Pharmacological evaluation of selective α2c-adrenergic agonists in experimental animal models of nasal congestion.

Nasal congestion is one of the most troublesome symptoms of many upper airways diseases. We characterized the effect of selective α2c-adrenergic agonists in animal models of nasal congestion. In porcine mucosa tissue, compound A and compound B contracted nasal veins with only modest effects on arteries. In in vivo experiments, we examined the nasal decongestant dose-response characteristics, pharmacokinetic/pharmacodynamic relationship, duration of action, potential development of tolerance, and topical efficacy of α2c-adrenergic agonists. Acoustic rhinometry was used to determine nasal cavity dimensions following intranasal compound 48/80 (1%, 75 µl). In feline experiments, compound 48/80 decreased nasal cavity volume and minimum cross-sectional areas by 77% and 40%, respectively. Oral administration of compound A (0.1-3.0 mg/kg), compound B (0.3-5.0 mg/kg), and d-pseudoephedrine (0.3 and 1.0 mg/kg) produced dose-dependent decongestion. Unlike d-pseudoephedrine, compounds A and B did not alter systolic blood pressure. The plasma exposure of compound A to produce a robust decongestion (EC(80)) was 500 nM, which related well to the duration of action of approximately 4.0 hours. No tolerance to the decongestant effect of compound A (1.0 mg/kg p.o.) was observed. To study the topical efficacies of compounds A and B, the drugs were given topically 30 minutes after compound 48/80 (a therapeutic paradigm) where both agents reversed nasal congestion. Finally, nasal-decongestive activity was confirmed in the dog. We demonstrate that α2c-adrenergic agonists behave as nasal decongestants without cardiovascular actions in animal models of upper airway congestion.

Substituted imidazole of 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine Inactivates cytochrome P450 2D6 by protein adduction.

5-Fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine (SCH 66712) is a potent mechanism-based inactivator of human cytochrome P450 2D6 that displays type I binding spectra with a K(s) of 0.39 ± 0.10 µM. The partition ratio is ~3, indicating potent inactivation that addition of exogenous nucleophiles does not prevent. Within 15 min of incubation with SCH 66712 and NADPH, ∼90% of CYP2D6 activity is lost with only ~20% loss in ability to bind CO and ~25% loss of native heme over the same time. The stoichiometry of binding to the protein was 1.2:1. SDS-polyacrylamide gel electrophoresis with Western blotting and autoradiography analyses of CYP2D6 after incubations with radiolabeled SCH 66712 further support the presence of a protein adduct. Metabolites of SCH 66712 detected by mass spectrometry indicate that the phenyl group on the imidazole ring of SCH 66712 is one site of oxidation by CYP2D6 and could lead to methylene quinone formation. Three other metabolites were also observed. For understanding the metabolic pathway that leads to CYP2D6 inactivation, metabolism studies with CYP2C9 and CYP2C19 were performed because neither of these enzymes is significantly inhibited by SCH 66712. The metabolites formed by CYP2C9 and CYP2C19 are the same as those seen with CYP2D6, although in different abundance. Modeling studies with CYP2D6 revealed potential roles of various active site residues in the oxidation of SCH 66712 and inactivation of CYP2D6 and showed that the phenyl group of SCH 66712 is positioned at 2.2 Å from the heme iron.

Pharmacokinetics of pseudoephedrine in rats, dogs, monkeys and its pharmacokinetic-pharmacodynamic relationship in a feline model of nasal congestion.

The objectives of these studies were to characterize the pharmacokinetics (PK) of the nasal decongestant pseudoephedrine (PSE) in rats, dogs, and monkeys, and to evaluate its lower gastrointestinal tract regional bioavailability in rats. An LC-MS/MS assay with a lower limit of quantification (LLOQ) of 0.4 ng/mL of plasma was developed for the analysis of PSE in animal plasma. The total body clearance (CL) was the highest in rats (78 mL/min/kg), lowest in monkeys (15 mL/min/kg) and the dog averaged in between (33 mL/min/kg). The volume of distribution at steady state (Vdss) ranged from 3-5 L/kg in all species. In rats and dogs, the mean half-lives (t1/2) was ≈1.5 hr, while in monkeys the mean t1/2 was 4.6 hr, comparable to that observed in adult humans (4-8 hr). The oral bioavailability was 38, 58 and 78% in rats, dogs and monkeys. The bioavailability following intra-ileum or intra-colonic administration in rats was superior to that following oral dosing (66% and 78%, respectively) suggesting that colonic absorption may be compensating for the short half-life, thus enabling successful QD sustained release formulations of PSE. The pharmacokinetic/pharmacodynamic relationship (PK/PD) of PSE was also investigated in a feline model of nasal congestion to establish efficacious trough concentrations in cats for a comparison with that in humans. The PK/PD in the cat model followed a sigmoid Emax model with an EC50 (plasma concentration that elicits 50% of the maximum response) of 0.32 ±0.05 (SD) µM consistent with human plasma concentrations required for efficacy.

