Evaluation of a new immunoassay to measure sirolimus blood concentrations compared to a tandem mass-spectrometric chromatographic analysis.

A new immunoassay of sirolimus based on the microparticle enzyme immunoassay (MEIA) principle has been developed with collaboration of Abbott Diagnostics. Laboratories and Axis-Shield. Our laboratory evaluated this new assay on 153 whole blood samples (EDTA) drawn from a population of renal (n = 141) and hepatic (n = 12) transplant patients. Each blood sample was analyzed simultaneously by MEIA (Y) and by a reference method (X) used routinely in our laboratory, namely, liquid chromatography tandem mass spectrometry (LC-MS/MS). The statistical analysis of Passing-Bablok produced the following results: Spearman r value = 0.95, slope = 1.15 and intercept with the Y axis = +0.2 ng/mL. The observed global overestimation of 15% compared to the reference method could be explained by the cross-reactivity of sirolimus metabolites with the antibody. A complementary analysis taking into account the transplanted organ (kidney versus hepatic) did not show any significant difference between the populations, most likely owing to the low number of hepatic transplantation samples. The analytical performance of the MEIA method showed CV < or =10% and a limit of quantification of 1.5 ng/mL, which were acceptable for routine clinical monitoring. In conclusion, the MEIA method has shown robust, stable, reproducible, features with an excellent correlation with the reference LC-MS/MS method.

Comparative clinical pharmacokinetics of tacrolimus in paediatric and adult patients.

Tacrolimus is a potent immunosuppressive agent used to prevent allograft rejection. The pharmacokinetics of tacrolimus have been studied in healthy volunteers and transplant recipients, mostly by using immunoassays to measure tacrolimus in plasma or blood. However, because of the cross-reactivity for certain tacrolimus metabolites of the antibodies used, these methods often lack specificity. This should be carefully taken into account when interpreting pharmacokinetic results for tacrolimus. In adult patients, tacrolimus is generally rapidly absorbed following oral administration (the time to reach maximum concentration is 1 to 2 hours), but in some patients absorption is slow or even delayed. Because of presystemic elimination, the oral bioavailability is low (around 20%) but may vary between 4 and 89%. Tacrolimus is highly bound to erythrocytes. Its binding to plasma proteins varies between 72 and 98% depending on the methodology used. Because of the extensive partitioning of tacrolimus into erythrocytes, its apparent volume of distribution (Vd) based on blood concentrations is much lower (1.0 to 1.5 L/kg) compared with values based on plasma concentrations (about 30 L/kg). Tacrolimus is metabolised by cytochrome P450 (CYP) 3A4 to at least 10 metabolites, some of which retain significant activity. Biliary excretion is the route of elimination of the tacrolimus metabolites. Systemic plasma clearance of tacrolimus is very high (0.6 to 5.4 L/h/kg), whereas blood clearance is much lower (0.03 to 0.09 L/h/kg). The terminal elimination half-life (t1/2beta) of tacrolimus is approximately 12 hours (with a range of 3.5 to 40.5 hours). Only limited information is available on the pharmacokinetics of tacrolimus in paediatric patients. The rate and extent of tacrolimus absorption after oral administration do not seem to be altered in paediatric patients. The Vd of tacrolimus based on blood concentrations in paediatric patients (2.6 L/kg) is approximately twice the adult value. Blood clearance of tacrolimus is also approximately twice as high in paediatric (0.14 L/h/kg) compared with adult (0.06 L/h/kg) patients. Consequently, t1/2beta does not appear modified in children, but oral doses need to be generally 2-fold higher than corresponding adult doses to reach similar tacrolimus blood concentrations. More pharmacokinetic studies in paediatric patients are, however, needed to rationalise the use of therapeutic drug monitoring for optimisation of tacrolimus therapy in this patient population.

Hyperhomocysteinemia in type 2 diabetes: relationship to macroangiopathy, nephropathy, and insulin resistance.

