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Immunosuppressive Drug Therapy in Lupus Nephritis
American Journal of Kidney Diseases
IF
The Official Journal of
The National Kidney Foundation
VOL 21, NO 3, MARCH 1993
IN-DEPTH REVIEW
Immunosuppressive Drug Therapy in Lupus Nephritis James V. Donadio, Jr, MD, and Richard J. Glassock, MD • During the past two decades, the immunosuppressive drugs azathioprine and cyclophosphamide have been widely used in the treatment of patients with lupus nephritis. Their toxicities are well known and are mostly dose- and timedependent. Complications that arise from these therapies stem from their immunosuppressive (susceptibility for infection) or pharmacologic (hemorrhagic cystitis, bladder cancer, and fibrosis from the alkylating agents) effects, or both. Uncontrolled studies reporting good results in treating patients with various combinations of corticosteroids and azathioprine and, especially, cyclophosphamide cannot be conclusively confirmed by the few controlled clinical trials that are available for review. Part of the problem of inconclusiveness has to do with timing treatment to different phases of the disease and the vast heterogeneity of lupus nephritis. Although these immunosuppressive agents may have favorable effects on the overall activity of systemic lupus erythematosus, their long-term effects per se on renal disease are in question and could be attributed to lower prednisone dosage and better medical management of hypertension, hyperlipidemia, infection, and other metabolic consequences of the disease. © 1993 by the National Kidney Foundation, Inc. INDEX WORDS: Immunosuppressive therapy; lupus nephritis.
F
OR THE PAST two decades, physicians have used immunosuppressive drugs to treat patients with lupus nephritis. The vast majority of both uncontrolled studies and prospective therapeutic trials have used two agents, the antimetabolite azathioprine and the alkylating agent cyclophosphamide. Both drugs are immunosuppressive in that they interfere with the growth or survival of immune cells. In this review, we focus on these two immunosuppressive drugs and discuss immunosuppressive properties, toxicities, and clinical studies pertaining to their use in the treatment of patients with lupus nephritis.
patients with lupus nephritis. Excellent reviews of the pharmacology of the antimetabolites are available to the interested reader. loS The immunosuppressive activities of azathioprine and 6-MP depend on the timing of their administration relative to antigenic challenge. Suppression is achieved only if these drugs are administered in the interval shortly after the immunologic stimulation. 6 Immune responses that require cell proliferation, such as the clonal expansion of T-helper cells driven by antigen exposure, may be inhibited; therefore, antibody production, graft rejection, and the induction of autoimmune disease may be suppressed. Aza-
ANTIMETABOLITES
Antimetabolites interfere with protein synthesis by competing for and blocking specific receptors. They include the purine antagonist 6-mercaptopurine (6-MP}-the parent compound of azathioprine; the pyrimidine antagonist 5-fluorouracil; cytosine arabinoside; and the folic acid antagonist methotrexate. Azathioprine has been the antimetabolite used most frequently to treat
From the Department ofNephrology and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN; and the Department of Medicine, University of Kentucky Medical School, Lexington, KY. Received May 19, 1992; accepted in revised form October 1,1992. Address reprint requests to James V. Donadio, MD, Mayo Clinic, 200 First St SW; Rochester, MN 55905. © 1993 by the National Kidney Foundation, Inc. 0272-6386/93/2103-0001$3.00/0
American Journal of Kidney Diseases, Vol 21, No 3 (March). 1993: pp 239-250
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DONADIO AND GLASSOCK
thioprine and 6-MP also exert a nonspecific, antiinflammatory effect, but this is probably not an important part of their immunosuppressive actions. 7 There is a lack of agreement on the immunosuppressive superiority of azathioprine over 6MP. Some investigators believe that the parent compound (6-MP) has greater activity,8 whereas others regard azathioprine as the more effective immunosuppressive drug. 9 In part, differences may be due to such factors as the test animal species, experimental models, routes of administration, and doses of the compounds used. Nevertheless, evidence suggests that azathioprine may be the more active form. Such evidence includes (l) azathioprine is absorbed better in the gastrointestinal tract than is 6-Mp lO ; (2) azathioprine has a lower incidence of toxicity at equivalent immunosuppressive concentrations ll ; (3) azathioprine can interfere with in vitro tests of lymphocyte reactivity at lower concentrations than 6_Mp I2 ; and (4) different, but as yet unknown, metabolism of azathioprine and 6-MP may occur in the body, which results in further immunosuppressive compounds. 9 Further studies are required to define clearly the superiority of one compound over the other. ALKYLATING AGENTS
Cyclophosphamide, the first clinically useful compound of the generically termed "oxazaphosphorines," is an alkylating agent whose cytotoxic activity is mediated through covalent bonding of alkyl groups (one or more saturated carbon atoms) to cellular molecules. 13 Cyclophosphamide itself is without alkylating or cytotoxic activity and must be metabolized in the body to its alkylating metabolites. Again, excellent pharmacologic reviews of the alkylating agents are available. 5,12-14 The immunosuppressive activity of alkylating agents was first described by Hektoen and Corper, 15 who reported that sulfur mustard depressed the serum antibody response of rabbits and dogs to heterologous erythrocytes. The most immunosuppressive drug of the alkylating agents, both on a molar basis and relative to other toxicities, is cyclophosphamide. 16 Selective effects of cyclophosphamide on different components of the lymphoid system have been described. In vivo, it has been reported to selectively suppress Blymphocyte function and to deplete B lympho-
cytes morphologically.17 However, cyclophosphamide can also suppress lymphocyte functions that are mediated by T cells, such as graft-versushost response and delayed hypersensitivity. 18 Properly timed and appropriate doses of cyclophosphamide in vivo or of activated cyclophosphamide in vitro can enhance immunologic responses by selective inhibition of the function of suppressor T cells. 19.20 Cyclophosphamide has therapeutic efficacy in the treatment of certain cancers and for the purpose of immunosuppression. Relative to other cross-linking agents, cyclophosphamide seems to be more useful in the treatment of various autoimmune disorders. Why this should be is not clearly understood. That cellular elements of the humoral and cell-mediated immune systems are differentially sensitive to the cytotoxic action of the oxazaphosphorines may be important. 21 Also important could be the fact that differentiated blood cells seem to be more sensitive to the cytotoxic action of the oxazaphosphorines than are pluripotent or committed progenitor hematopoietic cells. 22 Differential sensitivity could be the result of differential rates of biotransformation. Seemingly paradoxic is the fact that low doses of cyclophosphamide may effect an augmentation of the cell-mediated immune response. This may be because T-suppressor cells are apparently more sensitive to these agents than are other T-cell subsets?3 Clearly, more information is needed to explain fully the clinically observed immunosuppressive effects of cyclophosphamide. In the treatment of cancer, the most clinically useful alkylating agents have been the bischloroethylamines or nitrogen mustards. 13 Mechlorethamine was used in early studies of cytotoxic drugs in lupus,24 but it has been replaced for the most part in clinical use by cyclophosphamide. Chlorambucil, another analog of the nitrogen mustards, has been used in only one short-term (10-week) clinical trial oflupus nephritis,25 having been used most widely in the treatment of chronic lymphocytic leukemia, lymphomas, and ovarian carcinoma. 13 TOXICITIES OF IMMUNOSUPPRESSIVE DRUG THERAPY
Toxicities from immunosuppressive drugs are mostly dose- and time-dependent, and there are complications of these therapies that stem from the immunosuppressive (susceptibility for infec-
IMMUNOSUPPRESSIVES IN LUPUS NEPHRITIS
tion) or pharmacologic (hemorrhagic cystitis, bladder fibrosis due to alkylating agents) effects, or both. The most common toxic effect of each drug is depression of normal hematopoiesis (Table I).! In clinical studies with azathioprine, cytopenias occur 7 to 14 days after drug administration, the delay being due primarily to the interval required for precursor cells to differentiate into mature elements. The degree of hematopoietic toxicity is dose-dependent and is reversible on discontinuing therapy. A mild macrocytic anemia and megaloblastic changes in the bone marrow can be seen with chronic use. For the alkylating agents, the usual dose-limiting toxicity is also suppression of hematopoiesis. This suppression includes all the formed elements of the bloodleukocytes, platelets, and erythrocytes. With cyclophosphamide, the level of circulating leukocytes decreases as a function of the dose and frequency of administration. There is a relative platelet-sparing effect. Focusing further on toxicities attributable to both drugs (Table 1) and germane to azathioprine and cyclophosphamide treatment of patients with lupus nephritis, there are two long-term potential complications: development of malignancy26-29 and opportunistic infection.2 7•28 ,3o A prospective study29 in the United Kingdom of 1,634 patients without transplants treated with immunosuppressive drugs (68% with azathioprine, 28% with cyclophosphamide) found an excess of non-Hodgkin's lymphoma and squamous cell skin cancer (both drugs) and bladder cancer (cyclophosphamide). The relative risks for the three types of malignancy (observed/expected cases) were 10.9, 5.0, and 3.7, respectively. Only 38 of the 1,634 patients studied had lupus nephritis and polyarteritis, whereas 643 had rheumatoid arthritis. A separate analysis of the patients with rheumatoid arthritis found a 13-fold increase in non-Hodgkin's lymphoma, which was not significantly different from the excess in similarly treated patients with other disorders in the study. Four cancers, one fatal (myelocytic leukemia), in azathioprine-treated patients, and three malignancies, including one transitional cell carcinoma of the bladder in cyclophosphamidetreated (oral administration) patients, were reported in the long-term controlled studies of lupus nephritis at the National Institutes of Health (NIH).28 One fatal carcinoma of the tongue de-
241 Table 1. Toxicities Attributed to Immunosuppressive Drugs Antimetabolites
Abnormal hepatic function Pancreatitis
Both Drugs
Alkylating Agents
Hematopoietic suppression Carcinogenesis Opportunistic infection Teratogenesis Pulmonary fibrosis
Nausea and vomiting Reproductive tissue Renal and bladdercystitis, fibrOSis, carcinoma Alopecia Allergic reactions Water intoxication Cardiac failure
veloped in a patient treated for 6 months with orally administered cyclophosphamide in another randomized, long-term clinical trial from the Mayo Clinic. 3! The second major long-term problem is the increased risk of infection. Susceptibility to infections is due to a decrease in granulocyte production and to chronic, intrinsic impairment of the immune defense system with the superimposed effect of corticosteroid and immunosuppressive drugs. 30 Opportunistic viral and fungal infections are particularly common in patients receiving long-term therapy, and similar frequencies of major infections have developed in treatment groups of patients with lupus nephritis ( -15%) receiving either prednisone alone or prednisone combined with either azathioprine or cyclophosphamide. 27 •28 Pulmonary fibrosis and interstitial pneumonitis have been described in several patients receiving long-term therapy with either azathioprine or cyclophosphamide. 32 ,33 Of course, diffuse pulmonary fibrosis may also be caused by Pneumocystis carinii or cytomegalovirus under conditions of chronic immunosuppression, and they must be ruled out in order not to miss the opportunity for effective antimicrobial therapy. Virtually all the alkylating agents are teratogenic, whereas the teratogenic potential of azathioprine appears to be low, based on the infrequent occurrence of congenital anomalies in the offspring of renal transplant recipients receiving long-term therapy with azathioprine. The teratogenic action appears to be due to direct cytotoxicity to the developing embryo by the same mechanisms that operate in tumor cells. 34,35 Because of the demonstrated teratogenesis of the alkylating agents in animals, there has been appropriate concern about the potential effects of
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their administration to patients during pregnancy. In a report 36 that included 25 instances in which the alkylating agents were administered during the first trimester of pregnancy and the status of the fetus was recorded, there were four fetal malformations. There were no reported instances of malformed fetuses when alkylating agents or other cytotoxic drugs were administered during the second or third trimester. Since this 1968 report, there have been additional reports of malformed children born to mothers who had received chlorambucil,37 cyclophosphamide,38-40 or combined nitrogen mustard and procarbazine41 during the first trimester. Included is a single case report40 of multiple anomalies in an infant born to a woman with systemic lupus erythematosus not known to be in the first trimester of pregnancy who received cyclophosphamide intravenously for exacerbation of skin lesions. In the reported clinical trials in which cyclophosphamide and azathioprine were used in the treatment of patients with lupus nephritis, pregnancy was an exclusion criterion for the entry of patients into study, and to date no cases of teratogenesis have been reported. 27,31,42,43 Uroepithelial toxicity appears to be unique to cyclophosphamide and its analogs, ranging from mild cystitis to severe bladder damage with massive hemorrhage to invasive carcinoma (Table 1).27,44-46 Acrolein, a noncytotoxic metabolite, may be responsible for cyclophosphamide-induced cystitis. Hemorrhagic cystitis developed in 17% of the patients with lupus nephritis treated with long-term oral cyclophosphamide (median duration, 4 years) in controlled studies at the NIHY The largest experience with long-term oral administration of cyclophosphamide has been in the treatment of patients with Wegener's granulomatosis. In a prospective study of 158 patients studied at the NIH47 in which 133 patients (84%) received therapy with daily low-dose cyclophosphamide and glucocorticoids, the actual duration of cyclophosphamide treatment was not given, but the treatment protocol called for the continuation of cyclophosphamide for at least 1 year after the patient achieved complete remission. Cyclophosphamide-related hemorrhagic cystitis developed in 43% of the patients. In another series46 of 111 patients with Wegener's granulomatosis who were given cyclophosphamide therapy, hemorrhagic cystitis developed in 17 (15%).
