Sensitivity and specificity of isolated perfused guinea pig heart to test for drug-induced lengthening of QTc.

INTRODUCTION: The purpose of this study was to determine the sensitivity and specificity for predicting the liability of a compound to lengthen QTc using isolated, perfused guinea pig hearts (Langendorff preparation). METHODS: QTc (Fridericia correction) was calculated from bipolar transventricular electrograms. Hearts were exposed to escalating concentrations of 26 compounds thought to lengthen, and 13 compounds thought not to lengthen, QTc in humans. RESULTS: In this preparation, QTc was found to lengthen in 26 of 26 compounds thought to be positive (sensitivity 1.00) and not to lengthen or to lengthen insignificantly in 13 of 13 compounds thought to be negative (specificity 1.0) in man. Probucol and ontazolast could not be studied because of limited solubility. Successful experiments were conducted on over 98% of guinea pigs anesthetized. DISCUSSION: We believe that the isolated perfused guinea pig heart is an in vitro preparation that could be utilized early in preclinical testing for identifying a liability to lengthen QTc in humans, but we do not believe--as is true also for other in vitro methods--that the concentration at which the liability is demonstrated in vitro necessarily predicts the concentration at which a liability exists in man.

Use of short high-performance liquid chromatography columns and tandem-mass spectrometry for the rapid analysis of a prostaglandin analog, fluprostenol, in rat plasma.

A short reversed-phase HPLC column and a tandem mass spectrometer were used to develop a stable-isotope-dilution assay for the rapid and sensitive analysis of fluprostenol, a prostaglandin analog, in rat plasma. A Waters Symmetry ODS column (2.1x10 mm) afforded rapid isocratic elution of fluprostenol (t(R)=40 s) but still provided a relatively large k' value of 4. The use of tandem mass spectrometry allowed the interference-free detection of fluprostenol under the rapid elution conditions, with a limit of quantitation of 25 pg ml(-1) fluprostenol, using 0.2 ml plasma sample volumes. The method was linear over three orders of magnitude, yielded accurate and precise results and allowed the pharmacokinetic profile of fluprostenol to be defined following intravenous administration in rats.

Rapid and selective method for norepinephrine in rat urine using reversed-phase ion-pair high-performance liquid chromatography-tandem mass spectrometry.

A rugged, high-throughput HPLC-MS-MS-based method, suitable for quantitation of norepinephrine (NE) in urine, has been developed. A rapid, batch-mode procedure utilizes alumina to isolate NE and its deuterated internal standard from urine. After release of NE, using dilute formic acid, samples are analyzed by isocratic reversed-phase ion-pair HPLC, with electrospray ionization (ESI) and MS-MS detection. The ion-pair reagent, heptafluorobutyric acid, is compatible with the ESI interface and permits use of mobile phases with relatively high methanol content, enhancing ESI sensitivity. Furthermore, no significant drop in sensitivity is observed throughout more than 15 h of instrument operation. The selectivity of this approach permitted simplification of the extraction procedure and reduced run times (under 4 min), making single batch-run sizes of more than 200 samples practical. The lower limit of quantitation is 5 ng per 0.5 ml sample, with analytical recoveries of 97-100% and overall method precision of better than 4% relative standard deviation verified up to 500 ng ml(-1). This method was initially applied to study the diurnal rhythm in sympathetic nervous system activity of spontaneously hypertensive rats.

Analysis of a novel antiinflammatory agent, 1-(7-tert.-butyl-2,3-dihydro-3,3-dimethylbenzo[b]furan-5-yl)-4- cyclopylbutan-1-one (PGV-20229), in plasma matrices by stable-isotope-dilution gas chromatography-mass spectrometry.

A sensitive and selective GC-MS method was developed for the determination of low levels of a novel antiinflammatory agent, 1-(7-tert.-butyl-2,3-dihydro-3,3-dimethylbenzo[b]furan-5-yl)-4- cyclopropylbutan-1-one (I), in small volumes of animal plasma. The method involved the addition of 13C6-labeled-I to plasma samples, followed by a simple liquid-liquid extraction with hexane to isolate the analytes from matrix components. The levels of I in the sample extracts were determined by isotope-dilution GC-MS analysis using selected-ion monitoring. The method was linear over three orders of magnitude, with a limit of quantitation of 1.8 ng/ml I, using plasma sample volumes of 0.1 ml. The method was utilized to determine the pharmacokinetic parameters of I in rats and dogs, following intravenous administration.

