Development and validation of LC-MS/MS assays for the quantification of E7080 and metabolites in various human biological matrices.

To support clinical pharmacokinetic studies with the anticancer agent E7080 (lenvatinib), liquid chromatography tandem mass spectrometry (LC-MS/MS) methods were developed for the quantification of E7080 and four of its metabolites in human plasma, urine and faeces and of E7080 in whole blood. Cross-analyte interferences between metabolites and parent compound were expected and therefore accounted for early in the method development. Plasma, urine and faeces samples were extracted with acetonitrile. Chromatographic separation was achieved on a 50 mm × 2.1 mm I.D. XTerra MS C18 column, with a 0.2 mL/min flow and gradient elution starting with 100% formic acid in water, followed by an increasing percentage of acetonitrile. Whole blood samples were extracted with diethyl ether and extracts were injected on a 150 mm × 2.1mm I.D. Symmetry Shield RP8 column. Detection was performed using an API3000 triple quadrupole mass spectrometer, with a turbo ion spray interface, operating in positive ion mode. Using 250 µL of plasma, E7080 and its metabolites could be quantified between 0.25 and 50.0ng/mL. The quantifiable ranges of E7080 in whole blood, urine and faeces were 0.25-500 ng/mL, 1.00-500 ng/mL and 0.1-25µg/g, using sample volumes of 250 µL, 200 µL and 250 mg, respectively. Calibration curves in all matrices were linear with a correlation coefficient (r(2)) of 0.994 or better. At the lower limit of quantification, accuracies were within ±20% of the nominal concentration with CV values less than 20%. At the other concentrations the accuracies were within ±15% of the nominal concentration with CV values below 15%. The developed methods have successfully been applied in a mass balance study of E7080.

Captive and wild orangutan (Pongo sp.) survivorship: a comparison and the influence of management.

For managers of captive populations it is important to know whether their management provides a species with the physical and social environment that maximizes its survivorship. To determine this, survivorship comparisons with wild populations and long-term evaluations of captive populations are important. Here we provide both for orangutans. We show that survivorship has increased during the past 60 years for captive orangutan populations in zoos. In addition, we show that survivorship of captive orangutans in the past used to be lower than for wild orangutans, but that for recently born (1986-2005) orangutans survivorship is not significantly different from the wild. This indicates that captive management in the past was suboptimal for orangutan survivorship, but that modern management of captive orangutans has increased their survivorship. We discuss the possible factors of modern management that could have influenced this.

Spontaneous use of magnitude discrimination and ordination by the orangutan (Pongo pygmaeus).

The ability to discriminate quantity is descriptive of general cognitive ability. In this study, the authors presented 2 orangutans (Pongo pygmaeus) with a quantity judgment task. Each trial consisted of 2 choices, ranging from 1 to 6 food items in each. The orangutan chose 1 of the quantities, which was removed, and the remaining array was given as a reward. In contrast to chimpanzees previously tested on the same task (S. T. Boysen & G. G. Berntson, 1995; S. T. Boysen, G. G. Berntson, M. B. Hannan, & J. T. Cacioppo, 1996; S. T. Boysen, K. L. Mukobi, & G. G. Berntson, 1999), the orangutans optimized their performance. Orangutans, therefore, attend to differences in magnitude and can spontaneously use ordinality. Results also suggest a cognitive difference between chimpanzees and orangutans.

Coadministration of phenytoin and felbamate: evidence of additional phenytoin dose-reduction requirements based on pharmacokinetics and tolerability with increasing doses of felbamate.

