradiographs was similar to that in quantitative tissue distribution measurements. The excretion of radioactivity in the urine and feces up to 168 h were 4.5 and 94.9%, respectively. CONCLUSIONS: After the ocular instillation, FK506 is first absorbed in the cornea, conjunctiva, and nasolacrimal duct, and then the rest is distributed to digestive tract through the nasal meatus and esophagus, after which it is excreted mainly into the feces.

The disposition of 14C-labeled tacrolimus after intravenous and oral administration in healthy human subjects.

Tacrolimus is a macrolide lactone with potent immunosuppressive properties. It has been shown in clinical studies to prevent allograft rejection. The pharmacokinetics of tacrolimus in healthy subjects and transplant patients has been described in earlier studies using immunoassay methods; however, detailed information on the absorption, distribution, metabolism, and excretion of tacrolimus using a radiolabeled drug is lacking. The objective of the present study was to characterize the disposition of tacrolimus after single i.v. (0.01 mg/kg) and oral (0.05 mg/kg) administration of 14C-labeled drug in six healthy subjects. Tacrolimus was absorbed rapidly after oral dosing with a mean Cmax and Tmax of 42 ng/ml and 1 h, respectively. The oral bioavailability was about 20%. After i.v. and oral dosing, most of the administered dose was recovered in feces, suggesting that bile is the principal route of elimination. Urinary excretion accounted for less than 3% of total administered dose. In systemic circulation, unchanged parent compound accounted for nearly all the radioactivity; however, less than 0.5% of unchanged drug was detectable in feces and urine. The excretion of the metabolites was formation-rate-limited. The mean total body clearance at 37.5 ml/min was equivalent to about 3% of the liver blood flow. Renal clearance was less than 1% of the total body clearance. The mean elimination half-life was 44 h.

Simplified high-performance liquid chromatography-mass spectrometry assay for measurement of tacrolimus and its metabolites and cross-validation with microparticle enzyme immunoassay.

In this study, a modified, specific assay for measurement of tacrolimus and its metabolites in blood and urine from transplant patients using high-performance liquid chromatography (HPLC) linked to mass spectrometry (MS) is described. Samples were prepared for HPLC-MS by modified solid-liquid extraction. The original two-step washing procedure was replaced by a single washing step, and samples were eluted with acetonitrile/water instead of dichloromethane, thus avoiding an evaporation step. Samples were injected automatically every 3 min into the HPLC-MS system. Time-consuming gradient elution was replaced by isocratic elution. This procedure resulted in a lower limit of quantitation of 0.2 microgram/L. The interassay variability was 14.5% for 5 micrograms/L and 15.8% for 25 micrograms/L. The intrassay variability was 11.2% for 5 micrograms/L and 4% for 25 micrograms/L. The recovery for tacrolimus in blood was 90.4% for 1 microgram/L, 78.9% for 10 micrograms/L, and 81.3% for 25 micrograms/L. Measurement of tacrolimus and its metabolites in samples from various transplant patients showed that the main metabolites found in blood and urine are demethyl-tacrolimus, di-demethyl-tacrolimus and demethyl-hydroxy-tacrolimus. Cross validation of the modified HPLC-MS assay with a microparticle enzyme immunoassay showed a significant correlation between the two assays, with r = 0.915.

Specific and sensitive measurement of FK506 and its metabolites in blood and urine of liver-graft recipients.

A specific and sensitive assay for quantifying the immunosuppressant FK506 and its metabolites in blood and urine was developed. 32-O-Acetyl FK506 was synthesized and used as internal standard. FK506 and its metabolites were purified from the samples by solid-liquid extraction and were injected into a high-performance liquid chromatographic (HPLC) system linked to a mass spectrometer (MS) by particle-beam interface. The FK506 derivatives were separated from interfering material by use of a 100 x 4 mm C8 analytical column and water/acetonitrile or water/methanol gradient elution; they were detected by negative chemical ionization with methane as reagent gas. The limit of detection was 25 pg in a standard solution, and the limit of quantification in blood was 250 pg (extracted from 1 mL of blood). The CV was 11.3% at 5 ng, and no interferences with other drugs were found.