Int. J. lmmunopharmac., Vol. 12, No. 5, pp, 469-479, 1990. Primed in Great Britain.

0192-0561/90 $3.00 + .00 © 1990 International Society for lmmunopharmacology.

REVIEW ARTICLE

THE POTENTIAL OF CYCLOSPORIN A AS A N ANTI-TUMOUR AGENT G. McLACHLAN,*t L. M. SMART,* H. M. WALLACE~ and A. W. THOMSON*° Departments of *Pathology, *Medicine and Therapeutics and *Pharmacology, University of Aberdeen, Medical School, Aberdeen AB9 2ZD, Scotland, U.K.

(Received 22 January 1990 accepted 13 February 1990) Abstract - - Cyclosporin A (CsA) has become established as the agent of choice for the prevention of organ

allograft rejection and has shown considerable promise in the clinical management of certain autoimmune disorders. The impact of CsA as an immunotherapeutic agent of major importance is attributable to its powerful, selectiveinhibitory action on T-lymphocyte activation and proliferation. Moreover, CsA lacks the myelotoxic and other major side effects associated with cytotoxic immunosuppressive agents, such as cyclophosphamide or azathioprine. It is now clear that CsA has a potential therapeutic role in the treatment of malignancies, especially T-cell cancers. Recent studies suggest that there may be several areas of application for CsA, either as a direct antiproliferative agent or in combination with other drugs, including inhibitors of polyamine biosynthesis or cytotoxic anti-tumour agents, including vincristine and adriamycin. In addition, CsA and non-immunosuppressive analogues have been shown to restore mniti-drug sensitivityin cancer cells with acquired drug resistance. A further application of CsA may be to prevent the induction of human immune responses to therapeutic mouse monoclonal antibodies directed against turnout antigens, thereby enhancing the efficiency and safety of this form of cancer immunotherapy. Due to our incomplete understanding of the antiproliferative properties of CsA, further exploration of its potential as an antitumour agent must be accompanied by detailed studies aimed at elucidating its action on subcellular molecular events in both normal and malignant cells.

PROPERTIES OF CYCLOSPORIN A

The fungal metabolitc Cyclosporin A (CsA), a cyclic endecapeptide, is well-established as a potent immunosuppressive agent (Kahan, 1984; Borel, 1986; Thomson, 1989) and is widely used in the clinical management of allograft rejection. In addition, it is presently being evaluated for use in the management of certain autoimmune disorders, including juvenile onset (type 1) diabetes, severe psoriasis, sight-threatening uveitis and rheumatoid arthritis. CsA was first isolated by workers at Sandoz Ltd, Basle, Switzerland in 1970 from extracts of two strains of fungi imperfecti, Tolypocladium inflatum Gains and Cylindrocarpon lucidum Booth. Purification of CsA in 1973 and structural analysis of the compound in 1975 (Wenger, 1982) allowed evaluation of its immunosuppressive properties. This revealed that CsA was a powerful, selective inhibitor

of T lymphocyte activation (Borel, Feurer, Gubler & Stahelin, 1976; Borel, Feurer, Magnee & Stahelin, 1977), at doses which were not toxic to the bone marrow. The complex chemical structure of CsA has a number of distinctive features. The molecule is hydrophobic in nature, since 10 of the 11 amino acids are aliphatic and 7 nitrogens are methylated. It has a rigid structure, forming a/3-pleated sheet and an open loop due to hydrogen bonding of carbonyl groups of the 4 non-methylated nitrogens. Notably, it has a unique 9 carbon amino acid containing an ethylene bond at position 1. The active site is believed to be in the hydrophilic region involving amino acids 1,2,3 and 11 (Wenger, 1983). In contrast to other longer established pharmacological immunosuppressive agents, CsA has little, if any, direct depressive effect on normal bone marrow function, B-lymphocyte or natural killer cell activities, or on immunity to microbial pathogens. Administration of CsA however, is not without side

