Inhibitory effects of bisbenzylisoquinolines on synthesis of the inflammatory cytokines interleukin-1 and tumour necrosis factor-alpha

Synthesis of IL-1β and TNFα by human monocytesmacrophages was significantly inhibited by eleven bisbenzylisoquinolines and one half-molecule (benzylisoquinoline), with IC50 values in the μM range. The results indicate that these compounds may have value in the therapy of human diseases where these inflammatory cytokines have a central role in pathogenesis.


Introduction
The pro-inflammatory cytokines interleukin-1 (IL-1) and tumour necrosis factor (TNF) are potent molecular mediators with wide-ranging effects on a large array of cells, tissues and organs. 1'2 They are produced by a wide variety of cell types, but monocytes-macrophages are the major source of these polypeptides which share structural similarities and have overlapping activities. 3 '4 They have been implicated in the pathogenesis of chronic inflammatory (e.g. rheumatoid arthritisS), autoimmune (e.g. insulin-dependent diabetes melli-tus6), allergic (e.g. asthma7), infectious (e.g. leprosys) and malignant (e.g. ovarian carcinoma 9) diseases.
There is therefore a great interest in drugs that can control their generation and/or action because of the potential for therapeutic application. It has recently been shown that tetrandrine, a novel bisbenzylisoquinoline alkaloid, has potent in-(C) 1993 Rapid Communications of Oxford Ltd hibitory effects on the production of IL-lfl and TNFcz by human monocyte-macrophage cultures. [1][2] In addition, it has been shown to have therapeutic efficacy in a number of animal models of human disease, such as relapsing experimental allergic encephalitis in rats (multiple sclerosis), 13 spontaneous diabetes in BB rats (insulin-dependent diabetes mellitus), 4 air-pouch inflammation in rats (rheumatoid arthritis) is and airways microvascular leakage in guinea-pigs (asthma). 16 In an attempt to gain some mechanistic insight into the relationship between the bisbenzylisoquinoline structure and the extent of inhibition of the synthesis of TNF-z and IL-lfl, the inhibitory potencies of tetrandrine and a series of ten other bisbenzylisoquinolines and one benzylisoquinoline (half-molecule) are now compared. The bisbenzylisoquinolines vary in the type of substituent, location of their ether bridges and the stereochemistry at a chiral centre. The sole benzylisoquinoline tested is a monomeric component of the dimeric benzylisoquinoline, tetrandrine, W. Kim Seow et al. and serves to identify whether the dimeric Shoyaku Company, Tokyo, Japan; berbamineand bisbenzylisoquinolines are active as a result of their oxyacanthine were purchased from Carl Roth, inherent macrocyclic structure or because of their Germany; aromoline was isolated from root constituent functional groups which are also cultures of Stephania cepharantha, as described present in their smaller monomeric fragments, previously; 2 homoaromoline and obaberine were converted from aromoline and oxyacanthine, Materials and Methods respectively, by methylation with diazomethane; N-methylcoclaurine was prepared by chemical Compounds: Four types of compounds were used in synthesis. 21 these experiments (Fig. 1). Three of these were All compounds (>97% pure) were identified macrocyclic ether compounds of isoquinolines for chemical structure by mass spectroscopy and bridged by alpha-benzyloxy substituents. These 1H-NMR spectroscopy against reference samples compounds differ in the position of the linkages and literature values. 22 The HC1 salts of the between the two monomeric benzylisoquinoline compounds were dissolved in RPMI 1640 medium components, and the nature and number of at a concentration of 2 mg/ml, and these stock substituents on the aromatic rings. The fourth solutions further diluted in RPMI 1640 medium for type of compound was represented by Nthe experiments. methylcoclaurine, a monomeric benzylisoquinoline.
The twelve compounds used in this study were Production of IL-I[J and TNFe: The study was obtained from diverse sources. 7 Tetrandrine and performed with monocytes-macrophages isolated fangchinoline, were extracted from the tubers of from human blood. -12 Blood was drawn by Stephania tetrandra, 8 which were purchased from venepuncture from three healthy non-smoking Maruzen Seiyaku Company, Onomichi, Japan; adults not on any medication for at least 2 weeks cepharanoline, cepharanthine, isotetrandrine and prior to the study. The heparinized blood was cycleanine, extracted from the tuberous root of layered onto Hypaque-Ficoll medium of density Stephania cepharantha, 9 were purchased from Kaken 1.114 and centrifuged at 600 x g" for 30  Preparation of adherent MNCs, containing predominantly monocytes, were made in sterile plastic Petri dishes. After 3h at 37C, the non-adherent cells were washed off, and the adherent cells were later removed with the aid of a sterile rubber policeman, washed and re-suspended in RPMI 1640 medium containing 5% foetal calf serum, and the cell concentration adjusted by counting in a Neubauer haemocytometer. To 1 ml of 2 x 106 cells/ml was added 0.5 ml of drug solution. After incubation at 37C for 15 min, 0.5 ml of 4 x 10: heat-killed, formalin-fixed, Staphylococcus aureus as a stimulant, were added into wells of 24-well cluster plates. The supernatants were harvested after 24 h, and kept frozen at -70C for up to 2 weeks before being assayed for the presence of IL-lfl and TNF0. The cells were checked for viability by Trypan-blue dye exclusion (>94%).
Quantitation of IL-lfl and TNF: Both IL-1/g and TNF in the culture supernatants were measured by a competitive binding radioimmunoassay (Amersham, UK). 12 The tracer was [12sI]IL-1fl or TNF0 (human, recombinant), and the antibody to human recombinant IL-1/3 or TNF was made in rabbits. The antibody-bound IL-lfl or TNF0 was then reacted with the Amerlex-M second antibody reagent (donkey anti-rabbit serum) which contains a second antibody that is bound to magnetizable polymer particles. Separation of the antibodybound fraction is et:fected 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-1/3 or TNF0 in the bound fraction to be calculated. The concentration of unlabelled IL-lfl or TNF0 in the sample is then determined by interpolation from a standard curve.
Determination of ICso values: The concentration of drug (/IM) which causes 50% inhibition of cytokine production (ICs0) was determined from graphical plots of inhibitor concentration vs. percentage inhibition.

