From Bench to Bedside
The Hurdles of Discovering a New Leukotriene Receptor Antagonist

IAN W. RODGER

Worldwide Human Health, Merck & Company, Whitehouse Station, New Jersey

	INTRODUCTION

TOP INTRODUCTION THE GOALS OF DRUG... THE PROCESS OF DISCOVERY NEGOTIATING THE FINAL HURDLE:... CONCLUDING REMARKS REFERENCES

The discovery of leukotrienes dates back to the period between 1938 and 1940. During this period Kellaway, Feldberg, Trethewie, and Holden published the results of a series of experiments on snake venoms that led them to conclude that "there is formed from tissue constituents by enzymic action of the venoms a `slow-reaction smooth muscle-stimulating substance' which may largely determine the nature of the responses" (1, 2). It was, however, in their seminal paper in 1940, using guinea pigs sensitized to egg albumin, that Kellaway and Trethewie (3) coined the term SRS and concluded that "SRS was formed by the action of antigen upon sensitized tissue." It was not until some 20 yr later, when Brocklehurst was performing elegant experiments on perfused guinea pig lungs, that he confirmed the release of SRS-like activity on anaphylactic challenge of sensitized tissue (4). In his article Brocklehurst named this biological material "slow-reacting substance of anaphylaxis" (SRS-A) to differentiate it from other material that could induce "slow-reacting" contractions. Thus, the term SRS-A was born. Despite intensive research efforts by a wide variety of scientists it was not until 1979 that the chemical constituents of SRS-A were identified, by B. Samuelsson and colleagues at the Karolinska Institute in Stockholm, as the cysteinyl leukotrienes (LTs), namely LTC₄, LTD₄, and LTE₄ (5, 6).

In the period up until the chemical elucidation of SRS-A it had become progressively more widely accepted that SRS-A played a pivotal role in the bronchoconstriction that is one of the hallmarks of acute asthma attacks. Thus, the seminal discovery that the cysteinyl leukotrienes were the active principle of SRS-A and the elucidation of their biosynthetic pathway from arachidonic acid were key events that provided a rational basis for the design of molecules designed to interfere with these events. Not surprisingly, several pharmaceutical companies (e.g., Merck, ICI [now Zeneca], Abbott, Smith Kline & French [now SmithKline Beecham], Bayer, Lilly, Ono, Ciba Geigy [now Novartis]) mounted research programs that were designed to discover molecules that either inhibited leukotriene biosynthesis or blocked the receptors via which the leukotrienes exerted their effects.

The purpose of this article is to review some of the hurdles that are an inevitable component of the drug discovery process. In the interests of brevity, focus is directed particularly at the discovery of drugs by Merck designed to antagonize the cysteinyl leukotriene receptors. This focus, however, in no way diminishes or dismisses the significance of the complementary approach of inhibiting leukotriene biosynthesis, far from it. Indeed, 5-lipoxygenase-activating protein (FLAP) inhibitors have been developed by several companies including Merck (7, 8), and a direct 5-lipoxygenase inhibitor, zileuton, by Abbott (9).

	THE GOALS OF DRUG DISCOVERY AND DEVELOPMENT

TOP INTRODUCTION THE GOALS OF DRUG... THE PROCESS OF DISCOVERY NEGOTIATING THE FINAL HURDLE:... CONCLUDING REMARKS REFERENCES

The establishment of leukotriene receptor antagonist programs by Merck and other companies followed quickly after the revelation of the chemical identity of SRS-A. In the case of Merck, the decision was made to concentrate the program at the Merck Frosst Centre for Therapeutic Research in Montreal, Canada, where exploratory research examining biologically derived SRS-A had been in place for several years in the latter part of the 1970s. One of the first priorities of this group was to manufacture significant quantities of synthetic leukotrienes to support the preclinical biological screening efforts. Furthermore, it was well appreciated that the synthetic leukotrienes could serve as a foundation for antagonist chemistry efforts since modification of agonist molecules was a tried and tested approach in other discovery programs. Within 1 year of the discovery of the biologically synthesized leukotrienes, chemists at Merck Frosst (10) and Harvard (11) successfully achieved laboratory synthesis of pure leukotrienes. The decision by Merck to supply academic researchers throughout the world with free quantities of leukotrienes (which were otherwise commercially available but at extremely high cost) was widely applauded and catalyzed an explosion of research in the field.