~To whom correspondence should be addressed. 469

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effects, the most important of which is dose-related nephrotoxicity. Other side effects encountered in clinical practice include hypertension, hirsutism and gum hypertrophy. CsA is not mutagenic, although in animal studies, it has been reported to enhance metastatic spread of a murine T-cell lymphoma (Eccles, Heckford & Alexander, 1980) and to accelerate the development of spontaneous and radiation-induced thymic lymphomas (Hattori, Perera, Witkowski, Kunz, Gill & Shinozuka, 1986; Shinozuka, Gill, Kunz, Witkowski, Demetris & Perera, 1986). There is however, no evidence that CsA increases the risk of lymphoma or other tumour development in man compared with other immunosuppressive agents, such as azathioprine and steroids (Beveridge, Krupp & McKibbin, 1984; Thompson, Allen, Morris & Ward, 1985). The immunosuppressive action of CsA is attributed to its ability, in vitro, to interfere with early triggering events in CD4 + T-helper cell activation and lymphokine production. The molecular basis of this fortuitous, selective effect on T-lymphocytes is as yet poorly understood. In animal models, CsA has been shown to inhibit preferentially, T-lymphocyte-mediated immune reactions, rather than humoral (B-cell) responses. CsA is thus a powerful suppressant of graft-versushost reactions, delayed-type hypersensitivity responses, and skin, kidney and heart allograft rejection, as well as experimental autoimmune disorders, e.g. diabetes (BB rat), uveitis, collageninduced arthritis and lupus disease in NZB/W or MRLlpr mice. In keeping with its mode of action in vitro, CsA is most effective in all these conditions when administered early during the course of immune-mediated events. Thus, in established autoimmune disease, or in treatment of on-going graft rejection it may be poorly or ineffective (for reviews see Borel, 1986; Schindler, 1986; Thomson, 1989). CsA always shows a striking specificity for T-cells and it has been proposed that it binds to an, as yet, undefined surface membrane binding protein on T-lymphocytes (perhaps the prolactin receptor). It is then internalized and bound to a novel basic protein (mol.wt 16,000) which specifically binds cyclosporin, termed cyclophilin, present in the cytoplasm. CsA is then transported to the nucleus, where it interacts with specific sites on the DNA and interferes specifically with transcription of mRNAs coding for various lymphokines, in particular T-cell growth factor or interleukin-2 (IL-2). Recent reports show that CsA inhibits production of IL-3, IL-4, IL-5 and those lymphokines which affect macrophage

function, i.e. macrophage chemotactic factor, macrophage migration inhibition factor, procoagulant inducing factor and macrophage activating factor, which is believed to share identity with interferon-), (IFN-),). The effects of CsA on production of other cytokines, e.g. colony stimulating factors and tumour necrosis factors (a/p) are less clearly established (for reviews see Hess & Colombani, 1986b; Thomson & Duncan, 1989). The specificity of CsA for helper T-cells has prompted studies on the possible effects of CsA on other T-lymphocytes, for example malignant cells. Several studies have demonstrated that CsA is selectively cytostatic or cytolytic to a number of freshly-derived leukaemic cells and permanent cell lines of T-lymphocyte lineage, but not to those of B-lymphocyte lineage. Consequently, it has been suggested that CsA may prove useful as a highly selective anti-tumour agent in T-cell malignancies, either on its own or in combination with other chemotherapeutic agents, providing a more selective and effective therapy than that provided by chemotherapeutic agents currently in use. Presently, an increasing problem associated with the use of chemotherapeutic agents is the development of drug resistance in malignant cells. After an initial exposure, cells become increasingly desensitized to the drug, until the treatment becomes totally ineffective. Such resistance has been demonstrated against alkylating agents, platinums and adriamycin. In addition, multidrug or pleotropic drug resistance (where cells exposed to one drug in increasing concentrations in vitro develop cross resistance to that and other drugs related to it, i.e. vinca alkaloids, anthracyclines) is a continuing problem for the oncologist. Significantly, a number of reagents, such as verapamil, have been found to modify the response of resistant cells; now CsA too has been shown to enhance the efficacy of certain cytotoxic drugs.