Highly sensitive high-performance liquid chromatographic-tandem mass spectrometric method for the analysis of dextromethorphan in human plasma.

A stable-isotope-dilution HPLC-tandem mass spectrometry-based method was developed for the determination of dextromethorphan in human plasma. Plasma samples were prepared for analysis by solid-phase extraction on octadecylsilane extraction cartridges. Dextromethorphan and the deuterium-labeled dextromethorphan internal standard were chromatographed on a short reversed-phase column and detected by a selected-reaction-monitoring scheme. Linear standard curves were obtained over three orders of magnitude and the limit of quantitation for dextromethorphan was 50 pg/ml, using a 1-ml plasma sample. The combination of HPLC and electrospray tandem mass spectrometry resulted in a rapid, selective and sensitive method for the analysis of dextromethorphan in plasma. The method was applied for the evaluation of the pharmacokinetic profile of dextromethorphan in human volunteers following peroral administration.

Preclinical skin sensitization testing of antihistamines: guinea pig and local lymph node assay responses.

Preclinical test methods for allergic contact sensitivity have been widely used for sensitization hazard identification and, with consideration of human exposure conditions, have also been valuable tools for sensitization risk assessment. For many years, the guinea pig has been the test species of choice with a variety of test methods developed to assess the sensitization response. More recently the local lymph node assay (LLNA) in mice has been developed to provide a more objective index of sensitization potential. The standardized methods have proven to be very well suited to most situations in which potential skin sensitization of a chemical needs to be assessed before human exposure. A potential difficulty with all these relatively limited exposure preclinical test methods, however, is in the ability to detect weak contact allergens that prove to be significant clinical allergens due to chronic topical exposure, exposure to compromised skin, and/or highly exaggerated exposure through transdermal delivery. This has been shown with the transdermal drug clonidine and might also be the case for topical antihistamines. The latter are considered significant clinical contact allergens, although predictive preclinical test data are minimal or lacking. A series of guinea pig (modified Buehler) tests with two common antihistamine compounds (triprolidine and diphenhydramine) and LLNA on these and two other compounds (chlorpheniramine and promethazine) was conducted. Positive Buehler test results required use of penetrating vehicle systems and a modified nine-induction patch regimen. Positive LLNA responses were obtained with all four materials (to varying degrees) only if the application site was pre-abraded or a penetrating vehicle (dimethylformamide) was used. These data support the notion that preclinical sensitization test methods can be modified to increase sensitivity. This may be critical for preclinical assessment of topical/transdermal drugs or other materials with chronic or high-concentration exposures in man.

Interspecies scaling of tebufelone pharmacokinetic data and application to preclinical toxicology.

PURPOSE: Retrospective application of allometric scaling techniques to tebufelone, a nonsteroidal antiinflammatory agent, in order to better understand the systemic exposure relationships between the doses administered to the species used in toxicology studies and the doses given to human subjects and patients in clinical studies. METHODS: Non-compartmental estimates of tebufelone's total body volume of distribution during the terminal phase (Vz) and clearance (CL) obtained from intravenous dosing to rat, monkey, dog, and human were allometrically scaled to body weight, and body weight and brain weight, respectively. AUCs determined from single or multiple dose pharmacokinetic studies and from preclinical toxicology studies were plotted versus dose adjusted for bioavailability and divided by allometrically scaled clearance to produce an allometric relationship suggesting a non-linear increase in AUC with dose across the four species. RESULTS: Segmental linear regression analysis of this relationship indicates a change point associated with an AUC of approximately 2,300 ng-hr/mL. Elevations in serum levels of various liver enzymes or associated signs of hepatic toxicity occur in some, but not all of the animals exposed for more than three weeks in repeat dosing studies at the actual dose that this represents. CONCLUSIONS: The analysis suggests that doses producing tebufelone plasma levels above a certain threshold AUC and duration of exposure to parent tebufelone are associated with increased risks of hepatic effects. Whether this is because metabolic shifts occur at these doses cannot be determined from these data.

