Azathioprine, cyclophosphamide and chlorambucil.

8 Azathioprine, cyclophosphamide and chlorambucii R. A. L U Q M A N I R. G. P A L M E R P. A. BACON

The traditional pyramidal approach to the treatment of rheumatoid arthritis (RA) starting with the least toxic, least effective drugs, and then moving down to more aggressive therapy, is being increasingly challenged with more widespread knowledge of the underlying disease mechanisms in RA. The success of experimental T cell depletion therapies, such as thoracic duct drainage (Paulus et al, 1977) and lymph node irradiation (Kotzin et al, 1981) reinforce the need to develop more routine therapies based on suppression of lymphocytes. The concept that T cells play a key role in the underlying disease process provides the basis for using cytotoxic drugs to damage these cells. The major cytotoxic drugs used in rheumatological practice are capable of inhibiting cell division and or causing cell death. The effect is most marked in rapidly dividing cells such as in active immune sites; hence they are immunosuppressive. There are two discrepancies associated with these drugs. The first is that it is not clear whether their immunosuppressive function is responsible for their effect in the rheumatic diseases. The second is that their use has been largely in complicated systemic diseases rather than the chronic synovitis which is most firmly T cell based.

AZATHIOPRINE Pharmacology Azathioprine is an oral purine analogue which interferes with the synthesis of adenosine and guanine. It is biologically inactive until metabolized (chiefly in erythrocytes and in the liver) to 6-thioinosinic acid and 6thioguanylic acid; excretion is via the kidney. It would be expected to interfere with D N A synthesis and thus to act on all rapidly dividing cells. However, the exact mechanism by which it acts is not clear. Clinically it has a slower onset of action that cyclophosphamide and is generally regarded as a Baillidre"s Clinical Rheumatology--

Vol. 4, No. 3, December1990 ISBN0-7020-1484-2

595 Copyright9 1990,by Bailli6reTindall All rightsof reproductionin any formreserved

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safer but less effective drug. It may be better to regard them as having different indications. The usual dose is 1.5-2.5mgkg-aday -1 and for treatment of many conditions it may be initially combined with prednisolone in an attempt to suppress the inflammatory response rapidly. Paradoxically, in chronic use its 'steroid sparing' effect has been widely promoted. Use in rheumatoid arthritis

The initial use was in uncontrolled studies in severe late arthritis, where it appeared to be of benefit. The first controlled study showed improvement in the arthritis, although the most striking finding was a steroid sparing effect of azathioprine (2.5 mg kg-1 day-1) in a double-blind placebo-controlled study in RA (Mason et al, 1969). All patients were previously on at least 5 mg of prednisolone per day, and the azathioprine-treated group achieved a mean reduction in steroid dose of 36%. In a 30-month follow-up study of these patients, over one-third were still on the drug (Harris et al, 1971). Maximum steroid reduction was achieved in the first year, and was maintained after stopping the azathioprine. Toxic effects were all reversible and there were no deaths due to the drug. No effect on radiological progression of the disease could be shown. A series of studies have compared azathioprine with conventional second-line drugs. Dwosh et al (1977) compared it with gold and hydroxychloroquine in patients with RA of less than 5 years' duration. All three groups improved in a similar fashion at 12 and 24 weeks, but there were significantlymore side-effects in the azathioprine group (chiefly nausea and leukopenia). In contrast, a comparison with i>penicillamine (1020mgkg -1 day -1) showed fewer side-effects in the azathioprine-treated (1.25-1.5 mg kg -1 day -1) group (Paulus et al, 1984). In the 134 patients who completed 24 weeks of treatment, both drugs achieved favourable results. In a 12-month study comparing azathioprine with o-penicillamine, both drugs achieved satisfactory results, with a higher rate of withdrawal due to sideeffects in the azathioprine group (Berry et al, 1976). In severe rheumatoid arthritis a double-blind study of azathioprine, cyclophosphamide and gold was performed in 121 patients with RA, of whom 50% had disease of less than 3 years' duration (Currey et al, 1974). Results at 18 months showed that both cytotoxics were similar in efficacy to gold therapy but allowed additional steroid reduction and seemed to reduce the rate of radiological progression of the disease. Azathioprine was slower to act but less toxic than cyclophosphamide, which often led to azoospermia in the males. The Toronto group, in a cross-over trial, showed improvement in a number of variables as compared to placebo at 16 weeks (Urowitz et al, 1973). At 40 months, those cases who had still remained on azathioprine (12 out of the original 17) were in well-maintained remission (Hunter et al, 1975). An important study established that the beneficial effects of azathioprine were dose related (2.5 mg/kg was more effective than 1.25 mg/kg). There appeared to be a threshold since l m g k g -1 day -1 or less was ineffective (Woodland et al, 1981). In an attempt to improve efficacy, combination

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therapy has been reported by several authors. The Cambridge group used azathioprine and gold over a prolonged period (De Silva and Hazleman, 1981) and reported efficacy with minimal toxicity. In the United States, azathioprine was combined with cyclophosphamide and antimalarials in 31 cases of severe RA, of whom 11 had vasculitis (Csuka et al, 1986). After 43 months, remission was reported in over 50% with only 1 patient who had no response at all. However, four patients developed malignancies, suggesting that alkylating agents might best be excluded from further trials of combination therapy. These studies established the use of azathioprine in severe rheumatoid arthritis as a drug equivalent in effectiveness to other established slow-acting antirheumatic drugs. Importantly, they also established that it had no more toxicity. Indeed, it often appears to be more acceptable to patients over a prolonged period, especially in the elderly. It has also been used in the treatment of juvenile chronic arthritis. In a study of 32 children (Kvien et al, 1986), the azathioprine treated group were significantlyimproved at 8 weeks as assessed by a number of clinical parameters, but by 16 weeks only functional status and subjective total assessment were better. However it is still not a conventional therapy. Use in rheumatoid vasculitis Typical nail edge/nailfold vasculitis clearly involving small vessels is a common feature of RA and does not p e r se usually require cytotoxic therapy. A recent trial compared azathioprine and prednisolone with other disease-modifying drugs for the treatment of isolated small-vessel skin vasculitis (Heurkens, AHM, Westedt ML and Breedveld FC, submitted for publication). Progression of the vasculitis to larger vessels was only seen in 2 cases out of 19, after at least 2 years follow-up. There was no significant advantage in the use of azathioprine and steroids as compared to conventional therapy for rheumatoid arthritis. Uncontrolled data from the same authors also suggested that azathioprine could be useful in systemic rheumatoid vasculitis. In contrast, the only double-blind placebo-controlled study in systemic rheumatoid vasculitis which utilized azathioprine was abandoned owing to deaths in both groups (Nicholls et al, 1973). Many groups now use cyclophosphamide for this systemic disease. Use in systemic lupus erythematosus Azathioprine is widely used in this condition even though the hard evidence for benefit is less than ideal. An early 3-year study of 16 cases of lupus nephritis (Donadio et al, 1974) failed to find any benefit from azathioprine in addition to prednisolone in either renal or systemic manifestations of the disease. The dose of azathioprine used was 2 mg kg -1 day -1 and the average steroid dose was 20rag/day of prednisone for the first 6 months, with subsequent reduction in both agents. Six cases that flared despite their treatment were managed either with higher doses, or by switching to cyclophosphamide and prednisolone. Sztejnbok et al (1971) in a controlled

