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Oxidation of toluene derivatives was investigated in the housefly and the Egyptian cotton leafworm. Methyl group in o-, m-, p-aminotoluene (toluidines), m-, and p-nitrotoluene was converted to carboxylic acid in vivo in both insect species, but not in o-nitrotoluene. Oxidizing system in the housefly adults was more active than that in the cotton leafworm larvae. The rate of in vivo oxidation in the adults was affected by the substituted group in the following order: p-nitro > p-amino > m-nitro > m-amino > o-amino, while in the cotton leafworm larvae was in the following order: p-nitro > m-nitro > m-amino > o-amino > p-amino. The p-nitrotoluene gave the highest rate in both insect species. Also the nitro-group facilitated the oxidation reaction more than the amino-group. Thanite and piperonyl butoxide where tested as inhibitors to this oxidation system. It was found that Thanite completely inhibited the methyl group oxidation in the two insect species, while piperonyl butoxide inhibited completely this reaction in the cotton leafworm larvae and only decreased the amount of oxidation of methyl group in the housefly adults. A linear relationship was found between Hammett's substitution constant and the rate of in vivo oxidation in the cotton leafworm larvae. Zusammenfassung Oxydation der Methylgruppe von Toluen-Derivaten bei Musca domestica und Spodoptera littoralis Es wurde die Oxydation von Toluen-Derivaten im Korper der Stubenfliege und der agyptischen Baumwolleule Spodoptera littoralis untersucht. Die Methylgruppe im o-, m- und p-Aminotoluen sowie im m- und p-Nitrotoluen, nicht aber im o-Nitrotoluen, wurde zu Carboxylsaure in vivo bei beiden Insektenarten umgesetzt. Das oxydierende System in der imaginalen Stubenfliege war aktiver als das in den Eulenraupen. Die Rate der in vivo-Oxydation wurde in den Fliegen durch die substituierte Gruppe in der Reihenfolge beeinflust: p-nitro > p-amino > m-nitro > m-amino > o-amino, wahrend die Reihenfolge bei den Raupen lautete: p-nitro > m-nitro > m-amino > o-amino > p-amino. Das p-Nitrotoluen ergab die hochste Rate bei beiden Insekten. Die Nitrogruppe forderte die Oxydation mehr als die Aminogruppe. Thanit und Piperonylbutoxyd wurden als Hemmer des Oxydsystems befunden. Es zeigte sich, das Thanit die Methyl-Oxydation bei beiden Insekten vollkommen hemmte, wahrend Piperonylbutoxyd dies bei den Raupen vollstandig tat, die Oxydation bei den Fliegen dagegen nur herabsetzte. Es wurde eine lineare Beziehung zwischen Hammetts Substitutionskonstante und der Rate der in vivo-Oxydation bei den Eulenraupen gefunden.

Cytochrome P450s (CYPs) are heme-containing enzymes that metabolize small organic molecules including drugs. CYP3A4 and CYP2D6 are responsible for more than 70% of pharmaceutical drug metabolism an...

The KCNN4 potassium-ion channel has been reported to play an important role in regulating antigen-induced T cell effector functions in vitro. This study presents the first evidence that a selective KCNN4 blocker, TRAM-34, confers protection against experimental autoimmune encephalomyelitis (EAE) in the mouse model. Treatment with the KCNN4 blocker did not prevent infiltration of T cells in the spinal cord, but resulted in the reduction of both the protein and the message levels of TNF-alpha and IFN-gamma as well as the message levels of several other pro-inflammatory molecules in the spinal cord. Plasma concentrations of TRAM-34 within a 24-h period were between the in vitro IC(50) and IC(90) values for the KCNN4 channel. The effect of TRAM-34 was reversible, as indicated by the development of clinical EAE symptoms within 48 h after withdrawal of treatment. In summary, our data support the idea that KCNN4 channels play a critical role in the immune response during the development of MOG-induced EAE in C57BL/6 mice.

