1. Presenter:Dr. Mukesh Sharma Preceptor:Dr. Manish Soneja

2. Introduction Renal disease occurs approximately in 40–75% of patients with SLE and is associated with significant short- and long-term morbidity.  Lupus Nephritis is a marker of poor prognosis.  Search for an ideal treatment still surreal.  An alternative is to search from among the available drugs.  Initial treatment is with steroids.  Graduated to Cyclophosphamide in the landmark NIH trials.  Present regime advocates steroids and cyclophosphamide.

3. Introduction Despite Cyclophosphamide (CYC) and corticosteroid combination being the gold standard therapy, the regimen is not exempt of significant side effects, and only up to 50% of patients will achieve complete response.

4. Newer Therapies Efficacy of Mycophenolate Mofetil in diffuse proliferative lupus nephritis. NEJM 2000;343:1156-1162 Mycophenolate Mofetil versus Cyclophosphamide for induction treatment of Lupus Nephritis. J Am Soc Nephrol 20:1103-1112,2009 Tacrolimus for treatment of SLE with pure Class V nephritis. Rheumatology 2008;47:1678-1681

5. Why this Article ? Chan et al demonstrated that Mycophenolate mofetil in combination with prednisone was as effective as the regimen containing oral cyclophosphamide for the induction of remission of Lupus Nephritis but with a more favourable toxicity profile. NEJM 2000;343:1156-1162 Since then, several further clinical trials and metaanalysis have provided supplementary evidence of its safety and effficacy both as induction and maintenance therapy for the treatment of active nephritis. Despite treatment,a significant proportion of patient will be refractory to this regime. Whether MMF is also efficacious for preventing flares and prolonging survival is still uncertain in view of the short duration of previous studies and small number of patient included in the only published long term study.

6. Mycophenolate Mofetil Acts by reversibly inhibiting inosine monophosphate dehydrogenase,the rate- limiting step in a de novo purine synthesis,resulting in selective blockade of B and T Lymphocytes. Anti fibrotic properties. Ability to reduce mesangial cell proliferation.

7. Tacrolimus Tacrolimus binds to immunophilins(FK506-binding protein [FKBP], respectively), forming a complex that binds the phosphatase calcineurin and inhibits the calcineurin- catalyzed dephosphorylation essential to permit movement of the nuclear factor of activated T cells (NFAT) into the nucleus. NFAT is required for transcription of interleukin-2 (IL-2) and other growth and differentiation-associated cytokines (lymphokines).

8. Study Protocol November 2000 to December 2006. 70 Caucasian patients. Diagnosed as SLE by ARA criteria. Biopsy proven class III/IV/V lupus nephritis. Prospectively followed up for a mean of 65 ± 24 months.

9. Definitions Active Lupus – Persistent urinary protein excretion ≥0.5 g/day, – Active urinary sediment, – Biopsy-proven renal disease. Renal biopsy specimens examined by light and Immunofluorescence. Biopsy staged according to WHO/ISN RPS 2003.

10. Immunosuppression Protocol Induction therapy –iv methyl-prednisolone pulses (MP) (1000 mg daily for three days) –MMF (1 g two times a day orally) for at least 6 months. Dosage adjustments were performed when WBC counts decreased below 2.5 × 10 9 /L and reviewed within the following 2 weeks.

11. Immunosuppression Protocol In the event of gastrointestinal side effects, MMF dosage was distributed throughout the day (0.5 g four times a day) and substituted by enteric- coated mycophenolate sodium (EC-MPS) at equivalent doses. Following pulses, oral prednisone (1 mg/kg/day) was given for 4 weeks and tapered progressively by 10 mg/day every week to a maintenance dose of 5–7.5 mg/day.

12. Immunosuppression Protocol Once complete response (CR) was achieved, MMF treatment was gradually tapered by 500 mg every 6 months, MMF was increased up to 3 g/day if no clinical response occurred after the induction period, and In the event of intolerance or lack of efficacy after at least 3 months of treatment, tacrolimus was added (0.075 mg/kg/day orally divided into two doses). Dose adjustment was done within the target trough level of 5–10 ng/mL and maintained throughout, unless clinically contraindicated. Tacrolimus was not initiated in those patients with severe hypertension or serum creatinine levels ≥3 mg/mL.