MODE OF ACTION OF CsA (i) Binding and influence on intracellular events in normal T-cells Based on physico-chemical properties, it seemed likely that hydrophobic CsA molecules simply partitioned into the cell surface phospholipid bilayer, thus perturbing the homeostatic control of membrane function. However, Ryffel, G6tz & Heuberger (1982) using radiolabelled dihydrocyclosporin C (3H-CsC) reported the

Anti-tumour Effects of Cyclosporin A existence of a specific, high affinity receptor for CsA on human lymphocytes. This observation has subsequently been disputed by Le Grue, Friedman & Kahan (1983) who described this as a low-affinity 'acceptor site.' CsA competes for binding with prolactin (Russell, Kibler, Matrison, Larson, Poule & Magun, 1985) and the prolactin receptor may be the CsA binding site. In this way, CsA may interfere with triggering of intracellular synthetic pathways. The OKT3 (CD3) recognition site, closely associated with the T-cell antigen receptor, has also been implicated as the 'CsA receptor' (Palacios, 1982), but further studies have shown that OKT3 monoclonal antibody does not affect CsA binding (Ryffel, Willard-Gallo, Tammi & Loken, 1984). Paradoxically, Hess & Colombani (1986a) showed that T-suppressor cells had greater affinity for a fluorescent, dansylated derivative of CsA than T-helper cells. This they explained by the proposal that cells with fewer receptors required less CsA to inhibit their proliferative response. Further studies, to investigate the possibility that non-specific integration of molecules into the plasma membrane occurred, were carried out by Rossaro, Dowd, Ho & Van Thiel (1988), who found that, in a concentration-dependent manner, CsA decreased motion of the inner leaflet lipids. They inferred that CsA could affect membrane-bound enzyme systems, possibly by increasing viscosity of the membrane. Foxwell, Frazer, Winters, Hiestand, Wenger & Ryffel (1988), on the other hand, found that CsA diffused passively into erythrocytes and bound to the high affinity 16,000 tool, wt protein, 'cyclophilin', causing cytoplasmic accumulation of CsA. Some years earlier, Merker & Handschumacher (1984) first raised the possibility that CsA may be specifically localized internally by binding to cyclophilin. There is indeed good correlation between the immunosuppressive activity and cyclophilin binding affinity of CsA analogues (Handschumacher, Harding, Rice, Drugge & Speicher, 1984; Durette, Boger, Damont, Firestone, Frankshun, Koprak, Lin, Melino, Pessolano, Pisano, Schmidt, Sigal, Staruch & Wibzel, 1988). Further insight was provided by Quesniaux, Schreier, Wenger, Hiestand, Harding & Van Regenmortel (1987) who showed that cyclophilin bound exclusively to the active site on CsA and postulated that this interaction was central to inhibition of T-cell activation. Hess & Colombani (1986b) however, offered an alternative suggestion that since cyclophilin levels were higher in CsAresistant T-suppressor cells, the higher relative amounts of free CsA within T-helper cells may prevent Ca 2÷ binding to the calcium-binding protein,

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calmodulin, leading to inhibition of lymphocyte activation. In support of this latter concept, Hess & Colombani (1986a) found that inhibitors of calmodulin activity prevented the action of CsA. Le Grue, Turner, Weisbredt & Dedman (1986) however, reported that less immunosuppressive CsA derivatives were equally effective inhibitors of calmodulin and argued that since calmodulin was not specific to lymphocytes, it was unlikely to play a central role in CsA action. In addition to the calmodulin binding effect, Gelfand, Mills, Cheung, Lee & Grinstrein (1987) showed that CsA suppressed the early, transient rise in intracellular Ca 2+ following lymphocyte stimulation and may therefore inhibit other Ca 2*dependent events leading to expression of the IL-2 gene and growth factor receptors, such as the interleukin-2 receptor. Another possible mechanism of cell growth retardation by CsA is CsA-induced inhibition of polyamine biosynthesis. Polyamines are cellular polycations essential for optimal rates of cell growth and proliferation (Heby, 1981). Polyamine biosynthesis is regulated by the first enzyme in the pathway, ornithine decarboxylase (ODC) and induction of ODC is known to be an early event in T cell activation. Several studies have indicated that CsA may enter the cell via the prolactin receptor and intracellular CsA may lead to inhibition of the polyamine biosynthetic pathway (Hiestand, Mekler, Nordmann, Grieder & Permmongkol, 1986). Indeed, Fidelus, Laughter, Twomey, Taffet & Haddox (1984) showed that CsA inhibited the induction of ODC. Other studies have suggested that polyamine biosynthesis may be inhibited by CsA, the CsA again having entered the cell via prolactin receptormediated uptake. The principal antilymphocytic action of CsA in vitro is inhibition of the de n o v o synthesis of IL-2 in response to stimulation, e.g. in the mixed lymphocyte culture, or in response to mitogen or antigen (Hess, Donneberg, Engel, Tutschka & Santos, 1983; Andrus & Lafferty, 1982), by down regulation of lymphokine mRNA transcription (Elliot, Lin, Mizel, Bleackley, Harnish & Paetkan, 1984; Granelli-Piperno, Inaba & Steinman, 1984). Cell proliferation can be restored, at least in part, by the addition to cultures of exogenous IL-2. CsA acts only on the antigen-sensitive precursor cells, i.e. immunocompetent lymphocytes, which are blocked at the Go and G~ phases of the cell cycle. Since these are periods of physiological arrest, ceils can still be induced to proliferate, therefore the