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non-blinded study compared two groups of patients with severe lupus, 16 of whom received azathioprine and prednisolone, and 19 were treated with prednisolone alone. Seven other patients died in hospital before they could be entered into the study. In the treatment groups, there were six deaths in the controls but none in the azathioprine group who also showed reduced morbidity, without any increased incidence of infection. Renal function was better in the azathioprine group and toxicity was minimal. When the drug was stopped however, the disease often flared. Sharon et al (1973) highlighted this problem of relapse on stopping azathioprine. In their series of 16 patients given the drug for at least 18 months, 7 out of 9 who stopped therapy relapsed compared with 1 out of 7 who continued. Barnett et al (1978) reviewed 47 cases of lupus nephritis, all treated with azathioprine and prednisolone and followed for up to 12 years. There was a 5-year survival of 82%, 10-year survival of 74%, eight deaths and two patients on chronic dialysis. They tended to continue the azathioprine for at least 12 months after the relevant laboratory tests and measures were stable and there had been no signs of active renal inflammation. In a study of 71 patients with lupus nephritis (Cameron et al, 1979), azathioprine was preferred to cyclophosphamide because of bladder toxicity on the latter. Although benefit from azathioprine was shown in terms of steroid sparing and reduction of cardiovascular morbidity, the cost was high in terms of infection. Hayslett et al (1972) found azathioprine at a dose of 2-3 mg kg -1 day -1 to be useful in reducing proteinuria and improving renal function concurrent with improvement on renal biopsy appearance in 16 patients with lupus nephritis. Azathioprine may be useful in improving laboratory abnormalities such as double-stranded DNA binding as well as clinical parameters (Swaak et al, 1984). However, this is not a universal finding since no additional benefit over steroids alone has been reported in life-threatening lupus after an 18-24-month follow-up (Hahn et al, 1975). None of these longer-term studies was controlled or even randomized at entry. This contrasts with the NIH comparison with cyclophosphamide, which tended to favour the latter. Cade et al (1973) treated 50 cases of lupus nephritis with either azathioprine, prednisolone, azathioprine and prednisolone or azathioprine with heparin. Renal function and wellbeing were best on all groups given azathioprine (particularly in combination with heparin) compared to the group on prednisolone alone. Felson and Anderson (1984) collated data from all the randomized trials then published on treatment of lupus nephritis involving the use of either azathioprine or cyclophosphamide. In their analysis of 250 patients, there were significant differences between the cytotoxic-treated groups and the comparators, with less renal deterioration, less development of end-stage renal disease and nephritis-related death. They were unable to determine whether azathioprine or cyclophosphamide was the preferred agent. Small numbers in individual studies may have been the reason for previous failure to find consistent differences between steroids alone and the addition of immunosuppressive agents. However, it must be stressed that there has to be careful case selection when considering immunosuppressive agents in this disease, with particular regard for the presence of active nephritis.

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The use of cytotoxic agents in the treatment of lupus remains controversial. Some authors (Lieberman and Schatten, 1988) would reserve them for disease which was life threatening, unresponsive to other measures and with active organ damage not improving on steroids, or to reduce unacceptably high doses of steroids. Steinberg (1986), in a recent review, put an alternative view that azathioprine had a role only in cases of intermediate severity if long-term outcome was considered (i.e. prevention of end-stage renal disease). Use in other connective-tissue diseases

Azathioprine may serve a useful role in a steroid-sparing capacity in the treatment of resistant polymyalgia rheumatica/giant cell arteritis (De Silva and Hazleman, 1986). In a double-blind placebo-controlled study, the addition of azathioprine to steroids alone resulted in significant reduction in mean steroid dose after 1 year of therapy. Although it was used successfully in the induction of remission of polyarteritis nodosa in the study by Leib et al (1979), azathioprine has largely been superseded by cyclophosphamide in necrotizing vasculitides. Many groups are using it as a maintenance therapy, after cyclophosphamide, and this is currently the subject of a controlled, randomized prospective study in our unit. A trial of azathioprine compared with placebo in 73 patients with Beh~et's disease (of whom 48 had eye disease) showed that the drug was effective, both in terms of eye disease and the other manifestations (Yazici et al, 1990). In polymyositis, there has been a role for azathioprine in steroid-resistant cases (Bunch, 1981), but the use of methotrexate in this context is more firmly accepted. In a study of intensive immunosuppression, 12 patients with polymyositis all did well on antilymphocyte globulin, azathioprine and prednisolone, although this study was largely uncontrolled (Hollingworth et al, 1982). An uncontrolled study found a beneficial effect from azathioprine in 8 out of 21 patients with progressive systemic sclerosis (Jansen et al, 1968) but this has not been substantiated by other centres. Side-effects

The most important side-effect is reversible marrow suppression. In the event of marrow depression, it is our practice to temporarily stop therapy and then re-introduce at a 25% reduction of the previous dose when the total white-cell count and platelet count recover to within the normal range. Other effects include gastrointestinal intolerance and rarely hepatotoxicity (Zarday et al, 1972). Long-term effects may include the induction of lymphoid tumours (Rosman and Bertino, 1973). The drug has been used safely in pregnancy (Schein and Winokur, 1975), despite the fact that it can cross the placental barrier (Saarikoski and Seppala, 1973) and also appears in breast milk (Platzker et al, 1980). McKendry and colleagues (McKendry and Cyr, 1989) compared the toxicity profile of azathioprine with that of methotrexate in the treatment of rheumatoid arthritis. In a survey of nonrandomized cases, 94 were receiving methotrexate, and 37 were given

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azathioprine. Toxic effects were defined as major if they led to withdrawal of the drug or resulted in a significant number of abnormal laboratory tests. Major toxic effects were seen in about one-third of both groups, and non-major effects in approximately 80% of both groups. The chief inhibition to the use of cytotoxic agents is the potential risk of malignancies. In a series of 393 cases of rheumatoid arthritis treated with azathioprine, there were no reported malignancies, and the main reasons for stopping the drug were gastrointestinal upset in 60% of those who withdrew (Singh et al, 1989). The question of malignancy induced by azathioprine is still unresolved. A large study recently described the outcome after 20 years of treating 202 patients with 300 mg/day (Silman et al, 1988). Rheumatoid arthritis alone was associated with a 5-fold increased risk of lymphoma compared to controls. This was increased to 10-fold by the azathioprine treatment. In other words, despite a high dose of azathioprine, the lymphoma risk was only 2-fold above disease controls and no other significant malignancies were detected.

CYCLOPHOSPHAMIDE Pharmacology Cyclophosphamide is derived from nitrogen mustard, and was first made in 1958 (Arnold and Bourseaux, 1958). The drug is not bound to plasma proteins to any significant extent, although many of its metabolites are (Bagley et al, 1973). It is widely distributed throughout the body because of its solubility in both aqueous and lipid compartments. The plasma half-life varies between 2 and 10 hours before cellular metabolism occurs, chiefly in the liver but also to some extent in the kidney and lung (Friedman et al, 1979). Cyclophosphamide is a potent stimulator of hepatic microsomal enzymes and therefore shortens its own plasma half-life with continuing use (Bagley et al, 1973). However, because of increased production of active metabolites there is no change in the biologically important effect of the drug over time. The parent cyclophosphamide molecule has no cytotoxic properties, which are entirely due to its metabolites. It is metabolized via 4-hydroxycyclophosphamide and aldophosphamide into phosphoramide mustard. Acrolein, the other main metabolite, is thought to be responsible for the bladder toxicity, but has no useful activity in rheumatic diseases. Under 20% of cyclophosphamide is excreted directly into urine; 65% is excreted as carboxyphosphamide or 4-ketocyclophosphamide. In renal failure, there can be reduced excretion (Mouridsen and Jacobsen, 1975). High-dose intravenous cyclophosphamide (Cheigh and Reidenberg, 1978) may be particularly hazardous in these situations, suggesting that dose reduction would be prudent. Our recommendations in clinical practice are outlined in Table 1. Patients on dialysis may achieve 30-60% lower drug levels, although this has only been studied in patients with malignant diseases (Galleti et al, 1966). The mechanism of action of cyclophosphamide is uncertain. It is toxic for resting as well as dividing cells, by damaging DNA repair mechanisms. This

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Table 1.

