1. When and How to Use Therapeutic Drug Monitoring in IBD Patients Bruce E. Sands, MD, MS Chief of the Dr. Henry D. Janowitz Division of Gastroenterology Dr. Burrill B. Crohn Professor of Medicine Icahn School of Medicine at Mount Sinai New York, NY

2. Disclosures AbbVie Amgen Celgene Forest Research Institute Janssen Biotech Luitpold Pharmaceuticals MedImmune Millennium Pharmaceuticals Pfizer Prometheus Laboratories Takeda

3. Overview To understand the relationship between pharmacokinetics (levels and distribution of drug) to pharmacodynamics (therapeutic and adverse effects) To determine the roles for therapeutic drug monitoring in treating IBD, as well as limitations of current knowledge

4. Duration of action Therapeutic Range C max Onset time t max Time MTC MEC TOXICITY LACK OF EFFICACY AUC

5. Association of 6-thioguanine nucleotide levels and IBD activity: a meta-analysis ·Osterman MT. Gastroenterology 2006;130:1047-53.

6. Prospective Data Supporting Metabolite-Based Dose Optimization of Thiopurines are Inconclusive Dassopoulos T, et al. Aliment Pharmacol Ther 2014;39: 163-175 Intention To Treat Per Protocol p=0.11p=0.12 Multicenter RCT comparing AZA dosing - weight-based (2.5 mg/kg/day) versus individualized (stratified by TPMT, then optimized to target 6TGN – 250-400 pmol/8 x 10 8 RBC) Powered for 226 subjects, 50 subjects randomized, 27 subjects completed

7. Metabolite-Based Optimization of Thiopurines Improves Outcomes with Combination Therapy p=0.009 p=0.008 Single-center, observational cohort of 123 CD patients commencing adalimumab from 2006-2013 Compared outcomes of monotherapy, combination therapy, and optimized combination therapy Kariyawasam VC et al. Abstract No. 343; DDW 2014

8. Higher 6-TGN Levels are Associated with Higher Infliximab Trough Levels in Patients on Combination Therapy Cross-sectional study of 72 patients on combination therapy 6TGN levels (but not thiopurine dose or lymphocyte count) correlated with IFX levels (rho:0.466, p<0.001) 6TGN > 125 pmol/8 x 10 8 RBC predicted higher IFX levels – ROC: 0.82, p=0.002 6TGN < 125 pmol/8 x 10 8 RBC predicted higher likelihood of ATIs – OR: 1.3, 95% CI 2.3 – 72.5, p<0.01 Yarur A et al. DDW 2014, Abstract 788 6TGN level – pmol/8 x 10 8 RBC P=0.02

9. Thiopurine Metabolites Help Clarify Reasons for Inefficacy or Intolerance to Thiopurines Metabolite measurements are indicated in patients not responding or experiencing adverse events to adequate weight-bases doses of thiopurines Absent 6-TGN/ Absent 6-MMP Non- Adherence Education = Under- dosing Increase dose Low 6-TGN/ Low 6-MMP = Adapted from Gearry RB et al J Gastroenterol Hepatol 2005; 20:1149-57 High 6-TGN High 6-MMP Over- dosing Decrease dose = Low 6-TGN/ High 6-MMP Thiopurine Resistance Allopurinol = Ther 6-TGN/ Ther 6-MMP Thiopurine Refractory Another drug =

10. Questions about therapeutic drug monitoring (TDM) with biologic therapy  What is the relationship of exposure to response?  Can we define a minimum effective concentration?  Can we define a maximum therapeutic concentration above which there is –no additional benefit? –increased risk of toxicity?  What is the relationship between drug concentration and anti-drug antibodies, and how should this affect treatment strategy?  Is TDM cost-effective?  In what clinical situations should TDM be used? –Induction? –Maintenance? –Loss of response? –Toxicity? –Retreatment after drug holiday?

