1 2 3 4 5 6 7 8 F. HOFFMANN-LA ROCHE LTD 9 CLINICAL STUDY PROTOCOL 10 PROTOCOL NUMBER NP25620 11 RO4917523 12 EUDRACT NUMBER 2011-001436-33 13 IND NUMBER 103001 14 15 Sponsor: F. HOFFMANN-LA ROCHE LTD 16 Grenzacherstrasse 124, 17 4070 Basel, Switzerland 18 19 20 21 22 23 24 25 26 27 PROTOCOL APPROVAL 28 29 NP25620 / C 30 Protocol Number / Version: 31 32 Date: See date in electronic signature manifestation below. 33 Name Reason for Signing Date and Time (UTC) Quiroz,Jorge Translational Medicine Leader 04-Apr-2012 18:04:29 34 35 36 37 38 39 40 41 42 Confidentiality Statement 43 44 The information contained in this document, especially unpublished data, is the property 45 of F. Hoffmann-La Roche Ltd (or under its control), and therefore provided to you in 46 confidence as an investigator, potential investigator or consultant, for review by you, your 47 staff and an applicable Independent Ethics Committee/Institutional Review Board. It is 48 understood that this information will not be disclosed to others without written 49 authorization from Roche except to the extent necessary to obtain informed consent from 50 those persons to whom the drug may be administered.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 51 52 53 SYNOPSIS OF PROTOCOL NUMBER NP25620 54 55 56 TITLE A randomized, double-blind, parallel-group study of the safety and efficacy of 57 RO4917523 versus placebo, as adjunctive therapy in patients with major 58 depressive disorder with inadequate response to ongoing antidepressant 59 treatment. 60 61 SPONSOR F. Hoffmann-La Roche Ltd CLINICAL 64 IIb 62 PHASE 6563 INDICATION Major Depressive Disorder (MDD) 66 OBJECTIVES Primary 67 To evaluate the efficacy of two fixed doses of RO4917523 compared to 68 placebo in a confirmatory manner over 6 weeks as adjunctive therapy in 69 patients with MDD with inadequate response to ongoing antidepressant 70 treatment, based on mean change in the Montgomery Asberg Depression 71 Rating Scale (MADRS) total score from baseline to end of treatment. 72 73 Secondary 74 The secondary objectives are to evaluate change after 6 weeks of treatment 75 with RO4917523 versus placebo as adjunctive therapy on the following: 76 • Clinical Global Impression Scores: Severity (CGI-S) from baseline to end 77 of treatment, and Improvement (CGI-I) at end of treatment 78 • Safety and tolerability of RO4917523 79 • Proportion of patients exhibiting remission (a MADRS score of less than 80 or equal to 10) 81 • Proportion of patients exhibiting response (reduction in MADRS score 82 equal to or greater than 50% of the baseline score) 83 • Quick Inventory of Depressive Symptomatology Self Report (QIDS-SR16) 84 • Patient Global Impression of Improvement (PGI-I) score at end of 85 treatment 86 87 Exploratory 88 Key Exploratory Objective 89 To evaluate the effect size (ES) of two fixed doses of RO4917523 compared to 90 placebo over 6 weeks as adjunctive therapy in patients with MDD with 91 inadequate response to ongoing antidepressant treatment, based on mean 92 change in the Montgomery Asberg Depression Rating Scale (MADRS) total 93 score from baseline to end of treatment. 94 95 Other Exploratory Objectives 96 To investigate the differential effect of RO4917523 vs. placebo in: 97 • Proportion of patients who meet MADRS responder criteria plus CGI-I 98 score of 1) “very much improved” or 2) “much improved” 99 • Proportion of patients who meet MADRS remission criteria plus CGI-I 100 score of 1) “very much improved” or 2) “much improved 101 • Speed of onset of antidepressant effect based on change over time in any 102 of the depression symptom scales 103 • CANTAB Cognitive Test Battery subset 104 • Quality of Life Enjoyment and Satisfaction Questionnaire-Short Form (Q- 105 LES-Q-SF) 106 • Sheehan Disability Scale (SDS)

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 107 108 Pharmacokinetics 109 • Pharmacokinetics of RO4917523 in the target population with the 110 objective of performing a population pharmacokinetic analysis with non- 111 linear mixed effect model 112 Biomarkers 113 • Identify biomarkers that are predictive of response to RO4917523 114 treatment 115 • Increase our knowledge and understanding of the pathogenesis, course and 116 outcome of depression and related diseases 117 • Develop biomarker or diagnostic assays, and establish the performance 118 characteristics of these assays 119 TRIAL DESIGN This is an outpatient trial consisting of three consecutive periods: screening (up 120 to 14 days), 6-week double-blind treatment, and a 21 day follow-up period. 121 122 NUMBER OF SUBJECTS Approximately 300 patients, randomized in equal proportions to the three 123 treatment arms. 124 125 TARGET POPULATION The study will include male and female outpatients from 18 to 70 years of age 126 with a primary diagnosis of major depressive disorder (MDD) without 127 psychotic features as defined by DSM-IV-TR criteria, and having inadequate 128 response to ongoing antidepressant therapy. Patients must have had at least one 129 but no more than three treatment failures (of adequate dose and duration 130 according to the Massachusetts General Hospital Antidepressant Treatment 131 History Questionnaire [MGH ATRQ]). Failure to ongoing antidepressant 132 treatment in the current episode is counted as one treatment failure. 