DONADIO AND GLASSOCK
The average dose of cyclophosphamide was 101 g (range, 5 to 531 g) given for a mean duration of 38 months (range, 4 to 144 months), and both drug dose and duration of treatment were significantly larger and longer than for patients in whom hemorrhagic cystitis did not develop. In addition, bladder cancer developed in 2.8% of the patients in the NIH study, calculated as a 33fold increase in bladder cancer (observed/expected cases; the latter derived from the National Cancer Institute Registry).47 Three patients had bladder carcinoma in the Mayo Clinic series of patients with Wegener's granulomatosis who were given cyclophosphamide in doses of 56, Ill, and 530 g for 1,4, and 12 years, respectively.46 Thus, the long-term oral administration of cyclophosphamide presents a significant hazard for major urotoxicity, including invasive bladder cancer. The incidence and severity of this complication can be lessened by adequate hydration and frequent bladder emptying or by concomitant use of sodium 2-mercaptoethane sulfonate (mesna).46 In addition, no cases of hemorrhagic cystitis or bladder cancer have yet been reported in the various treatment studies27 ,48,49 oflupus nephritis in which intermittent, intravenously administered cyclophosphamide therapy was used. Alkylating agents have profound toxic effects on reproductive tissue (Table 1). The depletion of testicular germ cells but preservation ofSertoli cells and absence of mature or primordial ovarian follicles are the principal effects on gonadal tissues. 50-52 These effects are dose-related; the cumulative threshold dose above which oligospermia may be expected to occur is between 150 and 250 mg/kg body weight. 53 There may be recovery of spermatogenesis, indicating reversibility after cessation of therapy with alkylating agents. 44 Amenorrhea and ovarian atrophy appear to be more permanent and develop after lower doses in older patients than in younger patients, with less likelihood of reversibility in older patients. 52 For example, in the long-term clinical trials from the NIH,28 in 18 of33 patients (55%) treated with cyclophosphamide, secondary amenorrhea developed: in eight of eight patients when treatment was started in the fourth decade, nine of 17 patients when therapy was started in the third decade, and in only one of eight patients less than age 20 years when cyclophosphamide therapy was started.
IMMUNOSUPPRESSIVES IN LUPUS NEPHRITIS
Many of the toxicities reported with immunosuppressive drugs, especially the alkylating agents, relate to the anticancer doses used with these agents. However, there are some data relating plasma concentrations to either toxicity or therapeutic efficacy in patients treated with these drugs. For cyclophosphamide, in general, anticancer doses are 15 to 25 times the order of magnitude of orally administered doses and three to five times higher than intravenously administered doses reported in the clinical trials for lupus nephritis. 27 ,3J,48,49 Nonetheless, adverse drug reactions and severe complicating illnesses such as opportunistic or reactivated latent infections, hemorrhagic cystitis, ovarian failure, malignancies, and pulmonary fibrosis have been reported in patients receiving these agents for immunosuppressive therapy of autoimmune disorders, including systemic lupus erythematosus. CLINICAL STUDIES IN LUPUS NEPHRITIS
The major difficulty in interpreting the literature on the use of immunosuppressive drugs in the treatment of patients with lupus nephritis, or all human glomerular disease for that matter, is the paucity of suitably controlled studies. Uncontrolled studies reporting good results in treating patients with various combinations of corticosteroids and immunosuppressive agents cannot be conclusively confirmed in the few controlled clinical trials that are available for review. Part of the problem in reconciling differences in published reports and one's own experience of treating patients with lupus nephritis has to do with the status of the disease at the time treatment is begun and the great heterogeneity of morphologic renal lesions responsible for lupus nephritis. An obvious example is attempting to influence advanced late renal disease-in other words, the nephrosclerotic state-with corticosteroid and immunosuppressive drugs. This is exemplified in the controlled studies54 performed at the NIH in which histologic features relating to morphologic activity and chronicity were tested for their predictive value for the development of renal failure. The strongest predictor of a poor renal outcome was a high chronicity index made up of glomerular sclerosis, fibrous crescents, tubular atrophy, and interstitial fibrosis-features of irreversible lupus nephritis. On the other hand, Schwartz et al 55 did not find the chronicity index to be predictive of a renal failure outcome in the
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assessment of 84 biopsy specimens taken from 81 patients with lupus nephritis who entered a randomized clinical trial of plasmapheresis. In lupus nephritis, and as indicated by these two morphologic studies that came to opposite conclusions, this important problem is confounded by the lack of agreement regarding the amount and value of prognostic information that can be gleaned from renal biopsy over and above that from a careful analysis of clinical and laboratory data. This controversy is beyond the scope of the current review and the reader is referred to several studies examining this important topic. 56,57 Related to the problem oftiming treatment in human lupus nephritis are the studies of various murine strains with spontaneous autoimmune disease that show prolonged survival after various interventions with cyclophosphamide, azathioprine, and methylprednisolone alone and in various combinations. 58 Different animal strains develop diverse histologic lesions at different ages, with varying degrees of proteinuria and mortality. Survival studies58 appear to give the best information on the therapeutic intervention. Significant prolongation of survival has been observed only in groups of animals that received cyclophosphamide in high daily or intermittent dosages administered intravenously or in combined therapy with other immunosuppressants when started early in the course of disease, for example, during the early increase of anti-ds DNA titers and before clinical evidence of nephritis, ie, before the appearance of proteinuria. Also, doses were many times higher than those that can be safely used in human clinical trials. For example, NZB/ NZW F J hybrid mice with lupus nephritis were given cyclophosphamide intravenously at a dose of 90 mg/kg/mo for 3 to 8 months. Treatment given for 3 months represents one eighth of a mouse's life span, which is analogous to treating a female patient for approximately 10 years. 58 Thus, the influence of immunosuppressive treatment on murine lupus is quite apart from what can be obtained in clinical practice, in which clinicians must manage patients who have established glomerulonephritis. Moreover, the amount of immunosuppressants and duration of treatment must be modified to avoid major toxicities. Cameron59 addressed in detail the major issues of the complexities of clinical presentation, patient selection, and interpretation of outcome in the study of human glomerular disease.
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It is not surprising, therefore, that much uncertainty remains concerning the added value of immunosuppressive drugs in therapy in human lupus nephritis. In the early and mid 1970s, clinical studies designed to examine the effectiveness of various combinations of prednisone and azathioprine60-62 or prednisone and cyclophosphamide 63-65 in the treatment of lupus nephritis showed that those patients who improved generally had severe and progressive nephritis. Each of these studies had important flaws in design, was small, or had only a short-term follow-up. Histologic descriptions of the renal disease were only given in some cases, and there were wide variations in the degree of impaired renal function and proteinuria. These descriptions of clinical and renal morphologic findings often led the reader to assume that these patients would not have improved had therapy been continued only with prednisone. The presence of hypertension was often ignored, and details of treatment toxicities and complications and their possible contribution to the steroid treatment "failures" were not accounted for in these reports. These uncontrolled observations formed the basis for subsequent prospective clinical trials in patients with lupus nephritis. Two early investigations indicated favorable results from azathioprine treatment either combined with prednisone or heparin or used alone. Sztejnbok et al 66 noted improved renal function and better survival in patients treated with prednisone and azathioprine compared with survival of patients treated with prednisone alone. Patients who received azathioprine achieved better symptomatic control with lower doses of prednisone than did those treated with prednisone only. It is uncertain how many patients with glomerulonephritis were treated, because the investigators did not obtain renal tissue. Cade et al 67 showed improved survival and better renal function in patients with proliferative lupus nephritis who were treated with combinations of azathioprine and prednisone or azathioprine and heparin, or azathioprine alone compared with patients who were treated with prednisone alone. There were frequent toxic complications among the patients treated with prednisone, including hypertension, thromboembolic episodes, and septicemia, and there were also bleeding complications reported in the anticoagulant-treated group, all of which
DONADIO AND GLASSOCK
contributed to renal failure and death in many patients. In these studies, it is possible that the favorable effects may have been more related to avoidance of side effects of corticosteroids, eg, hypertension, rather than to any specific beneficial effects of azathioprine. Clinical improvements also have been reported in more recent uncontrolled studies involving patients with various types of renal involvement who received differing regimens of corticosteroid and immunosuppressive drugs. 48 ,49,68 For example, Ponticelli et al 68 reported 95% patient survival free of renal failure at 5 years in patients with diffuse glomerulonephritis who were treated aggressively with intravenous methylprednisolone usually given with various immunosuppressive drugs, including cyclophosphamide, azathioprine, and chlorambucil. They concluded that the long-term prognosis of diffuse proliferative lupus nephritis is becoming considerably better due to the combination of corticosteroids (short course of high-dose methylprednisolone administered intravenously followed by low-dose prednisone orally) and low doses ofimmunosuppressive agents. Unfortunately, this conclusion cannot be substantiated in an appropriately controlled study. Two additional studies, involving a small number of children9 in one and adults 16 in the other, reported the use of varying doses of prednisone and monthly intravenous administration of cyclophosphamide, and clinical, serologic, and renal laboratory improvements were noted. 48 ,49 In the early to mid 1970s, three prospective controlled investigations showed no benefits of treatment combinations with either prednisone and azathioprine or prednisone and orally administered cyclophosphamide. In one of these clinical trials, 16 patients with diffuse proliferative glomerulonephritis were randomly allocated to treatment with either prednisone (average dosage, 40 mg/d for 6 months) or prednisone (same dosage) and azathioprine (2 to 3 mg/kg body weight/ d).42 After 6 months,42 the dose of prednisone was reduced gradually in both groups and azathioprine therapy was continued for 1 to 3 years. After 3 years,69 there was no difference in the control of systemic or renal manifestations between the two treatment groups and no difference in the frequency of renal relapse. The incidence and duration of systemic nonrenal flares were greater in the group given combined prednisone
IMMUNOSUPPRESSIVES IN LUPUS NEPHRITIS
and azathioprine, whether azathioprine was being given or whether its use had been discontinued, and the dosage of prednisone required to control systemic manifestations was no different in the two groups. As will be pointed out later, the 3year follow-up may have been too short to have discerned a therapeutic advantage of azathioprine treatment even if one existed. In the first of a series of clinical trials from the NIH, Decker et aCo reported on 38 patients with diffuse proliferative glomerulonephritis and membranous nephropathy who were randomly assigned to a regimen of either orally administered cyclophosphamide or azathioprine (up to 4 mg/kg/d) plus low-dose prednisone, or of low-dose prednisone alone (0.5 mg/kg/d). After 28 months, these investigators concluded that the immunosuppressive drugs added only marginally to the control of the disease, and renal function gradually deteriorated in all three groups without significant differences among treatment groups. Hahn et al43 studied 24 patients with mesangial changes, diffuse glomerulonephritis, and membranous nephropathy who were randomly assigned to therapy with prednisone (60 mg/d initially) or with azathioprine (3 to 4 mg/kg/d) plus prednisone. After 18 to 24 months, there were no significant differences between the two groups in clinical, serologic, or renal manifestations. Each of the three studies mentioned above had follow-up periods of only 1112 to 3 years, and in 25% or fewer of the patients death occurred or renal failure developed. Additional reports of the original NIH clinical trials by Dinant et al in 1982 71 and by Carette et al in 1983 72 showed that patient survival and renal function were not substantially different among patients who received either immunosuppressive drug therapy or prednisone alone. In the continuum of the clinical trials from the NIH, two further treatment groups were added: one used a triple-drug regimen with orally administered prednisone, cyclophosphamide, and azathioprine (the last two drugs at dosages of <1 mg/kg/d), and one combined orally administered prednisone with intermittent intravenously administered cyclophosphamide (0.5 to 1.0 g/m 2 every 3 months).2 7,71,72 In 1984, Balow et al 73 reported on the evolution of renal histopathologic findings in 62 of the 111 patients who entered the various clinical trials at the NIH between 1969 and 1980 in which ef-
