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K L Napoli

Publications and source records attributed to K L Napoli.

At least 37 records · Page 2Linked to original sources
PubMed1996

Radioreceptor assay for sirolimus.

OBJECTIVES: To develop a radioreceptor assay (RRA) for sirolimus (rapamycin, RAPA). METHODS: A direct methanol extraction was used to prepare 45 patient samples for the RRA. Results were compared to the results obtained previously using high-performance liquid chromatography (HPLC). Between-run precision, recovery, and drug interference studies were also performed. RESULTS: The RRA is sensitive to 1.0 microgram/L RAPA equivalents in whole blood. Comparison with HPLC yielded a correlation coefficient for 45 patient samples of 0.977. Between-run precision at 2.5, 7.5, 12.5, and 20 micrograms/L showed coefficients of variation (CVs) of 12.9, 9.2, 8.5, and 5.9%, respectively. Recoveries from the extraction procedure were 93% at 7.5 micrograms/L and 103% at 12.5 micrograms/L. Drug interference studies showed no interference in the RRA by cyclosporine (CsA), dexamethasone, prednisone, or methotrexate. CONCLUSION: We have demonstrated that the RRA for RAPA correlates well with HPLC, and has excellent precision and recovery. The procedure is far less time-consuming and complex than HPLC and has potential for automation.

Carrier Proteins↗
PubMed1996

Safety and efficacy of chloral hydrate sedation in children undergoing echocardiography.

The purpose of this prospective study was to determine the safety and efficacy of chloral hydrate sedation in children with known or suspected congenital heart disease. The study population included 405 children with a median age of 13 months (3 weeks to 14 years). Cyanotic heart disease was present in 64 of the children. The median dosage of chloral hydrate given was 77 mg/kg, with a range of 25 to 125 mg/kg. Sedation was achieved in 397 (98%) of the children. The complete study time averaged 2.2 hours (range, 1.6 to 5.2 hours). The time to achieve sedation was 30 minutes or less in 82%, more than 30 but less than 60 minutes in 12%, and more than 60 minutes in 4%; 2% failed to achieve sedation. Children aged 3 years or younger were more likely to be successfully sedated with chloral hydrate (p = 0.003). The type of heart disease did not affect the success of sedation. No child had a clinically significant change in heart rate or blood pressure during sedation; however, oxygen saturation decreased in 24 (6%) of 397 children successfully sedated. Decreases in oxygen saturation occurred more commonly in children with trisomy 21 (7/13) than in children without genetic syndromes (17/384). Vomiting occurred in 23 (6%) of the 405 study subjects, usually immediately after drug administration. Chloral hydrate is a safe and effective agent for sedation of children with known or suspected congenital heart disease who are undergoing echocardiography in the outpatient cardiology clinic.

Adolescent↗
PubMed1996

Sensitive and specific quantification of sirolimus (rapamycin) and its metabolites in blood of kidney graft recipients by HPLC/electrospray-mass spectrometry.

Sirolimus (rapamycin) has a macrolide structure and is under clinical investigation as an immunosuppressant after organ transplantation. An HPLC/mass spectrometry assay to quantify sirolimus in blood was developed. 28-O-Acetyl sirolimus was used as internal standard. Blood samples were extracted with C18 columns. The extracts were injected into an HPLC system and isocratically eluted with methanol/1% formic acid (90/10 by vol) from a 150 X 4 mm C18 analytical column. The HPLC system was connected to a triple-stage quadrupole mass spectrometer with an electrospray interface and positive ions were detected. The limit of quantification in 1 mL of blood was 0.25 microgram/L and the calibration curve in blood was linear up to 250 microgram/L. The recovery from blood was 88 +/- 26% and interassay variation at 1 microgram/L was 19% and at 15 microgram/L 9.3%. Hydroxy, dihydroxy, demethyl, and didemethyl sirolimus as well as sirolimus were detected in blood of kidney graft patients.

Acetic Anhydrides↗
PubMed1996

Routine clinical monitoring of sirolimus (rapamycin) whole-blood concentrations by HPLC with ultraviolet detection.

