Inhibition of cutaneous and pkt to platelet-activating factor by orai in man James P. Hayes, MRCP, Susan M. Ridge, SRN, Susan Griffith, MRCGP,* Peter J. Barnes, DM, and Kian F. Chung, MD London and Bracknell, England WEB 2086, an antagonist of platelet-activating factor (PAF), was assessed against the cutaneous response in vivo and the aggregation of platelets ex vivo induced by PAF in a double-blind, placebo-controlled, crossover study in 12 normal male volunteers. Wheal-and-flare responses to intradermal PAF (200 ng) and histamine (1 t.~g)were measured 1 hour after placebo or control administration. Wheal volumes were signtjicantly reduced by 66% z? 8.7% (mean 2 SEM), 86% h 8.3%, and 90% i 6.0% after 10, 40, and 100 mg of WEB 2086 compared to placebo administration (p < 0.005). Flare responses were correspondingly inhibited by 66% ? 8.3%, 82% 2 8.7%, and 84% 5 7.8% at 10, 40, ami 100 mg of WEB 2086, respectively. Cutaneous responses to histamine were utuzffected. Ex viva platelet responses to PAF were significantly inhibited at all three doses of WEB 2086. Oral WEB 2086 is a potent and specijc inhibitor of cutaneous and platelet responses to PAF in man, (J ALLERGYCLINIMMUNOL 1991;88:83-8.) Key words: PAF, PAF antagonist, WEB 2086, histamine

PAF is an ether-linked phospholipid that has been implicated in pathophysiologic states, including asthma, allergic inflammation, and anaphylactic shock.’ Recently, several natural and synthetic compounds have been developed as PAF receptor antagonists and have beenusedin animal modelsof disease to test for the involvement and role of PAF in various pathophysiologic conditions.‘. 3 Only a few PAF antagonists have been tested in man, such as the gingkolide mixture, BN 52063,4andthe synthetic analogue of PAF, CV 3988.5 A recently developed synthetic antagonist of PAF resembling the benzodiazepines, WEB 2086,‘j is potent in inhibiting many of the activities of PAF in vitro and in laboratory animals and is also devoid of any sedativeactions.‘-” Theseeffects include inhibition of platelet aggregation, broncho-

From the Departmentof Thoracic Medicine, National Heart & Lung Institute, Royal Brompton and National Heart Hospital, London, and *Boehringer Ingelheim, Bracknell, England. Supportedby Boehringer Ingelheim, Bracknell, England. Received for publication Nov. 7, 1990. Revised Feb. 21, 1991. Accepted for publication March 12, 1991. Reprint requests:K. F. Chung,MD, Departmentof Thoracic Medicine, National Heart & Lung Institute, DovehouseSt., London SW3 6LY, England. 111129408

Abbreviations used

PAF: PRP: PPP: ADP: IC,,:

Platelet-activating factor Platelet-rich plasma Platelet-poor plasma Adenosine diphosphate Serum concentration of drug required for 50% inhibition

constriction, and influx and activation of eosinophils. To test the activity of WEB 2086 as a PAF antagonist in man, we conducted a double-blind, placebocontrolled, crossover study to examine the inhibitory effectsof WEB 2086 on the wheal-and-flareresponses to intradermal PAF and on platelet aggregation induced by PAF ex vivo. On the basis of previous studies,” we evaluatedthe effects of three oral dosesof WEB 2086, 10, 40, and 100 mg that we determined elicited a dose-relatedinhibition of platelet aggregation to PAF ex vivo. MATERIAL AND METHODS Subjects Twelve healthy nonatopic male volunteers (aged, 22 to 42 years) gave written informed consent to participate in this study, approvedby the Ethics Committee of the Royal 83