PURPOSE: This open-label study investigated the pharmacokinetic interaction of phenytoin (PHT) and felbamate (FBM). METHODS: Ten subjects with epilepsy receiving PHT monotherapy were administered increasing doses of FBM (1,200, 1,800, 2,400-3,600 mg/day) at 2-week intervals. PHT doses were reduced by 20% on an individual basis when evidence of clinically significant intolerance was present. With intolerance, the PHT dose was reduced before the next incremental FBM dose. Blood samples were analyzed for FBM, PHT, and PHT metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH). RESULTS: Total PHT plasma concentrations increased with coadministered FBM. PHT Cmax increased from 15.9 microg/ml at baseline to 20.9 microg/ml after 1,200 mg/day FBM and to 26.8 microg/ml after 1,800 mg/day FBM. Four subjects required a 20% PHT dose reduction after 1,800 mg/day FBM and six after the administration of 2,400 mg/day FBM. All subjects required further 20% PHT reductions before 3,600 mg/day FBM. FBM Cmax and AUCT were reduced, and apparent clearance increased compared with data from FBM monotherapy. CONCLUSIONS: With the initiation of FBM therapy in subjects receiving PHT, the PHT dosage should be reduced by 20%. Further PHT dose reductions are likely to be necessary if the FBM dose is increased. The requirements for reductions in dose might be predicted by clinical signs of PHT intolerance.

Evaluation of the potential interaction between felbamate and erythromycin in patients with epilepsy.

Effects of erythromycin on hepatic CYP450 3A4 isozymes can profoundly influence the metabolism of many therapeutic agents. An open-label, randomized, two-period, crossover study was therefore conducted to evaluate the pharmacokinetics of felbamate before and after a concurrent 10-day regimen (333 mg three times daily) of erythromycin. Patients were receiving either 3,000 or 3,600 mg/day felbamate monotherapy for treatment of epilepsy. Mean dose-normalized values for maximum concentration (Cmax) and area under the concentration-time curve (AUC tau) of felbamate were not statistically different in patients taking felbamate as monotherapy than in patients after erythromycin coadministration. Estimates of time to Cmax (tmax), minimum concentration (Cmin), apparent clearance (Cl/kg), average concentration (Cav), and degree of fluctuation (DFss) were likewise unchanged. The incidence of mild and moderate adverse events increased during coadministration of the two drugs. Because patients with epilepsy can not be treated with erythromycin alone, it could not be determined whether the adverse events were attributable to erythromycin or to the combination of the two drugs. Steady-state pharmacokinetic parameters of felbamate were not influenced by erythromycin coadministration.

Tolerability and pharmacokinetics of monotherapy felbamate doses of 1,200-6,000 mg/day in subjects with epilepsy.

PURPOSE: Felbamate (FBM) pharmacokinetic parameters, safety and tolerability in the dose range of 1,200-6,000 mg/day were assessed in two open-label studies with similar designs. METHODS: In study A, newly diagnosed subjects with epilepsy receiving FBM monotherapy at a starting dose of 1,200 mg/day (400 mg/three times daily, t.i.d.) and increased 1,200 mg/day, if tolerated, at 14-day intervals to 3,600 mg/day were investigated. In study B, epilepsy subjects with prior FBM monotherapy exposure received ascending FBM doses in five consecutive 14-day periods with a starting dose of 3,600 mg/day (1,200 mg t.i.d.) FBM. In each successive period, if FBM was well tolerated, the dose was increased by 600 mg/day to a maximum of 6,000 mg/day (2,000 mg t.i.d.). RESULTS: The pharmacokinetic parameter estimates maximum observed concentration (Cmax), area under the concentration-time curve (AUCtau) Ctrough, and Cav showed a linear dependence to dose above the 1,200-6,000 mg/day FBM dose range (F-tests; p < 0.0001) with apparent clearance (Cl/kg) and Tmax (time to Cmax) independent of dose. When AUCtau, Cmax and Ctrough were adjusted for dose, there were no significant differences between the dosing periods. CONCLUSIONS: The data establish that plasma concentrations of FBM are linear with respect to dose to 6,000 mg/day. In addition, FBM was safely administered at these doses for periods as long as 14 days to epileptic subjects with prior exposure to FBM. FBM-naive subjects appeared to report more adverse experiences (generally of mild to moderate severity) than did subjects with prior FBM exposure.

The effect of felbamate on valproic acid disposition.

