Springer-Verlag 1984

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Diabetlgia (1984) 26:55-59 Diabetlgia @ Springer-Verlag 1984 Binding f hypglycaemic sulphnylureas t an artificial phsphlipid bilayer M. Deleers and W.J.

1 Diabetlgia (1984) 26:55-59 Springer-Verlag 1984 Binding f hypglycaemic sulphnylureas t an artificial phsphlipid bilayer M. Deleers and W.J. Malaisse Labratry f xperimental Medicine, Brussels University Medical Schl, Brussels, Belgium Summary. Hypglycaemic sulphnylureas bind t multilamellar lipsmes frmed f egg ylk phsphatidylchline. In this artificial mdel, bth specific and nn-specific cmpnents f the binding phenmenn can be characterized by the same criteria as thse used in studies perfrmed with natural membranes. The relative ability f distinct sulphnylureas t inhibit the binding f 3H-glibenclamide r 3H-gliquidne t the lipsmes parallels their relative ptency as insulin secreta- ggues. It is prpsed that the insertin f hypglycaemic sulphnylureas int the phsphlipid dmain f the B cell membrane culd represent a primary event in the mechanism by which these agents stimulate insulin release. Key wrds: Lipsmes, phsphlipid, hypglycaemic sulphnylurea, glibenclamide, gliquidne, gliclazide, tlbutamide, chlrprpamide. Hypglycaemic sulphnylureas stimulate insulin release, apparently by facilitating the inflw f Ca 2+ int the pancreatic B cell [1, 21. Mst authrs favur the view that the stimulatin f Ca 2+ inflw is secndary t a decrease in K + cnductance, leading t deplarizatin f the plasma membrane and gating f vltage-sensitive calcium channels [3-6]. The mlecular mechanism respnsible fr the change in K permeability is nt knwn [7]. Radiistpic studies f sulphnylurea uptake by islated islets suggest that these hypglycaemic agents are bund t the plasma membrane f pancreatic B cells [8-10], which culd be equipped with sulphnylurea receptrs [111. The cell bundary [12] culd therefre represent the primary site f actin f hypglycaemic sulphnylureas. The aim f the present study was t investigate hw far the binding f sulphnylureas t natural membranes culd be simulated in an artificial mdel f phsphlipid bilayer. Materials and methds gg ylk phsphatidylchline was purchased frm Sigma (St. Luis, Missuri, USA). 3H-glibenclamide (31.1 Ci/mml) was kindly prvided by M.H. Mertens (Hechst Belgium, Brussels, Belgium), and 3H-gliquidne (4.8 Ci/mml) by Dr N. Kaubisch (Behringer, Ingelbeim, FRG) and Dr Rupprecht (Thmae, Biberach, FRG). Bth sulphnylureas were tritiated in the cyclhexyl ring and their purity assessed by chrmatgraphic analysis t be >96%. Unlabelled glibenclamide and tlbutamide were btained frm Hechst, gliquidne frm Thmae, gliclazide frm Servier Benelux, Brussels, Belgium and chlrprpamide frm Pfizer, Brussels, Belgium. Multilamellar lipsmes f egg ylk phsphatidylchline were frmed as described elsewhere [13] in a Tris-HCl buffer (50 retl/l, ph 7.30) yielding a final cncentratin f 2 mg/ml. The sulphnylureas were added t this buffer frm stck slutins prepared in a mixture f ethanl and 0.1 N NaOH (1/1, v/v), the final cncentratin f these slvents nt exceeding 0.2% (v/v). Aliquts (0.25 ml) f the lipsmal suspensin were placed in plyethylene micrcentrifuge tubes (Beckman, Pal Alt, Califrnia, USA), incubated fr 5-60 min at 37~ and centrifuged fr 30min at 4~ and 3,000 g. The supernatant slutin was then remved and the bttm f the tube cntaining the pellet f lipsmes cut, placed in a cunting vial cntaining 6 ml f scintillatin fluid (Aquasl-2; New ngland Nuclear, Bstn, Massachusetts, USA), and eventually examined fr its radiactive cntent. The specific radiactivity f 3H-glibenclamide r 3H-gliquidne was kept unchanged in all experiments, except fr measurement f the nn-specific binding in which case unlabelled sulphnylurea was added at a final cncentratin f 0.1 mml/l. Such a nn-specific binding was measured fr each cnditin under study. Results are expressed as mean + SM values tgether with the numbers f individual determinatins (n). Results At a 20 nml/1 cncentratin f 3H-gliquidne, the apparent binding f the sulphnylurea t the lipsmes reached an equilibrium value within 15 min f expsure t the drug (Figure 1). At the same cncentratin f the radiactive cmpund but in the presence f a much