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fectiveness of azathioprine or cyclophosphamide, combined with prednisone, was tested against the efficacy of prednisone alone in terms of morphologic activity and chronicity. The pretreatment renal biopsy specimens showed various renal morphologic patterns ranging from mesangial to diffuse proliferative glomerular lesions. In the follow-up renal biopsy specimens taken at various intervals and in selected patients (62 of the 111 patients entering the clinical trials), less renal scarring-defined as glomerulosclerosis, fibrous crescents, interstitial fibrosis, and tubular atrophy-was found in patients treated with the combination of immunosuppressive drugs than in the follow-up biopsy specimens taken from patients who were treated with prednisone alone, suggesting less progressive disease. The investigators concluded that renal failure was more likely to develop in patients treated with prednisone alone. Importantly, this conclusion is based on renal biopsy information secured from patients treated with prednisone alone for longer periods than those treated with combined immunosuppressives and corticosteroids. Thus, the worse prognosis for the prednisone-only group could have been related to the duration of observation. In 1986, Austin et al 27 examined clinical outcomes in 107 patients entered into the NIH therapeutic trials. Even though the majority of patients entered into these trials had diffuse proliferative nephritis, patients with membranous and focal proliferative nephritis were also included. Using life-table analysis, the investigators showed an improved probability of survival free of renal failure in each of the groups that received immunosuppressive drug regimens compared with the group treated with prednisone alone, but only beyond 5 years of follow-up (Fig 1). A statistically significant difference was achieved only between intravenously administered cyclophosphamide combined with low-dose prednisone versus prednisone alone when all patients who received prednisone treatment were included. Twenty-eight of the 47 patients (60%) who were treated with prednisone alone were not randomized concurrently with the intravenous cyclophosphamide group and were entered into the earlier clinical trials before start-up of the intravenous cyclophosphamide treatment arm. Also, at the 5-year follow-up, there were only 11 to 16 at-risk patients in each treatment group for sur-
246
DONADIO AND GLASSOCK
PRED AZA POCY AlCY IVCY
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Fig 1. Probability of surviving free of renal failure in 107 patients with lupus nephritis according to treatment group. PRED, prednisone; AZA, azathioprine; POCY, oral cyclophosphamide; AZCY, combined oral azathioprine and cyclophosphamide; IVCY, intravenous cyclophosphamide. PRED (group 1), all patients receiving prednisone; PRED (group IA), subset of patients receiving prednisone who were randomly assigned concurrently with the group IVCY. In the table at the top of the figure (Patients at Risk), numbers in parentheses refer to group 1A. (Reprinted, by permission of the New England Journal of Medicine [314; 614-619, 1986].27)
vival analysis; therefore, a change in outcome for only a few patients would have voided the statistical superiority of cyclophosphamide administered intravenously. In a more recent report of the same cohort of patients studied at the NIH in which follow-up was extended by 40 months, Steinberg and Steinberg74 showed that the probability of progression to end-stage renal disease was less in the groups treated with cyclophosphamide administered intravenously, as well as orally, and combined oral administration of azathioprine and cyclophosphamide. Changes in the survival curves are based on few patients monitored beyond 5 years, and it is misleading to draw strong conclusions about the different survival rates. In the actuarial life-table method of statistical estimation, the error of the estimate achieved increases sharply when small numbers are considered. 75 This point again underscores the requirement for larger numbers of patients entered into clinical trials, with further emphasis on long-term follow-up when considering outcome determinants such as renal failure. It is also important to emphasize that the effect of therapy on patient survival by life-table analysis did not differ among any of the five treatment groups in the NIH therapeutic trialS. 27 ,28,74
In another randomized clinical trial from the Mayo Clinic, treatment with prednisone administered orally (average dosage, 40 mg/d) was compared with treatment that combined lower doses of oral prednisone (average daily dose, 25 mg) and oral cyclophosphamide (average daily dose, 107 mg) for 6 months in 50 patients with diffuse proliferative glomerulonephritis. 31 Rapid decline of renal function, which ended primarily in end-stage renal disease, was equally frequent in the two treatment groups in patients with established advanced disease as defined by renal morphologic and functional criteria. After 4 years, the incidence and average rate of clinical recurrence of nephritis were less in the patients who were initially treated with combined prednisone and cyclophosphamide than in those given prednisone alone. 31 However, the proportion of patients whose renal function had stabilized or improved was similar in the two treatment groups. In a later follow-up of the study group, by actuarial life-table analysis, the cumulative survival rate was 76% at 2.5 years and 60% at 5 years after study entry (Fig 2A).76 Mortality includes death unrelated to renal failure and the development of end-stage renal disease. In analysis of the cumulative survival rates of the two treatment groups, there were no significant differences in the sum of deaths plus end-stage renal disease or in the end-stage renal disease outcome per se between the group treated with prednisone alone (Fig 2B) and the group treated with combined prednisone and cyclophosphamide administered orally (Fig 2C). An important point for physicians who are contemplating the use of combinations of corticosteroids and immunosuppressive drugs in patients with lupus nephritis is the potential for morbid complications from these treatments. The pertinent toxicities of these treatments have already been discussed. For example, in the NIH clinical trials, complications developed in all treatment groups as a direct consequence of the long-term use of prednisone and immunosuppressive drugs. 27 The median duration of treatment with the four immunosuppressive drug regimens ranged from 4 to 7 years. Major infections occurred in 18 of 111 (16%) patients and in all treatment groups; also, malignancies in azathioprine- and cyclophosphamide-treated groups, herpes zoster in all groups, secondary amenorrhea in all groups, but especially in those
IMMUNOSUPPRESSIVES IN LUPUS NEPHRITIS
80
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B 100 80
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60
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Years Fig 2. Cumulative survival rates in 50 patients with diffuse proliferative glomerulonephritis for (A) the total group, (8) those treated with prednisone alone, and (C) those treated with combined prednisone and cyclophosphamide. "All deaths" refers to the sum of deaths unrelated to renal failure plus the development of endstage renal failure (ESRD); the latter is also plotted separately. (Reprinted with permission of the National Kidney Foundation, Inc/I)
treated with cyclophosphamide, and hemorrhagic cystitis in the group treated with oral cyclophosphamide were reported. Although fewer toxic manifestations may have been observed in the group that received cyclophosphamide intravenously, these differences did not achieve statistical significance unless other groups were pooled.
247
In the Mayo Clinic trial,31,76 a myriad ofproblems added to the variability that is so highly characteristic of systemic lupus erythematosus. In addition to renal disease, other manifestations of severe lupus developed and various drug-related problems also were noted, including aseptic necrosis of the femoral head, medial femoral condyle, and tibial plateau; herpes zoster infections in one third of the patients and equally distributed between treatment groups; hypercortisonism; insulin-dependent diabetes mellitus (in one patient); alopecia, especially in the group treated with cyclophosphamide; and transient leukopenia. Patients in the cyclophosphamidetreatment group received the drug for only 6 months and neither hemorrhagic cystitis nor bladder cancer developed in any of them. Another significant problem was the development of hypertension, which occurred in nearly half of the patients and was severe enough to require two or more antihypertensive agents to control blood pressure. Hypertension that is related to steroid treatment and the renal disease can be addressed more effectively today with the array of antihypertensive agents that are available to the clinician. The relationship of progressive renal failure secondary to uncontrolled hypertension is well known to clinical nephrologists and undoubtedly plays a role in the progressive renal disease of lupus nephritis. The influence of hypertension on progressive renal failure and the role of steroid therapy in its genesis have not been adequately addressed in the clinical studies of patients with lupus nephritis. The poor outcomes are quite similar when one examines total numbers of patients in whom endstage renal disease developed, requiring dialysis or transplantation, and deaths, both occurring after renal failure and unassociated with renal failure, reported in the clinical trials from the NIH and the Mayo Clinic (Table 2). It is important to emphasize again that in the NIH clinical trials, four long-term immunosuppressive drug regimens that included azathioprine and cyclophosphamide were compared with treatment by prednisone alone,27,7o-72 whereas in the Mayo Clinic trial,31 a 6- month course of cyclophosphamide plus prednisone was compared with prednisone alone. From the viewpoint of surviving patients, survival free of renal failure was 64% in the NIH study and 60% in the Mayo Clinic trial after long-term follow-up (medians, 7 years NIH,
248
DONADIO AND GLASSOCK
Table 2. Outcomes of Deaths and End-Stage Renal Failure in the NIH and Mayo Clinic Trials of Cytotoxic Drug Treatment of Patients With Lupus Nephritis Patients in the Clinical Trials
Outcome
(n
End-stage renal failure Deaths after renal failure Nonrenal deaths Combined mortality:j:
23
NIH' = 107)
Mayot (n
= 50)
16
~~} 24%
~}22%
39(36%)
20(40%)
• Various renal morphologic lesions; eight patients did not have pretreatment renal biopsies; median follow-up, 7 years. t All patients had diffuse proliferative glomerulonephritis; median follow-up, 6.8 years. :j: Mortality is the combination of end-stage renal failure and nonrenal deaths.
6.8 years Mayo) compared with a 5-year patient survival of25% before 1970, calculated by Pollak et al,77 who pooled their data 78 with that of Baldwin et aC 9 before the immunosuppressive drug treatment era. The central issue from this comparison is that despite a 2 l h-fold improvement in overall survival and survival free ofrenal failure, as reported in the clinical trials that tested immunosuppressive drugs, such improvement cannot be convincingly attributed to the immunosuppressive drugs azathioprine and cyclophosphamide. Although these immunosuppressive agents may have salutary effects on the overall activity of disease and may induce remission in some patients, their long-term effects on renal disease could in a major way be attributed to lower prednisone dosage, better control of hypertension and hyperlipidemia, and better general management of infections and metabolic complications of the disease. A critical analysis of the available literature does not provide convincing support for the widely held and often practical view that intravenously administered cyclophosphamide given regularly over months to years offers promise of a superior outcome in terms of protection from renal failure in patients with diffuse proliferative lupus nephritis and other renal vasculitic diseases. 8o It is clear that long-term followup (~5 years) is required and that the cumulative long-term toxicity of immunosuppressive regimens, particularly those involv~ng prolonged use of alkylating agents, is uncertain at best and clearly harmful at worst. Even though intrave-
nously administered cyclophosphamide may have a better profile of toxic reactions, this could be due to a lower cumulative dosage compared to orally administered regimens. Thus, longer periods of observation may be required to fully evaluate the proposed safety index for the intermittent, intravenously administered regimens compared with the continuous, orally administered regimens. Finally, few studies have addressed the important issue of remissioninduction therapy as contrasted with remission-consolidation and remission-maintenance therapy. Like cancer chemotherapy regimens, optimal use of immunosuppressive agents in lupus nephritis may require specific induction regimens. Future trials should evaluate this issue, lest immunosuppressive agents fail to realize their potential to modify the natural history of lupus nephritis.
