Life Sciences, Vol. 50, pp. PL-53 - PL-58 Printed in the U S A
PHARMACOLOGY Accelerated
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COMPARATIVE EFFEC'q'S OF TETRANDRINE AND BERBAMINE ON PRODUCTION OF THE INFLAMMATORY C Y T O K I N ~ INTERLEUKIN-1 AND TUMOR NECROSIS FACTOR
W.K. Scow (1), A. Ferrante (2), A. Summors (1), Y.H. Thong * (1) Immunobiology Laboratory, Department of Child Health, University of Queensland, Mater Childrens Hospital, South Brisbane 4101, Australia (1), and Department of Immunology, University Department of Paediatrics, Adelaide Childrens Hospital, North Adelaide 5006, Australia (2) (Submitted September 23, 1991; accepted October 30, 1991; received in final form December 17, 1991)
ABSTRACT: Tetrandrine and berbamine are bisbenzylisoquinoline compounds which differ from each other in a minor way in terms of chemical structure, yet tetrandrine is 6-18 times more potent than berbamine in terms of inhibitory effects on production of interleukin-1 and tumor necrosis factor (TNFa) by monocytes and macrophages, and TNFB production by lymphocytes. Moreover, tetrandrine significantly suppressed phosphoinositide turnover while berbamine did not. These findings may provide important insights into structure-activity relationships and the design of novel analogues and congeners useful in the therapy of chronic inflammatory and auto-immune diseases.
Tetrandrine is the major alkaloid of hanfangji, the tuberous root of the creeper Stephania tetrandra used since antiquity by Chinese physicians for the treatment of rheumatic diseases (1,2). Its ability to retard and even reverse the lesions of pulmonary silicosis in open clinical trials and controlled rat experiments (3,4) was attributed to inhibition of collagen synthesis (5). However, we conjectured that its mode of action may be on a more fundamental aspect of inflammation, and experiments in our laboratory have borne out this contention (6-14). Berbamine is a natural analogue of tetrandrine isolated from the root of Berberis soulieana, also used in Chinese ethnopharmacy for therapy of rheumatic diseases (2), but it was noted to be less effective for silicosis. However, recent studies in our laboratory showed that berbamine was more
~ N
OCH 3 OCH3
cH . ~
l NCH3
Figure 1. The chemical structure of tetrandrine OCH3) and berbamine (R = OH)
o
* To whom correspondence should be addressed. 0024-3205/92
$5.00
Copyright © 1992 Pergamon Press plc
+ .00
All rights reserved.
(R =
PL-54
Tetrandrine and Berbamine
Vol. 50, No. 8, 1992
effective in suppression of relapsing experimental allergic encephalitis (EAE) in rats (15). Since inflammatory cytokines are important mediators of immunologicaUy mediated tissue damage, we compared the effects of these two bisbenzylisoquinoline compounds on the production of interleukin-1 (IL-1) and tumor necrosis factor (TNF) from human monocytes-macrophages and lymphocytes. Methods
Tetrandrine and berbamine: Tetrandrine, empirical formula C38H4206N2 and molecular weight 622.73, was obtained from Jin Hua Pharmaceutical Company, Jijang Province, China as a pure powder (> 98%). It was dissolved in dilute HCI (1 X N) and its pH adjusted to 7.0 with dilute NaOH; this aqueous stock solution of tetrandrine (2mg/ml) was diluted in RPMI 1640 medium for the experiments. Berbamine, empirical formula C37Ha006N2 and molecular weight 608.71, was dissolved in sterile normal saline (2 mg/ml), and the stock solution diluted in RPMI 1640 medium for the experiments. The chemical structure of these two bisbenzylisoquinoline analogues are shown in Figure 1. The final concentrations (20 ug/ml) of these compounds used in the experiments were previously determined to be non-toxic to neutrophils, monocytes, lymphocytes and other cells, by means of trypan blue dye exclusion and the tetrazolium colorimetric assay.