Serum creatinine (p~mol/1)

Initialintravenous bolus

Subsequentoral bolus

(a) Renal failure on bolus therapy.

< 150 150-250 251-500 > 500

15 mg/kg 10mg/kg 7.5 mg/kg 5 mg/kg

5mgkg-1 day 1• 3 mg kg-I day 1• 2.5mgkg 1day 1• 2mgkg 1day 1x3

(b) Renal failure on continuous oral therapy

500

2mgkg-1 day 1 1.75 mg kg 1day 1 1.5mgkg-1 day 1

* The bolus doses of cyclophosphamide are given together with high doses of intermittent prednisolone (10 mg/kgper bolus, with some reduction in severe renal failure). Treatment is commencedat fortnightly intervals with gradual lengthening out of the interval as the remission is obtained. We usually treat for a total of 18-24 months in this fashion. t Continuous oral cyclophosphamideis given in conjunction with high doses of oral prednisolone (starting at 0.85 mgkg 1day l and gradually reducing as the patient improves) for approximately 6 months before switching to azathioprine and prednisolone for a further 6 months. We usually continue with maintenance lowdose prednisolone for a further 6-12 months beyond this. may account for its more rapid action and increased toxicity when compared with azathioprine. The presence of large amounts of thiol groups or oxidative enzymes renders some cells less susceptible to damage by cyclophosphamide. Both immunosuppressive and apparent immunostimulatory effects of cyclophosphamide metabolites have been demonstrated (Turk and Parker, 1979). Miller and North (1981) reviewed the interaction between infections and immunosuppressive agents. They describe enhanced antibody responses with pulse cyclophosphamide, due to its differential effects on T cell subsets, which in some circumstances might lead to eradication of some bacterial infections. The modes of administration, either high-dose intermittent or continuous low-dose may have different mechanisms of action. Chronic oral low-dose therapy has more of an effect on cell-mediated responses than the high-dose intermittent therapy, which predominantly affects humoral immunity (Hersh et al, 1966). This might be explained by differential sensitivities to damage of T and B cells respectively. U s e in r h e u m a t o i d

arthritis

Its first major use in rheumatological practice was in an uncontrolled study of 38 patients with rheumatoid arthritis over 6-40 months. There was significant clinical improvement in 75% of the cases and 18 patients were able to stop their steroids (Fosdick et al, 1968). The dose of cyclophosphamide used was adjusted to achieve a sustained leukopenia, or clinical improvement in the absence of toxicity. Cyclophosphamide has not been successful in all studies. Lidsky and colleagues (1973) found no benefit over placebo in using 0.87-1 mg kg -1 day -1

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for at least i year in 10 patients on the drug out of the 22 in the study. The low dose was chosen in order to avoid its toxic effects but this is now accepted to be below the useful dose threshold. In the Co-operating Clinics study (1970) of daily oral cyclophosphamide, 48 patients were treated with either a higher (up to 150 mg) or lower (up to 15 rag) dose for 32 weeks. Clinical improvement, fall in immunoglobulin levels, reduced levels of rheumatoid factor and slowing of radiographic changes were found only in the group on high-dose cyclophosphamide. There was no relationship between improvement and the white-cell count. Side-effects including herpes zoster, alopecia and cystitis were common, especially in the high-dose group (90% compared to 40% in the low-dose group). Williams et al (1980) in a double-blind study showed the efficacy of 150 mg cyclophosphamide daily compared to 75 mg daily in 88 patients treated for 32 weeks. Both groups had improvement. Interestingly, there were similar numbers of side-effects, so the lower doses are not necessarily safer. Because of its toxicity (both potential and actual), continuous oral cyclophosphamide has not found a place in the routine therapy of uncomplicated rheumatoid arthritis. Intermittent high-dose cyclophosphamide and prednisolone has been successful in a small series of patients with refractory rheumatoid arthritis (Horslev-Petersen et al, 1983). However, a recent study (Waiters and Cawley, 1988) could not find any benefit from the use of pulsed intravenous cyclophosphamide in the treatment of severe synovitis in RA, confirming an earlier report (Hall et al, 1979). Its main use has been in the systemic complications of rheumatoid arthritis.

Use in systemic necrotizing rheumatoid vasculitis (SRV) We use the term SRV to refer to patients with rheumatoid arthritis who have clinical and/or histological evidence of vasculitis. Nailfold infarcts alone are not included (Scott et al, 1981a). SRV is a serious condition which is often relapsing and has a significant and cumulative mortality. Daily cyclophosphamide may be efficacious (Weisman and Zvaifler, 1975) but it is often given in the form of intermittent pulses (Scott and Bacon, 1984). These are combined with steroids during the pulse to lessen toxicity, although steroids alone may actually be a factor inducing the vasculitis of SRV (Kemper et al, 1957; Vollertsen et al, 1986). In our experience of over 100 cases, cyclophosphamide is the drug of choice in SRV, but no formal comparison has been performed.

Use in other systemic vasculitides This group includes the syndromes in which serious end-organ damage or death are common in the untreated case. Necrotizing vascular infammation can involve almost any body system alone or in combination, and there is an overlap in many of the clinical, laboratory and histological features in these disorders. Indeed at one time, rheumatoid vasculitis and polyarteritis nodosa were thought to be part of the same entity (Ellman and Ball, 1948). Novack and Pearson (1971) were the first to point out that cyelo-


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phosphamide had a dramatic effect in Wegener's granulomatosis. This was later extensively confirmed by the NIH group (Fauci and Wolff, 1973; Wolff et al, 1974). The survival time, in their untreated cases was 5 months, and in those treated with steroids alone was 12 months. With continuous low-dose oral cyclophosphamide there was an 80% 5-year survival. The same group is now talking about cures in this hitherto uniformly fatal disease (Fauci et al, 1983). In our experience of 22 patients studied over an 8-year period (mean follow-up 2 years), the overall mortality was 37.5% despite the aggressive use of cytotoxic agents, prednisolone and plasma exchange. More recently, 12 cases of Wegener's (7 of whom had not responded to oral low-dose cyclophosphamide and prednisolone) were given a regime of monthly bolus intravenous cyclophosphamide (0.5-1 g/m2) and 150mg oral prednisolone (Steppat and Gross, 1989). After achieving remission, these patients were switched to trimethoprim/sulphamethoxazole. However, their relapse rate off cyclophosphamide was again high. Therefore, the prognosis in this disease remains serious; cyclophosphamide, or other cytotoxic agents, are not a panacea. Earlier referral for therapy is one part of the answer since tissue necrosis inevitably leaves scars and functional impairment. This is particularly true of renal involvement, where damage is often halted by cyclophosphamide but is rarely reversed. Polyarteritis nodosa (PAN) is a severe multisystem disease similar to Wegener's but without the consistent involvement of the upper and lower respiratory tract (Rose and Spencer, 1957). The untreated survival is very poor in many series, particularly in the elderly. The use of steroids on their own improved the 1-year survival to 71% in an early study but had much less effect on the long-term survival (Pickering et al, 1960). Leib et al (1979) have emphasized the important therapeutic role of cytotoxic drugs (using mainly azathioprine). Their series consisted of 64 cases seen over a 22-year period, the treatments being non-randomized, with cytotoxic agents being used in only the most severely affected group. They were able to demonstrate significantly longer mean survival (149 months) and an 80% 5-year survival in the patients given cytotoxic agents as compared to 53% on steroids alone (mean survival 63 months) or 12% on no treatment (mean survival 3 months). Our own early experience from a district general hospital supported this (Scott et al, 1982). The mortality was high in the untreated group but in the small group who were given cyclophosphamide together with steroids the 2-year mortality was only 25 %. Comparison of these cases from 1974 to 1980 with our more recent data from our renal and rheumatological units gives a clear view of the overall change in prognosis obtained by using cyclophosphamide. The group who presented to a rheumatology unit had much less renal involvement and had no deaths after a mean follow-up of 3.9 years. Those who presented to a nephrology unit had a 40.5% mortality, chiefly in patients with severe renal impairment prior to the onset of therapy. The main cause of death in the pre-cyclophosphamide era was active vasculitis, supplemented by complications of vasculitis such as hypertension. In the cyclophosphamide group, sepsis was the biggest single cause of death, especially in the presence of renal failure. Thus, while mortality due to vasculitis has diminished, the problem of sepsis has become more

