15-deoxyspergualin (DSG; Gusperimus) shows immunosuppressive activity both in vitro and in vivo, affecting B-lymphocyte, T-lymphocyte and macrophage/monocyte function. In rodents and human cell systems, DSG shows a dose-dependent inhibition of primary and secondary responses to T-, B- and antigen-presenting cell dependent reactions [10-19]. It has been demonstrated that DSG binds with high affinity to heat shock protein c (hsc) 73 [20,21]. DSG also blocks nuclear translocation of NF-κB in a pre-B-cell line, thereby affecting NF-κB-driven transcription of the kappa light chain [20-23]. Finally, Nishimura et al. [24] reported that DSG inhibits desoxyhypusine synthase, the first enzyme in the formation of active eukaryotic translation initiation factor 5A. This factor is important for the stabilization of certain mRNA transcripts (TNF-α and others).

The immunosuppressive properties of DSG have been demonstrated in preclinical animal studies including SLE models [25-31]. In humans with glucocorticoid-resistant kidney transplant rejection, DSG shows the same efficacy rate as the strongly T-cell depleting anti-CD3 monoclonal antibody [32,33]. DSG has been licensed in Japan for acute renal allograft rejection since 1994. In 2003, an open clinical trial successfully tested DSG in patients with persistent ANCA-associated vasculitis [34-36]. Adverse events (AE) were common but rarely led to treatment discontinuation. Against this background, DSG was granted an orphan drug status for the treatment of Wegener's granulomatosis by the European Medicines Agency (EMA).

As DSG induces a reversible maturation block of granulocytes, it needs to be administered in cycles with intermittent wash-out periods. In the previous studies, it was concluded that the degree of the clinical response does not correlate to the severity or duration of leukopenia elicited in the individual patient. This was an important influence on the protocol for our current SLE study: for safety reasons, we shortened the treatment intervals and started with lower dosages, as SLE patients are more prone to leuko- and lymphocytopenia than patients with Wegener's granulomatosis. In human studies on cancer treatment, in contrast, DSG was applied intravenously at much higher dosages and was still generally well tolerated [37,38]. The study presented here was also encouraged by beneficial results achieved when three patients with active LN were treated with DSG using the same protocol as used here [39]. All three patients had been treated with various immunosuppressives including cyclophosphamide; after informed consent, we started treating with DSG along with corticosteroids, which could be gradually reduced within the first cycles. Indicators of response were a decrease of proteinuria, hematuria and an improvement in the serological parameters of lupus activity [39].

DSG dose remained unchanged in one patient over nine cycles; in three patients, DSG was reduced to 0.35 mg/kg/day (all patients were excluded from the study after five to six cycles). Fifteen patients received 0.7 mg/kg/day of DSG starting at cycle 5; in five patients DSG could be increased to 1.0 mg/kg/day in cycles 7 to 9.

Intermittent leukopenia (a known side effect of DSG) of grade 3 according to WHO classification (< 1.0 to 1.9 × 10⁹/L) was observed in seven patients during the course of the trial; however, it was observed in two cycles in only one patient. Importantly, neither the severity of leukopenia nor the DSG dosage correlated to the frequency and severity of side effects.

Overall, 329 AEs were reported in the 21 patients (Table ​(Table4,4, ​,5,5, ​,6).6). The most frequently reported AEs were infections and infestations (59 reports in 18 patients; Table ​Table5),5), followed by gastrointestinal disorders (52 reports in 16 patients) and general disorders/injection site reaction (39 reports in 17 patients). A total of 218 of 329 AEs were of mild intensity. A relationship with the administration of DSG was assessed as possible in 86 AEs (18 patients), as probable in 37 AEs (13 patients), and definite in 6 AEs (4 patients). In most of the AEs (299), the patients remained in the trial. Sixteen patients received additional therapy due to 81 AEs. Eight patients experienced 18 SAEs (Table ​(Table6),6), seven patients were hospitalized and five patients terminated the study due to SAEs including fever, leukopenia, oral candidiasis, herpes zoster or pneumonia with a consecutive SLE-flare (Table ​(Table6).6). No deaths occurred during the study and the follow-up period. Again, DSG dosage and number or severity of side effects did not correlate.

