Nonlinear mixed effects pharmacokinetic/pharmacodynamic analysis of the anticonvulsant ameltolide (LY201116) in a canine seizure model.

The anticonvulsant ameltolide (LY201116) is a novel potential therapy for the treatment of canine epilepsy. Eight dogs were administered five different oral doses of ameltolide and clinical scoring of the maximal electroshock (MES) induced seizures at 3 and 24 h postdosing were determined in two separate crossover design studies. Plasma ameltolide concentrations were determined at the time of seizures in all dogs and complete plasma concentration-time profiles were also determined in a separate study. A nonlinear mixed effects PK/PD model was fit to the resulting data. A one compartment open model with first order absorption was determined to best fit the ameltolide pharmacokinetics. An effect compartment with a cumulative logistic regression equation was used to establish the PK/PD relationship. The mean bioavailability normalized volume of distribution and the elimination half-life were estimated at 1.20 L/kg and 5.46 h, respectively. The fitted model estimated that from 2 to 15 h following a single 3 mg/kg oral ameltolide dose the mean probability of obtaining a 1 unit reduction in the seizure clinical score severity was greater than 0.80. The utilized PK/PD analysis combined with the canine MES model allowed for the rapid and efficient determination of the plasma ameltolide concentration-anticonvulsant relationship preclinically in dogs.

Development and validation of the maximal electro-shock seizure model in dogs.

The development and validation of the maximal electro-shock (MES) model using phenobarbital (Pb) as the positive control is described. This approach builds on previous work in rodent model systems, and has been adapted to dogs as a tool for pharmaceutical dose selection. Dogs, like rodents, exhibit generalized convulsions which manifest as progressive clinical signs in a dose (electrical current) dependent fashion. At the limit (300 mA, 200 msec) animals underwent clonic-tonic convulsions consistent with complete generalized (Grand Mal) seizures with a grade 3 clinical score (CS) and a menace response time of 98.5 +/- 24.4 sec (n = 8). Pretreatment of animals with Pb at 3, 10, and 30 mg/kg, in a 4-by-4 complete block crossover design (Latin-Square), resulted in a dose-dependant reduction in CS and menace response time. Estimates of plasma Pb concentration taken prior to MES induction showed a similar dose-dependent reduction in CS and menace response time with concentration. Using a cumulative logistic regression model, a predicted 50% probability of a CS = 1 was approximately 11.4 mg/kg. In addition, plasma Pb concentrations predicted a 50% probability of a CS = 1 occurs at plasma Pb concentration of approximately 16.0 mug/mL. Combined these data suggest that MES is a useful model for evaluating generalized convulsions in canines and may provide a tool for dose selection of novel pharmaceutical compounds.

Residues of zeta-cypermethrin in bovine tissues and milk following pour-on and spray application.

The depletion of zeta-cypermethrin residues in bovine tissues and milk was studied. Beef cattle were treated three times at 3-week intervals with 1 ml 10 kg-1 body weight of a 25 g litre-1 or 50 g litre-1 pour-on formulation (2.5 and 5.0 mg zeta-cypermethrin kg-1 body weight) or 100 mg kg-1 spray to simulate a likely worst-case treatment regime. Friesian and Jersey dairy cows were treated once with 2.5 mg zeta-cypermethrin kg-1 in a pour-on formulation. Muscle, liver and kidney residue concentrations were generally less than the limit of detection (LOD = 0.01 mg kg-1). Residues in renal-fat and back-fat samples from animals treated with 2.5 mg kg-1 all exceeded the limit of quantitation (LOQ = 0.05 mg kg-1), peaking at 10 days after treatment. Only two of five kidney fat samples were above the LOQ after 34 days, but none of the back-fat samples exceeded the LOQ at 28 days after treatment. Following spray treatments, fat residues were detectable in some animals but were below the LOQ at all sampling intervals. Zeta-cypermethrin was quantifiable (LOQ = 0.01 mg kg-1) in only one whole-milk sample from the Friesian cows (0.015 mg kg-1, 2 days after treatment). In whole milk from Jersey cows, the mean concentration of zeta-cypermethrin peaked 1 day after treatment, at 0.015 mg kg-1, and the highest individual sample concentration was 0.025 mg kg-1 at 3 days after treatment. Residues in milk were not quantifiable beginning 4 days after treatment. The mean concentrations of zeta-cypermethrin in milk fat from Friesian and Jersey cows peaked two days after treatment at 0.197 mg kg-1 and 0.377 mg kg-1, respectively, and the highest individual sample concentrations were 2 days after treatment at 0.47 mg kg-1 and 0.98 mg kg-1, respectively.