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Bromptonand National Heart Hospital. They abstainedfrom caffeine-containing beveragesfor at least 8 hours before attending the laboratory. Protocol Each subject attendedthe laboratory on five separateoccasions, each separatedby at least 1 week. The first visit was a screening visit, followed by entry to the study. The order of the four treatments(placebo or WEB 2086 at 10, 40, or 100 mg) was administered according to three Latin squaresbalancedfor order effects. At 1 hour after the ingestion of placebo or active tablet, blood (30 ml) was obtained from the antecubital fossafor routine hematology,biochemistry, platelet aggregation,and analysisof WEB 2086 levels. Skin provocation to PAF and histamine was then performed. Pulse and blood pressurewere monitored before and after ingestion of tablets. Skin studies Four sites for intradermal injections were chosen on the flexor surfaceof both forearms, two sites on each forearm. One hour after ingestion of the capsule, the subject was administered intradermal injections of 50 p,l each of four solutions with a 27-gaugeneedle. Thesesolutions contained PAF (200 ng), the vehicle for PAF containing 18% ethanol in 150 mmol/L of NaCl, histamine (1 pg), and 150 mmol/L of NaCl (the diluent for histamine). Solutions were coded for the investigator who performed the injection and measuredthe cutaneousresponses.The injections were randomized according to a Latin squaredesign to ensure that no injection was on the samesite on consecutivevisits and that sites were balanced between subjects and treatments. The flare area was measuredat 5 minutes. Skin fold thicknessat the site of injection before and 15 minutes after the injection was measuredfrom the central portion of the wheal with a low-tension, spring-loaded, thickness gauge (Mitutoyo Manufacturing Co., Ltd., Tokyo, Japan). Wheal area wasmeasuredat 15minutes. The wheal-and-flareareaswere calculated by planimetry by meansof an IBM compatible computer(Dell, Inc., Ltd., Berkshire, England) with a Sigmascanprogram (Jandel Scientific, Corte Madera, Calif.). The wheal volume was the primary end point of the study and was calculated according to the formula: (0.5) X (wheal area) x (increase in skin fold thickness). The correction factor of 0.5 was used to allow for the skin folding on itself. Platelet

aggregation

Approximately 15 ml of the blood samplecollected was mixed with 3.28% of trisodium citrate (vol/vol 9: 1) and was transferredinto plastic tubesin 1.5 ml aliquots and spun on a bench Eppendorf centrifuge (Hamburg, Germany) (model 1914; maximal centrifugal force, 8200 g) for time periods of 2 secondsto obtain PRP. PPP was obtained by centrifugation of 3 ml of anticoagulatedblood for 5 minutes on the Eppendorf centrifuge. Platelet aggregation was determined by measuring the light transmissionthrough PRP

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with a dual channelPaytonaggregometer(Centronics, Croydon, England) with the PPP sample used to define 100% light transmission. PRP (450 ~1) was placed in siliconized glasscuvettes containing nickel, stirring bars, and incubated at 37” C for 1 minute. PAF (10 pmol to 10 ~1) or ADP (10 pl) was added, and light transmission was recorded. Aggregationof plateletsby PAF was expressedin arbitary units as a percentageof the aggregationinduced by ADP in the samesampleof PRP. Serum WEB 2086 A heparinized blood sample (8 ml) was centrifuged at for 20 minutes and stored in aliquots of 3 ml at -20” C. Serum levels of WEB 2086 were assayedwith an RIA technique basedon competition for binding sites to a specific antibody betweenWEB 2086 in a heparinizedblood sample and ‘H-labeled WEB 2086. Nonbound WEB 2086 was separatedfrom the bound WEB 2086 by C-dextran absorption. The radioactivity of the blood products (supernatants)was measuredon a scintillation counter. The count was inversely proportional to the concentration of WEB 2086 in reference samples. With this method, the crossreactivity with known metabolites of WEB 2086 is negligible. Twelve calibration samples (all in triplicate) with concentrationsof the active ingredient from 0.02 mg/ml to 200 rig/ml were used for obtaining the calibration curve. 3000 rpm

Chemicals &,-PAF was obtained from Bachem, Bubendorf, Switzerland, and stock solutions of 1 mg/ml were made in absolute ethanol and stored at -70” C. On the study day, this stock was diluted in 150 mmol/L saline containing 0.25% bovine serumalbumin (Sigma Chemical Co., Poole, Dorset, England) for ex vivo studies and in saline only for cutaneous studies. Histamine diphosphate was prepared fresh in 150mmol/L of NaCl. ADP (Sigma Chemical Co. ,) was prepared in sterile water for ex vivo platelet studies. WEB 2086 capsules,including matchedplacebos,were provided by Boehringer Ingelheim, BracknelI, England. Statistical

analysis

All values are listed as mean and SEM. Wilcoxon’s matched-pairs, signed-rank test was used to compare the effects of each treatment with placebo on wheal-and-flare responsesto intradermal PAF and histamine. A p value of 0.02) in the wheal-and-flare responsesin ducedby 1 wg of intradermal histamine on the activetreatment days compared to the placebo-treatment day.

