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D W Holt

Publications and source records attributed to D W Holt.

At least 37 records · Page 2Linked to original sources
PubMed2000

Measurement of sirolimus in whole blood using high-performance liquid chromatography with ultraviolet detection.

BACKGROUND: Sirolimus, a potent immunosuppressive drug, exhibits intrapatient and interpatient variability of absorption and metabolism. Thus, therapeutic drug monitoring is important. OBJECTIVE: This paper describes a reverse-phase high-performance liquid chromatography (HPLC) method, using ultraviolet (UV) absorption for detection, for measuring sirolimus levels in human whole-blood samples. METHODS: The stability of sirolimus in whole blood was assessed under conditions likely to be encountered during transport of study samples to a central laboratory. The performance of the HPLC-UV assay in measuring sirolimus was compared with that of 3 established, validated HPLC assays with tandem mass-spectrometric (MS/MS) detection. Results of the HPLC-UV assay also were compared with results produced by a prototype microparticle enzyme immunoassay (MEIA). RESULTS: Inaccuracy for 3 in-house control samples was < or =4%, whereas within-assay repeatability (coefficient of variation [CV]) was < or =5% and between-assay reproducibility was < or =6.6%. Mean recovery of sirolimus from blood was 81.5%+/-4.3%. The lower limit of quantification was set at 6.5 ng/mL, and the repeatability CV at this concentration was 4.2% (n = 6). Sirolimus-containing whole-blood samples were stable for 3 freeze/thaw cycles when stored at -20 degrees C and for > or =2 days when stored at ambient temperature. The sample extract was shown to be stable for up to 54 hours at ambient temperature (approximately 22 degrees C) after extraction. Results of the HPLC-UV assay were consistent with those of the HPLC/ MS/MS assays but lower than those produced by MEIA. CONCLUSION: This HPLC-UV method is considered suitable for therapeutic drug monitoring of sirolimus.

Calibration↗
PubMed2000

An immunoassay for the measurement of sirolimus.

OBJECTIVE: This study assessed the performance characteristics of a new microparticle enzyme immunoassay (MEIA) for the determination of sirolimus in whole blood. BACKGROUND: In clinical investigatory studies, dose adjustments of the immunosuppressive drug sirolimus have been carried out using either high-performance liquid chromatography (HPLC) or, more recently, this investigational immunoassay kit based on the MEIA technique. METHODS: Calibration was made over the linear range 0 to 30 ng/mL. Inaccuracy and imprecision were assessed by means of 3 control samples supplied with the kit (5, 11, and 22 ng/mL) and dilution of an above-quantitation-limit sample (154 ng/mL). Specificity was determined by the addition of 2 sirolimus metabolites to sirolimus-free human whole blood or to I of the control samples supplied with the kit. In addition, whole-blood samples from patients receiving either cyclosporine or tacrolimus (N = 24) were analyzed for sirolimus. A comparison of the MEIA and a validated HPLC/MS/MS assay analyzed both pooled samples from patients receiving sirolimus and spiked samples (sirolimus 2-60 ng/mL). In a more extensive comparison of patient samples measured by the MEIA assay, a validated HPLC assay with UV detection (HPLC-UV) was used (HPLC-UV sirolimus 7-64 ng/mL). RESULTS: Inaccuracy (between-run) was < or =16.2% at all 4 concentrations (N = 5). Within-assay imprecision (repeatability) was <6% (N = 5), and between-assay imprecision (reproducibility) for the same samples was < 11% (N = 5). Recovery, assessed by means of 3 in-house control samples prepared in both fresh and previously frozen sirolimus-free human whole blood, ranged from 93.9% to 109.5%. The limit of detection, determined by dilution of the lowest nonzero calibrator (3 ng/mL), was set at 1 ng/mL, at which repeatability was 20.5% (N = 5). Five ng/mL of hydroxysirolimus cross-reacted with the assay by a mean of between 44% and 50% (N = 4); 5 ng/mL of 41-O-demethylsirolimus cross-reacted with the assay by a mean of between 86% and 127% (N = 4). Assay specificity was further challenged by ethylenediamine-tetraacetic acid (EDTA)-whole-blood samples from transplant patients not receiving sirolimus. These samples had tacrolimus and cyclosporine concentrations of 7.8 to 15.9 ng/mL and 38 to 485 microg/L, respectively. The median result was 0 ng/mL (third quartile, 0.7 ng/mL; maximum, 1.4 ng/mL); no value was above the lowest nonzero calibrator. The results of the comparison between the MEIA and the HPLC/MS/MS assay showed mean positive biases of 21% and 8% for the MEIA in measuring sirolimus in pooled patient samples and spiked samples, respectively. The results of the comparison of the MEIA and HPLC-UV median sirolimus concentrations were 18.2 and 20.1. Whole-blood samples anticoagulated with EDTA and containing sirolimus were stable for analysis by MEIA for 3 freeze-thaw cycles when stored at -20 degrees C and for 10 days when stored at 4 degrees C or at ambient temperature. A decline in sirolimus concentration occurred when samples were stored at 37 degrees C. CONCLUSION: The MEIA showed suitable precision across a clinically relevant concentration range. In terms of patient management, the practical significance of cross-reactivity with sirolimus metabolites remains to be assessed.