133 134 LENGTH OF STUDY • 14 day screening period 135 • 6 week double-blind treatment 136 • 21 day follow-up 137 END OF STUDY The date of the last visit (including the follow-up period) of the last patient in 138 the study 139 INVESTIGATIONAL 143 Doses of RO4917523 0.5 mg or 1.5 mg supplied as pellets in capsules, in 140 MEDICAL PRODUCT(S)144 combinations of Ro 491-7523/F18 0.5 mg, Ro 491-7523/F19 1.0 mg, taken 141 DOSE/ ROUTE/ 145 orally once a day 146142 REGIMEN 147 148 149 COMPARATOR “DRUG”154 Matching capsules of placebo Ro 491-7523/F21, taken orally once a day 150 DOSE/ ROUTE/ 151 REGIMEN 152 ASSESSMENTS OF: 155153 156 -EFFICACY • MADRS total score 157 • Remission (a MADRS score less than or equal to 10) 158 • Response (reduction in MADRS score equal to or greater than 50% from 159 baseline) 160 • CGI-S 161 • CGI-I 162 • PGI-I 163 • QIDS-SR16 164 • Q-LES-Q-SF 165 • SDS 166 • CANTAB

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 167 168 • Adverse events (AEs) and concomitant medications will be monitored 169 -SAFETY 170 throughout the entire study (screening through follow-up). Intensity of AEs 171 will be graded on a 3 point scale (mild, moderate, or severe) 172 • A subscale (4 items) of the BPRS will be completed only to follow up on 173 treatment emergent psychotic-related and mania-related (mania or 174 hypomania) adverse events. The symptom constructs included are 175 conceptual disorganization, suspiciousness, hallucinatory behavior, and 176 unusual thought content 177 • The YMRS (item 1 only for mood elevation) will be completed only to 178 follow up on treatment emergent psychotic-related and mania-related 179 (mania or hypomania) adverse events 180 • Columbia-Suicide Severity Rating Scale (C-SSRS) 181 • Physical examination 182 • Vital signs (pulse, blood pressure) 183 • 12-lead ECG 184 • Laboratory parameters 185 o Chemistry panel 186 o Hematology panel with differential 187 o Free T4 and TSH 188 o Viral Serology 189 o Urinalysis (dipstick) 190 o Urine drug screen 191 o Pregnancy test in females 192 • Data Safety Monitoring Board (DSMB) 193 o An independent Data Safety Monitoring Board (DSMB) will review 194 potential safety signals of concern. The DSMB will be composed of 195 non-sponsor members who are not involved in the conduct of this 196 study. The DSMB will review available safety data from this trial at 197 regularly scheduled intervals as specified in the DSMB Charter 198 • Statistical Interim Analyses 199 o No statistical interim analyses are planned 200 -PHARMACOKINETICS PK sampling will be obtained from all patients according to the Schedule of 201 Assessments. 202 -EXPLORATORY - 206 Specimens for the Roche Clinical Repository (RCR) will be collected for 203 BIOMARKERS (non- 207 dynamic (non-inherited) biomarker discovery and validation. The RCR 204 inherited) 208 sampling is optional. Blood specimens will be collected as per Schedule of 205 209 Assessments, with details as follows: 210 ƒ Plasma and Serum assays: Blood samples for plasma and serum isolation 211 will be obtained at baseline and the end of treatment. A total of 4 samples 212 (2x6 mL for serum and 2x6 mL for plasma; 24 mL total) for each patient 213 will be collected during the study. These samples will be used for 214 biomarker assays for candidate depression biomarkers. 215 ƒ Blood for RNA expression profiling: Blood samples for RNA isolation 216 will be obtained at baseline and the end of treatment (5 mL each; 10 mL 217 total). 218 ƒ Blood sample for epigenetic analysis: Blood samples for DNA isolation 219 will be collected at baseline and the end of treatment (6 mL each; 12 mL 220 total). 221 222 These specimens will be stored for up to 15 years after the end of the study.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 223 224 -EXPLORATORY 250 Additional specimens for the Roche Clinical Repository (RCR) will be 225 BIOMARKERS (inherited)251 collected from consenting patients for genetic biomarker (inherited) discovery 226 227 252 and validation. The RCR sampling (inherited) is optional. A single blood 228 253 specimen will be collected from consenting patients, with details as follows: 229 254 ƒ Blood sample for genetic analysis: A blood sample (approx. 6 mL) for 230 231 255 DNA isolation will be collected at a single study visit as per Schedule of 232 256 Assessments. If however, the RCR genetic blood sample is not collected 233 257 during the scheduled visit, it may be collected at any time (after 234 258 randomization) during the conduct of the clinical study The sample may be 235 259 processed using techniques such as sequencing or microarray profiling. 236 237 260 These specimen(s) will be stored for up to 15 yearsafter the end of the study. 