838 Notes Biol. Pharm. Bull. 24(7) 838—840 (2001) Vol. 24, No. 7

Inhibitory Effects of Ethaverine, a Homologue of , on Monoamine Oxidase Activity in Mouse Brain

a a a b a a Sang Seon LEE, Jae Joon LEE, Mi Jeong CHEONG, Young Ho KIM, Youngsoo KIM, Yeo Pyo YUN, a ,a Chong Kil LEE, and Myung Koo LEE* College of Pharmacy, Chungbuk National University,a San 48, Kaeshin-Dong, Heungduk-Ku, Cheongju 361–763, Republic of Korea and College of Pharmacy, Chungnam National University,b 220, Gung-Dong, Yoosung-Ku, Taejeon 305–764, Republic of Korea. Received December 11, 2000; accepted March 12, 2001

The effects of compounds such as ethaverine, , and tetrahydropapaverine on monoamine oxidase (MAO, EC 1.4.3.4) activity in mouse brain were investigated. Ethaverine showed an inhibi- tion of MAO activity in a concentration-dependent manner (57.6% inhibition at 40 mM). Papaverine also inhib- ited MAO activity (38.1% inhibition at 40 mM). However, laudanosine and tetrahydropapaverine did not inhibit

MAO activity. The IC50 value of ethaverine for MAO was 25.5 mM. Ethaverine non-competitively inhibited MAO activity with a substrate kynuramine. The Ki value for ethaverine was 11.9 mM. In addition, ethaverine proved to preferentially inhibit type B MAO activity in a concentration-dependent manner, with an IC50 value of 32.8 mM. These results suggest that ethaverine partially contributes to the regulation of catecholamine content. Key words ethaverine; papaverine; laudanosine; tetrahydropapaverine; kynuramine; monoamine oxidase

Ethaverine is a benzylisoquinoline alkaloid and a tetra- MATERIALS AND METHODS ethoxy homologue of papaverine from Papaver somniferum L. (Papaveraceae) (Fig. 1), and is used therapeutically as a Materials Ethaverine hydrochloride, papaverine hydro- 1) potent vasodilator. This action is mediated to inhibit the L- chloride, laudanosine, tetrahydropapaverine hydrochloride, type Caϩϩ channels responsible for Caϩϩ influx in arterial kynuramine dihydrobromide, 4-hydroxyquinoline, and zinc and cardiac muscle cells.2) Ethaverine is also known to re- sulfate were purchased from Sigma Chemical Company (St. ϩϩ duce catecholamine secretion through blocking L-type Ca Louis, MO, U.S.A.). All other reagents were of reagent channels in PC12 cells.3) These data suggest that ethaverine grade. regulates catecholamine content, depending upon such bio- MAO Preparation and Assay for MAO Activity Mice logically active sites as the central nervous system and pe- (male, ICR, 25—30 g) were obtained from the Samyook Ani- ripheral tissues. mal Laboratory, Inc. (Osan, Korea). Mice received laboratory

