Molecular investigations of orbivirus/vector interactions.

Defining predictors for insect-transmitted virus (arbovirus) disease cycles requires an understanding of the molecular interactions between the virus and vector insect. Studies of orbiviruses from numerous geographic regions have indicated that virus genes are affected by insect population differences. Therefore, the authors have initiated genetic studies of Culicoides sonorensis, isolating cDNAs for characterisation of differential insect gene expression, as well as a gene discovery project. Previous work identified insect transcripts elevated in orbivirus-infected female midguts at one day post infection (pI). Here, we report cDNAs that were more abundant in midguts two days following an epizootic haemorrhagic disease virus feeding, as well in head/salivary glands at three days pI. Of the cDNAs identified in midguts at two days pI, three encode translational machinery components, and three encode components that affect cellular structural features. Of the differentially expressed salivary gland cDNAs, only one was homologous to a previously identified gene, a putative odorant binding protein.

Pharmacokinetics and tissue distribution of the nonadecapeptide Moli1901 in rats and mice.

1. Administration of aerosolized, radiolabelled Moli1901 (duramycin, 2622U90), a 19 amino acid polycyclic peptide, to rats resulted in the deposition of high amounts of radiolabel in the respiratory tract, with deposited radiolabel persisting almost unchanged through 7 days after dosing. Little to no radiolabel was present in the bloodstream of these rats. 2. Rats absorbed little radiolabel after p.o. administration, with nearly all of the dose excreted in the faeces by 2 days after dosing. 3. At 7 days following an intravenous dose, rats excreted 54% of the radiolabel in faeces and 5.4% in the urine, with 44% remaining in the carcass, primarily in the liver (33%). 4. Following an intratracheal instillation dose to rats, radiolabel was eliminated from the pulmonary system with a half-life of 64 days. Excretion was almost exclusively via faeces, with an elimination half-life of 52 days. Plasma and blood concentrations in these animals were uniformly <1 ng eq. ml(-1) at all sampling times. 5. Results in mice given intravenous and oral doses were consistent with those observed in rats. 6. Prolonged retention of Moli1901 in pulmonary tissue supports its use in the treatment of respiratory diseases.

Pharmacokinetics and hematological effects of the PEGylated thrombopoietin peptide mimetic GW395058 in rats and monkeys after intravenous or subcutaneous administration.

GW395058, a potent PEGylated peptide human thrombopoietin receptor (HuTPOr) agonist in vitro, is being evaluated for the treatment of thrombocytopenia. GW395058 shares no sequence homology with TPO. In this report the pharmacokinetics and hematological effects of GW395058 in rats and monkeys are described. Doses eliciting thrombocytosis in rodents (2 or 10 microg/kg s.c.) produced insufficient plasma concentration data for pharmacokinetic parameter estimate calculations. At higher i.v. doses in rats (500, 1,000 or 2,000 microg/kg) serum t1/2 (half-life) values were >20 h, and the area under the concentration time curve increased proportionally with dose. In cynomolgus monkeys GW395058 plasma t1/2 values ranged from 37 to 68 h after s.c. or i.v. dosing, and similar values were observed in rhesus monkeys following s.c. dosing. Rat platelet counts increased following 2 (1.6-fold) or 10 microg/kg (fourfold) s.c. doses. Cynomolgus and rhesus monkey platelet counts did not change significantly at comparable s.c. doses, but did increase slightly (

Development of a novel scintillation proximity competitive hybridization assay for the determination of phosphorothioate antisense oligonucleotide plasma concentrations in a toxicokinetic study.

A novel scintillation proximity competitive hybridization assay was developed for determining plasma concentrations of compound 4003W94, a 15-base phosphorothioate antisense deoxyribonucleotide that is currently under preclinical evaluation for the treatment of restenosis following coronary artery angioplasty. The principle of the assay involves the hybridization binding of antisense (4003W94) to a biotinylated sense oligonucleotide to form a double-stranded nucleic acid complex on the surface of scintillation proximity beads derivatized with streptavidin. As in a competitive radioimmunoassay, there is an inverse relationship between the amount of radioactivity in the final binding complex and the amount of 4003W94 present in the sample being analyzed. Because this is a homogenous assay, no physical separation of bound from free radioligand is necessary. Conventional cross-reactivity studies with either 3'- or 5'-deletion oligomers of 4003W94 indicated that cross-reactivity generally decreased with each base deletion. The assay was used to determine plasma concentrations of 4003W94 equivalents in rhesus monkeys during an exploratory 14-day toxicity study. This method conceivably could be adapted for use as an effective in vitro screening tool for the identification of potential antisense oligonucleotide drug candidates or as a diagnostic tool for the detection of pathological disorders.