One of the major components of modern drug discovery is the structural determination and the assessment of biological activity of plasma metabolites. LC-MS/MS has played a prominent role in the identification of metabolites; however, fragmentation patterns alone may not be sufficient for identification. Consequently, it may be necessary to isolate the metabolites for NMR or LC-NMR analysis. This report describes the isolation and identification of the major plasma metabolites of two lead compounds (SCH X and SCH Y). The major metabolite of SCH X in monkey plasma constituted 65% of total compound-derived materials. incubation of rat liver microsomes with SCH X gave the mono-hydroxylated metabolite found in monkey plasma; however, the yield was low. Incubations with microsomes from rats pre-treated with various cytochrome P450 inducers showed that the highest yield was obtained from pregnenolone 16a-carbonitrile (PCN)-induced animals. For SCH Y, two metabolites were found in bile and plasma of both rats and monkeys. Various in vitro systems did not produce amounts sufficient for isolation. Therefore, the metabolites of SCH X and SCH Y were isolated from PCN-induced rat liver microsomal incubation and rat bile, respectively. The chemical structures of the metabolites were unambiguously determined using LC-NMR analyses.

Rapid, generic gradient liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays, designed to accelerate sample analyses, have been developed to keep pace with the productivity of advanced synthetic procedures. In this study, LC/MS/MS was combined with an in vitro, cell-based, blood-brain barrier (BBB) model to evaluate the potential of new chemical entities (NCEs) to cross the BBB. This in vitro assay provides the permeability of discovery compounds across a monolayer of a primary culture of bovine brain microvessel endothelial cells in a fraction of the time that is required for in vivo studies (brain/plasma concentrations), using only 2 mg of the compound. The results are consistent with in vivo brain/plasma concentration ratio data.

Genaconazole (SCH 39304) is a potent triazole antifungal agent that is active both orally and topically. Genaconazole is a racemic mixture which contains 50% of the RR (SCH 42427) and 50% of the SS (SCH 42426) enantiomers. The RR isomer accounts for most of the antifungal activity of genaconazole. Serum concentrations of the RR and SS enantiomers were analyzed by a chiral HPLC method which involved extraction of serum with organic solvent followed by separation on a Cyclobond I column and quantification by UV absorbance at 205 nm. The bioavailability and pharmacokinetic profiles of the two enantiomers after oral administration of the racemate (genaconazole) were very similar in cynomolgus monkeys. In rats following dosing with genaconazole, the RR enantiomer had a lower Cmax and a longer t1/2 than the SS enantiomer, while the AUC(I) values of the two enantiomers were similar. Based on chiral HPLC analysis, there was no evidence for the inversion of the RR to the SR isomer, or of the SS to the SR isomer, indicating that there was no chiral inversion of the RR or SS enantiomers in either species. Genaconazole at 20 mg/kg and the RR (SCH 42427) enantiomer at 10 mg/kg had very similar serum concentration–time profiles and Cmax, AUC(I), and t1/2 values for the RR enantiomer in both rats and monkeys, indicating that the two treatments were equivalent with respect to the bioavailability of the RR enantiomer. Chirality 14:436–441, 2002. © 2002 Wiley-Liss, Inc.

This report addresses the continuing need for increased throughput in the evaluation of new chemical entities (NCEs) in terms of their pharmacokinetic (PK) parameters by describing an alternative procedure for increasing the throughput of the in vivo screening of NCEs in the oral rat PK model. The new approach is called "cassette-accelerated rapid rat screen" (CARRS). In this assay, NCEs are dosed individually (n = 2 rats/compound) in batches of six compounds per set. The assay makes use of a semi-automated protein precipitation procedure for sample preparation in a 96-well plate format. The liquid chromatography/atmospheric pressure ionization tandem mass spectrometry (LC/API-MS/MS) assay is also streamlined by analyzing the samples as "cassettes of six". Using this new approach, a threefold increase in throughput was achieved over the previously reported "rapid rat screen".