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prominent and is related to therapy, as highlighted in the recent series reported from Indianapolis (Bradley et al, 1989). One group which appears to have a less good response to cyclophosphamide is patients with microscopic PAN. Here, there is still a mortality of over one-third despite aggressive therapy (Adu et al, 1987). Risk factors for poor outcome include age, significantly elevated serum creatinine (greater than 500 txmol/1) at diagnosis, oliguria and the presence of crescents on renal histology. Churg-Strauss syndrome was shown to be a separate disease from polyarteritis nodosa. Lanham et al (1984) attempted to define it as a triad of asthma, eosinophilia, and a systemic vasculitis involving two or more extrapulmonary organs. In their experience, steroids alone were beneficial, although 4 of their series of 16 cases were given cytotoxic agents, and 3 had additional plasma exchange. Chumbley et al (1977) also found that steroids alone were useful. However, their 5-year survivalis only 62%, suggesting that the long-term prognosis might have been improved by the addition of cytotoxic agents. A recent large study from France (Guillevin et al, 1988) combined data on polyarteritis nodosa and Churg-Strauss syndrome, which the authors considered to be inseparable. In their experience there was no statistically significant treatment benefit from the use of cyclophosphamide in terms of outcome; the mortality was high in their steroid-only group and they did not use cyclophosphamide as aggressively as we do. In our more limited experience of four cases, there was a poor response to steroids alone, and all our patients have done well on intermittent high-dose cyclophosphamide and prednisolone.

Use in systemic lupus erythematosus The first recorded use of cyclophosphamide therapy in lupus was a single case report in 1964 (Hill and Scott, 1964). In a large 7-year study of patients with lupus nephritis treated with either steroids alone, in combination with azathioprine, or in combination with cyclophosphamide, the group on cyclophosphamide had the best outcome in terms of maintaining renal function and preventing progression to end-stage renal disease (Carette et al, 1983). The toxicity of the drug was considerable. Progressive lymphopenia may be seen on continuous low-dose use, which may complicate that due to the disease. Intermittent intravenous therapy has been used as an alternative to oral cyclophosphamide. Monthly pulsed intravenous cyclophosphamide (McCune et al, 1988) resulted in a reduction in peripheral blood B and T cells (both CD4 and CD8). There was a delay in the reappearance of the T cells compared to the more rapid return of B cell numbers when therapy was stopped. Interestingly there was no reduction in some forms of in vitro T cell activation induced by cyclophosphamide (response to stimulation by phytohaemagluttinin or anti-CD3 antibody, which act via the T cell antigen receptor), whereas there was a reduction in response to others such as anti-CD2, which acts via the sheep erythrocyte receptor. It clearly has a selective effect on the immune system, but exactly how the drug works in SLE is not known. Cyclophosphamide alone may not


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be helpful, whereas in combination with steroids the results are better (Fries et al, 1983). Steinberg and Decker (1974) commenced a double-blind controlled trial in patients with lupus nephritis who in addition to oral steroids were randomly allocated to placebo, azathioprine or cyclophosphamide. Using a number of variables (including renal function, sediment abnormalities, proteinuria, extrarenal clinical disease, complements and double-stranded D N A levels) they found that cyclophosphamide was superior to both azathioprine and placebo, with little difference between the latter except on combining all six variables together in the assessment, when there was benefit from azathioprine in addition. At 28 months, when they had enrolled 38 patients with lupus nephritis (Decker et al, 1975), they could demonstrate only marginal benefit from the use of these agents. Although there was evidence for the prevention of severe renal damage, the risk of serious infection was very high. Klippel and co-authors reviewed the NIH study in 1979 (Decker et al, 1979). They concluded that there was less progression to renal disease in their 87 patients at 2 years in the groups treated with either intravenous cyclophosphamide or combined cyclophosphamide and prednisolone. A further review after 5 years showed that intravenous intermittent cyclophosphamide and prednisolone was the most effective therapy both in terms of efficacy and side-effects (Austin et al, 1986). Use in other connective-tissue diseases

Fries et al (1973) could not demonstrate any response from cyclophosphamide alone compared to steroids in polymyositis, and its use in this condition is uncommon. Although there are anecdotal reports of improvement in mixed connective-tissue disease, there are no formal studies which can confirm its benefit. Side-effects

Schein and Winokur (1975) extensively reviewed the complications of these agents in diseases where the long-term outlook was not necessarily poor. They discussed the problems of toxicity to several organs by these agents, as well as their oncogenic potential. Apart from the more minor problems of alopecia and gastrointestinal intolerance, many of the toxic effects are more serious. The bladder toxicity is well known, due chiefly to the effects of acrolein, one of its urinary metabolites. As well as causing haemorrhagic cystitis and bladder fibrosis, it has been associated with bladder carcinoma. Plotz et al (1979) described bladder complications on cyclophosphamide in SLE and RA. Stillwell et al (1988) reviewed their group of patients with Wegener's disease treated with cyclophosphamide. Out of their 111 cases, they found an incidence of haemorrhagic cystitis in 15 %, of whom 3 patients later developed bladder carcinoma. All these cases were given continuous oral therapy, and this is in contrast to the experience of using intermittent high-dose intravenous cyclophosphamide, where bladder toxicity is rare (Austin et al, 1986; Bacon, 1987). In any patient taking cyclophosphamide,

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general advice on maintaining a high oral fluid intake is important and is easier to achieve during intermittent therapy. Suppression of the immune system inevitably exposes patients to potentially serious infections, and was recently reviewed by Bradley et al (1989). In their 15 patients with various types of vasculitis, treated with daily oral cyclophosphamide and steroids, 10 patients developed significant infections, of whom 2 died. There did not appear to be any clear relationship between dose or degree of leukopenia and the infective episodes. Although this paper highlighted the potential for severe infections, the proportion of patients with problems was considerably higher than our own experience and those of others. Hellman et al (1987) found that prior use of either steroids or other cytotoxic agents was a more likely cause of fatal infections rather than current use of cytotoxic agents in their patients with SLE. Herpes zoster has been described as a complication of cytotoxic therapy of SLE (Moutsopoulos et al, 1978). In 83 cases of lupus nephritis there was a 21% incidence of herpes zoster infection. In only two cases did the infection disseminate, and most cases were successfully managed without specific antiviral therapy. It should be noted that there was no difference in the infection rate between those given any form of cytotoxic agent (cyclophosphamide, azathioprine or both) and those patients given steroids alone. Long-term effects of cyclophosphamide administration may be even more worrying. In vitro work has demonstrated an increased frequency of hypoxanthine-guanine phosphoribosyl transferase mutants in T cell clones (Palmer et al, 1988) and of increased sister chromatid exchange in peripheral blood mononuclear cells derived from patients treated with cyclophosphamide (Palmer et al, 1986a). The end result of these observed abnormalities of DNA has not been established at a clinical level, but are clearly worrying. Kinlen et al (1979) found an increase in the risk of malignancy for patients receiving at least 3 months' treatment with cyclophosphamide, azathioprine or chlorambucil. In 3823 renal transplantees, there was a 60-fold increase in the incidence of malignancies, mainly non-Hodgkin's lymphoma, squamous cell carcinoma of the skin and mesenchymal tumours. In 1349 patients being treated for other conditions (including rheumatoid arthritis) there was a smaller increase in non-Hodgkin's lymphoma over age and sex-matched controls. Their later report (Kinlen et al, 1981) suggested that the risks were greater for cyclophosphamide than for azathioprine. Baker et al (1987) described the delayed onset of a variety of malignant diseases including bladder carcinoma in patients with rheumatoid arthritis treated with cyclophosphamide. There appeared to be a relationship of total dose and duration of therapy with the incidence of malignancy. Some earlier reports even suggested that the risk of bladder malignancy was too great to justify the use of cyclophosphamide in systemic lupus erythematosus (Elliott et al, 1982; Baltus et al, 1983). In the work by Baltus et al, 15 out of 81 patients on cyclophosphamide developed malignancies (mainly lymphoid and haematological), representing a greater than 14-fold risk compared to disease controls. In the treatment of malignant diseases, cyclophosphamide has been linked to the development of subsequent malignancies. The relevance of this to the treatment of rheumatic diseases is unclear.


