1. REDOX: A secondary analysis What did we learn? Daren K. Heyland MD Professor of Medicine Queen’s University, Kingston, ON Canada On behalf of the REDOXS Study Investigators

2. Disclosures Research grants and speaking honorarium from Fresenius Kabi, biosyn, Baxter, Abbott and Nestle None of these companies have a decisional role in the conception, design, conduct, analysis, interpretation of results or decision to publish.

3. A RANDOMIZED TRIAL OF HIGH-DOSE GLUTAMINE AND ANTIOXIDANTS IN CRITICALLY ILL PATIENTS WITH MULTIORGAN FAILURE The REDOXS study Daren K. Heyland MD Professor of Medicine Queen’s University, Kingston, ON Canada On behalf of the REDOXS Study Investigators N Engl J Med 2013;368:1489-97.

4. 1200 ICU patients Evidence of Multi-organ failure R glutamine placebo Concealed Stratified by site R R antioxidants placebo Factorial 2x2 design Double blind treatment placebo antioxidants The REDOXS study

5. The Research Protocol Adults (>18) With 2 or more organ failures related to their acute illness : – Requiring mechanically ventilation (P/F<300) – Clinical evidence of hypoperfusion defined by need for vasopressor agents for more than 2 hour –Renal dysfunction : Cr>171 or <500ml/24 hrs –platelet < 50 Inclusion Criteria

6. Optimizing the Dose of Glutamine Dipeptides and Antioxidants In Critically Ill Patients: A Phase I dose finding study High dose appears safe High dose associated with –no worsening of SOFA Scores –greater resolution of oxidative stress –greater preservation of glutathione –Improved mitochondrial function Heyland JPEN Mar 2007 ParenterallyEnterally Glutamine/day0.35 gms/kg30 gms Antioxidants per day 500 mcg Selenium Vit C 1500 mg Vit E 500 mg B carotene 10 mg Zinc 20 mg Se 300 ug

7. Primary outcome of 28 day mortality using all 1218 evaluable patients (ITT) Glutamine (glut) Antioxidants (AOX) YesNo AOX OR conditioned on Glut Overall adjusted OR of AOX Yes 101/310 (32.6%)97/301 (32.2%) 1.02 (0.72, 1.43) 1.09 (0.86-1.40; p=0.48*) No 89/307 (29.0%)76/309 (25.3%) 1.20 (0.84, 1.72) Glut OR conditioned on AOX 1.18 (0.83-1.66)1.40 (0.98-2.00) Overall adjusted OR for glut 1.28 (1.00-1.64; p=0.049*) AOX by glut interaction p=0.49 OR=odds ratio. ORs are presented with 95% confidence intervals in parentheses. An OR>1 indicates increased mortality with treatment. All odds ratios adjust for presence of shock at baseline. Overall ORs also adjust for other treatment factor. *To account for the two interim analyses, we pre-specified statistical significance of the final analysis at a two-sided p<0.044 in our protocol. Thus, our primary outcome did not reach statistical significance for either intervention. N Engl J Med 2013;368:1489-97.

8. Mortality Outcomes P=0.07 P=0.049 P=0.02 Note: all P values pertain to GLN vs No GLN; no significant differences between AOX vs. No AOX

9. Pre-specified Sub-group Analysis Glutamine vs. No Glutamine 28 day mortality, OR with 95% CI)

10. Other Clinical Outcomes No differences between groups –SOFA –Need for dialysis –Duration of mechanical ventilation –PODS –infections –ICU and Hospital LOS

11. Plasma Levels of Glutamine in Subset of Patients P <0.001

12. Plasma Levels of Selenium in Subset of Patients P <0.001

13. Post-hoc Secondary Analyses

14. Letter to NEJM “…Major concerns in this study are the statistical adjustment of combining the glutamine groups, showing an imbalance in baseline variables. The number of patients with more than two failing organs at baseline was much higher in the new defined glutamine group compared to the group without glutamine (n=187 vs. n=148 respectively), obviously resulting in higher mortality... In conclusion, we suggest that more severely ill patients were allocated to the glutamine groups as a result of randomization error and patients were not adequately fed. This may explain the observed higher mortality in the new defined glutamine group. Complementary data is needed to support the scientific value of this study.” by Buijs NEJM 2013

