Defining the Contribution of CYP1A1 and CYP1A2 to Drug Metabolism Using Humanized CYP1A1/1A2 and Cyp1a1/Cyp1a2 Knockout Mice.

Cytochrome P450s CYP1A1 and CYP1A2 can metabolize a broad range of foreign compounds and drugs. However, these enzymes have significantly overlapping substrate specificities. To establish their relative contribution to drug metabolism in vivo, we used a combination of mice humanized for CYP1A1 and CYP1A2 together with mice nulled at the Cyp1a1 and Cyp1a2 gene loci. CYP1A2 was constitutively expressed in the liver, and both proteins were highly inducible by 2,3,7,8-tetrachlorodibenzodioxin (TCDD) in a number of tissues, including the liver, lung, kidney, and small intestine. Using the differential inhibition of the human enzymes by quinidine, we developed a method to distinguish the relative contribution of CYP1A1 or CYP1A2 in the metabolism of drugs and foreign compounds. Both enzymes made a significant contribution to the hepatic metabolism of the probe compounds 7-methoxy and 7-ehthoxyresorufin in microsomal fractions from animals treated with TCDD. This enzyme kinetic approach allows modeling of the CYP1A1, CYP1A2, and non-CYP1A contribution to the metabolism of any substrate at any substrate, inhibitor, or enzyme concentration and, as a consequence, can be integrated into a physiologically based pharmacokinetics model. The validity of the model can then be tested in humanized mice in vivo. SIGNIFICANCE STATEMENT: Human CYP1A1 and CYP1A2 are important in defining the efficacy and toxicity/carcinogenicity of drugs and foreign compounds. In light of differences in substrate specificity and sensitivity to inhibitors, it is of central importance to understand their relative role in foreign compound metabolism. To address this issue, we have generated mice humanized or nulled at the Cyp1a gene locus and, through the use of these mouse lines and selective inhibitors, developed an enzyme kinetic-based model to enable more accurate prediction of the fate of new chemicals in humans and which can be validated in vivo using mice humanized for cytochrome P450-mediated metabolism.

Development of (6 R)-2-Nitro-6-[4-(trifluoromethoxy)phenoxy]-6,7-dihydro-5 H-imidazo[2,1- b][1,3]oxazine (DNDI-8219): A New Lead for Visceral Leishmaniasis.

Discovery of the potent antileishmanial effects of antitubercular 6-nitro-2,3-dihydroimidazo[2,1- b][1,3]oxazoles and 7-substituted 2-nitro-5,6-dihydroimidazo[2,1- b][1,3]oxazines stimulated the examination of further scaffolds (e.g., 2-nitro-5,6,7,8-tetrahydroimidazo[2,1- b][1,3]oxazepines), but the results for these seemed less attractive. Following the screening of a 900-compound pretomanid analogue library, several hits with more suitable potency, solubility, and microsomal stability were identified, and the superior efficacy of newly synthesized 6 R enantiomers with phenylpyridine-based side chains was established through head-to-head assessments in a Leishmania donovani mouse model. Two such leads ( R-84 and R-89) displayed promising activity in the more stringent Leishmania infantum hamster model but were unexpectedly found to be potent inhibitors of hERG. An extensive structure-activity relationship investigation pinpointed two compounds ( R-6 and pyridine R-136) with better solubility and pharmacokinetic properties that also provided excellent oral efficacy in the same hamster model (>97% parasite clearance at 25 mg/kg, twice daily) and exhibited minimal hERG inhibition. Additional profiling earmarked R-6 as the favored backup development candidate.

Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion.