For most pharmaceutical companies the goals of drug discovery and development are remarkably similar. The goals of the leukotriene receptor antagonist program at Merck were, first, to discover a drug that provided significant clinical benefit to patients with asthma. Given the developing understanding that the cysteinyl leukotrienes seemed to have little, or no, physiological role and that their effects were principally confined to pathophysiological changes in the lung, the notion that an antagonist could be administered orally was advanced as a second program goal. Such an approach obviated the need for complicated inhaled drug regimens such as existed with -agonist bronchodilators and glucocorticosteroids. In addition, there was an acceptance that patient compliance was decreased with such aerosol medications and, in general, the driving force behind the inhaled route of administration was the necessity to limit or avoid systemic side effects. Furthermore, the most reliable, convenient, and successful method of delivering a drug to the target tissue is via the bloodstream. In the case of the lungs penetration of inhaled drug particles to the small airways, which are the major site of obstruction in asthma, is confounded by the presence of mucous plugging in addition to the bronchoconstriction that narrows the airway lumen. Drug access to these small airways via the bronchial (or indeed pulmonary) circulation, after oral ingestion, circumvents such problems. The third goal was to produce a potent drug so that the dose administered to patients could be as low as possible. With the correct pharmacokinetic and pharmacodynamic profile a drug could be produced that would be taken only once a day. This would markedly improve patients' compliance with their medication. This was established as a fourth objective. It went almost without saying that the drug had to be specific in its mechanism of action in order to decrease, or ideally eliminate, side effects. Any drug that satisfied these optimal properties could then be considered safe for use in the therapy of pediatric asthma, which remains a significant problem.

	THE PROCESS OF DISCOVERY

TOP INTRODUCTION THE GOALS OF DRUG... THE PROCESS OF DISCOVERY NEGOTIATING THE FINAL HURDLE:... CONCLUDING REMARKS REFERENCES

In the absence of a detailed molecular structure of the receptor (which even today remains unknown) the starting points for the design of receptor antagonist molecules can be either the known agonist ligand structure (the cysteinyl leukotrienes), existing antagonists (such as FPL-55712, which was discovered by Fisons through a broad screening effort against an SRS-A assay) (12), or via random screening of a company's chemical sample collection. All the pharmaceutical companies mentioned above used one or more of these approaches. For example, at Merck all three approaches were employed. Zeneca, on the other hand, focused on modifications to FPL-55712 and the native leukotriene ligands. Bayer and SmithKline Beecham focused principally on converting the agonist ligands into antagonists.

The first Merck compound that was administered to human subjects was L-649,923, a hydroxyacetophenone-containing antagonist derived from FPL-55712. In clinical trials this compound was orally absorbed and antagonized the bronchoconstrictor effects of inhaled leukotriene D₄ but not those of inhaled histamine, thus demonstrating a specificity of action (13). The compound also inhibited antigen-induced bronchoconstriction to a small degree (14). Notwithstanding, the clinical efficacy of the compound was inadequate and failed to meet the criteria that had been established. Hence it was dropped. In an effort to test the leukotriene receptor antagonist hypothesis more rigorously a second compound, L-648,051, was developed as an inhaled powder formulation. The hope was that via this route the compound (which was not orally bioavailable) might provide more potent antagonism at the leukotriene receptors in the lungs. In clinical testing, however, L-648,051 behaved almost exactly like its predecessor. Thus, it blocked LTD₄- induced, but not histamine-induced, bronchoconstriction and weakly inhibited antigen-induced bronchoconstriction (15, 16). Like its forerunner, L-648,051 failed to meet several of the established criteria and further development of the compound was abandoned. The Lilly compound LY-171,883 was similarly unconvincing in challenge studies in humans (17, 18). As a consequence of these failures, many people began to question the putative role of the leukotrienes in asthma. The view within Merck, and in several of the other companies involved in the search for antagonists, was that the receptor antagonists in question were quite simply not potent enough to adequately test the hypothesis.

