Pre-clinical assessment of a water-in-fluorocarbon emulsion for the treatment of pulmonary vascular diseases.

Hypoxic pulmonary vasoconstriction (HPV) is a well-characterized vascular response to low oxygen pressures and is involved in life-threatening conditions such as high-altitude pulmonary edema (HAPE) and pulmonary arterial hypertension (PAH). While the efficacy of oral therapies can be affected by drug metabolism, or dose-limiting systemic toxicity, inhaled treatment via pressured metered dose inhalers (pMDI) may be an effective, nontoxic, practical alternative. We hypothesized that a stable water-in-perfluorooctyl bromide (PFOB) emulsion that provides solubility in common pMDI propellants, engineered for intrapulmonary delivery of pulmonary vasodilators, reverses HPV during acute hypoxia (HX). Male Sprague Dawley rats received two 10-min bouts of HX (13% O2) with 20 min of room air and drug application between exposures. Treatment groups: intrapulmonary delivery (PUL) of (1) saline; (2) ambrisentan in saline (0.1 mg/kg); (3) empty emulsion; (4) emulsion encapsulating ambrisentan or sodium nitrite (NaNO2) (0.1 and 0.5 mg/kg each); and intravenous (5) ambrisentan (0.1 mg/kg) or (6) NaNO2 (0.5 mg/kg). Neither PUL of saline or empty emulsion, nor infusions of drugs prevented pulmonary artery pressure (PAP) elevation (32.6 ± 3.2, 31.5 ± 1.2, 29.3 ± 1.8, and 30.2 ± 2.5 mmHg, respectively). In contrast, PUL of aqueous ambrisentan and both drug emulsions reduced PAP by 20-30% during HX, compared to controls. IL6 expression in bronchoalveolar lavage fluid and whole lung 24 h post-PUL did not differ among cohorts. We demonstrate proof-of-concept for delivering pulmonary vasodilators via aerosolized water-in-PFOB emulsion. This concept opens a potentially feasible and effective route of treating pulmonary vascular pathologies via pMDI.

Challenges in the development of an M4 PAM preclinical candidate: The discovery, SAR, and biological characterization of a series of azetidine-derived tertiary amides.

Herein we describe the continued optimization of M4 positive allosteric modulators (PAMs) within the 5-amino-thieno[2,3-c]pyridazine series of compounds. In this letter, we disclose our studies on tertiary amides derived from substituted azetidines. This series provided excellent CNS penetration, which had been challenging to consistently achieve in other amide series. Efforts to mitigate high clearance, aided by metabolic softspot analysis, were unsuccessful and precluded this series from further consideration as a preclinical candidate. In the course of this study, we found that potassium tetrafluoroborate salts could be engaged in a tosyl hydrazone reductive cross coupling reaction, a previously unreported transformation, which expands the synthetic utility of the methodology.

Assessment of pesticide residues in strawberries grown under various treatment regimes.

The dynamics of pesticide residues in strawberries that involved quantification of pesticide residues in ripe fruits after model treatment was evaluated in repeated field trials conducted over 3 years. Sixteen commercial pesticide formulations in various combinations were employed in applications from 7 to 44 days before harvest. Altogether 21 active ingredients and some of their metabolites were determined in treated strawberries using LC-MS and GC-MS methods. Except for propargite, the concentrations of all active ingredients declined below the respective MRLs (Regulation (EC) No. 396/2005); nevertheless, most of the tested fungicides often persisted above the 0.01 mg kg⁻¹ limit required by baby food producers to avoid the risk of exceeding the 'baby food limit' established in Commission Directive 2006/141/EC. On the other hand, residues of the majority of tested insecticides, namely spinosad, pymetrozine, deltamethrin, lambda-cyhalothrin and azadirachtin, declined below this limit.

4-Hydroxypyridazin-3(2H)-one derivatives as novel D-amino acid oxidase inhibitors.

D-Amino acid oxidase (DAAO) catalyzes the oxidation of d-amino acids including d-serine, a coagonist of the N-methyl-d-aspartate receptor. We identified a series of 4-hydroxypyridazin-3(2H)-one derivatives as novel DAAO inhibitors with high potency and substantial cell permeability using fragment-based drug design. Comparisons of complex structures deposited in the Protein Data Bank as well as those determined with in-house fragment hits revealed that a hydrophobic subpocket was formed perpendicular to the flavin ring by flipping Tyr224 in a ligand-dependent manner. We investigated the ability of the initial fragment hit, 3-hydroxy-pyridine-2(1H)-one, to fill this subpocket with the aid of complex structure information. 3-Hydroxy-5-(2-phenylethyl)pyridine-2(1H)-one exhibited the predicted binding mode and demonstrated high inhibitory activity for human DAAO in enzyme- and cell-based assays. We further designed and synthesized 4-hydroxypyridazin-3(2H)-one derivatives, which are equivalent to the 3-hydroxy-pyridine-2(1H)-one series but lack cell toxicity. 6-[2-(3,5-Difluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-one was found to be effective against MK-801-induced cognitive deficit in the Y-maze.