Evaluation of CYP1A1 and CYP2B1/2 m-RNA induction in rat liver slices using the NanoString technology: a novel tool for drug discovery lead optimization.

Cytochrome P450 (CYP) induction in rodents and humans is considered a liability for new chemical entities (NCEs) in drug discovery. In particular, CYP1A1 and CYP2B1/2 have been associated with the induction of liver tumors in oncogenicity studies during safety evaluation studies of potential drugs. In our laboratory, real time PCR (Taqman) has been used to quantify the induction of rat hepatic CYP1A1 and CYP2B1/2 in precision -cut rat liver slices. A novel technology that does not require m-RNA isolation or RT-PCR, (developed by NanoString Technologies) has been investigated to quantify CYP1A1 and CYP2B1/2 induction in rat liver slices. Seventeen commercially available compounds were evaluated using both Taqman and NanoString technologies. Precision-cut rat liver slices were incubated with individual compounds for 24 hr at 37 degrees C in a humidified CO(2) incubator and CYP1A1 and CYP2B1/2 m-RNA were quantified. The results from the NanoString technology were similar to those of the Taqman(R) with a high degree of correlation for both CYP isoforms (r(2)>0.85). Therefore, NanoString provides an additional new technology to evaluate the induction of CYP1A1 and 2B1/2, as well as potentially other enzymes or transporters in rat liver slices.

High-throughput evaluation of CYP1A1 and 2B1 induction in rat liver slices using a semi-automated system.

Drug candidates with the propensity to induce rat CYP1A1 or 2B1 isoforms are believed to possess a greater tendency to induce hepatic tumors in oncogenicity studies. We have previously published on a manual rat liver slice assay that showed a satisfactory relationship between in vitro CYP2B1 m-RNA induction using real time PCR and the ex vivo pentoxyresorufin O-dealkylase (PROD) activity in liver microsomes prepared from rats treated daily via the oral route for 14 consecutive days with inducers or non-inducers. We now describe this automated in vitro high throughput liver slice technique to screen out drug candidates that are potent rodent CYP1A1 and/or CYP2B1 inducers. A good concordance between in vitro and in vivo data was observed for both CYP1A1 (100 %) and CYP2B1 (90%) isoforms. Automation of key steps has enabled us to increase the annual screening throughput from 200 (manual) to 1500 compounds. The increase in throughput allowed the quick development of structure-induction relationships (SIR's) for multiple drug discovery programs in a facile manner.

Assessment of temporal biochemical and gene transcription changes in rat liver cytochrome P450: utility of real-time quantitative RT-PCR.

PURPOSE: A conventional approach to assess cytochrome P450 (CYP) induction in preclinical animal models involves daily dosing for a least a week followed by Western blot and/or enzyme activity analysis. To evaluate the potential benefit of a third more specific and sensitive assay, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), with the objective of reducing the duration of the conventional 1-week study, we simultaneously assessed gene expression by qRT-PCR along with Western blots and enzyme activity assays as a time course in an in vivo model. METHODS: Rats were dosed daily for 8 days with model inducers of CYP1A, CYP2B, CYP3A, or CYP4A. Liver P450 levels were measured after 0.5, 1, 2, 4, and 8 days of dosing by qRT-PCR, Western blot, and enzyme activity. RESULTS: CYP1A, CYP3A, and CYP4A genes were maximally induced very rapidly (0.5-1 day), whereas the CYP2B gene was maximally induced after a lag time of 4 days. In all cases, fold changes in induction detected by qRT-PCR were greater than fold changes in protein levels and enzyme activities. CONCLUSIONS: Maximal persistent and larger fold changes observed by qRT-PCR either preceded or occurred simultaneously with maximal sustained fold changes in protein levels as measured by Western blots and enzyme activity assays. Our data show that qRT-PCR provides increased sensitivity and specificity over conventional assays and may be key information for reliable assessment of drug-related changes in CYP induction during the transition from discovery to toxicology studies.