Drug Candidate-Induced Changes in the Thyroid Gland: Contrasting Case Studies Joan Lane, Katie Zokowski, Jeffrey Horrigan, Daniel Aleksandrowicz, Doriana Froim, Ken Loveday, David Peters, Evelyne Polacke1 1Biogen Idec, Inc., Cambridge, MA, United States Ref. No. 0099 Poster No. 36.5" wide x 20.5"H Introduction Results- Program 1 Results- Program 2 Program 2 Lead: Control vs Treated Thyroid Gland Histomorphology Cynomolgus Monkey Investigative Plan Lead compound Back up compound: Develop testable mechanistic hypothesis (on/off target) Identify potential structural basis of effect by comparison of compounds Provide screening recommendations for new compound selection Understand protein binding/free fraction/local concentration/effect relationships Identify any species differences that may be relevant to predicting human risk Introduction: While xenobiotics that depress thyroid function by inhibition of thyroid hormone synthesis or enhancement of peripheral metabolism are fairly common, those that stimulate thyroid hormone synthesis are rare. Recently, we identified histomorphologic and functional thyroid changes associated with lead and follow on compounds for two preIND-stage programs with distinct pathways. Program 1 targeted a tyrosine kinase expressed in lymphoid and myeloid cells; Program 2 targeted a G protein-coupled receptor with a broad expression profile. Findings indicated thyroid suppression in Program 1 and thyroid stimulation in Program 2. Experimental Design: For Program 1, 28 day oral repeat dose toxicology studies for the lead and 14 day dose range finding studies for the backup compound were completed in rats and dogs. For Program 2, 28 day oral repeat dose toxicology studies for both lead and back up molecules were completed in rats and cynomolgus monkeys. Methods: Standard toxicologic end points, serum Thyroid Stimulating Hormone (TSH), Triiodothyronine (T3), and Thyroxine (T4) were evaluated in 28 day studies. Results: For Program 1, increased thyroid weight in rats, increased TSH in rats and dogs, and decreased T4 in rats were observed. T3 was unchanged in both species. For Program 2, increased thyroid weight, elevated T3 and T4, and decreased TSH were noted in monkeys, but not in rats, and the pattern was conserved between lead and back up. Conclusion/Impact statement: Histopathologic changes in the thyroid correlating with the hormonal effects are described and the investigative activities and impact for each program are discussed. Lead compound, discontinued due to (nonthyroid) dose limiting toxicity Dose-related increases in T3 and T4 levels and decrease in TSH levels were observed in ≥ 5 mg/kg/day. Three 25 mg/kg and one (female) 1 mg/kg animal were noted to have thyroid enlargement grossly. Greater absolute and relative thyroid/parathyroid gland weights were observed at ≥ 5.0 mg/kg/day in males and at 1.0 and 25 mg/kg/day in females. Minimal to moderate dose dependent follicular cell hypertrophy/hyperplasia was observed microscopically in the thyroid gland at ≥ 5.0 mg/kg/day. By Day 85, all thyroid changes returned to baseline levels,demonstratin g reversibility. Back up compound (clinical candidate): T4 was statistically elevated ≥15 mg/kg; T3 was statistically elevated at 30 mg/kg on D14 only (data not shown), and TSH was statistically decreased at 30 mg/kg. No correlating microscopic or gross observations in thyroid gland, no correlating changes in thyroid weights, All thyroid hormone values were normal during the drug-free recovery period. Discussion/Conclusions Program 1: Program 2: Increased T3 and T4 increase and decreased TSH indicated signaling through the TSHR with negative feedback suppression of TSH, therefore the finding was considered an off target effect . The selected back up compound fortuitously had a less potent thyroid effects with good exposure margins and minimal structural modification. Thyroid hormones were none the less built in to the Phase I safety assessment plan. Rescreening of the human TSH receptor was performed in attempt to determine an EC50 for both compounds; however, no specific binding was observed up to 30 uM, well in excess of planned clinical exposures. Table x) Program 2 Lead: Microscopic Findings, Thyroid Gland, Cynomolgus Monkey   Males Females Group 1 2 3 4 Dose (mg/kg/day) 5 25 No. animals examined Thyroid:Hypertrophy/ hyperplasia: follicular cell Minimal Slight Moderate Table 1.) Summary Comparison of Thyroid Findings Program 1 Lead vs Back up Observed Thyroid Effects* Program 1 lead Program 1 back up Increased TSH Unchanged T3 Decreased T4 Increased Organ Weight Gross Organ Enlargement Follicular cell enlargement, decreased colloid Table 2.) Summary Comparison of Thyroid Findings Program 2 Lead vs Back up Observed Thyroid Effects* Program 2 lead (discontinued program) Program 2 back up (clinical candidate) Decreased TSH √ √** Elevated T3 Elevated T4 Increased Organ Weight - Gross Organ Enlargement Follicular Hyperplasia *Cynomolgus monkey only ** Changes significant only when expressed as change from prestudy baseline values Table x.) Program 2 Back up (Clinical Candidate) Average (Pooled Male and Female) Thyroid Hormones Change from Baseline, Cynomolgus Monkey (Mean ± SD) *p ≤ 0.05 Parameter 0 mg/kg 1.5 mg/kg 5 mg/kg 15 mg/kg 30 mg/kg T3 (ng/mL) 0.06 ± 0.4 0.12 ± 0.25 0.13 ± 0.36 0.22 ± 0.33 0.27 ±0.21 T4 (mg/dL) ± 1.33 -0.4 ± 1.24 0.19 ± 1.97 *2.23 ± 1.49 *2.61 ±2.11 TSH (mIU/mL)  0.22 ± 0.54 0.21 ± 0.49 0.45 ± 54 -0.10 ± 0.68 *-0.78 ± 0.91 Table x.) Program 2 Lead: Mean Thyroid Weights(g) Cynomolgus Monkey Sex Males Females Dose (mg/kg/day) 1 5 25 Thyroid gland 0.31 0.30 0.41 0.57 0.29 0.45 0.26 0.52 sd 0.06 0.14 0.25 0.24 0.09 N/A % body weight 0.01 0.02 0.00 % brain weight 0.44 0.43 0.56 0.81 0.46 0.68 0.40 0.70 0.07 0.19 0.18 0.35 0.23 0.37 0.12 Thank you to xxxxx for scientific review/technical support