Synthesis and HIV-1 RT inhibitory action of novel (4/6-substituted benzo[d]thiazol -2-yl)thiazolidin-4-ones. Divergence from the non-competitive inhibition mechanism.

Reverse transcriptase (RT) inhibitors play a major role in the therapy of human immunodeficiency virus type 1 (HIV-1) infection. Although, many compounds are already used as anti-HIV drugs, research on development of novel inhibitors continues, since drug resistant strains appear because of prolonged therapy. In this paper, we present the synthesis and evaluation of HIV-1 RT inhibitory action of eighteen novel (4/6-halogen/MeO/EtO-substituted benzo[d]thiazol-2-yl)thiazolidin-4-ones. The two more active compounds (IC50 : 0.04 µM and 0.25 µM) exhibited better inhibitory action than the reference compound, nevirapine. Docking analysis supports a stable binding of the most active derivative to the allosteric centre of RT. Kinetic analysis of two of the most active compounds indicate an uncompetitive inhibition mode. This is a desired characteristic, since mutations that affect activity of traditional non-competitive NNRTIs may not affect activity of compounds of this series. Interestingly, the less active derivatives (IC50 > 40 µM) exhibit a competitive mode of action.

Preclinical absorption, distribution, metabolism, excretion, and pharmacokinetic-pharmacodynamic modelling of N-(4-(3-((3S,4R)-1-ethyl-3-fluoropiperidine-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide, a novel MET kinase inhibitor.

GNE-A (AR00451896; N-(4-(3-((3S,4R)-1-ethyl-3-fluoropiperidine-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide) is a potent, selective MET kinase inhibitor being developed as a potential drug for the treatment of human cancers. Plasma clearance was low in mice and dogs (15.8 and 2.44 mL/min/kg, respectively) and moderate in rats and monkeys (36.6 and 13.9 mL/min/kg, respectively). The volume of distribution ranged from 2.1 to 9.0 L/kg. The mean terminal elimination half-life ranged from 1.67 h in rats to 16.3 h in dogs. Oral bioavailability in rats, mice, monkeys, and dogs were 11.2%, 88.0%, 72.4%, and 55.8%, respectively. Allometric scaling predicted a clearance of 1.3-7.4 mL/min/kg and a volume of distribution of 4.8-11 L/kg in human. Plasma protein binding was high (96.7-99.0% bound). Blood-to-plasma concentration ratios (0.78-1.46) indicated that GNE-A did not preferentially distribute into red blood cells. Transporter studies in MDCKI-MDR1 and MDCKII-Bcrp1 cells suggested that GNE-A is likely a substrate for MDR1 and BCRP. Pharmacokinetic-pharmacodynamic modelling of tumour growth inhibition in MET-amplified EBC-1 human non-small cell lung carcinoma tumour xenograft mice projected oral doses of 5.6 and 13 mg/kg/day for 50% and 90% tumour growth inhibition, respectively. Overall, GNE-A exhibited favourable preclinical properties and projected human dose estimates.

Bioactivation of isothiazoles: minimizing the risk of potential toxicity in drug discovery.

Compound 1, (7-methoxy-N-((6-(3-methylisothiazol-5-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)-1,5-naphthyridin-4-amine) is a potent, selective inhibitor of c-Met (mesenchymal-epithelial transition factor), a receptor tyrosine kinase that is often deregulated in cancer. Compound 1 displayed desirable pharmacokinetic properties in multiple preclinical species. Glutathione trapping studies in liver microsomes resulted in the NADPH-dependent formation of a glutathione conjugate. Compound 1 also exhibited very high in vitro NADPH-dependent covalent binding to microsomal proteins. Species differences in covalent binding were observed, with the highest binding in rats, mice, and monkeys (1100-1300 pmol/mg/h), followed by dogs (400 pmol/mg/h) and humans (144 pmol/mg/h). This covalent binding to protein was abolished by coincubation with glutathione. Together, these in vitro data suggest that covalent binding and glutathione conjugation proceed via bioactivation to a chemically reactive intermediate. The cytochrome (CYP) P450 enzymes responsible for this bioactivation were identified as cytochrome P450 3A4, 1A2, and 2D6 in human and cytochrome P450 2A2, 3A1, and 3A2 in rats. The glutathione metabolite was detected in the bile of rats and mice, thus demonstrating bioactivation occurring in vivo. Efforts to elucidate the structure of the glutathione adduct led to the isolation and characterization of the metabolite by NMR and mass spectrometry. The analytical data confirmed conclusively that the glutathione conjugation was on the 4-C position of the isothiazole ring. Such P450-mediated bioactivation of an isothiazole or thiazole group has not been previously reported. We propose a mechanism of bioactivation via sulfur oxidation followed by glutathione attack at the 4-position with subsequent loss of water resulting in the formation of the glutathione conjugate. Efforts to reduce bioactivation without compromising potency and pharmacokinetics were undertaken in order to minimize the potential risk of toxicity. Because of the exemplary pharmacokinetic/pharmacodynamic (PK/PD) properties of the isothiazole group, initial attempts were focused on introducing alternative metabolic soft spots into the molecule. These efforts resulted in the discovery of 7-(2-methoxyethoxy)-N-((6-(3-methyl-5-isothiazolyl)[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)-1,5-naphthyridin-4-amine (compound 2), with the major metabolic transformation occurring on the naphthyridine ring alkoxy substituent. However, a glutathione conjugate of compound 2 was produced in vitro and in vivo in a manner similar to that observed for compound 1. Furthermore, the covalent binding was high across species (360, 300, 529, 208, and 98 pmol/mg/h in rats, mice, dogs, monkeys, and humans, respectively), but coincubation with glutathione reduced the extent of covalent binding. The second viable alternative in reducing bioactivation involved replacing the isothiazole ring with bioisosteric heterocycles. Replacement of the isothiazole ring with an isoxazole or a pyrazole reduced the bioactivation while retaining the desirable PK/PD characteristics of compounds 1 and 2.