11. Higher Serum Infliximab Concentration is Associated with a Higher Response Rate in CD Study design: prospective, cohort study N=125, 30% Rx for fistula Median follow-up: 36 months Efficacy –Infliximab concentrations ≥12 μg/mL were associated with greater median duration of response –Immunosuppressant use was associated with IFX concentrations ≥12 μg/mL Baert F, et al. N Engl J Med. 2003;348:601. Duration of Response Based on IFX Concentrations P<0.01

12. ·Proportion of Patients (%) IFX Concentration (mcg/ml) at Wk 30 Clinical Remission Without Corticosteroids by Trough IFX Concentration at Week 26: SONIC Primary Endpoint ·*IFX and IFX+AZA patients who had 1 or more PK samples obtained after their first study agent administration were included in the analysis 31/4336/4943/5913/2319/32 ·Colombel JF, et al. N Engl J Med. 2010.

13. Higher Serum Infliximab Level is Associated with Longer Remission and Better Endoscopy Score in CD Study design: prospective cohort in moderate-severe CD N=105 Median follow-up: 88 weeks Efficacy – Infliximab concentrations were positively correlated with the interval of clinical remission and change in endoscopic score Maser EA, et al. Clin Gastroenterol Hepatol. 2006;4:1248. 40 50 60 70 80 90 100 Remission (%) Serum Infliximab (µg/ml) R 2 = 0.61 P<0.001 024681012 -100 50 0 100 Endoscopic Improvement (%) Serum Infliximab (µg/ml) R 2 = 0.46 P<0.001 024681012

14. High Infliximab Levels are Associated with Mucosal Healing in Crohn’s Disease Serum samples in 210 CD patients undergoing treatment with infliximab were collected Infliximab trough levels were correlated with endoscopic healing (complete, partial or none) ·Van Moerkercke W. et al. DDW 2010. Abs #405

15. ACCENT I: Week 54 Sustained Clinical Outcome and Week 14 Serum Infliximab Level in CD Sustained Clinical Outcome <3.5 μg/mL Week 14 Serum IFX Level ≥3.5 μg/mL Week 14 Serum IFX LevelP-value* Subjects included in analysis9651 Subjects with sustained response 17 (17.7%)20 (39.2%)0.0042 Subjects without sustained response 79 (82.3%)31 (60.8%) *Chi-square test Cornillie F, et al. Presented at the 19 th Annual United European Gastroenterology Week (UEGW); October 25, 2011. Stockholm, Sweden. Abstract P0919.

16. Infliximab Trough Between Weeks 14 and 22 May Predict Sustained Response in Crohn’s Disease Retrospective adult cohort 84 patients IFX trough level measured at 14 or 22 wks Sustained clinical response IFX Trough level > 3 μg/ml Increase in ATI IFX Trough level < 3 μg/ml Bortlik M, et al. J Crohns Colitis 2013;7:736-743.

17. Prospective cohort study of relationship between serum infliximab level and efficacy in luminal CD Levesque BG, et al. Aliment Pharmacol Ther. 2014;39:1126-35.

18. Detectable Serum Trough Infliximab Associated with Higher Remission Rate and Endoscopic Improvement in UC Study design: cohort study N=115 patients with moderate to severe UC Follow-up time: median 13.9 months Efficacy – Detectable serum IFX was associated with Higher remission rates (69% vs. 15%; P<0.001) Endoscopic improvement (76% vs. 28%; P<0.001) Seow CH, et al. Gut. 2010;59:49-54. P<0.001

19. Presence of Detectable Trough Infliximab Levels Reduces Colectomy Rates in Ulcerative Colitis Seow CH, et al. Gut. 2010;59:49. P < 0.001