2 56 higher cncentratin f unlabelled gliquidne (0.1 retl/l), the radiactivity assciated with the lipsmes als rapidly reached equilibrium. Further experiments were perfrmed ver 30 rain incubatin. In experiments perfrmed in the presence f a 10nml/1 cncentratin f 3H-sulphnylurea and a 0.1 mml/1 cncentratin f the unlabelled drug, the ttal binding f glibenclamide and gliquidne averaged and nml/mg f phsphlipid, respectively (n = 6-10). This crrespnds t apprximately 1.00_ and tl sulphnylurea/100 ml phsphlipid. If the binding f 3H-sulphnylurea at this high cncentratin f the unlabelled agent is as- 2O '? 16 0,~,12 "ID C "~ 8 " " 4 i "r I i 5 ljo 1~5 2 0 // ;0 9 Crn,n) Fig.l. Time curse fr the ttal (9 9 and nn-specific ( binding f 3H-gliquidne (20 nml/1) t lipsmes as measured in the absence and presence f unlabelled gliquidne (0.1 mml/1), respectively. Mean _+ SM values refer t fur individual determinatins and are expressed as cpm. 10-3/rag phsphlipid M. Deleers and W. J. Malaisse: Binding f hypglycaemic sulphnylureas sumed t represent the nn-specific binding, it shuld be substracted frm ther readings in rder t characterize the specific binding f 3H-sulphnylurea. In a range f cncentratins frm 0.2 t nml/1, such a specific binding was nt quite prprtinal t the drug cncentratin (Fig. 2), the specific binding recrded at the highest cncentratin amunting t nly 63% (glibenclamide) and 77% (gliquidne) f the theretical values btained by linear extraplatin f the data cllected at the fur lwest cncentratins f sulphnylurea (i. e. between 0.2 and 2.0 nml/1). Figure 3 illustrates the effect f increasing cncentratins f unlabelled gliquidne and gliclazide upn the specific binding f 3H-glibenclamide (10.0 nml/1). Within the range f cncentratins under investigatin (10.0 nml/1 t 0.] mml/1), the binding f 3H-glibenclamide was much mre severely inhibited by gliquidne than by gliclazide. Tlbutamide als slightly decreased 3H-glibenclamide binding (p<0.01), whereas chlrprpamide failed t affect 3H-glibenclamide binding significantly (p> 0.1; data nt shwn). The inhibitin f 3H-gliquidne-specific binding by unlabelled sulphnylureas is illustrated in Figure 4. The three drugs tested, namely gliclazide, tlbutamide, and chlrprpamide, all caused significant inhibitin. The slpes f the regressin lines fr the inhibitry effect were nt vastly different frm ne anther. Hwever, the cncentratins f sulphnylurea required t cause 50% inhibitin f 3H-gliquidne-specific binding spanned a range f at least tw rders f magnitude, averaging 0.6 lxml/1 with gliclazide, 6.4.Ltm1/l with tlbutamide and 0.1 retl/1 with chlrprpamide. Discussin In this study we have investigated the binding f hypglycaemic sulphnylureas t lipsmes frmed f egg /// --~" 0.6 // 0.6 ////~ "t].c 0.4 "1:] ~: 0.2 / O / / + ///. O4 //I 4- ( g ~ 0.2._g O ; I'O 0 0 GL,BNCLAMID Gmil0 / /// GLIQUIDON ~ml/b Fig. 2. Relatinship between cncentratin and specific binding f 3H-glibenclamide and 3H-gliquidne t lipsmes. Mean + SM values refer t six t ten individual determinatins and are expressed as pml sulphnylurea bund/mg phsphlipid. The dtted lines illustrate relatinships f prprtinality with a slpe derived frm the data cllected at the fur lwest cncentratins f sulphnylurea ( nml/1)

M. Deleers and W. J. Malaisse: Binding f hypglycaemic sulphnylureas 57 ~ MJ r 100 80 "0 " 6C "0 -~ 40 u c 0~.Q ~ 20-1- 9 "2. 0 i i I i i 10-8 10-7 10-6 10-5 10-4 SULPHONYLURA (m,/,) Fig.3.