Evaluation of the primary skin irritation and allergic contact sensitization potential of transdermal triprolidine.

A transdermal patch for the OTC antihistamine, triprolidine (TP), might provide benefits in terms of increased efficacy and reduced sedative side effects. However, concerns over potential irritant or allergic contact sensitization (ACS) skin reactions necessitated through skin toxicity testing before and during initial clinical development. Initial effort was expended on development of a binary vehicle delivery system comprised of TP in 0.5% oleic acid (OA) in propylene glycol (PG). Rabbit skin irritation and Buehler guinea pig skin sensitization testing indicated that this TP/OA/PG formula had both skin irritation and ACS potential. Both tests underestimated, to some degree, the skin toxicities observed in later clinical testing. In clinical tests, skin irritation was due mainly to the OA/PG vehicle, but was enhanced in the presence of high TP concentrations. Of 26 subjects enrolled in a rising dose clinical pharmacokinetics study, one subject exposed twice to TP/OA/PG presented with delayed skin reactions suggestive of ACS. Positive diagnostic patch test results for this subject and four out of five other twice-exposed study subjects suggested that the TP/OA/PG formula had a very high ACS potential. Subsequent predictive clinical patch testing was conducted with a buffered aqueous TP formula which provided in vitro skin penetration of the drug equivalent to the TP/OA/PG formula. These clinical studies demonstrated that TP itself had no significant irritation potential but still induced ACS reactions in a high proportion of test subjects. The incidence of adverse skin reactions to TP was considered to be too high relative to the degree of improved therapeutic benefit of this delivery form. On this basis, all technology development effort was discontinued.

Evaluation of compounding accuracy and aseptic techniques for intravenous admixtures.

Intravenous admixtures containing potassium collected from three hospital pharmacies were analyzed for compounding accuracy, sterility and pyrogenicity. The study was performed in two stages. During stage I, pharmacists and technicians were not informed of the study, but during stage II they were informed. In each stage 10 samples were collected from each person in the two personnel groups, analyzed and the results compared between the two personnel groups and the two stages. Results of the study showed that without monitoring (stage I) pharmacists had a higher mean percent error and contamination level than technicians. With monitoring, however, pharmacists showed a lower mean percent error and contamination level than technicians. Both personnel groups showed a decline in their mean percent error in the second stage, but there were still 83 (39.5%) errors in compounding accuracy greater than +/- 6%. No positive results with the Limulus test for pyrogens were obtained. It is recommended that a planned program of quality control be instituted for the preparation of i.v. admixtures by both pharmacists and technicians.

Estimation of drug dosage regimens with a pharmacokinetic slide rule.

A pharmacokinetic slide rule to facilitate the computations based on relatively simple pharmacokinetic principles involved in the development of individualized drug dosage regimens is described. The calculations are based on the assumption that the body can be conceived as a one-compartment open model with drug elimination proceeding by apparent first-order kinetics. Examples are presented (1) to illustrate the clinical application of a slide rule to compute the time-course of drug in the body, (2) to calculate steady-state maximum and minimum levels, and accumulation during multiple dosage and (3) to estimate appropriate maintenance doses and intravenous infusion rates.

Binding of salicylate and sulfathiazole by whole blood constituents.

The binding of salicylic acid and sulfathiazole to bovine whole blood, plasma proteins, and purified albumin fraction was investigated using a dynamic dialysis system. The binding profiles for salicylic acid were quite similar in bovine plasma and 4% bovine serum albumin. In contrast, the binding of sulfathiazole was significantly greater in the plasma than in solutions of fraction V bovine serum albumin. Data from dynamic dialysis binding studies of the compounds, conducted in whole blood and suspended erythrocyte systems, did not lend themselves to analysis by classical methods. Hemolysis and alteration in the nature of the protein binding sites during the binding studies were shown to be factors that could explain the unusual binding observed in the whole blood system.