In the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single drug candidate can be identified for development. In order to accelerate the drug discovery process, we have developed higher-throughput enzyme assays to evaluate the inhibition of cytochrome P450 isoforms 2D6 (CYP2D6) and 3A4 (CYP3A4) in human liver microsomes. The assays are based on high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) techniques. The analysis time for each sample was reduced from approximately 20 minutes for the conventional HPLC assay to 30 seconds for the LC/MS/MS assay. For both LC/MS/MS assays, the linearity (r(2) > 0.99), precision (%CV < 15%) and accuracy (% bias

SCH 56592 is a novel triazole antifungal agent that is active both orally and intravenously in animal models of infection. This compound is in Phase II-III clinical trials for the treatment of systemic fungal infections. SCH 56592 is a single enantiomer with four stereogenic centers; therefore, it was necessary to evaluate the possible chiral inversion of this drug candidate in animals and humans. Thus, chiral high-performance liquid chromatographic (HPLC) methods have been developed to separate SCH 56592 from its diastereomers and to evaluate its chiral inversion in rats, dogs, cynomolgus monkeys, and humans. Chiral HPLC analysis involved the use of a Chiralcel OD column set at 39 degrees C with a mobile phase of hexane-ethanol-diethylamine and a fluorescence detector set at an excitation wavelength of 270 nm and an emission wavelength of 390 nm. Plasma or serum samples were subjected to solid phase extraction on a C(2) cartridge followed by HPLC analysis. The method was sensitive with a limit of quantitation of 0.1 microg/ml in dog serum. The linearity was satisfactory, as shown by correlations of >0.997 and by visual examination of the calibration curves. The precision and accuracy were satisfactory, as indicated by coefficients of variation (CV) ranging from 1.1 to 12.1% and bias values ranging from -11.0 to 9.0%. Chiral HPLC analysis indicated that SCH 56592 was not subjected to chiral inversion in rats, dogs, cynomolgus monkeys, and humans.

Aims The objective of this study was to compare the pharmacokinetics, safety and tolerability of the antiepileptic drug felbamate in young and elderly healthy vounteers. Methods The single and multiple dose pharmacokinetics of felbamate were examined in an open-label two-dose level parallel group study in 24 elderly (66 to 78-year-old) and 11 young (18 to 45-year-old) healthy volunteer subjects. Pharmacokinetics were determined from blood samples obtained over 120 h after administration of single 600 mg or 1200 mg doses, and after multiple doses of 600 mg or 1200 mg administered every 12 h. Safety and tolerability were assessed through laboratory tests, ECGs, vital signs and reported adverse events. Results Single dose felbamate pharmacokinetic parameters differed between young and elderly subjects; compared with young subjects, elderly subjects had lower mean clearance (31.2 vs 25.1 ml min−1; 90% CI −11.4 to −0.9; P=0.02) and a trend towards a greater half-life (18.6 vs 21.0 h; 90% CI −0.6 to 5.4; P=0.11). Mean AUC and Cmax values were also higher in elderly subjects. No gender differences were noted for weight-adjusted pharmacokinetic variables. Felbamate was less well tolerated in elderly subjects compared with young subjects, as shown by higher rates of adverse event reporting and dropouts at the higher dose level. This may be due to age-related pharmacokinetic differences, to the rapid dose titration schedule used in this study, and/or to altered sensitivity to felbamate’s pharmacodynamic effects. Conclusions These findings imply that elderly subjects require lower initial dosing and slower dose titration of felbamate than non-elderly subjects.

Papillary muscle rupture is an uncommon, catastrophic complication of myocardial infarction that is potentially correctable if promptly diagnosed. Two cases are described in which the diagnosis of papillary muscle rupture was complicated by unusual initial presentations, absence of audible murmurs, and technically difficult, nondiagnostic transthoracic echocardiograms. In both cases, a high index of clinical suspicion for papillary muscle rupture led to transesophageal echocardiography and a rapid, definitive diagnosis. Since early surgery may be important in this disorder, a rapid, reliable diagnosis is essential and transesophageal echocardiography is the diagnostic procedure of choice.

