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Pharmacotherapy of airway disease in cystic fibrosis
TIPS -August 1987 [Vol. 83
316 19 Heldman, J. and Lancet, D. (1986) J. Neuwhem. 47,1527-1533 20 Nakamua, T. and Gold, G. H. (1987) Nature 325.4424 21 Trot&, D. and MacLeod, P. (1983) Brain Res. 268.225-237 22 Nagabama, S., Kobatake, Y. and Kurihara. K. (1982) J. Gen. Physiol. 80, 78%
800 23 Taylor, C. W. and Merritt, J. E. (1987) Trends Pharmacol. Sci. 7,238-242 24 Lefkowitz, R. J., Benovic, J. L., Kobilka, B. and Caron, M. G. (1986) Trends Pharmacof. Sci. 7, 444-448 25 Nathans, J., Thomas, D. and Hogness, D. S: (1986) Science 232, 193-202
Pharmacotherapy of airway disease in cystic fibrosis W. E. Waltner, R. C. Boucher, J. T. Gatzy and M. R. Knowles Current therapy for CF airway disease slows, but does not stop, the inexorable of airway damage. Recent research suggests that abnormal regulation of ion transport in CF ainvay epithelial cells leads to excessive Na+ absorption across a relatively Cl--impermeable airway epithelium. This pattern of dysfunction predisposes to dehydration of airway secretions and adverse effects on mucociliay clearance and lung defense. W. E. Waltner et al; aplain the rationale behind potential new approaches to therapy involving
progression
26 Sldar, P. B., Anholt, R. H. and Snyder, S. H. (1986) J. Biol. Chem. 261, 1553815543 27 Stern, J., Kaupp, U. B. and MacLeish, P. R. (1986) Pm. Nutl Acud. Sci. USA 83, 1163-1167 28 Conner, J. and Hockberger, P. (1984) 1. Physiol. (London) 354.139-162
events occur in conducting airways, and alveolar involvement is present only in the latter phases of the disease. Abnormal airway secretions adversely affect mucociliary clearance and contribute to persistent airways infection, but CF patients exhibit a relatively unique sputurn microbiologicai flora that is not explained solely b abnormal mucociliary clearance2,s Staphylococcus aureus is a common pathogen early in life, later being replaced as the predominant organism by Pseudomonas aeruginosa, frequently of the mucoid variety. Because patients with immotile cilia syndrome do not typically harbor these organisms4, mnchrnicmc . .._w _.-._ “__.” nF .,_ lwno ‘-_-p rl~kno~ ______I_ n+hnr ___-__
Cystic fibrosis (CF) is an inherited disease involving multiple organ systems. The major clinical manifestations occur in the respiratory and gastrointestinal tracts, and the organ-system effects appear to reflect, in part, abnormal secretiOW on epithelial surfaces. Although sweat ductal and gastrointestinal function has long been recognized as abnormal, the major cause of morbidity and mortality results from airway disease. This review will focus on several topics that relate to respiratory tract disease in CF: (1) conventional concepts of airway pathophysiology; (2) new insights into CF respiratory epithelial cell dysfunction; (3) current therapy of CF lung disease; and (4) new approaches to the treatment of CF airway disease.
z:
. pathophysiolog
in cystic
The pathophysiologic event that initiates CF airway disease is not knowx Although mucopurulent W. E. Wakner is Fellow in Medicine, R. C. Boucher is Professor of Medicine, and M. R. Knowles is Assistant Professor of Medicine it: the School of Medicine, Department of Pulmonu y Dis:ases and 1. T. G&y is Professor of Pharmacology in the Department of Fharmncology at the University of North Carolina, Chapel Hill, NC 27514, USA. 8 19.47, usevier
PublIoHons,
CambrIdge
secretions are a hallmark of established CF airway disease, the lungs tend to be morphologically normal and withctlt evidence of infection at birthl,‘. It is not clear from available pathological material whether a primary abnormality of airway secretions leads to chronic bacterial infection or bacterial infection leads to hypertrophy of the mucus secreting elements of the airways (Fig. 1). Most observers agree that small airways inflammation is an early event, in conjunction with bacterial infection. After bacterial infection becomes established, a vicious cycle ensues: thick mucopurulent secretions obstruct airways, leading to persistent infection with a high concentration of microbial flora (>107 organisms ml-‘), which perpetuates the inflammatory response. Bacterial exoproducts, neutrophil-derived lytic enzymes (elastase, cathepsin G) and inflammatory mediators (arachidonic acid metabolites), and immunologic processes (hypersensitivity and antigen-antibody complexes) lead to airway damage, and ultimately, bronchiectasis’. Despite reDetitive treatment with antibiotics, progressive airway disease leads to respiratory failure and death. It is important to note that the major pathophysiological
0165 -6147/87/$02.1X1
than mucociliary clearance are likely to be abnormal in CF (see below). Role of epithelial ion transport in airway defense The liquid layer that lines the airways is composed of a watery ‘sol’ phase that bathes the periciliary region and a glycoprotein rich ‘gel’ phase that resides on top of the sol layer. As airway surface liquid generated in distal lung regions is swept from distal to proximal airway surfaces by ciliomotion, the volume of surface liquid is reduced in parallel with the reduction in the surface area of distal versus proximal conducting airways (-4000-fold change) . Studies of segmental human and other mammalian bronchi indicate that active ion transport mechanisms located within the superficial epithelium contribute to the reduction of the volume of airway surface liquid6p7.Studies of excised airway epithelium in the presence of a normal transepithelial electrical potential difference indicate that Na+ is absorbed via an active transcellular process whereas Cl- is the major accompanying counterion. Observations from a number of studies&l’ have led to a model for salt and water transport across proximal human airways (Fig.