OBJECTIVE: The aim of this study was to determine the distribution of plasma total homocysteine (tHcy) concentrations in type 2 diabetic patients and to assess whether high tHcy values were related to chronic complications (particularly macroangiopathy and nephropathy) and/or the degree of insulin resistance. RESEARCH DESIGN AND METHODS: Fasting tHcy levels were measured in 122 type 2 diabetic patients in whom the presence of chronic complications (e.g., macroangiopathy, microalbuminuria, macroproteinuria, decreased creatinine clearance, hypertension, retinopathy, and neuropathy) was recorded alongside an assessment of insulin resistance by the homeostasis model assessment (HOMA). RESULTS: We found that 31% of the cohort (group 1) had raised tHcy (mean +/- 1 SD) values (20.8 +/- 5.1 micromol/l), whereas 69% (group 2) had normal values (10.2 +/- 2.0 micromol/l). The prevalence of macroangiopathy was higher in group 1 than in group 2 subjects (70 vs. 42%, P < 0.01); the prevalence of coronary artery disease was particularly higher in group 1 (46 vs. 21%, P < 0.02). The prevalence of impaired renal function, evidenced by decreased creatinine clearance, was higher in group 1 (32 vs. 10%, P < 0.005). Other clinical and biological characteristics of both groups were comparable, although group 1 had lower levels of folic acid than group 2 (5.2 +/- 2.9 vs. 7.0 +/- 3.4 ng/ml, P < 0.01). No differences were found for microalbuminuria (33 vs. 31%), retinopathy (45 vs. 42%), or neuropathy (70 vs. 59%) between groups 1 and 2, respectively The degree of insulin resistance was similar in groups 1 and 2 (46 +/- 21 and 42 +/- 20% of HOMA-insulin sensitivity) as was the assessment of beta-cell function (63 +/- 28 and 65 +/- 46%, respectively). No differences in tHcy levels were found between subjects receiving metformin and those not receiving metformin. In contrast, the plasma tHcy level was higher in diabetic patients treated with fibrates (P = 0.0016). CONCLUSIONS: Elevated plasma tHcy levels in type 2 diabetes is associated with a higher prevalence of macroangiopathy and nephropathy when assessed from creatinine clearance indexes and is not associated with different degrees of insulin resistance.

Simultaneous detection and quantification of acidic and basic drugs in serum toxicological screening: use of ion-pairing technique in liquid-liquid extraction.

The toxicological screening, using the combination of high performance liquid chromatography with diode array ultraviolet detector and ion-pairing technique in liquid-liquid extraction, is an effective tool in the identification and quantification of the acidic and basic substances in a single run. The use of an ion-pairing technique in the conventional extraction shows the co-extraction of the uncharged and charged form of the analytes present in a serum sample. The stationary phase used is C-18-bonded phase. The mobile phase is acetonitrile--phosphate buffer (pH 3; 25 mM) containing 25 mM triethylammonium as ion-pairing agent. The analytical validation shows reproducible recoveries, good day-to-day repeatability and sensible results compatible with clinical and forensic use.

Pharmacokinetics of tacrolimus (FK506) in paediatric liver transplant recipients.

The pharmacokinetics of intravenous and oral tacrolimus was assessed in paediatric liver transplant patients at two centers in Europe. Sixteen patients, age 0.7 to 13 years, participated in the study; 12 patients were evaluable for intravenous pharmacokinetics, and 16 for oral. Intravenous tacrolimus was given as a continuous 24 h infusion (mean 0.037+/-0.013 mg/kg/day), and oral tacrolimus was given in 2 doses per day (mean 0.152+/-0.015 mg/kg). Whole blood samples for the intravenous pharmacokinetic profile were taken before initiation of the first infusion, 4, 8, 12 and 24 h post-infusion, and every 24 h thereafter until intravenous administration was discontinued. During the 12 h wash-out period between intravenous and oral administration, samples were taken every 3 h. Samples for the oral pharmacokinetic profile were taken immediately before the first oral dose and 0.5, 0.75, 1, 2, 2.5, 3, 4, 6, 8, 10 and 12 h post-administration. Non-compartmental procedures were used to characterise the pharmacokinetic parameters. Mean estimates for clearance and terminal half-life were 2.3+/-1.2 ml/min/kg and 11.5+/-3.8 h, respectively, following intravenous tacrolimus. The mean bioavailability of oral tacrolimus was 25+/-20%. A strong correlation was observed between AUC and trough whole blood levels of tacrolimus (r=0.90). The clearance was approximately 2-fold higher than that previously observed in adults; this could explain the higher dosage requirements in children.