Excess mineralocorticoid receptor (MR) activation promotes target organ dysfunction, vascular injury and fibrosis. MR antagonists like eplerenone are used for treating heart failure, but their use is limited due to the compound class-inherent hyperkalemia risk. Here we present evidence that AZD9977, a first-in-class MR modulator shows cardio-renal protection despite a mechanism-based reduced liability to cause hyperkalemia. AZD9977 in vitro potency and binding mode to MR were characterized using reporter gene, binding, cofactor recruitment assays and X-ray crystallopgraphy. Organ protection was studied in uni-nephrectomised db/db mice and uni-nephrectomised rats administered aldosterone and high salt. Acute effects of single compound doses on urinary electrolyte excretion were tested in rats on a low salt diet. AZD9977 and eplerenone showed similar human MR in vitro potencies. Unlike eplerenone, AZD9977 is a partial MR antagonist due to its unique interaction pattern with MR, which results in a distinct recruitment of co-factor peptides when compared to eplerenone. AZD9977 dose dependently reduced albuminuria and improved kidney histopathology similar to eplerenone in db/db uni-nephrectomised mice and uni-nephrectomised rats. In acute testing, AZD9977 did not affect urinary Na+/K+ ratio, while eplerenone increased the Na+/K+ ratio dose dependently. AZD9977 is a selective MR modulator, retaining organ protection without acute effect on urinary electrolyte excretion. This predicts a reduced hyperkalemia risk and AZD9977 therefore has the potential to deliver a safe, efficacious treatment to patients prone to hyperkalemia.

Clinical pharmacokinetics and pharmacodynamic target attainment of pazufloxacin in prostate tissue: Dosing considerations for prostatitis.

The present study examined the clinical pharmacokinetics of pazufloxacin in prostate tissue and estimated the probability of target attainment for tissue-specific pharmacodynamic goals related to treating prostatitis using various intravenous dosing regimens. Patients with prostatic hypertrophy received prophylactic infusions of pazufloxacin (500 mg, n = 23; 1000 mg, n = 25) for 0.5 h prior to transurethral prostate resection. Drug concentrations in plasma (0.5-5 h) and prostate tissue (0.5-1.5 h) were measured by high-performance liquid chromatography and used for subsequent noncompartmental and three-compartmental analysis. Monte Carlo simulation was performed to evaluate the probability of target attainment of a specific minimum inhibitory concentration (MIC) in prostate tissue: the proportion that achieved both area under the drug concentration over time curve (AUC)/MIC = 100 and maximum concentration (Cmax)/MIC = 8. Prostatic penetration of pazufloxacin was good with mean Cmax ratios (prostate tissue/plasma) of 0.82-0.99 and for AUC, 0.80-0.98. The probability of reaching target MIC concentrations in prostate tissue was more than 90% for dosing schedules of 0.25 mg/L for 500 mg every 24 h (500 mg daily), 0.5 mg/L for 500 mg every 12 h (1000 mg daily), 1 mg/L for 1000 mg every 24 h (1000 mg daily), and 2 mg/L for 1000 mg every 12 h (2000 mg daily). Importantly, the 2000 mg daily regimen of pazufloxacin produced a profile sufficient to have an antibacterial effect in prostate tissue against clinical isolates of Escherichia coli and Klebsiella pneumonia with MIC values less than 2 mg/L.

Discovery of BI 135585, an in vivo efficacious oxazinanone-based 11ß hydroxysteroid dehydrogenase type 1 inhibitor.

A potent, in vivo efficacious 11ß hydroxysteroid dehydrogenase type 1 (11ß HSD1) inhibitor (11j) has been identified. Compound 11j inhibited 11ß HSD1 activity in human adipocytes with an IC50 of 4.3nM and in primary human adipose tissue with an IC80 of 53nM. Oral administration of 11j to cynomolgus monkey inhibited 11ß HSD1 activity in adipose tissue. Compound 11j exhibited >1000× selectivity over other hydroxysteroid dehydrogenases, displays desirable pharmacodynamic properties and entered human clinical trials in 2011.

In vitro and in vivo activities of the nitroimidazole TBA-354 against Mycobacterium tuberculosis.