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effects of CsA can be reversed. CsA does not inhibit the response to IL-2 in lymphocytes which have developed the capacity to respond to this lymphokine. Thus a key question is: does CsA have any effect on expression of the IL-2 receptor? This area remains highly controversal, with some workers reporting inhibition of receptor expression (Palacios, 1982; LiUehoj, Malek & Shevach, 1984; Aiello, Maggiano, Lavocca, Piantelli & Musani, 1986) and others showing no effect (Larsson; 1980, Kronke, Leonard, Depper, Arya, Weng-staU, Gallo, Waldmann & Greene, 1984; Ferrini, Moretta, Biassoni, Nicolin & Moretta, 1986; Gelfand et al., 1987). The fact however, that the high affinity IL-2 receptor is complex, requiring two components (a and/J), has led to the hypothesis that CsA could interfere at a post translational level, i.e. with the combination/assembly of the surface molecules essential for formation of the high affinity receptor (Thomson, 1989). Recent evidence supports the view that interleukin 1 (IL-1) production by macrophages is CsAinsensitive (Wagner, 1983; Granelli-Piperno, Andrus & Steinman, 1986), despite initial reports (Bunjes, Hardt, RoUinghoff & Wagner, 1981; Andrus & Lafferty, 1982) to the contrary. This discrepancy is probably related to the nature of the stimulus used to elicit IL-I production and to whether or not the response is T cell dependent. CsA also influences other T-cells; inhibition of clonal proliferation and activation of cytotoxic T-cells is probably indirect, the result of insufficient IL-2 production. Lymphokine depression may also be the underlying reason for reported inhibitory effects on B-cell function (immunoglobulin production). Suppressor cells appear to be resistant to CsA, perhaps because their amplification is permitted via a non IL-2-dependent mechanism (Hess et al., 1983). Although inhibition of lymphokine mRNA is an attractive proposal for the action of CsA on normal T-cells, other mechanisms are probably required to explain inhibition of proliferation of other cells by the drug. These mechanisms may be less specific, but also constitute inhibition of early events in cells stimulated to proliferate (e.g. tumour cells). Many of the actions of CsA have been determined by in-vitro studies and have now been supported by in-vivo evidence. However, the presence of lymphocytes within human grafts under cover of CsA treatment and findings in experimental models that T-cell proliferation may continue, despite the presence of CsA, suggests the existence of CsA-independent, proliferative pathways (Chisholm & Bevan, 1988).