INTRODUCTION: Felbamate is a new antiepileptic drug approved for partial and secondarily generalized seizures. DESIGN: Subjects with epilepsy (three men and seven women; age range, 20 to 39 years; weight range, 53 to 88 kg) who were previously stabilized with valproic acid, 9.5 to 31.7 mg/kg/day, received both 600 and 1200 mg felbamate twice a day in an open-label, randomized, crossover study. RESULTS: Coadministration of 1200 or 2400 mg felbamate increased the mean valproic acid area under the curve (from 802.2 to 1025.4 and 1235.9 mg/hr/ml, respectively), peak concentrations (from 86.1 to 115.1 and 133.4 mg/ml, respectively), and average steady-state concentrations (from 66.9 to 85.5 and 103.0 mg/ml, respectively). No changes were observed in valproic acid time to peak concentration or protein binding. Average steady-state felbamate concentrations were 34.7 mg/ml for 600 mg administered twice daily and 61.2 mg/ml for 1200 mg administered twice daily. CONCLUSION: When felbamate is added to a regimen of valproic acid, valproic acid doses may require reduction because coadministration of felbamate decreased steady-state valproic acid clearance (28% and 54%, respectively; p < 0.01).

Simultaneous determination of felbamate, primidone, phenobarbital, carbamazepine, two carbamazepine metabolites, phenytoin, and one phenytoin metabolite in human plasma by high-performance liquid chromatography.

An isocratic liquid-chromatographic method employing one extraction step has been developed for the quantitation of five drugs and three metabolites in human plasma. The method uses 0.100-ml aliquots of human plasma and two internal standards. Chromatographic conditions include a 4.6 mm x 150 mm Spherisorb ODS2, 3 microns a high-performance liquid chromatography, (HPLC) column, a phosphate buffer-acetonitrile-methanol (700:160:140) mobile phase, and ultraviolet (UV) absorbance detection at 210 nm. Analytes and linear quantitation ranges (microgram/ml) were felbamate (FBM) 0.391-200; primidone (PRIM), 0.098-100; phenobarbital (PHENO), 0.195-100; carbamazepine (CBZ), 0.195-100; phenytoin (PHT), 0.195-200. For CBZ-transdiol (CBZ-TR) CBZ-epoxide (CBZ-EP), and the PHT metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), the range was 0.049-25.0 micrograms/ml. Ethosuximide, methsuximide, 2-methyl-2-phenyl-succinimide (methsuximide metabolite), 2-ethyl-2-phenyl malonamide (PRIM metabolite, 5-ethyl-5-(4-hydroxyphenyl)-barbituric acid (PHENO metabolite), and mephenytoin do not interfere with quantitation of the above compounds.

Human trophoblast xenografts in athymic mice: a model for peripheral aromatization.

A novel procedure was developed for evaluating aromatase inhibitors using human enzyme in a rodent model. Human choriocarcinoma trophoblast (JAr line) cells injected subcutaneously into athymic nude mice develop into tumor xenografts in 7-14 days which represent sites for peripheral aromatization of androgens. The rapid growth of these trophoblast tumors is estrogen independent. The tumors provide a source of nonovarian human tissue which has relatively high levels of enzyme activity (248 +/- 12 pmol estrogen/g/h) for biochemical determination of in vivo aromatase inhibition. These are major advantages for pharmacological evaluations in comparison to the slow tumor growth response of most carcinogen-induced rodent mammary cancers, which are usually devoid of aromatase activity. In addition, the hormonal dependent components of rodent mammary tumors require several weeks to regress as a result of the indirect effects of estrogen deprivation on tumor growth via inhibition of prolactin dependency, a minor component relative to the role estrogen occupies in hormonally-dependent breast cancer in humans. This model of peripheral aromatization was utilized to evaluate in vivo pharmacological parameters of MDL 18,962 (10-(2-propynyl)estr-4-ene-3,17-dione) such as bioavailability of several formulations, time course and dose responses following different routes of drug administration, pharmacokinetics and tissue distribution of [14C]MDL 18,962. Tumor aromatase activities of trophoblast xenografts were significantly (P less than or equal to 0.05) inhibited when MDL 18,962 was administered intravenously, orally, subcutaneously, or via subcutaneous silastic implants. The ED50 of MDL 18,962 for tumor aromatase inhibition at 6 h after a single treatment was 1.4 mg/kg, s.c. and 3.0 mg/kg, orally. MDL 18,962 blocked aromatase activity more effectively in human trophoblast than in mouse ovarian tissue. Human trophoblast aromatase activity was inhibited by 70% following a single oral dose of 100 mg/kg of MDL 18,962, while the host's ovarian aromatase activity exhibited only marginal inhibition. In vitro, the addition of 10 microM MDL 18,962 to trophoblast tumor cytosol or mouse ovarian cytosol resulted in 99.6 and 91.4% inhibition of aromatase activity, respectively. Tissue distribution of [14C]MDL 18,962 was predominantly associated with endocrine tissues with aromatase activity and organ systems involved in steroid metabolism and excretion. These in vivo data show that MDL 18,962 an enzyme-activated aromatase inhibitor, causes prolonged aromatase inhibition in the absence of saturating levels of inhibitor.

