Richard R. Rosenthal

The Effect of Inhibition of 5-Lipoxygenase by Zileuton in Mild-to-Moderate Asthma

Although intermittent episodic airway narrowing occurs in persons with asthma, the biochemical basis of this obstruction has not been elucidated. Nonetheless, inflammatory cells present in the airways of persons with asthma [1, 2] release various substances that narrow airways. Among these are cysteinyl leukotrienes, which are formed from arachidonic acid in part by the enzyme 5-lipoxygenase [3]. The evidence favoring a role for leukotrienes in asthma is that they are produced by various airway cells including eosinophils and mast cells [4, 5], they are potent bronchoconstrictor agonists [6-8], and they can be recovered from biological fluids during asthma attacks [9-11]. Recently, the salutary effects of specific leukotriene-receptor antagonists or synthesis inhibitors in persons with asthma have suggested that interventions in the 5-lipoxygenase pathway may be of therapeutic use in the treatment of asthma [12-20]. These observations are particularly interesting because of the increasing concerns about asthma therapies such as -agonists and theophylline [21-24] and the known toxicity of long-term steroid use [25, 26]. However, the conclusions about the efficacy of these new drugs in persons with asthma largely derive from studies in laboratory-induced, rather than spontaneously occurring, asthma. Because cases of spontaneously occurring asthma may differ from those of laboratory-induced asthma, in mechanism or in response to therapy, we examined the effects of zileuton (N-1-[benzo(b)thien-2-ylethyl]-N-hydroxyurea), an investigational inhibitor of 5-lipoxygenase [27] currently in phase III trials of efficacy, in persons with asthma. In a double-blind, placebo-controlled trial in patients with mild-to-moderate airflow obstruction, we investigated the effects of inhibition of 5-lipoxygenase with zileuton (Leutrol; Abbott Laboratories, North Chicago, Illinois), during a 4-week period, on airway function, asthma symptoms, and the bronchodilator response to -agonists. We found that a dose of 600 mg four times per day (2.4 g/d), which produces more than 35% inhibition of leukotriene production as indicated by excretion of leukotriene E4 (LTE4) in the urine, had a salutary effect on airway function and asthma symptoms. Methods Patient Selection Patients with mild-to-moderate asthma were recruited at 14 centers, which included university hospitals and private allergy and pulmonary practices. Patients with symptoms that corresponded with the American Thoracic Society definition of asthma [28] were screened. All patients had to have a forced expiratory volume in 1 second (FEV1) of 40% to 75% of predicted value and a 15% or greater increase in FEV1 30 minutes after inhalation of two puffs of albuterol. Additionally, patients were required to be 18 to 65 years old; women of childbearing potential were excluded. Before enrollment in the study, none of the patients had used oral or inhaled steroids or cromolyn sodium for 4 weeks. Beta-blockers, calcium-channel blockers, and nonsteroidal anti-inflammatory drugs could not have been used for at least 1 week before entry into the study. All patients were required to be able to achieve adequate symptomatic asthma control without using theophylline, oral -agonists, or antihistamines; none of these medications was permitted throughout the entire study period. Study Design and Intervention A randomized parallel design was used in this double-blind, placebo-controlled study. Patients were chosen randomly to receive either 600 mg of zileuton (four times a day), 800 mg of zileuton (twice a day), or placebo. A 1-week, single-blind, placebo lead-in qualification period (dummy lead-in period) was followed by random allocation to one of the three treatment groups for a 4-week, double-blind phase. During the single-blind, dummy lead-in and the double-blind study periods, all patients took capsules four times a day. Self-determined peak expiratory flow rates were recorded in the morning (before medication) and evening (2 hours after the third set of capsules) in a study diary. Albuterol inhaler use and asthma symptoms were recorded in the diary as well. Daytime asthma symptoms were self-rated on a scale of 1 to 5 (1 = no symptoms, 5 = severe symptoms; maximum weekly score of 35). After the 1-week dummy lead-in period, patients returned to their study center. Inhaled albuterol was withheld for at least 8 hours before the study visit. Spirometry was done on patients who had no clinically significant laboratory abnormalities, who had successfully completed their diary card, who had moderately symptomatic asthma (a total score of 12 but 28 in the previous 7 days), and who had used their albuterol inhaler at least 7 times during the dummy lead-in week. If the FEV1 was 40% to 75% of the predicted value, the patient was assigned randomly to a group according to a predetermined code. All patients took visually identical capsules four times per