Monoamine oxidase [MAO, monoamine: O2 oxidoreduc- chow and tap water ad libitum and were killed by cervical tase (deaminating), EC 1.4.3.4] plays an important role in dislocation by the guidelines for the care and use of labora- the catabolism of catecholamines, serotonin, and dietary tory animals. For MAO enzyme preparation, a crude mito- amines.4) MAO is classified into two types, type A and B chondrial fraction was isolated from mouse whole brain ac- (MAO-A and -B), according to different sensitivities to cording to the method of Naoi et al.7) MAO activity was inhibition by selective MAO inhibitors and substrate prefer- measured fluorimetrically using kynuramine as an amine ences.5,6) MAO-A is inhibited by low concentrations of clor- substrate according to the method of Kraml15) with a slight gyline, and MAO-B by low concentrations of deprenyl and modification.7,14) 4-Hydroxyquinoline formed by the enzyme pargyline. The inhibitors of these selective MAO are applied reaction for 30 min at 37 °C was measured using a fluores- to the treatment of some neuropsychiatric disorders such as cence spectrophotometer (lex, 315 nm/lem, 380 nm, Hitachi depression, alcoholism and schizophrenia, which are closely Model F-3000, Tokyo, Japan). To differentiate type A and B related to the catecholamine content in the central nervous activities, MAO activities were measured in the presence of 6) system. 1 m M clorgyline and deprenyl with a pre-incubation time of Many inhibitors of MAO activity are reported: isoquino- 15 min.5,16,17) lines and tetrahydroisoquinolines,7,8) 4-(2-benzofuranyl)- Data Analysis The values of the Michaelis constant 9) 10) 11) piperidines, oxadiazoles, phenoxathin-10,10-dioxides, (Km) and the maximum velocity (Vmax) were obtained by and natural xanthones.12) In addition, tetrahydropapaveroline Lineweaver–Burk’s plot using various concentrations of and higenamine belong to a group of benzylisoquinoline de- kynuramine. The amount of protein was determined by the rivatives and exhibit an inhibitory effect on MAO activ- method of Lowry et al.18) using bovine serum albumin as a ity.13,14) standard. The results represent meansϮS.E.M. of five experi- The present study was, therefore, undertaken to examine ments. Statistical significance was determined at the pϽ0.05 the effects of ethaverine and its structural analogues, pa- level by Student’s t-test. paverine, laudanosine and tetrahydropapaverine, on MAO ac- tivity in mouse whole brain using kynuramine as a substrate RESULTS AND DISCUSSION to determine whether these compounds have an ability to regulate catecholamine content. Many isoquinoline compounds such as salsolinol, N- methylisoquinoline, tetrahydropapaveroline, and higenamine inhibit MAO activity.7,8,13,14) Among them, tetrahydropapa-

∗ To whom correspondence should be addressed. e-mail: [email protected] © 2001 Pharmaceutical Society of Japan July 2001 839

Table 1. Effects of Benzylisoquinoline Compounds on Monoamine Oxi- dase (MAO) in Mouse Brain

MAO activity IC values Compounds (nmol/min/mg protein) 50 (m M) (% of control)

Control 0.320Ϯ0.012 (100) Iproniazid 12.5 1 m M 0.301Ϯ0.021 (94.0) 10 m M 0.163Ϯ0.016 (50.9)** 20 m M 0.125Ϯ0.013 (39.1)*** Clorgyline 1.4 0.1 m M 0.207Ϯ0.014 (70.1)* 1 m M 0.174Ϯ0.018 (54.3)** 10 m M 0.062Ϯ0.009 (19.5)*** Fig. 1. Structures of Various Benzylisoquinoline Compounds Ethaverine 25.5 20 m M 0.166Ϯ0.011 (51.8)** 40 m M 0.136Ϯ0.009 (42.4)*** 80 m M 0.121Ϯ0.012 (38.2)*** Papaverine 20 m M 0.255Ϯ0.018 (79.7)* 40 m M 0.198Ϯ0.011 (61.9)** 80 m M 0.161Ϯ0.012 (50.3)** Laudanosine 25 m M 0.391Ϯ0.006 (122) 50 m M 0.356Ϯ0.003 (111) 100 m M 0.345Ϯ0.004 (107) Tetrahydropapaverine 25 m M 0.328Ϯ0.005 (102) 50 m M 0.334Ϯ0.007 (105) 100 m M 0.324Ϯ0.010 (101)

The control of MAO activity, 0.320 nmol/min/mg protein, was taken as 100. Iproni- azid and clorgyline were used as the positive control. The data are expressed as meansϮ S.E.M of five experiments. As compared with control value: *, pϽ0.05; **, pϽ0.01; ***, pϽ0.001 (Student’s t-test).