15. Adjusted Analysis Imbalance in organ failures at baseline?

16. Kaplan-Meier Survival Curve by Treatment Arm

17. Adjusted Analysis The 28-day mortality rates in the placebo, glutamine, antioxidant and combination groups were 25%, 32%, 29% and 33% respectively. Compared to placebo, the unadjusted OR (95% CI) of mortality was Glutamine 1.4 (1.0-2.0, P =0.063), Antioxidant 1.2 (0.8-1.7, P =0.31), Both 1.4 (1.0-2.0, P=0.049). After adjusting for all statistically significant baseline characteristics, the corresponding adjusted ORs remained virtually unchanged at: Glutamine 1.4 (1.0-2.1, P =0.054) Antioxidant 1.2(0.8-1.8, P =0.34) Both 1.4 (0.9-2.0, P =0.10)

18. Selected Subgroup Analyses OR (95% CI) compared to placeboP-values* SubgroupDeaths/n (%)GLN aloneAOX aloneGLN+AOX Overall 363/1218 (30%)1.40 (0.98-2.00)1.20 (0.84-1.72)1.42 (1.00-2.03) Study Setting Region 0.37 Canada303/1044 (29%)1.41 (0.96-2.07)1.14 (0.77-1.67)1.29 (0.88-1.89) USA44/131 (34%)1.56 (0.51-4.81)1.43 (0.47-4.38)3.43 (1.17-10.07) Europe16/43 (37%)0.86 (0.12-5.9)2.40 (0.39-14.88)0.89 (0.14-5.48) Baseline Patient Characteristics Admission category 0.52 Surgical 59/255 (23%) 2.16 (0.91-5.15)1.94 (0.78-4.82)1.58 (0.67-3.76) Medical 304/963 (32%) 1.28 (0.87-1.89)1.08 (0.73-1.60)1.43 (0.97-2.12) Cancer patients 0.74 No 297/1048 (28%) 1.48 (1.01-2.18)1.15 (0.77-1.71)1.42 (0.97-2.10) Yes 66/170 (39%) 1.05 (0.41-2.73)1.43 (0.60-3.40)1.38 (0.58-3.27) Etiology of Shock 0.71 Cardiogenic 74/240 (31%) 1.24 (0.56-2.79)1.62 (0.75-3.51)2.19 (1.03-4.67) Septic 256/826 (31%) 1.43 (0.93-2.19)1.06 (0.69-1.63)1.21 (0.79-1.86) Other/Unkown/None 33/152 (22%) 1.45 (0.46-4.57)1.45 (0.43-4.86)1.83 (0.60-5.78) Vasopressors 0.37 <15 mcg/min162/595 (27%)1.58 (0.92-2.70)1.66 (0.97-2.84)1.50 (0.87-2.58) >=15 mcg/min201/623 (32%)1.32 (0.82-2.13)0.92 (0.57-1.51)1.39 (0.87-2.22) Renal dysfunction 0.035 No216/776 (28%)0.93 (0.59-1.46)0.90 (0.58-1.40)1.14 (0.74-1.77) Yes147/442 (33%)2.75 (1.50-5.03)2.16 (1.15-4.07)2.15 (1.17-3.94) OR-odds ratio; CI-confidence interval; GLN-Glutamine; AOX-antioxidants

19. Age BMI Comorbidities Diabetes Number of organ failures Additional Subgroup Analyses Subgroup analyses based on variable occurring post randomization not valid

20. Examination of Treatment Effect by Baseline Renal Dysfunction and Post-Baseline Dialysis Multivariable SubgroupOR (95% CI) Compared To Placebo Arm Renal Dysfunction Ever On Dialysisdeaths/n (%)GLN aloneAOX aloneGLN+AOX No 158/634 (25%) 1.1 (0.6-1.8) 1.3 (0.8-2.2) NoYes58/142 (41%) 0.4 (0.2-1.2)0.5 (0.2-1.3)0.6 (0.3-1.6) YesNo76/240 (32%) 3.9 (1.7-9.0)3.3 (1.4-7.8)1.6 (0.7-3.8) Yes 71/202 (35%)1.8 (0.7-4.4) 1.4 (0.6-3.5)3.1 (1.2-7.6) OR-odds ratio; CI-confidence interval; GLN-glutamine; AOX-antioxidants Cells in bold indicate treatment arm had significantly higher 28 day mortality than placebo at p<0.05.