Based on the predefined response criteria, 11 of 20 (55%) patients achieved PR (four) or CR (seven) on their final visits (one patient with PR after cycle 5, all other patients after cycle 9), eight (40%) were judged as TF on their final visit. Importantly, of these eight patients, two first responded well and achieved PR or CR, but consequently experienced a flare of their LN. Both patients had incompliantly stopped application of DSG, thus they had to be rated as TF. In one patient, response was not assessable due to missing data. Figure ​Figure11 shows the responses at cycles 4, 6 and 9. Sixteen patients completed cycle 6 (four in CR, five in PR, four as TF; for three patients, data were missing for the definition of response). Twelve patients were treated for the full nine cycles, with seven patients finishing DSG therapy in CR, three in PR. Six patients never improved, however, 14 of 20 (70%) patients improved to at least PR at some point during the study.

Proteinuria decreased significantly: at screening, the patients in the study population had a mean protein excretion of 5.124+/-4,379 g/day (range 0.248 to 20.880; n = 20; missing entry data on proteinuria for one patient), this decreased to 3.374+/-4,787 g/day in those 12 patients who were treated for all nine cycles. Table ​Table77 summarizes the average proteinuria at entry and at cycles 4, 6 and 9 for the overall study population. In the 12 patients who were treated through all cycles, proteinuria fell from 5.883+/-5,503 g/day to 3.374+/-4,787 g/day (P = 0.028). The increase from cycle 6 to cycle 9 is mainly due to a 6- to 10-fold increase in proteinuria in the two patients who had incompliantly stopped application of DSG (patients with CRF 10 and 31 in Table ​Table8).8). In 13 of 20 patients, proteinuria decreased by 50% (Table ​(Table8);8); in 7 patients, to less than 1 g/day (levels on entry: 1.13 to 20.88 g/day); and in 9 patients, proteinuria fell below the baseline values before onset of the recent LN flare. Only one of four patients with WHO type V LN responded (partially) to DSG; the patient with WHO type III did not improve.

The analysis of urinary erythrocyte and granular casts revealed casts at screening and study entry for eight patients. In all but one patient, casts disappeared at the latest by cycle 9. At screening, patients of the ITT population had a mean EGFR of 83.75 ml/minute (range 34 to 179 ml/minute). By the end of cycle 2, mean EGFR increased to 91.57 ml/minute. During the subsequent treatment cycles, EGFR was generally stable with mean values ranging between 88.45 ml/minute (cycle 5) and 107.81 ml/minute (cycle 9). Due to the high variability and the low number of patients in this trial this did not reach statistical significance.

Interestingly, SLE-associated rashes improved in six out of eight of the affected patients (completely in four patients, partially in two). Selena-SLEDAI scores were calculated at entry, on the last day of cycles 4, 6 and 9 and at each follow-up visit. The overall scores decreased from a mean of 16.9 (12 to 32; n = 20) at screening to 12.9 (4 to 21; n = 20), 13.7 (4 to 22; n = 15) and 11.7 (6 to 21; n = 12) at the end of cycles 4, 6 or 9, respectively (again, due to the high variability and the low number of patients in this trial this did not reach statistical significance). In the 12 patients who were treated through all nine cycles, Selena-SLEDAI score decreased from 17.6 at entry to 11.7 at the end of cycle 9. The most frequent parameters scoring for the Selena-SLEDAI at the end of the study were low complements, positive dsDNA Ab titers, pyuria, hematuria, rash and arthritis, The response was maintained: at follow-up visits 1, 2 and 3, the average scores were 11.7 (n = 16), 12.2 (15) and 12.0 (13), respectively.

Steroid dosage, an indirect measure of treatment efficacy, could be decreased throughout the cycles as shown in Figure ​Figure2.2. The number of days on which the predniso(lo)ne dose was lower than 7.5 mg/day increased continuously with treatment cycle, from an average of 2.8 days during cycle 1 to 18 days during cycle 9. Complement C3 (screening: 0.70 +/- 0.23 g/L, cycle 9: 0.76 +/- 0.25 g/L) and C4 (screening: 0.08 +/- 0.05 g/L, cycle 9: 0.15 +/- 0.20 g/L) concentrations tended to increase, C-reactive protein (CRP) (screening: 4.59 +/- 6.88 g/L; cycle 9: 2.58 +/- 3.21 g/L) and dsDNA antibody levels decreased (screening: 287.6 +/- 277 U/ml, cycle 9: 160.15 +/- 134 U/ml) (P > 0.05 for CRP and dsDNA Ab titers).