These observations highlight the fact that in-vitro work cannot fully explain the actions of CsA in vivo. (ii) Selective action o f CsA on malignant T-ceils in vitro and in vivo The striking specificity of CsA for normal T-cells has prompted investigation of its effects on malignant human and animal T-ceU lines both in vitro and in vivo (Table 1). Early work by Totterman, Scheynius, Killander, Danersund & Aim (1982) on a panel of human leukaemia/lymphoma cell lines showed CsA to have selective cytotoxicity to leukaemic T-cells but not B-cells or non malignant control cells. As well as these established cell lines, they also showed cytotoxicity against malignant T-cells freshly isolated from patients with a variety of leukaemias/ lymphomas. Again the activity was only observed against T-cells and not B-cells. Yanagihara & Adler (1983) demonstrated a direct, cytolytic effect of CsA on T-cell lines, the effect being greatest at low cell densities; CsA inhibited growth in both T and non T-cell lines, but did not affect the viability of the latter. They suggested that the presence of the Thy-1 antigen indicated the susceptibility of a cell line to CsA-induced lysis by comparing a Thy 1-negative subline with its Thy 1positive parent line. In a study of several routine and human T-cell lines, Fidelus et al. (1984) confirmed the T-specificity of CsA but interestingly, also documented enhanced growth of an EBVtransformed B cell line. Further work by Fidelus and Laughter (1986) showed inhibition of cell proliferation by CsA in murine thymic T-cell lymphoma cells and suggested that inhibition of polyamine biosynthesis via ODC was involved. More recently, Foa, Mallo, Baldini, Quarto, Di Palo, Starace & Polli (1986) have demonstrated dosedependent inhibition of the growth of a human T-leukaemia cell line, with irreversible and fatal damage at doses similar to those achieving immunosuppression. They postulated that CsA inhibited growth of these cells in the Go phase of the cell cycle and prevented progression to S-phase. Another study by Mahajan & Thompson (1987) reported inhibition by CsA of growth and rDNA transcription in p1798 lymphosarcoma cells. There have been fewer reports of the influence of CsA on tumour growth in vivo. The earliest in-vivo studies by Kreis & Soricelli (1979) showed CsA to have no effect on growth of a variety of solid murine tumours. However, some prolongation of survival was noted with ascitic tumours, although lymphoid

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Anti-turnout Effects of Cyclosporin A Table 1. Selective action of CsA on malignant T-cells in vitro and in vivo Cells

Dose

Effect

Authors

In vitro

Murine T-cell lymphoma lines 5 ~g/ml Murine macrophage, monocyte myeloma cell lines 5 pg/ml

Growth retardation and cytocidal effects

Human and routine T-cell lines 1 pg/ml

Inhibition of cell proliferation in 4/5

Human and routine B-cell lines I/ag/ml

No inhibition in 7/8

Murine thymic T-cell lymphoma 0.1/ag/ml cells

Inhibition of growth + ornithine decarboxylase activity

Human T-leukaemia cell line

Dose dependent inhibition of growth rate Foa et al., 1986

Yanagihara & Adler, 1983 Non cytocidal growth retardation Fidelus et al., 1983

5 pg/ml, 10/ag/ml

Panel of human leukaemia/ lymphoma cells lines. Freshly isolated cells from patients with a variety of leukaemia/ lymphomas 0.1 - 10/~g/ml Lymphosarcoma P1798 cells

1/ag/ml

Fidelus & Laughter, 1986

Selective cytotoxicity to leukaemic T-cells but not B-cells Totterman et al., 1982 Inhibition of growth and rDNA transcription

Mahajan & Thompson, 1987

In vivo

Panel of transplantable routine turnouts Solid intradermal T-cell tumour Ascites (Lymphoid) leukaemia (L5178) T-cell tumour (EL4) Leukaemia (L1210)

5 0 - 100 mg/kg No response 5 0 - 100 mg/kg No response

Kreis & Soricelli, 1979

5 0 - 100 mg/kg Marginal response 5 0 - 100 mg/kg Significant response (no t survival) Significant t' survival

T-cell lymphoma (EL4)

7.5 mg/kg

Roser T-cell leukaemia

12.5, 25 mg/kg Decreased development of leukaemic phase, dose-dependent

ascites responded poorly compared to non lymphoid counterparts and host toxicity was noted at 75 mg/kg. On the other hand, Eccles et ai. (1980) observed no inhibitory effect of CsA on a range of mouse and rat tumours; indeed enhanced metastases were noted with the more immunogenic tumours. Preliminary studies by Yanagihara & Adler (1983), administering low dose CsA (7.5 mg/kg) intraperitoneally (i.p.) to mice from the time of tumour inoculation, showed 8007o long term survival after i.p. injection of EL4 lymphoma cells. These findings however, must be interpreted with caution, as 3007o of controls were also still alive at 8 weeks. In a recent study o f T-cell leukaemia, Thomson, Forrest, Smart, Sewell, Whiting & Davidson (1988) demonstrated a dose dependent inhibitory effect of CsA on the development o f the leukaemic phase of