REFERENCES 1. Elion GB, Burgi E, Hitchings GH: Studies on condensed pyrimidine systems. IX. The synthesis of some 6-substituted purines. J Am Chern Soc 74:411-414, 1952 2. Elion GB, Callahan S, Bieber S, Hitchings GH, Rundles RW: A summary of investigations with 6-[(1-methyl-4-nitro5-imidazolyl)thio)purine (B.W. 57-322). Cancer Chemother Rep 14:93-115, 1961 3. Bertino JR: Chemical action and pharmacology of methotrexate, azathioprine and cyclophosphamide in man. Arthritis Rheum 16:79-83, 1973 4. Elion GB, Hitchings GH: Azathioprine, in Sartorelli AC, Johns DG (eds): Antineoplastic and Immunosuppressive Agents, Part II. Berlin, Germany, Springer-Verlag, 1975, pp 404-425 5. Spreafico F, Anaclerio A: Immunosuppressive agents, in Hadden JW, Coffey RG, Spreafico F (eds): Immunopharmacology. New York, NY, Plenum, 1977, pp 245-278 6. Berenbaum MC: Time-dependence and selectivity of immunosuppressive agents. Immunology 36:355-365, 1979 7. Winkelstein A: The effects of azathioprine and 6 MP on immunity. J Immunopharmacol 1:429-454, 1979 8. Berenbaum MC: Is azathioprine a better immunosuppressive than 6-mercaptopurine? Clin Exp Immunol 8: 1-8, 1971 9. Elion GB: Biochemistry and pharmacology of purine analogues. Fed Proc 26:898-903, 1967 10. Spreafico F, Donelli MG, Bossi A, Vecchi A, Standen S, Garattini S: Immunodepressant activity and 6-mercaptopurine levels after administration of 6-mercaptopurine and azathioprine. Transplantation 16:269-278,1973 11. Bach J-F: Immunology (ed 2). New York, NY, Wiley, 1982, pp 942-965 12. Elion GB: Immunosuppressive agents. Transplant Proc 9:975-979, 1977 13. Colvin M: The alkylating agents, in Chabner B (ed): Pharmacologic Principles of Cancer Treatment. Philadelphia, PA, Saunders, 1982, pp 276-308
IMMUNOSUPPRESSIVES IN LUPUS NEPHRITIS
14. Grochow LB, Colvin M: Clinical pharmacokinetics of cyclophosphamide, in Ames MM, Powis G, Kovach JS (eds): Pharmocokinetics of Anticancer Agents in Humans. Amsterdam, The Netherlands, Elsevier, 1983, pp 135-154 15. Hektoen L, Corper HJ: The effect of mustard gas (dichlorethylsulphid) on antibody formation. J Infect Dis 28: 279-285, 1921 16. Makinodan T, Santos GW, Quinn RP: Immunosuppressive drugs. Pharmacol Rev 22:189-247, 1970 17. Turk JL, Poulter LW: Selective depletion oflymphoid tissue by cyclophosphamide. Clin Exp Immunol 10:285-296, 1972 18. Turk JL: Studies on the mechanism of action of methotrexate and cyclophosphamide on contact sensitivity in the guinea pig. Int Arch Allergy Appl Immunol 24: 191-200, 1964 19. Maguire HC Jr, Ettore VL: Enhancement of dinitrochlorbenzene (DNCB) contact sensitization by cyclophosphamide in the guinea pig. J Invest Dermatol 48:39-43, 1967 20. Stevenson HC, Fauci AS: Activation of human B lymphocytes. XII. Differential effects of in vitro cyclophosphamide on human lymphocyte subpopulations involved in B-cell activation. Immunology 39:391-397, 1980 21. Smith PC, Sladek NE: Sensitivity of murine B- and Tlymphocytes to oxazaphosphorine and non-oxazaphosphorine nitrogen mustards. Biochem Pharmacol 34:3459-3463, 1985 22. Sladek NE: Oxazaphosphorines, in Powis G, Prough RA (eds): Metabolism and Action of Anti-Cancer Drugs. New York, NY, Taylor & Francis, 1987, pp 48-90 23. Hilgard P, Pohl J, Stekar J, Voegeli R: Oxazaphosphorines as biological response modifiers-Experimental and clinical perspectives. Cancer Treat Rev 12:155-162, 1985 24. Dillard MG, Dujovne I, Pollak VE, Pirani CL: The effect of treatment with prednisone and nitrogen mustard on the renal lesions and life span of patients with lupus glomerulonephritis. Nephron 10:273-291, 1973 25. Ivanova MM, Nassanova VA, Solovyor SK, Akhnazarova VD, Speransky AI, Decker JL, Steinberg AD: Controlled trial of cyclophosphamide, azathioprine and chlorambucil in lupus nephritis. Rheumatisma 2:11-18, 1981 26. Penn I: The incidence of malignancies in transplant recipients. Transplant Proc 7:323-326, 1975 27. Austin HA III, Klippel JH, Balow JE, Ie Riche NGH, Steinberg AD, Plotz PH, Decker JL: Therapy of lupus nephritis: controlled trial of prednisone and cytotoxic drugs. N Engl J Med 314:614-619, 1986 28. Balow JE, Austin HA III, Tsokos GC, et al: Lupus nephritis. Ann Intern Med 106:79-94, 1987 29. Kinlen U: Incidence of cancer in rheumatoid arthritis and other disorders after immunosuppressive treatment. Am J Med 78 Suppl 1A:44-49, 1985 30. Karnofsky DA: Late effects of immunosuppressive anticancer drugs. Fed Proc 26:925-932, 1967 31. Donadio JV Jr, Holley KE, Ferguson RH, Iistrup DM: Treatment of diffuse proliferative nephritis with prednisone and combined prednisone and cyclophosphamide. N Engl J Med 299:1151-1155,1978 32. Rubin G, Baume P, Vandenberg R: Azathioprine and acute restrictive lung disease. Aust NZ J Med 2:272-274, 1972 33. Patel AR, Shah PC, Rhee HC, Sassoon H, Rao KP: Cyclophosphamide therapy and interstitial pulmonary fibrosis. Cancer 38:1542-1549, 1976
249 34. Klein NW, Vogler MA, Chatot CL, Pierro U: The use of cultured rat embryos to evaluate the teratogenic activity of serum: Cadmium and cyclophosphamide. Teratology 21: 199208, 1980 35. Gibson JE, Becker BA: Teratogenicity of structural truncates of cyclophosphamide in mice. Teratology 4: 141150,1971 36. Nicholson HO: Cytotoxic drugs in pregnancy: Review of reported cases. J Obstet Gynaecol Br Commonw 75:307312, 1968 37. Steege JF, Caldwell DS: Renal agenesis after first trimester exposure to chlorambucil. South Med J 73:14141415, 1980 38. Toledo TM, Harper RC, Moser RH: Fetal effects during cyclophosphamide and irradiation therapy. Ann Intern Med 74:87-91, 1971 39. Garrett MJ: Teratogenic effects of combination chemotherapy. Ann Intern Med 80:667, 1974 (letter) 40. Kirshon B, Wasserstrum N, Willis R, Herman GE, McCabe ERB: Teratogenic effects of first-trimester cyclophosphamide therapy. Obstet Gynecol 72:462-464, 1988 41. Hochberg MC, Shulman LE: Acute leukemia following cyclophosphamide therapy for Sjogren's syndrome. Johns Hopkins Med J 142:211-214, 1978 42. Donadio JV Jr, Holley KE, Wagoner RD, Ferguson RH, McDuffie FC: Treatment of lupus nephritis with prednisone and combined prednisone and azathioprine. Ann Intern Med 77:829-835, 1972 43. Hahn BH, Kantor OS, Osterland CK: Azathioprine plus prednisone compared with prednisone alone in the treatment of systemic lupus erythematosus: Report of a prospective controlled trial in 24 patients. Ann Intern Med 83:597-605, 1975 44. Philips FS, Sternberg SS, Cronin AP, Vidal PM: Cyclophosphamide and urinary bladder toxicity. Cancer Res 21: 1577-1589, 1961 45. Plotz PH, Klippel JH, Decker JL, Grauman D, Wolff B, Brown BC, Rutt G: Bladder complications in patients receiving cyclophosphamide for systemic lupus erythematosus or rheumatoid arthritis. Ann Intern Med 91:221-223, 1979 46. Stillwell TJ, Benson RC Jr, DeRemee RA, MacDonald TJ, Weiland LH: Cyclophosphamide-induced bladder toxicity in Wegener's granulomatosis. Arthritis Rheum 31:465-470, 1988 47. Hoffman GS, Kerr GS, Leavitt RY, Hallahan CW, Lebovics RS, Travis WD, Rottem M, Fauci AS: Wegener granulomatosis: An analysis of 158 patients. Ann Intern Med 116: 488-498, 1992 48. Lehman TJA, Sherry DD, Wagner-Weiner L, McCurdy DK, Emery HM, Magilavy DB, Kowalesky A: Intermittent intravenous cyclophosphamide therapy for lupus nephritis. J Pediatr 114: 1055-1060, 1989 49. McCune WJ, Golbus J, Zeldes W, Bohlke P, Dunne R, Fox DA: Clinical and immunologic effects of monthly administration of intravenous cyclophosphamide in severe systemic lupus erythematosus. N Engl J Med 318:1423-1431, 1988 50. Spitz S: The histological effects of nitrogen mustards on human tumors and tissues. Cancer 1:383-398, 1948 51. Miller DG: Alkylating agents and human spermatogenesis. JAMA 217:1662-1665,1971
250 52. Koyama H, Wada T, Nishizawa T, Iwanaga T, Aoki Y, Terasawa T, Kosaki G, Yamamoto T, Wada A: Cyclophosphamide-induced ovarian failure and its therapeutic significance in patients with breast cancer. Cancer 39:1403-1409, 1977 53. Trompeter RS, Evans PR, Barratt TM: Gonadal function in boys with steroid-responsive nephrotic syndrome treated with cyclophosphamide for short periods. Lancet I: 1177-1179,1981 54. Austin HA III, Muenz LR, Joyce KM, Antonovych TT, Balow JE: Diffuse proliferative lupus nephritis: Identification of specific pathologic features affecting renal outcome. Kidney Int 25:689-695, 1984 55. Schwartz MM, Bernstein J, Hill GS, Holley K, Phillips EA, and the Lupus Nephritis Collaborative Study Group: Predictive value of renal pathology in diffuse proliferative lupus glomerulonephritis. Kidney Int 36:891-896, 1989 56. McCluskey RT: The value of the renal biopsy in lupus nephritis. Arthritis Rheum 25:867-875, 1982 57. Ballou SP, Kushner I: How important is renal biopsy in managing patients with renal disease? The case against its utility: Lupus nephritis, in Narins RG (ed): Controversies in Nephrology and Hypertension. New York, NY, Churchill Livingstone, 1984, pp 271-284 58. Steinberg AD: The treatment oflupus nephritis. Kidney Int 30:769-787, 1986 59. Cameron JS: The long-term outcome of glomerular diseases, in Schrier RW, Gottschalk CW (eds): Diseases of the Kidney (ed 5), vol 2. Boston, MA, Little Brown, 1992, pp 1895-1958 60. Drinkard JP, Stanley TM, Dornfeld L, Austin RC, Barnett EV, Pearson CM, Vernier RL, Adams DA, Latta H, Gonick HC: Azathioprine and prednisone in the treatment of adults with lupus nephritis: Clinical, histological, and immunological changes with therapy. Medicine (Baltimore) 49: 411-432, 1970 61. Shelp WD, Bloodworth JMB Jr, Rieselbach RE: Effect of azathioprine on renal histology and function in lupus nephritis. Arch Intern Med 128:566-573, 1971 62. Hayslett JP, Kashgarian M, Cook CD, Spargo BH: The effect of azathioprine on lupus glomerulonephritis. Medicine (Baltimore) 51:393-412,1972 63. Cameron JS, Boulton-Jones M, Robinson R, Ogg C: Treatment oflupus nephritis with cyclophosphamide. Lancet 2:846-849, 1970 64. Hadidi T: Cyclophosphamide in systemic lupus erythematosus. Ann Rheum Dis 29:673-676, 1970 65. Feng PH, Jayaratnam FJ, Tock EPC, Seah CS: Cyclophosphamide in treatment of systemic lupus erythematosus: 7 years' experience. Br Med J 2:450-452, 1973 66. Sztejnbok M, Stewart A, Diamond H, Kaplan D: Azathioprine in the treatment of systemic lupus erythematosus: A controlled study. Arthritis Rheum 14:639-645, 1971
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67. Cade R, Spooner G, Schlein E, Pickering M, deQuesada A, Holcomb A, Juncas L, Richard G, Shires D, Levin D, Hackett R, Free J, Hunt R, Fregly M: Comparison of azathioprine, prednisone, and heparin alone or combined in treating lupus nephritis. Nephron 10:37-56, 1973 68. Ponticelli C, Zucchelli P, Moroni G, Gagnoli L, Banfi G, Pasquali S: Long-term prognosis of diffuse lupus nephritis. Clin NephroI28:263-271, 1987 69. Donadio JV Jr, Holley KE, Wagoner RD, Ferguson RH, McDuffie FC: Further observations on the treatment of lupus nephritis with prednisone and combined prednisone and azathioprine. Arthritis Rheum 17:573-581, 1974 70. Decker JL, Klippel JH, Plotz PH, Steinberg AD: Cyclophosphamide or azathioprine in lupus glomerulonephritis. A controlled trial: Results at 28 months. Ann Intern Med 83: 606-615, 1975 71. Dinant HJ, Decker JL, Klippel JH, Balow JL, Plotz PH, Steinberg AD: Alternative modes of cyclophosphamide and azathioprine therapy in lupus nephritis. Ann Intern Med 96:728-736, 1982 72. Carette S, Klippel JH, Decker JL, Austin HA, Plotz PH, Steinberg AD, Balow JH: Controlled studies of oral immunosuppressive drugs in lupus nephritis: A long-term followup. Ann Intern Med 99:1-8,1983 73. Balow JE, Austin HA III, Muenz LR, Joyce KM, Antonovych TT, Klippel JH, Steinberg AD, Plotz PH, Decker JL: Effect of treatment on the evolution of renal abnormalities in lupus nephritis. N Engl J Med 311:491-495, 1984 74. Steinberg AD, Steinberg SC: Long-term preservation of renal function in patients with lupus nephritis receiving treatment that includes cyclophosphamide versus those treated with prednisone only. Arthritis Rheum 34:945-950, 1991 75. Peto R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard SV, Mantel N, McPherson K, Peto J, Smith PG: Design and analysis of randomized clinical trials requiring prolonged observation of each patient. II. Analysis and examples. Br J Cancer 35:1-39, 1977 76. Donadio JV Jr, Holley KE, Iistrup DM: Cytotoxic drug treatment of lupus nephritis. Am J Kidney Dis 2: 178-181, 1982 (suppl I) 77. Pollak VE, Pirani CL, Dujovne I, Dillard MG: The clinical course of lupus nephritis: Relationship to the renal histologic findings, in Kincaid-Smith P, Mathew TH, Becker EL (eds): Glomerulonephritis: Morphology, Natural History, and Treatment. New York, NY, Wiley, 1973, pp 1167-1181 78. Pollak VE, Pirani CL, Schwartz FD: The natural history of the renal manifestations of systemic lupus erythematosus. J Lab Clin Med 63:537-550, 1964 79. Baldwin DS, Lowenstein J, Rothfield NF, Gallo G, McCluskey RT: The clinical course of the proliferative and membranous forms oflupus nephritis. Ann Intern Med 73: 929-942, 1970 80. Cupps TR: Cyclophosphamide: To pulse or not to pulse? Am J Med 89:399-402, 1990 (editorial)
IF
The Official Journal of
The National Kidney Foundation
VOL 21, NO 3, MARCH 1993
IN-DEPTH REVIEW
Immunosuppressive Drug Therapy in Lupus Nephritis James V. Donadio, Jr, MD, and Richard J. Glassock, MD • During the past two decades, the immunosuppressive drugs azathioprine and cyclophosphamide have been widely used in the treatment of patients with lupus nephritis. Their toxicities are well known and are mostly dose- and timedependent. Complications that arise from these therapies stem from their immunosuppressive (susceptibility for infection) or pharmacologic (hemorrhagic cystitis, bladder cancer, and fibrosis from the alkylating agents) effects, or both. Uncontrolled studies reporting good results in treating patients with various combinations of corticosteroids and azathioprine and, especially, cyclophosphamide cannot be conclusively confirmed by the few controlled clinical trials that are available for review. Part of the problem of inconclusiveness has to do with timing treatment to different phases of the disease and the vast heterogeneity of lupus nephritis. Although these immunosuppressive agents may have favorable effects on the overall activity of systemic lupus erythematosus, their long-term effects per se on renal disease are in question and could be attributed to lower prednisone dosage and better medical management of hypertension, hyperlipidemia, infection, and other metabolic consequences of the disease. © 1993 by the National Kidney Foundation, Inc. INDEX WORDS: Immunosuppressive therapy; lupus nephritis.