20. Proportion of Patients Achieving Clinical Remission by Serum IFX Concentration: ACT 1 and 2 IFX Conc. (% patients) 1st Quartile 2ndQuartile3rdQuartile 4th Quartile P-values Week 8 26.3% (<21.3μg/mL) 37.9% (≥21.3-<33μg/mL) 43.9% (≥33-<47.9μg/mL) 43.1% (>47.9μg/mL) P=0.0504 Week 30 14.6% (<0.11μg/mL) 25.5% (≥0.11-<2.4μg/mL) 59.6% (≥2.4-<6.8μg/mL) 52.1% (>6.8μg/mL) P<0.0001 Week 54 21.1% (<1.4μg/mL) 55.0% (≥1.4-<3.6μg/mL) 79.0% (≥3.6-<8.1μg/mL) 60.0% (>8.1μg/mL) P=0.0066 At wks 8, 30 and 54, the proportion of patients achieving clinical remission increased with increasing quartiles of IFX concentrations. Reinisch W et al., Gastro Vol 142, Issue 5, suppl-1, May 2012, page S-114

21. Week 54 Outcome IFX14 median level (μg/ml) P-value* Persistent Remission (Y vs. N) 4.7 vs. 2.6 P = 0.03 Clinical Remission (Y vs. N) 3.2 vs. 2.2 P = 0.07 Deep Remission (Y vs. N) 4.2 vs. 3.0 P = 0.07 Sustained Durable Remission 14 (Y vs. N) 5.5 vs. 3.1 P = 0.05 Sustained Durable Remission 22 (Y vs. N)5.1 vs. 3.0 P = 0.04 Week 14 IFX levels predict week 54 outcomes Singh N, et al. Inflamm Bowel Dis 2014;10:1708-13

22. * 6 discontinued IFX, 3 continued same dose 3, 3 proceeded to surgery, 5 patients could not be assessed Clinical outcomes in patients with detectable HACA (n=35)* * 10 continued same dose, 9 discontinued IFX, 8 proceeded to surgery and 7 patients could not be assessed Clinical outcomes in patients with sub- therapeutic concentrations (n=69)* Increasing dose of infliximab in the presence of ATI formation is inferior to changing anti-TNF Complete / partial response (%) P<0.004 Complete / partial response (%) P<0.016 Afif W, et al. Am J Gastroenterol 2010;105:1133-9.

23. Proposed algorithm for patients with loss of response to infliximab Positive HACA Change to another anti-TNF agent Change to non– anti-TNF agent persistent disease Increase infliximab dose or frequency Change to non– anti-TNF agent Change to different anti-TNF agent Change to different anti-TNF agent Subtherapeutic IFX concentration Therapeutic IFX concentration Active disease on endoscopy/radiology? Change to different anti-TNF agent Investigate alternate etiologies yes no Afif W et al. Am J Gastroenterol 2010;105:1133.

24. 66 patients: 27% with detectable ATA  59 (89%) CD, 7 (11%) UC Mean ADA levels were higher in patients with  Undetectable ATA  Mucosal healing  Concomitant use of immunosuppressants ADA level ≥ 5 µg/ml is associated with lower CRP and healed mucosa Variables associated with positive ATA (≥ 1.7 µg/ml)  ADA < 5µg/ml: OR 8.6, 95%CI (2.3-31)  Mucosal inflammation: OR 3.8, 95% CI (1.1-13)  Steroid use: OR 3.7, 95% CI (1.1-13) Serum Adalimumab (ADA) Levels and Anti-Adalimumab Antibodies (ATA) Correlate with Endoscopic Inflammation and Inflammatory Markers Yarur, AJ, et al. Presented at DDW; May 21, 2013. Abstract Tu1147.

25. Cross-sectional study of ADA drug level as predictor of clinical response and CRP in CD ADA level of 5.85 μg/mL was optimal -Sensitivity 68% -Specificity 71% -Positive LR 2.3 AAA were inversely related to ADA drug levels Mazor Y, et al. Aliment Pharmcol Ther 2014;40:620-8.

26. Higher trough levels of adalimumab are associated with higher rates of mucosal healing Roblin X, et al. Clin Gastroenterol Hepatol 2014;12:80-84.