TIPS -August 1987 /Vol. 81 2a). Sodium is absorbed in a twostep process; it enters the cell through a conductive channel in the apical membrane down a favorable electrochemical gradient and is extruded from the cell across the basolateral membrane by the activity of a Na+/K+ATPase pump. The entry step for Na+ is inhibited in a dosedependent manner by a relatively low concentrations (EDsO= 104 M) of arniloride in the lumen. Little is known about regulation of the rate of Na+ transport in airway epithelia. Most modulators of Na+ transport in other target epithelia (e.g. mineralocorticoids) appear to be ineffective in human airway epithelia6*‘,‘*. Under basal conditions, Cl- secretion does not occur across epithelia of human large airways, but it can be induced under certain circumstances in the presence of a Cl- permeable huninal membrane. Chloride secretion can be enhanced by agents that increase luminal Cl- permeability through the generation of CAMP, such as p-adrenoceptor agonists. Maintenance of Cl- secretion may require CAMP stimulation of other ion transport paths, i.e. the Na+ pump** and the co-transporter=. Studies of avian erythrocytes have provided the strongest evidence that CAMP regulates an electroneutral co-transporter’3. A similar regulation has been described in several epithelia’*, but there has not been z rigorous search for this process in airway epithelia. Abnormal regulation of ion transport acr0ss CF epithelia
Although the pa~ophysiologic consequences are not fully defined, it is clear that the rates of Na+ and Cl- transport across CF airway epithelia differ markedly from normal (Fig. 2b)lpB. A reduced Cl- permeability is exhibited by both CF airway and sweat ductal epithelialg. Furthermore, Cl- channels in CF airwa and sweat gland acinar to rAMI’cells*dy m r:+fan, dependent activation. Recent patch clamp studies*‘*” indicate that chloride channels exist in CF respiratory epithelia, and have single-channel conductances that are not different from those of normal ce!b. Because patch clamp experiments have shown that conductive chloride channels in CF cells can be activated by Ca2” but
bacterialinfection
airway obstruction
neutmphilproducts
Fig. 1. Pathophysiologicalschema of progrwsive airwaysdamagein CF. Thegenetic defect may involve abnormal airway secretions aMor predispasitf0n tu bsctefia~ infection of the airway. The #nit&kg events feed into a vicious cj&@ ot mucus h~et~ end ~~a~ f&ted by ~~ end host Mom @estext).
not by epinephrine, it appears that cAMP-dependent activation mechanisms (or inhibition of these mechanisms) are defective in CF. Transepithelial Na’ absorption across CF respiratory epithelia is two-fold greater than that of normal human epithelium, but Na+ transport of the sweat ductal epithelia does not appear to be affected by the diseaselg. The greater basal NaS transport across a CF respiratory epithelia is the consequence of both a raised lumirtal membrane Na+ conductance and an increased number of Na” pumps (ouabain binding sites). The increased Na+ transport cannot be explained simply by the electrical gradient resulting from the low Cl- conductance because the luminal membrane in
CF is depolarized. Net Na+ transport is still balanced by the flow of the counterion Cl-, but most of the flux is through paracellular, paths. cellular, than rather Moreover, whereas @-adrenergic agents induce Cl- secretion by normal airway epithelia, the major effect of these agents in CF epithelia is to further increase net Na+ absorption without affecting the low Cl- permeability. It is not clear whether this response reflects a primary effect on the luminal, basolateral, or both, membranes. These findings cannot be explained by different concentrations or time courses of intracelldar CAMP production. The responses of normal and CF airway epithelia are comparableis. Excessive Na+ absorption and an inability to secrete Cl- suggest
TIPS - August 1987 iVot. 81
38 (b)
(a)
LUMEN
/ Amiloride
_Amiloride
K
F 2c1-
suBMucosA
I
Fi$. 2. Modet of ion and wafer transport acrvss a nomral (A) and CF (6) respiratory eptthettatcett.AMor&-sensitiveNa+ entrysitesam shownon the tuminatmembranes. Ef6ctroneutratNa%‘K*xI- oo-transport systems and Na*/K+-ATPase pumps are depkfed on the basotatetatmembranes,ParaoBthdarpaths for ion and water flaw are shown. tntracetkftarpaths for cAMP-mediatedregulation are depicted [see text).
that the pa~oph~iolo~ of CP airway disease may reflect, in part, an increased net removal of salt and water from airway surfaces (pig. 3)1624Z. This dysfunction would tend to concentrate macromolecules in airway secretions with a resultant adverse effect on viscoelastic properties and clearance of airway secretions. It is not known whether the increased rate of Nai transport is linked to other epithelial cell influences on the composition of airway surface liquid, e.g., H+, Ca*+, or other ion concentrations. It is also not known whether the defective regulatory mechanism affects airway surface cell functions unrelated to ion transport (macromolecular secretion, receptor function) or the secretory function of submucosal glandular epithelial cells. g5zet .
clearance of retained secretions2. This approach is used as a method of ‘maintenance’ therapy, and is intensified during exacerbations of airway disease. Chest percussion and postural drainage have been demonstrated to improve function acutely. pulmonary Voluntary coughing and vigorous exercise are also efficacious in the acute clearance of secretions in selected patients. ~timicrobi~s are an important therapy in CF. Oral antibiotics are used for mild exacerbations of airway infection, even in the presence of P. aeruginosa that is resistant to oral drugs. The benefit of oral antibiotics in the presence
of drug-resistant P. aeruginosa may reflect polymicrobial airway infection with recognized pathogens (S. aureus, H. influenza) or (>lO’ high concentrations organisms ml-‘) of bacteria that are not usual pathogens3. Parenteral antibiotic therapy usually provides clinical and functional benefit for severe pulmonary exacerbations2zz6. Parenteral therapy is guided by sputum microbial flora and drug sensitivity. Usual therapy consists of an aminoglycoside plus a semisynthetic penicillin or a third generacephalosporin effective tion against pseudomonas. Combination therapy is advocated to provide antibacterial synergism and to protect against the emergence of resistant bacterial strains. Because the rate of clearance of aminoglycosides and penicillins is increased in CF patients, larger than conv~tion~ doses are necessa$6. The requirements for aminoglycosides are -SOoh higher than in non-CF patients. Thus, peak and trough serum levels of aminoglycoside are monitored to allow optimal therapy without toxicity. The localization of the infection in the airways suggests that aerosolized antibiotics may have a role in the prevention or treatment of pulmonary exacerbations. Studies of the efficacy of aerosolized antibiotics have produced variable results2~26, which may reflect the use of aerosol systems that generate particles of differing size. Aerosol antibiotics are presently being studied using existing aero-