13. Immunosuppression Protocol Following CR, MMF would be the first drug to be reduced and withdrawn, followed by tacrolimus in steps of 25% of the original dose according to clinical response. For renal relapse, patients received one 3-day MP pulse and had the MMF dose increased up to a maximum dose of 3 g daily unless contraindicated. All patients received an angiotensin-converting enzyme inhibitor or an angiotensin II-receptor antagonist.

14. Definitions Complete Remission No urinary sediments Urine Protein Excretion <0.3g/24h Normal or stable renal function (within 10% of normal eGFR if previously abnormal renal function). Partial response (PR) Urinary protein excretion within the range of 0.3–2.9 g/day with a stable or improved renal function. Early response (ER) 30% improvement in at least two measures of renal function (serum creatinine, proteinuria or urine sediment) when compared with baseline after 3 months therapy. Treatment failure Urinary protein excretion ≥3 g/24 h or Increase in the serum creatinine concentration ≥0.6 mg/dL or Creatinine clearance >15% below the baseline value Discontinuation of treatment due to side effects or Renal relapse while on full treatment.

15. Definitions Renal flares were defined according to the two types –Proteinuric flare was defined as -Persistent increase in proteinuria >0.5–1.0 g/day after CR - Doubling of proteinuria with values >1 g/day after achieving PR. – Nephritic flare was defined as »Recurrence of active urinary sediment and/or »Increase of ≥25% in serum creatinine.

16. Primary outcome Complete response at 6, 12 and 24 months and at the end of the follow-up period. Secondary end points Partial remission Improvements in anti-DNA and complement levels, adverse effects, renal flare, doubling of the serum creatinine level. Progression to end-stage renal disease (ESRD) and death. Prognostic factors of remission and relapse were assessed at the end of the follow-up.

17. Follow up Patients were seen fortnightly during the first 2 months, then monthly for 6 months and 3-monthly afterwards. At each visit, blood pressure measurement, disease activity index (SLEDAI score) and occurrence of adverse events were recorded. Laboratory tests included : –24-h urine for proteinuria and creatinine clearance, –Full blood count, –Renal and liver function, –Fasting sugar, lipids, –Complement levels, –anti-dsDNAantibody titres –Serum tacrolimus levels.

18. Study treatment algorithm

19. Class III/IV/V Lupus Nephritis n=70 MMF 2g/d+MP Pulse (1g 3days) (3 patients excluded as follow up finished before 3 months) N=67 61 patients 6 patients Clinical Response Treatment Failure 13 CR+48PR at 6 months 36CR+11 PR at 65 months 24 developed Renal fare/Relapse MP pulses +MMF 2-3g/day N=17 No Response/ Intolerance Complete Remission N=6 N=15 N=2 Tacrolimus(0.075mg/kg/day) IV Cyclophosphamide Anti CD-20 Partial Response N=6 Treatment Failure IVIG

20. Demographics

21. Statistical methods Descriptive statistics are reported as frequency and percentage for categorical variables and as mean and standard deviation for continuous variables. Continuous data were compared by means of the t-test and Mann–Whitney test as appropriate. Cox regression models were used to estimate the association between times to relapse and failure. For failure and relapse, Kaplan–Meier estimates of cumulative probability were used.

22. Results Thirty-two patients (46%) presented with nephrotic syndrome, and 14 (20%) had creatinine levels ≥1.5 mg/dL. Eighteen (26%) patients were considered to have severe disease. Fifty-six (80%) patients had raised serum anti- dsDNA antibodies, and 54 (77%) had low serum C3 concentrations.

23. Response after induction therapy

24. Clinical response (combined CR and PR) occurred in 61 (88%) and 55 (78%) patients at 6 and 12 months, respectively. At the equivalent times, CR was achieved in 13 (19%) and 24 (34%) patients. Forty-eight (69%) patients met criteria for an early response at 12 weeks. Rates of ER tended to be higher in patients with newly diagnosed nephritis (74% de novo vs 61% relapsing), but the difference was not statistically significant (P=0.298).

25. Response after induction therapy Despite being a small group, patients with membranous glomerulonephritis were compared with those with diffuse proliferative nephritis. Higher rates of PR, although not significant, were observed in patients with membranous glomerulonephritis (82% vs 66%, P=0.240 at 6 months and 64% vs 41%, P=0.135 at 12 months). Treatment failure occurred more in those with proliferative glomerulonephritis (15% vs 0%, P=0.336 and 24% vs 9%, P=0.435 at 6 and 12 months, respectively).

