New aspects of the therapeutic potential of theophylline in asthma

ew aspects of the therapeutic eophylline in asthma main A. Pauwels, MD Ghent, Belgium In addition to its bronchodilating activity, the antiasthmatic drug theophylline also reduces microvascular permeability and increases mucociliary clearance, diaphragmatic contractility, and circulating catecholamine levels. Both experimental and clinical data now suggest that at therapeutic serum levels, theophylline is also of value during the delayed asthmatic reaction by inhibiting airway injammation and the associated bronchial responsiveness. In vitro, high theophylline concentrations inhibit histamine release from mast cells and basophils. This drug also suppresses polymorphonuclear leukocyte activation and stabilizes a variety of injammatory cells at therapeutically relevant concentrations. (J ALLERGY CLINIMMUNOL1989;83:548-53.)

Theophylline hasdifferent pharmacologicactivities that may contribute to its therapeutic value in the managementof asthma.lABesidesits bronchodilating activity, theophylllneenhancesmucociliary clearance, diminishes the microvascularpermeability in the airway mucosa, increasesthe level of circulating catecholamines, and enhancesthe contractility of the diaphragm.The purposeof this article is to demonstrate that at therapeuticserum concentrationstheophylline inhibits the developmentof airway inflammation. THEOPHYLLINE INHIBITS BOTH IMMEDIATE AND LATE ASTHMATIC REACTIONS AFTER ALLERGEN CHALLENGE At least two studies have now demonstratedthat theophylline inhibits the late asthmaticreaction after bronchial challenge with either an allergen or an occupational agent. The effect of theophylline on allergen-induced bronchoconstriction had not been extensively studied until quite recently and only the effect on the immediate bronchoconstriction had been investigated. We studied the effect of theophylline and enprofylline on the allergen-induced immediate and late bronchoconstrictionin nine asthmatic patients,5Enprofylline is a xanthine derivative, 3-propylxanthine,that has negligible ability to antagonize adenosine,and thereforethe resultsof our study may be relevantto the modeof action of theophylline. The patientswere challengedthree times at weekly intervals with the same dose of allergen. This dose had previously been chosenas causing an immediate bronchoconstrictionwith a decreasein forced expiFrom the Department of Respiratory Diseases, University Hospital, Ghent, Belgium. Reprint requests: Romain Pauwels, MD, University Hospital De Pintelaan, Number 185, B9000 Ghent, Belgium.

ynarmc compliance Forced expiratory volume in 1 second

5HT: 5-Hydroxytryptamine R,: Total lung resistance TDI: Toluenediisocyanate

ratory volume in 1 second(FEV,) between20% and 50% of the prechallengevalue. On the 3 days FEV, and specific airway conductancewere followed as long as 6 hours after challenge.The drugswere given intravenously.Placebowasgiven on the first occasion. Theophylline and enprofylline were administe~cdon test days 2 and 3 according to a double-blind, randomized, cross-overprotocol. One hour before the allergen challenge, a loading dose was given during 60 minutes, followed by a constantinfusion over 6 hours. The loading infusion was 7.2 mglkg of theophylline and 2.7 mgikg of enprofylline. The maintenancedoseswere 74 and 71 mgihr, respectively. Both theophylline andenprofyllline caused a minor initial bronchodilation.Theophylline ande~p~o~y~~~~e slightly but significantly inhibited the immediatebronchoconstrictivereactionafter allergeninhalation. Both drugs had a significant inhibitory effect on the late reaction (Fig. 1). The mean serumco~ce~tra~io~s of theophylline were 0, 10.8, 10.5, and 10.5 mg/L at 0, 1, 4, and 7 hours, respectively, after the start of the loading infusion. The correspondingmean serum concentrationsof enprofylline were 0. 2.6, 2.7, and 2.7 mg/L. Thus our data showedthat at plasmalevels considered therapeutic, theophylline inhibits both the immediateand late bronchoconstrictivereactionafter al-

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FIG. 1. Relative changes in FEV, after bronchial allergen challenge in six patients who developed a significant late reaction on the placebo (p/ treatment day. The effect of a continuous infusion with either’theophylline (T) or enprofylline (E) was investigated on two separate days in a singleblind, randomized study. (From Pauwels R, Van Renterghem D, Van Der Straeten M, Johannessen N, Persson CGA. J ALLERGY CLIN IMMUNOL 1985;76:583.)