Antibody Specificity↗
PubMed2000

Proficiency-testing issues relating to sirolimus.

BACKGROUND: A need exists to document laboratory proficiency to (1) compare results produced by different analytical techniques and (2) ensure consistency of results from multiple testing sites. OBJECTIVES: To enable concentration-controlled studies of sirolimus to proceed with confidence, proficiency-testing schemes were put in place at laboratories selected to act as reference laboratories. The feasibility of establishing an ongoing proficiency-testing scheme was addressed with respect to sample stability. The scheme was then used to test proficiency for the measurement of sirolimus in 3 blinded samples each month. METHODS: The method chosen for measurement of sirolimus was a prototype microparticle enzyme immunoassay. Initially, 15 laboratories were asked to analyze a series of 85 blinded samples that tested their inaccuracy, repeatability, and reproducibility for the measurement and their ability to dilute over-range samples competently. Both blood samples spiked with sirolimus and pooled blood samples from patients receiving the drug were circulated to a maximum of 50 laboratories. RESULTS: Overall, both inaccuracy and imprecision were acceptable by predefined criteria. Inaccuracy for the immunoassay (percentage difference of the measured value against the nominal value) averaged -5% (95% CI, -9% to -1%). The mean percentage difference between the immunoassay and a high-performance liquid chromatographic assay with mass-spectrometric detection for the measurement of sirolimus in pooled samples (n = 5) from patients receiving the drug was 29% (95% CI, 24% to 34%). CONCLUSION: The techniques documented here as part of the International Sirolimus Proficiency Testing Scheme could be applied to other clinical studies requiring protocol-driven dosing adjustments based on sirolimus measurements, irrespective of analytical technique used.

Chromatography, High Pressure Liquid↗
PubMed2000

Pharmacokinetics of mycophenolate mofetil in patients with end-stage renal failure.

BACKGROUND: Mycophenolate mofetil (MMF) acts as a prodrug for the immunosuppressive drug mycophenolic acid (MPA). It is rapidly converted to MPA following oral ingestion. MPA is metabolized to MPA glucuronide (MPAG), which is renally excreted. This study examines the pharmacokinetics of MPA and MPAG in patients with end-stage renal failure who were on hemodialysis (N = 10) or peritoneal dialysis (N = 10) treatment. METHODS: After an overnight fast, a single oral dose of 1 g MMF was given. Plasma concentrations of MPA and MPAG were measured from 0 (predose) to 36 hours after administration, using high-performance liquid chromatography (HPLC). The area under the concentration time curve (AUC) from 0 to 36 hours was calculated using the trapezoidal rule. RESULTS: Mean (+/- SD) AUC for MPA was 55.7 +/- 32.6 mg/L.h for hemodialysis patients and 44.7 +/- 14.7 mg/L.h for peritoneal dialysis patients, which is similar to expected values for subjects with normal renal function. The mean (+/- SD) maximum plasma concentration (Cmax) for MPA was lower than would be expected for subjects with normal renal function (16.01 +/- 10.61 mg/L for hemodialysis, 11.48 +/- 4.98 mg/L for peritoneal dialysis). MPAG clearance was prolonged with AUC approximately five times what would be expected in subjects with normal renal function (1565 +/- 596 mg/L.h for hemodialysis, 1386 +/- 410 mg/L.h for peritoneal dialysis). There was no significant difference for any of the pharmacokinetic parameters between subjects on hemodialysis and those on peritoneal dialysis. Plasma concentrations of MPA and MPAG did not fall significantly during hemodialysis. No MPA was detectable in hemodialysis or peritoneal dialysis fluid, but small amounts of MPAG were detected in hemodialysis fluid in 1 out of 10 subjects and in peritoneal dialysis fluid in 3 out of 10 subjects. CONCLUSIONS: The accumulation of MPAG may be responsible for the poor gastrointestinal tolerance of this drug in dialysis patients and probably limits the maximum dose of MMF that can be tolerated.

Adult↗
PubMed2000

Accuracy of cyclosporin measurements made in capillary blood samples obtained by skin puncture.