26. Long-term follow-up Clinical response occurred in 50 (72%) patients at 24 months and remained in 47 (67%) after 65 months of follow-up. At the equivalent periods, CR was attained by 32 (46%) and 36 (51%) patients, respectively. The mean time to achieve CR was 26 ± 23 months. MMF duration of treatment was 35 ± 11 months. Newly diagnosed patients and those with more moderate disease achieved higher CR rates compared with those with relapsed disease at both 24 (57% vs 25%, P=0.013) and 65 months (64% vs 32%, P=0.014) of follow-up and those with severe disease (48% vs 22%, P=0.028 at 24 months and 56% vs 33% at 65 months, P=0.171).

27. Long-term follow-up RemissionRelapse

28. Long-term follow-up CreatinineUrinary Protein

29. Long-term follow-up Anti dsDNA antibody titre Serum C3 levels

30. Relapse Twenty-four (34%) patients suffered a renal relapse. Eleven of them (16%) had an extra-renal flare. The mean time to flare was 24 ± 21 months.

31. Relapse and Flares Nephritic flares account for 58% (14 out of 24) of all flares. Time to flare was associated with persistent anti- dsDNA titres (HR=1.001; 95%CI, 1.001–1.003, P=0.005) and younger age at renal flare (HR=0.36; 95%CI, 0.14–0.90, P=0.029). Although relapsers had also lower rates of ER and CR and persistent hypocomplementaemia, these variables did not reach statistical significance. Out of the 17 patients who had a MMF-non- responding renal flare, 15 received tacrolimus, and two required cyclophosphamide.

32. Tacrolimus for MMF-resistant nephritis Seventeen (24%) patients, 15 with type IV and 2 with type V glomerulonephritis, received tacrolimus in addition to MMF. Fifteen of them had a MMF-non-responding biologically active flare, and the other two patients had persistent proteinuria (>2.9 g/day) despite 3 g of MMF with a second renal biopsy showing active disease. After a mean follow-up period of 24 ± 3 months, 12 patients (70%) achieved clinical response [combined CR (35%) and PR (35%)]. Time to CR was 10.5 ± 6.2 months. A significant reduction of proteinuria was already observed at 3 months (P=0.002), mirrored by an increase in serum albumin levels (P=0.008).

33. Tacrolimus for MMF-resistant nephritis Anti-dsDNA antibody titres also decreased significantly over time (P=0.0362 at 9 months), and it was mirrored by an upward trend in serum C3 levels (P=0.03). Two patients bore a 23% and >30% increase in serum creatinine level after 6 months treatment. Time to flare was 19 ± 6 months, and at the time of flare the mean dose of MMF and Tacrolimus was 1500 mg (500–3000 mg/day) and 4.25 mg (4–5 mg/day), respectively. Five patients did not respond to this combination therapy (29%). Three patients required further anti-CD20.

34. Adverse effects Gastrointestinal side effects occurred in 16 (23%) patients, 11 (69%) of whom had substituted MMF with EC-MPS at the equivalent dose. No effect on blood pressure, glycemic control or lipid profile was observed.

35. Discussion Good long-term results in patients with LN can be achieved with continuous MMF induction- maintenance therapy. Rate is slightly lower compared with the 73% described by Chan et al. in the only reported long-term study. Not yet clear what the optimal dose for induction is nor the dose for maintenance of remission what the treatment duration should be.

36. Discussion Addition of tacrolimus was safe and effective for MMF-non- responsive cases. Clinical response was achieved in 70% of patients. These results are similar to previous results described with tacrolimus in the treatment of proliferative and pure class V LN but lower compared with the 90% obtained at 9 months in the only study that uses the same multitarget therapy for induction of remission. Outcome differences between the two studies might be related to the different genetic background, the definition of complete remission and to the fact that only patients with Class IV+V LN were included in Bao’s study, whereas only non-MMF respondents were included in ours. Tacrolimus use continues to be hampered by the risk of nephrotoxicity, development of severe hypertension and high rates of renal relapse after drug discontinuation.

37. Conclusion More aggressive strategy should be adopted for patients who do not achieve an early response. MMF and Tacrolimus combination therapy may be an alternative for nonresponsive cases. Tacrolimus is a good alternative as a rescue therapy due to it dual action of immunosuppression and proteinuria reduction.

38. Limitations of the study Small number of patients were included in the study. The mean follow up period after adding Tacrolimus was only 24 months. Cost of therapy is increased with use of Mycophenolate Mofetil