lergen challenge.The protective effect was most pronouncedduring the late asthmaticreaction, suggesting that mechanismsother than bronchodilation are involved in the protective activity againstthe allergeninduced late asthmaticreaction. THEOPHYLLINE DURING TOLUENE DIISOCYANATE CHALLENGE Mapp et a1.6investigatedthe effect of different antiasthmaticdrugs on the immediateand late bronchoconstrictive reaction after challenge with toluene diisocyanate(TDI) in sensitiveasthmaticpatients. The drugs were administeredfor 1 week before challenge. The last dose was administereda short time before the TDI exposure.Theophylline was administeredas a dose of 6.5 mg/kg of a slow-releasepreparation twice daily. The last dose of 10 mg/kg was taken 2 hoursbeforechallenge.The bronchialresponseto TDI was followed, and the bronchial responsivenessto methacholinewas studiedbefore and 8 hoursafter the TDI challenge. Theophylline significantly inhibited the late reaction after TDI challengebut did not have a significant effect on the increasein methacholine responsiveness.The meantheophylline concentration in the six patients was 18 mg / L before and 20 mg/ L 8 hours after the TDI challenge. Animal studies are in accordancewith human ob-

servations. In a guinea pig model of the allergeninduced bronchial reactions, Anderssonet al.7 demonstratedthat the intravenousinjection of a low dose of theophylline immediately before the antigen challenge significantly inhibited the immediate bronchoconstrictionand the late reaction. The late reactionin the guinea pig is characterizedby the influx of neutrophils, eosinophils, and lymphocytes. pretreatment with theophylline significantly reduced the neutrophilic airway inflammation. The intravenousinjection of theophylline 90 minutes after the antigen~ind~ced immediatebronchoconstrictionalso had an inhibitor effect on the late reaction. Similar findings were obtained in an allergic sheepmodel8 Theo~~ylii~~ at a serum concentration of 10 mg/L inhibited the late reaction after allergenchallengewhen given bot fore and after the allergen challenge. THE LATE ASTHMATIC REACTION The late asthmaticreaction is characterizedby the associationbetween a slowly developing and poorly reversible airways obstruction and an inflammato~ reaction in the airways.’ The airway inflammatory reaction during the late reaction is ch~acteri~ed by an influx of eosinophils and neutrophils, depending on the type of stimulus that causesthe reactionl’, ‘I The late reactionis equally associatedwith an increase

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FIG. 2. The effect of different antiasthmatic drugs on the endotoxininduced airway hyperresponsiveness, expressed as PC,& and PC,,-Cdyn for 5HT, and airway inflammation, expressed as percentage of neutrophils in the bronchoalveolar lavage fluid. The results are expressed as mean k SE. As&risk indicates significant (p < 0.05) difference compared with saline pretreatment in similarly exposed animals. S, saline exposed; E, endotoxin exposed; KEIO, ketotifen; THEO, theophylline; M-PRED, methylprednisolone; SCG, sodium cromogiycate; TERB, terbutaline.

in the bronchial responsiveness to bronchoconstrictory agents such as histamine and methacholine.‘*x l3 The association of airway inflammation and increased bronchial responsiveness has been considered as evidence for the hypothesis that airway inflammation may increase airway responsiveness.‘4

THEOPHYLLINE’S PROTECTIVE EFFECT ON THE LATE ASTHMATIC REACTION: The observation that theophylline, at therapeutic serum concentrations, had a more pronounced protective effect on the late asthmatic reaction after allergen challenge than on the immediate reaction suggested that theophylline may have an inhibitory effect on airway inflammation. I5 Therefore we have initiated studies on the effect of antiasthmatic drugs on the airway inflammation and increase in bronchial responsiveness after endotoxin exposure in the rat model that we have developed.16% I7 The animals were injected intraperitoneally, 15 minutes before the start of the endotoxin exposure, with the following solutions in a volume of 0.5 ml: saline solution; ketotifen, 0.1 mg /kg; ketotifen, 0.3 mg/kg; ketotifen, 1 mg/kg; aminophylline, 15 mg / kg; sodium cromoglycate, 1 mg / kg; terbutaline, 0.05 mgl kg; or methylprednisolone, 5 mg/kg. Each

treatment was given to a control (saline)- and an endotoxin-exposed group of rats. Each group consisted of nine animals. The animals were placed in a plastic box that could contain eight to 10 rats at a time. The experiment started in the morning around 8:30 AM, and the animals were left in the box for approximately half an hour for adapta~Gn purposes. After this, the animals were pretreated by the intraperitoneal injection and left in the box for another 15 minutes. Subsequently, the rats were exposed to an aerosol of either an endotoxin or a control solution during 30 minutes. The endotoxin solution contained 100 p,g of lipopolysaccharide/ml of saline solution. Saline was used as the control solution. The aerosol was duced with an ultrasonic nebulizer (~~crov~~eb~cr, Zurich, Switzerland) that generates particles with a diameter between 0:5 and 5 km and a mean diameter of 3.5 pm according to the specifications of the manufacturer. The output of the nebulizer was 0.5 ml/min, and the outlet of the aerosol was lead into the plastic box where the awake asaimds were running freely. 0ne and a half hours after the end of the aerosol exposure, bronchial responsiveness to Shydroxytryptamine (SHT) was assessedand bronchoalveolar lavage was performed. The animals were anesthetized, intubated, and ar-

