Peter R. Bernstein

Inhibitors of human leukocyte elastase.

Publisher Summary This chapter focuses on inhibitors of human leukocyte elastase (HLE). HLE is a strongly basic glycoprotein, which is produced by polymorphonuclear leukocytes (neutrophils) and is released from their azurophilic granules. HLE exists as at least four distinct isozymes, which range in molecular weight from 24 to 30 kDa and appear to differ only in carbohydrate content. Furthermore, human sputum elastase (HSE), which is isolated from purulent sputum as at least five distinct isozymes, is both immunologically and catalytically indistinguishable from HLE and is believed to be identical to it. The primary role of intracellular HLE appears to be the degradation of foreign proteins ingested by leukocytes during phagocytosis, whereas the main target for extracellular elastase appears to be elastin. Elastin is the primary elastic component of the lungs, blood vessels and other organs, and is degraded by HLE in a process called elastolysis. There are many high-molecular-weight, polypeptide, elastase inhibitors which have been isolated from animal or plant sources. Most of these, the non-human proteins, would probably induce an immunogenic response and are not suitable for clinical development. A subset of these inhibitors, predominantly human in origin, is being explored as a source for clinically useful elastase inhibitors.

Determination of SRS-A release from guinea-pig lungs by a radioimmunoassay

A sensitive radioimmunoassay for leukotrienes (LTs) has been developed. Rabbits were immunized with a conjugate of LTD4 and bovine serum albumin, prepared by using 1,5-difluoro-2,4-dinitrobenzene as the coupling agent. The assay can detect 0.045 pmol LTD4 at a final plasma dilution of 1:72. 50% displacement of bound 3H-LTD4 was obtained with 0.43 +/- 0.03 pmol LTD4. LTC4, LTE4 and LTF4 cross-react 159%, 57% and 85%, respectively, whereas LTB4, 5-HETE and prostaglandins did not. The assay was validated by measuring the antigen-induced release of LTs from sensitized guinea-pig chopped lungs. High correlation (0.9434, p less than 0.05) was found when LTs were simultaneously determined by this assay and a bioassay on guinea pig ileum.

Comparative potency and pharmacology of isomers of leukotriene D4 on guinea-pig trachea: Requirement for a 5(S)6(R) configuration

The relative contractile activity of C5 and C6 diastereomers of Leukotriene D4 (LTD4), as well as 11-trans stereoisomers were evaluated in guinea-pig tracheal smooth muscle. 5(S)6(R) LTD4 was 1000 times more potent than histamine as a contractile agent. While a change of the 11-ethylenic bond from cis to trans resulted in a four fold decrease in potency, a change in configuration of the 5-hydroxyl group and/or the 6-peptide adduct resulted in a decrease in potency of at least 2 to 3 orders of magnitude. The contractile activity of all LTD4 isomers was inhibited by FPL 55712, whereas indomethacin markedly enhanced the contractile activity of 5(S)6(R) LTD4, but appeared to have less of an effect on the other diastereomers. The results demonstrate the critical nature of configuration of the 5-hydroxyl and the 6-peptide adduct of eicosatetraenoic acid for maintenance of high affinity for receptors.

Airway Response to Leukotriene D4 in Rhesus Monkeys

We studied the effect of leukotriene D4(LTD4) given by aerosol challenge on asthmatic and normal monkeys. The response to LTD4 occurred within 2 min., was prolonged at higher concentrations, was reversed by epinephrine, and more closely resembled an antigen response than a histamine response. Since LTD4 concentrations of 30-100 micrograms/ml produced a response similar to 2-20 mg/ml of histamine, the LTD4 was 300-900 times as potent as histamine on a molar basis. Thus, LTD4, which appears to reproduce an antigen-induced response in rhesus monkeys, may be a mediator of allergic asthma in these animals.

De Novo Design of a Picomolar Nonbasic 5-HT1B Receptor Antagonist

We describe herein the discovery of novel, de novo designed, 5-HT(1B) receptor antagonists that lack a basic moiety and that provide improved hERG and in vitro phospholipidosis profiles. We used a known 5-HT(1B) antagonist template as our starting point and focused on replacing the piperazine moiety. Pyrazole-based ideas were designed and synthesized among a small library of piperazine replacements. To our knowledge, these are the first potent, nonbasic, functionally active antagonists of the 5-HT(1B) receptor.