F
OR THE PAST two decades, physicians have used immunosuppressive drugs to treat patients with lupus nephritis. The vast majority of both uncontrolled studies and prospective therapeutic trials have used two agents, the antimetabolite azathioprine and the alkylating agent cyclophosphamide. Both drugs are immunosuppressive in that they interfere with the growth or survival of immune cells. In this review, we focus on these two immunosuppressive drugs and discuss immunosuppressive properties, toxicities, and clinical studies pertaining to their use in the treatment of patients with lupus nephritis.
patients with lupus nephritis. Excellent reviews of the pharmacology of the antimetabolites are available to the interested reader. loS The immunosuppressive activities of azathioprine and 6-MP depend on the timing of their administration relative to antigenic challenge. Suppression is achieved only if these drugs are administered in the interval shortly after the immunologic stimulation. 6 Immune responses that require cell proliferation, such as the clonal expansion of T-helper cells driven by antigen exposure, may be inhibited; therefore, antibody production, graft rejection, and the induction of autoimmune disease may be suppressed. Aza-
ANTIMETABOLITES
Antimetabolites interfere with protein synthesis by competing for and blocking specific receptors. They include the purine antagonist 6-mercaptopurine (6-MP}-the parent compound of azathioprine; the pyrimidine antagonist 5-fluorouracil; cytosine arabinoside; and the folic acid antagonist methotrexate. Azathioprine has been the antimetabolite used most frequently to treat
From the Department ofNephrology and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN; and the Department of Medicine, University of Kentucky Medical School, Lexington, KY. Received May 19, 1992; accepted in revised form October 1,1992. Address reprint requests to James V. Donadio, MD, Mayo Clinic, 200 First St SW; Rochester, MN 55905. © 1993 by the National Kidney Foundation, Inc. 0272-6386/93/2103-0001$3.00/0
American Journal of Kidney Diseases, Vol 21, No 3 (March). 1993: pp 239-250
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240
DONADIO AND GLASSOCK
thioprine and 6-MP also exert a nonspecific, antiinflammatory effect, but this is probably not an important part of their immunosuppressive actions. 7 There is a lack of agreement on the immunosuppressive superiority of azathioprine over 6MP. Some investigators believe that the parent compound (6-MP) has greater activity,8 whereas others regard azathioprine as the more effective immunosuppressive drug. 9 In part, differences may be due to such factors as the test animal species, experimental models, routes of administration, and doses of the compounds used. Nevertheless, evidence suggests that azathioprine may be the more active form. Such evidence includes (l) azathioprine is absorbed better in the gastrointestinal tract than is 6-Mp lO ; (2) azathioprine has a lower incidence of toxicity at equivalent immunosuppressive concentrations ll ; (3) azathioprine can interfere with in vitro tests of lymphocyte reactivity at lower concentrations than 6_Mp I2 ; and (4) different, but as yet unknown, metabolism of azathioprine and 6-MP may occur in the body, which results in further immunosuppressive compounds. 9 Further studies are required to define clearly the superiority of one compound over the other. ALKYLATING AGENTS
Cyclophosphamide, the first clinically useful compound of the generically termed "oxazaphosphorines," is an alkylating agent whose cytotoxic activity is mediated through covalent bonding of alkyl groups (one or more saturated carbon atoms) to cellular molecules. 13 Cyclophosphamide itself is without alkylating or cytotoxic activity and must be metabolized in the body to its alkylating metabolites. Again, excellent pharmacologic reviews of the alkylating agents are available. 5,12-14 The immunosuppressive activity of alkylating agents was first described by Hektoen and Corper, 15 who reported that sulfur mustard depressed the serum antibody response of rabbits and dogs to heterologous erythrocytes. The most immunosuppressive drug of the alkylating agents, both on a molar basis and relative to other toxicities, is cyclophosphamide. 16 Selective effects of cyclophosphamide on different components of the lymphoid system have been described. In vivo, it has been reported to selectively suppress Blymphocyte function and to deplete B lympho-
cytes morphologically.17 However, cyclophosphamide can also suppress lymphocyte functions that are mediated by T cells, such as graft-versushost response and delayed hypersensitivity. 18 Properly timed and appropriate doses of cyclophosphamide in vivo or of activated cyclophosphamide in vitro can enhance immunologic responses by selective inhibition of the function of suppressor T cells. 19.20 Cyclophosphamide has therapeutic efficacy in the treatment of certain cancers and for the purpose of immunosuppression. Relative to other cross-linking agents, cyclophosphamide seems to be more useful in the treatment of various autoimmune disorders. Why this should be is not clearly understood. That cellular elements of the humoral and cell-mediated immune systems are differentially sensitive to the cytotoxic action of the oxazaphosphorines may be important. 21 Also important could be the fact that differentiated blood cells seem to be more sensitive to the cytotoxic action of the oxazaphosphorines than are pluripotent or committed progenitor hematopoietic cells. 22 Differential sensitivity could be the result of differential rates of biotransformation. Seemingly paradoxic is the fact that low doses of cyclophosphamide may effect an augmentation of the cell-mediated immune response. This may be because T-suppressor cells are apparently more sensitive to these agents than are other T-cell subsets?3 Clearly, more information is needed to explain fully the clinically observed immunosuppressive effects of cyclophosphamide. In the treatment of cancer, the most clinically useful alkylating agents have been the bischloroethylamines or nitrogen mustards. 13 Mechlorethamine was used in early studies of cytotoxic drugs in lupus,24 but it has been replaced for the most part in clinical use by cyclophosphamide. Chlorambucil, another analog of the nitrogen mustards, has been used in only one short-term (10-week) clinical trial oflupus nephritis,25 having been used most widely in the treatment of chronic lymphocytic leukemia, lymphomas, and ovarian carcinoma. 13 TOXICITIES OF IMMUNOSUPPRESSIVE DRUG THERAPY
Toxicities from immunosuppressive drugs are mostly dose- and time-dependent, and there are complications of these therapies that stem from the immunosuppressive (susceptibility for infec-
IMMUNOSUPPRESSIVES IN LUPUS NEPHRITIS
tion) or pharmacologic (hemorrhagic cystitis, bladder fibrosis due to alkylating agents) effects, or both. The most common toxic effect of each drug is depression of normal hematopoiesis (Table I).! In clinical studies with azathioprine, cytopenias occur 7 to 14 days after drug administration, the delay being due primarily to the interval required for precursor cells to differentiate into mature elements. The degree of hematopoietic toxicity is dose-dependent and is reversible on discontinuing therapy. A mild macrocytic anemia and megaloblastic changes in the bone marrow can be seen with chronic use. For the alkylating agents, the usual dose-limiting toxicity is also suppression of hematopoiesis. This suppression includes all the formed elements of the bloodleukocytes, platelets, and erythrocytes. With cyclophosphamide, the level of circulating leukocytes decreases as a function of the dose and frequency of administration. There is a relative platelet-sparing effect. Focusing further on toxicities attributable to both drugs (Table 1) and germane to azathioprine and cyclophosphamide treatment of patients with lupus nephritis, there are two long-term potential complications: development of malignancy26-29 and opportunistic infection.2 7•28 ,3o A prospective study29 in the United Kingdom of 1,634 patients without transplants treated with immunosuppressive drugs (68% with azathioprine, 28% with cyclophosphamide) found an excess of non-Hodgkin's lymphoma and squamous cell skin cancer (both drugs) and bladder cancer (cyclophosphamide). The relative risks for the three types of malignancy (observed/expected cases) were 10.9, 5.0, and 3.7, respectively. Only 38 of the 1,634 patients studied had lupus nephritis and polyarteritis, whereas 643 had rheumatoid arthritis. A separate analysis of the patients with rheumatoid arthritis found a 13-fold increase in non-Hodgkin's lymphoma, which was not significantly different from the excess in similarly treated patients with other disorders in the study. Four cancers, one fatal (myelocytic leukemia), in azathioprine-treated patients, and three malignancies, including one transitional cell carcinoma of the bladder in cyclophosphamidetreated (oral administration) patients, were reported in the long-term controlled studies of lupus nephritis at the National Institutes of Health (NIH).28 One fatal carcinoma of the tongue de-
241 Table 1. Toxicities Attributed to Immunosuppressive Drugs Antimetabolites
Abnormal hepatic function Pancreatitis
Both Drugs
Alkylating Agents
Hematopoietic suppression Carcinogenesis Opportunistic infection Teratogenesis Pulmonary fibrosis
Nausea and vomiting Reproductive tissue Renal and bladdercystitis, fibrOSis, carcinoma Alopecia Allergic reactions Water intoxication Cardiac failure
veloped in a patient treated for 6 months with orally administered cyclophosphamide in another randomized, long-term clinical trial from the Mayo Clinic. 3! The second major long-term problem is the increased risk of infection. Susceptibility to infections is due to a decrease in granulocyte production and to chronic, intrinsic impairment of the immune defense system with the superimposed effect of corticosteroid and immunosuppressive drugs. 30 Opportunistic viral and fungal infections are particularly common in patients receiving long-term therapy, and similar frequencies of major infections have developed in treatment groups of patients with lupus nephritis ( -15%) receiving either prednisone alone or prednisone combined with either azathioprine or cyclophosphamide. 27 •28 Pulmonary fibrosis and interstitial pneumonitis have been described in several patients receiving long-term therapy with either azathioprine or cyclophosphamide. 32 ,33 Of course, diffuse pulmonary fibrosis may also be caused by Pneumocystis carinii or cytomegalovirus under conditions of chronic immunosuppression, and they must be ruled out in order not to miss the opportunity for effective antimicrobial therapy. Virtually all the alkylating agents are teratogenic, whereas the teratogenic potential of azathioprine appears to be low, based on the infrequent occurrence of congenital anomalies in the offspring of renal transplant recipients receiving long-term therapy with azathioprine. The teratogenic action appears to be due to direct cytotoxicity to the developing embryo by the same mechanisms that operate in tumor cells. 34,35 Because of the demonstrated teratogenesis of the alkylating agents in animals, there has been appropriate concern about the potential effects of
242
their administration to patients during pregnancy. In a report 36 that included 25 instances in which the alkylating agents were administered during the first trimester of pregnancy and the status of the fetus was recorded, there were four fetal malformations. There were no reported instances of malformed fetuses when alkylating agents or other cytotoxic drugs were administered during the second or third trimester. Since this 1968 report, there have been additional reports of malformed children born to mothers who had received chlorambucil,37 cyclophosphamide,38-40 or combined nitrogen mustard and procarbazine41 during the first trimester. Included is a single case report40 of multiple anomalies in an infant born to a woman with systemic lupus erythematosus not known to be in the first trimester of pregnancy who received cyclophosphamide intravenously for exacerbation of skin lesions. In the reported clinical trials in which cyclophosphamide and azathioprine were used in the treatment of patients with lupus nephritis, pregnancy was an exclusion criterion for the entry of patients into study, and to date no cases of teratogenesis have been reported. 27,31,42,43 Uroepithelial toxicity appears to be unique to cyclophosphamide and its analogs, ranging from mild cystitis to severe bladder damage with massive hemorrhage to invasive carcinoma (Table 1).27,44-46 Acrolein, a noncytotoxic metabolite, may be responsible for cyclophosphamide-induced cystitis. Hemorrhagic cystitis developed in 17% of the patients with lupus nephritis treated with long-term oral cyclophosphamide (median duration, 4 years) in controlled studies at the NIHY The largest experience with long-term oral administration of cyclophosphamide has been in the treatment of patients with Wegener's granulomatosis. In a prospective study of 158 patients studied at the NIH47 in which 133 patients (84%) received therapy with daily low-dose cyclophosphamide and glucocorticoids, the actual duration of cyclophosphamide treatment was not given, but the treatment protocol called for the continuation of cyclophosphamide for at least 1 year after the patient achieved complete remission. Cyclophosphamide-related hemorrhagic cystitis developed in 43% of the patients. In another series46 of 111 patients with Wegener's granulomatosis who were given cyclophosphamide therapy, hemorrhagic cystitis developed in 17 (15%).