27. Trough adalimumab levels are higher in patients with mucosal healing 6.5 μg/mL 4.2 μg/mL P<0.005 Roblin X, et al. Clin Gastroenterol Hepatol 2014;12:80-84.

28. Adalimumab Trough Serum Levels < 0.33 µg/mL Predicts a Lower Rate of Sustained Complete Response in Patients with Crohn’s Disease Karmiris K, et al. Gastroenterology. 2009;137:1628. Patients with Sustained Clinical Response (%) 0.0 0.2 0.4 0.6 0.8 1.0 0210240 LogRank: P=.01 Sustained Clinical Response (weeks) 901203015060180 ADA TR>0.33 µg/mL, n=104 ADA TR<0.33 µg/mL, n=16

29. ADA levels in CLASSIC I (induction) and CLASSIC II (maintenance) Median ADA level significantly higher in patients with clinical remission at week 4 (8.10 vs. 5.05 mcg/mL, P<0.05) Clinical remission rate at week 24 of CLASSIC II for patients with undetectable ADA was 58% (7 of 12), compared with 53% (98 of 186) for patients with detectable concentrations (P=0.77) Clinical remission rates at week 56 were 50% (7 of 14) and 64% (98 of 154; P= 0.39), respectively Chiu YL, et al. Inflamm Bowel Dis 2013;19:1112-22.

30. Proposed algorithm incorporating pharmacokinetics of adalimumab in IBD AAA: antibodies against adalimumab ADA: adalimumab TRA: trough levels of adalimumab Low TRA: <4.9 μg/ml AAA present: >10 ng/ml Roblin X, et al. Am J Gastroenterol 2014;109:1250-6.

31. Single-center cohort of 125 steroid- refractory acute UC patients Standard infliximab induction/maintenance Fluid-phase assay for [IFX] and ATI Trough Infliximab >2 µg/ml is Associated with Clinical Remission in Steroid-Refractory UC Remission aOR [95%CI] Colectomy aOR [95%CI] Trough IFX > 2 µg/ml (vs. ≤ 2 µg/ml) 10 [3,35]0.18 [0.07,0.44] ATI (vs. no ATI)0.64 [0.2,2.4]1.0 [0.5,2.1] IFX, infliximab Murthy S, et al. Presented at DDW; May 19, 2012. Abstract Sa2047. Steroid-free Remission by IFX/ATI Status 100 0 60 20 Patients in Remission (%) 80 40 IFX+ ATI- 70.0 16.6 28.5 13.0 IFX+ ATI+ IFX- ATI- IFX- ATI+ P=0.84 P=0.073 P<0.001 Steroid-free Remission by IFX Trough Status 100 0 60 20 Patients in Remission (%) 80 40 Serum IFX ≤ 2µg/ml 17.5 69.4 Serum IFX > 2µg/ml P<0.001 Colectomy by IFX Trough Status 100 0 60 20 Colectomy (%) 80 40 Serum IFX ≤ 2µg/ml 55.5 17.7 Serum IFX > 2µg/ml P<0.001

32. Rapid IFX Clearance: Mechanism of Non-Response in UC Kevans D, et al. Presented at DDW; May 19, 2012.

33. Multicenter, propspective observational study in anti-TNF-naïve patients (N=19) with moderate-to-severe UC (Endoscopic Mayo 2/3) 1 – IFX measured at 10 time points during first 6 weeks of induction therapy – Nonlinear mixed-effects modelling No difference in IFX concentration area under the curve (AUC) between endoscopic responders and endoscopic non-responders at week 8 (P=0.65 ) Patients with CRP>50 µg/mL had lower IFX concentration (P=0.001) 7/19 had positive ATI (homogeneous mobility shift assay) – 6 of 8 endoscopic non-responders were ATI + – 2 of 8 endoscopic non-responders were ATI – Concomitant immunomodulator = 12/19 (P=NS) IFX presumed to be lost in stool in severe IBD colitis 2 Pharmacokinetics of Infliximab Induction Therapy in Patients With Moderate to Severe UC 1.Brandse JF, et al. Presented at DDW; May 5, 2014 A786. 2.Brandse JF, et al. Presented at DDW; May 18, 2013. A157.