WEB 2086 inhibited platelet aggregationat all three dosesused in this study (Fig. 2). There was no statistical difference among the three groups, and even the lowest dose of WEB 2086 was effective against the highest concentration of PAF (lo-’ mol/L) used. Serum levels of WEB 2086 increasedwith increasing oral dosing. The mean serum level of WEB 2086 for 10, 40, and 100 mg doses was 83.2 rig/ml f 13.1 rig/ml (mean + SEM), 356.7 rig/ml, and 1138.0 rig/ml 2 70.1 rig/ml, respectively (Fig. 3). There wasno significant correlation betweenthe serumWEB 2086 levels and the inhibition of either the wheal or flare response. DISCUSSION We have demonstratedthat oral WEB 2086 is an effective antagonistto the effects of PAF on skin and on circulating platelets. There was no significant difference in the efficacy of the three dosesused in this study; however, total inhibition of the wheal-and-flare responsesinduced by PAF was more likely to occur with WEB 2086 at doses of 40 and 100 mg. WEB 2086 had no effect on the histamine-induced whealand-flare responses,which suggeststhat this antagonist is specific againstthe effectsof PAF. Serumlevels of WEB 2086 increasedwith increasingdosesof WEB 2086, with a mean 13-fold difference between the mean serum level at the small dose versus the large dose. The lack of correlation betweenthe serumWEB

VOLUME NUMBER

Oral WEB 2086 inhibits

88 1

3

lOOO-

P 8

800'

m 5

600'

49

400

responses

to PAF

87

200

0

Dose of WEB 2086 (mg) FIG. 3. Serum levels of WEB 2086 (10,40, and 100 mg) measured dose.

2086 levels and the cutaneousresponsemay be explained by the fact that no further inhibition was obtained at the doses of WEB 2086 >40 mg. Similar results were obtained when inhibition of platelet aggregation ex vivo was examined, with all three doses of WEB 2086 used being very effective, confirming the results of a previous study.” Recently, Adamus et al.‘* have demonstratedthe effectivenessof an oral dose of 40 mg of WEB 2086 in inhibiting bronchoconstriction induced by inhalation of PAF. Comparedto other PAF antagoniststhat have been assessedin vivo in rnaq4, ’ WEB 2086 appearsthe most potent. Thus, in a previous study of the effectiveness of the oral gingkolide mixture, BN 52063, we found only a mean60% inhibition of PAF-induced wheal responsesby the 120 mg (2 mmol/L) dose. Even the smallest doseof WEB 2086 (0.02 mmol/L) used in the present study caused a greater degree of inhibition. In addition, ex vivo platelet aggregation induced by PAF was only partially inhibited by the oral BN 52063 (2 mol I L) . Thesein vivo observations are in agreementwith in vitro studies in which WEB 2086 has been demonstratedto be more potent than BN 52063, such as their effects against platelet aggregation in vitro.4. 7 Thus, the IC, against lOA mol/L of PAF for WEB 2086 was 117 nmol/ L and against lo-* mol/L of PAF for BN 52063, 2760

1 hour after ingestion

of each

nmol/L. The serum concentration after 10 mg WEB 2086 was 182 nmol / L, and this caused approximately 85% inhibition of platelet aggregationcausedby PAF (lo-’ mol/L) ex vivo. This inhibiting concentration of WEB 2086 correspondsclosely to the IC,Owe previously found for WEB 2086 of 117nmol/L. Because the oral doseswe chosein the current study were very effective, it is not possible to obtain an I& from serum WEB 2086 concentrations. In the study of Arnout et al.,5 plasma levels of CV 3988 during intravenous infusion reached approximately 8 kmol/L, causing an approximatedoubling of the threshold concentration of PAF for platelet aggregation ex vivo. This finding would suggestthat WEB 2086 is a more potent PAF antagonist than BN 52063 and CV 3988 in man. Although we have not studiedits duration of action, other studies have demonstrateda duration of effect of up to 8 hours against in vivo platelet aggregation for the 40 mg dosage.” Our studies demonstratethat a 40 mg dose is probably the best dose to use for a maximum PAF antagonist effect, at least in the skin and on platelet function, at a dose devoid of measurable side effects. The decreasein blood pressure at 100 mg is of interest, and whether this is a PAFmediatedeffect or other nonspecific effect is not clear. This finding is a paradoxical observationbecausePAF

