Jpn J ClinChem 19:350―353,1990. "A rticle" Action of Dilazep Dihydrochloride on Chemiluminescence from a Xanthine Oxidase/Acetaldehyde System Yasunobu Yoshinaka*, Toru Yokoyama* and Masaki Nakamura* *Tokyo Research Laboratories , Kowa Co., Ltd., 2-17-43 Noguchi-cho, Higashimurayama-city, Tokyo 189, Japan Received on Apr. 12, 1990, Accepted on Aug. 17, 1990 Key words: dilazep, chemiluminescence,xanthine oxidase, radical scavenger Summary Superoxide dismutase ISOD), catalase, hydroxyl radical (OH) scavengers and singlet oxygen (102) quenchers suppress the light signal from a xanthine oxidase/acetaldehyde system. Xanthine oxidase inhibitor allopurinol also suppresses the light signal from the system. Dilazep HCI (10-5-10-3M) suppresses the chemiluminescence in a concentration dependent manner without affecting the xanthine oxidase activity.These results suggest that dilazep exhibits a radical scavenger-like action. damage8).In this study,we determined the radi- Introduction cal scavenging effect of dilazep using chemilu- minescence from a xanthine oxidase/acet- Tetrahydro-1H-1,4-diazepine-1,4(5H)-dipro- aldehyde system. panol bis(3,4,5-trimethoxybenzoate) dihydroch- loride monohydrate (dilazep) has been success- Materials and Method fully employed in the treatment of patients with ischemic heart diseases and sequelae of cerebral The sources of materials used were as fol- vascular diseases.It exhibits unique pharmaco- lows:xanthine oxidase (grade ifi from butter logical effects,such as adenosine potentiation1,2), milk),superoxide dismutase(SOD from bovine platelet aggregation inhibition3),membrane stabi- erythrocytes)and catalase(from bovine liver) lization4) and improvement of red blood cell from Sigma,ethylenediaminetetraacetic acid dis- deformability5).Recent evidence indicates that odium salt (EDTA),1,4-diazabicyclo-(2,2,2)- dilazep prevents brain injury and brain lipid octane(DABCO),dimethylsulfoxide (DMSO) and peroxidation induced by reperfusion in rate and mannitol from Wako Pure Chemicals,dilazep decrement in cerebral blood flow caused by re- and sodium allopurinol from Kowa,sodium azide perfusion in dogs7) although the mechanism(s) is and benzoic acid from Showa Chemicals,acet- unclarified.Recently,oxygen radicals have been aldehyde from Merck,ethanol from Katayama suggested as mediators of ischemic brain Chemicals and methionine from Daiichi Chemi- 350 Fig.2.Dose-response curves of inhibition of drugs on chemiluminescence in a xanthine oxidase/ aeetaldehyde system Catalase(○);SOD(●);allopurinol(△); dilazep(▲);DMSO(□). Conditions are as in Fig.1. was started by the addition of 0.05ml of 1M .acetaldehyde to the reaction mixture,and the photon count was recorded for 7min.A net che- miluminescence value was calculated by the fol- Fig.1.Dilazep inhibition of the xanthine oxidase/ lowing equation: aeetaldehyde reaction Acetaldehyde(10mM)was added at zero time. Net chemiluminescence=Recorded count•| The xanthine oxidase concentration was 50 Background count mU/ml.Dilazep was added prior to acetal- The inhibition ratio of each tested substance dehyde addition. was determined at the peak time of the control Control(○― ○);10-6Mdilazep(● ― ●) reaction. 10.5Mdilazep(△― △);10-4M dilazep(▲― Xanthine oxidase activity with 1mM xanthine ▲);10-3Mdilazep(□― □). The sereactions were carried out in 5ml of as the substrate was assayed at 37•Ž by 0.05M potassium phospｈate,10-4M EDTA, measuring the absorption of uric acid at 292nm. pH7.8,30℃. Duplicate assays were performed for each experiment,and the result was expressed as the mean. cals. Chemiluminescence was measured by a chem- Results iluminescence analyzer(OX-7,Tohoku Electro- nics,Co.,Ltd.).One minute readings were made The time course of the xanthine oxidase/ for all experiments at 30•Ž.Five milliliters of aldehyde reaction and the effect of dilazep on potassium phosphate buffer(0.05M,pH7.8,con- the reaction are given in Fig.1.As reported by taining 0.1mM EDTA)was used in each stain- Arneson9),the chemiluminescence of the xanthine less steel dish and 0.05ml of 5U/ml xanthine ox- oxidase/acetaldehyde system first increased and idase and a small volume (0.05-0.25ml) of rea- then decreased. Dilazep inhibited the chemilu- gents were added.Each dish was incubated in- minescence dose-dependently.The IC50(the side the counting chamber and background concentration of each drug required to inhibit counts were read for 3 min before initiation of the chemiluminescence by 50%)of dilazep is2.9 the chemiluminescence reaction. The reaction 10-5 M(Fig.2).Catalase,SOD,allopurinol •~ Jpn J Clin Chem Vol.19,No.4,Dec.,1990.351 Fig.3.Inhibition of chemilumineseene einaxanth- Fig.4.Effeet of dilazep(○)and allopurinol(●)on. ine oxidase/aeetaldehyde system by1O2quen- xanthine oxidase activity chers and・OH seavengers Xanthine oxidase(3mU)was preincubated at Conditions are as in Fig.1. 37℃with dilazep or allopurinol in 0.9ml of Tris buffer(0.078M,pH8.0).The reaction and DMSO10),a hydroxyl radical scavenger,also was started by adding 0.1ml of 10mM xanth- ine solution.Thirty minutes after the begin- inhibited the chemiluminescence dose-depen- ning,1ml of 10%PCA was added in the reac- dently(Fig.2),and the IC50 values are 1.7× tion mixture and the formation of uricacid 10-9M,3.5×10-9M,3.8×10-6 M and 3.0× was measured. 