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tificially ventilated. The bronchial responsivenessto 5HT was measuredby intravenousadministration of increasingquantities of 5HT. Increasingquantitiesof 5HT were injected in thejugular vein as a bolus every 5 minutes until an increase in total lung resistance (R3 of at least 50% was observed. The following increasingconcentrationsof 5HT were used: 0.5, 1, 2.5, 5: and 10 kg per kg of body weight. Bronchoalveolarlavagewas performed after determining bronchial responsiveness.The rats were killed and bled by transsectionof the abdominal aorta. The airways were lavaged via the tracheal cannula with the use of five times 4 ml of Hanks’ balanced salt solution free of ionized calcium and magnesiumand supplementedwith 0.05 mmol/L sodium ethylenediamine ten-a-aceticacid. The lavage was performed with plastic syringes (5 ml), and the instillation and suction were done manually. The lavage fluid was centrifuged at 1000 r-pm, and the cells contained in the fluid were washed twice in calcium- and magnesium-freeHanks’ solution. After the final centrifugation, the cells were taken up in 1 ml of Hanks’ solution and counted manually in a Burker chamber. Finally, a cytocentrifuge preparation was made, the smearwas colored with a May-Grtinwald staining solution, and the differential count was madeon at least 1000 ceils. Endotoxin exposureinduced a significant increase in the bronchial responsivenessto 5HT in the control group (saline treatment intraperitoneally) (Fig. 2). Both the PC&-dynamiccompliance (Cdyn) (concentration that decreasedCdyn by 25%) and the PC&-R, (concentrationthat increasedRL by 50%) decreased significantly. Endotoxin causeda significant decrease in the percentageof macrophagesand a significant increasein the percentageof neutrophilsin the pooled bronchoalveolarlavage fluid. Endotoxin had no significant influence on the basal Cdyn and RL before challengewith 5HT. Pretreatmentwith aminophylline causeda significant increasein the PC,,-Cdyn after endotoxin exposurein comparisonwith the sameparameter after saline pretreatmentfollowed by endotoxin exposure. The same treatment also showed a significant influence on the cellular compositionof the bronchoalveolar fluid after endotoxin exposure. After aminophylline treatment there were significantly more macrophagesand less neutrophils than in the salinetreated, endotoxin-exposedgroup. Essentially, the samefindings were observedafter treatmentwith sodium cromoglycate.Terbutalinesignificantly inhibited the endotoxin-inducedincreasein airway responsiveness but had no significant effect on the neutrophil influx. Methylprednisolone pretreatment was followed by a significantly higher PC,,-Cdyn in the

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endotoxin-exposedgroup comparedwith pretreated,endotoxin-exposedgroup. ylprednisolonepretreatmentdid not causea sig~~~ca~t changein the cellular compositian of the b~~Rcb5~veolar fluid. Ketotifen pretreatmenthad no s~g~~fica~t influence on the increasein bronchial responsiveness or changesin cellular composition of the bronchoalveolar lavage fluid after endotoxin exposure. Therefore this experiment showed that theophylline, sodium cromoglycate,rne~y~pred~isolo~e~ and terbutalineadministeredintraperitoneallyhalf an hour before the exposure of inbred rats to an aerosol of endotoxinpartially inhibited the increasein bronchial responsivenessto 5HT. Theophylline, sodium moglycate,and terbutalinealso partially inhib~te neutrophilic infiltration of the airways after end~toxln exposure.At the dosesandtiming used,ketotifen had no significant protective effect. DOSE-RESPONSE STUDY WITH THEOPHYLLINE After this initial observation on the effect of theophylline on airway inflammation and responsiveness,we have now performe responsestudy with theophylline in the sameanimal model.” This time inbred F344 rats were used. Zncreasing doses of aminophylline were administered intraperitoneallyin different groupsof rats !5 minutes before the start of the saline or endotoxin expo Saline solution was used as control treatment. group consistedof nine animals. Bronchial responsivenessto 5MT and bronchoalveolar lavagewere performed 1Yzhours after the end of the aerosol exposurewith the samemethod as in the previousexperiment.At the samemoment, blood was taken for the analysis of the theophylline serum concentration.Pretreatmentwith theo~hylli~edid not modify the basal RL or Cdyn. Endotoxin exposure significantly increasedbronchial 5HT responsiveness and induced a neutrophil influx in the airways. The total number of cells in the lavage fluid did not increasesignificantly. Pretreatmentwith ~eophylli~~ in increasingdoseshad no significant influence on the bronchial responsivenessof the spine-exposed animals and did not modify the cellular composition of the bronchoalveolar lavage fluid in these animals. However,pretreatmentwith theophylline sig~~~ca~~y inhibited the endotoxin-inducedincreasein bronchial responsivenessand did so at serum concentrations consideredtherapeutic. Theophylline treatment also significantly inhibited the influx of ne~~op~i~§in the airways even at rather low serum concentrations. Therefore these experimentssuggestthat tbeophylline may exert part of its therapeuticactivity by the inhibition of airway inflammation and the associated