34. Factors Affecting the Pharmacokinetics of Monoclonal Antibodies Impact on Pharmacokinetics Presence of ADAs Decreases serum mAbs Threefold-increased clearance Worse clinical outcomes Concomitant use of IS Reduces formation Increases serum mAbs Decreases mAb clearance Better clinical outcomes High baseline TNF-α May decrease mAbs by increasing clearance Low albumin Increases clearance Worse clinical outcomes High baseline CRP Increases clearance Body size High BMI may increase clearance Gender Males have higher clearance Ordas I et al. Clin Pharmacol Ther. 2012;91:635. mAB, monoclonal antibody; ADA, antidrug antibody

35. Do early serial trough and antidrug antibody level measurements predict clinical outcome of infliximab and adalimumab treatment? Vande Casteele N, et al. Gut 2012;61:321

36. Retrospective cohort of patients in clinical remission, single physician practice – IFX dose optimization to trough concentrations 5–10ug/mL (n=48) – No IFX dose optimization (n=78) Evaluated probability of remaining on IFX, up to 5 years Prospective Therapeutic Drug Monitoring to Optimize Infliximab Maintenance Therapy in IBD Vaughn BP, et al. Presented at DDW; May 3, 2014 Abstract 209. Dose optimization increases probability of remaining on IFX therapy up to 5 years

37. IFX TL within optimal interval LB Group CB Group Randomized 1:1 IFX dosing based on clinical symptoms & CRP Maintenance phase (52 weeks) IFX dosing based on IFX TL (3-7 µg/mL) Dosing based on IFX TL (3-7 µg/mL) Optimization phase (n weeks) Prospective Controlled Trial of Trough Level Adapted Infliximab Treatment (TAXIT): Study outline ScreeningRandomization Primary end point Primary end point = rate of clinical (Harvey-Bradshaw or Partial Mayo score) and biological (C-reactive protein ≤5 mg/l) remission one year after randomization in each group CB Group= Clinically Based Group; LB Group= Level Based Group IFX maintenance therapy –> Stable clinical response Vande Casteele N, et al. United European Gastroenterology Journal 2013;1:A1

38. TAXIT Results: Optimization Phase Dose escalation (N=76) Dose escalation in Crohn’s disease patients with subtherapeutic levels results in a better disease control *five patients (1 CD and 4 UC) were excluded from analysis because of withdrawal of consent during optimization phase. CD: Harvey-Bradshaw ≤ 4 / UC: Partial Mayo ≤ 2 Vande Casteele N, et al. United European Gastroenterology Journal 2013;1:A1

39. Enrolled 52 IBD patients (34 CD and 18 UC) with secondary failure to IFX. Dose escalation to 10mg/kg in all. IFX trough, ATI, CRP, and calprotectin measured before dose optimization and at Week 8 Endpoint – Mucosal healing at Week 8 Conclusion: The change in infliximab trough levels after dose intensification (delta IFX) predicts mucosal healing in IBD patients. Therapeutic Drug Monitoring of Infliximab (IFX) Predicts Mucosal Healing Following Dose Intensification in IBD Multivariate Predictors of Mucosal Healing Factors LR95% CIP value Delta IFX >0.52.021.01-4.060.048 IFX<2μg/mL and ATI <200ng/mL (before) 5.280.82-34.30.08 UC3.10.41-23.60.27 Sex F/M0.580.13-30.52 Paul S, et al. Presented at DDW; May 19, 2013. Abstract 495.