Production of IL-I and TNF: Heparanized blood was layered onto Hypaque-Ficoll medium of density 1 .I 14 and centrifuged at 600g for 30 rain (16). The mononuclear leukocytes (MNC) in the top band (at the interface) were harvested and used for studies in TNF fi production. These leukocytes were seeded at a concentration of 4 x 106 cells/ml in 50 ul of RPMI 1640 medium in 96-well microtitre plates (Linbro, Flow Laboratories). These were treated with 50 ul of either drug or diluent for I hr at 37°C and then cultured in the presence of 1 ug/ml of phytohaemagglutinin (PHA, Wellcome, Sydney) in medium supplemented with 2.5% human AB serum, For studies of IL-1 and TNFa production, preparation of adherent MNCs, containing predominantly monocytes were made in 24-well cluster plates (Linbro, Flow Laboratories) as described previously (11, 14). To each well were added 2 ml of 1 X 106 MNC/ml and incubated at 37°C for 2 h. After incubation the non-adherent cells were removed and the adherent cells used for cytokine production. To 1 ml of 2 X 106 cells/ml was added 1 ml of 2 X 107 of heat-killed, formalin-fixed Staphylococcus aureus in wells of 24-well cluster plates. S. aureus acted as a stimulus and was prepared as described previously (14). PHA was also used as a stimulant at 1 ug/ml conc. in some monocytemacrophage experiments.
Quantitation of IL-I: IL-IB in the culture supernatants is measured by a competitive binding radioimmunoassay (Amersham, U.K.). The tracer is [125I] IL-II] (human, recombinant), and the antibody to human recombinant IL-11~ is made in rabbits. The antibody-bound IL-11~ is then reacted with the Amerlex-M second antibody reagent (donkey anti-rabbit serum) which contains second antibody that is bound to magnetizable polymer particles. Separation of the antibody-bound fraction is effected by centrifugation of the Amerlex-M suspension and decantation of the supernatant. Measurement of the radioactivity in the pellet enables the amount of labelled IL-ltl in the bound fraction to be calculated. The concentration of unlabelled IL-18 in the sample is then determined by interpolation from a standard curve.
Quantitation of TNFs: An enzyme immunoassay (EIA) using a monoclonal capture method was employed for the quantitation of TNFa and TNFft as previously described (14, 17). The mouse monoclonal antibody (TNF-E) against rHuTNF-e was of the immunoglobulin G1 (lgG1) isotype and reconstituted to 1.7 mg/ml of PBS (Genentech, San Francisco), and a murine monoclonal antibody (LTX-9) raised against human TNF-I~ was of the IgG1 isotype and reconstituted to 3.5 ug/ml in PBS (Ernst-Boehringer Institute). The rabbit antiserum to rHuTNF-a contained approximately 3 x 105 to 10 x 105 U of TNF-
Vol. 50, No. 8, 1992
Tetrandrine and Berbamine
PL-55
a-neutralizing capacity per ml, and rabbit antiserum to rHuTNF-I; contained 2.9 x 107 U of TNF-I~neutralizing capacity per ml. Both of the rabbit antisera were developed by Genentech. Details of the EIA procedure have been described elsewhere (14,17). The generation of phosphoinositides was performed as .previously described (18). MNCs in Earl's buffered salt solution (BSS), at the concentration of 5 x 10°/ml, were labelled overnight at 37°C with 41~Ci/ml of myo-3H-inositol (specific radioactivity 10-20 Ci/mM, Amersham Australia). The cells were then washed twice in Earl's BSS containing 41xM inositol and 5mM lithium chloride in order to remove excess isotope (6), and resuspended at a concentration of 5 x 106/ml in the same buffer. Aliquots (0.5 ml) of this cell suspension were pipetted into capped glass tubes and incubated at 37°C in a shaking water bath for 5 rain prior to the addition of 10 ~tl tetrandrine, berbamine or medium (control) and 10 ixl concanavalin A (Con A, Sigma Chemical Co., St. Louis, Me.). The reaction was terminated by the addition of 0.5 ml of ice-cold trichloraeetie acid (20% w/v). The samples were extracted with 4 x 2 ml water-saturated ether. The aqueous phases were retained and adjusted to pH 7-8 with phosphate buffer, and applied to 2.5 x 0.4 cm (Dowex l(x8 formate) chromatography columns. The inositol phosphates were eluted by stepwise additon of solution containing increasing levels of formate and the radioactivity in each fraction measured by liquid scintillation counting. Phosphoinositide turnover:
Results Effect on IL-I production: The effect of varying concentrations of tetrandrine and berbamine on ILlfi production by S. aureus stimulated human monocytes-macrophages was examined by the first set of
experiments. The results (figure 2) showed greater suppresion of IL-11~production by tetrandrine than berbamine (EDso 1.6. vs 28.5 ug/mi); although significant inhibition occurred at 10 and 20 ug/ml for both compounds, the effect of tetrandrine was greater. 1800 1~0 1~0 1200,
~0. ~0. 400. 200. 0
0.1
11o
.io
16.o
2(;.o
Drug conc. (ug/ml)
Ir!gure2. Effect of tetrandrine (open circles) and berbamine (closed circles) on IL-1B production by human MNCs. The MNC cultures were stimulated by heat-killed, formalin-treated S. aureus. The supematant was harvested ailer 72 hrs, and the cone. of IL-IB determined by radio-immunoassay. Results are shown as mean ± S.D. of 3 separate experiments, with MNCs obtained from 3 individuals.