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increasein bronchial responsiveness.Another in vivo animal experiment demonstratinga similar mode of action of theophylline has been performed by Page et al. l9 In the guinea pig, they investigatedthe influence of various antiasthmaticdrugs on the plateletactivating factor-induced bronchial hyperresponsiveness.A short-terminfusion of platelet-activatingfactor induced a long-lasting and nonselectiveincrease in airway responsivenessin these animals. This induction of hyperresponsiveness was inhibited by theophylline. Nelson et al.” observed that in mice and rats exposed to an aerosol of Gram-negativebacteria, the intraperitoneal administration of aminophylline decreasedthe pulmonary antibacterial defensemechanisms mainly by decreasingthe influx of neutrophils. The dosesof aminophylline used and the serumconcentrationsobtained in their study were higher than the dosesusedin the presentstudy and reachedlevels consideredtoxic. The anti-inflammatory effect of theophylline has been demonstratedin vitro with the use of several inflammatory cell types. Theophyllinehasbeenshown to inhibit histamine releasefrom mast cells and basophils.2’~” However, the concentrationrequired to obtain this inhibitory effect is one to two orders of magnitude above the therapeutic theophylline concentrationsand therefore is of little relevancefor the in vivo situation. At therapeuticconcentrations,thelline inhibits the adenosine-inducedenhanceof mediator release from mast cells, and for this reason, adenosine-receptorantagonismhas been put forward as a possible mode of action of theophylline .23,” Outside mast cells and basophils, theophylline has been found to stabilize or inactivate a variety of inflammatory cells including macrophages,neutrophils, andplatelets,andmay do so at therapeuticallyrelevant concentrations.25-27 Nielson et al.**,2greportedthe effect of theophylline on the generationof toxic oxygen metabolitesby humanpolymorphonuclearleukocytes. At therapeutic concentrations, theophylline significantly inbibited the polymorphonuclearactivationand potentiatedthe inhibitory activity of isoproterenol. A considerableamount of experimentaldata suggests that at therapeutic serum concentrations,theophylline inhibits airway inflammation and the associated increasein bronchial responsiveness.Human studiesthat use the late asthmaticreaction as model indirectly confirm this hypothesisand clearly indicate the need for further studieson this new aspectof an antiasthmaticdrug.

REFERENCES 1. Jenne JW. Physiology and pharmacodynamics of the xanthines. In: Jenne JW, Murphy S, eds. Drug therapy for asthma. New York: Marcel Dekker, 1987;297-334. 2. Mite&o PA, Ogilvie RI. Rational intravenous doses of theophylline. New Engl J Med 1973;289:600-3. 3. Pavia D, Sutton PP, Lopez-Vidriero MD, Agnew JE, Clark SW. Drug effects on mucociliary function. Eur J Respir Dis 1983;64(suppl):304-17. 4. Persson CGA, Ekman M, Erjeflt I. Vascular anti-permeability effects of beta-receptor agonists and theophylline in the lung. Acta Pharmacol Toxic01 1979;44:216-20. 5. Pauwels R, Van Renterghem D, Van Der Straeten M, Johannessen N, Persson CGA. The effect of theophylline and enprofylline on allergen-induced bronchoconstriction. J ALLERGY