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The risk of gonadal failure is considerable in both sexes treated with cytotoxic agents. A post-mortem of a 13-year-old girl with lupus (who died of infection) revealed severe ovarian atrophy, with no follicles or ova seen (Miller et al, 1971). She had received cyclophosphamide for 3 years. In males, sterility and testicular atrophy as a result of cyclophosphamide therapy have been well described (Fairley et al, 1972). In a study of 31 adult males treated with cyclophosphamide for nephrotic syndrome, the majority were azoospermic after 6 months of treatment, and in the 5 cases undergoing testicular biopsy no spermatogenesis was seen. In one case who underwent repeat biopsy 15 months later, there was some return of spermatogenesis. CHLORAMBUCIL Pharmacology

Chlorambucil, like cyclophosphamide, is a bifunctional alkylating agent. The term 'bifunctional' refers to the two chloroethyl radicals possessed by each drug. These radicals have a high affinity for DNA and irreversible crosslinking of D N A strands may occur, resulting in cell death at cell division. The chlorambucil molecule is active unchanged, unlike cyclophosphamide. Both compounds induce interstrand cross-links and it is likely that specific repair processes, such as excision repair, are used by cells to survive DNA damage induced by this drug (Ruhland et al, 1978). Chlorambucil is given as an oral preparation; the intravenous preparation is unstable because of rapid hydrolysis. The oral drug is rapidly absorbed and detectable in the plasma within 15 minutes, reaching peak plasma levels within i hour. With a half-life of 11/2 hours, little is detectable after 6 hours. The main metabolite is phenylacetic mustard, which is also an active alkylating agent. Chlorambucil and its metabolites are excreted in the urine (McLean et al, 1979, 1980). Hypersensitivity to chlorambucil is uncommon but when it occurs the skin rash may be florid (Hitchins et al, 1987); cross-reactivity to cyclophosphamide has also been recorded (Kritharides et al, 1987). Clinical use of chlorambucil

Chlorambucil has proved to be less popular than cyclophosphamide over the last two decades. In part this may be due to the availability of an intravenous preparation of cyclophosphamide, although more likely it reflects fashions, with no objective evidence to prove the benefit of one drug over the other (comparative studies are rare). Chlorambucil has been popular in France for the treatment of most connective-tissue diseases, and more specifically this drug is being used extensively in Europe and North America to treat inflammatory eye disease associated with these disorders. Use in rheumatoid arthritis

Chlorambucil became a popular choice of treatment for severe, resistant rheumatoid arthritis in France in the 1960s. Although clinical benefit was

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observed, studies were not controlled. Doses ranged between 0.1 and 0.2 mg kg -1 day -1 and treatment was continued for many months or years. Forty patients with rheumatoid arthritis who had not responded to conventional second-line therapy including prednisolone were given chlorambucil for 3--4 months with a significant improvement in more than half the patients, there being a concomitant fall in rheumatoid factor (Kahn et al, 1967). Chlorambucil was reported to be helpfulin the treatment of 23 out of 40 severe cases of rheumatoid arthritis, including three patients with biopsy proven vasculitis complicating their arthritis. Similar results have been reported in other series (Kahn and de Seze, 1974). It is common practice to start with a loading dose and then gradually reduce the amount given. Frequently there is a time delay of up to 2-3 months before clinical improvement is detected and the improvement may persist for many months or years after discontinuing therapy, although after a year off treatment relapses are common. More recent experience, again uncontrolled, has shown that approximately three-quarters of patients with extensive rheumatoid arthritis respond to chlorambucil therapy, some patients being given continuous low-dose therapy for several years (Cannon et al, 1985). Use in vasculitis and connective-tissue diseases

Reports on the use of chlorambucil for vasculitis complicating rheumatoid arthritis are limited. Cutaneous lesions, neuropathic changes and systemic manifestations of rheumatoid disease may respond to chlorambucil treatment (Thorpe et al, 1976). Chlorambucil has been used in patients with Wegener's granulomatosis. Israel and Patchefsky (1975) treated 12 cases, of whom the majority had non-renal disease. Chlorambucil was the least toxic agent used in their experience but was less effective than cyclophosphamide. Although anecdotal evidence hints that chlorambucil helps some patients with scleroderma, a recent double-blind placebo-controlled study of 65 patients indicated that it did not help the outcome of this disease; doses of drug ranged between 0.05 and 0.1 mg kg- 1 day- 1 (Furst et al, 1989). There appears to be no benefit from long-term chlorambucil on lung function in scleroderma (Greenwald et al, 1987). Data concerning the use of chlorambucil in the treatment of SLE are limited. Small numbers of patients have been noted to have improvement of renal function and prolongation of remission when treated with this drug (Abuelo et al, 1984). Use in amyloidosis

Chlorambucil has an established role in the treatment of amyloidosis complicating connective-tissue diseases, especially juvenile chronic arthritis. Continuous low-dose therapy administered daily for many years led to greater survival than intermittent treatment and both regimens led to a better prognosis than for the untreated in children (Schnitzer and Ansell, 1977). Adults with rheumatoid arthritis or ankylosing spondylitis who had


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developed amyloidosis also had a significant improvement in renal function and prolonged survival after treatment with chlorambucil; even in those patients who deteriorated it was noted that the rate of worsening was slower with treatment than with no treatment (Berglund et al, 1987). Use in inflammatory eye disease complicating connective-tissue diseases Chlorambucil has been used more frequently than other alkylating agents to treat eye disease complicating connective-tissues diseases. Again it is likely that it is a reflection of trends rather than being based upon objective evidence that this is a more effective drug. There are several reports concerning the uveitis of Beh~et's disease--frequently patients have failed to respond adequately to oral steroids and chlorambucil leads to an improvement of vision. Unfortunately, most studies are not controlled (Mamo and Azzam, 1970; Abdalla and Bahgat, 1973). Chlorambucil was superior to colchicine and was given at 0.1 mgkg -1 day -1 with a gradual tapering of the dose (O'Duffy et al, 1984). In a trial comparing this regimen with prednisolone, chlorambucil was found to be more effective. However, since then the latter drug has been re-evaluated and not thought to be useful in the most significant and common lesion of Beh~et's, namely the uveitis. Tabbara (1983) reported that in 10 male patients with ocular Beh~et's, 7 were rendered oligospermic and 3 azoospermic by chlorambucil, without any significant improvement in their eye lesions (75% of their eyes had a visual acuity of 20/200 or worse). The treatment of choice currently for eye disease complicating Beh~et's appears to be cyclosporin A. The meningoencephalitis of Beh~et's syndrome and the scleritis and uveitis that sometimes complicates rheumatoid arthritis may also respond favourably to chlorambucil (Godfrey et al, 1974). But there is no consensus of opinion on the likelihood of benefit of chlorambucil given to children with uveitis complicating juvenile chronic arthritis, some responding but others having no improvement. It is possible that benefit is only short lived (Mehra et al, 1981; Palmer et al, 1985a). Side-effects The advantages of chlorambucil have to be balanced against the risks of inducing adverse effects, including neoplasia. Cytotoxic drugs are used to treat non-malignant diseases, which is a particular concern when chlorambucil is given to children many of whom have a good life expectancy. It is generally accepted that chlorambucil will induce malignancies in both adult patients and children with connective-tissue diseases (Kahn et al, 1979; Buriot et al, 1979). There are no data to indicate that there are differences between the frequency of tumours induced by chlorambucil compared with those by cyclophosphamide. Some reports suggest that these drugs induce malignancy in a dose-dependent manner. Chlorambucil precipitated leukaemia in polycythaemia with a risk that was four times greater in those patients whose daily dose exceeded 4 mg than in those who had 4 mg or less, and five times greater.in those who had received the drug for at least 50% of the time during