day that contained either 600 mg of zileuton four times daily, 800 mg of zileuton twice daily (active drug first and last dose daily), or placebo, which were supplied by Abbott Laboratories in a blind manner. The first dose of study medication was administered at the study center, and spirometry was done 30, 60, and 120 minutes later. In the 800-mg group, each days drug card contained both placebo and active drug, and as a result, on the first day, an undetermined number of patients received placebo instead of 800 mg of zileuton as their first dose of drug. Therefore, the 800-mg group was not included in the analysis of the acute response to the first dose of drug. During the 4-week double-blind period, patients returned to the study center at the same time of day on a weekly basis to have spirometry done and to review diary cards and medication use. During the second and third weeks of the double-blind randomization period, spirometry was also repeated 30 minutes after inhalation of two puffs of albuterol. Urine Collection and Analysis Urine was collected for 4 hours beginning at 8:00 a.m. before the dummy lead-in period and on day 28 of the study. Urinary LTE4 levels were determined by reverse-phase high-performance liquid chromatography and enzyme immunoassay using minor modifications of established procedures [29]. The recovery of the internal LTE4 standard was 74% 6%. The LTE4 content of the urine was expressed as picograms of immunoreactive LTE4 per milligram of creatinine. Adverse Events Routine complete blood counts, serum chemistries, urinalyses, and electrocardiograms were obtained throughout the study. Adverse symptoms were elicited daily through a diary question and were reviewed at the weekly visit to the study site. Statistical Analysis All values were expressed as means with associated 95% CIs; all outcome indicators were normally distributed. Paired t-tests were used to assess the statistical significance of any within-group changes from the baseline dummy lead-in phase. The statistical significance of differences among the placebo and active treatment groups during the 4 weeks of double-blind treatment was evaluated using a two-way analysis of variance model with effects for center, treatment, and center-treatment interaction. When statistical differences were noted among groups in the dummy lead-in, the groups were compared using an analysis of covariance adjusting for baseline differences. Available data were analyzed up to the point of withdrawal for patients who did not complete the study protocol. The Fisher test for the protected least significant difference was used to make pair-wise comparisons. Results Patients A total of 188 patients entered the single-blind dummy lead-in period; 143 fulfilled the enrollment criteria and were randomly assigned to receive study drug or placebo (46 patients received 2.4 g/d, 49 patients received 1.6 g/d, and 48 patients received placebo). Two patients withdrew during the first week of the double-blind study1 for personal reasons and the other because of worsening asthma (both received 1.6 g/d of zileuton). Two patients were not included in the final analysis, because they were enrolled in a center that did not have representation in all three treatment groups (1 received 1.6 g/d of zileuton and 1 received placebo). The characteristics of the 139 evaluated patients are given in Table 1. Of the 139 patients who were still in the trial after 1 week, 12 evaluated patients left the study before completing the trial protocol (all their data were included up to the point of termination): 4 patients because of worsening asthma (1 received 2.4 g/d, 2 received 1.6 g/d, and 1 received placebo); 2 patients because of upper respiratory infections (1 received 2.4 g/d and 1 received placebo); 1 patient because of sinusitis (placebo); 1 patient because of urticaria (1.6 g/d); 3 patients because of personal reasons (1 in each group); and 1 patient because of headaches that had begun before randomization (2.4 g/d). Table 1. Characteristics or Evaluated Patients* Acute Effects on Airway Obstruction A single 600-mg dose of zileuton produced rapid bronchodilation (Figure 1). Compared with the mean FEV1 measured just before study drug ingestion (0 minutes), the mean FEV1 improved 30 minutes after a single 600-mg dose of zileuton and remained increased for the entire 2-hour observation period (P < 0.005 for all observation points). The maximum increase (14.6%) in the mean FEV1 was 0.35 L (CI, 0.25 to 0.45 L) (P < 0.001), which occurred at 60 minutes. No improvement of the FEV1 occurred in the placebo group (0.09 L [CI, 0.01 to 0.19 L]; P = 0.075). The improvement in the mean FEV1 after zileuton was greater than that after placebo at 60 and 120 minutes (P < 0.001 and P = 0.01, respectively). Figure 1. Change in the forced expiratory volume during the 2 hours after administration of zileuton or placebo. P P P Effects of 4 Weeks of Zileuton Administration on Airway Obstruction All three groups of patients had an initial impr