CLM IMMUNOL1985;76:583-90. 6. Mapp C, Boschetto P, da1 Veccho L, et al. Protective effect of antiasthma drugs on late asthmatic reactions and increased airway responsiveness induced by toluene diisocyanate in sensitized subjects. Am Rev Respir Dis 1987;136:1403-7. 7. Andersson P, Brange C, Sonmark B, et al. Anti-anaphylactic and antiinflammatory effects of xanthines in the iung. In: Andersson K-E, Persson CGA, eds. Anti-asthma xanthines and adenosine. Amsterdam: Excerpta Medica, 1985:187-92. 8. Perruchoud AP, Yerger L, Abraham W. Differential effects of aminophylline on the early and late antigen-induced bronchial obstruction in allergic sheep. Respiration 1984;46:44. 9. O’Bryne PM, Dolovich J, Hargreave FE. Late asthmatic responses. Am Rev Respir Dis 1987;136:740-5 1. 10. De Monchy JGR, Kaufmann HF, Venge P, et al. Bronchoalveolar eosinophilia during allergen induced late asthmatic reactions. Am Rev Respir Dis 1985;131:373-76. 11. Metzger WJ, Richardson HB, Worden K, Monick M, Hunninghake GW. Bronchoalveolar lavage of allergic asthmatic patients following allergen bronchoprovocation. Chest 1986; 89:47?-83. 12. Cockcroft DW, Ruflin RE, Dolovich J, Hargreave FE. Ahergen induced increase in non-allergic bronchial reactivity. Clin Allergy 1977;7:503-13. 13. Cattier A, Thomson MC, Frith PA, Roberts R, Hargreave FE. Allergen-induced increase in bronchial responsiveness to histamine: relationship to the late asthmatic response and change in airway caiiber. J ALLERGYCL~ IMMUNOL198%;70:170-7. 14. Nadel J. Inflammation and asthma. J ALLERGYCUN IMMUNOL 1984;73:651-3. 15. Pauwels R. The effects of theophylline on airway inhammation. Chest 1987;92:32s-7s. 16. Pauwels R, Peleman R, Van Der Straeten M. Airway inflammation and nonallergic bronchial responsiveness. Eur J Respir Dis 1986;68(suppl):137-62. 17. Pauwels R, Peleman R, Van Der Straeten M. The effect of endotoxin inhalati.on on airway responsiveness and ceil&r influx in rats (submitted for publication). Pauwels- R, Van Der Straeten M. The effect of theophylline on the endotoxin-induced airway inllammation and bronchial hyperresponsiveness (submitted for publication). 19. Page CP, Sanjar S, Alvemini D, Morley J. Intlammatory mediators of asthma. Eur J Respir Dis 19~6;68(su~p~~:l63-~9. 20. Nelson S, Summer WR, Jakab GJ. A~nophyliine-deiced suppression of pulmonary antibacterial defenses. Am Rev Respir Dis 1985;131:923-7. 21. Lichtenstein LM, Margolis S. Histamine release in vitro: Inhibition by catecholamines and methylxanthines. Science 1968:161:902.

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22. Pauwels R, Van Der Straeten M. The anti-allergic effect of theophylline. In: Jonkman JHG, Jenne JW, Simons FER, eds. Sustained release theophylline in the treatment of CRAO. Amsterdam: Excerpta Medica, 1984:9-13. 23. Marquardt DL, Grnber HE, Wasserman SI. Adenosine release from stimulated mast cells. Proc Nat1 Acad Sci USA 1984;81:192-6. 24. Wehon AF, Sin&o BA. Regulatory role of adenosine in antigen-induced histamine release from the lung tissue of actively sensitized guinea pigs. Biochem Pharmacol 1980;29: 108592. 25. Kaliner M, Austen F. Cyclic nucleotides and modulation of effector systems of inflammation. Biochem Pharmacol 1974; 23~763-71.

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26. Bussolino F, Benveniste. J. Pharmacological modulation of platelet-activating factor (PAF) release from rabbit leucocytes. I. Role of CAMP. Immunol 1980;40:367-‘76. 27. Weissman G, Dukor P, Zarier RB. Effect of cyclic AMP on release of lysosomal enzymes from phagocytes. Nature 1971;231:131-3. 28. Nielson CP, Crowley JJ, Cusack BJ, Vestal RE. Therapeutic concentrations of theophylline and enprofylline potentiate catecholamine effects and inhibit leukocyte activation. J ALLERGY CLANIMMUNOL1986;78:660-7. 29. Nielson CP, Crowley JJ, Morgan ME, Vestai RE. Polymorphonuclear leukocyte inhibition by therapeutic concentrations of theophylline is mediated by cyclic-3’,-5’-adenosine monophosphate. Am Rev Respir Dis 1988;137:25-30.