DONADIO AND GLASSOCK
The average dose of cyclophosphamide was 101 g (range, 5 to 531 g) given for a mean duration of 38 months (range, 4 to 144 months), and both drug dose and duration of treatment were significantly larger and longer than for patients in whom hemorrhagic cystitis did not develop. In addition, bladder cancer developed in 2.8% of the patients in the NIH study, calculated as a 33fold increase in bladder cancer (observed/expected cases; the latter derived from the National Cancer Institute Registry).47 Three patients had bladder carcinoma in the Mayo Clinic series of patients with Wegener's granulomatosis who were given cyclophosphamide in doses of 56, Ill, and 530 g for 1,4, and 12 years, respectively.46 Thus, the long-term oral administration of cyclophosphamide presents a significant hazard for major urotoxicity, including invasive bladder cancer. The incidence and severity of this complication can be lessened by adequate hydration and frequent bladder emptying or by concomitant use of sodium 2-mercaptoethane sulfonate (mesna).46 In addition, no cases of hemorrhagic cystitis or bladder cancer have yet been reported in the various treatment studies27 ,48,49 oflupus nephritis in which intermittent, intravenously administered cyclophosphamide therapy was used. Alkylating agents have profound toxic effects on reproductive tissue (Table 1). The depletion of testicular germ cells but preservation ofSertoli cells and absence of mature or primordial ovarian follicles are the principal effects on gonadal tissues. 50-52 These effects are dose-related; the cumulative threshold dose above which oligospermia may be expected to occur is between 150 and 250 mg/kg body weight. 53 There may be recovery of spermatogenesis, indicating reversibility after cessation of therapy with alkylating agents. 44 Amenorrhea and ovarian atrophy appear to be more permanent and develop after lower doses in older patients than in younger patients, with less likelihood of reversibility in older patients. 52 For example, in the long-term clinical trials from the NIH,28 in 18 of33 patients (55%) treated with cyclophosphamide, secondary amenorrhea developed: in eight of eight patients when treatment was started in the fourth decade, nine of 17 patients when therapy was started in the third decade, and in only one of eight patients less than age 20 years when cyclophosphamide therapy was started.
IMMUNOSUPPRESSIVES IN LUPUS NEPHRITIS
Many of the toxicities reported with immunosuppressive drugs, especially the alkylating agents, relate to the anticancer doses used with these agents. However, there are some data relating plasma concentrations to either toxicity or therapeutic efficacy in patients treated with these drugs. For cyclophosphamide, in general, anticancer doses are 15 to 25 times the order of magnitude of orally administered doses and three to five times higher than intravenously administered doses reported in the clinical trials for lupus nephritis. 27 ,3J,48,49 Nonetheless, adverse drug reactions and severe complicating illnesses such as opportunistic or reactivated latent infections, hemorrhagic cystitis, ovarian failure, malignancies, and pulmonary fibrosis have been reported in patients receiving these agents for immunosuppressive therapy of autoimmune disorders, including systemic lupus erythematosus. CLINICAL STUDIES IN LUPUS NEPHRITIS
The major difficulty in interpreting the literature on the use of immunosuppressive drugs in the treatment of patients with lupus nephritis, or all human glomerular disease for that matter, is the paucity of suitably controlled studies. Uncontrolled studies reporting good results in treating patients with various combinations of corticosteroids and immunosuppressive agents cannot be conclusively confirmed in the few controlled clinical trials that are available for review. Part of the problem in reconciling differences in published reports and one's own experience of treating patients with lupus nephritis has to do with the status of the disease at the time treatment is begun and the great heterogeneity of morphologic renal lesions responsible for lupus nephritis. An obvious example is attempting to influence advanced late renal disease-in other words, the nephrosclerotic state-with corticosteroid and immunosuppressive drugs. This is exemplified in the controlled studies54 performed at the NIH in which histologic features relating to morphologic activity and chronicity were tested for their predictive value for the development of renal failure. The strongest predictor of a poor renal outcome was a high chronicity index made up of glomerular sclerosis, fibrous crescents, tubular atrophy, and interstitial fibrosis-features of irreversible lupus nephritis. On the other hand, Schwartz et al 55 did not find the chronicity index to be predictive of a renal failure outcome in the
243
assessment of 84 biopsy specimens taken from 81 patients with lupus nephritis who entered a randomized clinical trial of plasmapheresis. In lupus nephritis, and as indicated by these two morphologic studies that came to opposite conclusions, this important problem is confounded by the lack of agreement regarding the amount and value of prognostic information that can be gleaned from renal biopsy over and above that from a careful analysis of clinical and laboratory data. This controversy is beyond the scope of the current review and the reader is referred to several studies examining this important topic. 56,57 Related to the problem oftiming treatment in human lupus nephritis are the studies of various murine strains with spontaneous autoimmune disease that show prolonged survival after various interventions with cyclophosphamide, azathioprine, and methylprednisolone alone and in various combinations. 58 Different animal strains develop diverse histologic lesions at different ages, with varying degrees of proteinuria and mortality. Survival studies58 appear to give the best information on the therapeutic intervention. Significant prolongation of survival has been observed only in groups of animals that received cyclophosphamide in high daily or intermittent dosages administered intravenously or in combined therapy with other immunosuppressants when started early in the course of disease, for example, during the early increase of anti-ds DNA titers and before clinical evidence of nephritis, ie, before the appearance of proteinuria. Also, doses were many times higher than those that can be safely used in human clinical trials. For example, NZB/ NZW F J hybrid mice with lupus nephritis were given cyclophosphamide intravenously at a dose of 90 mg/kg/mo for 3 to 8 months. Treatment given for 3 months represents one eighth of a mouse's life span, which is analogous to treating a female patient for approximately 10 years. 58 Thus, the influence of immunosuppressive treatment on murine lupus is quite apart from what can be obtained in clinical practice, in which clinicians must manage patients who have established glomerulonephritis. Moreover, the amount of immunosuppressants and duration of treatment must be modified to avoid major toxicities. Cameron59 addressed in detail the major issues of the complexities of clinical presentation, patient selection, and interpretation of outcome in the study of human glomerular disease.
244
It is not surprising, therefore, that much uncertainty remains concerning the added value of immunosuppressive drugs in therapy in human lupus nephritis. In the early and mid 1970s, clinical studies designed to examine the effectiveness of various combinations of prednisone and azathioprine60-62 or prednisone and cyclophosphamide 63-65 in the treatment of lupus nephritis showed that those patients who improved generally had severe and progressive nephritis. Each of these studies had important flaws in design, was small, or had only a short-term follow-up. Histologic descriptions of the renal disease were only given in some cases, and there were wide variations in the degree of impaired renal function and proteinuria. These descriptions of clinical and renal morphologic findings often led the reader to assume that these patients would not have improved had therapy been continued only with prednisone. The presence of hypertension was often ignored, and details of treatment toxicities and complications and their possible contribution to the steroid treatment "failures" were not accounted for in these reports. These uncontrolled observations formed the basis for subsequent prospective clinical trials in patients with lupus nephritis. Two early investigations indicated favorable results from azathioprine treatment either combined with prednisone or heparin or used alone. Sztejnbok et al 66 noted improved renal function and better survival in patients treated with prednisone and azathioprine compared with survival of patients treated with prednisone alone. Patients who received azathioprine achieved better symptomatic control with lower doses of prednisone than did those treated with prednisone only. It is uncertain how many patients with glomerulonephritis were treated, because the investigators did not obtain renal tissue. Cade et al 67 showed improved survival and better renal function in patients with proliferative lupus nephritis who were treated with combinations of azathioprine and prednisone or azathioprine and heparin, or azathioprine alone compared with patients who were treated with prednisone alone. There were frequent toxic complications among the patients treated with prednisone, including hypertension, thromboembolic episodes, and septicemia, and there were also bleeding complications reported in the anticoagulant-treated group, all of which
DONADIO AND GLASSOCK
contributed to renal failure and death in many patients. In these studies, it is possible that the favorable effects may have been more related to avoidance of side effects of corticosteroids, eg, hypertension, rather than to any specific beneficial effects of azathioprine. Clinical improvements also have been reported in more recent uncontrolled studies involving patients with various types of renal involvement who received differing regimens of corticosteroid and immunosuppressive drugs. 48 ,49,68 For example, Ponticelli et al 68 reported 95% patient survival free of renal failure at 5 years in patients with diffuse glomerulonephritis who were treated aggressively with intravenous methylprednisolone usually given with various immunosuppressive drugs, including cyclophosphamide, azathioprine, and chlorambucil. They concluded that the long-term prognosis of diffuse proliferative lupus nephritis is becoming considerably better due to the combination of corticosteroids (short course of high-dose methylprednisolone administered intravenously followed by low-dose prednisone orally) and low doses ofimmunosuppressive agents. Unfortunately, this conclusion cannot be substantiated in an appropriately controlled study. Two additional studies, involving a small number of children9 in one and adults 16 in the other, reported the use of varying doses of prednisone and monthly intravenous administration of cyclophosphamide, and clinical, serologic, and renal laboratory improvements were noted. 48 ,49 In the early to mid 1970s, three prospective controlled investigations showed no benefits of treatment combinations with either prednisone and azathioprine or prednisone and orally administered cyclophosphamide. In one of these clinical trials, 16 patients with diffuse proliferative glomerulonephritis were randomly allocated to treatment with either prednisone (average dosage, 40 mg/d for 6 months) or prednisone (same dosage) and azathioprine (2 to 3 mg/kg body weight/ d).42 After 6 months,42 the dose of prednisone was reduced gradually in both groups and azathioprine therapy was continued for 1 to 3 years. After 3 years,69 there was no difference in the control of systemic or renal manifestations between the two treatment groups and no difference in the frequency of renal relapse. The incidence and duration of systemic nonrenal flares were greater in the group given combined prednisone
IMMUNOSUPPRESSIVES IN LUPUS NEPHRITIS
and azathioprine, whether azathioprine was being given or whether its use had been discontinued, and the dosage of prednisone required to control systemic manifestations was no different in the two groups. As will be pointed out later, the 3year follow-up may have been too short to have discerned a therapeutic advantage of azathioprine treatment even if one existed. In the first of a series of clinical trials from the NIH, Decker et aCo reported on 38 patients with diffuse proliferative glomerulonephritis and membranous nephropathy who were randomly assigned to a regimen of either orally administered cyclophosphamide or azathioprine (up to 4 mg/kg/d) plus low-dose prednisone, or of low-dose prednisone alone (0.5 mg/kg/d). After 28 months, these investigators concluded that the immunosuppressive drugs added only marginally to the control of the disease, and renal function gradually deteriorated in all three groups without significant differences among treatment groups. Hahn et al43 studied 24 patients with mesangial changes, diffuse glomerulonephritis, and membranous nephropathy who were randomly assigned to therapy with prednisone (60 mg/d initially) or with azathioprine (3 to 4 mg/kg/d) plus prednisone. After 18 to 24 months, there were no significant differences between the two groups in clinical, serologic, or renal manifestations. Each of the three studies mentioned above had follow-up periods of only 1112 to 3 years, and in 25% or fewer of the patients death occurred or renal failure developed. Additional reports of the original NIH clinical trials by Dinant et al in 1982 71 and by Carette et al in 1983 72 showed that patient survival and renal function were not substantially different among patients who received either immunosuppressive drug therapy or prednisone alone. In the continuum of the clinical trials from the NIH, two further treatment groups were added: one used a triple-drug regimen with orally administered prednisone, cyclophosphamide, and azathioprine (the last two drugs at dosages of <1 mg/kg/d), and one combined orally administered prednisone with intermittent intravenously administered cyclophosphamide (0.5 to 1.0 g/m 2 every 3 months).2 7,71,72 In 1984, Balow et al 73 reported on the evolution of renal histopathologic findings in 62 of the 111 patients who entered the various clinical trials at the NIH between 1969 and 1980 in which ef-
245
fectiveness of azathioprine or cyclophosphamide, combined with prednisone, was tested against the efficacy of prednisone alone in terms of morphologic activity and chronicity. The pretreatment renal biopsy specimens showed various renal morphologic patterns ranging from mesangial to diffuse proliferative glomerular lesions. In the follow-up renal biopsy specimens taken at various intervals and in selected patients (62 of the 111 patients entering the clinical trials), less renal scarring-defined as glomerulosclerosis, fibrous crescents, interstitial fibrosis, and tubular atrophy-was found in patients treated with the combination of immunosuppressive drugs than in the follow-up biopsy specimens taken from patients who were treated with prednisone alone, suggesting less progressive disease. The investigators concluded that renal failure was more likely to develop in patients treated with prednisone alone. Importantly, this conclusion is based on renal biopsy information secured from patients treated with prednisone alone for longer periods than those treated with combined immunosuppressives and corticosteroids. Thus, the worse prognosis for the prednisone-only group could have been related to the duration of observation. In 1986, Austin et al 27 examined clinical outcomes in 107 patients entered into the NIH therapeutic trials. Even though the majority of patients entered into these trials had diffuse proliferative nephritis, patients with membranous and focal proliferative nephritis were also included. Using life-table analysis, the investigators showed an improved probability of survival free of renal failure in each of the groups that received immunosuppressive drug regimens compared with the group treated with prednisone alone, but only beyond 5 years of follow-up (Fig 1). A statistically significant difference was achieved only between intravenously administered cyclophosphamide combined with low-dose prednisone versus prednisone alone when all patients who received prednisone treatment were included. Twenty-eight of the 47 patients (60%) who were treated with prednisone alone were not randomized concurrently with the intravenous cyclophosphamide group and were entered into the earlier clinical trials before start-up of the intravenous cyclophosphamide treatment arm. Also, at the 5-year follow-up, there were only 11 to 16 at-risk patients in each treatment group for sur-
246
DONADIO AND GLASSOCK
PRED AZA POCY AlCY IVCY
1ft ~ :::l
~
0;
c
~ 0
c
'0 ~
:s e'"
28(19) 19 18 22 20
100 80
24(18) 15 15 20 16
Patients at Risk 16(11) 10(6) 5(2) 13 13 13 14 13 12 13 12 6 12 10 5
4(1) 11 8 3 1
__.
1
8 7
[~~~-~:--~~~F' ~.:=.='~ ..!!CY --'I --------------------i>;z.CY ----1 __ ----I
POCY
AZA
PRED (group 1)
40
Mantel·Haenszel PRED (group 1) vs. IVCY P=0.027 PRED (group lA) vs. IVCY P=0.07
20 0
2 6
PRED (group lA)I---.