As shown in Fig. 3, tetrandrine was also superior to berbamine with regard to inhibition of TNFa production. For PHA stimulated cultures, the EDso of tetrandrine and berbamine was 2.8 and 20.2 ug/ml, respectivdy; for $. aureus stimulated cultures, the EDso was 1.8 and 10.3 ug/ml, respectively. Effect on TNFproduction:
PL-56
Tetrandrine
and B e r b a m i n e
50, No.
8, 1992
PHA
1=
0.1
10
4.0 Drug COne. (uo/ml)
10.0
20.0
S. aureus
Figure 3. Effect of tetrandrine (open circles) and berbamine (closed circles) on TNFa production by human MNCs. The 2 stimulants
25000-
20000-
used w e r e P H A (upper) and S. aureus (lower). Results are e x p r e s s e d as mean + S.D. of 3 separate experiments with M N C s obtained from 3 donors.
15000tm v
J
Vol.
10000-
Z
I--
S000-~
0
01
10 Drug ~ n c
40 (ug/ml)
00
O
TNFI~ production by lymphocytes was also more sensitive to tetrandrine than berbamine (Fig. 4). Again, the EDso of tetrandrine was better than that of berbamine (2.6 vs 28.3 ug/ml).
10000 9000 8000 7000
Figure. 4.
6000
Effect of tetrandrine (open circles) berbamine (closed circles) on TNFfl production by human MNCs. The stimulant was heat-kiUed,
5000 4000 I-
formalin treated S. aureus. Results are shown as mean + S.D. of 3 separate experiments with MNCs from different donors.
3000 2000 1000 0.1
1.0
4.0
10.0
20.0
Drug conc. (ug/ml)
Effect on phosphoinositide
turnover: As shown in Fig 5, there was significant inhibition of phosphoinositide turnover by 20 ug/ml of tetrandrine (63.0 4- 4.1% of control, p < 0.001). By contrast, berbamine actually increased phosphoinositide turnover (111.4 4- 9.7% of control, p > 0 . 1 ) , although this was not statistically significant.
Vol. 50, No. 8, 1992
Tetrandrine and Berbamine
PL-57
i00
F'~mre5. Effect of tetrandrine and berbamine of phosphoinositide turnover in human MNCs. The stimulant was ConA. Results are shown a s percent of control 5- S.D. of 3 experiments with MNCs from differentdonors.