During phase I/II clinical trials of sirolimus (rapamycin; SRL), therapeutic drug monitoring was performed with a multistep liquid-liquid extraction of 1-mL aliquots of whole blood followed by reversed-phase HPLC with ultraviolet detection. Blood was sampled according to a standardized protocol and clinical status. SRL concentrations were interpolated from calibration curves with a linear range of 0-50 micrograms/L and 1 microgram/L lower limit of quantification. Quality control was monitored over 68 consecutive analytical runs by using frozen aliquots of SRL-supplemented pooled whole blood at 4, 12, and 32 micrograms/L. These samples showed mean concentrations of 3.7 +/- 0.6, 10.9 +/- 1.1, and 29.6 +/- 2.6 micrograms/L, respectively. This method for therapeutic drug monitoring of SRL permits one full-time technician to analyze 100 clinical specimens per week with a 24-h turnaround time. With this method, a strong linear relation (r2 = 0.946, Sy/x = 0.41, n = 115) between the average SRL concentration over a 24-h period and the SRL concentration at the 24th h was revealed.

Chromatography, High Pressure Liquid↗
PubMed1995

Oxygen radical formation during cytochrome P450-catalyzed cyclosporine metabolism in rat and human liver microsomes at varying hydrogen ion concentrations.

The role of pH in uncoupling the electron-flux between oxidoreductase and cytochrome P450 (P450) or P450 and cyclosporine (CyA) and resulting in the generation of oxygen radicals was investigated in vitro in rat and human liver microsomal preparations. Since the electron-flux from NADPH to cytochrome c via oxidoreductase showed a fairly constant reduction activity from pH 7.0-9.5, the generation of oxygen radicals at the level of P450-Cyclosporine (instead of oxidoreductase-P450) was investigated. The effects of increasing pH on oxygen radical formation was measured by the thiobarbituric acid assay (TBA) and the adrenochrome reaction. The trends in oxygen radical production were correlated with benzphetamine metabolism (production of formaldehyde) and CyA metabolism (analyzed by high performance liquid chromatography). The TBA assay showed increased MDA-detected lipid peroxidation (unrelated to autooxidation) at pH < 8.0 and pH > 8.0 (rat and human, respectively) while the adrenochrome reaction showed decreased oxygen radical production. When these results were compared to benzphetamine (a substrate of P450 2B and 3A) metabolism and CyA (a substrate of P450 3A) metabolism, increased metabolism followed the pH-dependent trend of MDA-detected lipid peroxidation. Benzphetamine metabolism with formaldehyde production and depletion of parent compound during CyA metabolism were increased at pH < 8.0 in the rat samples and at pH > 8.0 in the human samples. This parallel relation suggests that the increased metabolism of CyA at lower pH in rats and higher pH in humans may be the result of favorable interactions of P450 with Cyclosporine that also result in increased oxygen radical-related lipid peroxidation.

Adrenochrome↗
PubMed1994

Sample clean-up and high-performance liquid chromatographic techniques for measurement of whole blood rapamycin concentrations.

Determination of rapamycin (RAPA), a potential adjunct to cyclosporin immunosuppressive therapy in solid organ transplantation, presents a challenge because of the agent's unusual chemical properties. RAPA is not readily extracted from biological matrices, is light-sensitive, and exists in numerous isomeric forms. Using liquid-liquid extraction techniques with tert.-butyl methyl ether followed by ethanol, one achieves 96% recovery of RAPA and 70% recovery of the internal standard, beta-estradiol-3-methyl ether, from human whole blood. Subsequent high-performance liquid chromatography (HPLC) utilizing two heated reversed-phase C18 columns in tandem and an 85% methanol-water mobile phase with ultraviolet detection at 276 nm provides measurement of RAPA from 1-ml samples of human whole blood. The chromatography requires less than 40 min per sample. The assay exhibits < or = 10% standard error of the mean and < or = 12% coefficient of variation over the concentration range 2-100 ng/ml. The method has been tested using pharmacokinetic profiles from renal transplant recipients receiving bolus intravenous RAPA infusions.

Chromatography, High Pressure Liquid↗
PubMed1993

Generation of oxygen free radicals during the metabolism of cyclosporine A: a cause-effect relationship with metabolism inhibition.