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the treatment period than in those who had received it for a shorter duration (Berk et al, 1981). In addition, it was noted that leukaemia following chlorambucil therapy for rheumatoid arthritis occurred in those patients who had received a total dose of at least 1 g and had been treated for at least 6 months (Kahn et al, 1979). For cyclophosphamide it was observed that the group of patients with rheumatoid arthritis who developed malignancies following this drug had received a higher mean total dose than those without tumours (Baltus et al, 1983). A retrospective survey (Patapanian et al, 1988) of 39 patients receiving chlorambucil for a mean of 25 months as treatment for rheumatoid arthritis revealed a high incidence of cutaneous (8 cases) and haematological malignancies (3 cases). In the control group of 30 patients treated for a similar period with either azathioprine or 6-mercaptopurine for their rheumatoid arthritis there was only one case of skin malignancy. The authors felt that the oncogenic risk of chlorambucil was too high to justify its use in rheumatoid arthritis. An extensive review of the literature concerning chlorambucil discussed the possibility that tumours may be induced by this drug in less than 1% of patients. The majority of the malignancies were leukaemias, with a particularly high incidence of acute myeloid leukaemia. It was not possible to exclude the possibility that in part the disease process itself accounted for the problem. However, there were hints that patients with connective-tissue diseases could be more sensitive to the leukaemogenic effects of chlorambucil than patients with other non-malignant conditions (Palmer and Denman, 1984). The mechanism of tumour induction by alkylating agents is not known, although damage to D N A is likely to be an important component. This damage may be reflected by chromosomal aberrations. There were more chromosome abnormalities in eight patients with rheumatoid arthritis treated with cyclophosphamide than in a group of untreated patients with this disease (Tolchin et al, 1974). However, significant chromosome damage was not apparent in SLE patients receiving chlorambucil (Snaith et al, 1973) and abnormalities were unusual in patients with Beh~et's uveitis treated with this drug (Reeves et al, 1975). A more sensitive measure of chromosome damage may be found in sister chromatid exchange; increased frequencies have been found in lymphocytes of patients with connective-tissue diseases treated with chlorambucil, the increase being dose-dependent and cumulative (Palmer et al, 1984). Similar findings occur in both adults and children (Palmer et al, 1985b). Cyclophosphamide may have a greater potential than chlorambucil for inducing this type of chromosome damage in patients with these diseases than chlorambucil (Palmer et al, 1986a). In addition this drug is responsible for a high incidence of specific mutations in T-cells of lymphocytes from these patients (Palmer et al, 1986b). Much of the published work on the effects of chlorambucil on gonadal function has concerned damage to testicular cells; ovarian function is probably equally at risk. Children with nephrotic syndrome who have been treated with chlorambucil frequently have oligo- or azoospermia in association with abnormalities of hormone function, especially elevated FSH levels (Guesry et al, 1978; Callis et al, 1980). Testicular damage may be apparent on biopsy (Reichter et al, 1970). For many of these reported


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patients, the doses of chlorambucil have been much higher than would be given as immunosuppressive therapy in connective-tissue diseases; nevertheless, it is reasonable to assume that suppression of testicular function will occur to a significant degree. MECHANISMS OF ACTIONmSIMILARITIES AND DIFFERENCES

Azathioprine is toxic to cells in the S phase of the mitotic cycle, which is the time of DNA synthesis (Spina, 1984). Thus, its effect is not really cytotoxic; rather it reduces the rate of cell division. Azathioprine is reported to inhibit both T cell and B cell function (Yu et al, 1974); there was no selectivity of T or B cells in these experiments so that the T/B ratio was unaltered. Azathioprine inhibits production of immunoglobulin by B cells (Levy et al, 1972) (reduced synthesis of IgM and IgG after 4 months' treatment in 9 patients) as well as B lymphocyte proliferation (Abdou et al, 1973). The effects of azathioprine on T cell function are not clear: Fournier et al (1973) reported a specific effect, whereas other studies have found none. Azathioprine has been shown to have a cumulative effect in vivo on natural killer cell function and number (Spina, 1984). A comparison between cyclophosphamide and steroid effect in vivo was performed by Bast et al (1983). In 5 patients with malignant melanoma who were treated with intravenous cyclophosphamide, there was a dose-related response. At low doses, B cells were affected; higher doses were required to reduce CD8 numbers, and CD4 cells were the most resistant. On the other hand, prednisolone given to healthy volunteers had no effect on peripheral blood phenotype. Hengst and Kempf (1984) reviewed the effects of cyclophosphamide on immunocompetent cells. The drug appears to act predominantly on the premitotic phase (G2) of the cell cycle, but also interferes with a number of metabolic pathways, so that its precise mechanism of action is not clear (Wheeler and Alexander, t969). Humoral immunity is most easily suppressed by cyclophosphamide at moderate doses, whereas T cells are more resistant, and this appears to be most pronounced in the CD4+ cells. There may be an inhibition of interleukin-2 production. Paradoxically, cyclophosphamide enhances many of the functions of CDS+ cells. We have shown benefit from cyclophosphamide in patients with the histologically similar necrotizing vasculitis of PAN and SRV despite the fact that the pathogenesis of these two diseases appears different. Cyclophosphamide even appears to favourably influence the histologically different giant cell arteritis described by Takayasu. There is thus confusion as to the mechanism of action. In SRV, following therapy, levels of both the IgG rheumatoid factor and the immune complexes fall, suggesting immunosuppressant action at the B-cell level with diminished formation of autoantibodies and immune complexes (Scott et al, 1981b). Elevated levels of circulating activated T cells again fall with therapy. By contrast, in PAN, relevant complement-fixing immune complexes are rare (except in the few cases associated with hepatitis B), as are elevated levels of activated T cells.

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Therefore, the mechanism(s) of action of cyclophosphamide in PAN is unlikely to be through these immunological pathways. It may be through a final common pathway of vascular inflammation, perhaps at the endothelial cell level. The cytotoxic effect of cyclophosphamide is via the phosphoramide mustard which covalently bonds to nucleic acids within DNA, thereby preventing replication of the cell (Fischer, 1982). It is likely that the cyclophosphamide is affecting the cells mediating the vascular damage in these diseases rather than the underlying trigger factors. In necrotizing vasculitis, polymorph-mediated damage may be stimulated by either immune complexes or free radicals (Cochrane and Aiken, 1966; Cochrane, 1968), indicating that polymorphs may also be a target of cyclophosphamide. A final interesting effect of cyclophosphamide in rheumatoid disease is the way it appears to shift patients across the clinical and immune spectrum. In SRV we have observed a number of patients who have developed a flare-up in their joint disease simultaneously with improvement in vasculitis following intermittent bolus cyclophosphamide (Bacon, 1987). The reverse is also seen, since the use of steroids and gold for rheumatoid synovitis has on occasion been associated with the development of rheumatoid vasculitis. This is not the only pathway, since it has been noted that methotrexate for rheumatoid synovitis can be associated with the development of rheumatoid nodules (Segal et al, 1988) and our experience is similar. This effect may not depend on pre-existing synovitis, since we have seen five patients whose joint disease developed during or soon after the treatment of vasculitis with cyclophosphamide. CONCLUSIONS We have reviewed the use of three agents in rheumatological practice. There are clear similarities and differences between them, and to some extent this reflects their use in different disorders. As with all chronic diseases, long-term toxicity is a serious consideration and must be balanced against the potential therapeutic benefits. In the case of refractory but uncomplicated rheumatoid arthritis, the evidence is in favour of azathioprine (and increasingly also methotrexate). The development of systemic manifestations heralds a different phase of the disease and requires more aggressive therapy and our choice would be cyclophosphamide. For vasculitis this is probably best given as pulsed therapy combined with prednisolone. Systemic vasculitides often require cytotoxic agents in combination with steroids, and there is strong evidence in favour of cyclophosphamide as the drug of choice. In view of the potential problems of low-dose oral continuous therapy both in the short term and in the long term, we have tended to favour the use of high-dose intermittent cyclophosphamide. Interestingly, there is very little known about the effects of cyclophosphamide on Beh~et's disease, where traditionally chlorambucil has been widely used until the more recent advent of cyclosporin A. In treating lupus, one has to balance the amount of activity of the disease against the irreversible