-I" 50
Tetrandrine Berbamine
Discussion The inflammatory cytokines IL-1 and TNF are potent molecular mediators of inflammation with wide ranging effects on a large array of cell types, tissues and organs (19). They are produced by a variety of cells, but monocytes-macrophages are major sources of these polypeptides which have overlapping activities and are regarded as central to the inflammatory process. Our previous finding that tetrandrine has inhbitory effects on IL-1 production was based on a biological assay, and this is confirmed by a more specific radio-immunoassay used in the present study. Our results also show that berbamine is a less potent inhibitor of IL-1 production than tetrandrine, although both are active at the non-toxic concentration range of 4-20 ug/ml (6-8). The results confirm and extend our previous observations on the the ability of tetrandrine to suppress TNFa (cachectin) production by monocytes-macrophages. Since TNFg is a product of T lymphocytes, it is worhwhile noting that tetrandrine is also able to suppress the production of this cytokine and suggests that it may also mediate its anti-inflammatory effects by suppressing T-cell function. In addition, the results of the present study show that berbamine is less potent than tetrandrine in suppression of production of both TNFs. The inflammatory cytokines IL-1 and TNF are not preformed, but are synthesized and released in reponse to external stimuli. One way in which tetrandrine can inhibit the production of these cytokines is by interference with transmembrane signal transduction, and this was shown in a previous report (18). Results of the present study show the capacity of tetrandrine but not berbamine to suppress the phosphoinositide second messenger system. Thus, berbamine has a different mode of action to tetrandrine in this regard, and may explain the less potent effects of berbamine on inhibition of inflammatory cytokine production. Tetrandrine and berbamine are bisbenzylisoquinoline analogues which differ from each other only in the methoxyl substitution of the hydroxyl group in one of the side chains of one of the benzene rings, and also in chirality at that point. It is well known that even minor differences in chemical structure can have profound effects on the biological activity of a compound. In this connection, berbamine has greater solubility in water than tetrandrine, and while both can inhibit prostaglandin synthesis, only tetrandrine can inhibit leukotriene synthesis (13). Also, berbamine appears to be more potent in terms of immunosuppression in mice (20), and has a greater effect on relapsing experimental allergic encephalitis (EAE) in rats (15). Autoimmune and chronic inflammatory diseases are heterogeneous with diverse etiologies and mechanisms of pathogenesis. Taken together, these findings suggest that tetrandrine may be superior to berbamine for chronic inflammatory diseases where inflammatory mediators and cytokines have a major role in pathogenesis (eg. silicosis), while berbamine may be somewhat superior for the treatment of autoimmune disease where immunological mechanisms have a greater role in pathogenesis (eg. EAE). There is at present a dearth of compounds able to inhibit inflammatory cytokine synthesis. Comparative data regarding these 2 analogues can provide important insights into structure-activity
PL-58
Tetrandrine
and
Berbamine
relationships, and the design and development of synthetic treatment of chronic inflammatory and autoimmune diseases.
vol.
analogues
50,
and congeners
No.
8,
useful
199;
in the
Acknowledgement This work was supported in part by grants from the JP Kelly Foundation (Mater Hospital), the Mayne Bequest Fund (University of Queensland) and the National Health and Medical Research Council (Australia). References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
