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Background: Excess activity of neutrophil elastase (NE) in the lung of COPD patients results in the degradation of extracellular matrix (ECM) proteins and an increased activation of other proteases, leading to the destruction of alveolar septa and, ultimately, emphysema development. Direct inhibition of NE is expected to reduce lung parenchyma destruction, emphysema formation and progression in patients with COPD. Aim: We investigated the in vitro and in vivo profile of the novel NE inhibitor BI 1323495. Methods: Inhibition of human NE and the related proteases cathepsin G and proteinase 3 was determined in enzymatic assays using purified enzymes. In addition, inhibition of NE in plasma from zymosan-stimulated human whole blood was measured. An acute lung injury model in mice was used to monitor the in vivo inhibition of NE in the lung by BI 1323495. Lung injury was induced by intratracheal application of 25 µg human NE and haemoglobin concentration as a read-out for lung injury, and neutrophil counts as a read-out for inflammation were measured in bronchioalveolar lavage fluid after 4 hours. Results: BI 1323495 blocked NE in vitro with an IC50 of 0.4 nM and demonstrated a > 4000-fold selectivity versus related proteases cathepsin G and proteinase 3. BI 1323495 fully inhibited the activity of NE released by zymosan-stimulated human neutrophils with a mean IC50 of 1 nM. Treatment of mice with an oral formulation of BI 1323495 attenuated lung damage and inflammation induced by intratracheal instillation of human recombinant NE with an ED50 of 1.9 mg/kg. Conclusions: BI 1323495 is a very potent and highly selective inhibitor of human NE with in vivo efficacy.

A potent, in vivo efficacious 11β hydroxysteroid dehydrogenase type 1 (11β HSD1) inhibitor (11j) has been identified. Compound 11j inhibited 11β 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 11β 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.

Inhibition of local cortisol regeneration from circulating cortisone by blocking 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) has been shown to ameliorate the risk factors associated with the metabolic syndrome. Chronic modulation of glucocorticoid homeostasis may result in hypothalamic-pituitary-adrenal (HPA) axis stimulation. HPA axis over-activation leading androgen excess would be undesirable in a therapeutic intervention designed to treat a chronic condition such as the metabolic syndrome. To address whether 11β-HSD1 inhibition would lead to excess androgens, we treated female cynomolgus monkeys with a selective inhibitor, BI 135558, for 4 weeks. Continual action of the compound over the dosing period was confirmed by constant plasma exposure, and a maintained change in urinary glucocorticoid metabolites consistent with 11β-HSD1 inhibition. No significant changes in adrenal function, as evidenced by an adrenocorticotropic hormone (ATCH) challenge, were observed. An examination of androgenic hormones revealed a slight increase in dehydroepiandrosterone sulfate (DHEA-S), while other hormones such as testosterone remained within reference values. Overall, treatment with BI 135558 in monkeys did not result in obvious over-activation of the HPA axis.

CYP2A6 (cytochrome P450 2A6), which was first identified as the human coumarin 7-hydroxylase, is the most important enzyme in nicotine C-oxidation. The enzyme also metabolically activates the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in vitro. Polymorphisms in the CYP2A6 gene may thus impact on both smoking behavior and lung cancer susceptibility. Several different genotyping methods have been reported with conflicting results in the frequencies of CYP2A6 polymorphic variants. Thus we decided to perform a sequence analysis of the entire CYP2A6 gene. Sequencing confirmed the published CYP2A6 cDNA sequence. However, intron sequences differed considerably from the reported sequence of the CYP2A6*3 (v2) variant. Our analyses revealed that parts of introns shared homologies with the published sequence of CYP2A13. Based on our sequence data we developed a one step protocol for specific amplification of exon 3 of CYP2A6. The resulting PCR product can be used directly for restriction endonuclease digestion with XcmI and DdeI to determine the frequencies of the reported variant alleles CYP2A6*2 and CYP2A6*3. In a population of 305 African-Americans and 145 Caucasians, we found allele frequencies of 0.003 (2/610) for CYP2A6*2 and 0 (0/610) for CYP2A6*3 in African-Americans and allele frequencies of 0.014 (4/290) and 0 (0/290) in Caucasians. We conclude that both alleles are considerably less frequent in populations than previously reported.

Zygotic expression of the BMP-4 gene in Xenopus embryos is regulated by an auto-regulatory loop. Since AP-1 is known as a mediator of auto-regulatory loops both in the case of the Drosophila dpp and the mammalian TGF-b genes, we have analysed the potential of Xenopus cJun (AP-1) as a mediator of BMP-4 expression during Xenopus development. RNA injection experiments revealed that both heteromeric cFos/c-Jun and homodimeric c-Jun/c-Jun strongly activate BMP-4 transcription, whereas BMP signaling was found to activate the Xenopus cJun gene only at a rather low extent. In addition, the lack of zygotic c-Jun transcripts until the end of gastrulation should exclude a role of AP1 in the activation and the early expression of BMP-4 during gastrulation in vivo. However, at later stages of Xenopus development, we find a spatial overlap of c-Jun and BMP-4 transcripts which suggests that AP-1 might serve as an additional activatory component for the autoregulation of BMP-4. Promoter/reporter and gel mobility shift assays demonstrate multiple responsive sites for AP-1 in the 5 0 flanking region and two in the second intron of the BMP-4 gene. We further demonstrate that AP-1 acts independently of Xvent-2 which has recently been shown to mediate the early expression of BMP-4 in gastrula stage embryos. q 2000 Elsevier Science Ireland Ltd. All rights reserved.

