Improved pharmacokinetic and bioavailability support of drug discovery using serial blood sampling in mice.

Pharmacokinetic studies in mice traditionally require one animal per time point, resulting in dosing and euthanizing a large number of animals and producing suboptimal quality of pharmacokinetic data due to inter-animal variability and dosing error. These studies are time-consuming and labor-intensive. To improve the throughput and quality of pharmacokinetic evaluation in mice, we have developed a serial blood sampling methodology using the lateral saphenous vein puncture technique. Two marketed drugs, indinavir and rosuvastatin, were selected for this validation study because of their distinctly different physicochemical and pharmacokinetic properties. Each compound was dosed orally and intravenously in mice using both discrete and serial blood sampling methods. The pharmacokinetic results from serial bleeding are in excellent agreement with those from discrete sampling for both compounds. Compared to the discrete sampling, the serial sampling procedure is a more humane method, allowing for rapid and repeated sampling from the same site without the need for anesthesia. The application of this new method has led to a remarkable reduction in animal and compound usage, a significant increase in throughput and speed, and a drastic improvement in pharmacokinetic data quality. This approach is especially useful for the first-tier in vivo pharmacokinetic screening of discovery compounds.

Synthesis and biological evaluation of novel pyridazinone-based alpha4 integrin receptor antagonists.

A novel series of pyridazinone-functionalized phenylalanine analogues was prepared and evaluated for inhibition of cellular adhesion mediated by alpha4beta1/VCAM-1 and alpha4beta7/MAdCAM-1 interactions. Concise syntheses were developed and applied for exploration of structure-activity relationships pertaining to the pyridazinone ring as well as the N-acyl phenylalanine scaffold. Potent dual antagonists of alpha4beta1 and alpha4beta7 were generated from an amide subseries; antagonists selective for alpha4beta7 were identified from urea and carbamate-based subseries. The pharmacokinetic properties of selected members of the series have been determined in rats and demonstrate that the use of ester prodrugs and alterations to the amide linkage can lead to improved oral bioavailability in this series. An alpha4beta7-selective member of the carbamate subseries (36c), upon oral administration, demonstrated in vivo efficacy in the mouse DSS colitis model.

Characterization of RWJ-351647, a novel nonpeptide vasopressin V2 receptor antagonist.

1. Antagonists of the V(2) vasopressin (AVP) receptor are aquaretic agents, inhibiting water resorption without stimulating electrolyte excretion. In this set of experiments, a novel V(2) receptor antagonist, RWJ-351647, was characterized in vitro and in vivo. 2. RWJ-351647 displaced (3)H-AVP binding from cloned human V(2) and V(1A) receptors with Ki values of 1 nmol/L and 24 nmol/L. In assays using transfected HEK293 cells expressing either human or rat V(2) receptors, RWJ-351647 inhibited AVP-induced cAMP accumulation with Ki values of 3 nmol/L and 6 nmol/L, respectively. 3. RWJ-351647 was very selective in binding assays and showed only weak functional antagonist activity at either the cloned human V(1B) and oxytocin receptors or the human platelet V(1A) receptor. No agonist activity was seen with the compound at any receptor. 4. Pharmacokinetic studies in rats showed RWJ-351647 to be 41.9% bioavailable after a single oral administration. After repeated daily dosing over 5 days, the oral bioavailability remained at 43.9% with no change in the compound peak plasma levels or clearance rate. 5. In efficacy studies, RWJ-351647 increased urine output and decreased urine osmolality with oral doses as low as 0.1 mg/kg and 1.0 mg/kg in rats and cynomolgus monkeys, respectively. In a multiple dose study in primates, RWJ-351647 maintained a consistent aquaretic effect over 10 days without increasing sodium or potassium excretion. 6. In summary, RWJ-351647 was shown to be a selective and potent V(2) receptor antagonist with sustainable aquaretic activity in both rats and primates. The preclinical data suggest that RWJ-351647 is a potent and effective aquaretic agent with potential for use in diseases characterized by water retention.

The use of the suicide CYP450 inhibitor ABT for distinguishing absorption and metabolism processes in in-vivo pharmacokinetic screens.