60
.0
0..
26(19) 17 17 22 19
0
20
40
60
80
100
120
140
160
Follow·up. mo
Fig 1. Probability of surviving free of renal failure in 107 patients with lupus nephritis according to treatment group. PRED, prednisone; AZA, azathioprine; POCY, oral cyclophosphamide; AZCY, combined oral azathioprine and cyclophosphamide; IVCY, intravenous cyclophosphamide. PRED (group 1), all patients receiving prednisone; PRED (group IA), subset of patients receiving prednisone who were randomly assigned concurrently with the group IVCY. In the table at the top of the figure (Patients at Risk), numbers in parentheses refer to group 1A. (Reprinted, by permission of the New England Journal of Medicine [314; 614-619, 1986].27)
vival analysis; therefore, a change in outcome for only a few patients would have voided the statistical superiority of cyclophosphamide administered intravenously. In a more recent report of the same cohort of patients studied at the NIH in which follow-up was extended by 40 months, Steinberg and Steinberg74 showed that the probability of progression to end-stage renal disease was less in the groups treated with cyclophosphamide administered intravenously, as well as orally, and combined oral administration of azathioprine and cyclophosphamide. Changes in the survival curves are based on few patients monitored beyond 5 years, and it is misleading to draw strong conclusions about the different survival rates. In the actuarial life-table method of statistical estimation, the error of the estimate achieved increases sharply when small numbers are considered. 75 This point again underscores the requirement for larger numbers of patients entered into clinical trials, with further emphasis on long-term follow-up when considering outcome determinants such as renal failure. It is also important to emphasize that the effect of therapy on patient survival by life-table analysis did not differ among any of the five treatment groups in the NIH therapeutic trialS. 27 ,28,74
In another randomized clinical trial from the Mayo Clinic, treatment with prednisone administered orally (average dosage, 40 mg/d) was compared with treatment that combined lower doses of oral prednisone (average daily dose, 25 mg) and oral cyclophosphamide (average daily dose, 107 mg) for 6 months in 50 patients with diffuse proliferative glomerulonephritis. 31 Rapid decline of renal function, which ended primarily in end-stage renal disease, was equally frequent in the two treatment groups in patients with established advanced disease as defined by renal morphologic and functional criteria. After 4 years, the incidence and average rate of clinical recurrence of nephritis were less in the patients who were initially treated with combined prednisone and cyclophosphamide than in those given prednisone alone. 31 However, the proportion of patients whose renal function had stabilized or improved was similar in the two treatment groups. In a later follow-up of the study group, by actuarial life-table analysis, the cumulative survival rate was 76% at 2.5 years and 60% at 5 years after study entry (Fig 2A).76 Mortality includes death unrelated to renal failure and the development of end-stage renal disease. In analysis of the cumulative survival rates of the two treatment groups, there were no significant differences in the sum of deaths plus end-stage renal disease or in the end-stage renal disease outcome per se between the group treated with prednisone alone (Fig 2B) and the group treated with combined prednisone and cyclophosphamide administered orally (Fig 2C). An important point for physicians who are contemplating the use of combinations of corticosteroids and immunosuppressive drugs in patients with lupus nephritis is the potential for morbid complications from these treatments. The pertinent toxicities of these treatments have already been discussed. For example, in the NIH clinical trials, complications developed in all treatment groups as a direct consequence of the long-term use of prednisone and immunosuppressive drugs. 27 The median duration of treatment with the four immunosuppressive drug regimens ranged from 4 to 7 years. Major infections occurred in 18 of 111 (16%) patients and in all treatment groups; also, malignancies in azathioprine- and cyclophosphamide-treated groups, herpes zoster in all groups, secondary amenorrhea in all groups, but especially in those
IMMUNOSUPPRESSIVES IN LUPUS NEPHRITIS
80
'#.
60
::J
40
f
CIJ
All deaths
20
0
3
0
Years
B 100 80
'#.
60
::J
40
f
CIJ
ESRD All deaths
20
0
0
2
3
4
Years
C 100 80
'#.
60
::J
40
f
CIJ
All deaths
20
0
0
3
2
4
Years Fig 2. Cumulative survival rates in 50 patients with diffuse proliferative glomerulonephritis for (A) the total group, (8) those treated with prednisone alone, and (C) those treated with combined prednisone and cyclophosphamide. "All deaths" refers to the sum of deaths unrelated to renal failure plus the development of endstage renal failure (ESRD); the latter is also plotted separately. (Reprinted with permission of the National Kidney Foundation, Inc/I)
treated with cyclophosphamide, and hemorrhagic cystitis in the group treated with oral cyclophosphamide were reported. Although fewer toxic manifestations may have been observed in the group that received cyclophosphamide intravenously, these differences did not achieve statistical significance unless other groups were pooled.
247
In the Mayo Clinic trial,31,76 a myriad ofproblems added to the variability that is so highly characteristic of systemic lupus erythematosus. In addition to renal disease, other manifestations of severe lupus developed and various drug-related problems also were noted, including aseptic necrosis of the femoral head, medial femoral condyle, and tibial plateau; herpes zoster infections in one third of the patients and equally distributed between treatment groups; hypercortisonism; insulin-dependent diabetes mellitus (in one patient); alopecia, especially in the group treated with cyclophosphamide; and transient leukopenia. Patients in the cyclophosphamidetreatment group received the drug for only 6 months and neither hemorrhagic cystitis nor bladder cancer developed in any of them. Another significant problem was the development of hypertension, which occurred in nearly half of the patients and was severe enough to require two or more antihypertensive agents to control blood pressure. Hypertension that is related to steroid treatment and the renal disease can be addressed more effectively today with the array of antihypertensive agents that are available to the clinician. The relationship of progressive renal failure secondary to uncontrolled hypertension is well known to clinical nephrologists and undoubtedly plays a role in the progressive renal disease of lupus nephritis. The influence of hypertension on progressive renal failure and the role of steroid therapy in its genesis have not been adequately addressed in the clinical studies of patients with lupus nephritis. The poor outcomes are quite similar when one examines total numbers of patients in whom endstage renal disease developed, requiring dialysis or transplantation, and deaths, both occurring after renal failure and unassociated with renal failure, reported in the clinical trials from the NIH and the Mayo Clinic (Table 2). It is important to emphasize again that in the NIH clinical trials, four long-term immunosuppressive drug regimens that included azathioprine and cyclophosphamide were compared with treatment by prednisone alone,27,7o-72 whereas in the Mayo Clinic trial,31 a 6- month course of cyclophosphamide plus prednisone was compared with prednisone alone. From the viewpoint of surviving patients, survival free of renal failure was 64% in the NIH study and 60% in the Mayo Clinic trial after long-term follow-up (medians, 7 years NIH,
248
DONADIO AND GLASSOCK
Table 2. Outcomes of Deaths and End-Stage Renal Failure in the NIH and Mayo Clinic Trials of Cytotoxic Drug Treatment of Patients With Lupus Nephritis Patients in the Clinical Trials
Outcome
(n
End-stage renal failure Deaths after renal failure Nonrenal deaths Combined mortality:j:
23
NIH' = 107)
Mayot (n
= 50)
16
~~} 24%
~}22%
39(36%)
20(40%)
• Various renal morphologic lesions; eight patients did not have pretreatment renal biopsies; median follow-up, 7 years. t All patients had diffuse proliferative glomerulonephritis; median follow-up, 6.8 years. :j: Mortality is the combination of end-stage renal failure and nonrenal deaths.
6.8 years Mayo) compared with a 5-year patient survival of25% before 1970, calculated by Pollak et al,77 who pooled their data 78 with that of Baldwin et aC 9 before the immunosuppressive drug treatment era. The central issue from this comparison is that despite a 2 l h-fold improvement in overall survival and survival free ofrenal failure, as reported in the clinical trials that tested immunosuppressive drugs, such improvement cannot be convincingly attributed to the immunosuppressive drugs azathioprine and cyclophosphamide. Although these immunosuppressive agents may have salutary effects on the overall activity of disease and may induce remission in some patients, their long-term effects on renal disease could in a major way be attributed to lower prednisone dosage, better control of hypertension and hyperlipidemia, and better general management of infections and metabolic complications of the disease. A critical analysis of the available literature does not provide convincing support for the widely held and often practical view that intravenously administered cyclophosphamide given regularly over months to years offers promise of a superior outcome in terms of protection from renal failure in patients with diffuse proliferative lupus nephritis and other renal vasculitic diseases. 8o It is clear that long-term followup (~5 years) is required and that the cumulative long-term toxicity of immunosuppressive regimens, particularly those involv~ng prolonged use of alkylating agents, is uncertain at best and clearly harmful at worst. Even though intrave-
nously administered cyclophosphamide may have a better profile of toxic reactions, this could be due to a lower cumulative dosage compared to orally administered regimens. Thus, longer periods of observation may be required to fully evaluate the proposed safety index for the intermittent, intravenously administered regimens compared with the continuous, orally administered regimens. Finally, few studies have addressed the important issue of remissioninduction therapy as contrasted with remission-consolidation and remission-maintenance therapy. Like cancer chemotherapy regimens, optimal use of immunosuppressive agents in lupus nephritis may require specific induction regimens. Future trials should evaluate this issue, lest immunosuppressive agents fail to realize their potential to modify the natural history of lupus nephritis.