CYSTICFIBROSIS I
f Amiloride
therapy of Cl? airway
The goal of treatment is to prevent progressive damage to the airways. Therapy is directed at removing viscous secretions, decreasing the bacteriological burden of the airways, and inhibiting the inflammatory response. The therapeutic approach has recently been reviewed elsewhere2J~26. Therefore, we will address only general principles in this article. A cornerstone in the therapy of CP airway disease is the daily
Na+
Q-
Na+
Na+
Fig.3. Schematicillustration of the relattmship between ion {and water) transportana mU&?Ciiiafycleamnce. Under normal &cumstances, a gtycoprotein-richmucus tayer resides on the tips of &a that beat rapidly in a watery peM/iary phase. In CF, excessive NaGt absorption across airway epithelia impairs viscoatastkity and rn~~ ctearancaof ahway secretions. The ~~ ofexcess&eNaQ ante with ium~l a~ is depictad as corraoting ant ~~ in CF,
I
TlPS - August 1987 [Vol. S] technology to establish their role in the management of CF airway disease. Many CF patients exhibit reactive airflow obstruction, and some patients benefit from treatment with bronchodilators. However, some patients demonstrate a reduc+ion in airflow after bronchodilators3,a7. This phenomenon is compatible with a Sadrenoceptor agonist-induced increase in absorption of Na+ (and volume) across airway surfaces and a consequent worsening of the viscoelastic properties of CF secretions, but the mechanism for the reduced airflow is not established. In general, bronchodilators should be used with caution in CF patientsz6. Corticosteroids are not routinely used in cystic fibrosis because immunosuppressive effects of the drugs may impair defense mechanisms. However, steroids are potent inhibitors of inflammation and edema, and amelioration of these processes might improve lung function. Furthermore, an improved prognosis for CF patients with low-normal serum imm~oglobu~ns**, and the presence of circulating immune complexes in patients with severe diseasez9, suggests that a hyper-immune response may play a pathophysiological role in the airway disease. A study using alternate-day steroids in a small number of CF patients did not demons&ate clear pulmonary benefitso. Thus, the routine use of steroids is precluded until further study is made. ~01
Future directions The current empirical therapy for CF airway disease is helpful, but it does not prevent the inexorable progression of the disease. The identification of respiratory epithelial ion transport dysfunctions has spurred efforts to exploit these findings in a therapeutic manner. If excessive volume absorption plays a role in the pathogenesis of CF airway disease, then modalities to either induce Cl- (and volume) secretion or block excess Na+ absorption may be beneficial (Fig. 3). No established drug activates Cl- channels in CF airway epithelia. Further, simple activation of the Cl’ channel may not induce secretion because CF epithelia are not characterized by
319 an appropriate electrochemical driving force for Cl- secretion across the apical membrane*B*23. A second approach to counteract the functions abno~~i~ of CF airway mucosa would inhibit the excessive absorption of Na+ (Fig. 3). Amiloride, a pyrazine diuretic, inhibits Na+ absorption across normal and CF respiratory epithelia when ap lied to the mucosal surfacers- R . However, oral adminis~ation of amiloride does not achieve an effective concentration of drug in the airway surface liquid of CF or normal subjects3*. Because diuretic agents had not been delivered to the airway via aerosol, we assessed the efficacy and safety of aerosol delivery of amiloride. In sheep, amiloride aerosol increased the volume of airway surface li uid with no evidence of toxici jrs 2. Subsequent studies of amiloride aerosol were performed in humans using a standard pressure-driven liquid nebulizer (DeVilbiss 646)33. Amiloride aerosol was administered to CF and normal subjects from a nebulizer solution with 5 x BY3 M drug. Samples of airway surface liquid obtained by bronchoscopy demonstrated that a maximally effective concentration of amiloride could be achieved (~1~ M), and that a concentration of at least lob M persisted for 3-5 hours in the airway surface liquid compartment. No toxicity was evident in either group after acute and chronic exposure to amilorido aerosol. A blinded crossover trial is underway at the University of North Carolina to compare the chronic effects of amiloride aerosoi with those of saline aerosol (placebo) in CF adults. Although there is a clear rationale for amiloride aerosol therapy in CF airway disease, an increase in the volume of airway secretions may not be sufficient for clinical benefit if other abnormalities of lung defense are linked to the cell defect but are not modified by inhibition of Na+ transport. Finally, the CF gene has been linked to the long arm of chromosome 7 (Refs 34 and 351, but the abnormal gene product and its pathophysiological consequences have not been described. Recent advances in molecular biology suggest that the abnormal
gene product will soon be identified, and the application of available electrophysiologic techniques make it likely that the abnormal gene product can be linked to the cellular pathobiology of CF. These advances may provide the basis for other therapies that are designed to correct abnormal regulatory mechanisms.
The research documented in this article is supported by grants HL34322, PHSRR46, and CFRRDI’ RO02 7-04.
Davis, P. B. (1985) Semin. Respir. Med. 6, 261-270 Taussig, L. M., Landau, L. I. and Marks, M. I (1984) in Cysfic Fibmsis (Taussig, L., ed.), pp. 1X5-174, Springer-Vedag Myers, M. G., Koontz, F. P. and Weinberger, M. (1983) in Textbook of Cystic Fibrosis (Lloyd-Still, J., ea.), pp. 91-109, John Wright Miller, R. D. and Divertie. M. B. (1972) . Chest 62,130-135 Ktlburn, K. H. (1968) Am. Rev. Respir. Dis. 9&,449-463 Knowles, M., Murray, G., ShaUal, J., Asldn, F., Ranga, V., Gatzy, J. and Boucher, R. (1964) f. Appl. Pkysiol. 56, 860-877 Boucher, R. C., Narvarte, J., Cotton, C. Stutts. M. 1.. Knowles. M. R.. Finn. A. L. and Gatz$; J. T. (1982) in Fluid and Electrolyte Abnormalities in Exocrine Glands h Cysfic Fibrosis (Quinton, P. M., Martinez, J. R. and Hopfer, U., eds), pp. 271-288, San Francisco Press Al-bazraz, F. (1986) C&r. Chesf Med. 7, 259-272 Welsh, Michael J. (1986) Ctin. Chest. Med. 7,273-2&S Knowles, M. R., Gamy, J. T. and Boucher, R. C. (1985) Pedintr. Rcs. 19, 676-679 Butt, A. G. and Frizaell, R. A. (1987) Fed. Proc. 46,635