Since drug candidates with low oral systemic exposure may be due to either or both absorption and metabolism factors, determining what factors limit the oral systemic exposure is not always obvious in a single in-vivo pharmacokinetic (PK) assay. A rapid rat in-vivo PK screen where the oxidative drug metabolism has been attenuated using the suicide CYP450 inhibitor aminobenzotriazole (ABT) is described. We have shown that the roles of absorption and metabolism for drug candidates with low oral systemic exposure can be determined by comparing the PK parameters of drug candidates orally administered to non-treated and ABT-treated rats. Propranolol, metoprolol and climetidine are used as model drugs. Propranolol and metoprolol have low oral systemic exposures in rats primarily due to metabolism factors while the oral systemic exposure of climetidine is high in rats. For propranolol and metoprolol, large increases in the systemic exposure of these drugs were observed between non-treated and ABT-treated rats. ABT appeared not to increase or decrease significantly the rate and extent of absorption or metabolism of cimetidine since that oral systemic exposure of non-treated and ABT-treated rats did not significantly change. These experiments suggest that for drug candidates with low systemic exposures in rats an observation of no change in the oral systemic exposure in ABT-treated rats when compared to the non-treated rats imply that absorption (or formulation) factors limit the systemic exposure of the drug while an increase in the systemic exposure in ABT-treated rats imply that metabolism factors limit the systemic exposure. Due to the ease of preparing and interpreting PK data from ABT-treated rats, is suggested that this assay could be used as an alternative to in vivo cannulation assays.

The application of nuclear magnetic resonance-based metabonomics to the dominant-submissive rat behavioral model.

Nuclear magnetic resonance (NMR) methods were used to study whether there are differences in the urine content between behaviorally distinct groups of rats: dominant and submissive. The dominant-submissive relationships (DSRs) were established in rat pairs competing for access to the feeder filled with sweetened milk. Dominant rats spend significantly longer amounts of time at the feeder than do their submissive partners. During a 2-week period, rats were tested for the DSR. At the end of the second week, behavioral groups of rats were selected and urine was collected during a 3.5-h time period. Principal component analysis revealed a metabolite from milk sugar, galactose, as a discriminating factor between rats classified as dominant and those classified as submissive. Measurements of galactose showed that the amount present in the urine correlated with the time spent in the feeder zone, thereby supporting the time criterion established for the DSR model.

1,2,3,4-Tetrahydroquinoline-containing alphaVbeta3 integrin antagonists with enhanced oral bioavailability.

Reduction of the quinoline ring in an alpha(v)beta(3) antagonist yielded a 1,2,3,4-tetrahydro derivative as two diastereomers, the four isomers of which were separated by sequential chiral HPLC. Two isomers had significant alpha(V)beta(3) antagonist activity with improved oral bioavailability, relative to the corresponding quinoline derivative.

Liquid chromatographic and tandem mass spectrometric assay for evaluation of in vivo inhibition of rat brain monoamine oxidases (MAO) A and B following a single dose of MAO inhibitors: application of biomarkers in drug discovery.

A simple and selective assay for the evaluation of in vivo inhibition of rat brain monoamine oxidases (MAO) A and B following a single dose of MAO inhibitors was developed through the simultaneous determination of endogenous 5-hydroxy tryptamine, 5-hydroxyindole-3-acetic acid (5-HIAA), tryptophane, and 2-phenethylamine (PEA) in rat brain using liquid chromatography-tandem mass spectrometry (LC/MS/MS). These analytes were separated on a Zorbax SB-C18 column using a gradient elution with acetonitrile and 0.2% formic acid and detected on an electrospray ionization mass spectrometer in positive-ion multiple-reaction-monitoring mode. The susceptibility and variability of these analytes as potential biomarkers in response to MAO inhibition in vivo were evaluated after application to three MAO inhibitors, tranylcypromine, clorgyline, and pargyline. A dramatic increase (about 40-fold) in PEA brain level and a decrease in 5-HIAA by more than 90% were observed after administration of 15 mg/kg of the nonselective MAO inhibitor tranylcypromine. As expected, the brain level of PEA escalated to about 6-fold, while the 5-HIAA level remained unchanged following a dose of the MAO B inhibitor pargyline at 2mg/kg. In contrast, the brain level of 5-HIAA reduced by approximately 53%, but the PEA level was unaffected following the same dose of the MAO A inhibitor clorgyline. The results indicated that 5-HIAA and PEA were susceptible and effective biomarkers in the rat brain in response to MAO A and B inhibition, respectively. The LC/MS/MS method is useful not only for the determination of inhibitory potency but also for the differentiation of the selectivity of a MAO inhibitor against rat brain MAO A and B in vivo.

Allometric scaling of pharmacokinetic parameters in drug discovery: can human CL, Vss and t1/2 be predicted from in-vivo rat data?