Development and validation of a chiral LC method for analysis of the four stereoisomers of 1045U85 in plasma.

A method for analysis of the four stereoisomers of 1045U85 in rat plasma was developed and validated. The method involved liquid extraction of 1045U85 and an internal standard (propranolol) from plasma, followed by reaction with a chiral derivatizing reagent, GITC. The diastereoisomeric products were then separated by reversed-phase LC. The range of quantitation was 9.828-0.121 micrograms ml-1 for total 1045U85 (3.440-0.042 micrograms ml-1 for the RR and SS isomers, and 1.474-0.018 micrograms ml-1 for the RS and SR isomers). Specificity of the method for 1045U85 was demonstrated using spiked plasma samples as well as plasma samples from dosed animals. Extraction recovery of 1045U85 and propranolol was greater than 95%, and the derivatization reaction was shown to be complete. Accuracy (% bias) ranged from -2.6 to 3.9% for total 1045U85 and from -4.7 to 14.1% for the individual stereoisomers. Precision (% RSD) was 3.8-8.7% for total 1045U85 and 2.9-16.5% for the individual isomers. Plasma samples stored at -70 degrees C were stable for 19 weeks. The method has been used to determine plasma 1045U85 concentrations in nonclinical studies with this compound.

The pharmacokinetics and pharmacodynamics of the prostacyclin analog 15AU81 in the anesthetized beagle dog.

15AU81, a tricyclic benzindene analog of prostacyclin, is currently under preclinical evaluation as a potential treatment for congestive heart failure. The cardiovascular effects of 15AU81 were evaluated in anesthetized beagle dogs given 4-h infusions at rates of either 0.1, 0.3, 1.0, or 3.0 micrograms/kg/min. Plasma samples taken from these dogs prior to, during, and after the infusion, were analyzed for 15AU81 by a radioimmunoassay (RIA). This report integrates the vasodilatory effects and plasma concentration data from the 15AU81 infusion study. Pharmacokinetic analysis of mean data indicated a biphasic decay of 15AU81 in plasma, with an initial half-life of approximately 2 min, and a terminal half-life of approximately 20 min. Visual inspection of plots of drug effect and drug concentration against time indicated a close relationship between plasma concentration of 15AU81 and the onset of decreases in total peripheral resistance (TPR) and pulmonary vascular resistance (PVR). In general, the decreases in TPR and PVR induced by 15AU81 were maintained during infusion. Concentration-effect plots indicated some hysteresis in TPR vs plasma concentrations of 15AU81 after termination of the infusion; possible explanations for this hysteresis include the presence of saturating concentrations of 15AU81 at the effect site, with a delay in the clearance of 15AU81 from the effect site compared to its clearance from plasma, and/or the presence of active metabolites at the effect site. A fit of the TPR and PVR data to the Emax pharmacodynamic model predicted that the maximum decrease in TPR achievable with 15AU81 in anesthetized dogs was 66%, and that the concentration of 15AU81 producing 50% of the maximum effect (EC50) was 8.6 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioimmunoassay for the chemical stable prostacyclin analog, 15AU81: a preliminary pharmacokinetics study in the dog.

15AU81 is a chemically stable tricyclic benzindene analog of prostacyclin, with possible application to the treatment of congestive heart failure. Effective pharmacological doses in a dog model are in the microgram to sub-microgram/kg range, necessitating an analytical method of high sensitivity for determination of the drug in plasma. This report describes the development and validation of a radioimmunoassay for 15AU81, and its application to a pharmacokinetic study in the beagle dog. An antiserum elicited by immunization with a 15AU81-bovine thyroglobulin conjugate was employed, along with 3H-15AU81, in the radioimmunoassay. Analogs of 15AU81, as well as a glucuronide metabolite produced by a dog liver subcellular fraction in vitro, were used to demonstrate the specificity of the radioimmunoassay. Specificity was confirmed by comparative analysis by radioimmunoassay and by a quantitative GC/MS procedure of plasma samples from dogs dosed with 15AU81. The limit of quantitation in dog plasma was 1.6 ng/ml; accuracy and precision were both acceptable. The assay was applied to a study of the pharmacokinetics of 15AU81 in the beagle dog after intravenous or intratracheal administration of a single 20-micrograms/kg dose. Following intravenous dosing, 15AU81 was eliminated rapidly from plasma (t1/2, 2.8 min), while after intratracheal administration, clearance appeared to be somewhat slower and bioavailability was appreciable (mean, 46%), suggesting that this route of administration may be worthy of further evaluation.

Disposition and metabolism of triprolidine in mice.