Only one patient had decreased leukocyte counts when entering the study. During treatment with DSG, this low leukocyte count did not decrease further under the expected limits during DSG treatment. As expected by the known side effects of DSG, 13 of the 21 patients suffered from leucocytopenia at at least one point during the treatment period. As in DSG trials in patients with Wegener's granulomatosis, the incidence of infections did not correlate to the degree of leukopenia.

Overall, treatment with DSG as proposed in the study protocol seems to be reasonably safe. The drop-out rate is partially explained by the early phase of clinical development of DSG, in which one needs to be cautious and withdraw patients early if there is uncertainty about the causes of AEs. As seen with other immunosuppressants, an increased rate of infections needs to be envisioned. However, it is important to remember that most of the patients had received other potent immunosuppressants in their disease history, and it is known that such patients are particularly prone to infections [40]. The treatment duration, a maximum of 27 weeks, is too short to estimate the long-term effects of DSG. Thus, safety must be considered in the future trials with DSG. With this proviso, however, DSG seems to be reasonably well tolerated.

Another aim of the study was to get an idea of the required dosage of DSG in the treatment of LN. The protocol involved treating patients with 0.5 mg/kg/d s.c. for two weeks, followed by a seven-day break to give the bone marrow time to compensate for the DSG-related intermittent leukocyte maturation block. This was an adaptation of the protocol for the treatment of Wegener's granulomatosis, in which DSG was injected daily until leucocyte counts dropped below 4,000 cells/μl. As SLE patients are prone to leukopenia per se, we decreased the starting dosage of DSG to 0.5 mg/kg/d and limited the injection period of DSG to 14 days. Thus, therapy was easy to handle without the frequently required blood count controls. This protocol might, therefore, offer advantages over the 'Wegener protocol', at least for the initial cycles. In terms of efficacy; however, the initial dosage of 0.5 mg/kg/d might have been too low, as for 16 of 20 patients who tolerated the drug, the dosage subsequently had to be increased to at least 0.7 mg/kg/d. Therefore, in further trials, we recommend either starting with higher dosages or increasing the dosage to a 0.7 or 1.0 mg/kg/d (or even higher if required and tolerated) faster and earlier than after cycle 4 (as in this protocol). We aimed to treat patients for a maximum of nine cycles (two weeks on drug, one week off drug = one cycle). Thus, in the best scenario in this study, patients were treated with DSG (+ low-dose glucocorticoids) for a maximum of 27 weeks. Of the 21 patients, we excluded one patient after the first injection. Of the remaining 20 patients, 12 were indeed treated for 27 weeks according to the protocol, with 5 patients reaching the maximal dosage of 1.0 mg/kg/d.

Efficacy was defined according to the response criteria detailed above and in Table ​Table1.1. This method of determining the response in LN allows the improvement to be assessed individually, as patients differ in their baseline settings. Based on these parameters, only 4 of the 16 patients completing at least cycle 6 were defined as TF and taken off the study. Of the 12 patients reaching cycle 9, 7 finished as CR, 3 as PR, and 2 further patients reached CR or PR after cycle 6, then experienced a flare with increasing proteinuria due to incompliance in cycles 7 to 9 (rated as TF; Table ​Table7).7). Of course, we cannot attribute this therapeutic response to DSG alone, as all patients started with elevated dosages of corticosteroids along with DSG. However, 10 out of 21 patients had been unsuccessfully treated with at least 20 mg/day of corticosteroids before the start of DSG during this SLE flare. Nonetheless, the low number of patients and the lack of a control group with an alternative treatment strategy prohibit any definite conclusion to be drawn from this trial on the efficacy of DSG in the treatment of LN.

Only a controlled randomized trial can help to define the efficacy of DSG in therapy of LN.

During treatment, renal function was stable in all patients, despite active LN at inclusion. Interestingly, in one patient, creatinine concentrations normalized from values of 1.8 mg/dl at the end of cycle 1, remained normal throughout the study and again increased during follow-up. In three patients, renal function was impaired after termination of DSG-treatment despite treatment with MMF or CYC, AZA and immunoglobulins, respectively.

Most patients suffered from WHO type IV nephritis; only four patients had WHO type V glomerulonephritis (GN). Proteinuria is one of the best predictors for end stage renal failure [41]. In 13 patients of the ITT population, proteinuria decreased by at least 50% (all WHO type IV GN), indicating that DSG (in combination with OCS) seems to affect protein excretion. Remarkably, in nine patients, proteinuria fell below the baseline values from before the onset of the recent LN flare. Amongst patients with WHO type V GN, DSG improved proteinuria in one patient only.