K.K. CHEN and A.L. CHEN. J. Biol. Chem. m 681685 (1935). H.M. CHANG and PPH BUT. Pharmacology and applications of Chinese Materia Medica. World Scientific Publishing Company Singapore (1987). LI QUANLU, XU YONGHOU and ZHOU ZESHEN, et al. Chin. J. Tuberc. Respir. Dis. 4 321328 (1981). YU XIUFEN, ZOU CHANGGI and LIH MUBIN. Ecotoxicol. Environ. Safety. Z 306-312 (1983). LIU BINGCI, ZOU CHANGGI and LI YURUI. Ecotoxicol. Environ. Safety.? 323-329 (1983). W.K. SEOW, S.Y. LI and Y.H. THONG. Immunol. Letters. l3, 83-88 (1986). W.K. SEOW, A. FERRANTE, S.Y. LI, and THONG YH. Int. Archs. Allergy Appl. Immunol. a 404409 (1988). W.K. SEOW, A. FERRANTE, D.B.H. GOH, A.H. CHALMERS, S.Y. LI and Y.H. THONG. Int. Archs. Allergy Appl. Immunol. @, 410415 (1988). B.S. TEH, W.K. SEOW, A.H. CHALMERS, B. IOANNONI, S. PLAYFORD and Y.H. THONG. Int. Archs. Allergy Appl. Immunol. f& 220-224 (1988) B.S. TEH, B. IOANNONI, W.K. SEOW, J.G. McCORMACK, and Y.H. THONG. Int. Archs. Allergy Appl. Immunol. 88 272-272 (1989). W.K. SEOW, A. FERRANTE, S.Y. LI and Y .H. THONG. Clin. Exp. Immunol. 75 47-51 (1989). S.Y. LI, L.H. LING, B.S. TEH, W.K. SEOW and Y.H. THGNG. Int. J. Immunopharmacol. 11 395-401 (1989). B.S. TEH, W.K. SEOW, S.Y. LI, and Y .H. THONG. Int. J. Immunopharmacol. 12. 321-326 (1990). A. FERRANTE, W.K. SEOW, E. ROWAN-KELLY and Y.H. THONG. Clin. Exp. Immunol. 8Q, 232-235 (1990). C.W. WONG, W.K. SEOW and Y.H. THONG. Int. Archs. Allergy Appl. Immunol (in press). A. FERRANTE, Y.H. THONG. J. Immunol. Methods 3fj 109-l 16 (1980). A. FERRANTE, R.E.M. STANGAS, B. ROWAN-KELLY, S. BRESATZ, L.M. KUMARATILAKE, C.M. RZEPCZYK and G.R. ADOLF. Infect. Immun. 58 3996-4003 (1990). B. IOANNONI, A.H. CHALMERS, W.K. SEOW, J.G. McCORMACK and Y.H. THONG. Int. Archs Allergy Appl. Immun. 89 349-354 (1989). C.A. DINARELLA. J. Infect. Dis. m 1177-1184 (1991). C.W. WONG, W.K. SEOW, T.S. ZENG, W.J. HALLIDAY, AND Y.H. THONG. Int. J. Immunopharmacol. 1991; 13 579-585.
PHARMACOLOGY Accelerated
Pergamon P r e s s
LETTERS
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COMPARATIVE EFFEC'q'S OF TETRANDRINE AND BERBAMINE ON PRODUCTION OF THE INFLAMMATORY C Y T O K I N ~ INTERLEUKIN-1 AND TUMOR NECROSIS FACTOR
W.K. Scow (1), A. Ferrante (2), A. Summors (1), Y.H. Thong * (1) Immunobiology Laboratory, Department of Child Health, University of Queensland, Mater Childrens Hospital, South Brisbane 4101, Australia (1), and Department of Immunology, University Department of Paediatrics, Adelaide Childrens Hospital, North Adelaide 5006, Australia (2) (Submitted September 23, 1991; accepted October 30, 1991; received in final form December 17, 1991)
ABSTRACT: Tetrandrine and berbamine are bisbenzylisoquinoline compounds which differ from each other in a minor way in terms of chemical structure, yet tetrandrine is 6-18 times more potent than berbamine in terms of inhibitory effects on production of interleukin-1 and tumor necrosis factor (TNFa) by monocytes and macrophages, and TNFB production by lymphocytes. Moreover, tetrandrine significantly suppressed phosphoinositide turnover while berbamine did not. These findings may provide important insights into structure-activity relationships and the design of novel analogues and congeners useful in the therapy of chronic inflammatory and auto-immune diseases.
Tetrandrine is the major alkaloid of hanfangji, the tuberous root of the creeper Stephania tetrandra used since antiquity by Chinese physicians for the treatment of rheumatic diseases (1,2). Its ability to retard and even reverse the lesions of pulmonary silicosis in open clinical trials and controlled rat experiments (3,4) was attributed to inhibition of collagen synthesis (5). However, we conjectured that its mode of action may be on a more fundamental aspect of inflammation, and experiments in our laboratory have borne out this contention (6-14). Berbamine is a natural analogue of tetrandrine isolated from the root of Berberis soulieana, also used in Chinese ethnopharmacy for therapy of rheumatic diseases (2), but it was noted to be less effective for silicosis. However, recent studies in our laboratory showed that berbamine was more
~ N
OCH 3 OCH3
cH . ~
l NCH3
Figure 1. The chemical structure of tetrandrine OCH3) and berbamine (R = OH)
o
* To whom correspondence should be addressed. 0024-3205/92
$5.00
Copyright © 1992 Pergamon Press plc
+ .00
All rights reserved.