Results
Effect on ILfl and TNF synthesis: Human monocytesmacrophages were incubated with 0, 1, 4, 10, 20 and 40/lg/ml of each of the twelve compounds. The results (

Discussion
Currently available drugs for therapy of chronic inflammatory and autoimmune diseases are unsatisfactory because of significant toxic and/or immunosuppressive side effects. There is therefore a great need to develop better and safer drugs to control inflammatory tissue destruction. The discovery of the central role of inflammatory cytokines in the pathogenesis of these diseases 1-9 opens up a new strategic target for drug development. The authors recently showed that the bisbenzylisoquinoline alkaloids, tetrandrine and berbamine, have the capacity to inhibit both the generation and action of IL-1/ and TNFo, 1-12 and control disease progression in a number of animal models. 1>6 Although the mechanism of action of bisbenzylisoquinolines is not well understood, studies with tetrandrine have shown its capacity to block calcium channels, 24 interfere with transmembrane signalling, is and induce apoptosis. 26 Moreover, it is largely devoid of immunosuppressive properties, 2v'28 and is non-toxic at therapeutic doses (20mg/kg per day for 84 days) as assessed by appearance, behaviour, weight change, blood chemistry and organ histology of BB rats. 4 This compares well with the oral LDs0 of 2 230 mg/kg in rats, 29 which is more than 100 times the therapeutic dose. Furthermore, tetrandrine has been used empirically in humans for the treatment of silicosis at a dose of 300 mg/day for 3 years without serious toxic side effects. 3 Thus, bisbenzylisoquinolines may have significant advantages over existing drugs used in the treatment of chronic inflammatory and autoimmune diseases, such as corticosteroids, cytotoxic and anti-rejection drugs, which have substantial toxic and immunosuppressive side effects. As such, they may become the fore-runner of a new class of safe and effective drug suitable for long term clinical usage. The results of the present study showed that all eleven bisbenzylisoquinoline alkaloids have inhibitory effects on IL-lfl and TNFo synthesis by human monocytes-macrophages. Calculation of ICs0 values showed that inhibitory concentrations of these eleven compounds were within the same order of magnitude. This was surprising as the structures depicted in Fig. 1 vary considerably. It was expected A very significant finding may be that Nmethylcoclaurine, a non-cyclic benzylisoquinoline quite different from the cyclic bisbenzylisoquinolines, has equivalent inhibitory effects on IL-1/ and TNF production. There are at least two possible interpretations of these findings in terms of structure-activity relationships. One interpretation would be that the bisbenzylisoquinolines and benzylisoquinolines act on different receptors, even though both are equipotent inhibitors of inflammatory cytokine production. A more likely interpretation is that the monomers, benzylisoquinolines, may be the active forms of the bisbenzylisoquinolines. Bisbenzylisoquinolines like N-methylcoclaurine are known to chemically assemble in plants to form bisbenzylisoquinolines like tetrandrine. However, it is not known whether bisbenzylisoquinolines transform biologically into 202 Mediators of Inflammation. Vol 2.1993 Drug inhibition of IL-1 and TNF monomeric benzylisoquinolines, although it has been shown chemically that cleavage of tetrandrine can be achieved using strong reducing conditions (metallic sodium in liquid ammonia) into two monomers, one of which is N-methylcoclaurine. 31 One avenue for further investigation would be to determine whether the bisbenzylisoquinolines are metabolized under the conditions of these experiments and, if so, to identify the metabolites after in vitro and in vivo degradation. Another would be to determine whether bisbenzylisoquinolines and benzylisoquinolines have similar or different molecular modes of actions. Because of the importance of" inflammatory cytokines in health and disease, further development of these classes of compounds may provide insights into the biology of inflammatory cytokines as well as to a better and safer drug for therapy of inflammatory diseases.