MLTIDOSE: a multiple-dose simulation program for linear systems characterized by exponential functions.

MLTIDOSE is a multiple-dose simulation program for use on IBM PC (and compatible) computers. It assumes dose-independent disposition and absorption (i.e., a linear system) and simulates blood concentration-time profiles (over a range of times or at specific times) upon administration of any combination of intravascular (i.v.) (bolus and/or intermittent constant rate infusions) and extravascular (e.v.) doses of fixed or variable size, administered at fixed or variable intervals. Input requirements include pharmacokinetic parameters obtained following single-dose administration (entered from the keyboard or from a data file). Options for printing data to files (e.g., ASCII and DIF) for further use are also provided.

PKCALC: a BASIC interactive computer program for statistical and pharmacokinetic analysis of data.

PKCALC, an interactive computer program written in BASIC, facilitates: initial statistical analysis of multisubject data sets, pharmacokinetic analysis of experimental data, transfer of data between commercially available electronic spread-sheets (e.g., LOTUS 123) and the pharmacokinetic programs ESTRIP and PCNONLIN, and rapid preparation of graphs of data. Concentration versus time data can be entered into the program manually, from a data interchange format (DIF) file, or from a data file previously generated by PKCALC. The program has a main menu listing nine options. These include, but are not limited to, setting up a data file which can subsequently be read by PCNONLIN, chaining to a copy of ESTRIP augmented to read PKCALC data files, stripping curves manually, and utilizing the CRT and a line printer to graph polyexponential equations and/or subject data on linear or semilogarithmic axes. PKCALC runs on the IBM PC, IBM XT, IBM AT, and COMPAQ personal computers.

Increasingly Ruth: toward understanding sex reassignment.

Both the clinician and the patient present accounts of a 44-year-old male's search for a comfortable gender identity. The juxtaposition of these two perspectives illustrates many of the dilemmas inherent in the therapy of gender dysphoria. Ruth underwent sex reassignment surgery in 1976--one year after the assumption of a full-time female gender role. Six months after surgery, she made a serious suicide attempt. At age 50, she has now consolidated her feminine gender identity and has become a thoughtful, unusually honest, articulate person. The physician's "objective" and the patient's retrospective perspectives provide evidence of the psychodynamic nature of transsexualism and the limitations of evaluation criteria for sex reassignment surgery.

The response of manual motor functioning in Parkinsonians to frontal EMG biofeedback and progressive relaxation.

The purpose of the current investigation was to determine the effects of frontal EMG biofeedback and progressive relaxation training on manual motor functioning in Parkinsonians. Twenty patients were matched and randomly assigned to two groups. All subjects were administered a brief manual motor assessment. The experimental group then underwent weekly sessions of frontal EMG and relaxation training for a period of 15 weeks. At the conclusion of the training period, both experimental and control groups were again administered the manual motor tasks. The results indicated that Parkinsonian patients are capable of significantly lowering frontal EMG activity levels. The motor task results, however, yielded no statistically significant differences between the two groups as a result of the biofeedback training.