Comparative degradation of [14C]-2,4-dichlorophenoxyacetic acid in wheat and potato after Foliar application and in wheat, radish, lettuce, and apple after soil application.

The fate of 2,4-dichlorophenoxyacetic acid (2,4-D) applied foliarly as the 2-ethylhexyl ester (EHE) to wheat and potatoes, to the soil as the dimethylamine (DMA) salt under apple tree canopies, and preplant as the free acid for wheat, lettuce, and radish was studied to evaluate metabolic pathways. Crop fractions analyzed for (14)C residues included wheat forage, straw, and grain; potato vine and tubers; and apple fruit. The primary metabolic pathway for foliar application in wheat is ester hydrolysis followed by the formation of base-labile 2,4-D conjugates. A less significant pathway for 2,4-D in wheat was ring hydroxylation to give NIH-shift products 2,5-dichloro-4-hydroxyphenoxyacetic acid (4-OH-2,5-D), 4-OH-2,3-D, and 5-OH-2,4-D both free and as acid-labile conjugates. The primary metabolic pathway in potato was again ester hydrolysis. 2,4-D acid was further transformed to 4-chlorophenoxyacetic acid and 4-OH-2,5-D. For the soil applications, (14)C residues in the crops were low, and characterization of the (14)C residues indicated association with or incorporation into the biochemical matrix of the tissue. The degradative pathways observed in wheat are similar to those characterized in other intact plant studies but differ from those in studies in wheat cell suspension culture in that no amino acid conjugates were observed.

Activation of intracellular serine proteinase in Bacillus subtilis cells during sporulation.

Cells of Bacillus subtilis 168 (trpC2) growing and sporulating in a single chemically defined medium carried out intracellular protein degradation and increased their levels of intracellular serine protease-1 in a manner very similar to what had previously been reported for cells sporulating in nutrient broth. The results were interpreted to mean that these processes are intrinsic to sporulation rather than medium dependent. To determine the cause of these increases in specific activity of proteinases, we purified the protease, prepared rabbit immunoglobulins directed against it, and monitored changes in protease antigen levels by performing rocket immunoelectrophoresis. In cells sporulating in nutrient broth, the protease antigen levels increased about 7-fold, whereas the specific activity increased about 150-fold, for an activation of about 20-fold. In cells sporulating in the single chemically defined sporulation medium, the protease antigen increased about 10-fold, whereas the specific activity increased at least 400-fold, for an activation of about 40-fold. These results were interpreted to mean that a posttranslational event activated the protease in vivo; a previously described endogenous proteinase inhibitor was confirmed to be present in the strain used. Chloramphenicol added to the cultures inhibited both the increases in antigen levels and in the specific activity of the proteinase.

Assaying proteinases with azocoll.

Azocoll, an insoluble, ground collagen to which a bright-red azodye is attached has been widely used for the assay of proteolytic enzymes. Earlier studies showed that hydrolysis of azocoll progressed linearly as a function of proteinase concentration but in an exponentially increasing manner as a function of time. No explanation for the latter behavior has been offered. We have found that assays of both crude extracts of Bacillus subtilis and commercial preparations of subtilisin BPN' gave linear rates of hydrolysis of azocoll as a function of protease concentration; however, both gave increasing rates of hydrolysis of azocoll as a function of time. In attempting to improve and standardize proteolytic assays using azocoll we have found: (a) the absorption maximum of solubilized azocoll at pH 7.8 is 516 nm and is not significantly altered at acid pH; (b) assays which are perfectly linear as a function of time can be obtained by using azocoll that has been vigorously prewashed with buffer; (c) the soluble filtrate removed by prewashing can regenerate the nonlinear time courses previously observed; and (d) the rate of hydrolysis of azocoll can be varied by a factor of 3 by varying the rates of agitation of the assay tubes. In summary, to obtain reproducible, linear assays it was essential to prewash commercial azocoll and agitate reaction tubes vigorously.