The search for more potent compounds led Merck to screen its extensive chemical sample collection for possible new structural leads. Ultimately, this led to the identification of MK-0571, an orally bioavailable compound, that entered clinical trials testing in 1989. In subjects with chronic asthma MK-0571 increased baseline FEV₁ by 8-14% (clearly indicating that individuals with asthma have an ongoing production of cysteinyl leukotrienes contributing to bronchomotor tone) and decreased daytime symptom scores and -agonist use by approximately 30% (19). In antigen challenge studies MK-0571 was highly effective in reducing both early-phase (by approximately 88%) and late-phase (by approximately 63%) bronchoconstrictor responses (20). In exercise challenge studies in subjects with asthma the compound was also remarkably effective in abrogating the fall in FEV₁ and hastening the return of lung function to baseline after a period of vigorous exercise (21). Thus, the report card on MK-0571 was the best to date. It possessed good clinical efficacy, was orally bioavailable, and was highly specific in its mechanism of action. On the downside, it lacked the necessary potency and was unsuitable for dosing once daily. Critically, however, in safety studies in mice MK-0571 was found to induce peroxisome proliferation. This toxicity meant that further development of the compound was precluded. Notwithstanding, the clinical data generated with MK-0571, along with similar results obtained by Zeneca using ICI-204,219 (later to be called zafirlukast) (22) served to underscore the significance of the cysteinyl leukotrienes in allergic asthma and, somewhat remarkably, in exercise-induced bronchoconstriction. Furthermore, these data effectively provided proof of principle that the cysteinyl leukotrienes were important, indeed pivotal, chemical mediators involved in the pathophysiological events underlying an asthmatic attack.

Given the significant promise of MK-0571 its toxicity was closely examined by Merck scientists. Importantly, the compound was a racemic mixture of R- and S-enantiomers. Thus, these enantiomers were separated and characterized for their biological and peroxisomal proliferative activity. Remarkably, it was discovered that the biological (antiasthmatic) activity resided in the R-enantiomer while the peroxisomal proliferative activity was a property of the S-enantiomer. The R-enantiomer (MK-0679), subsequently called verlukast, was carried forward into development and entered clinical trials in 1990. Not surprisingly (being the biologically active half of MK-0571), MK-0679 possessed efficacy in studies of patients with chronic asthma that was almost identical to that seen with MK-0571 (23). Verlukast was also studied, for the first time, in aspirin-intolerant subjects with asthma. Remarkably, it increased baseline FEV₁ in these patients by approximately 18% and induced a highly significant shift in the bronchoconstrictor dose-response curve to inhaled lysine-aspirin (24, 25). These data served to highlight the fact that in this subset of subjects with asthma there was an exaggerated involvement of the cysteinyl leukotrienes in their disease process. Thus, in terms of its overall profile MK-0679 possessed significant clinical efficacy and specificity of action, and was orally bioavailable. Like MK-0571, however, it still lacked the desired potency and required administration at least twice daily. Thus, it still failed to measure up to the goals established at the origination of the program. It was nonetheless a potentially viable drug candidate that moved forward into advanced clinical trials. It was during these late-stage studies that a low incidence (approximately 3%) of elevations in serum transaminases was reported as an adverse effect. In light of the fact that asthma is a chronic disease and that Merck sought a drug that could be safely administered prophylactically to control the disease in both adults and children the decision was taken in 1991 to abandon the development of MK-0679 and continue the search for a superior compound. Yet again another promising clinical candidate had failed to negotiate a late-stage hurdle. At this point the leukotriene program had been running at Merck Frosst for more than 11 yr. In the minds of some, the seeds of doubt were beginning to grow as to whether the right compound would ever be discovered.