The role of ragweed pollen in autumnal asthma

Thirty-nine ragweed-allergic seasonal asthmatics were studied from 1972 to 1974. After quantitative skin tests, antigen E-induced leukocyte histamine release, quantitative inhalation bronchial challenge with ragweed extract to determine PD35 (provocation dose of allergen causing 35% decrease in specific airways conductance), and radioallergosorbent test (RAST) determinations were done, patients were paired based on PD35 values and randomly assigned to treatment or placebo groups, receiving either aqueous ragweed extract or placebo prior to the 1973 ragweed season. Treated patients received a mean cumulative dose of extract equivalent to 11.7 microng antigen E (4,180 protein nitrogen units [PNU]). Twenty-nine patients were followed through the ragweed season with daily symptom diaries and biweekly physician examinations. Severity of disease was not predictable by PD35 data, skin tests, leukocyte histamine release, or radioallergosorbent test (RAST) values. Although all patients were ragweed-allergic by objective tests, only 13/29 had asthma symptoms correlating with ragweed counts. Mold spore counts were related significantly to symptoms in some patients. Asthma and hay fever symptoms correlated significantly in 24/29 patients. This dose of immunotherapy caused no significant difference to be found in asthma or hay fever symptoms in treated versus placebo patients for the 1973 reporting period as determined by physician evaluations or daily symptom diaries. No patients showed significant improvement in PD35 values after treatment in 1973. Similar findings were obtained for a smaller group of patients followed through the 1974 ragweed season who received a mean dose of 31.2 microng antigen E (11,140 PNU). The failure of these patients to show a response to immunotherapy could be due to a combination of the relatively low dose of ragweed extract and their sensitivity to other allergens.

Inhalation challenge with ragweed pollen in ragweed-sensitive asthmatics

We reexamined the ability of inhaled ragweed pollen to induce bronchoconstriction in ragweed-sensitive asthmatic patients using a turbo-inhaler to administer pollen quantitatively. Adult subjects were selected for study on the basis of fall season asthmatic attacks, positive skin test, histamine release, RAST, and bronchial challenge responses to ragweed extract. Not one of 12 such subjects had any bronchial response to oral inhalation of whole pollen grains even when the dose was increased to 7640 pollen grains (more than the estimated maximum daily exposure in season), whereas nasal challenge by the same method produced brisk hay fever responses without bronchospasm. On the other hand, when the pollen was ground to fragments with a size range of 1 to 8 micrometers, oral inhalation produced a 35% fall in airways conductance in six of seven subjects in doses ranging from 59 to 20,000 pollen grain equivalents. Atropine pretreatment did not modify the response to pollen fragments, making an irritant response unlikely. These data, coupled with earlier observations that no more than a few pollen grains penetrate further than the larynx, raise further questions about the role of whole ragweed pollen in fall asthma in allergic patients. In addition, ragweed-allergic asthmatics appear not to have their symptoms at the time of maximum pollen load in the air. We believe that small-particle allergens other than ragweed pollen should be considered in most cases of fall seasonal asthma.

Asthma Improved by Nonsteroidal Anti-Inflammatory Drugs

A patient who claimed benefit from aspirin for her reversible bronchospasm was challenged orally in a placebo-controlled study with aspirin and other aspirin-like drugs. Specific airways conductance and spirometry were monitored for up to 150 minutes after oral challenge. Aspirin, mefenamic acid, and ibuprofen administration resulted in marked (45% to 80%) improvement in forced expiratory volume in 1 second (FEV1) compared to lactose placebo. Indomethacin, sodium salicylate, and tartrazine resulted in modest (15% to 25%) FEV1 improvement, while phenylbutazone produced a 25% decrease. These results are discussed here in terms of the ability of these drugs to inhibit the prostaglandin synthetase enzyme system. This case suggests that aspirin and other nonsteroidal anti-inflammatory drugs may be beneficial rather than harmful in some asthmatic patients.

The effect of vitamin C on antigen-induced bronchospasm

The effect of vitamin C pretreatment in preventing ragweed-induced bronchospasm was evaluated in 6 ragweed-sensitive asthmatics studied in a double-blind randomized fashion. The patients received either lactose capsules or 500 mg of ascorbic acid and were studied out of season. Antigen dose-response curves were determined prior to the administration of lactose or ascorbic acid in each individual subject and subsequently after administration of ascorbic acid or lactose. Bothe PD20FEV1 (provocation dose necessary for a 20% reduction in forced expiratory volume in 1 second) and PD35SGaw (provocation dose necessary for a 35% reduction in specific airways conductance) were determined. In none of the six patients was there a change in baseline FEV 1 (p greater than 0.70) nor the overall average baseline specific airways conductance (rho greater than 0.90). Additionally, no statistically significant difference (p greater than 0.60) was noted between log PD35SGaw vitamin C day and lactose day. Likewise, no statistically significant difference (p greater than 0.60) was evident when comparing log PD20FEV1 lactose and ascorbic acid days. Vitamin C (500 MG) HAS NO PROTECTIVE EFFECT AGAINST RAGWEED ANTIGEN-INDUCED BRONCHOSPASM.