Palfrey, H. C. and Greengard, F. (1981) A_ Ann. N.Y. Acad. Sci. 372,291~308 13 McManus, 7: J. and Schmidt, W. F. (1978) in Me~kr~n~ Tr~~~~ Processes (Hoffman, J. F., ed.), pp. 79196, Raven Press 14 Knowles, M. R., Gatay, J-T. and Boucher, R. C. (1981) N. Eugl. J. Med. 305.1489-1495 15 Knowles, M. R., Stutts, M. J., Speck, A., Fischer, N., Gamy, J. T. and Boucher, R. G. (1983) Science 221,1067-1070 16 Knowles, M. R., Stutts, M. J., Yankashas, J. R., G&.y, J. and Boucher, R. C. (1986) C&t. Ckesf. P&d. 7,285-296 17 Boucber, R. C., Stutts, M. J.. Knowles, M. R., Cantiey, L. and Gatay, T. (1986) J. Clin. Invest. 78, 1245-1252 18 Cotton, C. U., Stutts. M. J., Knowles, M. R., Gamy, J. T. and Boucher, R. C. (1987) J, Ckn. Invest. 79,8m5 19 Quinton, P. M. (1983) Nuture 301, 421422 20 Sato, K. (1984) Am. J. Physiol. 247, R644-
TIPS - August 2987 [Vol. 81
320 R&l9 21 FrizzeU. R. A., Rechkemmer, G. and 22 23 24 25 26 27
Shoemaker, R. (19%) Stience 233, 5% 560 Welsh, M. J. and Liedtke, C. (1986) Nature 322,467470 Wiiumsen, N. J., Davis, C. W. and Boucher, R. C. (1987) Fed. Proc. 46,1236 Chemick, W. S. and Barbero, G.J. (1959) Pediatrics 24.739-745 Potter, J. L., Matthews, L. W., Spector, S. and Lemm, J. (1967) Am. Rev. Respir. Dis. 96, 83-87 Mischler, E. H. (1985) Semin. Respir. Med. 4.271-282 Landau, L. I. and Phelan, P. D. (1973)
The meaning of numbers Statistics for Toxicologists by Dad S. Salsburg, Marcel Dekker, 1986. US$49.75 (xiii + 296 paged ISBN 0 8247 7590 2 David Salsburg h&s long been a provocative reviewer of the statistical methods used to assess toxicological studies. He was first to question the extrapolation to humans of the results of long-term animal studies of the feeding of potential carcinogens. He claimed that only 37% of a set of chemicals known to cause cancer in humans were carcinogenic in an animal test. His comment that ‘we would have been better off to toss a coin’ has been widely quoted. Anyone expecting his book Statistics for Toxicologists to be a polemic, chaIlenging established ideas will be disappointed. Instead Salsburg has written a thoughtful and reflective text, probing rather than overturning the many statistical issues in toxicology. The editors of the series Drug and C~e~icu~ To~c~~o~, of which this is the fourth volume, have highlighted in their introduction that this is a field that is both confusing and little understood. Salsburg provides a nonmathematical description of statistical issues relating to three areas of toxicology: the LDs, the subchronic and the chronic toxicity study. In each case he first discusses how the methods are applied, then how to critically evaluate the results and then provides a wider description of the
I. Dediatr. 82.863-M 28 ivheeier, W. B., WiRiams, M., Matthews, W. T. and Cotter, H. R. (1984) 1. Fediatr. 104,695-699 29 Moss, R. (1983) in Textbook of Cystic Fibrosis (Lloyd-Still, J., ed.), pp. 109-151, John Wright 30 Auerbach, H. 5, Williams, M., Kirkpatrick, J. A. and Colter, H. R. (1985) Lancet
ii,
686-688
31 Knowles, M., FuRton. J., Jones, D. Gamy, J. and Boucher, R; (1633) Cystic Fibrosis Ciub Abstracts 24,3 32 Mentz, W. M., Brown, J. B., Friedman, M., Stutts, M. J., Gamy, J. T. and Boucher, R. A. (1986) Am. Rev. Respir.
statistical theory underlying the methods. There is no assumption of mathematical knowledge beyond school level or of statistical theory except an aquaintance with the methods used in toxicology. This means that the book is not a ‘how statistics’ to do textbook. However, the toxicologist who puzzles over the large data sets and even larger computer printouts that these sets generate should find the book stimulating. Some questions will be answered but others, more profound, will replace them. Nevertheless, a deeper appreciation of the role, potential and limitation of statistics should be achieved. If nothing else a realization that there are no come easy answers should through. The apparent similarity of the testing of hypotheses by statistical methods and the decision making processes has at times tempted the regulator ts overemphasize statistical conclusions. In fact, statistics is Only one of many inputs into the final assessment of a chemical
A step nearer classification Perspectives on Receptor Classification: Receptor Biochemistry and Methodology Vol. 6 edited by 1. W. Black, D. H. Jackson apld V. P. ~erskowitck‘ AIan tiss 1987, E47.00 (xi -!- 295) ISBN 0 8451 3795 0 The organizers of the Schild Memorial Festschrift held in Lon-
Dis. 134,938-943 33 WaItier, W. E., Church, N. L., Gatzy, J. T., Boucher, R. C. and Rnowtes, M. R. (1986) Cystic Fibrosis Club Abstracts 27, 121 Robert G., Cohen34 Knowlton, Haguenauer, O., Van Cong, N., Frezal, J., Brown, V. A., Barker, D., Braman, J. C., Schamm, J. W., Tscii, L-C., Bachwald, M. and Donis-Keller (1985) Natare 318,380-382 35 Wainwright, B. J., Scambler, P. J., Schmidtke, J., Watson, E. A., Law HaiYang, Faval, M., Cooke, H. J., Eiberg, H. and Williamson, R. (1985) Nature 318, 381-385
in what is a subjective decision based upon the judgements of a range of experts. The book brings out this relationship between statistical significance and biological importance together with the increasing emphasis on the use of statistics for exploration and interpretation of data. The book is well produced in the standard Dekker format, clear text and diagrsms and no obvious mathematical errors. The index is a useful reference for the statistical tests described. The list of references while not exhaustive has a helpful brief commentary on the paper or book. The statistics will enjoy the book as a ‘good read’ and will like the historical perspective to the issues described. Toxicologists are, however, the real target of this book and I wonder if it will reach them. Too many, I fear, will say ‘I haven’t got time to read a statistics book’. These are the toxicologists who most need to read and reflect on the issues raised in this excellent little book. D.P. LOVELL British Industrial Biologists Research Association, Woodtnansterne Road, Carskalton, Swrrey SH5 4DS, UK.