Yanagihara et al., 1983 Thomson et al., 1988

the disease in rats bearing the Roser T-cell leukaemia. No significant prolongation o f host survival however was observed, there being no effect of treatment on the extent of organ infiltration by tumour cells. With respect to non-lymphoid tumours, Saydjari, Townsend, Barranco, James & Thompson (1986) and Saydjari, Townsend, Barranco & Thompson (1988) have reported that CsA has inhibitory effects on the growth o f mouse colon cancer cells (MC26) in vitro and in vivo and also on growth of hamster pancreatic cancer cells (H2T) in vitro. In addition, work by Twentyman (1988) has shown dose-dependent inhibition of cell growth in three human lung cell malignancies. From these experimental studies (summarized in Table 2), it is clear that the antitumour potential of CsA is not restricted to T-cells or even to lymphoid cells.

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G. MCLACHLANet al. Table 2. Inhibitory action of CsA on non-lymphoid cancer cells Cells

Non-Lymphoid ascites Taper Liver 5180J sarcoma Ehrlich ascites P815 sarcoma Mouse colon carcinoma (MC 26) Hamster pancreatic cancer (H2T)

Dose 50505050-

100 mg/kg 100 mg/kg 100 mg/kg 100 mg/kg

Effect Significant response Increased survival Significant response toxic at 85 mg/kg Significant response

Authors

Kreis & Soricelli, 1979

8.3 x 10-3Dose dependent inhibition of cell growth 8.3 x 10-4mM + survival (10mg/kg in vivo) Saydjari et al., 1986, 1988 (10mg/kg in vivo)

Growth + survival

Human small cell lung carcinoma (NCI-H60)

1 - 8 ~g/ml

Dose dependent inhibition of cell growth

Human large cell lung carcinoma (COR L23)

1 - 8/~g/ml

Dose dependent inhibition of cell growth

Human lung adenocarcinoma (MOR)

1 - 8 ~g/ml

Dose dependent inhibition of cell growth

D R U G C O M B I N A T I O N STUDIES

Even though CsA has been shown to have marked anti-proliferative properties on malignant cells in vitro and in some cases in vivo, the results of in-vivo studies suggest that on its own, CsA does not produce a marked increase in host survival. It may be possible however, to combine CsA with other drugs thereby enhancing its effects. Reports by Fidelus et al. (1984) that polyamine biosynthesis was inhibited by CsA stimulated interest in combining CsA with other drugs that interfere with polyamine biosynthesis, in particular, a-difhioromethylornithine (DFMO), an irreversible inhibitor of ODC. Saydjari et al. (1986) reported that CsA and D F M O both inhibited growth of a mouse colon carcinoma (MC-26) and of a hamster pancreatic carcinoma (H2T) in vitro. Moreover, they found that the inhibition could be overcome in both cases by the addition of putrescine. This suggested that CsA and DFMO were blocking the conversion of ornithine to putrescine by ODC. What was more interesting however, was that a combination of the drugs produced a synergistic effect. Further work by Saydjari et al. (t988) revealed a similar, potent inhibitory effect of each drug on the same cells in vivo. However, the drug combination did not exhibit an effect over that observed with either C.sA or DFMO alone in vivo. In our own work, w e have been studying the effects of CsA and D F M O alone and in combination, on growth of the Roser acute T-cell

Twentyman, 1988

leukaemia in PVG rats and on growth of the EL4 T-cell lymphoma in C57BL/6 mice. We have shown that either drug (Smart, Davidson, Wallace & Thomson, 1989; Smart, McLachlan, Wallace & Thomson, 1990) markedly reduced numbers of circulating lymphoblasts in leukaemic rats, although survival was prolonged only in the animals given DFMO. Drug combination further decreased bloodborne tumour cell numbers but had no additional effect on host survival. Neither CsA nor D F M O had any effect on peritoneal growth of the EL4 lymphoma. Whilst DFMO did decrease polyamine levels in vivo, the anti-turnout effect of CsA was not accompanied by decreased polyamine biosynthesis. It may be however, that by decreasing polyamine synthesis, DFMO may have enhanced the susceptibility of the malignant T-cells to the as yet unexplained inhibitory action of CsA in vivo. Further work is in progress to evaluate the influence of CsA, in combination with DFMO (or other inhibitors of polyamine biosynthesis) or with other anti-T-cell agents, on tumour growth.