REFERENCES 1. Elion GB, Burgi E, Hitchings GH: Studies on condensed pyrimidine systems. IX. The synthesis of some 6-substituted purines. J Am Chern Soc 74:411-414, 1952 2. Elion GB, Callahan S, Bieber S, Hitchings GH, Rundles RW: A summary of investigations with 6-[(1-methyl-4-nitro5-imidazolyl)thio)purine (B.W. 57-322). Cancer Chemother Rep 14:93-115, 1961 3. Bertino JR: Chemical action and pharmacology of methotrexate, azathioprine and cyclophosphamide in man. Arthritis Rheum 16:79-83, 1973 4. Elion GB, Hitchings GH: Azathioprine, in Sartorelli AC, Johns DG (eds): Antineoplastic and Immunosuppressive Agents, Part II. Berlin, Germany, Springer-Verlag, 1975, pp 404-425 5. Spreafico F, Anaclerio A: Immunosuppressive agents, in Hadden JW, Coffey RG, Spreafico F (eds): Immunopharmacology. New York, NY, Plenum, 1977, pp 245-278 6. Berenbaum MC: Time-dependence and selectivity of immunosuppressive agents. Immunology 36:355-365, 1979 7. Winkelstein A: The effects of azathioprine and 6 MP on immunity. J Immunopharmacol 1:429-454, 1979 8. Berenbaum MC: Is azathioprine a better immunosuppressive than 6-mercaptopurine? Clin Exp Immunol 8: 1-8, 1971 9. Elion GB: Biochemistry and pharmacology of purine analogues. Fed Proc 26:898-903, 1967 10. Spreafico F, Donelli MG, Bossi A, Vecchi A, Standen S, Garattini S: Immunodepressant activity and 6-mercaptopurine levels after administration of 6-mercaptopurine and azathioprine. Transplantation 16:269-278,1973 11. Bach J-F: Immunology (ed 2). New York, NY, Wiley, 1982, pp 942-965 12. Elion GB: Immunosuppressive agents. Transplant Proc 9:975-979, 1977 13. Colvin M: The alkylating agents, in Chabner B (ed): Pharmacologic Principles of Cancer Treatment. Philadelphia, PA, Saunders, 1982, pp 276-308
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14. Grochow LB, Colvin M: Clinical pharmacokinetics of cyclophosphamide, in Ames MM, Powis G, Kovach JS (eds): Pharmocokinetics of Anticancer Agents in Humans. Amsterdam, The Netherlands, Elsevier, 1983, pp 135-154 15. Hektoen L, Corper HJ: The effect of mustard gas (dichlorethylsulphid) on antibody formation. J Infect Dis 28: 279-285, 1921 16. Makinodan T, Santos GW, Quinn RP: Immunosuppressive drugs. Pharmacol Rev 22:189-247, 1970 17. Turk JL, Poulter LW: Selective depletion oflymphoid tissue by cyclophosphamide. Clin Exp Immunol 10:285-296, 1972 18. Turk JL: Studies on the mechanism of action of methotrexate and cyclophosphamide on contact sensitivity in the guinea pig. Int Arch Allergy Appl Immunol 24: 191-200, 1964 19. Maguire HC Jr, Ettore VL: Enhancement of dinitrochlorbenzene (DNCB) contact sensitization by cyclophosphamide in the guinea pig. J Invest Dermatol 48:39-43, 1967 20. Stevenson HC, Fauci AS: Activation of human B lymphocytes. XII. Differential effects of in vitro cyclophosphamide on human lymphocyte subpopulations involved in B-cell activation. Immunology 39:391-397, 1980 21. Smith PC, Sladek NE: Sensitivity of murine B- and Tlymphocytes to oxazaphosphorine and non-oxazaphosphorine nitrogen mustards. Biochem Pharmacol 34:3459-3463, 1985 22. Sladek NE: Oxazaphosphorines, in Powis G, Prough RA (eds): Metabolism and Action of Anti-Cancer Drugs. New York, NY, Taylor & Francis, 1987, pp 48-90 23. Hilgard P, Pohl J, Stekar J, Voegeli R: Oxazaphosphorines as biological response modifiers-Experimental and clinical perspectives. Cancer Treat Rev 12:155-162, 1985 24. Dillard MG, Dujovne I, Pollak VE, Pirani CL: The effect of treatment with prednisone and nitrogen mustard on the renal lesions and life span of patients with lupus glomerulonephritis. Nephron 10:273-291, 1973 25. Ivanova MM, Nassanova VA, Solovyor SK, Akhnazarova VD, Speransky AI, Decker JL, Steinberg AD: Controlled trial of cyclophosphamide, azathioprine and chlorambucil in lupus nephritis. Rheumatisma 2:11-18, 1981 26. Penn I: The incidence of malignancies in transplant recipients. Transplant Proc 7:323-326, 1975 27. Austin HA III, Klippel JH, Balow JE, Ie Riche NGH, Steinberg AD, Plotz PH, Decker JL: Therapy of lupus nephritis: controlled trial of prednisone and cytotoxic drugs. N Engl J Med 314:614-619, 1986 28. Balow JE, Austin HA III, Tsokos GC, et al: Lupus nephritis. Ann Intern Med 106:79-94, 1987 29. Kinlen U: Incidence of cancer in rheumatoid arthritis and other disorders after immunosuppressive treatment. Am J Med 78 Suppl 1A:44-49, 1985 30. Karnofsky DA: Late effects of immunosuppressive anticancer drugs. Fed Proc 26:925-932, 1967 31. Donadio JV Jr, Holley KE, Ferguson RH, Iistrup DM: Treatment of diffuse proliferative nephritis with prednisone and combined prednisone and cyclophosphamide. N Engl J Med 299:1151-1155,1978 32. Rubin G, Baume P, Vandenberg R: Azathioprine and acute restrictive lung disease. Aust NZ J Med 2:272-274, 1972 33. Patel AR, Shah PC, Rhee HC, Sassoon H, Rao KP: Cyclophosphamide therapy and interstitial pulmonary fibrosis. Cancer 38:1542-1549, 1976
249 34. Klein NW, Vogler MA, Chatot CL, Pierro U: The use of cultured rat embryos to evaluate the teratogenic activity of serum: Cadmium and cyclophosphamide. Teratology 21: 199208, 1980 35. Gibson JE, Becker BA: Teratogenicity of structural truncates of cyclophosphamide in mice. Teratology 4: 141150,1971 36. Nicholson HO: Cytotoxic drugs in pregnancy: Review of reported cases. J Obstet Gynaecol Br Commonw 75:307312, 1968 37. Steege JF, Caldwell DS: Renal agenesis after first trimester exposure to chlorambucil. South Med J 73:14141415, 1980 38. Toledo TM, Harper RC, Moser RH: Fetal effects during cyclophosphamide and irradiation therapy. Ann Intern Med 74:87-91, 1971 39. Garrett MJ: Teratogenic effects of combination chemotherapy. Ann Intern Med 80:667, 1974 (letter) 40. Kirshon B, Wasserstrum N, Willis R, Herman GE, McCabe ERB: Teratogenic effects of first-trimester cyclophosphamide therapy. Obstet Gynecol 72:462-464, 1988 41. Hochberg MC, Shulman LE: Acute leukemia following cyclophosphamide therapy for Sjogren's syndrome. Johns Hopkins Med J 142:211-214, 1978 42. Donadio JV Jr, Holley KE, Wagoner RD, Ferguson RH, McDuffie FC: Treatment of lupus nephritis with prednisone and combined prednisone and azathioprine. Ann Intern Med 77:829-835, 1972 43. Hahn BH, Kantor OS, Osterland CK: Azathioprine plus prednisone compared with prednisone alone in the treatment of systemic lupus erythematosus: Report of a prospective controlled trial in 24 patients. Ann Intern Med 83:597-605, 1975 44. Philips FS, Sternberg SS, Cronin AP, Vidal PM: Cyclophosphamide and urinary bladder toxicity. Cancer Res 21: 1577-1589, 1961 45. Plotz PH, Klippel JH, Decker JL, Grauman D, Wolff B, Brown BC, Rutt G: Bladder complications in patients receiving cyclophosphamide for systemic lupus erythematosus or rheumatoid arthritis. Ann Intern Med 91:221-223, 1979 46. Stillwell TJ, Benson RC Jr, DeRemee RA, MacDonald TJ, Weiland LH: Cyclophosphamide-induced bladder toxicity in Wegener's granulomatosis. Arthritis Rheum 31:465-470, 1988 47. Hoffman GS, Kerr GS, Leavitt RY, Hallahan CW, Lebovics RS, Travis WD, Rottem M, Fauci AS: Wegener granulomatosis: An analysis of 158 patients. Ann Intern Med 116: 488-498, 1992 48. Lehman TJA, Sherry DD, Wagner-Weiner L, McCurdy DK, Emery HM, Magilavy DB, Kowalesky A: Intermittent intravenous cyclophosphamide therapy for lupus nephritis. J Pediatr 114: 1055-1060, 1989 49. McCune WJ, Golbus J, Zeldes W, Bohlke P, Dunne R, Fox DA: Clinical and immunologic effects of monthly administration of intravenous cyclophosphamide in severe systemic lupus erythematosus. N Engl J Med 318:1423-1431, 1988 50. Spitz S: The histological effects of nitrogen mustards on human tumors and tissues. Cancer 1:383-398, 1948 51. Miller DG: Alkylating agents and human spermatogenesis. JAMA 217:1662-1665,1971
250 52. Koyama H, Wada T, Nishizawa T, Iwanaga T, Aoki Y, Terasawa T, Kosaki G, Yamamoto T, Wada A: Cyclophosphamide-induced ovarian failure and its therapeutic significance in patients with breast cancer. Cancer 39:1403-1409, 1977 53. Trompeter RS, Evans PR, Barratt TM: Gonadal function in boys with steroid-responsive nephrotic syndrome treated with cyclophosphamide for short periods. Lancet I: 1177-1179,1981 54. Austin HA III, Muenz LR, Joyce KM, Antonovych TT, Balow JE: Diffuse proliferative lupus nephritis: Identification of specific pathologic features affecting renal outcome. Kidney Int 25:689-695, 1984 55. Schwartz MM, Bernstein J, Hill GS, Holley K, Phillips EA, and the Lupus Nephritis Collaborative Study Group: Predictive value of renal pathology in diffuse proliferative lupus glomerulonephritis. Kidney Int 36:891-896, 1989 56. McCluskey RT: The value of the renal biopsy in lupus nephritis. Arthritis Rheum 25:867-875, 1982 57. Ballou SP, Kushner I: How important is renal biopsy in managing patients with renal disease? The case against its utility: Lupus nephritis, in Narins RG (ed): Controversies in Nephrology and Hypertension. New York, NY, Churchill Livingstone, 1984, pp 271-284 58. Steinberg AD: The treatment oflupus nephritis. Kidney Int 30:769-787, 1986 59. Cameron JS: The long-term outcome of glomerular diseases, in Schrier RW, Gottschalk CW (eds): Diseases of the Kidney (ed 5), vol 2. Boston, MA, Little Brown, 1992, pp 1895-1958 60. Drinkard JP, Stanley TM, Dornfeld L, Austin RC, Barnett EV, Pearson CM, Vernier RL, Adams DA, Latta H, Gonick HC: Azathioprine and prednisone in the treatment of adults with lupus nephritis: Clinical, histological, and immunological changes with therapy. Medicine (Baltimore) 49: 411-432, 1970 61. Shelp WD, Bloodworth JMB Jr, Rieselbach RE: Effect of azathioprine on renal histology and function in lupus nephritis. Arch Intern Med 128:566-573, 1971 62. Hayslett JP, Kashgarian M, Cook CD, Spargo BH: The effect of azathioprine on lupus glomerulonephritis. Medicine (Baltimore) 51:393-412,1972 63. Cameron JS, Boulton-Jones M, Robinson R, Ogg C: Treatment oflupus nephritis with cyclophosphamide. Lancet 2:846-849, 1970 64. Hadidi T: Cyclophosphamide in systemic lupus erythematosus. Ann Rheum Dis 29:673-676, 1970 65. Feng PH, Jayaratnam FJ, Tock EPC, Seah CS: Cyclophosphamide in treatment of systemic lupus erythematosus: 7 years' experience. Br Med J 2:450-452, 1973 66. Sztejnbok M, Stewart A, Diamond H, Kaplan D: Azathioprine in the treatment of systemic lupus erythematosus: A controlled study. Arthritis Rheum 14:639-645, 1971
DONADIO AND GLASSOCK
67. Cade R, Spooner G, Schlein E, Pickering M, deQuesada A, Holcomb A, Juncas L, Richard G, Shires D, Levin D, Hackett R, Free J, Hunt R, Fregly M: Comparison of azathioprine, prednisone, and heparin alone or combined in treating lupus nephritis. Nephron 10:37-56, 1973 68. Ponticelli C, Zucchelli P, Moroni G, Gagnoli L, Banfi G, Pasquali S: Long-term prognosis of diffuse lupus nephritis. Clin NephroI28:263-271, 1987 69. Donadio JV Jr, Holley KE, Wagoner RD, Ferguson RH, McDuffie FC: Further observations on the treatment of lupus nephritis with prednisone and combined prednisone and azathioprine. Arthritis Rheum 17:573-581, 1974 70. Decker JL, Klippel JH, Plotz PH, Steinberg AD: Cyclophosphamide or azathioprine in lupus glomerulonephritis. A controlled trial: Results at 28 months. Ann Intern Med 83: 606-615, 1975 71. Dinant HJ, Decker JL, Klippel JH, Balow JL, Plotz PH, Steinberg AD: Alternative modes of cyclophosphamide and azathioprine therapy in lupus nephritis. Ann Intern Med 96:728-736, 1982 72. Carette S, Klippel JH, Decker JL, Austin HA, Plotz PH, Steinberg AD, Balow JH: Controlled studies of oral immunosuppressive drugs in lupus nephritis: A long-term followup. Ann Intern Med 99:1-8,1983 73. Balow JE, Austin HA III, Muenz LR, Joyce KM, Antonovych TT, Klippel JH, Steinberg AD, Plotz PH, Decker JL: Effect of treatment on the evolution of renal abnormalities in lupus nephritis. N Engl J Med 311:491-495, 1984 74. Steinberg AD, Steinberg SC: Long-term preservation of renal function in patients with lupus nephritis receiving treatment that includes cyclophosphamide versus those treated with prednisone only. Arthritis Rheum 34:945-950, 1991 75. Peto R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard SV, Mantel N, McPherson K, Peto J, Smith PG: Design and analysis of randomized clinical trials requiring prolonged observation of each patient. II. Analysis and examples. Br J Cancer 35:1-39, 1977 76. Donadio JV Jr, Holley KE, Iistrup DM: Cytotoxic drug treatment of lupus nephritis. Am J Kidney Dis 2: 178-181, 1982 (suppl I) 77. Pollak VE, Pirani CL, Dujovne I, Dillard MG: The clinical course of lupus nephritis: Relationship to the renal histologic findings, in Kincaid-Smith P, Mathew TH, Becker EL (eds): Glomerulonephritis: Morphology, Natural History, and Treatment. New York, NY, Wiley, 1973, pp 1167-1181 78. Pollak VE, Pirani CL, Schwartz FD: The natural history of the renal manifestations of systemic lupus erythematosus. J Lab Clin Med 63:537-550, 1964 79. Baldwin DS, Lowenstein J, Rothfield NF, Gallo G, McCluskey RT: The clinical course of the proliferative and membranous forms oflupus nephritis. Ann Intern Med 73: 929-942, 1970 80. Cupps TR: Cyclophosphamide: To pulse or not to pulse? Am J Med 89:399-402, 1990 (editorial)