3 M. Deleers and W. J. Malaisse: Binding f hypglycaemic sulphnylureas 57 ~ MJ r "0 " 6C "0 -~ 40 u c 0~.Q ~ "2. 0 i i I i i SULPHONYLURA (m,/,) Fig.3. Inhibitin f the specific binding f 3H-glibenclamide (10 nml/1) t lipsmes by increasing cncentratins f unlabelled gliquidne ( ) r gliclazide (O O). ach value refers t the pint-mving mean derived frm quintuplate measurements perfrmed at each cncentratin f sulphnylurea. The SM fr individual measurements averaged 9%. Q G 100 "... ; " 80 "".. "'. ~ \ \\ ~ \ 9.. N % \ " \\\ 60 ~ \ "IO c "% ~ " g ~ 2 I "r 0 I l I I I 10 "6 10 " SULPHONYLURA (m I/I) Fig. 4. Inhibitin f the specific binding f 3H-gliquidne (I 0 nmi/1) t lipsmes by increasing cncentratins f unlabelled gliclazide ( tlbutamide ( A zx ) and chlrprpamide (9169 Same presentatin as in Fig.3. The SM fr individual measurement averaged 17%. ylk phsphatidylchline. Our results refer t a binding phenmenn rather than penetratin f the drug int the vesicular lumen. Indeed, at the cncentratin f phsphlipid used here (0.5 rag/250 gl), the intravesicular space f multilamellar lipsmes des nt exceed 1% f the vlume f incubatin medium [14], where- as the lipsme-assciated radiactivity represented 34%-64% f the ttal radiactivity present in each sample. Three majr analgies can be fund between the present results and thse btained with natural membranes. First, in ur system just as in brain membranes [11] r intact islets [15-17], the ttal binding f [3H] sulphnylurea increased prgressively, albeit nt prprtinally, as the cncentratin f sulphnylurea was raised in a range between 0.2 nml/1 and 0.1 mml/1. At the latter cncentratin, the equilibrium value fr ttal binding t intact islets averaged 0.15, 0.67 and 3.15 mml/kg dry weight in the case f tlbutamide [15], glibrnuride [16] and glibenclamide [17], respectively. If these values are representative f binding t the plasma membrane, as prpsed by the Ume5 grup, they wuld crrespnd, in the case f glibenclamide, t a sulphnylurea/ phsphlipid mlar rati clse t unity. This estimatin is based n the knwledge that, in pancreatic islets, the plasma membrane/dry weight rati is clse t 1700 m2/kg dry weight [18, 19] and that the cntent in phsphlipids (averaged mlecular weight taken as 750) f bilgical membranes is clse t 1.4 mg/m 2 [20, 21]. Hwever, if the hypglycaemic sulphnylureas were lcated nt nly in the plasma membrane but als in the membrane f intracellular rganelles - a view which cannt yet be ruled ut - the glibenclamide/ phsphlipid mlar rati culd fall frm 1.0 t a value as lw as 0.02, the ttal phsphlipid cntent f islets averaging 230 mml/kg prtein [22]. In the present system and at the same initial cncentratin f sulphnylurea (0.1 retl/l), the sulphnylurea/phsphlipid rati was clse t The latter values culd be smewhat underestimated. Indeed, if allwance is made fr the fact that we are dealing with multilamellar lipsmes (apprximately 5-15 layers) [23], the true binding t the uter bilayer f phsphlipids culd be higher than the value calculated by reference t the ttal amunt f phsphlipid present in each sample. There appears, therefre, t be a fair agreement between data cllected in living cells and ur artificial mdel. A secnd analgy between the artificial and bilgical systems cnsists in the fact that it is pssible, in bth mdels, t islate a cmpnent f the binding phenmenn which can be peratinally defined as specific binding. The saturatin f this specific binding appeared t be reached at much lwer cncentratins f sulphnylurea in brain membranes (1.0 nml/l) than in lipsmes. It culd be disputed that ur artificial membrane cntains n specific receptrs and hence the terms 'specific' and 'nn-specific' binding were used here incrrectly. Hwever, the aim f the present study was t test precisely whether artificial membranes devid f specific receptr might behave phenmenlgically in the same manner as natural membranes. Our experimental data demnstrate that, within limits, such is the case. A similar situatin was recently bserved fr the binding f tumur-prmting phrbl esters t lip-