88 Hayes et al.

itself is potentially a hypotensive agent. This hypotensive effect of WEB 2086 at large doses needsto be further investigated. Other characteristics of the pharmacokinetics obtained from other studies demonstrate that WEB 2086 is rapidly absorbedfrom the gastrointestinal tract, reaching maximal plasma levels within 1 to 2 hours in man. It is also 60% protein bound, and its plasma half-life is 5 to 6 hours with linear pharmacokinetics.* In summary, our results indicate that WEB 2086 is the most potent PAF receptor antagonist that has so far beentestedin man. An oral doseof 40 mg is likely to be ideal, perhapstaken at a twice daily dose. This compound can therefore be used to investigate the role of PAF in the various diseasesin which it has been postulated to play an important role, such as allergic inflammation, anaphylactic shock, and asthma.In addition, WEB 2086 may prove to be therapeutically effective in certain diseasestates.’ *Boehringer Ingelheim Pharmacokineticsof WEB 2086; 1987data on file.

REFERENCES 1. Barnes PJ, Chung KF, Page CP. Platelet-activating factor as a mediator of allergic disease[Review]. J ALLERGY CLIN IMMUNOL 1988;81:919-34. 2. Hosford D, Page CP, Barnes PJ, Braquet P. PAF-receptorantagonists. In: Barnes PJ, Page CP, HensonPM, eds. Plateletactivating factor and human disease, Oxford: Blackwell, 1989:82-l16.

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3. Chung KF, Barnes PJ. PAF antagonists:their potential role in asthma. Drugs 1988;35:93-101. 4. Chung KF, McCusker M, PageCP, Dent G, Guinot P, Barnes PJ. Effect of a gingkolide mixture (BN 52063) in antagonising skin and platelet-activating factor in man. Lancet 1987;1:24851. 5. Amout J, Hecken AV, Delpelaire I, Miyamoto Y, et al. Effectiveness and tolerability of CV 6988, a selective PAF antagonist, after intravenous administration in man. Br J Clin Pharmacol 1988;25:445-52. 6. Casals-StenzelJ, Weber KH. Triazolodiazepines:dissociation of their PAF (platelet-activating factor) antagonistic and CNS activity. Br J Pharmacol 1987;90:139-46. I. Evans TW, Dent G, Rogers DF, Aursudkij B, Chung KF, BarnesPJ. Effect of the PAF antagonist,WEB 2086, on airway microvascular leakage in the guinea pig and platelet aggregation in man. Br J Pharmacol 1988;94:164-8. 8. Casals-StenzelJ, Muacevic Cl, Weber GH. Pharmacological actions of WEB 2086, a new specific antagonist of plateletactivating factor. J PharmacolExp Ther 1987;241:974-81. 9. Leilouch-TubianaA, Lefort J, Simon MT, Ptister A, Vargaftig BB. Eosinophil recruitment into guinea pig lungs after PAF acether and allergen administration: modulation by prostaglandin, platelet depletion, and selective antagonists.Am Rev Respir Dis 1988;137:948-54. 10. Kroegel C, Yukama T, Dent G, Chanez P, Chung KF, Barnes PJ. Platelet-activating factor induceseosinophil peroxidaserelease from peripheral human eosinophils. Immunology 1988;64:559-62. 11. Adamus WS, Heuer H, Meade CJ, Frey G, Brecht HM. Inhibitory effect of oral WEB 2086, a novel selectivePAF-acether antagonist on ex vivo platelet aggregation. Eur J Clin Pharmacol 1988;35:237-40. 12. Adamus WS, Heuer HO, Meade CJ, Schilling JC. Inhibitory effects of the new PAF acetherantagonistWEB2086 on pharmacologicchangesinducedby PAF inhalation in humanbeings. Clin PharmacolTher 1990;47:456-62.