don, in December 1985, conceived a fasting memorial to Heinz Schild - a gathering of eminent phsrraand cologists, biochemists physiologists who could pool their knowledge and focus their differing intellectual directions fruitfully onto the problems of receptor classification. It was hoped that the meeting would form the impetus for the setting up of an International Commission on Receptor Classification,anal-
316 19 Heldman, J. and Lancet, D. (1986) J. Neuwhem. 47,1527-1533 20 Nakamua, T. and Gold, G. H. (1987) Nature 325.4424 21 Trot&, D. and MacLeod, P. (1983) Brain Res. 268.225-237 22 Nagabama, S., Kobatake, Y. and Kurihara. K. (1982) J. Gen. Physiol. 80, 78%
800 23 Taylor, C. W. and Merritt, J. E. (1987) Trends Pharmacol. Sci. 7,238-242 24 Lefkowitz, R. J., Benovic, J. L., Kobilka, B. and Caron, M. G. (1986) Trends Pharmacof. Sci. 7, 444-448 25 Nathans, J., Thomas, D. and Hogness, D. S: (1986) Science 232, 193-202
Pharmacotherapy of airway disease in cystic fibrosis W. E. Waltner, R. C. Boucher, J. T. Gatzy and M. R. Knowles Current therapy for CF airway disease slows, but does not stop, the inexorable of airway damage. Recent research suggests that abnormal regulation of ion transport in CF ainvay epithelial cells leads to excessive Na+ absorption across a relatively Cl--impermeable airway epithelium. This pattern of dysfunction predisposes to dehydration of airway secretions and adverse effects on mucociliay clearance and lung defense. W. E. Waltner et al; aplain the rationale behind potential new approaches to therapy involving
progression
26 Sldar, P. B., Anholt, R. H. and Snyder, S. H. (1986) J. Biol. Chem. 261, 1553815543 27 Stern, J., Kaupp, U. B. and MacLeish, P. R. (1986) Pm. Nutl Acud. Sci. USA 83, 1163-1167 28 Conner, J. and Hockberger, P. (1984) 1. Physiol. (London) 354.139-162
events occur in conducting airways, and alveolar involvement is present only in the latter phases of the disease. Abnormal airway secretions adversely affect mucociliary clearance and contribute to persistent airways infection, but CF patients exhibit a relatively unique sputurn microbiologicai flora that is not explained solely b abnormal mucociliary clearance2,s Staphylococcus aureus is a common pathogen early in life, later being replaced as the predominant organism by Pseudomonas aeruginosa, frequently of the mucoid variety. Because patients with immotile cilia syndrome do not typically harbor these organisms4, mnchrnicmc . .._w _.-._ “__.” nF .,_ lwno ‘-_-p rl~kno~ ______I_ n+hnr ___-__
Cystic fibrosis (CF) is an inherited disease involving multiple organ systems. The major clinical manifestations occur in the respiratory and gastrointestinal tracts, and the organ-system effects appear to reflect, in part, abnormal secretiOW on epithelial surfaces. Although sweat ductal and gastrointestinal function has long been recognized as abnormal, the major cause of morbidity and mortality results from airway disease. This review will focus on several topics that relate to respiratory tract disease in CF: (1) conventional concepts of airway pathophysiology; (2) new insights into CF respiratory epithelial cell dysfunction; (3) current therapy of CF lung disease; and (4) new approaches to the treatment of CF airway disease.
z:
. pathophysiolog
in cystic
The pathophysiologic event that initiates CF airway disease is not knowx Although mucopurulent W. E. Wakner is Fellow in Medicine, R. C. Boucher is Professor of Medicine, and M. R. Knowles is Assistant Professor of Medicine it: the School of Medicine, Department of Pulmonu y Dis:ases and 1. T. G&y is Professor of Pharmacology in the Department of Fharmncology at the University of North Carolina, Chapel Hill, NC 27514, USA. 8 19.47, usevier
PublIoHons,
CambrIdge
secretions are a hallmark of established CF airway disease, the lungs tend to be morphologically normal and withctlt evidence of infection at birthl,‘. It is not clear from available pathological material whether a primary abnormality of airway secretions leads to chronic bacterial infection or bacterial infection leads to hypertrophy of the mucus secreting elements of the airways (Fig. 1). Most observers agree that small airways inflammation is an early event, in conjunction with bacterial infection. After bacterial infection becomes established, a vicious cycle ensues: thick mucopurulent secretions obstruct airways, leading to persistent infection with a high concentration of microbial flora (>107 organisms ml-‘), which perpetuates the inflammatory response. Bacterial exoproducts, neutrophil-derived lytic enzymes (elastase, cathepsin G) and inflammatory mediators (arachidonic acid metabolites), and immunologic processes (hypersensitivity and antigen-antibody complexes) lead to airway damage, and ultimately, bronchiectasis’. Despite reDetitive treatment with antibiotics, progressive airway disease leads to respiratory failure and death. It is important to note that the major pathophysiological
0165 -6147/87/$02.1X1
than mucociliary clearance are likely to be abnormal in CF (see below). Role of epithelial ion transport in airway defense The liquid layer that lines the airways is composed of a watery ‘sol’ phase that bathes the periciliary region and a glycoprotein rich ‘gel’ phase that resides on top of the sol layer. As airway surface liquid generated in distal lung regions is swept from distal to proximal airway surfaces by ciliomotion, the volume of surface liquid is reduced in parallel with the reduction in the surface area of distal versus proximal conducting airways (-4000-fold change) . Studies of segmental human and other mammalian bronchi indicate that active ion transport mechanisms located within the superficial epithelium contribute to the reduction of the volume of airway surface liquid6p7.Studies of excised airway epithelium in the presence of a normal transepithelial electrical potential difference indicate that Na+ is absorbed via an active transcellular process whereas Cl- is the major accompanying counterion. Observations from a number of studies&l’ have led to a model for salt and water transport across proximal human airways (Fig.
