Trolox contributes to Nrf2-mediated protection of human and murine primary alveolar type II cells from injury by cigarette smoke.

Cigarette smoke (CS) is a main risk factor for chronic obstructive pulmonary disease (COPD). Oxidative stress induced by CS causes DNA and lung damage. Oxidant/antioxidant imbalance occurs in the distal air spaces of smokers and in patients with COPD. We studied the effect of oxidative stress generated by CS both in vivo and in vitro on murine primary alveolar type II (ATII) cells isolated from nuclear erythroid 2-related factor-2 (Nrf2)(-/-) mice. We determined human primary ATII cell injury by CS in vitro and analyzed ATII cells isolated from smoker and non-smoker lung donors ex vivo. We also studied whether trolox (water-soluble derivative of vitamin E) could protect murine and human ATII cells against CS-induced DNA damage and/or decrease injury. We analyzed oxidative stress by 4-hydroxynonenal expression, reactive oxygen species (ROS) generation by Amplex Red Hydrogen Peroxide Assay, Nrf2, heme oxygenase 1, p53 and P53-binding protein 1 (53BP1) expression by immonoblotting, Nrf2 nuclear translocation, Nrf2 and p53 DNA-binding activities, apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and cytokine production by ELISA. We found that ATII cells isolated from Nrf2(-/-) mice are more susceptible to CS-induced oxidative DNA damage mediated by p53/53BP1 both in vivo and in vitro compared with wild-type mice. Therefore, Nrf2 activation is a key factor to protect ATII cells against injury by CS. Moreover, trolox abolished human ATII cell injury and decreased DNA damage induced by CS in vitro. Furthermore, we found higher inflammation and p53 mRNA expression by RT-PCR in ATII cells isolated from smoker lung donors in comparison with non-smokers ex vivo. Our results indicate that the Nrf2 and p53 cross talk in ATII cells affect the susceptibility of these cells to injury by CS. Trolox can protect against oxidative stress, genotoxicity and inflammation induced by CS through ROS scavenging mechanism, and serve as a potential antioxidant prevention strategy against oxidative injury of ATII cells in CS-related lung diseases.

Comparison between the genotoxicity of cis-Pt(ll) complex of 3-aminoflavone and cis-DDP in lymphocytes evaluated by the comet assay.

cis-bis(3-aminoflavone)dichloroplatinum(II) [cis-Pt(II) complex of 3-aminoflavone] is an analog of cis-DDP characterized by low cytotoxicity and anticancer properties in vivo. In order to evaluate genotoxic properties of this chemical compound, the comet assay in human lymphocytes was used. The analysis of DNA damage after 1-h cell incubation with cis-Pt(II) complex of 3-aminoflavone and cis-DDP was carried out, and DNA repair kinetics were evaluated after 0.5-h, 1-h, and 1.5-h postexposure incubation. It has been shown that cis-Pt(II) complex of 3-aminoflavone causes the increase in tail moments in comparison with cis-DDP. On the other hand, the decrease in these values caused by cis-DDP was connected mainly with the presence of DNA and DNA-protein crosslinks. The decrease in tail moments after cis-Pt(II) complex of 3-aminoflavone lymphocyte treatment was already observed after 0.5-h postexposure incubation, whereas in the variant with hydrogen peroxide application these values after cis-Pt(II) complex of 3-aminoflavone addition were higher in comparison with cis-DDP. Results obtained on the basis of the comet assay could confirm the occurrence of DNA breaks and cross-links induced by cis-Pt(II) complex of 3-aminoflavone.

Discovery of novel, orally active dual NK1/NK2 antagonists.

Exploration of the SAR around selective NK2 antagonists, SR48968 and ZD7944, led to the discovery that naphth-1-amide analogues provide potent dual NK1 and NK2 antagonists. ZD6021 inhibited binding of [3H]-NKA or [3H]-SP to human NK1 and NK2 receptors, with high-affinity (K(i)=0.12 and 0.62nM, respectively). In functional assays ZD6021 had, at 10(-7)M, in human pulmonary artery pK(B)=8.9 and in human bronchus pK(B)=7.3, for NK1 and NK2, respectively. Oral administration of ZD6021 to guinea pigs dose-dependently attenuated ASMSP induced extravasation of plasma proteins, ED(50)=0.5mg/kg, and NK2 mediated bronchoconstriction, ED(50)=13mg/kg.

