Biosafety Level 3 setup for multiphoton microscopy in vivo.

Multiphoton microscopy has revealed important insights into cellular behavior in vivo. However, its application in infectious settings often encounters technical, safety and regulatory limitations that prevent its wider use with highly virulent human pathogens. Herein, we present a method that renders multiphoton microscopy in vivo compatible with biosafety level 3 regulations and present an example of its application and potential to visualize a Mycobacterium tuberculosis infection of the mouse lung.

Pharmacokinetics, toxicokinetics, distribution, metabolism and excretion of linezolid in mouse, rat and dog.

1. Linezolid (ZYVOX), the first of a new class of antibiotics, the oxazolidinones, is approved for treatment of Gram-positive bacterial infections. 2. The aim was to determine the absorption, distribution, metabolism and excretion (ADME) of linezolid in mouse, rat and dog in support of preclinical safety studies and clinical development. 3. Conventional replicate study designs were employed in animal experiments, and biofluids were assayed by HPLC or HPLC-MS. 4. Linezolid was rapidly absorbed after p.o. dosing with an p.o. bioavailability of > 95% in rat and dog, and > 70% in mouse. Twenty-eight-day i.v./p.o. toxicokinetic studies in rat (20-200mg kg(-1) day(-1)) and dog (10-80 mg kg(-1) day(-1)) revealed neither a meaningful increase in clearance nor accumulation upon multiple dosing. 5. Linezolid had limited protein binding (<35%) and was very well distributed to most extravascular sites, with a volume of distribution at steady-state (V(ss)) approximately equal to total body water. 6. Linezolid circulated mainly as parent drug and was excreted mainly as parent drug and two inactive carboxylic acids, PNU-142586 and PNU-142300. Minor secondary metabolites were also characterized. In all species, the clearance rate was determined by metabolism. 7. Radioactivity recovery was essentially complete within 24-48 h. Renal excretion of parent drug and metabolites was a major elimination route. Parent drug underwent renal tubular reabsorption, significantly slowing parent drug excretion and allowing a slow metabolic process to become rate-limiting in overall clearance. 8. It is concluded that ADME data were relatively consistent across species and supported the rat and dog as the principal non-clinical safety species.

Venous irritation, pharmacokinetics, and tissue distribution of tirilazad in rats following intravenous administration of a novel supersaturated submicron lipid emulsion.

PURPOSE: To compare the venous irritation, pharmacokinetics, and tissue distribution of tirilazad in rats after intravenous administration of a submicron lipid emulsion with that of an aqueous solution. METHODS: Venous irritation was determined by microscopic evaluation of injury to the lateral tail veins of rats. Pharmacokinetic parameters were determined by following plasma concentrations of drug. Tissue distribution of [14C]-tirilazad was determined by quantitative whole body autoradiography. RESULTS: Single dose injections of tirilazad as an emulsion at doses ranging from 1.52 mg to 13.5 mg were non-irritating whereas the solution was irritating at a dose of 1.3 mg. The pharmacokinetic parameters were not statistically different between the emulsion and the solution (p > 0.2) at doses of 6 mg/kg/day and 20 mg/kg/day. However, at 65 mg/kg/day dose, a higher AUC(0,6) (4-fold) and lower V(ss), (18-fold) and CL(5-fold) were observed for the lipid emulsion as compared to the solution (p < 0.05). Tissue distribution showed higher initial concentrations (two fold or more) in most tissues for the solution. These values, however, equilibrated by 4 h and AUC(0,4) differences were less than two fold in most tissues. CONCLUSIONS: Formulating tirilazad in the lipid emulsion significantly reduces the venous irritation without changing the pharmacokinetics and tissue distribution at low doses.

Modeling the effects of varicella vaccination programs on the incidence of chickenpox and shingles.

Two possible dangers of an extensive varicella vaccination program are more varicella (chickenpox) cases in adults, when the complication rates are higher, and an increase in cases of zoster (shingles). Here an age-structured epidemiologic-demographic model with vaccination is developed for varicella and zoster. Parameters are estimated from epidemiological data. This mathematical and computer simulation model is used to evaluate the effects of varicella vaccination programs. Although the age distribution of varicella cases does shift in the simulations, this does not seem to be a danger because many of the adult cases occur after vaccine-induced immunity wanes, so they are mild varicella cases with fewer complications. In the simulations, zoster incidence increases in the first three decades after initiation of a vaccination program, because people who had varicella in childhood age without boosting, but then it decreases. Thus the simulations validate the second danger of more zoster cases.

Absorption, distribution, metabolism, and excretion of atevirdine in the rat.