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effects of previous scarring, particularly with respect to the renal lesions. The large NIH study suggested that pulsed intravenous cyclophosphamide alone resulted in the least toxicity and least likelihood of progression to end-stage renal disease. Against this background is the ever-improving management of end-stage renal disease. In the miscellaneous connective-tissue diseases, some of these agents have usefully been employed, although there are too few numbers in any series to determine which is the drug of choice in these diseases. SUMMARY

Immunosuppressive agents serve a major role in the management of oncefatal conditions such as the systemic necrotizing vasculitides, but they are also being used in more common, chronic inflammatory disorders such as rheumatoid arthritis. The drugs are all capable of reducing cell division but they differ in their modes of action. This is in keeping with their differing rates of action, and different indications. Azathioprine is a valuable alternative to slow-acting antirheumatic drugs in older patients with rheumatoid arthritis. Cyclophosphamide has transformed the outlook of many forms of vasculitis. Chlorambucil is particularly useful in improving the prognosis for children with amyloidosis secondary to juvenile chronic arthritis. We have tried to highlight the role of these drugs in a number of rheumatic diseases. We have emphasized their clinical applications, with some laboratory evidence for their effects. The major side-effects are reviewed. Finally, we have discussed their possible mechanisms of action. REFERENCES Abdalla MI & Bahgat NE (1973) Long-lasting remission of Beh~et's disease after chlorambucil therapy. British Journal of Ophthalmology 57: 706-711. Abdou NI, Zweimann B & Casella SR (1973) The effects of azathioprine therapy on bone marrow dependent and thymus dependent cells in man. Clinical and Experimental Immunology 13: 55-64. Abuelo JG, Esparza AR & Garella S (1984) Steroid dependent nephrotic syndrome in lupus nephritis--response to chlorambucil. Archives oflnternal Medicine 144: 2411-2412. Adu D, Howie AJ, Scott DGI et al (1987) Polyarteritis and the kidney. Quarterly Journal of Medicine 62: 221-237. Arnold H & Bourseaux F (1958) Synthese und Abbau cytostatisch wirksamer cyclischer N-Phosphamidester des Bis-(13-chlorathyl)-amins. Angewandte Chernie 70: 539-544. Austin HA, Klippel JH, Balow JE et al (1986) Therapy of lupus nephritis: controlled trial of prednisolone and cytotoxic drugs. New England Journal of Medicine 314: 614-619. Bacon PA (1987) Vasculitis---clinical aspects and therapy. Acta Medica Scandinavica Supplementum 715: 157-163. Bagley CM Jr, Bostick FW & DeVita VT (1973) Clinical pharmacology of cyclophosphamide. Cancer Research 33: 226-233. Baker GL, Kahl LE, Zee BC et al (1987) Malignancy following the treatment of rheumatoid arthritis with cyclophosphamide. Long term cases control follow up study. American Journal of Medicine 83: 1-9. Baltus JAM, Boersma JW, Hartman AP & Vandenbroucke JP (1983) The occurrence of malignancies in patients with rheumatoid arthritis treated with cyclophosphamide: a controlled retrospective follow-up. Annals of the Rheumatic Diseases 42: 368-373.

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Barnett EV, Dornfield L, Lee DBN & Liebling MR (1978) Long term survival of lupus nephritis patients treated with azathioprine and prednisolone. Journal of Rheumatology 5: 275-287. Bast RC Jr, Reinherz EL, Maver C et al (1983) Contrasting effects of cyclophosphamide and prednisolone on the phenotype of human peripheral blood lymphocytes. Clinical Immunology and Immunopathology 28: 101-114. Berglund K, Keller C & Thysell H (1987) Alkylating cytostatic treatment in renal amyloidosis secondary to rheumatic disease. Annals of the Rheumatic Diseases 46: 757-762. Berk PD, Goldberg JD, Silverstein MN et al (1981) Increased incidence of acute leukemia in Polycythemia vera associated with Chlorambucil therapy. New England Journal of Medicine 304: 441-447. Berry H, Liyanage SP, Durance RA et al (1976) Azathioprine and penicillamine in the treatment of rheumatoid arthritis: a controlled trial. British Medical Journal 7: 1052-1054. Bradley JD, Brandt KD & Katz BP (1989) Infectious complications of cyclophosphamide treatment for vasculitis. Arthritis and Rheumatism 32: 45-53. Bunch TW (1981) Prednisone and azathioprine for polymyositis. Arthritis and Rheumatism 24: 45-48. Buriot D, Prieur A, Lebranchu Yet al (1979) Acute leukemia in three children after treatment of juvenile chronic arthritis with chlorambucil. Archives Francaises de Pediatrie 36: 592-598. Cade R, Spooner G, Schlein E et al (1973) Comparison of azathioprine, prednisone and heparin alone or combined in treating lupus nephritis. Nephron 10: 37-56. Callis L, Nieto J, Vila A & Rende J (1980) Chlorambucil treatment in minimal lesion nephrotic syndrome: A reappraisal of its gonadal toxicity. Journal of Pediatrics 97: 653-656. Cameron JS, Turner DR, Ogg CS et al (1979) Systemic lupus with nephritis: A long term study. Quarterly Journal of Medicine 48: 1-24. Cannon GW, Jackson CG, Samuelson CO et al (1985) Chlorambucil therapy in rheumatoid arthritis: clinical experience in 28 patients and literature review. Seminars in Arthritis and Rheumatism 15: 106--118. Carette S, Klippel JH, Decker JL et al (1983) Controlled studies of oral immunosuppressive drugs in lupus nephritis. A long term follow up. Annals oflnternal Medicine 99: 1-8. Cheigh JS & Reidenberg MM (1978) Cyclophosphamide dose in renal failure (letter). American Journal of Medicine 64: 726. Chumbley LC, Harrison R A & DeRemee RA (1977) Allergic granulomatous angiitis (ChurgStrauss syndrome). Report and analysis of 30 cases. Mayo Clinic Proceedings 52: 477-484. Cochrane CG (1968) Immunologic tissue injury mediated by neutrophilic leukocytes. Advances in Immunology 9" 97-162. Cochrane CG & Aiken BS (1966) Polymorphonuclear leukocytes in immunologic reactions. The destruction of vascular basement membrane in vivo and in vitro. Journal of Experimental Medicine 124: 733-752. Cooperating Clinics Committee of the American Rheumatism Association (1970) A controlled trial of cyclophosphamide in rheumatoid arthritis. New England Journal of Medicine 283: 883-889. Csuka M, Carrera GF & McCarty D3 (1986) Treatment of intractable rheumatoid arthritis with combined cyelophosphamide, azathioprine and hydroxychloroquine. A follow up study. Journal of the American Medical Association 255: 2315-2319. Currey HLF, Harris J, Mason RM et al (1974) Comparison of azathioprine, cyclophosphamide and gold in the treatment of rheumatoid arthritis. British Medical Journal 3: 763-766. Decker JL, Klippel JH, Plotz PH & Steinberg AD (1975) Cyclophosphamide or azathioprine in lupus glomerulonephritis. A controlled trial results at 28 months. Annals of Internal Medicine 38: 606-615. Decker JL, Steinberg AD, Reinertsen JL et al (1979) Studies in the treatment of lupus nephritis. Annals of lnternal Medicine 91: 587-604. De Silva M & Hazleman B (1981) Long-term azathioprine in rheumatoid arthritis: a doubleblind study. Annals of the Rheumatic Diseases 40: 560-563. De Silva M & Hazleman B (1986) Azathioprine in giant cell arteritis/polymyalgia rheumatica: a double blind study. Annals of the Rheumatic Diseases 45: 136-138. Donadio JV, Holley KE, Wagoner RD et al (1974) Further observations on the treatment of lupus nephritis with prednisolone and combined prednisolone and azathioprine. Arthritis and Rheumatism 17: 573-581.