Cor triatriatum is a rare congenital cardiac anomaly consisting of a left atrial membrane dividing the atrium into proximal and distal chambers. The anomaly usually presents in childhood, but can present later in life, in which case it is often associated with mitral regurgitation. We report a patient in whom cor triatriatum was found coincidentally by intraoperative transesophageal echocardiography before surgery for severe mitral regurgitation. An interesting finding was preservation of antegrade systolic pulmonary vein flow by Doppler possibly due to shielding of the pulmonary veins from the regurgitant jet by the left atrial membrane.

Sixteen patients with progressive systemic sclerosis were evaluated for cardiopulmonary manifestations of their disease and for serologic immune abnormalities. Twenty-five percent of patients had abnormal echocardiograms, and 81% had a significant reduction in pulmonary function by spirometry. Circulating immune complexes (IC) were detected in 44% of patients by using a fluorescent Raji cell assay, and these patients were more likely to have an abnormal echocardiogram (P less than 0.02). Seventy-five percent of the patients had fluorescent antinuclear antibodies (FANA) and these patients were more likely to have pulmonary disease (P less than 0.01).

Initial studies from Bowman Gray School of Medicine showed that 18 of 30 patients with classic rheumatoid arthritis (RA) had cardiac involvement from their disease. These abnormalities were detected by echocardiography and consisted of mitral valve and/or pericardial abnormalities. All patients were followed for 4 years from the initial workup. Mitral valve abnormalities were seen on followup in 63% of the patients who initially showed this abnormality, while pericardial effusion remained in 20%. Pericardial thickening persisted in 6 of 7 patients. None of the patients developed constrictive pericarditis or heart failure. There was no definite correlation between persistence of these abnormalities and other clinical data, but it was noticed that patients who had persistent pericardial effusion and mitral valve abnormalities showed a higher number of involved joints and a higher erythrocyte sedimentation rate. The cardiac abnormalities described in this study have remained clinically insignificant in this population of RA patients.

An improved high performance liquid chromatography (HPLC) method of gossypol analysis was developed and compared with the current spectrophotometric method. Gossypol was determined in four samples of cottonseeds ofGossypium hirsutum with varying contents of gossypol, as well as in the stems and roots ofG. arboreum andG. thurber. The roots of both species each contained equally high amounts of gossypol, while the stems of each species varied in content. Those ofG. thurber contained slightly higher amounts of gossypol than those ofG. arboreum. Among the seeds studied, the glanded cottonseeds contained the highest concentration of gossypol, followed by low gossypol seeds, then glandless cottonseeds, and finally the roasted glandless cottonseeds. The stability of gossypol was studied in five solvents at six different storage temperatures. Both the type of solvent and the temperature were found to affect the rate of decomposition of gossypol. In all the solvents studied, gossypol was found to be highly unstable at 37 C and at room temperature, while its stability increased as the storage temperature decreased. At all temperatures, depending on the solvent used, the rate of decomposition increased in the following order: acetone < acetonitrile < chloroform < ethanol < methanol. Although the decomposition products of gossypol were not identified, their formation was found to be dependent on the solvent used.

The rare occurrence of mitral stenosis and coexistent left atrial myxoma is reported. The patient had a 25-year history of rheumatic heart disease and was referred for evaluation of progressive mitral stenosis without clinical suspicion of left atrial myxoma. The tumor was discovered by routine echocardiography in the course of evaluation of mitral stenosis. However, prior to surgery the patient experienced an episode of embolization of the tumor without major clinical sequelae. The utility of echocardiography in this case and in patients with mitral stenosis is discussed as well as the patient's spontaneous "cure."