A better understanding of the mechanism of lipid peroxidation during the metabolism of cyclosporine A (CsA) might help explain the toxicities of this immunosuppressive drug on various organs. Our in vitro work used microsomes prepared from livers of phenobarbital-induced male rats. The incubations (total volume 1ml) also contained a NADPH regenerating system and substrate (i.e., CsA, carbon tetrachloride, or aminopyrine) dissolved in ethanol. Lipid peroxidation was inferred from the presence of malondialdehyde (MDA) which was detected by the thiobarbituric acid assay. The formation of CsA hydroxylated metabolites (AM9 and AM1) was monitored by liquid chromatography. The activity of the microsomal incubation was confirmed by measurements of MDA and formaldehyde production caused by increasing concentrations of CsA, carbon tetrachloride, and aminopyrine. The occurrence of hydroxylated metabolites was not coupled to the production of MDA. Aminopyrine could inhibit MDA production by CsA, but CsA could not reduce the formation of formaldehyde by aminopyrine. Erythromycin, a competitor for the binding site of CsA on cytochrome P450, reduced MDA production by CsA, and CsA inhibited formaldehyde production by erythromycin. Interaction studies with SKF 525A, ketoconazole, superoxide dismutase, catalase, alpha-tocopherol, and reduced glutathione confirmed the role of cytochrome P450 and the presence of activated oxygen species as a source of microsomal peroxidation which in return may explain the inhibitory effect of CsA on cytochrome P450 itself.

Aminopyrine↗
PubMed1993

Adrenochrome reaction implicates oxygen radicals in metabolism of cyclosporine A and FK-506 in rat and human liver microsomes.

The role of oxygen radicals in the metabolism of cyclosporine A (CyA), FR900506 (FK-506) and carbon tetrachloride (CCl4) catalyzed by the cytochrome P450 system was investigated in vitro in rat and human microsomal preparations. Varying concentrations of CyA, FK-506 and CCl4 (100 microM-1.0 mM) were added to microsomal preparations, and lipid peroxidation was measured by malondialdehyde (MDA) formation as detected by the thiobarbituric acid assay. The effects of oxygen radical scavengers [superoxide dismutase (SOD) and catalase (CAT)] and an antioxidant [glutathione (GLUT)] were tested on various incubations of CyA, FK-506 and CCl4 to assess the role of oxygen radicals in lipid peroxidation. CyA-dependent MDA formation was moderately inhibited by SOD in the rat model and increased by SOD in the human model. In both models, CAT slightly inhibited CyA-dependent MDA formation and GLUT significantly inhibited MDA formation. FK-506-dependent MDA formation, studied only in the rat model, paralleled CyA-induced MDA formation but showed greater inhibition with CAT and less inhibition with SOD or GLUT. In both models, CCl4-dependent MDA formation was significantly inhibited by GLUT and showed no sensitivity to SOD or CAT. In addition, the adrenochrome reaction, which measures the oxidation of epinephrine to adrenochrome, was used to measure the increased oxygen radical-flux resulting from the metabolism of CyA, FK-506 and CCl4. CyA with epinephrine showed the highest oxidative activity, followed by FK-506 and then CCl4, which showed the least formation of adrenochrome. These results indicated a role for oxygen radicals in CyA and FK-506 metabolism.

Adrenochrome↗
PubMed1991

Prediction of acute graft rejection in renal transplantation: the utility of cyclosporine blood concentrations.