The monoethylglycinexylidide test does not correctly evaluate lidocaine metabolism after ischemic liver injury in the rat.

Although the monoethylglycinexylidide (MEGX) test defined as a single determination of MEGX plasma concentration after lidocaine injection has been proposed as a liver function test, some discrepancies appeared in assessing the quality of liver donor for transplantation as well as the severity of liver disease. The present study used a severe ischemia-reperfusion liver injury (IRI) in rat to evaluate the various factors able to influence the level of MEGX. The metabolism of lidocaine was studied on microsomes isolated from intact rats and from rats submitted to this liver injury. A significant reduction of the various pathways transforming lidocaine but also MEGX was demonstrated. Lidocaine inhibited the MEGX transformation both in intact and injured liver microsomes. In vivo, plasma MEGX concentrations, determined by high-performance liquid chromatography (HPLC), were lower in IRI than in controls up to 80 minutes after lidocaine injection but not later. By contrast, using the usual commercial fluorescence polarization immunoassay (FPIA), MEGX concentrations were paradoxically higher in IRI than in controls. Moreover, MEGX values obtained using FPIA were threefold higher in controls and ninefold higher in IRI than with HPLC. It was shown that these differences were related to the detection by FPIA of free and mainly of conjugated hydroxy-MEGX that accumulated in plasma from rats submitted to an IRI. These data emphasize the complexity of factors influencing the appearance and disappearance of MEGX because of delayed MEGX formation with liver injury but also to inhibition of its further metabolization. The choice of the sampling time for MEGX determination is critical and has to be optimized in every type of liver injury. Moreover, a specific technique, such as HPLC, will avoid cross-reactivity with other metabolites, which may be particularly abundant when the biliary excretion is impaired.

Excretion of tacrolimus glucuronides in human bile.

Tacrolimus is extensively metabolized by the cytochrome P-450 system. Hepatic metabolic phase I reactions of tacrolimus include mainly demethylation and/or hydroxylation. No valid data have been published on phase II pathways (glucuronide- or sulfo-conjugation). In order to investigate these pathways, different beta-glucuronidase/sulfatase enzyme preparations were used to hydrolyse the conjugates potentially present in human bile extracts. Two analytical methods were used: a non-specific method, MEIA, and a specific combined HPLC/MEIA method. The influence of the extraction pH was investigated. After beta-glucuronidase hydrolysis and extraction at pH 5, tacrolimus concentrations, obtained either from HPLC-MEIA or MEIA, always appeared significantly higher, suggesting the presence of glucuronides in the bile. When the extraction was performed at pH 1.5, only the HPLC-MEIA concentrations appeared higher after hydrolysis. MEIA concentrations obtained before and after hydrolysis were similar. These data are consistent with the fact that glucuronides are extracted at pH 1.5 but not at pH 5 and suggest first that, without hydrolysis, the extracted glucuronides are separated from the tacrolimus fraction in the HPLC-MEIA procedure, and second, that the glucuronides are cross-detected by the monoclonal antibody in the immunoassay. From these data, it is concluded that clues have been found, suggesting the presence in human bile of tacrolimus glucuronides, which cross-react with the monoclonal antibody, provided they are extracted in the sample tested.

Clinical pharmacokinetics of Neoral in pediatric recipients of primary liver transplants.