The disposition of the antihistamine, triprolidine, was studied in male and female CD-1 mice after a single oral 50 mg/kg dose of [14C]triprolidine HCl. Urine and feces collected over 72 hr postdosing were analyzed for total radiocarbon, and for parent drug and metabolites by radiochromatography. Structures of metabolites were determined by GC/MS, direct probe MS, FAB/MS, LC/MS, NMR, and IR techniques. More than 80% of the dose was recovered in the urine, with the remainder recovered in the feces. The carboxylic acid analog of triprolidine (219C69) was found to be the major metabolite in urine and feces, accounting for an average of 57.6% of the administered dose. Three minor metabolites were identified as a gamma-aminobutyric acid analog of triprolidine, a pyrrolidinone analog of 219C69, and a pyridine-ring hydroxylated derivative of triprolidine. Parent drug could only be detected in urine and accounted for 0.3% (females) to 1.1% (males) of the dose. The results of this study showed that triprolidine was absorbed well but extensively metabolized when administered orally to mice.

Disposition of triprolidine in the male beagle dog.

Three male beagle dogs were given 2.5 mg/kg doses of [14C]triprolidine HCl monohydrate (2.09 mg/kg of the free base) by intravenous and oral routes, in a nonrandomized cross-over experiment. After either route of administration, approximately 75% of the dose was excreted in the urine, and the remainder was excreted in the feces. Triprolidine was extensively metabolized, with less than 1% of the parent drug recovered in the excreta after either route of administration. Three metabolites were isolated from excreta and identified, including the major metabolite (metabolite 1, 219C69), in which the toluene ring methyl group was oxidized to a carboxylic acid, a metabolite (metabolite 2) in which the pyrrolidine ring was opened with oxidation of the terminal carbon to a carboxylic acid (a gamma-aminobutyric acid), and a metabolite (metabolite 3) that was a pyrrolidinone derivative of 219C69. Other metabolites in urine and feces were present in amounts too small for quantitation or identification. Route of administration had little effect on the metabolic pattern of triprolidine. Thus, after oral administration of triprolidine, a mean of 49.1% of the dose was excreted as 219C69, 12.0% as metabolite 2, 3.4% as metabolite 3, and 0.6% as triprolidine, while after intravenous administration, a mean of 50.8% of the dose was excreted as 219C69, 11.1% as metabolite 2, 4.2% as metabolite 3, and 0.8% as triprolidine. Plasma contained triprolidine, 219C69, and metabolite 2, as well as other apparent metabolites that were present at levels too low for quantitation. Mean pharmacokinetic parameters calculated for triprolidine after intravenous dosing were: CL = 24.4 ml/min/kg, Vdss = 5.8 liters/kg, and Vc = 1.6 liters/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Disposition of acrivastine in the male beagle dog.

Three male beagle dogs were given 10 mg/kg iv and oral doses of [14C]acrivastine, a novel nonsedating antihistaminic agent, in a nonrandomized crossover experiment. Urine and feces were collected for 72 hr after dosing. After iv dosing, a mean of 34% was recovered in the urine, and 63% was recovered in the feces. After po dosing, a mean of 29% of the radiocarbon was recovered in the urine, and 63% was recovered in the feces (dose adjusted for 14% lost in vomitus). Acrivastine and three major metabolites were detected in the excreta. The metabolites were identified as a side-chain-reduced analog of acrivastine (metabolite 3, 270C81), a gamma-aminobutyric acid analog of 270C81 (metabolite 2), and a benzoic acid analog of 270C81 (metabolite 1). After iv dosing, 34% of the dose was excreted as parent drug, 21% as metabolite 3, 15% as metabolite 2, and 6% as metabolite 1, while after po dosing, 35% of the dose was excreted as parent drug, 18% as metabolite 3, 11% as metabolite 2, and 7% as metabolite 1. Pharmacokinetic analysis of acrivastine plasma concentration-time curves after both routes of administration indicated a mean total body clearance of 17.3 ml/min/kg, a Vss of 0.93 liter/kg, a terminal half-life of 0.7 hr, and an oral bioavailability of 40%. The apparent plasma half-life of the metabolite, 270C81, was 1.5 hr. Analysis of AUC values indicated that greater amounts of 270C81 than acrivastine circulated in plasma after both iv and po dosing, and that first-pass metabolism of acrivastine to 270C81 occurred. The results indicated that acrivastine was extensively metabolized in the dog to 270C81 and suggested that 270C81 itself underwent further metabolism to metabolites 1 and 2.

Disposition and pharmacokinetics of inhaled dimethylamine in the Fischer 344 rat.