While cyclosporine is recommended to be used only in conjunction with monitoring of its blood concentrations, the utility of these measurements in preventing treatment failure is not established. In a group of 52 patients trough levels and steady-state concentrations were monitored in serum and whole blood by specific (SP) and nonspecific (NS) assays (polyclonal radioimmunoassay, PR; fluorescence polarization immunoassay, FP; high-pressure liquid chromatography, HP). From as many as 10 determinations of trough level and steady state concentrations during the first 40 days after renal transplantation, the lowest measurement was selected. In the case of an acute rejection episode within that time period, only values until that event were considered. Trough level measurements in serum by PR/NS and by FP/NS and in whole blood by HP/SP were not significantly different between patients with and patients without rejection episodes. However, simultaneously measured steady-state values (serum/PR/NS and serum/FP/NS) were significantly lower in patients suffering from rejection (with rejection SS/serum/PR/NS mean = 127 ng/ml, SD = 41 ng/ml; without rejection mean = 163 ng/ml, SD = 60 ng/ml; P = 0.027, t test). This difference could not be demonstrated for steady state/whole blood/HP/SP measurements. A logistic regression analysis demonstrated that the probability of rejection can be decreased by up to 40% if steady state/serum/PR/NS or steady state/serum/FP/NS values never drop below 250 ng/ml early after renal transplantation.

Adolescent↗
PubMed1991

Considerations for monitoring cyclosporine in transplant recipients.

Cyclosporine (CsA) monitoring has remained controversial since the advent of the use of CsA in organ transplantation in the early 1980s. This article attempts to clarify for the clinical laboratory worker many of the vagaries associated with CsA monitoring by considering choice of sample matrix, the nature of metabolites, the technical specifications of the assay techniques used for therapeutic monitoring of CsA, comparison of results assessed by the various assays, and the pharmacokinetic considerations associated with choice of sampling time. Expectations for the degree of clinical usefulness achievable for CsA levels through further improvement in assay methodologies are also discussed.

Chromatography, High Pressure Liquid↗
PubMed1990

Area-under-the-curve monitoring of cyclosporine therapy: performance of different assay methods and their target concentrations.

The measurement of areas under the concentration-time curve (AUC) was recently introduced as an alternative to trough level monitoring of cyclosporine therapy. The AUC is divided by the oral dosing interval to calculate an average concentration. All measurements are performed at clinical steady state. The initial evaluation of AUC monitoring showed advantages over trough level monitoring with concentrations of cyclosporine measured in serum by the polyclonal radioimmunoassay of Sandoz. This assay technique is no longer available and the following assays were performed in parallel during up to 173 AUC determinations in 51 consecutive renal transplant patients: polyclonal fluorescence polarization immunoassay of Abbott in serum, specific and nonspecific monoclonal radioimmunoassays using 3H and 125I tracers in serum and whole blood, and high performance liquid chromatography in whole blood. Both trough levels and average concentrations at steady state measured by those different techniques were significantly correlated with the oral dose. The best correlation (r2 = 0.54) was shown by average concentrations measured in whole blood by the specific monoclonal radioimmunoassay of Sandoz (3H tracer). This monitoring technique was also associated with the smallest absolute error between repeated observations in the same patient while the oral dose rate remained the same or was changed. Both allegedly specific monoclonal radioimmunoassays (with 3H and 125I tracer) measured significantly higher concentrations than the liquid chromatography. (ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, High Pressure Liquid↗
PubMed1990

Nonselective measurement of cyclosporine for therapeutic drug monitoring by fluorescence polarization immunoassay with a rabbit polyclonal antibody: I. Evaluation of the serum methodology and comparison with a sheep polyclonal antibody in an 3H-tracer mediated radioimmunoassay.

Among the new techniques available for CyA monitoring, the FPIA offers the advantages of rapidity and simplicity. The present communication assesses the technical performance of this test in comparison with a 3H tracer-based PC-RIA for serum CyA levels using 971 samples obtained during the first 6 posttransplant months from 14 kidney transplant recipients. The FPIA evaluation included verification of CyA concentrations in manufacturer-supplied calibrators and controls by reference methods, determination of intraassay/interassay precision and accuracy, feasibility of specimen dilution and assessed assay sensitivity, and range of linearity. Comparison of FPIA with PC-RIA indicated that trough samples, when assessed by FPIA, averaged 1.3-fold greater than the PC-RIA, whereas non-trough FPIA measurements indicated similarity between the two methods. Although the two assays showed similar trends in most renal transplant recipients, two subjects demonstrated discrepancies, presumably reflecting the differing specificities of the polyclonal antibodies used in each assay. Thus, the FPIA appears to be a useful addition to CyA monitoring technology.

Administration, Oral↗