Nitroimidazoles are a promising new class of antitubercular agents. The nitroimidazo-oxazole delamanid (OPC-67683, Deltyba) is in phase III trials for the treatment of multidrug-resistant tuberculosis, while the nitroimidazo-oxazine PA-824 is entering phase III for drug-sensitive and drug-resistant tuberculosis. TBA-354 (SN31354[(S)-2-nitro-6-((6-(4-trifluoromethoxy)phenyl)pyridine-3-yl)methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine]) is a pyridine-containing biaryl compound with exceptional efficacy against chronic murine tuberculosis and favorable bioavailability in preliminary rodent studies. It was selected as a potential next-generation antituberculosis nitroimidazole following an extensive medicinal chemistry effort. Here, we further evaluate the pharmacokinetic properties and activity of TBA-354 against Mycobacterium tuberculosis. TBA-354 is narrow spectrum and bactericidal in vitro against replicating and nonreplicating Mycobacterium tuberculosis, with potency similar to that of delamanid and greater than that of PA-824. The addition of serum protein or albumin does not significantly alter this activity. TBA-354 maintains activity against Mycobacterium tuberculosis H37Rv isogenic monoresistant strains and clinical drug-sensitive and drug-resistant isolates. Spontaneous resistant mutants appear at a frequency of 3 × 10(-7). In vitro studies and in vivo studies in mice confirm that TBA-354 has high bioavailability and a long elimination half-life. In vitro studies suggest a low risk of drug-drug interactions. Low-dose aerosol infection models of acute and chronic murine tuberculosis reveal time- and dose-dependent in vivo bactericidal activity that is at least as potent as that of delamanid and more potent than that of PA-824. Its superior potency and pharmacokinetic profile that predicts suitability for once-daily oral dosing suggest that TBA-354 be studied further for its potential as a next-generation nitroimidazole.

Fostamatinib, a Syk inhibitor prodrug for the treatment of inflammatory diseases.

Rigel Pharmaceuticals Inc is developing fostamatinib, a prodrug of the spleen tyrosine kinase (Syk) inhibitor R-406, for the potential treatment of autoimmune diseases such as rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP) and B-cell lymphomas. Syk is a key mediator of Fc and B-cell receptor signaling in inflammatory cells, such as B-cells, mast cells, macrophages and neutrophils. Preclinical studies of R-406 or fostamatinib demonstrated a significant reduction in major inflammatory mediators such as TNFalpha, IL-1, IL-6 and IL-18, leading to reduced inflammation and bone degradation in models of RA. In a phase II clinical trial, fostamatinib treatment effectively improved American College of Rheumatology response rates in patients with RA. Preclinical studies and phase II trials also suggested the potential of using fostamatinib for the treatment of ITP and B-cell lymphomas, by increasing platelet counts and inducing response rates, respectively. Fostamatinib is orally bioavailable and was well tolerated in phase I and II trials, with the most common side effect being gastrointestinal symptoms. At the time of publication, phase II trials for fostamatinib were ongoing in patients with RA, ITP and B-cell lymphomas. The Syk inhibitor appears to be a promising therapeutic for immunological diseases, but further data are required to establish the efficacy and long-term safety of the drug in humans.

Preclinical pharmacokinetics and interspecies scaling of ragaglitazar, a novel biliary excreted PPAR dual activator.

Allometric scaling has been used as an effective tool for the prediction of human pharmacokinetic parameters. Allometry has been a useful approach for the analysis of compounds that are eliminated unchanged in the urine and/or exhibit similar metabolic patterns across species. However, it has been a challenging issue to correctly predict human pharmacokinetic parameters for drugs that are eliminated intact and/or as conjugates in the bile. Ragaglitazar is a novel, non-thiazolidinedione peroxisome proliferator-activated receptor (PPAR) alpha- and gamma-agonist. In our investigation, preclinical pharmacokinetic data on ragaglitazar were gathered for several animal species (mice, rats, rabbits and dogs). Ragaglitazar when administered orally has shown a low clearance rate (Cl/F; < 5% of hepatic blood flow) in mice, rats and rabbits and a moderately high Cl/F in dogs (> 15% of hepatic blood flow). A qualitative estimation of rat bile has unequivocally confirmed the elimination of ragaglitazar in the bile. The human pharmacokinetic data are also indicative of the involvement of enterohepatic biliary recycling. In order to predict key parameters such as Cl/F and volume of distribution (V/F), simple allometry was the approach adopted at the onset. Although V/F scaled adequately, it failed to accurately predict human Cl/F. Therefore, standard correction factors such as maximum life span potential (MLP) and brain weight were also included. Although such modifications improved the linearity (r2 > 0.9), they failed to predict the investigated values. Further incorporation of correction factors particularly relevant to biliary excreted drugs improved the prediction of these values. Interestingly, the exclusion of dog data from the interspecies scaling considerably improved the prediction of both Cl/F and V/F.

R406, an orally available spleen tyrosine kinase inhibitor blocks fc receptor signaling and reduces immune complex-mediated inflammation.