The disposition and pharmacokinetics of [14C]dimethylamine [( 14C] DMA) following 6-hr inhalation of either 10 or 175 ppm were determined in male Fischer 344 rats. Seventy-two hours after termination of exposure, the disposition of recovered radioactivity was similar for each airborne concentration, with more than 90% in the urine and feces, 7 to 8% in selected tissues and the carcass, and 1.5% exhaled as 14CO2. Over 98% of the radioactivity in the urine was unmetabolized DMA. Analysis of tissue radioactivity immediately after exposure to [14C]DMA showed that the respiratory nasal mucosa contained the highest concentration of 14C, followed by the olfactory nasal mucosa; concentrations of 14C in liver, lung, kidney, brain, and testes were approximately 2 orders of magnitude less than in the nasal mucosal tissues. Radioactivity in plasma of rats exposed by inhalation to 175 ppm of [14C]DMA decayed in a biphasic manner. The terminal half-life for plasma radioactivity was similar to the half-lives of some plasma proteins, suggesting incorporation of 14C into proteins subsequent to metabolism of [14C]DMA. The results indicate that, while most of the inhaled DMA is excreted unchanged, a small amount of oxidative metabolism of DMA occurs.

Metabolism of dimethylamine in the nasal mucosa of the Fischer 344 rat.

The metabolism of dimethylamine (DMA) in the nasal mucosa of the male Fischer 344 rat was investigated in vitro and in vivo. Microsomes were prepared from liver, and from respiratory and olfactory nasal mucosa. All microsomal preparations metabolized DMA to formaldehyde (CH2O), though DMA was a poor substrate for the N-demethylation reaction when compared to benzphetamine. Phenobarbital-induced microsomes metabolized DMA at a rate less than that of control. The results indicated that DMA was a substrate for both cytochrome P-450 and FAD-containing monooxygenase, and that both enzyme activities were present in all microsomal preparations. Finally, unextractable radioactivity was observed in DNA, RNA, and protein isolated from respiratory and olfactory mucosa of rats exposed to either 10 or 175 ppm of [14C]DMA, suggesting metabolism of [14C]DMA to 14CH20 with subsequent incorporation of 14C into macromolecules. The results demonstrate that the respiratory and olfactory nasal mucosa have the capability to metabolize DMA to CH2O, and indicate that such metabolism occurs in vivo.

Sulfhydryl oxidation in rat nasal mucosal tissues after chlorine inhalation.

Male F-344 rats were exposed to target concentrations of 2.5, 5, or 10 ppm Cl2 for various amounts of time, after which respiratory and olfactory nasal mucosal tissues were analyzed for changes in total sulfhydryl (TSH) content. No changes in olfactory mucosa TSH content were observed at the maximum Cl2 exposure. Decreases in content of respiratory mucosa TSH were seen after 6 hr exposures to 5 ppm and 10 ppm, but not 2.5 ppm. The concentration of TSH returned to control values 19 hr after termination of exposure. Analysis of TSH changes in concentration X time (CT) studies suggested that decreases in TSH were dependent on the airborne concentration of Cl2 and not on 'dose' (the CT product). The results suggest that inhaled Cl2 can oxidize tissue sulfhydryl groups at the point of entry, but not at deeper regions of the respiratory tract.

Iprindole-induced phospholipidosis in rat alveolar macrophages: alterations in oxygen consumption and release of oxidants.

Rats chronically treated with the cationic amphipilic drug iprindole developed a phospholipid storage disorder in their pulmonary alveolar macrophages (AMs). AMs from these iprindole-treated rats (IP-AMs) were compared to AMs from control rats (C-AMs) regarding oxygen consumption and the release of two reactive oxygen species, superoxide anion and hydrogen peroxide. Responses of C-AMs and IP-AMs were compared at rest and when stimulated by unopsonized or opsonized zymosan. Opsonization was not necessary in order to induce respiratory burst-associated phenomena in either cell type; in fact in all cases, for given cell type responses to unopsonized zymosan were virtually identical to those of opsonized zymosan. When at rest, IP-AMs consumed oxygen at a rate nearly identical to that of C-AMs. When stimulated with zymosan particles, IP-AMs consumed more oxygen than controls. However, when superoxide anion and hydrogen peroxide, two products of the respiratory burst were measured, IP-AMs released less of these species than C-AMs when at rest and when particle stimulated. Despite the lower total release of these species by the IP-AMs, the zymosan-induced release (stimulated minus resting levels) was greater for these cells than C-AMs. Therefore, the IP-AMs were found to be more responsive to the zymosan particle than C-AMs. The results indicate marked changes in the release of reactive oxygen species from the AMs following induction of phospholipidosis.