TIPS -August 1987 /Vol. 81 2a). Sodium is absorbed in a twostep process; it enters the cell through a conductive channel in the apical membrane down a favorable electrochemical gradient and is extruded from the cell across the basolateral membrane by the activity of a Na+/K+ATPase pump. The entry step for Na+ is inhibited in a dosedependent manner by a relatively low concentrations (EDsO= 104 M) of arniloride in the lumen. Little is known about regulation of the rate of Na+ transport in airway epithelia. Most modulators of Na+ transport in other target epithelia (e.g. mineralocorticoids) appear to be ineffective in human airway epithelia6*‘,‘*. Under basal conditions, Cl- secretion does not occur across epithelia of human large airways, but it can be induced under certain circumstances in the presence of a Cl- permeable huninal membrane. Chloride secretion can be enhanced by agents that increase luminal Cl- permeability through the generation of CAMP, such as p-adrenoceptor agonists. Maintenance of Cl- secretion may require CAMP stimulation of other ion transport paths, i.e. the Na+ pump** and the co-transporter=. Studies of avian erythrocytes have provided the strongest evidence that CAMP regulates an electroneutral co-transporter’3. A similar regulation has been described in several epithelia’*, but there has not been z rigorous search for this process in airway epithelia. Abnormal regulation of ion transport acr0ss CF epithelia
Although the pa~ophysiologic consequences are not fully defined, it is clear that the rates of Na+ and Cl- transport across CF airway epithelia differ markedly from normal (Fig. 2b)lpB. A reduced Cl- permeability is exhibited by both CF airway and sweat ductal epithelialg. Furthermore, Cl- channels in CF airwa and sweat gland acinar to rAMI’cells*dy m r:+fan, dependent activation. Recent patch clamp studies*‘*” indicate that chloride channels exist in CF respiratory epithelia, and have single-channel conductances that are not different from those of normal ce!b. Because patch clamp experiments have shown that conductive chloride channels in CF cells can be activated by Ca2” but
bacterialinfection
airway obstruction
neutmphilproducts
Fig. 1. Pathophysiologicalschema of progrwsive airwaysdamagein CF. Thegenetic defect may involve abnormal airway secretions aMor predispasitf0n tu bsctefia~ infection of the airway. The #nit&kg events feed into a vicious cj&@ ot mucus h~et~ end ~~a~ f&ted by ~~ end host Mom @estext).
not by epinephrine, it appears that cAMP-dependent activation mechanisms (or inhibition of these mechanisms) are defective in CF. Transepithelial Na’ absorption across CF respiratory epithelia is two-fold greater than that of normal human epithelium, but Na+ transport of the sweat ductal epithelia does not appear to be affected by the diseaselg. The greater basal NaS transport across a CF respiratory epithelia is the consequence of both a raised lumirtal membrane Na+ conductance and an increased number of Na” pumps (ouabain binding sites). The increased Na+ transport cannot be explained simply by the electrical gradient resulting from the low Cl- conductance because the luminal membrane in
CF is depolarized. Net Na+ transport is still balanced by the flow of the counterion Cl-, but most of the flux is through paracellular, paths. cellular, than rather Moreover, whereas @-adrenergic agents induce Cl- secretion by normal airway epithelia, the major effect of these agents in CF epithelia is to further increase net Na+ absorption without affecting the low Cl- permeability. It is not clear whether this response reflects a primary effect on the luminal, basolateral, or both, membranes. These findings cannot be explained by different concentrations or time courses of intracelldar CAMP production. The responses of normal and CF airway epithelia are comparableis. Excessive Na+ absorption and an inability to secrete Cl- suggest
TIPS - August 1987 iVot. 81
38 (b)
(a)
LUMEN
/ Amiloride
_Amiloride
K
F 2c1-
suBMucosA
I
Fi$. 2. Modet of ion and wafer transport acrvss a nomral (A) and CF (6) respiratory eptthettatcett.AMor&-sensitiveNa+ entrysitesam shownon the tuminatmembranes. Ef6ctroneutratNa%‘K*xI- oo-transport systems and Na*/K+-ATPase pumps are depkfed on the basotatetatmembranes,ParaoBthdarpaths for ion and water flaw are shown. tntracetkftarpaths for cAMP-mediatedregulation are depicted [see text).
that the pa~oph~iolo~ of CP airway disease may reflect, in part, an increased net removal of salt and water from airway surfaces (pig. 3)1624Z. This dysfunction would tend to concentrate macromolecules in airway secretions with a resultant adverse effect on viscoelastic properties and clearance of airway secretions. It is not known whether the increased rate of Nai transport is linked to other epithelial cell influences on the composition of airway surface liquid, e.g., H+, Ca*+, or other ion concentrations. It is also not known whether the defective regulatory mechanism affects airway surface cell functions unrelated to ion transport (macromolecular secretion, receptor function) or the secretory function of submucosal glandular epithelial cells. g5zet .
clearance of retained secretions2. This approach is used as a method of ‘maintenance’ therapy, and is intensified during exacerbations of airway disease. Chest percussion and postural drainage have been demonstrated to improve function acutely. pulmonary Voluntary coughing and vigorous exercise are also efficacious in the acute clearance of secretions in selected patients. ~timicrobi~s are an important therapy in CF. Oral antibiotics are used for mild exacerbations of airway infection, even in the presence of P. aeruginosa that is resistant to oral drugs. The benefit of oral antibiotics in the presence
of drug-resistant P. aeruginosa may reflect polymicrobial airway infection with recognized pathogens (S. aureus, H. influenza) or (>lO’ high concentrations organisms ml-‘) of bacteria that are not usual pathogens3. Parenteral antibiotic therapy usually provides clinical and functional benefit for severe pulmonary exacerbations2zz6. Parenteral therapy is guided by sputum microbial flora and drug sensitivity. Usual therapy consists of an aminoglycoside plus a semisynthetic penicillin or a third generacephalosporin effective tion against pseudomonas. Combination therapy is advocated to provide antibacterial synergism and to protect against the emergence of resistant bacterial strains. Because the rate of clearance of aminoglycosides and penicillins is increased in CF patients, larger than conv~tion~ doses are necessa$6. The requirements for aminoglycosides are -SOoh higher than in non-CF patients. Thus, peak and trough serum levels of aminoglycoside are monitored to allow optimal therapy without toxicity. The localization of the infection in the airways suggests that aerosolized antibiotics may have a role in the prevention or treatment of pulmonary exacerbations. Studies of the efficacy of aerosolized antibiotics have produced variable results2~26, which may reflect the use of aerosol systems that generate particles of differing size. Aerosol antibiotics are presently being studied using existing aero-
