Neoplastic transformation in tissues of rats exposed to monocrotaline or dehydroretronecine.

Male Sprague-Dawley rats received sc injections biweekly of either the pyrrolizidine alkaloid monocrotaline or its metabolite dehydroretronecine for 1 year. The animals were then observed for an additional 12 months for the induction of neoplasms. Of 60 rats that received dehydroretronecine, 39 developed rhabdomyosarcomas at the injection site, and 5 of these neoplasms metastasized. In the 60 monocrotaline-treated rats, 31 widely dispersed tumors of various cell types were recorded. The reason suggested for the variation in tissue response was that the metabolite dehydroretronecine is a proximate carcinogen, whereas monocrotaline must first be metabolized before its carcinogenic potential is realized.

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).

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.

Fibrin thrombosis in monocrotaline pyrrole-induced cor pulmonale in rats.

Investigations were carried out to determine the lung lesions responsible for the development of pulmonary heart disease, cor pulmonale, in rats treated with monocrotaline pyrrole or monocrotaline. Animals with right ventricular hypertrophy showed microscopic lung alterations consisting of alveolar edema; fibrin thrombi with partial to complete occlusion of arteries, arterioles, capillaries, and veins; connective tissue proliferation of alveolar septae; cellular hyperplasia of septae; and medial hypertrophy of arterioles. Due to the high incidence of fibrin thrombi in animals with right ventricular hypertrophy, we believe that formation of fibrin thrombi plays a decisive role in the development of chemically induced cor pulmonale.

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.

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.

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.

Localisation and tissue effects of tritiated dehydroretronecine in young rats.

Sixteen male Spraque-Dawley rats were injected with 3H-dehydroretronecine. The rats were subsequently evaluated for gross and microscopic changes. Scintillation counts and autoradiographic studies of the various organs demonstrated that the pyrrole accumulates in organs which were shown here and by others (Peterson et al; Allen and Hsu) to be affected most severely by dehydroretronecine and dehydroheliotridine. In the present experiment 3H-dehydroretronecine was shown to decrease the growth rate of the rats, decrease the percentage of circulating neutrophils and alter the hepatic mitotic index. Histologically, accumulations of label were seen in the glandular region of the stomach, in the liver, in Huxley's and Henle's layers of the hair follicles and in the epithelial cells lining the convoluted tubules of the kidney. Scintillation counts on the various tissues and autoradiographic evaluations of tissue sections indicate that there is a preferential localisation of radioactivity in the gastric mucosa which is postulated to be related to the pH in this area.

Disenecioyl dehydroretronecine--synthesis and acute hepatic toxicity of a pyrrolizidine alkaloid pyrrole analog.

Disenecioyl dehydroretronecine (DSDR), a semi-synthetic analog of the highly reactive pyrrole metabolites of the pyrrolizidine alkaloids, has been synthesized from disenecioyl retronecine. Rats which received 40 mg DSDR/kg body weight via the mesenteric vein showed multiple depressed areas (less than 2 mm in diameter) on the surface of the median lobe of the liver. Microscopically these areas consisted of hepatic venous occlusion, necrosis, and proliferation of fibroblasts and bile ducts in and around the portal triad. These hepatic lesions were similar to those produced by dehydroretrorcine.

Binding of tritiated dehydroretronecine to macromolecules.

In vivo and in vitro experiments have shown that the pyrrolizidine alkaloid metabolite dehydroretronecine binds readily to macromolecules. In the in vivo experiment there was a preferential binding of dehydroretronecine to the gastric mucosa. Further extraction of the mucosa revealed a large percentage of the 3H was bound to the protein fraction and to a much lesser extent to DNA and RNA. The influence of pH on the binding of dehydroretronecine was substantiated in the in vitro experiment. Dehydroretronecine bound to calf thymus DNA and bovine serum albumin most readily under acidic conditions. These data suggest a direct correlation of the levels of dehydroretronecine binding to cellular macromolecules with the lesions that develop in affected organs.

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.

Pathophysiology of dehydromonocrotaline-induced pulmonary fibrosis in the beagle.

The purpose of this study was to characterize the sequential hemodynamic alterations and pulmonary vascular lesions produced by a single pulmonary artery injection of the vasotoxic pyrrolic alkaloid dehydromonocrotaline in the young beagle. Normotensive pulmonary pressure was replaced by hypertension 21 days after injection. By 28 days, the pulmonary pressure and total pulmonary vascualr resistance of the experimental animals were significantly greater than the controls (p less than 0.01). Right ventricular work increased from a baseline mean of 0.58 to 1.40 kg . m/min. Morphological and morphometrical analyses revealed alveolar edema, increased numbers of alveolar macrophages, cellular hyperplasia in the alveolar septa, and a progressive interstitial fibrosis. The precise mechansims by which dehydromonocrotaline injection initiates and promotes pulmonary hypertension and pulmonary fibrosis still needs clarification; however, our data indicate that the fraction of air space is reduced relative to the fraction of tissue space, and this change occurs with concurrent fibrosis in the alveolar septa and an increased pulmonary arterial pressure although hypoxia was not clinically detectable.

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.

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.