21. Discussion Increased harm associated with glutamine administration have persisted despite adjustment for random imbalances in baseline covariates. In both the pooled analysis where both glutamine-receiving groups were combined or whether considering the effect of glutamine alone vs. placebo, we confirm a trend towards increased mortality and 28 days and a significant increase in 6-month mortality associated with glutamine administration. Our unadjusted subgroup analysis showed that the trend for a harmful glutamine effect existed among the 879 patients with ≤2 organ failures but also among the 335 patients with 3 or 4 organ failures. Thus, the random imbalance in the number of organ failures across groups does not affect our main inference that high-dose glutamine supplementation was not beneficial, and perhaps harmful.

22. Conclusions Glutamine and antioxidants at doses studied in this study do not improve clinical outcomes in critically ill patients with multi-organ failure Glutamine may be harmful For both glutamine and antioxidants, the greatest signal of harm was in patients with multi-organ failure that included renal dysfunction upon study enrollment. Patients with multi-organ failure not uniformly associated with low plasma glutamine levels

23. e Experimental Diet enriched with Glutamine, AOX, and Omega 3 FFAs A van Zanten, unpublished data GLN-enriched EN

24. Experimental Diet enriched with Glutamine, AOX, and Omega 3 FFAs A van Zanten, unpublished data

25. GLN-enriched EN A van Zanten, unpublished data

26. Future Trials Require Bedside Testing?

27. Where does that leave Glutamine?

28. Updated Meta-analysis of IV Glutamine (n=28 RCTs) Overall Mortality Note: Does not include EN GLN studies nor REDOXS study RR=0.87 (0.75,1.02) P=0.08 In press Critical Care

29. Updated Meta-analysis of IV Glutamine (n=28 RCTs) Hospital Mortality RR=0.68 (0.51,0.89) P= 0.005 In press Critical Care

30. Updated Meta-analysis of IV Glutamine (n=28 RCTs) Hospital Mortality Influence of the number of study sites involved in the trial In press Critical Care

31. Updated Meta-analysis of IV Glutamine (n=28 RCTs) Infection RR=0.86 (0.73,1.03) P=0.10

32. Updated Meta-analysis of IV Glutamine (n=28 RCTs) ICU Length of Stay Note: Does not include EN GLN studies nor REDOXS study

33. Updated Meta-analysis of IV Glutamine (n=28 RCTs) Hospital Length of Stay WMD=-2.42 (-4.60, -0.24) P=0.03

34. Double-blind, multicenter RCT 142 trauma patients (excluded renal failure) 0.5 gm/kg of Ala-Gln dipeptide x 5 days unrelated to PN vs. saline placebo (pharmaconutrition) Overall, no effect on infection (primary endpoint), LOS, or mortality No effect in subgroup of severe trauma (ISS>24) Of treated patients, 39% had low plasma levels at END of treatment – day 6 levels associated with worse outcomes

35. Pre-specified subgroup in patients with low basal levels of glutamine Perez-Barcena ICM 2014

36. Canadian Nutrition CPGs: IV Glutamine Recommendation: When parenteral nutrition is prescribed to critically ill patients, parenteral supplementation with glutamine should be considered*. However, we strongly recommend that glutamine NOT be used in critically ill patients with multi-organ failure. here are insufficient data to generate recommendations for intravenous glutamine in critically ill patients receiving enteral nutrition. *downgraded from ‘strongly recommend’

37. Canadian Nutrition CPGs: EN Glutamine No new studies since 2009 Conclusions are: –1) Glutamine supplemented enteral nutrition may be associated with a reduction in mortality in burn patients, but inconclusive in other critically ill patients. –2) Glutamine supplemented enteral nutrition may be associated with a reduction in infectious complications in burn and trauma patients. –3) Glutamine supplemented enteral nutrition is associated with a significant reduction in hospital length of stay in burn and trauma patients. Recommendation: Enteral glutamine should be considered in burn and trauma patients. There are insufficient data to support the routine use of enteral glutamine in other critically ill patients.* *warning against use in multi-organ failure

38. Canadian Nutrition CPGs: Combined IV+ EN Glutamine Recommendation: Based on one level 1 study (REDOXS), we strongly recommend that high dose combined parenteral and enteral glutamine supplementation NOT be used in critically ill patients with multi-organ failure.

39. Is the patient in shock or have multi-organ failure, particularly renal failure? Patient is PN dependent Is the patient: Burns? Trauma? Is EN possible? Give EN Glutamine 0.35-0.5 gm/kg/day as long as they are on EN Do not give any glutamine, neither EN or PN Give IV Glutamine 0.35 gm/kg/day as long as they are on PN Yes No Yes Do not give glutamine No

41. Questions?