CYSTICFIBROSIS I
f Amiloride
therapy of Cl? airway
The goal of treatment is to prevent progressive damage to the airways. Therapy is directed at removing viscous secretions, decreasing the bacteriological burden of the airways, and inhibiting the inflammatory response. The therapeutic approach has recently been reviewed elsewhere2J~26. Therefore, we will address only general principles in this article. A cornerstone in the therapy of CP airway disease is the daily
Na+
Q-
Na+
Na+
Fig.3. Schematicillustration of the relattmship between ion {and water) transportana mU&?Ciiiafycleamnce. Under normal &cumstances, a gtycoprotein-richmucus tayer resides on the tips of &a that beat rapidly in a watery peM/iary phase. In CF, excessive NaGt absorption across airway epithelia impairs viscoatastkity and rn~~ ctearancaof ahway secretions. The ~~ ofexcess&eNaQ ante with ium~l a~ is depictad as corraoting ant ~~ in CF,
I
TlPS - August 1987 [Vol. S] technology to establish their role in the management of CF airway disease. Many CF patients exhibit reactive airflow obstruction, and some patients benefit from treatment with bronchodilators. However, some patients demonstrate a reduc+ion in airflow after bronchodilators3,a7. This phenomenon is compatible with a Sadrenoceptor agonist-induced increase in absorption of Na+ (and volume) across airway surfaces and a consequent worsening of the viscoelastic properties of CF secretions, but the mechanism for the reduced airflow is not established. In general, bronchodilators should be used with caution in CF patientsz6. Corticosteroids are not routinely used in cystic fibrosis because immunosuppressive effects of the drugs may impair defense mechanisms. However, steroids are potent inhibitors of inflammation and edema, and amelioration of these processes might improve lung function. Furthermore, an improved prognosis for CF patients with low-normal serum imm~oglobu~ns**, and the presence of circulating immune complexes in patients with severe diseasez9, suggests that a hyper-immune response may play a pathophysiological role in the airway disease. A study using alternate-day steroids in a small number of CF patients did not demons&ate clear pulmonary benefitso. Thus, the routine use of steroids is precluded until further study is made. ~01
Future directions The current empirical therapy for CF airway disease is helpful, but it does not prevent the inexorable progression of the disease. The identification of respiratory epithelial ion transport dysfunctions has spurred efforts to exploit these findings in a therapeutic manner. If excessive volume absorption plays a role in the pathogenesis of CF airway disease, then modalities to either induce Cl- (and volume) secretion or block excess Na+ absorption may be beneficial (Fig. 3). No established drug activates Cl- channels in CF airway epithelia. Further, simple activation of the Cl’ channel may not induce secretion because CF epithelia are not characterized by
319 an appropriate electrochemical driving force for Cl- secretion across the apical membrane*B*23. A second approach to counteract the functions abno~~i~ of CF airway mucosa would inhibit the excessive absorption of Na+ (Fig. 3). Amiloride, a pyrazine diuretic, inhibits Na+ absorption across normal and CF respiratory epithelia when ap lied to the mucosal surfacers- R . However, oral adminis~ation of amiloride does not achieve an effective concentration of drug in the airway surface liquid of CF or normal subjects3*. Because diuretic agents had not been delivered to the airway via aerosol, we assessed the efficacy and safety of aerosol delivery of amiloride. In sheep, amiloride aerosol increased the volume of airway surface li uid with no evidence of toxici jrs 2. Subsequent studies of amiloride aerosol were performed in humans using a standard pressure-driven liquid nebulizer (DeVilbiss 646)33. Amiloride aerosol was administered to CF and normal subjects from a nebulizer solution with 5 x BY3 M drug. Samples of airway surface liquid obtained by bronchoscopy demonstrated that a maximally effective concentration of amiloride could be achieved (~1~ M), and that a concentration of at least lob M persisted for 3-5 hours in the airway surface liquid compartment. No toxicity was evident in either group after acute and chronic exposure to amilorido aerosol. A blinded crossover trial is underway at the University of North Carolina to compare the chronic effects of amiloride aerosoi with those of saline aerosol (placebo) in CF adults. Although there is a clear rationale for amiloride aerosol therapy in CF airway disease, an increase in the volume of airway secretions may not be sufficient for clinical benefit if other abnormalities of lung defense are linked to the cell defect but are not modified by inhibition of Na+ transport. Finally, the CF gene has been linked to the long arm of chromosome 7 (Refs 34 and 351, but the abnormal gene product and its pathophysiological consequences have not been described. Recent advances in molecular biology suggest that the abnormal
gene product will soon be identified, and the application of available electrophysiologic techniques make it likely that the abnormal gene product can be linked to the cellular pathobiology of CF. These advances may provide the basis for other therapies that are designed to correct abnormal regulatory mechanisms.
The research documented in this article is supported by grants HL34322, PHSRR46, and CFRRDI’ RO02 7-04.
Davis, P. B. (1985) Semin. Respir. Med. 6, 261-270 Taussig, L. M., Landau, L. I. and Marks, M. I (1984) in Cysfic Fibmsis (Taussig, L., ed.), pp. 1X5-174, Springer-Vedag Myers, M. G., Koontz, F. P. and Weinberger, M. (1983) in Textbook of Cystic Fibrosis (Lloyd-Still, J., ea.), pp. 91-109, John Wright Miller, R. D. and Divertie. M. B. (1972) . Chest 62,130-135 Ktlburn, K. H. (1968) Am. Rev. Respir. Dis. 9&,449-463 Knowles, M., Murray, G., ShaUal, J., Asldn, F., Ranga, V., Gatzy, J. and Boucher, R. (1964) f. Appl. Pkysiol. 56, 860-877 Boucher, R. C., Narvarte, J., Cotton, C. Stutts. M. 1.. Knowles. M. R.. Finn. A. L. and Gatz$; J. T. (1982) in Fluid and Electrolyte Abnormalities in Exocrine Glands h Cysfic Fibrosis (Quinton, P. M., Martinez, J. R. and Hopfer, U., eds), pp. 271-288, San Francisco Press Al-bazraz, F. (1986) C&r. Chesf Med. 7, 259-272 Welsh, Michael J. (1986) Ctin. Chest. Med. 7,273-2&S Knowles, M. R., Gamy, J. T. and Boucher, R. C. (1985) Pedintr. Rcs. 19, 676-679 Butt, A. G. and Frizaell, R. A. (1987) Fed. Proc. 46,635