International consensus guidelines suggest that cyclosporin should be measured in whole blood. In some instances it may be advantageous to collect capillary blood, by a finger or ear prick method. However, drug concentrations in skin-puncture blood may not necessarily correlate with those measured in venous blood. This study compared cyclosporin concentrations in blood collected from the fingertip or earlobe with blood collected by standard venipuncture. Patient preference for each of the blood collection methods was also assessed. Specimens were obtained from organ transplant patients receiving cyclosporin, using each of the three methods: venipuncture, finger prick, and earlobe prick. The samples were assayed using a specific radioimmunoassay and the results were compared. In the 102 sets of samples collected, the mean difference (+/- standard deviation) in cyclosporin concentration between finger prick and venipuncture and ear prick and venipuncture was 2.6% (+/- 9.5%) and 2.7% (+/- 12.1%), respectively, while the comparable median (IQR) differences were 1.9% (-3.4% to +6.6%) and -1.1% (-2.8% to +7.2%), respectively. A high degree of correlation was observed between finger prick and venipuncture or ear prick and venipuncture or ear prick and finger prick (r2 > 0.86). Of the three methods of blood collection, finger prick was the patients' preferred method (P < 0.01). These data suggest that capillary blood collected by skin puncture is suitable for use in cyclosporin blood monitoring and acceptable to patients.

Area Under Curve↗
PubMed1999

Current opinions on therapeutic drug monitoring of immunosuppressive drugs.

The pharmacokinetics of the immunosuppressive drugs cyclosporine, tacrolimus, mycophenolate mofetil (MMF), and sirolimus are complex and unpredictable. A narrow therapeutic index unique to each patient, as well as variable absorption, distribution, and elimination, are characteristics of these drugs. Therapeutic drug monitoring plays a key role in helping clinicians maintain blood and plasma levels of immunosuppressive drugs within their respective therapeutic ranges. Variation in concentrations outside the narrow therapeutic ranges can result in adverse clinical outcomes. Therapeutic drug monitoring ensures that concentrations are not too high or too low, thereby reducing the risks of toxicity or rejection, respectively. Therapeutic monitoring of immunosuppressive drugs has been based on several choices of assay and biologic fluid (i.e., whole blood, plasma) appropriate for a particular drug. High-performance liquid chromatography (HPLC) remains the gold standard among assay methods used to monitor immunosuppressive drugs. Although HPLC is the assay of choice for cyclosporine, newer monoclonal assays are suitable as well for routine monitoring. HPLC is also widely used for therapeutic drug monitoring of mycophenolic acid, the active metabolite of MMF, and an immunoassay (used in European centers) has been developed. Therapeutic drug monitoring of tacrolimus has been improved with the recent development of assays with greater sensitivity and specificity for tacrolimus than those previously available. No commercial assays are currently available for the therapeutic monitoring of sirolimus. It is also important to identify a specific pharmacokinetic parameter for each individual drug, whether it is trough or area under the concentration-time curve, that may be most useful as a tool for optimal therapeutic drug monitoring in clinical practice. With an increased understanding of the pharmacokinetics of immunosuppressive drugs, therapeutic drug monitoring guidelines will be more clearly defined to ensure the safe and effective management of transplant recipients.

Cyclosporine↗
PubMed1999

Measurement of mycophenolate mofetil plasma levels after heart transplantation and a potential side effect of high levels.

Mycophenolate mofetil (MMF) is gaining momentum in its use as an immunosuppressant and in the field of heart transplantation because of its efficacy and ease of use without a reported need to monitor plasma levels. We describe a case in which standard dosage of MMF (initially 1.5 g twice daily) produced elevated trough levels of mycophenolic acid (MPA). Although organ rejection was eradicated by the use of MMF, the patient developed severe anemia, which required repeated blood transfusions while the patient was on therapy. This case illustrates the potential value of monitoring MPA concentrations.

Adult↗
PubMed1999

Neuropsychological change and S-100 protein release in 130 unselected patients undergoing cardiac surgery.

BACKGROUND AND PURPOSE: S-100 protein promises to be a valuable surrogate end point for cerebral injury. This is of particular interest within the context of cardiac surgery. We sought to explore the relationship between change in neurospychological performance attributable to cardiopulmonary bypass and the release of brain-specific S-100 protein. METHODS: In an observational comparative study in a University Hospital Cardiac Surgical Unit, S-100 protein release during and 5 hours after the onset of cardiopulmonary bypass was compared with change (from preoperative to 6 to 8 weeks postoperative) in neuropsychological tests in 130 patients undergoing the full range of cardiac surgical procedures. RESULTS: Neuropsychological performance usually improved, being significantly so in 10 of 25 parameters. S-100 area under the curve (AUC) protein release correlated with age (r=0.24, P<0.008) and bypass time (r=0.17, P<0.02). S-100 Cmax correlated with bypass times (r=0.29, P<0.0001). Bypass times correlated with memory performance (Rey R5; r=-0.21, P<0.03). Less S-100 protein release was associated with better neuropsychological performance, as indexed by significant correlations with the Rey Auditory Verbal Learning memory test, descending Critical Flicker Fusion thresholds, and the Hospital Anxiety and Depression rating scales, typically around r=0.2. Multiple regression models showed that neuropsychological tests accounted for 23% of the variance associated with S-100 AUC release, after partialing out the effects of age and bypass time. CONCLUSIONS: The correlation between S-100 protein release and neuropsychological function supports the belief that it is a measure of brain injury, which may be useful in future studies of mechanisms and prevention.

Affect↗