Atevirdine mesylate (U-87201E) is a highly specific nonnucleoside inhibitor of human immunodeficiency virus type 1 reverse transcriptase. The absorption, metabolism, and excretion of atevirdine were investigated in male and female Sprague-Dawley rats after oral administration of nonradiolabeled atevirdine mesylate at doses of 20 mg/kg/day or 200 mg/kg/day for 8 days, with [14C]atevirdine mesylate single doses of 10 mg/kg or 100 mg/kg on study days 1 and 10. The distribution of [14C]atevirdine mesylate was also evaluated by whole-body autoradiography in male and female Sprague-Dawley, pregnant Sprague-Dawley, and male Long-Evans rats after a single 10 mg/kg oral dose. Plasma levels of atevirdine and its N-desethyl and O-desmethyl metabolites were determined by high-performance liquid chromatography (HPLC) with ultraviolet detection, urine and feces were profiled for atevirdine and metabolites by HPLC with radiochemical detection, major metabolites in urine were isolated and identified by nuclear magnetic resonance and mass spectrometry, and minor urinary metabolites were identified by liquid chromatography/mass spectrometry. Atevirdine was rapidly absorbed. The pharmacokinetics of atevirdine were nonlinear. Gender differences in the pharmacokinetics and metabolism of atevirdine were observed, consistent with the involvement of cytochrome P450 3A. Atevirdine effectively crossed the blood-brain barrier and had a high rate of maternal-fetal transfer. At the low doses, <2% of the dose was excreted as unchanged parent drug, while atevirdine constituted 9%-25% of the dose at the high doses. The metabolism of atevirdine was extensive in the rat and involved N-deethylation, O-demethylation, hydroxylation at the C-6 position of the indole ring, and hydroxylation of the pyridine ring.

Pharmacokinetics of the individual enantiomers of cis-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzamide monohydrochloride (U-54494A) in the dog.

U-54494A, a racemic mixture of two enantiomers, is being developed in racemic form as an anticonvulsant drug candidate. A comparative pharmacokinetic study with intravenous and oral administration of the two individual enantiomers to the dog was conducted to evaluate the potential enantioselective pharmacokinetics of U-54494. Following i.v. administration, the (-)- and (+)-enantiomers showed no significant differences in plasma clearance (0.84 +/- 0.11 versus 0.86 +/- 0.06 l/hr/kg) and terminal elimination half-life (11.2 +/- 2.7 versus 8.0 +/- 2.6 hr) for the parent drug. However, the AUC of intact drug was two-fold lower with two-fold shorter elimination half-life following the oral administration for the (+)-enantiomer as compared to the (-)-enantiomer. Higher plasma levels of the four metabolites were also observed for the (+)-than for the (-)-enantiomer, particularly after oral administration. These results suggested that the (+)-enantiomer appeared to be more extensively metabolized by first-pass effect than the (-)-enantiomer after oral dosing, and as a result, oral bioavailability for the (+)-enantiomer is only one half of that for its antipode (12.0 +/- 1.5% versus 26 +/- 9%).

Evidence for an alpha-1 receptor-mediated central sympathoinhibitory action of ketanserin.

The effect of the serotonin receptor antagonist ketanserin on sympathetic nervous discharge recorded from the inferior cardiac and splanchnic nerves was studied in the baroreceptor-denervated cat. Intravenous ketanserin (0.05-0.8 mg/kg) produced dose-dependent decreases in mean arterial pressure, heart rate and sympathetic nervous discharge, with maximal decreases of 41, 7 and 55% of control, respectively. The decrease in mean arterial pressure could not be disassociated from a decrement in the pressor response to i.v. phenylephrine. The central sympatholytic effect of ketanserin was augmented in serotonin-depleted animals. In contrast, the centrally mediated reduction in sympathetic nervous discharge produced by ketanserin was blocked completely in catecholamine-depleted cats. In addition, the central sympatholytic action of the alpha-1 adrenergic receptor antagonist prazosin was blocked in catecholamine-depleted animals. These data indicate that ketanserin acts at central alpha-1 adrenergic receptors to reduce sympathetic outflow from the central nervous system. Central serotonergic pathways do not appear to be involved in the sympatholytic action of ketanserin. The nature of the interaction between central noradrenergic neurons and neurons involved in the genesis of sympathetic nerve activity is discussed.

Evidence for a central sympathoexcitatory action of alpha-2 adrenergic antagonists.

The effect of alpha-2 adrenergic receptor antagonists on sympathetic nerve discharge (SND) recorded from the external carotid and splanchnic nerves were studied in baroreceptor-denervated cats. Low i.v. doses of piperoxane and rauwolscine dramatically increased SND and produced a concomitant rise in mean arterial pressure and heart rate. High doses of piperoxane also resulted in an increase in SND. High doses of rauwolscine, however, markedly reduced mean arterial pressure, heart rate and SND. The pressor response to i.v. norepinephrine was greatly attenuated by high doses of rauwolscine and slightly reduced by piperoxane. Piperoxane failed to alter SND in catecholamine-depleted animals. In vitro binding experiments indicated that piperoxane and rauwolscine bound selectively to the alpha-2 adrenergic receptor and had little affinity for alpha-1 receptor sites. In addition, rauwolscine displaced [3H]LSD binding. These data indicate that low doses of the alpha-2 receptor antagonist piperoxane and rauwolscine act centrally to increase SND. In addition, the nature of the interactions between central noradrenergic neurons and neurons involved in the genesis of sympathetic nerve activity is discussed.