Pediatric liver transplant recipients constitute a population characterized by a particularly unpredictable and poor bioavailability of cyclosporin (CyA). Even though several adult studies show that the new oral formulation of CyA, Neoral (NEO), produces better bioavailability and blood level predictability, few data describe its pharmacokinetics in children. We performed a complete analysis of the pharmacokinetics of NEO in ten small children after primary liver transplantation. Three pharmacokinetic profiles were set up with data obtained from tests taken during i.v. administration of CyA, after the first oral NEO dose, and after the last NEO dose before discharge from the hospital. The mean half-lives obtained were 8.1, 7.7, and 6.9 h, respectively, and the bioavailabilities were 22% and 21% for the first and last NEO doses. A large interpatient variability was observed. This was due, in part, to episodes of diarrhea that interfered with the pharmacokinetic evaluation and, in part, to the variability of post-transplant hepatic function. There was a good correlation between CyA trough levels and their related AUCs for both NEO profiles (r = 0.93 and r = 0.74, respectively). We conclude that, even though the pediatric OLT population remains more unpredictable than that of adults, NEO has a relatively rapid half-life and a remarkably improved bioavailability.

HPLC-microparticle enzyme immunoassay specific for tacrolimus in whole blood of hepatic and renal transplant patients.

Tacrolimus is a relatively new immunosuppressant used in organ transplantation to prevent graft rejection. However, its use is not devoid of side effects, making it important to maintain blood concentrations within therapeutic ranges. Several analytical methods are currently available for routine drug monitoring. However, these methods are based on use of the same monoclonal antibody, which also cross-reacts with some metabolites, resulting in overestimation of some blood concentrations. Even though this antibody appears appropriate for therapeutic drug monitoring, no reference method measures only the parent drug, mainly because of the poor absorptivity of tacrolimus in ultraviolet light. We have developed a method displaying an increased specificity towards the unchanged drug, using conventional equipment available in most clinical laboratories. After chromatographic separation of the blood extract, the tacrolimus fraction is analyzed by an automated microparticle enzyme immunoassay (MEIA) performed on the IMx analyzer (Abbott Labs.). This method is linear from 0 to 40 micrograms/L, yields CVs from 8.5% to 18.2%, and has a detection limit of 5 micrograms/L. Tacrolimus concentrations obtained by HPLC-MEIA in hepatic and renal transplant patients are from 47.5% to 18.8% lower than those obtained by MEIA, according to liver function tests and metabolite accumulation, even though no significant differences were observed between the methods for drug-free blood samples supplemented with known amounts of tacrolimus.

Inhibitory effects of solvent and solid-phase extraction residues on mixed lymphocyte cultures.

Residues from eight solvents/solvent mixtures before and after their passage through C18- bonded phase columns were assayed for cytostatic activity using mixed lymphocyte cultures (MLC). All residues, except those from acetonitrile, exhibited cytostatic activity (15%-35% MLC inhibition as measured by 3H-thymidine incorporation). Passage of solvents through bonded phase columns contributed an additional and significant cytostatic effect (19%-69% MLC inhibition). Pretreatment of columns with methanol led to further increases in the release of cytostatic residues from the columns, only when followed by less polar solvents (hexane, ethylacetate, etc.). It is concluded that residues from solid-phase extraction columns may interfere with subsequent cell culture-based assays for proliferative/antiproliferative activity.

Conversion from cyclosporine to FK506 for salvage of immunocompromised pediatric liver allografts. Efficacy, toxicity, and dose regimen in 23 children.

Twenty-three pediatric liver transplant recipients (median age 3.9 years) were converted from cyclosporine A-based immunosuppression to FK506 for uncontrollable acute rejection (AR; n = 16), chronic rejection (n = 4), or predominantly nonspecific hepatitis (n = 3). Of these, 19 had received poly- or monoclonal anti-T lymphocyte antibodies either for AR prophylaxis or therapy before FK506 conversion. Full clinical and histologic responses to FK506 therapy were observed in 11/16 cases of AR compared with 0/7 cases of non-AR indications (P = 0.006). Acute FK506 toxicity included renal dysfunction in 12/23 children (52%), neurological disorders in 7/23 (30%), and isolated hyperkalemia in 2/23 (9%), with a poor correlation with the corresponding FK506 trough plasma level. Moreover, a significant impairment of glomerular filtration rate was recorded in the 12 children who received FK506 treatment for more than 6 months (P = 0.002). FK506 therapy had to be definitively withdrawn in 6 cases (fatal infections: n = 4; persistent tremor: n = 1; reason unrelated to FK506: n = 1). Five children developed a lymphoproliferative syndrome (LPS), leading to death in 3 cases despite cessation of the immunosuppressive therapy; in the other 2 patients, LPS was controlled, and the children were successfully retransplanted for chronic rejection under FK506. The occurrence of Epstein-Barr virus primary infection under FK506 therapy was found to constitute a significant risk factor for LPS (P = 0.027). In summary, full response to FK506 conversion was observed in 69% of uncontrollable AR cases; however, 74% and 22% of this probably over-immunosuppressed population experienced major adverse events and LPS under FK506 therapy, respectively.