Fig. 2. Lineweaver–Burk Plot of MAO Activity in Mouse Brain in the Ab- Table 2. Inhibitory Effects of Ethaverine on Type A and Type B sence and Presence of Ethaverine Monoamine Oxidase (MAO-A and -B) in Mouse Brain The reciprocal of MAO activities was plotted against the reciprocal of substrate con- centrations. Ethaverine concentrations: 1, 0 m M.; 2, 20 m M; 3, 40 m M. MAO-A or -B activity IC50 value Compounds (nmol/min/mg protein) ( m M) (% of control) veroline and higenamine are grouped into benzylisoquinoline Control 0.326Ϯ0.019 compounds (Fig. 1). We, therefore, investigated the effects of MAO-A (deprenyl-treated) ethaverine, papaverine, laudanosine and tetrahydropapaver- (Controlϩdeprenyl) 0.137Ϯ0.009 (100) ine, which are benzylisoquinoline derivatives, on MAO activ- Ethaverine — Ϯ ity. 20 m M 0.123 0.010 (89.8) 40 m M 0.112Ϯ0.008 (81.7) MAO was partially purified from mouse brain, and the val- 80 m M 0.106Ϯ0.011 (77.4)* ues of Km and Vmax, determined by using the substrate kynu- MAO-B (clorgyline-treated) ramine, were 78.2Ϯ4.0 m M and 0.65Ϯ0.05 nmol/min/mg pro- (Controlϩclorgyline) 0.162Ϯ0.010 (100) tein, respectively (nϭ5). In this experiment, ethaverine ex- Ethaverine 32.8 Ϯ hibited an inhibitory effect on MAO activity in a concentra- 20 m M 0.089 0.007 (54.9)** 40 m M 0.075Ϯ0.006 (46.3)*** M tion-dependent manner (57.6% inhibition at 40 m ) (Table 80 m M 0.059Ϯ0.009 (36.4)*** 1). Papaverine also showed a mild inhibitory effect on MAO activity (38.1% inhibition at 40 m M). However, laudanosine MAO-A or -B activity was measured in the presence of 1 m M deprenyl or clorgyline, respectively. For further comments, see Table 1. and tetrahydropapaverine did not inhibit MAO activity (Table