Recent compelling evidence has lead to renewed interest in the role of antibodies and immune complexes in the pathogenesis of several autoimmune disorders, such as rheumatoid arthritis. These immune complexes, consisting of autoantibodies to self-antigens, can mediate inflammatory responses largely through binding and activating the immunoglobulin Fc receptors (FcRs). Using cell-based structure activity relationships with cultured human mast cells, we have identified the small molecule R406 [N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine] as a potent inhibitor of immunoglobulin E (IgE)- and IgG-mediated activation of Fc receptor signaling (EC(50) for degranulation = 56-64 nM). Here we show that the primary target for R406 is the spleen tyrosine kinase (Syk), which plays a key role in the signaling of activating Fc receptors and the B-cell receptor (BCR). R406 inhibited phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 bound to the ATP binding pocket of Syk and inhibited its kinase activity as an ATP-competitive inhibitor (K(i) = 30 nM). Furthermore, R406 blocked Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and BCR-mediated activation of B lymphocytes. R406 was selective as assessed using a large panel of Syk-independent cell-based assays representing both specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models. Finally, we report a first-inhuman study showing that R406 is orally bioavailable, achieving exposures capable of inhibiting Syk-dependent IgE-mediated basophil activation. Collectively, the results show R406 potential for modulating Syk activity in human disease.

Evaluation of the excretion, and metabolism of the cardiotonic agent bemoradan in male rats and female beagle dogs.

The excretion and metabolism of (+/-) [6-(3,4-dihydro-3-oxo-1,4[2H]-benzoxazine-yl)-2,3,4,5-tetrahydro-5-methylpyridazin-3-one] (bemoradan; RWJ-22867) have been investigated in male Long-Evans rats and female beagle dogs. Radiolabeled [14C] bemoradan was administered to rats as a singkle 1 mg/kg suspension dose while the dogs received 0.1 mg/kg suspension dose. Plasma (0-24 h; rat and dog), urine (0-72 h; rat and dog) and fecal (0-72 h; rat and dog) samples were collected and analyzed. The terminal half-life of the total radioactivity for rats from plasma was estimate to be 4.3 +/- 0.1 h while for dogs it was 7.5 +/- 1.3 h. Recoveries of total radioactivity in urine and feces for rats were 49.1 +/- 2.4% and 51.1 +/- 4.9% of th dose, respectively. Recoveries of total radioactivity in urine and feces for dogs were 56.2 +/- 12.0% and 42.7 V 9.9% of the dose, respectively. Bemoradan and a total of nine metabolites were isolated and tentatively identified in rat and dog plasma, urine, and fecal extracts. Unchanged bemoradan accounted for approimately < 2% of the dose in rat urine and 20% in rat feces. Unchanged bemoradan accounted for approximately 5% of the dose in urine and 16% in feces in dog. Six proposed pathways were used to describe the metabolites found in rats and dogs: pyridazinyl oxidations, methyl hydroxylation, hydration, N-oxidation, dehydration and phase II conjugations.

Correlation of anti-inflammatory activity with peak tissue rather than peak plasma levels of BF389.

BF389 (Biofor 389) is a potent anti-inflammatory agent in various animal models including adjuvant and type II collagen arthritis in rats. In vitro assays indicate that this compound is a mixed inhibitor of arachidonic acid metabolism. The compound was evaluated for effects on the acute inflammatory response in the carrageenan paw edema assay in rats using a standard protocol in which the animals were given single or multiple (daily for 5 days) p.o. doses of the compound. Carrageenan was injected into the footpad of each animal 1 hr after dosing and volumes of both hind paws were determined 3 hr later. Basal serum prostaglandin (PG) E2 levels and PGE2 levels in arachidonate-stimulated blood from these same animals also were measured. No anti-inflammatory activity was observed in BF389-treated rats despite the occurrence of profound suppression of basal and stimulated PGE2 production. Animals treated with conventional nonsteroidal anti-inflammatory drugs in this assay had both suppressed PGE2 production and significant anti-inflammatory activity. In another study, groups of rats were given p.o. doses of 1, 10, 100 or 250 mg/kg of BF389 for 5 days in order to determine peak concentrations, T1/2s and total areas under the plasma (tissue) drug concentration curves in plasma and paws. Peak plasma concentrations occurred 2 to 4 hr postdosing and the half-life was 8 to 15 hr over the 1- to 250-mg/kg/day dose range. Peak paw concentrations occurred 6 to 12 hr postdosing and the levels were 9- to 14-fold greater in the paw than in the plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of quinolones in respiratory tract infections.