Palfrey, H. C. and Greengard, F. (1981) A_ Ann. N.Y. Acad. Sci. 372,291~308 13 McManus, 7: J. and Schmidt, W. F. (1978) in Me~kr~n~ Tr~~~~ Processes (Hoffman, J. F., ed.), pp. 79196, Raven Press 14 Knowles, M. R., Gatay, J-T. and Boucher, R. C. (1981) N. Eugl. J. Med. 305.1489-1495 15 Knowles, M. R., Stutts, M. J., Speck, A., Fischer, N., Gamy, J. T. and Boucher, R. G. (1983) Science 221,1067-1070 16 Knowles, M. R., Stutts, M. J., Yankashas, J. R., G&.y, J. and Boucher, R. C. (1986) C&t. Ckesf. P&d. 7,285-296 17 Boucber, R. C., Stutts, M. J.. Knowles, M. R., Cantiey, L. and Gatay, T. (1986) J. Clin. Invest. 78, 1245-1252 18 Cotton, C. U., Stutts. M. J., Knowles, M. R., Gamy, J. T. and Boucher, R. C. (1987) J, Ckn. Invest. 79,8m5 19 Quinton, P. M. (1983) Nuture 301, 421422 20 Sato, K. (1984) Am. J. Physiol. 247, R644-
TIPS - August 2987 [Vol. 81
320 R&l9 21 FrizzeU. R. A., Rechkemmer, G. and 22 23 24 25 26 27
Shoemaker, R. (19%) Stience 233, 5% 560 Welsh, M. J. and Liedtke, C. (1986) Nature 322,467470 Wiiumsen, N. J., Davis, C. W. and Boucher, R. C. (1987) Fed. Proc. 46,1236 Chemick, W. S. and Barbero, G.J. (1959) Pediatrics 24.739-745 Potter, J. L., Matthews, L. W., Spector, S. and Lemm, J. (1967) Am. Rev. Respir. Dis. 96, 83-87 Mischler, E. H. (1985) Semin. Respir. Med. 4.271-282 Landau, L. I. and Phelan, P. D. (1973)
The meaning of numbers Statistics for Toxicologists by Dad S. Salsburg, Marcel Dekker, 1986. US$49.75 (xiii + 296 paged ISBN 0 8247 7590 2 David Salsburg h&s long been a provocative reviewer of the statistical methods used to assess toxicological studies. He was first to question the extrapolation to humans of the results of long-term animal studies of the feeding of potential carcinogens. He claimed that only 37% of a set of chemicals known to cause cancer in humans were carcinogenic in an animal test. His comment that ‘we would have been better off to toss a coin’ has been widely quoted. Anyone expecting his book Statistics for Toxicologists to be a polemic, chaIlenging established ideas will be disappointed. Instead Salsburg has written a thoughtful and reflective text, probing rather than overturning the many statistical issues in toxicology. The editors of the series Drug and C~e~icu~ To~c~~o~, of which this is the fourth volume, have highlighted in their introduction that this is a field that is both confusing and little understood. Salsburg provides a nonmathematical description of statistical issues relating to three areas of toxicology: the LDs, the subchronic and the chronic toxicity study. In each case he first discusses how the methods are applied, then how to critically evaluate the results and then provides a wider description of the
I. Dediatr. 82.863-M 28 ivheeier, W. B., WiRiams, M., Matthews, W. T. and Cotter, H. R. (1984) 1. Fediatr. 104,695-699 29 Moss, R. (1983) in Textbook of Cystic Fibrosis (Lloyd-Still, J., ed.), pp. 109-151, John Wright 30 Auerbach, H. 5, Williams, M., Kirkpatrick, J. A. and Colter, H. R. (1985) Lancet
ii,
686-688
31 Knowles, M., FuRton. J., Jones, D. Gamy, J. and Boucher, R; (1633) Cystic Fibrosis Ciub Abstracts 24,3 32 Mentz, W. M., Brown, J. B., Friedman, M., Stutts, M. J., Gamy, J. T. and Boucher, R. A. (1986) Am. Rev. Respir.
statistical theory underlying the methods. There is no assumption of mathematical knowledge beyond school level or of statistical theory except an aquaintance with the methods used in toxicology. This means that the book is not a ‘how statistics’ to do textbook. However, the toxicologist who puzzles over the large data sets and even larger computer printouts that these sets generate should find the book stimulating. Some questions will be answered but others, more profound, will replace them. Nevertheless, a deeper appreciation of the role, potential and limitation of statistics should be achieved. If nothing else a realization that there are no come easy answers should through. The apparent similarity of the testing of hypotheses by statistical methods and the decision making processes has at times tempted the regulator ts overemphasize statistical conclusions. In fact, statistics is Only one of many inputs into the final assessment of a chemical
A step nearer classification Perspectives on Receptor Classification: Receptor Biochemistry and Methodology Vol. 6 edited by 1. W. Black, D. H. Jackson apld V. P. ~erskowitck‘ AIan tiss 1987, E47.00 (xi -!- 295) ISBN 0 8451 3795 0 The organizers of the Schild Memorial Festschrift held in Lon-
Dis. 134,938-943 33 WaItier, W. E., Church, N. L., Gatzy, J. T., Boucher, R. C. and Rnowtes, M. R. (1986) Cystic Fibrosis Club Abstracts 27, 121 Robert G., Cohen34 Knowlton, Haguenauer, O., Van Cong, N., Frezal, J., Brown, V. A., Barker, D., Braman, J. C., Schamm, J. W., Tscii, L-C., Bachwald, M. and Donis-Keller (1985) Natare 318,380-382 35 Wainwright, B. J., Scambler, P. J., Schmidtke, J., Watson, E. A., Law HaiYang, Faval, M., Cooke, H. J., Eiberg, H. and Williamson, R. (1985) Nature 318, 381-385
in what is a subjective decision based upon the judgements of a range of experts. The book brings out this relationship between statistical significance and biological importance together with the increasing emphasis on the use of statistics for exploration and interpretation of data. The book is well produced in the standard Dekker format, clear text and diagrsms and no obvious mathematical errors. The index is a useful reference for the statistical tests described. The list of references while not exhaustive has a helpful brief commentary on the paper or book. The statistics will enjoy the book as a ‘good read’ and will like the historical perspective to the issues described. Toxicologists are, however, the real target of this book and I wonder if it will reach them. Too many, I fear, will say ‘I haven’t got time to read a statistics book’. These are the toxicologists who most need to read and reflect on the issues raised in this excellent little book. D.P. LOVELL British Industrial Biologists Research Association, Woodtnansterne Road, Carskalton, Swrrey SH5 4DS, UK.
don, in December 1985, conceived a fasting memorial to Heinz Schild - a gathering of eminent phsrraand cologists, biochemists physiologists who could pool their knowledge and focus their differing intellectual directions fruitfully onto the problems of receptor classification. It was hoped that the meeting would form the impetus for the setting up of an International Commission on Receptor Classification,anal-