A semi-automated 96-well protein precipitation method for the determination of montelukast in human plasma using high performance liquid chromatography/fluorescence detection.

Fisher AL.

Merck Research Laboratories, WP75A-303, Sumneytown Pike, West Point, PA 19486, USA.

A simple, semi-automated, protein precipitation assay for the determination of montelukast (SINGULAIR, MK-0476) in human plasma has been developed. Montelukast is a potent and selective antagonist of the cysteinyl leukotriene receptor used for the treatment of asthma. A Packard MultiPROBE II EX is used to transfer 300 microl of plasma from sample, standard, and QC sample tubes to a microtiter plate (96-well). After addition of the internal standard by a repeating pipettor, a Tomtec QUADRA 96 adds 400 microl of acetonitrile to all plasma sample wells, simultaneously, in the microtiter plate. The Tomtec is also used to transfer the acetonitrile supernatant from the plasma protein precipitation step, batchwise, to another microtiter plate for analysis by HPLC with fluorescence detection. This assay has been validated and implemented for a clinical study of over 1300 plasma samples and is comparable to manual assays in the LLOQ (lower limit of quantitation, 3 ng/ml) and in stability. This is the first semi-automated protein precipitation assay published for the analysis of montelukast in human plasma and it results in significant time savings over the manual methods, both in sample preparation and in HPLC run time.

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Up-regulation of cysteinyl leukotriene 1 receptor by IL-13 enables human lung fibroblasts to respond to leukotriene C4 and produce eotaxin.

Fukuda T.

Department of Pulmonary Medicine and Clinical Immunology, Dokkyo University School of Medicine, Tochigi, Japan.

Cysteinyl leukotrienes (CysLTs) play an important role in eosinophilic airway inflammation. In addition to their direct chemotactic effects on eosinophils, indirect effects have been reported. Eotaxin is a potent eosinophil-specific chemotactic factor produced mainly by fibroblasts. We investigated whether CysLTs augment eosinophilic inflammation via eotaxin production by fibroblasts. Leukotriene (LT)C(4) alone had no effect on eotaxin production by human fetal lung fibroblasts (HFL-1). However, LTC(4) stimulated eotaxin production by IL-13-treated fibroblasts, thereby indirectly inducing eosinophil sequestration. Unstimulated fibroblasts did not respond to LTC(4), but coincubation or preincubation of fibroblasts with IL-13 altered the response to LTC(4). To examine the mechanism(s) involved, the expression of CysLT1R in HFL-1 was investigated by quantitative real-time PCR and flow cytometry. Only low levels of CysLT1R mRNA and no CysLT1R protein were expressed in unstimulated HFL-1. In contrast, stimulation with IL-13 at a concentration of 10 ng/ml for 24 h significantly up-regulated both CysLT1R mRNA and protein expression in HFL-1. The synergistic effect of LTC(4) and IL-13 on eotaxin production was abolished by CysLT1R antagonists pranlukast and montelukast. These findings suggest that IL-13 up-regulates CysLT1R expression, which may contribute to the synergistic effect of LTC(4) and IL-13 on eotaxin production by lung fibroblasts. In the Th2 cytokine-rich milieu, such as that in bronchial asthma, CysLT1R expression on fibroblasts might be up-regulated, thereby allowing CysLTs to act effectively and increase eosinophilic inflammation.

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Bladder disease: A report based on the smooth muscle function in health and disease satellite meeting to the International Congress of Physiology.

Doggrell SA.

Department of Physiology and Pharmacology, The University of Queensland, Brisbane, Queensland, Australia. s_doggrell yahoo.com

One of the satellite meetings to the International Congress of Physiological Sciences concerned smooth muscle function in health and disease. In this report I have highlighted one of the areas of disease that were discussed: namely, bladder disease. In the treatment of unstable bladder, antimuscarinic agents are effective but cause unacceptable side effects. Thus, in order to prevent the side effects, new antimuscarinic agents are being prepared that show selectivity for the bladder and new methods for local delivery of antimuscarinics to the bladder are being developed. Potential new targets for the unstable bladder include P2X receptors and the nerve growth factor pathway. In detrusor-external sphincter dyssynergia, there is evidence to support a clinical trial of nitric oxide donors. The unpleasant and painful condition of interstitial cystitis has, to date, proven difficult to treat. Preliminary clinical data suggests that the leukotriene D(4) receptor antagonist montelukast may be useful in the treatment of IC. (c) 2001 Prous Science. All rights reserved.

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Treatment of interstitial cystitis with montelukast, a leukotriene D(4) receptor antagonist.

Bouchelouche P.

Department of Urology, Herlev Hospital, University of Copenhagen, Denmark.

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CysLT1 receptor upregulation by TGF-beta and IL-13 is associated with bronchial smooth muscle cell proliferation in response to LTD4.

Rola-Pleszczynski M.

Immunology Division, Department of Pediatrics, Faculty of Medicine, Universite de Sherbrooke, Sherbrooke, Quebec, Canada.

BACKGROUND: Airway remodeling is a feature of chronic asthma. It involves a number of structural changes, including bronchial smooth muscle cell (BSMC) hyperplasia and hypertrophy. Cysteinyl leukotrienes (cysLTs) have been suggested to play a role in airway remodeling in addition to their numerous other physiopathologic effects. OBJECTIVES: This work was aimed at characterizing the potential modulation of CysLT1 receptor expression by cytokines and the eventual functional relevance of this modulation. METHODS: Expression of CysLT1 receptor was measured by flow cytometry and immunofluorescence microscopy. Transcripts were measured by RT-PCR and BSMC proliferation by crystal violet staining. RESULTS: When human BSMC were exposed to transforming growth factor (TGF)-beta, IL-13, or IFN-gamma, their expression of CysLT1 receptor was significantly augmented in a time- and concentration-dependent manner. Interestingly, IL-4 had no significant effect on CysLT1 receptor expression in BSMC. Moreover, IL-13 and IFN-gamma but not TGF-beta were able to increase CysLT1 mRNA levels. Finally, when BSMC were pretreated with TGF-beta or IL-13 but not IFN-gamma, their responsiveness to LTD(4) was markedly enhanced in terms of BSMC proliferation. Whereas TGF-beta, IL-13, or LTD(4) alone had little effect on BSMC proliferation, preexposure of the cells to TGF-beta or IL-13 for 24 hours resulted in a significant increase in proliferation in response to LTD(4). The enhanced proliferation was totally prevented by pretreating the cytokine-primed BSMC with the selective CysLT1 receptor antagonist Montelukast. CONCLUSIONS: Taken together, our findings indicate a synergy between certain cytokines and cysLTs, mediated by the augmented expression of the CysLT1 receptor and subsequent LTD(4)-triggered BSMC proliferation. These findings support a role for cysLTs in the airway remodeling observed in asthmatic patients and may provide a rationale for preventive and therapeutic intervention.

Online pharmacy ref source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12743568&dopt=Abstract montelukast, Singulair