In a prospective (and continuing) trial, a total of 271 patients with acute purulent exacerbations of chronic respiratory disease (bacteriologically confirmed) were treated with various new oral quinolones including enoxacin (26), pefloxacin (50), ciprofloxacin (80) and ofloxacin (115). Various therapeutic schedules were employed, with differing drug dosages, frequencies of administration and durations of treatment. All patients were investigated microbiologically during and immediately after treatment and after 7 days of follow-up. The best clinical results were noted after ofloxacin 800 mg once daily for 7 days, which showed excellent gastrointestinal absorption and rapid penetration through to the sputum. Some of the treatment failures with enoxacin and pefloxacin could be ascribed to the development of resistance during treatment, rises in minimal inhibitory concentrations (MICs) being noted with Streptococcus pneumoniae and Pseudomonas aeruginosa.

Ofloxacin versus trimethoprim-sulphamethoxazole in acute cystitis.

The clinical and bacteriological efficacy and adverse reactions of ofloxacin vs trimethoprim-sulphamethoxazole were investigated in a double-blind, randomised study in 250 female patients (125 in each group) with acute, uncomplicated lower urinary tract infections. The dosages of ofloxacin and trimethoprim-sulphamethoxazole were 100mg and 160mg + 800mg twice daily, respectively. The duration of therapy was 3 days. 81% of the patients had significant bacteriuria. Escherichia coli was isolated in 76% and Staphylococcus saprophyticus in 11% of the infections. The bacteriological elimination, clinical cure and improvement rates of the evaluable patients on ofloxacin treatment were 92 and 95%, respectively. The corresponding figures on trimethoprim-sulphamethoxazole therapy were 88 and 90%. Adverse reactions were clinically unimportant, and none of the patients had to stop treatment. Mild and transient side effects, mainly from the gastrointestinal tract, central nervous system and skin, were reported by 19 and 22% of the patients in the ofloxacin and trimethoprim-sulphamethoxazole groups, respectively. None of the differences in clinical and bacteriological efficacy and side effects of ofloxacin vs trimethoprim-sulphamethoxazole were statistically significant. Ofloxacin appears to be an appropriate antibiotic for short term therapy of acute, uncomplicated, lower urinary tract infections, comparing favourably with trimethoprim-sulphamethoxazole treatment in this study.

Steady-state kinetics of the quinolone derivatives ofloxacin, enoxacin, ciprofloxacin and pefloxacin during maintenance treatment with theophylline.

Some of the new quinolone derivatives may be of value in the treatment of respiratory tract infections. It has been demonstrated that enoxacin, pefloxacin and ciprofloxacin, but not ofloxacin, decreased the metabolic clearance of the bronchodilator theophylline. This resulted in elevated plasma theophylline concentrations and, in some of the patients, theophylline toxicity. When the pharmacokinetic parameters of enoxacin, pefloxacin, ciprofloxacin and ofloxacin obtained in the present study were compared with those obtained from other studies in healthy volunteers not given concomitant theophylline, there was no evidence of theophylline influencing the clearance of the investigated quinolones.

[Bronchopulmonary aminochlorthenoxycycline levels in experimental burns of the respiratory tree]. / Ricerche sul tropismo degli antibiotici a livello broncopolmonare nella ustione sperimentale dell'albero respiratorio.

A special technique was employed to obtain burns of the respiratory tree in 24 male rabbits, Increased bronchial secretion was observed during the burning. Aminochlorthenoxycycline values were higher in the secretion after 90' than in the controls, while blood and lung concentrations displayed a similar pattern. The usefulness of the drug in the prevention and treatment of infectious complications following burns in this area is stressed.

[Aminochlorthenoxycycline levels in experimental acute inflammation of the respiratory tree]. / Ricerche sulla distribuzione dell'aminoclortenoxiciclina nell'infiammazione sperimentale acuta dell'albero respiratorio.

A special technique used to obtain acute respiratory inflammation in the rabbit produced a considerable increase in expectorate by comparison with the controls. Bronchial, lung and blood aminochlorthenoxycycline levels were much higher than in the controls, showing that this antibiotic has a marked affinity for inflamed lung tissue.