Orally active trifluoromethyl ketone inhibitors of human leukocyte elastase.

This paper describes the development a series of peptidyl trifluoromethyl ketone inhibitors of human leukocyte elastase which are found to have excellent pharmacological profiles. Methods have been developed that allow for the synthesis of these inhibitors in stereochemically pure form. Two of these compounds, 1k and 1l, have high levels of oral bioavailability in several species. Compound 1l has entered development as ZD8321 and is presently undergoing clinical evaluation. These compounds demonstrate that peptidyl trifluoromethyl ketone inhibitors can achieve high levels of oral activity and bioavailability, and therefore they may prove useful as therapeutic agents in the treatment of diseases in which elastase is implicated.

Nonpeptidic inhibitors of human leukocyte elastase. 6. Design of a potent, intratracheally active, pyridone-based trifluoromethyl ketone.

Further modification of the 3-amino substituent in a trifluoromethyl ketone-based series of 3-amino-6-phenylpyridin-2-ones that had been optimized for oral activity led to analogs that were potent intratracheal inhibitors in a model of HLE-induced lung damage in the hamster. The best 3-amino substituent for intratracheal activity is [4-[N-[(4-chlorophenyl)sulfonyl]-carbamoyl]phenyl]sulfonyl. At a 30 min prechallenge interval, compound 9, which incorporates this substituent, had an ED50 of approximately 2 nmol/animal and, qualitatively, afforded a very similar dose-response relationship to that found with a peptidic trifluoromethyl ketone inhibitor, ICI 200,355.

Nonpeptidic inhibitors of human leukocyte elastase. 5. Design, synthesis, and X-ray crystallography of a series of orally active 5-aminopyrimidin-6-one-containing trifluoromethyl ketones.

The effects of changes in substitution in a series of 5-amino-2-pyrimidin-6-ones on both in vitro activity and oral activity in an acute hemorrhagic assay have been explored. These compounds contained either a trifluoromethyl ketone or a boronic acid moiety to bind covalently to the Ser-195 hydroxyl of human leukocyte elastase (HLE). Boronic acid-containing inhibitors were found to be more potent than the corresponding trifluoromethyl ketones in vitro but were less active upon oral administration. Compound 13b was found to offer the best combination of oral potency, duration of action, and enzyme selectivity and, as such, was selected for further biological testing. X-ray crystallography of a cocrystallized complex of compound 19m and porcine pancreatic elastase demonstrated that the inhibitor is bound to the enzyme in a manner similar to that found previously for a closely related series of pyridone-containing inhibitors of HLE.

RG 12561 (dalvastatin): a novel synthetic inhibitor of HMG-CoA reductase and cholesterol-lowering agent.

RG 12561 (dalvastatin) is a prodrug which converts to its open hydroxyacid form in the body. The Na salt of RG 12561 (RG 12561-Na) is a potent inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. It competitively inhibits rat liver HMG-CoA reductase with an IC50 value of 3.4 nmol/l. In the same assay, the IC50 values for other potent HMG-CoA reductase inhibitors, lovastatin-Na and pravastatin, were 2.3 and 8.9 nmol/l, respectively. In Hep G2 liver cells, RG 12561-Na, lovastatin-Na and pravastatin inhibited cholesterol biosynthesis from radiolabeled octanoate with IC50 values of 4 and 5 nmol/l and 1.1 mumol/l, respectively. In a rat ex vivo assay, orally administered RG 12561, lovastatin and pravastatin inhibited cholesterol biosynthesis in liver slices with ED50 values of 0.9, 0.5 and 12 mg/kg, respectively. In cholestyramine-fed hamsters, RG 12561 (0.1% in food for 18 days) reduced LDL cholesterol, whereas HDL was slightly increased. The reductions in the LDL/HDL ratio for RG 12561, RG 12561-Na, lovastatin and lovastatin-Na were 35, 76, 88 and 88%, respectively. At a higher dose, RG 12561 (0.4% in food) reduced serum cholesterol, LDL and LDL/HDL by 84, 97 and 91%, respectively. In WHHL rabbits, RG 12561 and lovastatin (5 mg/kg, b.i.d., 12 days) reduced serum cholesterol by 17 and 16%, respectively. These results demonstrate that RG 12561 is a potent cholesterol-lowering agent.