4 58 M. Deleers and W. J. Malaisse: Binding f hypglycaemic sulphnylureas Table 1. Relative bilgical ptency f hypglycaemic sulphnylureas (and a sulphnamide) Sulphnylurea Mlecular A B C D F G H K weight (daltns) Glibenclamide Glisxepide Glipizide Gliquidne Glibrnuride Gliclazide Tlazamide Carbutamide 271 Tlbutamide Glycdiazine a Chlrprpamide a Sulphnamide. The relative bilgical ptency f each drug was judged frm (A) the dse-actin relatinship fr insulin release by pieces f rat pancreatic tissue [26, 27]; (B) the dse-actin relatinship fr the maximal fall in bld glucse cncentratin after ral administratin t nrmal subjects [28]; (C) the dse-actin relatinship fr the maximal decrease in bld glucse cncentratin after intravenus administratin t nrmal cnscius dgs [29]; (D) the dses causing a 30% decrease in bld glucse cncentratin after intravenus administratin t nrmal healthy vlunteers [30, 31]; () the dses yielding cmparable integrated glycaemic prfiles after intravenus administratin t nrmal subjects [32]; (F) the dses prvking cmparable glycaemic decreases after ral r intravenus administratin t nrmal subjects [33, 34]; (G) the minimal therapeutic dses [11, 35], chlrprpamide being excluded frm this series because f its unusually lng half-life; (H) the increments in glucse fractinal remval rate (K value) bserved after intravenus administratin f a lw dse (5 mg/kg bdy weight) f each drug t anaesthetized dgs [36, 37]; and (K) the decrease in bld glucse cncentratin bserved 2 h after ral administratin f a lw dse (10 mg/kg bdy weight) f sulphnylurea t mnkeys [38]. In each clumn, the data are expressed relative t ne anther, accrding t the reference(s) cited. The abslute values in different clumns were adjusted t prvide cmparable data fr the same drug(s). Sme agents, which are nt cited elsewhere in this reprt, are listed in the Table because they were used t establish the crrespndence between the different clumns. All cmparisns refer t the cncentratin r dsage required t achieve a given secretry r glycaemic respnse, results being expressed in weight units rather than mlar units smes and bilgical membranes, respectively [24]. It shuld be emphasized that the distinctin between specific and nn-specific binding in the present wrk neither implies nr rules ut the existence f distinct mdalities f interactin between sulphnylureas and phsphlipids. The present data merely reflect a lack f prprtinality between ttal binding and drug cncentratin. Further wrk is required bth t characterize the mdality r mdalities f interactin (e. g. hydrphbic versus hydrphilic) between hypglycaemic sulphnylureas and phsphlipids and t distinguish, in bilgical membranes, between the insertin f these agents in the phsphlipid dmain and their pssible binding t membrane-assciated prteins. A third and striking analgy was fund in the ability f distinct sulphnylureas t inhibit the binding f a given drug. In the lipsmes, the Ki fr inhibitin f3h - glibenclamide by gliquidne was clse t 2.1 ~tml/1, whereas bilgically less ptent sulphnylureas (gliclazide, tlbutamide, chlrprpamide) tested at cncentratins up t 0.I mml/1 caused nly mdest t marginal decreases in 3H-glibenclamide-specific binding. The relative ptency f gliclazide, tlbutamide and chlrprpamide in inhibiting 3H-gliquidne binding t lipsmes paralleled their respective bilgical ptency as insulin secretaggues (Table 1). The Ki was clse t 6.4 t.tml/1 with tlbutamide, which cmpares favurably with the values f 2.7 ~tml/1 reprted by Kaubisch et al. in brain membranes [11]. Likewise, in intact islets frm besehyperglycaemic mice, the ttal binding f 3H-glibencla- mide is unaffected by tlbutamide [25], that f 3H-glibrnuride reduced by glibenclamide (-21%) but nt by tlbutamide [16], and that f 3H-tlbutamide inhibited (-34%) by glibenclamide [15], all experiments being carried ut at a 20 ~tml/1 cncentratin f 3H-sulphnylurea and a 0.1 mml/1 cncentratin f the unlabelled ptential inhibitr. Thus, whether in intact islets, brain membranes r lipsmes, there was a clse parallel between the relative bilgical ptency f distinct sulphnylureas as insulin secretaggues and their relative ability t cause reciprcal inhibitin f binding. These analgies raise the idea that the insertin f hypglycaemic sulphnylureas in the phsphlipid dmain f the B cell membrane may represent the first step in the sequence f cytphysilgical events leading t stimulatin f insulin release by these agents. Hence, the ability f distinct sulphnylureas t penetrate int the phsphlipid dmain f cell membrane(s) culd well represent ne f the main factrs cnditining their bilgical ptency. Acknwledgements. The authrs are grateful t M. Mahy fr technical assistance and C. Demesmaeker fr secretarial help. This wrk was supprted in part by grants frm the Belgian Fundatin fr Scientific Research and Belgian Ministry f Scientific Plicy. References 1. Malaisse WJ, Mahy M, Brissn GR, Malaisse-Lagae F (1972) The stimulus-secretin cupling f glucse-induced insulin release. VIII. Cmbined effects f glucse and sulfnylureas. ur J Clin Invest 2:85 90

M. Deleers and W. J. Malaisse: Binding f hypglycaemic sulphnylureas 59 2. Lebrun P, Malaisse WJ, Herchuelz A (1982) Mdalities f gliclazide-induced Ca 2+ influx int the pancreatic B cell.