FK506 (tacrolimus), a novel immunosuppressant in organ transplantation: clinical, biomedical, and analytical aspects.

The macrolide immunosuppressant FK506 (tacrolimus) is a powerful and selective anti-T-lymphocyte agent that was discovered in 1984. This agent, isolated from the fungus Streptomyces tsukubaensis, has a mechanism of action similar to that of cyclosporine. Experimental data were first published in 1987, and clinical trials were started 2 years later in Pittsburgh. The drug has a potent hepatotrophic effect, which could explain its success in liver transplantation. Particularly encouraging results were obtained in liver allograft recipients, suggesting a lower risk/benefit ratio than with other immunosuppressants. However, recent data show that the drug is not devoid of toxicity (mainly nephrotoxicity), which should the percent the need for careful blood monitoring. Several methods of analysis have been described, some satisfactory, others inadequate for routine monitoring. There is still a lack of specific methods to determine routinely the parent drug concentrations in biological fluids for clinical pharmacokinetics purposes. Despite greater experience in therapeutic drug monitoring, the correlation between FK506 concentrations and efficacy or toxicity is still unclear. More investigations are required to better understand and determine the appropriate use of FK506 in organ transplantation and treating autoimmune diseases.

Improvement and assessment of enzyme-linked immunosorbent assay to detect low FK506 concentrations in plasma or whole blood within 6 hours.

FK506, a promising new immunosuppressant, is currently under clinical investigation. Because dose-dependent toxicity is possible, blood concentrations of FK506 should be monitored. We improved the original ELISA of FK506 by shortening the incubation time. With some modification of materials, results are obtained within 6 h instead of 2 days, with similar or even better precision. Internal and external quality-control programs showed that our results correlated satisfactorily both with values determined by the original method and the theoretical expected values. Either plasma (detection limit 0.1 microgram/L) or whole-blood (detection limit 1 microgram/L) samples can be used. The sensitivity of the method makes it particularly useful for accurate pharmacokinetic studies or measurement of low blood concentrations. Twenty-four drugs and nine biological variables showed no significant interference on the assay. Study of the concentration- and temperature-dependent distribution of FK506 shows that the drug is largely bound to erythrocytes (ratio of blood to plasma concentrations is 10-40); as the erythrocytes become saturated, more of the drug is found in the plasma. Plasma concentrations may vary according to the blood temperature. We conclude that whole blood should be used for FK506 monitoring, as it is for monitoring cyclosporine.

Computerized approach to monitoring aminoglycosides.

Aminoglycosides are still used extensively in the treatment of nosocomial infections with Gram-negative bacteria. However, the treatment is associated with several adverse effects. Aminoglycosides monitoring is therefore essential to prevent toxic accumulations and to reach therapeutic concentrations. A computer program, PHARMONITOR, has been developed to optimize aminoglycosides monitoring, responding to the demands of most clinical daily situations. This program, based on a one-compartment open pharmacokinetics model, is developed for IBM PC-compatible computers, using D-Base III+. It can calculate t1/2, Vd, Cldrug, Cpmax, and the theoretical optimal dose and interval and also evaluates the creatinine clearance. The program has been conceived to allow maximal speed, flexibility, and reliability by the use of (e.g.) a linear least-squares analysis, the possible reference to previous protocols, the extensive use of keywords to classify and recall patients according to their pathologies, the development of messages recommending maximal dose or minimal dosing interval, and increasing the safety of the analysis. We consider the program a valuable tool for adjusting aminoglycoside dosage in individuals.