(R =
PL-54
Tetrandrine and Berbamine
Vol. 50, No. 8, 1992
effective in suppression of relapsing experimental allergic encephalitis (EAE) in rats (15). Since inflammatory cytokines are important mediators of immunologicaUy mediated tissue damage, we compared the effects of these two bisbenzylisoquinoline compounds on the production of interleukin-1 (IL-1) and tumor necrosis factor (TNF) from human monocytes-macrophages and lymphocytes. Methods
Tetrandrine and berbamine: Tetrandrine, empirical formula C38H4206N2 and molecular weight 622.73, was obtained from Jin Hua Pharmaceutical Company, Jijang Province, China as a pure powder (> 98%). It was dissolved in dilute HCI (1 X N) and its pH adjusted to 7.0 with dilute NaOH; this aqueous stock solution of tetrandrine (2mg/ml) was diluted in RPMI 1640 medium for the experiments. Berbamine, empirical formula C37Ha006N2 and molecular weight 608.71, was dissolved in sterile normal saline (2 mg/ml), and the stock solution diluted in RPMI 1640 medium for the experiments. The chemical structure of these two bisbenzylisoquinoline analogues are shown in Figure 1. The final concentrations (20 ug/ml) of these compounds used in the experiments were previously determined to be non-toxic to neutrophils, monocytes, lymphocytes and other cells, by means of trypan blue dye exclusion and the tetrazolium colorimetric assay.
Production of IL-I and TNF: Heparanized blood was layered onto Hypaque-Ficoll medium of density 1 .I 14 and centrifuged at 600g for 30 rain (16). The mononuclear leukocytes (MNC) in the top band (at the interface) were harvested and used for studies in TNF fi production. These leukocytes were seeded at a concentration of 4 x 106 cells/ml in 50 ul of RPMI 1640 medium in 96-well microtitre plates (Linbro, Flow Laboratories). These were treated with 50 ul of either drug or diluent for I hr at 37°C and then cultured in the presence of 1 ug/ml of phytohaemagglutinin (PHA, Wellcome, Sydney) in medium supplemented with 2.5% human AB serum, For studies of IL-1 and TNFa production, preparation of adherent MNCs, containing predominantly monocytes were made in 24-well cluster plates (Linbro, Flow Laboratories) as described previously (11, 14). To each well were added 2 ml of 1 X 106 MNC/ml and incubated at 37°C for 2 h. After incubation the non-adherent cells were removed and the adherent cells used for cytokine production. To 1 ml of 2 X 106 cells/ml was added 1 ml of 2 X 107 of heat-killed, formalin-fixed Staphylococcus aureus in wells of 24-well cluster plates. S. aureus acted as a stimulus and was prepared as described previously (14). PHA was also used as a stimulant at 1 ug/ml conc. in some monocytemacrophage experiments.
Quantitation of IL-I: IL-IB in the culture supernatants is measured by a competitive binding radioimmunoassay (Amersham, U.K.). The tracer is [125I] IL-II] (human, recombinant), and the antibody to human recombinant IL-11~ is made in rabbits. The antibody-bound IL-11~ is then reacted with the Amerlex-M second antibody reagent (donkey anti-rabbit serum) which contains second antibody that is bound to magnetizable polymer particles. Separation of the antibody-bound fraction is effected by centrifugation of the Amerlex-M suspension and decantation of the supernatant. Measurement of the radioactivity in the pellet enables the amount of labelled IL-ltl in the bound fraction to be calculated. The concentration of unlabelled IL-18 in the sample is then determined by interpolation from a standard curve.