CsA IN M O I H F I C A T I O N OF D R U G R E S I S T A N C E

Another possible role of CsA as an anticancer agent is currently being explored, i.e. the ability of CsA to modify drug resistance. These studies have yielded promising initial results and have been reviewed recently by Twentyman (1988).

Anti-tumour Effects of CyclosporinA Multidrug resistance (MDR) is induced in vitro by exposure of cancer cells to increasing concentrations of high molecular weight natural products, such as anthracyclines and vinca alkaloids. Induction of resistance to one of these agents generally leads to cross resistance to other agents within the group. One approach to overcoming this drug resistance is to use additional chemical compounds, "resistance modifiers" (RMs) which lead to restoration of sensitivity. The most common RMs are the calcium channel blockers, such as verapamil or the calmodulin inhibitors, such as trifluoroperazine. The mechanism by which sensitivity is restored is unclear. It may however, be due to altered drug efflux from cells. This may be a result of alteration of the physico-chemical properties of the cell membrane or effects on lipid metabolism resulting in changes in membrane fluidity. Significantly perhaps, CsA and calcium channel blockers are known to act synergistically in the inhibition of normal T-cell activation (McMillen, Tesi, Baumgarten, Jaffe & Wait, 1985; van Eendenburg, Brisson, Klatzmann & Gluckman, 1988). The interest in cyclosporins as modifiers of MDR stems from reports that CsA may bind to and inhibit the function of calmodulin (Hait, Harding, Rice, Drugge & Speicher, 1986) and also from the observation that a patient with T-cell leukaemia in relapse after multidrug chemotherapy given CsA in combination with etoposide (VP 16) exhibited a lethal depletion of the bone marrow (Kloke & Osieka, 1985). The same group of workers initiated in vitro studies which combined CsA with VP16 and investigated DNA damage due to VP16 treatment of leukaemic and non-leukaemic mononuclear cells. It was shown that CsA enhanced VP16 damage in both cell types. Slater, Sweet, Stupecky & Gupta (1986) examined the ability of CsA to restore sensitivity to vincristine (VCR) and cross resistance to daunorubicin (DNR) in a VCR resistant subline of GM3639 acute lymphatic leukaemia cells. CsA had little effect on the parent line but restored, in part, the cytotoxicity of the etoposide in the resistant subline. The same workers using Ehrlich ascites carcinoma cells (parent and DNR resistant) in vitro and in mice observed similar effects, i.e. that CsA enhanced the effects of DNR on the resistant cells. Further studies on the Ehrlich ascites carcinoma and on a murine hepatoma 129 (Meador, Sweet, Stupecky, Wetzel, Murray, Gupta & Slater, 1987) showed that prolonged survival of mice was achieved by treatment with CsA and DNR. It was also noted that ineffective DNR regimens became effective when CsA was added. It