In a drug discovery environment, reasonable go/no-go human in-vivo pharmacokinetic (PK) decisions must be made in a timely manner with a minimum amount of animal in-vivo or in-vitro data. We have investigated the accuracy of the in-vivo correlation between rat and human for the prediction of the total systemic clearance (CL), the volume of distribution at steady state (Vss), and the half-life (t1/2) using simple allometric scaling techniques. We have shown, using a large diverse set of drugs, that a fixed exponent allometric scaling approach can be used to predict human in-vivo PK parameters CL, Vss and t(1/2) solely from rat in-vivo PK data with acceptable accuracy for making go/no-go decisions in drug discovery. Human in-vivo PK predictions can be obtained using the simple allometric scaling relationships CL(Human) approximately = 40 CL(Rat) (L/hr), Vss(Human) approximately = 200 Vss(Rat) (L), and t1/2(Human) approximately = 4 t1/2(Rat) (hr). The average fold error for human CL predictions for N = 176 drugs was 2.25 with 79% of the drugs having a fold error less than 3. The average fold error for human Vss predictions for N = 144 drugs was 1.85 with 84% of the drugs having a fold error less than 3. The average fold error for human t1/2 predictions for N = 145 drugs was 2.05 with 76% of the drugs having a fold error less than 3. Using these simple allometric relationships, the sorting of drug candidates into a low/medium/high/very high human classification scheme was also possible from rat data. Since these simple allometric relationships between rat and human CL, Vss, and t1/2 are reasonably accurate, easy to remember and simple to calculate, these equations should be useful for making early go/no-go in-vivo human PK decisions for drug discovery candidates.

Synthesis and evaluation of nonpeptide substituted spirobenzazepines as potent vasopressin antagonists.

A series of substituted spirobenzazepines was prepared and evaluated as V(1a) and V(2) dual vasopressin receptor antagonists. Compounds 7p and 7q have been shown to be not only potent inhibitors of vasopressin receptors, but also have exhibited an excellent overall pharmaceutical suitability profile.

Potent nonpeptide vasopressin receptor antagonists based on oxazino- and thiazinobenzodiazepine templates.

Vasopressin receptor antagonists can elicit ion-sparing diuretic effects (i.e., aquaresis) in vivo by blunting the action of the circulating hypophyseal hormone arginine vasopressin. We have identified two new series of basic tricyclic benzodiazepines, represented by general structure 1, which contain compounds that bind with high affinity to human V2 receptors. For example, (S)-(+)-8 and 5 are potent and selective V2 receptor antagonists with pronounced aquaretic activity in rats on oral administration.

Synthesis and evaluation of spirobenzazepines as potent vasopressin receptor antagonists.

A novel series of spirobenzazepines was synthesized and evaluated for V1a and V2 receptor antagonist activity. Compounds 8b, 8i, and 8k have shown selective V1a receptor antagonist activity. Compounds 8p and 8q were shown to be dual V1a/V2 receptor antagonists.

Discovery of a dihydropyrimidine series of molecules that selectively mimic the biological actions of calcitonin.

The use of a multiplex mimetic assay led us to identify 1,4-dihydropyrimidines with potent and selective calcitonin receptor mimetic activity. Subsequent modification of the dihydropyrimidine scaffold led to a series of molecules that were efficacious in a neonatal mouse calvaria in vitro model. Dihydropyrimidine 5h, in particular, was identified as a calcitonin mimetic (EC(50)=6 microM), active in-vivo in the Weanling rat model when administered subcutaneously.

Synthesis and biological evaluation of novel indoloazepine derivatives as non-peptide vasopressin V2 receptor antagonists.

A series of novel 3,4,5,6-tetrahydro-1H-azepino[4,3,2-cd]indoles was synthesized and tested for vasopressin receptor antagonist activity. We identified compounds with high affinity for the human V2 receptor and good selectivity over the human V1a receptor. Compound 6c bound to V2 receptors with an IC(50) value of 20 nM, had >100-fold selectivity over V1a receptors, and inhibited cAMP formation in a cellular V2 functional assay with an IC(50) value of 70 nM.

Bridged bicyclic vasopressin receptor antagonists with V(2)-selective or dual V(1a)/V(2) activity.

The synthesis and biological testing of a novel series of nonpeptide vasopressin receptor antagonists, containing a bridged bicyclic nucleus, are reported. Variation of substituents (R(1)-R(3)) in general formula 3, and the configuration of the stereocenter, resulted in potent V(2)-selective (e.g., 5) and balanced dual V(1a)/V(2) (e.g., 10) compounds. Data from receptor binding, cell-based functional, and in vivo assays are presented [corrected]