40. TAXIT Results: Maintenance Phase Primary end point *Harvey-Bradshaw index score ≤4 (CD) or Partial MAYO score ≤2 (UC) and C-reactive protein level ≤5 mg/l. Primary end point could not be calculated for 3 Patients (1 CD from CB and 1 UC and 1 CD from LB group). Vande Casteele N, et al. United European Gastroenterology Journal 2013;1:A1

41. TAXIT Results: Maintenance Phase Secondary end point (loss of response and need for an intervention) 17.3% of CB Group vs. 5.5% of LB Group needed rescue therapy by the end of the maintenance phase N at risk 127126122 119116651 120118116109105100571 Vande Casteele N, et al. United European Gastroenterology Journal 2013;1:A1

42. Patients with secondary IFX failure randomized to: –IFX intensification group: IFX 5mg/kg every 4 weeks OR –Algorithm group: Low IFX and 0 ATI: IFX q4 ATI positive and low IFX :adalimumab High IFX +/- ATI: stop anti-TNF Endpoints –Costs related to treatment –Clinical response: reduction in CDAI≥70 or ≥50% reduction of active fistulas at 12 weeks Conclusions: Treatment of IFX failure using an algorithm significantly reduces average costs without compromising care Algorithm-Based Approach Based on PK for Secondary Non-Responders to Infliximab More Cost-Effective Intention to treat Algorithm n=33 IFX intensified n=36 PDifference [95%CI] Response – no. (%) 19 (58) 19 (53) 0.810 5% [-19 – 28] Costs – $ mean (SD) 7,806 (5,360) 11,867 (2,661) <0.001-4,060 [-5,969;-1,775] Steenholdt C, et al. Gut 2014;63:919-27.

43. - Consecutive cohort of patients (n=128); 105 CD, 23 UC where IFX was restarted after a median drug holiday of 15 months (at least >6 months). - Success at Week 14, 1 year, and end of follow-up (median 4 years); ATI and trough level (TL) assessed - Restarting IFX successful in 84.5% (short term), 70% (at 1 year) and in 61% (end of follow-up) - IFX discontinued in 12% due to infusion reaction. IMM at restart prevents infusion reactions. Multivariate analysis Conclusion: Restarting IFX after a drug holiday is safe, with success predicted by absence of early ATI formation, IMM at recommencement, and not having had previous infusion reactions Trough Levels and Anti-drug Antibodies Predict Safety and Success of Restarting IFX After Long Drug Holiday Response (%) Short- termYear 1 End of Follow- UpHR (95% CI) ATI (at 2 nd infusion) detectable N=31 71%54.8%38.7% 0.14 (0.026-0.74) P=0.021 IMM at restart N=8491.6%74.7%66.6%6 (1.3-2.7) P=0.019 Reason for discontinuation (remission &/or pregnancy) 90%77.5%66.6%2.70 (1.09-6.67) P=0.033 TL (at 2 nd infusion) > 2 μg/ml N=43 93%74%70% 2.94 (1.18-7.69) P=0.021 Baert FJ, et al. Clin Gastroenterol Hepatol. 2014;12:1474-1481.

44. Additional questions Should target trough levels differ at different phases of treatment? – Rate of early drug clearance seems important early in severe disease – Is a higher trough required in induction than in maintenance? – Would rate of early drug clearance be a clinically useful parameter? Should TDM be individualized, or do population means work? -Can accurate predictive models for individual PK be created? Is there a safety benefit to dose reduction for supratherapeutic levels? Questions of timing and frequency of TDM: maximizing the value

45. Conclusions TDM with biologics more complex than with thiopurines Minimum effective concentration roughly defined – IFX trough ~3 μg/mL – ADA trough ~5 μg/mL Role of TDM in assessing loss of response is best established TDM appears to be cost effective when dose-reduction incorporated into treatment algorithm, but will be highly dependent upon cost and frequency of assay Growing interest in early TDM to dose optimize in severe disease (especially UC)