	NEGOTIATING THE FINAL HURDLE: REALIZATION OF THE GOALS

TOP INTRODUCTION THE GOALS OF DRUG... THE PROCESS OF DISCOVERY NEGOTIATING THE FINAL HURDLE:... CONCLUDING REMARKS REFERENCES

Intensive analysis of MK-0679 failed to uncover any mechanistic basis for the elevation in serum transaminases observed during its clinical program. Thus, confidence existed in the basic structure of the molecule and key chemical substitutions were made to MK-0679 that ultimately produced MK-0476 (later named montelukast), a substantially more potent compound at the cysteinyl leukotriene 1 (cysLT₁) receptor (26). After rapid preclinical evaluation and safety testing montelukast entered phase 1 clinical studies in 1992. In a wide range of clinical trials montelukast has been shown to provide significant clinical benefit in both children (6-14 yr old) and adults with chronic asthma (27). In addition, like its predecessors, montelukast protects against exercise-induced bronchoconstriction (in both adults and children) (31, 32) and provides substantial clinical benefit to aspirin-intolerant patients with asthma. In addition, montelukast has been shown to require no dose titration, possess no significant interactions with either food or other commonly used medications, and have a side-effect profile that is not significantly different from placebo. Thus, in terms of the program objectives the report card for montelukast is excellent. The drug is potent, orally active and readily bioavailable, long acting (so that it need be administered only once daily), specific for the cysLT₁ receptor, and possesses an outstanding safety profile. Thus, 17 yr after initiation of the leukotriene program at Merck the worldwide marketing application for montelukast was filed, on February 23, 1997. In August 1997 montelukast (under its trade name Singulair) was approved for use in both adults and children with asthma, first in Finland and then in December in much of the remainder of the European Community. In February 1998 it was approved by the Food and Drug Administration (FDA) for use in the United States. The final hurdles had been negotiated successfully after many trials and tribulations. A leukotriene antagonist with all the desired properties had finally been realized.

	CONCLUDING REMARKS

TOP INTRODUCTION THE GOALS OF DRUG... THE PROCESS OF DISCOVERY NEGOTIATING THE FINAL HURDLE:... CONCLUDING REMARKS REFERENCES

Coincident with the final stages of the development program of montelukast, Zeneca was concluding its own development activities with ICI-204,219 (zafirlukast; Accolate) and Ono with pranlukast, that had been occurring in parallel with those in Merck. Thus, today there are three specific antagonists of the actions of the cysteinyl leukotrienes at the cysLT₁ receptor. Together these drugs form part of the first new class of therapy for asthma in more than 25 yr. For these three drugs, which have already demonstrated significant benefits in patients with asthma, their full clinical potential is yet to be realized. Only with the passage of time and wider experience of their use in clinical practice will this occur. Notwithstanding, their ultimate appearance as part of the medical armament in the therapy of asthma is a triumph for commitment and endurance in drug discovery. In discovering these drugs the pathway from bench to bedside has been a long, arduous, and tortuous one. It has taken more than a half-century of diligent scientific endeavor from the original days of SRS-A to get to where we are today. One cannot help but think that those early pioneers would be proud of what they initiated and what has ultimately been accomplished.

	Footnotes

Correspondence and requests for reprints should be addressed to Ian W. Rodger, Worldwide Human Health, Merck & Company, Inc., 1 Merck Drive, Whitehouse Station, NJ 08889-0100. E-mail: ian_rodger{at}merck.com.

	References

TOP INTRODUCTION THE GOALS OF DRUG... THE PROCESS OF DISCOVERY NEGOTIATING THE FINAL HURDLE:... CONCLUDING REMARKS REFERENCES

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