5 M. Deleers and W. J. Malaisse: Binding f hypglycaemic sulphnylureas Lebrun P, Malaisse WJ, Herchuelz A (1982) Mdalities f gliclazide-induced Ca 2+ influx int the pancreatic B cell. Diabetes 31: Henquin JC (1980) Tlbutamide stimulatin and inhibitin f insulin release: studies f the underlying inic mechanisms in islated rat islets. Diabetlgia 18: Henquin JC, Meissner HP (1982) Oppsite effects f tlbutamide and diazxide n 86Rb fluxes and membrane ptential in pancreatic B-cells. Bichem Pharmacl 31: Meissner HP, Preissler M, Henquin JC (1980) Pssible inic mechanisms f the electrical activity induced by glucse and tlbutamide in pancreatic B cells. In: Waldhfiusl WK (ed) Diabetes. xcerpta Medica, Amsterdam, pp Hellman B (1981) Tlbutamide-stimulatin f 45Ca fluxes in micrdissected pancreatic islets rich in B-cells. Mlec Pharmac120: Malaisse WJ, Hubinnt C, Lebrun P, Herchuelz A, Cuturier, Deleers M, Malaisse-Lagae F, Sener A (1983) Mde f actin f hypglycemic sulfnylureas in the pancreatic B cell: cinciding and cnflicting views. In: Serran-Ris M, Krall LP (eds) Clinical and pharmaclgical activities f sulfnylurea drugs. xcerpta Medica, Amsterdam pp Hellman B, Sehlin J, T/Jljedal IB (1971) The pancreatic fl cell recgnitin f insulin secretaggues. II. Site f actin f tlbutamide. Bichem Biphys Res Cmmun 45: Hellman B (1974) Factrs affecting the uptake f glibenclamide in micrdissected pancreatic islets rich in r-cells. Pharmaclgy 11 : Hellman B, T~ljedal IB (1979) ffects f sulfnylurea derivatives n pancreatic fl cells. In: Hasselblatt A, Bruchhausen FV (eds) Insulin II. Springer Verlag, Berlin Heidelberg New Yrk, pp Kaubisch N, Hammer R, Wllheim C, Renld A, Offrd R (1982) Specific receptrs fr sulfnylureas in brain and in a r-cell tumr f the rat. Bichem Pharmacl 31: Orci L, Ravazzla M, Amherdt M, Malaisse-Lagae F (1974) The fl cell bundary. In: Malaisse WJ, Pirart J (eds) Diabetes. xcerpta Medica, Amsterdam, pp ] Deleers M, Malaisse WJ (1980) Inphre-mediated calcium exchange diffusin in lipsmes. Bichem Biphys Res Cmmun 95: Szka F, Papahadjpuls D (1978) Prcedure fr preparatin f lipsmes with large internal aqueus space and high capture by reverse-phase evapratin. Prc Natl Acad Sci USA 75: Sehlin J (1973) vidence fr specific binding ftlbutamide t the plasma membrane f the pancreatic B cells. Acta Diabetl lat 5: T/iljedal IB (1974) Uptake f glibrnuride by micrdissected pancreatic islets. Hrmne Res 5: Hellman B, Sehlin J, T~iljedal IB (1973) The pancreatic B cell recgnitin f insulin secretaggues. IV. Islet uptake f sulfnylurea. Diabetlgia 9: Malaisse WJ, Lebrun P, Herchuelz A (1982) Inic determinants f bielectrical activity in the pancreatic B-cell. Pflfigers Arch 395: Deleers M, Gelbcke M, Malaisse WJ (1983) Transprt f Pr 3+ by hypglycemic sulfnylureas acrss lipsmal membranes. FBS Lett 151 : Philips MC (1972) The physical state f phsphlipids and chlesterl in mnlayers, bilayers and membranes. Prgr Surf Membrane Sci 5: Singer SJ (1975) Architecture