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appeared that CsA brought about a small increase in DNR uptake but had no observable effect on efflux, indicating that CsA enhancement of DNR activity was due to factors other than increased accumulation. Studies by Vayuvegula et al. (1988) showed a marked membrane depolarisation in drugresistant tumour cell sublines (human acute lymphatic leukaemia, murine P388 leukaemia, Ehrlich ascites carcinoma, hepatoma-129). CsA was found to restore these to the normal potentials found in the parental, drug sensitive cells. Twentyman, Fox & White (1987) compared the drug resistance modifying and immunosuppressive activities of several CsA analogues using an adriamycin-resistant, VCR cross-resistant, human small cell lung carcinoma cell line. A good correlation was found between the immunosuppressive ability and resistance modification. However, further studies revealed that two non-immunosuppressive cyclosporin analogues (B3-243 and W8-032) maintained the ability to modify resistance at a dose of 1 -2/Jg/ml at which CsA was ineffective (Twentyman, 1988). These studies highlight an interesting property of CsA and suggest that distinct mechanisms may underly the tumour cell drug resistance modifying and immunosuppressive activities of cyclosporins. Potentiation o f monoclonal antibody therapy using CsA as an immunosuppressant Repeated therapy of human cancer with mouse monoclonal antibodies (Mab) frequently leads to production of antibodies against the therapeutic agent. This leads to accelerated clearance of subsequent doses of the Mab, reduced binding to target tissue and hypersensitivity reactions. However, it has been shown that CsA given with each course of ultracentrifuged mouse antibodies to rabbits could completely prevent production of rabbit anti-mouse antibodies (Lederman, Begent & Bagshawe, 1988a). Subsequently, it has been shown that CsA permits repeated therapy with anti-tumour monoclonal antibodies in man by suppressing human anti-mouse antibodies (Lederman, Begent, Bagshawe, Riggs, Searle & Glaser, 1988b). Cyclosporin and human cancer To date, use of CsA in human T-cell malignancies has been confined to a few cases of cutaneous T-lymphomas, unresponsive to conventional chemotherapy. Puttick, Pollack & Fairburn (1983) in a case of late-stage S6zary syndrome achieved symptomatic relief but no elimination of abnormal

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O. MCLACHLANet al.

cells, whilst CateraU, Addis, Smith & Coode (1983) found that 2 weeks of CsA treatment in a patient with S6zary syndrome resulted in symptomatic relief and objective improvement. The patient went on to develop a high-grade lymphoma 8 months later, but as suggested by Totterman, Scheynius, Killander, Danersund & Aim (1985), this may represent the natural course of S6zary syndrome. In the case referred to, the patient continued in partial remission for 14 months, while receiving CsA treatment. Notably, this response to CsA was markedly greater than to the previous more conventional treatments used. In a report on the effect of CsA in Hodgkin's disease, Zwitter (1987) observed that out of the ten patients treated, seven subjective and five objective improvements were noted. It appears therefore, that CsA had a favourable therapeutic role. In three cases of mycosis fungoides (Kreis, Budman & Shapiro, 1988) two of the patients showed a partial response to CsA, but toxicity proved problematic. These studies included only patients who were all extensively pretreated and resistant to standard treatment procedures. Perhaps due to the selection of patients with aggressive disease and with poor prognosis, no complete responses were seen to CsA as a single agent. Nevertheless, these clinical observations provide encouragement for continued evaluation of CsA either alone or in combination therapy. Future prospects

The laboratory and clinical data published to date clearly indicate a potential therapeutic role of cyclosporin in the treatment of malignancies, especially T-cell cancers. Future evaluation of cyclosporin may be in several directions. First, it may be that cyclosporin will be used as a direct anti-tumour agent, with inhibition of polyamine biosynthesis being a factor involved in

this effect. There may be an even brighter prospect for CsA used in combination with other polyamine biosynthesis inhibitors and further work in this area is clearly necessary. Second, CsA might be used to potentiate the effects of (other) cytotoxic drugs such as VP16, daunorubicin, adriamycin and vincristine, since these effects have been reported both in vitro and in vivo. Although potentiation of drug activity is seen in both tumour cells and normal tissue, a differential, overall effect against tumour cells has been reported. Third, another avenue for exploration is the use of CsA and its analogues in the modification of drug resistance. CsA has been clearly shown to restore drug sensitivity in cells with acquired drug resistance. This effect can also be seen with nonimmunosuppressive cyclosporin analogues. Therefore, it would appear that the mechanisms for immunosuppression and resistance-modification are not the same. Further studies in this field are required to elucidate the mechanism of drug resistance modification. Fourth, evaluation of the use of CsA in cancer immunotherapy is also required since prevention of the development of human antibodies to the mouse monoclonal antibodies targeted to tumour antigens could lead to more effective therapy. It is clear that there is still much to be discovered about CsA and its mechanisms of action but its potential for useful therapeutic development remains high. A greater understanding of the molecular mechanisms by which CsA interferes with cell growth is however, required before large scale clinical trials can be considered. Acknowledgements -- The authors' work is supported by

grants from the Cancer Research Campaign and from the Grampian Health Board. We thank Mrs I. M. Watson for typing the manuscript.

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