The emerging role of bronchoprovocation

Bronchoprovocation may be defined as the administration of a stimulus in susceptible individuals, followed by measurements of the resulting bronchospasm. Inhalation challenge is a method of testing for bronchial reactivity following inhalation of specific antigen to which the patient may be sensitive or, alternatively, following more “nonspecific” pharmacologic agents such as histamine, methacholine, and carbacholine. The method of inhalation challenge has been refined in our laboratory to a semiquantitative technique, enabling us to identify dose-response relationships between inhaled agonists and selected pulmonary function parameters. Central to the technology was the development of the “Nebulization Dosimeter,” an automatic air metering device that delivers consistent and reproducible amounts of aerosolized material from a standard nebulizer (Fig. 1). The instrument consists of an electronic variable timing circuit that is used to open a solenoid valve. The timing valve opening is automatically initiated by patient inspiration or, alternatively, by an operator-controlled electronic trigger. The open valve allows air to flow at 20 psi from a compressed air tank to the nebulizer for an interval that is determined by the selected setting of the timing circuit. Once activated, the device delivers aerosol for the predetermined length of time, helping to ensure that the inhalation will contain a consistent amount of aerosol. An interval of 0.6 set was emperitally determined to be comfortable for adult patients to inhale to total lung capacity from functional residual capacity and so was selected. Since nebulization occurs during most of inhalation, the dispersion

Simplified eucapnic voluntary hyperventilation challenge

A simplified scheme for eucapnic voluntary hyperventilation ( EVH ) is described that requires only that a source of dry gas containing 5% CO2, 21% O2, and 74% N2 be hyperventilated at 40 L/min or greater. Refrigeration or measurement of end tidal CO2 is not required. When the ventilation pattern of a standardized treadmill exercise challenge in a group of exercise-sensitive asthmatics breathing dry air was matched according to this EVH procedure, the magnitude of the resultant bronchospasm and the exclusive nature of the postchallenge refractory period were similar, since patients in both cases were still fully responsive to methacholine. It is concluded that such an EVH challenge may be used to reproduce the bronchial heat and water fluxes that occur with exercise challenge as well as the consequential challenge-specific refractoriness that follows.

215 The effect of inspiratory flow rate regulation on the human airways response to methacholine aerosol

VARIOUS STORAGE CONDITIONS. J.M. Rosenfeld, M.D., E.F. Juniper, M.C.S.P. and F.E. Harweave, M.D., Hamilton, Ontario, Canada. Acetyl-p-methyl choline chloride (methacholine) in aqueous solution is extensively used to measure nonallergic bronchial responsiveness. When methacholine, a highly deliquescent powder, is dissolved in aqueous solution (water and 0.9% saline) the acetyl radical is hydrolyzed to acetic acid. We examined the effects of concentration of solution, temperature and diluent on the rate of degradation. Fifty ml aliquots of solutions (16 mg/ml, 2 mg/ml and 0.125 mg/ml) were made up both in normal saline and phosphate buffered saline and stored at 250C and 4oC. Concentrations of methacholine were determined using gas chromatography. The benzyl alcohol, included as a preservative in the diluent, was used as an internal standard. We have shown that all concentrations of methacholine used for measurement of bronchial responsiveness are stable for at least 2 months when made up in saline and phosphate buffered saline and when stored at 25% and 4oC.

Standards for the conduct of clinical trials and evaluation of bronchodilator drugs

The proper administration of clinical drug trials requires a highly integrated effort among the investigator, the sponsor (usually a pharmaceutical concern), and most often an institution such as a medical school or hospital. The current climate, moreover, has favored the breeding of new regulations and procedures by government, institutions, and the pharmaceutical industry. Factors influencing these new concerns are consumer awareness, the need for peer review, increasing liability risks, and the desire to help ensure patient safeguards as well as validity of data. It is encumbent upon the clinical investigator to be aware of his responsibilities in this regard so that trials may be conducted in accordance with contemporary standards. The Food and Drug Administration (FDA) has published in the