Selective reduction of N-oxides to amines: application to drug metabolism.

Phase I oxidative metabolism of nitrogen-containing drug molecules to their corresponding N-oxides is a common occurrence. There are instances where liquid chromatography/tandem mass spectometry techniques are inadequate to distinguish this pathway from other oxidation processes, including C-hydroxylations and other heteroatom oxidations, such as sulfur to sulfoxide. Therefore, the purpose of the present study was to develop and optimize an efficient and practical chemical method to selectively convert N-oxides to their corresponding amines suitable for drug metabolism applications. Our results indicated that efficient conversion of N-oxides to amines could be achieved with TiCl(3) and poly(methylhydrosiloxane). Among them, we found TiCl(3) to be a facile and easy-to-use reagent, specifically applicable to drug metabolism. There are a few reports describing the use of TiCl(3) to reduce N-O bonds in drug metabolism studies, but this methodology has not been widely used. Our results indicated that TiCl(3) is nearly as efficient when the reductions were carried out in the presence of biological matrices, including plasma and urine. Finally, we have shown a number of examples where TiCl(3) can be successfully used to selectively reduce N-oxides in the presence of sulfoxides and other labile groups.

Synthesis and biological evaluation of 6-aryl-6H-pyrrolo[3,4-d]pyridazine derivatives: high-affinity ligands to the alpha 2 delta subunit of voltage gated calcium channels.

A novel class of 6-aryl-6H-pyrrolo[3,4-d]pyridazine ligands for the alpha2delta subunit of voltage-gated calcium channels has been described. Substitutions in the aryl ring of the molecule were generally not tolerated, and resulted in diminished binding to the alpha2delta subunit. Modifications to the pyridazine ring revealed numerous permissive substitutions, and detailed SAR studies were carried out in this portion of the molecule. Replacement of the pyridazine ring methyl group with an aminomethyl functionality provided greatly improved potency over the initial lead. The initial lead compound displayed good rat pharmacokinetic properties, and was shown to be efficacious in the Chung model for neuropathic pain in rats.

Synthesis and biological evaluation of pyridazino[4,3-b]indoles and indeno[1,2-c]pyridazines as new ligands of central and peripheral benzodiazepine receptors.

A large number of pyridazino[4,3-b]indoles and indeno[1,2-c]pyridazines were synthesised and tested to evaluate their binding affinities at both central (CBR) and peripheral (PBR) benzodiazepine receptors. Relatively good PBR binding affinities were found for ligands belonging to the 3-arylmethyloxy-pyridazinoindole series, whereas only 2-aryl-indenopyridazines 7a, 8a and 10a display a weak binding affinity for CBR. To find out the main structural determinants affecting PBR affinity, a molecular modelling study based on the comparative analysis of the three-dimensional properties of four properly selected derivatives 24a, 3b, 18a and 10d, with those of highly active and selective PBR ligands, taken as reference, was performed.

Multiple benzodiazepine receptors in the ovine brain: ontogenesis, properties, and distribution of 3H-diazepam binding.

Benzodiazepine receptors in the ovine frontal cortex were present at 56 days gestation and developed slowly until 96 days when the number increased rapidly, reaching adult levels by 120 days gestation. Scatchard analysis of 3H-diazepam specifically bound to cortical membranes suggested high (KD approximately equal to 2.0 nM) and low (KD approximately equal to 20.0 nM) affinity benzodiazepine receptors at all stages of development. Whereas the affinity of these receptors for 3H-diazepam did not alter during development, the number of both high and low affinity receptors increased significantly between 56 and 120 days gestation. The number of low affinity receptors were higher in late gestation and early neonatal life than in adulthood. The functional state of these receptors as determined by sensitivity to GABA did not alter during development. However, in the adult, nitrazepam, flunitrazepam, midazolam, and 1-methylisoguanosine were more potent in displacing 3H-diazepam at the low affinity than the high affinity receptor, whereas chlordiazepoxide and diazepam had greater potency at the high affinity binding site. Development of the benzodiazepine receptor in the majority of other brain regions studied occurred primarily after 68 days gestation, as was the case in frontal cortex. In contrast, hindbrain and midbrain benzodiazepine receptors had reached adult levels by 68 days gestation.