Peter R. Farina

The role of 5-lipoxygenase products in preclinical models of asthma

BACKGROUND The action of 5-lipoxygenase on arachidonic acid generates potent inflammatory mediators that may contribute to the pathophysiology of asthma. METHODS Using the potent and selective 5-lipoxygenase inhibitor BI-L-239, we have examined the role of 5-lipoxygenase products in three animal models of asthma. RESULTS In vitro BI-L-239 inhibited 5-lipoxygenase product generation from human lung mast cells, alveolar macrophages, and peripheral blood leukocytes with a concentration that would provide 50% inhibition values of 28 to 340 nmol/L. A 36-fold selectivity for immunoreactive leukotriene C4 versus immunoreactive prostaglandin D2 inhibition was demonstrated in mast cells. In anesthetized cynomolgus monkeys, inhaled BI-L-239 provided dose-dependent inhibition of the inhaled Ascaris-induced immunoreactive leukotriene C4 release (maximum, 73%; bronchoalveolar lavage [BAL], 20 minutes), late-phase bronchoconstriction (maximum, 41%; +6 to 8 hours), and neutrophil infiltration (maximum, 63%; BAL, +8 hours). In conscious sheep, inhaled BI-L-239 provided dose-dependent inhibition of the inhaled Ascaris-induced late-phase bronchoconstriction (maximum, 66%; +6 to 8 hours) and increase in airway responsiveness (maximum, 82%; carbachol, +24 hours). The acute bronchoconstriction was shortened, and neutrophil infiltration diminished (maximum, 61%; BAL, +8 hours) in this model. Finally in conscious actively sensitized guinea pigs pretreated with pyrilamine and indomethacin, inhaled BI-L-239 attenuated acute bronchoconstriction (maximum, 80%; +5 to 15 minutes), leukocyte infiltration (58%; BAL, +3 days) and increase in airway responsiveness (100%; methacholine, +3 days) induced by three alternate-day ovalbumin inhalations. CONCLUSIONS In conclusion, results in these three animal models indicate that 5-lipoxygenase products may be major contributors to the bronchoconstriction (especially late phase), leukocyte infiltration, and airway hyperresponsiveness that characterize asthma.

Radioimmunoassay for clonidine in human plasma and urine using a solid phase second antibody separation.

A reliable, sensitive, and specific radioimmunoassay (RIA) procedure for the quantitation of clonidine in plasma and other biological fluids was developed. The detection limit of the assay is 2 pg based on a 200 microliters sample. Nine commonly used drugs were found not to interfere with the RIA. The utility of the assay was demonstrated in a bioavailability study of clonidine conducted with 24 healthy subjects. Clonidine was readily quantitated in plasma over 4 half-lives. This assay is suitable for pharmacokinetic and bioavailability studies as well as therapeutic drug monitoring of patients.

Analysis of chlorthalidone in biological fluids by high-performance liquid chromatography using a rapid column cleanup procedure.

A high-performance liquid chromatographic assay usable for clinical monitoring of chlorthalidone in biological fluids was developed. Extraction efficiency was greater than 80% for blood and urine using a rapid, disposable column cleanup procedure. Chlorthalidone could be reliably measured in the range of 100-4,000 ng/ml in biological fluids with excellent day-to-day reproducibility and within-day precision. Chlorthalidone was found to be stable at -20 degrees C in blood and urine for at least 1 year, permitting repeat assays and large clinical studies to be conducted. The pharmacokinetics of chlorthalidone was studied in 24 subjects over a 120-h time interval following a single dose. chlorthalidone has a long terminal half-life in whole blood of 49 h, with peak concentrations occurring 8-10 h after oral dosing. During the first 12 h after dosing, chlorthalidone was rapidly excreted into urine followed by a slower phase with a half-life of 49 h.

Antiinflammatory benzimidazole derivative with inhibitory effects on neutrophil function

Abstract5-Methyl-2,2,2-trifluoroethylsulfonyl-1H-benzimidazole (BI-L-45 XX) inhibits both neutrophil enzyme release and chemotaxisin vitro and also inhibits chemotaxisin vivo. BI-L-45 XX has an IC50 between 16μM and 25 μM in inhibiting lysosomal enzyme release from human peripheral blood neutrophils. In a Boyden chamber experiment, BI-L-45 XX inhibited migration in response to fMLP with an IC50 of 5 μM. When given orally to passively sensitized rats at doses of 0.1 to 1.0 mg/kg, it inhibited migration of neutrophils to the pleural cavity in response to an antigen (ovalbumin) challenge. BI-L-45 XX also shows activity in the developing adjuvant arthritis model, with an ED50 of 45 mg/kg, while exhibiting no significant inhibition of cyclooxygenase in a human platelet assay. This suggests the possibility that its antiinflammatory activity may be in part mediated by its effect on neutrophil function.

A selective radioimmunoassay for the determination of pirenzepine in plasma and urine.