Quantitation of TNFs: An enzyme immunoassay (EIA) using a monoclonal capture method was employed for the quantitation of TNFa and TNFft as previously described (14, 17). The mouse monoclonal antibody (TNF-E) against rHuTNF-e was of the immunoglobulin G1 (lgG1) isotype and reconstituted to 1.7 mg/ml of PBS (Genentech, San Francisco), and a murine monoclonal antibody (LTX-9) raised against human TNF-I~ was of the IgG1 isotype and reconstituted to 3.5 ug/ml in PBS (Ernst-Boehringer Institute). The rabbit antiserum to rHuTNF-a contained approximately 3 x 105 to 10 x 105 U of TNF-
Vol. 50, No. 8, 1992
Tetrandrine and Berbamine
PL-55
a-neutralizing capacity per ml, and rabbit antiserum to rHuTNF-I; contained 2.9 x 107 U of TNF-I~neutralizing capacity per ml. Both of the rabbit antisera were developed by Genentech. Details of the EIA procedure have been described elsewhere (14,17). The generation of phosphoinositides was performed as .previously described (18). MNCs in Earl's buffered salt solution (BSS), at the concentration of 5 x 10°/ml, were labelled overnight at 37°C with 41~Ci/ml of myo-3H-inositol (specific radioactivity 10-20 Ci/mM, Amersham Australia). The cells were then washed twice in Earl's BSS containing 41xM inositol and 5mM lithium chloride in order to remove excess isotope (6), and resuspended at a concentration of 5 x 106/ml in the same buffer. Aliquots (0.5 ml) of this cell suspension were pipetted into capped glass tubes and incubated at 37°C in a shaking water bath for 5 rain prior to the addition of 10 ~tl tetrandrine, berbamine or medium (control) and 10 ixl concanavalin A (Con A, Sigma Chemical Co., St. Louis, Me.). The reaction was terminated by the addition of 0.5 ml of ice-cold trichloraeetie acid (20% w/v). The samples were extracted with 4 x 2 ml water-saturated ether. The aqueous phases were retained and adjusted to pH 7-8 with phosphate buffer, and applied to 2.5 x 0.4 cm (Dowex l(x8 formate) chromatography columns. The inositol phosphates were eluted by stepwise additon of solution containing increasing levels of formate and the radioactivity in each fraction measured by liquid scintillation counting. Phosphoinositide turnover:
Results Effect on IL-I production: The effect of varying concentrations of tetrandrine and berbamine on ILlfi production by S. aureus stimulated human monocytes-macrophages was examined by the first set of
experiments. The results (figure 2) showed greater suppresion of IL-11~production by tetrandrine than berbamine (EDso 1.6. vs 28.5 ug/mi); although significant inhibition occurred at 10 and 20 ug/ml for both compounds, the effect of tetrandrine was greater. 1800 1~0 1~0 1200,
~0. ~0. 400. 200. 0
0.1
11o
.io
16.o
2(;.o
Drug conc. (ug/ml)
Ir!gure2. Effect of tetrandrine (open circles) and berbamine (closed circles) on IL-1B production by human MNCs. The MNC cultures were stimulated by heat-killed, formalin-treated S. aureus. The supematant was harvested ailer 72 hrs, and the cone. of IL-IB determined by radio-immunoassay. Results are shown as mean ± S.D. of 3 separate experiments, with MNCs obtained from 3 individuals.
As shown in Fig. 3, tetrandrine was also superior to berbamine with regard to inhibition of TNFa production. For PHA stimulated cultures, the EDso of tetrandrine and berbamine was 2.8 and 20.2 ug/ml, respectivdy; for $. aureus stimulated cultures, the EDso was 1.8 and 10.3 ug/ml, respectively. Effect on TNFproduction:
PL-56
Tetrandrine
and B e r b a m i n e
50, No.
8, 1992
PHA
1=
0.1
10
4.0 Drug COne. (uo/ml)
10.0
20.0
S. aureus
Figure 3. Effect of tetrandrine (open circles) and berbamine (closed circles) on TNFa production by human MNCs. The 2 stimulants
25000-
20000-
used w e r e P H A (upper) and S. aureus (lower). Results are e x p r e s s e d as mean + S.D. of 3 separate experiments with M N C s obtained from 3 donors.
15000tm v
J
Vol.
10000-
Z
I--
S000-~
0
01
10 Drug ~ n c
40 (ug/ml)
00
O
TNFI~ production by lymphocytes was also more sensitive to tetrandrine than berbamine (Fig. 4). Again, the EDso of tetrandrine was better than that of berbamine (2.6 vs 28.3 ug/ml).
10000 9000 8000 7000
Figure. 4.
6000
Effect of tetrandrine (open circles) berbamine (closed circles) on TNFfl production by human MNCs. The stimulant was heat-kiUed,
5000 4000 I-
formalin treated S. aureus. Results are shown as mean + S.D. of 3 separate experiments with MNCs from different donors.