Discovery of spirofused piperazine and diazepane amides as selective histamine-3 antagonists with in vivo efficacy in a mouse model of cognition.

A new series of potent and selective histamine-3 receptor (H3R) antagonists was identified on the basis of an azaspiro[2.5]octane carboxamide scaffold. Many scaffold modifications were largely tolerated, resulting in nanomolar-potent compounds in the H3R functional assay. Exemplar compound 6s demonstrated a selective profile against a panel of 144 secondary pharmacological receptors, with activity at only σ2 (62% at 10 μM). Compound 6s demonstrated free-plasma exposures above the IC50 (∼50×) with a brain-to-plasma ratio of ∼3 following intravenous dosing in mice. At three doses tested in the mouse novel object recognition model (1, 3, and 10 mg/kg s.c.), 6s demonstrated a statistically significant response compared with the control group. This series represents a new scaffold of H3 receptor antagonists that demonstrates in vivo exposure and efficacy in an animal model of cognition.

Azepines and piperidines with dual norepinephrine dopamine uptake inhibition and antidepressant activity.

Herein, we describe the discovery of inhibitors of norepinephrine (NET) and dopamine (DAT) transporters with reduced activity relative to serotonin transporters (SERT). Two compounds, 8b and 21a, along with nomifensine were tested in a rodent receptor occupancy study and demonstrated dose-dependent displacement of radiolabeled NET and DAT ligands. These compounds were efficacious in a rat forced swim assay (model of depression) and also had activity in rat spontaneous locomotion assay.

Effect of trifluoromethyl ketone‐based elastase inhibitors on neutrophil function in vitro

Neutrophils release elastase, which is known secondarily to cause tissue damage. However, it is rapidly inactivated by the endogenous α1‐proteinase inhibitor (α1Pi). Nevertheless, under pathological conditions, α1Pi is inactivated by oxidants released from neutrophils, resulting in an excess of elastase at the site of inflammation. This elastase/α1Pi imbalance has been implicated as a pathogenic factor in cystic fibrosis, acute respiratory distress syndrome, and emphysema. Elastase inhibitors, which do not interfere with the microbicidal activity of neutrophils and are resistant to neutrophil‐released oxidants, would undoubtedly represent an important advance in the management of neutrophil‐mediated tissue injury. We report that a new family of elastase inhibitors ICI200355 and ZD0892 was found to be resistant toward superoxide, hypochlorous acid, hydrogen peroxide, hydroxyl radical, and peroxynitrite mediated degradation as well as having no effect on the formation of these oxidants by activated neutrophils. More importantly, we found that these inhibitors did not interfere with the ability of human neutrophils to phagocytose and to kill Staphylococcus aureus. In conclusion, a new potent class of elastase inhibitors, while blocking the effects of neutrophil elastase, was found not to impede various physiological functions of human neutrophils, in particular the ability of these phagocytic cells to phagocytose and kill bacteria. J. Leukoc. Biol. 64: 322–330; 1998.

Preparation of a diketopiperazine analog of leukotriene D4 (LTD4)

Abstract A LTD 4 analog has been synthesized in which the peptidyl fragment has been replaced by a nonionic diketopiperazine. Biological evaluation showed agonist activity, giving key information on LTD 4 geometry to the receptor.

Effects of combined receptor antagonists of leukotriene D4 (LTD4) and platelet-activating factor (PAF) on rhesus airway responses to LTD4, PAF and antigen.

Rhesus monkeys with IgE-mediated asthmatic-type responses to Ascaris suum antigen were pretreated with a combination of 2 receptor antagonists to leukotriene D4 (LTD4) and platelet-activating factor (PAF). This combination of anti-LTD4 and anti-PAF was shown to inhibit airway responses to either LTD4 or PAF in separate experiments in these Ascaris airway-reactive animals. When the same combination of LTD4 and PAF receptor antagonists was used to pretreat the same animal prior to aerosol challenge with Ascaris antigen we could not demonstrate inhibition of the antigen-induced airway response. Thus, combined receptor blockade for LTD4 and PAF did not alter IgE-mediated acute airway responses to antigen in this species. We review the current status of rhesus asthma and LTD4 and PAF receptor antagonist activity in this model.