1). The IC50 value of ethaverine for MAO activity was 25.5 m M. Ethaverine showed a less inhibitory effect on MAO ac- with an IC50 value of 32.8 m M (Table 2). tivity than iproniazid and clorgyline (Table 1). According to the structural suggestion of Severina,5) a hy- Kinetic analysis by a Lineweaver–Burk reciprocal plot in- drophobic region, a nucleophilic locus and an electrophilic dicates that ethaverine is a non-competitive inhibitor of MAO group on MAO are required for binding with inhibitors or with respect to kynuramine (Fig. 2). The Ki value for ethaver- substrates. Ethaverine has a hydrophobic pocket (–OC2H5 ine was 11.9 m M. radicals) and a nucleophilic locus (N group of isoquinoline Next, the inhibitory effects of ethaverine on MAO-A and ring) (Fig. 1, upper). Ethaverine and papaverine showed more MAO-B were investigated. Ethaverine preferentially inhib- potent inhibitory effects on MAO activity than higenamine 14) ited MAO-B activity in a concentration-dependent manner, (IC50 value of higenamine, 159.0 m M), but laudanosine and 840 Vol. 24, No. 7 tetrahydropapaverine did not. These results suggest that the Chubb J. M., Angiology, 37, 342—351 (1986). molecular planarity of the isoquinoline ring plays an impor- 2) Greenslade F. C., Scott C. K., Newquist K. L., Krider K. M., Chasin M., J. Pharm. Sci., 71, 94—100 (1982). tant role and that the –OH, –OCH3 or –OC2H5 radicals of the 3) Suh B. C., Kim K. T., Biochem. Pharmacol., 47, 1262—1266 (1994). R1, R2, R3 and R4 positions play a minor role in the inhibition 4) Tipton K. F., O’Carroll A. M., McCrodden J. M., J. Neural Transm. of MAO activity (Fig. 1). (Suppl.), 23, 25—35 (1987). Ethaverine is reported to inhibit phosphodiesterase in 5) Severina I. S., “Mechanism of Selective Inhibition by Clorgyline and smooth muscle and to inhibit catecholamine secretion by Deprenyl of the Possible Nature of Its Form A and B,” in Monoamine ϩϩ 2,3) Oxidase: Structure, Function, and Altered Functions, ed. by Singer T. blocking the L-type Ca channel. In addition, Shin et al. P., Von Korff R. W., Murphy D. L., Academic Press, New York, 1979, have reported that ethaverine decreases content by pp. 169—183. reducing the tyrosine hydroxylase activity in PC12 cells.19) 6) Deniker P., “The Search for New Antidepressant and Related Drugs,” in Monoamine Oxidase and Diseases; Prospects for Therapy with Re- The IC50 value (1.4 m M) of ethaverine for dopamine biosyn- thesis is much lower than that for MAO inhibition (IC versible Inhibitors, ed. by Tipton K. F., Dostert D., Benedetti M. S., 50 Academic Press, New York, 1984, pp. 3—8. value, 25.5 m M). PC12 cells have an MAO activity with type 7) Naoi M., Takahashi T., Darrez H., Kabeya R., Taguchi E., Neurochem. A. However, our preliminary experiments have shown that Int., 15, 315—320 (1989). dopamine content was not apparently altered by treatment 8) Bembenek M. E., Abell C. W., Chrisey L. A., Rozwadowska M. D., with 1 m M of clorgyline or deprenyl, which are selective Gessner W., Brossi A., J. Med. Chem., 33, 147—152 (1990). MAO-A or MAO-B inhibitors, in PC12 cells. These data in- 9) Strolin-Benedetti M., Dostert P. L., J. Neural Transm. (Suppl.), 23, 103—119 (1987). dicate that ethaverine uses a different mechanism between 10) Mazouz F., Gueddari S., Burstein C., Mansuy D., Milcent R., J. Med. dopamine biosynthesis in PC12 cells and the inhibition of Chem., 36, 1157—1167 (1993). MAO activity. Similar results were obtained when hige- 11) White H. L., Scates P., Drug Dev. Res., 25, 191—199 (1992). namine was used in PC12 cells: higenamine has been found 12) Suzuki O., Katsumata Y., Oya M., Chari V. M., Vermes B., Wagner H., to lower the dopamine content in PC12 cells despite its inhi- Hostettmann K., Planta Med., 42, 17—21 (1981). 20) 13) Giovine A., Renis M., Bertolino A., Pharmacology, 14, 86—94 bition of MAO activity. (1976). In summary, our present study indicates that ethaverine 14) Lee S. S., Yun-Choi H. S., Kim E. I., Lee M. K., Med. Sci. Res., 27, non-competitively inhibits MAO activity in mouse brain, and 71—72 (1999). the possibility that ethaverine has a dual role in the regulation 15) Kraml M., Biochem. Pharmacol., 14, 1684—1686 (1965). 16) Naoi M., Nomura Y., Ishiki R., Suzuki H., Nagatsu T., J. Neurochem., of catecholamine content can be proposed. The in vivo physi- 50, 243—247 (1988). ological functions of ethaverine need to be studied further. 17) Naoi M., Matsuura S., Parves H., Takahashi T., Hirata Y., Minami M., Nagatsu T., J. Neurochem., 52, 653—655 (1989). Acknowledgements The authors wish to acknowledge 18) Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J., J. Biol. the financial support of the Ministry of Public and Welfare Chem., 193, 265—275 (1951). 19) Shin J. S., Lee J. J., Kim Y., Lee C. K., Yun Y. P., Lee M. K., Biol. (HMP-97-D-4-0022, 1999), and the Brain Korea 21 project Pharm. Bull., 24, 103—105 (2001). of the Ministry of Education, Republic of Korea. 20) Shin J. S., Yun-Choi H. S., Kim E. I., Lee M. K., Planta Med., 65, 452—455 (1999). REFERENCES

1) Trainer F. S., Phillips R. E., Michie D. D., Zellner S. R., Hogan L., Jr.,