3000 2000 1000 0.1
1.0
4.0
10.0
20.0
Drug conc. (ug/ml)
Effect on phosphoinositide
turnover: As shown in Fig 5, there was significant inhibition of phosphoinositide turnover by 20 ug/ml of tetrandrine (63.0 4- 4.1% of control, p < 0.001). By contrast, berbamine actually increased phosphoinositide turnover (111.4 4- 9.7% of control, p > 0 . 1 ) , although this was not statistically significant.
Vol. 50, No. 8, 1992
Tetrandrine and Berbamine
PL-57
i00
F'~mre5. Effect of tetrandrine and berbamine of phosphoinositide turnover in human MNCs. The stimulant was ConA. Results are shown a s percent of control 5- S.D. of 3 experiments with MNCs from differentdonors.
-I" 50
Tetrandrine Berbamine
Discussion The inflammatory cytokines IL-1 and TNF are potent molecular mediators of inflammation with wide ranging effects on a large array of cell types, tissues and organs (19). They are produced by a variety of cells, but monocytes-macrophages are major sources of these polypeptides which have overlapping activities and are regarded as central to the inflammatory process. Our previous finding that tetrandrine has inhbitory effects on IL-1 production was based on a biological assay, and this is confirmed by a more specific radio-immunoassay used in the present study. Our results also show that berbamine is a less potent inhibitor of IL-1 production than tetrandrine, although both are active at the non-toxic concentration range of 4-20 ug/ml (6-8). The results confirm and extend our previous observations on the the ability of tetrandrine to suppress TNFa (cachectin) production by monocytes-macrophages. Since TNFg is a product of T lymphocytes, it is worhwhile noting that tetrandrine is also able to suppress the production of this cytokine and suggests that it may also mediate its anti-inflammatory effects by suppressing T-cell function. In addition, the results of the present study show that berbamine is less potent than tetrandrine in suppression of production of both TNFs. The inflammatory cytokines IL-1 and TNF are not preformed, but are synthesized and released in reponse to external stimuli. One way in which tetrandrine can inhibit the production of these cytokines is by interference with transmembrane signal transduction, and this was shown in a previous report (18). Results of the present study show the capacity of tetrandrine but not berbamine to suppress the phosphoinositide second messenger system. Thus, berbamine has a different mode of action to tetrandrine in this regard, and may explain the less potent effects of berbamine on inhibition of inflammatory cytokine production. Tetrandrine and berbamine are bisbenzylisoquinoline analogues which differ from each other only in the methoxyl substitution of the hydroxyl group in one of the side chains of one of the benzene rings, and also in chirality at that point. It is well known that even minor differences in chemical structure can have profound effects on the biological activity of a compound. In this connection, berbamine has greater solubility in water than tetrandrine, and while both can inhibit prostaglandin synthesis, only tetrandrine can inhibit leukotriene synthesis (13). Also, berbamine appears to be more potent in terms of immunosuppression in mice (20), and has a greater effect on relapsing experimental allergic encephalitis (EAE) in rats (15). Autoimmune and chronic inflammatory diseases are heterogeneous with diverse etiologies and mechanisms of pathogenesis. Taken together, these findings suggest that tetrandrine may be superior to berbamine for chronic inflammatory diseases where inflammatory mediators and cytokines have a major role in pathogenesis (eg. silicosis), while berbamine may be somewhat superior for the treatment of autoimmune disease where immunological mechanisms have a greater role in pathogenesis (eg. EAE). There is at present a dearth of compounds able to inhibit inflammatory cytokine synthesis. Comparative data regarding these 2 analogues can provide important insights into structure-activity
PL-58
Tetrandrine
and
Berbamine
relationships, and the design and development of synthetic treatment of chronic inflammatory and autoimmune diseases.
vol.
analogues
50,
and congeners
No.
8,
useful
199;
in the
Acknowledgement This work was supported in part by grants from the JP Kelly Foundation (Mater Hospital), the Mayne Bequest Fund (University of Queensland) and the National Health and Medical Research Council (Australia). References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
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