A radioimmunoassay procedure for the determination of pirenzepine in either plasma or urine was demonstrated to be both sensitive and specific as well as highly reproducible. The assay could detect levels as low as 1.25 ng/ml. The sensitivity was sufficient to allow the analysis of biological samples from both pharmacokinetic and clinical studies. The two metabolites of pirenzepine, LS 75 and LS 822, did not cross-react with the antiserum. The assay was not affected by a change in anticoagulant or by the presence of several over-the-counter or prescription drugs, even at very high levels. Samples could be frozen and stored for at least a year without affecting the analysis. Repeat analysis could be performed on samples that had been re-frozen. Several thousand plasma and urine samples, including plasma samples from severely renally impaired patients, have been analyzed for pirenzepine by the RIA with no interferences having been detected.

Comparative purification of recombinant HIV-1 and HIV-2 reverse transcriptase: Preparation of heterodimeric enzyme devoid of unprocessed gene product

A procedure for producing and purifying recombinant HIV-1 and HIV-2 reverse transcriptase (RT) is described. These enzymes are produced by Escherichia coli-transformed with a plasmid containing the gene encoding for either the human immunodeficiency virus type 1 (HIV-1) or HIV-2 RT protein. Both proteins are partially processed by host cell proteases giving rise to a mixture of heterodimeric and nonheterodimeric products, which are subsequently resolved to near homogeneity by chromatography on phosphocellulose, Q-Sepharose, and hydrophobic interaction HPLC. Both HIV-1 (66/51 kDa) and HIV-2 (68/54 kDa) heterodimeric enzymes devoid of excess unprocessed (p66 or p68) precursors are isolated, enabling comparative enzymatic characterization of the fully active (and biologically relevant) heterodimeric forms. Homogenous HIV-1 and HIV-2 RT purified by this methodology exhibit near equivalent polymerase and RNase H activities.

Generation of leukotrienes by antigen and calcium lonophore A23187 stimuli from a mouse mastocytoma cell line, MMC-16

The generation of leukotrienes and histamine release by the mouse mastocytoma cell line MMC-16 was investigated. These cells produced leukotriene C4 (LTC4) and released histamine upon calcium ionophore A23187 and antigen stimulation. The ionophore also stimulated the biosynthesis of leukotriene B4 (LTB4) by MMC-16. Generation of LTC4 was confirmed by its characteristic UV absorption spectrum, fast atom bombardment-MS, equivalent HPLC retention time with an authentic standard and radioimmunoassay. Leukotriene B4 was characterized by its distinctive UV spectrum and HPLC retention time compared with synthetic material. IgE-mediated LTC4 generation was also observed in a dose dependent fashion with MMC-16 cells passively sensitized with monoclonal IgE specific for ovalbumin. LTC4 biosynthesis was effectively inhibited by the lipoxygenase inhibitor NDGA.

Pharmacological modulation of plasminogen activator secretion by P388D1 cell line

P388D1 is a murine macrophage cell line which spontaneously secretes plasminogen activator (PA; activated function) and lysozyme (LYS; constitutive function). Compounds which decrease PA secretion without affecting LYS secretion have potential as “down-regulators” of macrophage function and, hence, of the immune system. Glucocorticoids (e.g., dexamethasone, IC50<0.01 μM) and auranofin (IC50=1 μM) are positive in this model. In contrast, cyclooxygenase inhibitors (indomethacin, ibuprofen and piroxicam, all at 1 μM) boost PA secretion; lipoxygenase inhibitors (REV-5901, NDGA and piriprost, all at 10 μM) have little or no effect.Dexamethasone, but not auranofin, induces a urokinase-inhibitory activity which elutes between 0.13 and 0.19M NaCl upon anion exchange HPLC (TSK-DEAE-5-PW). Fibrin overlay following SDS-PAGE of the HPLC peak reveals a urokinase-inhibitory band at ∼90 Kd.

Discovery of BIRM 270: A New Class of Leukotriene Biosynthesis Inhibitors

Several events in the late 1970s and early 1980s helped to shape the research agenda for pharmaceutical companies engaged in the search for a new generation of anti-inflammatory agents. The structural identity of the arachi- donic acid (AA) metabolite leukotriene B4 (LTB4) (Borgeat and Samuels- son, 1979) and the discovery that it was a potent neutrophil chemoattractant (Ford-Hutchinson et al., 1980) helped to elucidate how leukocytes migrate to a site of inflammation. In addition, the identification of leukotriene C4 (LTC4) as a component of slow reacting substance of anaphylaxis (SRS- A) demonstrated that arachidonic acid-derived substances were also potent bronchoconstrictors (Murphy and Hammarstrom, 1979; Corey et al., 1980; Sirois and Borgeat, 1980). Common to both classes of leukotrienes is the enzyme 5-lipoxygenase (5-LO) which introduces a molecule of oxygen at the C-5 position of AA followed by cyclization to the epoxide intermediate ITA4. Further reaction with a glutathione S-transferase yields the peptidyl LTs, LTC4 and its metabolites LTD4 and LTE4. Conversion of the epoxide by LTA4 hydrolase produces LTB4. Given that AA also serves as a substrate for the cyclooxygenase (CO) pathway to produce pro-inflammatory prostaglandins and thromboxane, interest was certainly reinforced in inhibiting one or both arms of what is often called the arachidonic acid cascade.