A method based on liquid chromatography tandem mass spectrometry (LC–MSMS) applying atmospheric pressure chemical ionisation (APCI) in the positive ion mode was developed for the direct determination of nicotine, cotinine, trans-3′-hydroxycotinine, their corresponding glucuronide conjugates as well as cotinine-N-oxide, norcotinine, and nicotine-N′-oxide in the urine of smokers. The assay involves filtration of crude urine, fast liquid chromatography on a reversed-phase column and mass-specific detection using MSMS transitions. Deuterium-labeled nicotine, cotinine, and trans-3′-hydroxycotinine were used as internal standards. Glucuronides used as reference material were either chemically (cotinine-N-glucuronide) or enzymatically synthesized (nicotine-N-glucuronide and trans-3′-hydroxycotinine-O-glucuronide). Precision for the major nicotine analytes at levels observable in urine of smokers was better than 10%. Accuracy expressed in recovery rates in urine matrix for nicotine, cotinine, trans-3′-hydroxycotinine, and cotinine-N-glucuronide ranged from 87 to 113%. Quantitative results for the three glucuronides in urine samples of 15 smokers were compared to an indirect method in which the aglycons were determined with gas chromatography and nitrogen-selective detection (GC–NPD) before and after enzymatic splitting of the conjugates. Good agreement was found for cotinine-N-glucuronide (coefficient of variation, CV: 9%) and trans-3′-hydroxycotinine-O-glucuronide (CV: 20%), whereas the accordance between both methods was moderate for nicotine-N-glucuronide (CV: 33%). The described LC–MSMS method allows the simultaneous determination of nicotine and eight of its major metabolites in urine of smokers with good precision and accuracy. Since the method requires a minimum of sample clean-up and a very short time for chromatography (3 min), it is suitable for determining the nicotine dose in large-scale human biomonitoring studies.

Like other genes of the transforming growth factor-beta family, the BMP-4 gene is regulated by an autocatalytic loop. In Xenopus embryos this loop can be ectopically induced by injection of BMP-2 RNA. However, cycloheximide treatment subsequent to BMP-2 overexpression revealed that BMP signaling is not direct but requires additional factor(s). As putative mediator we have identified Xvent-2 which is activated by BMP-2/4 signaling and, in turn, activates BMP-4 transcription. Using promoter/reporter assays we have delineated Xvent-2 responsive elements within the BMP-4 gene. We further demonstrate that Xvent-2 which has recently been characterized as a transcriptional repressor can also act, context dependent, as an activator binding two copies of a 5'-CTAATT-3' motif in the second intron of the BMP-4 gene. Replacement of Xvent-2 target sites within the goosecoid (gsc) promoter by the BMP-4 enhancer converts Xvent-2 caused repression of gsc to strong activation. This switch is obviously due to adjacent nucleotides probably binding a transcriptional co-activator interacting with Xvent-2. A model is presented describing the mechanism of BMP-4 gene activation in Xenopus embryos at the early gastrula stage.

Inhibition of two key serine proteases in the coagulation cascade, thrombin (IIa) and factor Xa, are currently being exploited for direct, oral antithrombotic activity in the clinic. However, it is still unclear if one form of coagulation factor inhibition is more effective than the other. Thus, the objective of this study was to test the antithrombotic efficacy of the clinically advanced compounds, the potent direct thrombin inhibitor, dabigatran etexilate and rivaroxaban, a potent direct factor Xa inhibitor in the rabbit A-V shunt model of thrombosis. In addition, another internally developed factor Xa inhibitor, BI42551, with properties similar to those in clinical development was tested. All three compounds have affinities (Ki) for their respective coagulation factor in the low nM range, i.e. human thrombin with dabigatran or human factor Xa with rivaroxaban or BI42551. In addition, each is at least >700-fold selective for its human coagulation factor, dabigatran etexilate for IIa vs Xa and the factor Xa inhibitors for Xa vs IIa. These compounds are highly selective inhibitors not only of the human enzyme, but also have similar values for rabbit thrombin and Xa, respectively. All experiments were performed according to German animal ethics guidelines. The femoral artery and vein of anesthetised rabbits were connected with polyethylene tubing containing a fixed length of suture, pre-soaked in tissue factor. Blood flow through the shunt was maintained over 40 min, after which the suture with any thrombus was removed from the shunt and weighed. The prodrug dabigatran etexilate and the factor Xa inhibitors were given in doses of 3 and 10 mg/kg orally and the rabbits were anesthetised either 90 min or at the highest dose, also 6.5 hrs after drug administration. There was a dose-dependent reduction of thrombus formation with all three compounds as compared to control. Antithrombotic efficacy at 3 and 10 mg/kg is shown as % inhibition of control measured 2 hrs after drug administration (table, columns 2&3). These effects were long-lasting, as significant antithrombotic activity was also measured 7 hrs post administration (last column). Plasma levels of all compounds were dose-dependent and clotting tests correlated well with dose. | | 3 mg/kg–2 hrs | 10 mg/kg–2 hrs | 10 mg/kg–7 hrs | |:-------------------- | ------------- | -------------- | -------------- | | Dabigatran etexilate | 61.7 ± 8.7 | 82.1 ± 5.5 | 59.5 ± 17.6 | | Rivaroxaban | 43.2 ± 7.7 | 64.5 ± 8.1 | 41.0 ± 8.4 | | BI42551 | 31.1 ± 10.7 | 70.3 ± 3.3 | 39.9 ± 14.7 | These results show that both thrombin and factor Xa inhibition are effective methods of inhibiting thrombosis in a rabbit AV shunt model. All drugs had potent and long-lasting effects after a single oral administration in this model, though dabigatran showed a trend to elevated antithrombotic efficacy at both 2 and 7 hrs. However, in the clinical setting differences in antithrombotic treatment may also be related to differences in pharmacokinetic profiles, drug interactions or metabolism, or the individual side effect profiles of each compound.