1. Tumor Metabolism and Chemotherapy Resistance Glutamine a Paradox Michael B. Sawyer, MD, B.Sc. Phm. Associate Professor, Division of Medical Oncology FRCPC (Int. Medicine & Medical Oncology) Dip.ABIM (Int. Medicine & Medical Oncology) Dip.American Board of Clinical Pharmacology Fellow American College of Clinical Pharmacology

2. Potential Conflict of Interest Dr. Michael Sawyer –Abraxis, research funding (2006-present) –AstraZeneca, research funding (2006-present) –Roche research funding (2005-present)

3. Outline In vitro glutamine research In vivo glutamine research Clinical glutamine research –Oxaliplatin neuropathy and glutamine Future Research –Translational research into glutamine and oxaliplatin

4. What is Glutamine In health non essential amino acid Conditionally essential amino acid in illness Preferred fuel for enterocytes and immune cells Precursor for glutathione – major cellular antioxidant Involved in interorgan nitrogen exchange and maintenance of pH homeostasis

5. Altered Metabolism in Cancer Cells Warburg noted aerobic gycolysis in 1927 Glutamine similarly appeared to be metabolized inefficiently Many cancer cell lines appear to have a “glutamine” addiction –Withdrawal of glutamine causing cell death –Withdrawal of glutamine and substitution of alpha ketoglutarate rescues the cells from death but does not allow proliferation

6. Altered Metabolism in Cancer Cells Activation of the oncogene Myc activates many enzymes in glutamine metabolism –Glutamine transporters SLC38A5 and SLC1A5 –Glutaminase that converts glutamine to glutamate releasing free ammonia Glutamine replenishes alpha ketoglutarate Provides NADPH for biosynthetic reactions Provides nitrogen for synthesis of non essential amino acids

7. Putative in vitro role of gln

8. In Vivo Glutamine Models Surgeons and radiation oncologists have not had these concerns Klimberg, Rombeau and Coupland have studied glutamine in multiple in vivo models Radiation immunotherapy and methotrexate Demonstrated increased natural killer cell activity, decreased tumor glutathione, increased efficacy and decreased toxicity

9. Hypothesis Postulated that glutamine supplementation would improve the therapeutic index of irinotecan Glutamine would decrease reduced glutathione in tumor cells Glutamine would increase reduced glutathione in normal tissues such as the intestine decreased chemo toxicity Gln would not increase heat shock proteins in the tumor Gln would increase heat shock proteins in normal tissues Overall glutamine would increase the efficacy of chemotherapy in the tumor and decrease the toxicity of chemotherapy in the normal tissues

10. Dietary treatments Treatment groups Control diet Prebiotic enriched diet Fish oil enriched diet Bolus glutamine Timeline + irinotecan 125mg/kg×3days -28 1, 2, 3 Day irinotecan Control, prebiotic, fish oil diets start Glutamine bolus N=8-12 for each treatment group

11. Comparison of Effects of Different Dietary Treatments on Irinotecan-Induced Diarrhea 0 0.5 1 1.5 2 2.5 3 3.5 0246810 Time (day) diarrhea scoring control diet + irinotecan Gln bolus + irinotecan prebiotics + irinotecan fish oil + irinotecan

12. Comparison of Effects of Different Dietary Treatments on Irinotecan-Induced Diarrhea

13. Does Glutamine Treatment Potentiate HSP Response in Tumor as Well? Control diet + irinotecan Gln bolus + irinotecan + Hsp70 Hsp27 Hsp90α

14. Gln Bolus vs. Control- Colonic Mucosa Control diet + irinotecan Gln bolus + irinotecan Hsp70 Hsp27 Hsp90 α Hsc70 * * * *

15. Dietary Effects on Irinotecan/5FU Efficacy

16. Dietary Effects on Normal Tissue GSH/GSSG

17. Dietary Effects on Tumor Tissue GSSG/GSH

18. Wang et al. 86 metastatic colorectal cancer patients Randomized to glutamine (n=42) or control group (n=44) Treated with oxaliplatin (85) mg/m2 days 1 and 15 and weekly 5-flurouracil (5-FU; 500 mg/m2) and folinic acid (FA 20 mg/m2) on days 1, 8, and 15 Glutamine was administered 15 g twice a day for 7 days every two weeks

19. Wang, W.-S. et al. Oncologist 2007;12:312-319 Incidence of oxaliplatin-induced neuropathy in different patient groups

20. Wang, W.-S. et al. Oncologist 2007;12:312-319 Outcome of oral glutamine supplementation

21. Copyright ©2007 AlphaMed Press Wang, W.-S. et al. Oncologist 2007;12:312-319 Table 4. Response to oxaliplatin-based chemotherapy in different patient groups

22. Wang, W.-S. et al. Oncologist 2007;12:312-319 Survival curves of metastatic colorectal cancer patients receiving (filled circle) or not receiving (open circle) glutamine supplementation during oxaliplatin treatments plotted by the Kaplan-Meier method (p =.788; log-rank test)

23. Wang Study Interpretations Hard endpoint: survival Not placebo controlled No pharmacokinetics Did not look at diarrhea No mechanism of action –No glutathione levels –No HSP Started the day of chemotherapy

24. Sanofi’s Glutox Study Using FOLFOX Randomized placebo controlled Starting glutamine/placebo two days before chemotherapy Looking at diarrhea

25. Cross Cancer Parallel Glutox Patients randomized to glutamine first cycle or second cycle Oxaliplatin pharmacokinetics done in cycle 1 and 2 Measurement of plasma, red blood cell glutathione (GSSG and GSH) levels in cycle 1 and 2 Measurement of white blood cell heat shock protein levels

26. Take Home Messages Translational research matters In vitro models may not hold up in in vivo models In vitro models may not hold to human clinical trials Cancer cells are model systems An isolated cancer cell is not a syngenic tumor graft nor a human cancer patient

27. Acknowledgments Hongyu Xue MD PhD Co-Principal Investigators: Lab members: Dr. Vickie Baracos Abha Hoedl Dr. Catherine Field Michelle Mackenzie Dr. Leo Dieleman Dr. Marina Mourtzakis Stephanie Best Alberta Health Services Sue Goruk Cancer Care Celine CCI vivarium staff

28. Glutamine Effects on  -Glucuronidase vs. control diet + irinotecan Stress response study