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Dwosh IL, Stein HP, Urowitz MB et al (1977) Azathioprine in early rheumatoid arthritis. Comparison with gold and chloroquine. Arthritis and Rheumatism 20: 685-692. Elliott RW, Essenhigh DM & Morley AR (1982) Cyclophosphamide treatment of systemic lupus erythematosus: risk of bladder cancer exceeds benefits. British Medical Journal 284: 1160-1161. Ellman P & Ball RE (1948) Rheumatoid disease with joint and pulmonary manifestations. British Medical Journal 2" 816-820. Fairley KF, Barrie JG & Johnson W (1972) Sterility and testicular atrophy related to cyclophosphamide therapy. Lancet 1: 568--569. Fauci AS & Wolff SM (1973) Wegener's granulomatosis: studies in eighteen patients and a review of the literature. Medicine 52" 535-561. Fauci AS, Haynes BF, Katz P & Wolff SM (1983) Wegener's granulomatosis: prospective clinical and therapeutic experience with 85 patients for 21 years. Annals of Internal Medicine 98: 76-85. Felson DT & Anderson J (1984) Evidence for the superiority of immunosuppressive drugs and prednisone over prednisone alone in lupus nephritis. Results of a pooled analysis. New England Journal of Medicine 311: 1528-1533. Fischer DS (1982) Alkylating agents. In Fischer DS & Marsh JC (eds) Cancer Therapy, Boston: G K Hall. Fosdick WM, Parsons JL & Hill DF (1968) Long term cyclophosphamide therapy in rheumatoid arthritis. Arthritis and Rheumatism 11: 151-161. Fournier C, Bach MA, Dardenne M & Bach JF (1973) Selective action of azathioprine on T cells. Transplantation Proceedings 5: 523-526. Friedman OM, Myles A & Colvin M (1979) Cyclophosphamide and related phosphoramide mustards: Current status and future prospects. Advances in Cancer Chemotherapy 1: 143-204. Fries JF, Sharp GC, McDevitt HO & Hohnan HR (1973) Cyclophosphamide therapy in systemic lupus erythematosus and polymyositis. Arthritis and Rheumatism 16: 154-162. Furst DE, Clements PJ, Hillis S et al (1989) Immunosuppression with chlorambucil versus placebo for scleroderma. Arthritis and Rheumatism 32: 584-593. Galetti PM, Pasqualino A & Geering RG (1966) Haemodialysis in cancer chemotherapy. Transactions of the American Society for Artificial Organs 12: 20-25. Godfrey WA, Epstein WV, O'Connor GR et al (1974) The use of chlorambucil in intractable idiopathic uveitis. American Journal of Ophthalmology 78: 415-427. Greenwald GI, Tashkin DP, Gong H et al (1987) Longitudinal changes in lung function and respiratory symptoms in progressive systemic sclerosis. Prospective study. American Journal of Medicine 83: 83-92. Guesry P, Lenoir G & Broyer M (1978) Gonadal effects of chlorambucil given to prepubertal pubertal boys for nephrotic syndrome. Journal of Pediatrics 92- 299-303. Guillevin L, Le Thi Huong Du, Godeau P e t al (1988) Clinical findings and prognosis of polyarteritis nodosa and Churg-Strauss angiitis: a study in 165 patients. British Journal of Rheumatology 27: 258-264. Hahn BH, Kantor OS & Osterland CK (1975) Azathioprine plus prednisolone compared with prednisolone alone in the treatment of systemic lupus erythematosus. Report of a prospective trial in 24 patients. Annals oflnternal Medicine 83" 597-605. Hall ND, Bird HA, Ring EFJ & Bacon PA (1979) A combined clinical and immunological assessment of four cyclophosphamide regimes in rheumatoid arthritis. Agents and Actions 9" 97-102. Harris J, Jessop JD & Chaput du Saintoge DM (1971) Further experience with azathioprine in rheumatoid arthritis. British Medical Journal 4: 463-464. Hayslett JP, Kashgarian M, Cook CD & Spargo BH (1972) The effect of azathioprine on lupus glomerulonephritis. Medicine (Baltimore) 51: 393-412. Hellman DB, Petri M & Whiting-O'Keefe Q (1987) Fatal infections in systemic lupus erythematosus: The role of opportunistic organisms. Medicine (Baltimore) 66: 341347. Hengst JCD & Kempf RA (1984) Immunomodnlation by cyclophosphamide. Clinics in Immunology and Allergy 4: 199-216. Hersh EM, Wong VG & Freireich EJ (1966) Inhibition of the local inflammatory response in man by antimetabolites. Blood 27: 38-48.

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Comparison of chlorambucil, azathioprine or cyclophosphamide combined with corticosteroids in the treatment of lupus nephritis.

Immunosuppression with azathioprine, prednisone, and cyclophosphamide.

Latent carcinoma of the prostate in a 24-year-old man receiving cyclophosphamide and azathioprine.

Wegener's granulomatosis. Combined therapy with low-dosage systemic corticosteroids, azathioprine and cyclophosphamide in three patients.

Interstitial lung disease resistant to corticosteroid therapy. Report of three cases treated with azathioprine or cyclophosphamide.

The effects of cyclophosphamide and azathioprine on collagen in skin and granulation tissue in rats, and the effects of cyclophosphamide on collagen in human skin.

A controlled trial of cyclophosphamide and azathioprine in Nigerian children with the nephrotic syndrome and poorly selective proteinuria.

Combination of pulse cyclophosphamide and azathioprine in ocular manifestations of Behcet's disease: longitudinal study of up to 10 years.

The metabolism of chlorambucil.

Azathioprine and warts.

Long-term follow-up of cyclophosphamide compared with azathioprine for initial maintenance therapy in ANCA-associated vasculitis.

Kinetics and mechanism of chlorambucil hydrolysis.

Focal fits during chlorambucil therapy.

Studies of chlorambucil-DNA adducts.

Pancreatitis, amylase clearance, and azathioprine.

Fatal azathioprine toxicity.

Chlorambucil-induced acute interstitial pneumonitis.

Determination of plasma azathioprine and 6-mercaptopurine in patients with rheumatoid arthritis treated with oral azathioprine.

Azathioprine-induced atrial fibrillation.

Azathioprine in Behçet's syndrome.

Cytotoxicity of chlorambucil and chlorambucil-fatty acid conjugates against human lymphomas and normal human peripheral blood lymphocytes.

[Neutrophilic eccrine hidradenitis and azathioprine hypersensitivity syndrome].

AZT: zidovudine or azathioprine?

c mice.