and tpgraphy f bilgic membranes. In: Weissmann G, Clairbne R (eds) Cell membranes; bichemistry, cell bilgy and pathlgy. HP Publishing, New Yrk, pp Mntague W, Parkin N (1980) Changes in membrane lipids f the r-cell during insulin secretin. In: Malaisse WJ, Tfiljedal IB (eds) Bichemistry and biphysics f the pancreatic B-cell. Hrm Metab Res (Suppl) 10: Szka F, Papahadjpuls D (1980) Cmparative prperties and methds f preparatin f lipid vesicles. Ann Rev Biphys Bieng 9: Deleers M, Malaisse WJ (1982) Binding f tumr-prmting and bilgically inactive phrbl esters t artificial membranes. Cancer Lett 17: Hellman B (1974) Ptentiating effects f drugs n the binding f glibenclamide t pancreatic beta cells. Metablism 23 : Malaisse WJ, Leclercq-Meyer V (1972) Insulintrpic actin f a new sulphnylurea: gliclazide. Rev urp tud Clin Bil 17: Herchuelz A, Malaisse WJ (1973) Insulintrpic ptency f glipizide in vitr. Diabetlgia 9: Puls W, Keup U, Frerichs M (1974) Beeinflussung der Glykfimie und Insulinfimie unter Khlenhydratbelastung durch Pr-Diaban an gesunden Versuchspersnen. In: Schrffiing K, Krneberg G, Laudahn G (eds) Pr-Diaban. F. K. Schattauer, Stuttgart, pp Lubatieres A, Lubatieres-Mariani MM (1974) xperimental study f the betacyttrpic and betacyttrphic actin f glisxepide (Pr-Diaban). In: Sch6ffling K, Krneberg G, Laudahn G (eds) Pr-Diaban, F. K. Schattauer, Stuttgart, pp Haupt, KOberich W, Beyer J, Schrffling K (1971) Pharmacdynamic aspects f tlbutamide, gtibenclamide, glibrnuride and glisxepide. I. Dse respnse relatins and repeated administratin in diabetic subjects. Diabetlgia 7: Haupt K, Kiillmer G, Sch~Jffling K (1976) Pharmacdynamics f glurenrm. Diab Cratica 5: Raptis S, Laube H, Katsilambrs N, Hinz M, Rthmann G, Pfeiffer F (1974) Tierexperimentelle und klinische Untersuchungen mit dem neuen Sulfnylharnstff Glisxepid (Pr-Diaban). In: Schrffling K, Krneberg G, Laudahn G (eds) Pr-Diaban, F.K. Schattauer, Stuttgart, pp Zilker T, Li~ders L, Bttermann P (1975) Vergleichende Anwendung vn Gliquidne und Tlbutamid nach intraven6ser Gabe. Mtinch Med Wchenschr 117: Zilker T, Waldthaler A, rmler R, Bttermann P (1975) Wirkung vn Gliquidn und Glibenclamid nach raler Anwendung. Mfinch Med Wchenschr 117: Balant L (1981) Clinical pharmackinetics f sulphnylurea hypglycaemic drugs. Clin Pharrnackin 6: Bellens R (1961) Cntributin fi l'rtude des mrcanismes d'actin des drgues hypglycrmiantes. Acta ndcrinl (Kbh) supplem Malaisse W, Bellens R, Francksn JRM (1964) Cmparaisn de l'actin de deux drgues hypglycrmiantes sur la glycrmie et l'assimilatin glucidique du chien nrmal. Arch lnt Pharmacdyn Ther 149: Schneider JA, Salgad D, Jaeger D, Delahunt C (1959) The pharmaclgy f chlrprpamide. Ann NY Acad Sci 74: Received: 15 March 1983 and in revised frm: 24 June 1983 Prfessr W.J. Malaisse Labratry f xperimental Medicine Brussels University Medical Schl 115, Bulevard de Waterl B-1000 Brussels, Belgium

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