10-3M,respectively.As shown in Fig.3,DAB- CO(10-2M),a quencher of 1O211),inhibited the chemiluminescence by87%.Other quenchers of The chemiluminescence from the xanthine lO 211),histidine(His,10-2M)and sodium azide oxidase system has been attributed to the pro- (10-2M),inhibited the chemiluminescence by duction of1O2 by the interaction of O2- with 97%and62%,respectively.Mannitol(Man,0.1 H2O29).On the other hand,Nagano and M),methionine(Met,10-2M),benzoic acid Fridovichll) reported that there is no detectable (Benz,10-2M)and ethanol(EtOH,0.24M),which production of1O2by the xanthine oxidase reac- are known ･OH ScavengerS10,11),inhibited the Che- tion.Thus,we investigated the effect of 1O2 miluminescence by74%,99%,76%and52%,re- quenchers (DABCO,histidine and azide) on the spectively. chemiluminescence from the xanthine oxidase Fig.4 shows the effects of allopurinol and reaction.The results show that these1O2quen- dilazep on xanthine oxidase activity.Allopurinol chers inhibit chemiluminescence.We also deter- directly inhibited xanthine oxidase(IC50 value mined that SOD,catalase and •EHO scavengers is5•~10-6M),whereas dilazep had no signifi- (DMSO, mannitol,methionine,benzoic acid and cant effect.The IC50 of allopurinol on xanthine ethanol) inhibit chemiluminescence.These re- oxidase activity was about the same as on che- sults suggest that not only O2-,H2O2 and •EOH miluminescence. but also 1O2 is responsible for the chemilu- minescence.Dilazep inhibited the chemilu- Discussion minescence in such a system,whereas it has no significant effect on the xanthine oxidase reac- We did not use xanthine,but instead acetal- tion,indicating that dilazep exhibits a scaven- dehyde,as the substrate in the xanthine oxidase ger-like function.Further work is necessary to system,because the luminescense yield for the identify the radical species scavenged by xanthine oxidase/acetaldehyde system was high- dilazep.Allopurinol inhibited both the chemilu- er than that of xanthine oxidase/xanthine sys- minescence and the oxidation of xanthine at the tem,and the reaction with acetaldehyde was same concentration.This indicates that allopuri- highly reproducible. nol inhibited the chemiluminescence through the 352 inhibition of xanthine oxidase and not through a 4) Hirai K and Koketsu K: Effect of comelian on radical scavenging effect. bullfrog sympathetic ganglion cells, Kurume- Dilazep is reported to be effective in pre- Igakkai-Zasshi, 43: 460-467, 1980 (in Japanese). 5) Yamamoto S, Nakajima K and Nagakura M: A venting re-circulation impairment6)and lipid possible mechanism of dilazep (Comelian) on en- peroxi dation7)after cerebral ischemia.The hancement of red cell deformability, Jan Pharma- mechanisms of these actions have not been clar- col Ther, 11: 4273-4277, 1983 (in Japanese). ified.Recently,oxygen free radicals have been 6) Kondo S, Kawada M and Sano N: Effects of suggested as mediators of ischemic brain dilazep on cerebral blood flow under normal con- damage8).Therefore,we tested the efficacy of ditions and recirculation impairment after cere- dilazep in scavenging free radicals in a xanthine bral ischemia, Folia Pharmacol Japon, 77: oxidase system. The results show that dilazep 205-211, 1981 (in Japanese). has a radical scavenging function and the IC50 7) Yamauchi Y, Ikuta J, Shimizu S and Nakamura M value is 2.9•~10-5M.This inhibition of dilazep : Effect of dilazep dihydrochloride on ischemia is stronger than that of other radical scavengers and reperfusion-indused cerebral injury in spon- taneously hypertensive rats, Folia Pharmacol and quenchers used in this study.In addition, Japon, 95: 239-246, 1990 (in Japanese). Yonemitsu,et al12)reported that dilazep has a 8) Demopoulos HB, Flamm ES, Seligman ML, high affinity for blood vessels,and Tamaki,et Jorgensen E and Ransohoff J: Antioxidant effects al13)showed that dilazep protects endothelial of barbiturates in model membranes undergoing cells from hydroperoxide-induced injury in vitro. free radical damage, Acta Neurol Scand, 56: These lines of evidence suggest that the free Suppl. 64, 152-153, 1977. radical scavenging activity of dilazep may playa 9) Arneson RM: Substrate-induced chemiluminesc- role in its brain protective action. ence of xanthine oxidase and aldehyde oxidase, Arch Biochem Biophys, 136: 352-360, 1970. References 10) Nakagami K, Shingu M and Nobunaga M: Oxygen radical-induced endothelial cell damage, The 1) Sano N, Katsuki S and Kawada M: Enhancement Saishin-Igaku, 39 : 1334-1341, 1984(in Japan- of coronary vasodilator action of adenosine by ese). 1,4-bis [3-(3,4,5-trimethoxybenzoyloxy)-propyl]- 11) Nagano T and Fridovich I: Does the aerobic perhydro-1, 4-diazepine (Dilazep I.N.N), Arznei- mittelforschung, 22:1655-1657, 1972. xanthine oxidase reaction generate singlet ox- 2) Yoshinaka Y and Uchida Y: Inhibition of ygen? Photochem Photobiol, 41: 33-37, 1985.