238 239 -MANDATORY 261 Blood samples for serum and plasma protein biomarker discovery and 240 BIOMARKERS (MBS) 262 validation will be collected from all patients where permissible with local 241 263 regulations at baseline and the end of treatment. A total of 4 samples (2x6 mL 242 for serum and 2x6 mL for plasma; 24 mL total) for each patient will be 243 264 244 265 collected during the study. These specimens will be destroyed no later than 5 245 266 years after the end of the study. 246 267 247 - CLINICAL GENOTYPING268 A 3 mL whole blood sample will be taken for DNA extraction from all patients 248 (CG) 269 where permissible with local regulations at baseline. The DNA will be tested 249 270 for one or more of the following specific genes: the cytochrome P450 variant 271 1A2 gene (CYP1A2), the metabotropic glutamate receptor 5 (GRM5), the 272 serotonin 2A receptor (HTR2A), the serotonin transporter (SLC6A4), and brain 273 derived neurotropic factor (BDNF). This specimen will be destroyed 274 immediately after the analysis has been completed. 275 276 277 PROCEDURES (summary): Screening Period (up to 14 days) 278 279 During the screening period, informed consent will be obtained, and the investigator will 280 determine whether the candidate meets all inclusion criteria and does not meet any 281 exclusion criteria. 282 283 6-Week Double-blind Treatment Period 284 285 In order to be randomized into the double-blind treatment period, patients must have at 286 baseline: 287 288 – No significant risk of suicidal behavior (e.g., consider the Suicidal Ideation section of 289 the C-SSRS “Since Last Visit” for this evaluation) 290 291 – No significant change in medical or psychiatric condition, or change in medications 292 since screening (unless agreed with the Sponsor/Medical Monitor) 293 294 – Negative result on the Baseline pregnancy test (if applicable) 295 296 – No change in ongoing antidepressant therapy, and ability to continue for the duration 297 of the double-blind treatment period without modification to the dosing schedule 298 299 – An ESF/EAF approved by the Sponsor/Medical Monitor

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 300 301 The double-blind treatment period begins with the investigational site call into IVRS 302 confirming the patient’s eligibility. The patient will be randomized and receive their first 303 dose of study medication on Day 1. 304 305 Dose 1 of the blinded study medication is to be administered in the clinic immediately 306 after a meal and before 12 pm (noon), or soon thereafter upon consultation with the 307 Sponsor/Medical Monitor, once all baseline procedures and assessments are completed. 308 Patients will remain at the clinic for 6 hours after the first dose for safety monitoring and 309 for the PK samples (according to clinical observation and patient availability). 310 Subsequently, dosing will be once daily in the morning immediately after breakfast. 311 312 As with any experimental drug at this stage of development, it is advisable for patients 313 not to drive or operate dangerous machinery until known side effects (e.g., dizziness and 314 somnolence) can be adequately assessed on an individual basis during the trial. 315 316 Patients will arrive at each study visit without having taken their daily dose of study 317 medication, and site staff will record the time of their last dose. Following collection of 318 the pre-dose PK blood sample (if applicable) and a meal, patients will take their next dose 319 of study medication before 12 pm (noon) or soon thereafter upon consultation with the 320 Sponsor/Medical Monitor. The last dose of study medication will be administered on Day 321 42 (End-of-Treatment visit). 322 323 Early Discontinuers will be instructed to return as soon as possible for the End-of- 324 Treatment visit, and 21 days later for the Follow-up visit. 325 326 Follow-up Period (21 days) 327 328 A Follow-up Visit will take place 21 days after the End-of-Treatment visit. During the 329 follow-up period, adjustments to antidepressant treatments may be initiated if deemed 330 necessary by the investigator. 331 332 STATISTICAL ANALYSES: 333 334 Main efficacy analysis will be performed in a confirmatory manner based on ITT 335 population, using a mixed effects covariance pattern model (MMRM) to utilize all the 336 data collected over time. A closed testing procedure will be used to take multiple 337 comparisons into account. As supporting analysis, the analysis may be repeated on the 338 per-protocol population. Another supportive analysis will use an ANCOVA with LOCF 339 imputation for missing data. The primary efficacy variable is change in the Montgomery 340 Asberg Depression Rating Scale (MADRS) total score from baseline to end of treatment. 341 342 All safety variables (e.g., adverse events, lab tests, ECG, vital signs, BPRS, YMRS, 343 ASEX) will be summarized for each assessment time (including follow-up) using 344 descriptive statistics. The items of the C-SSRS will be presented by individual listings 345 and the outcomes from this scale will be classified using the C-CASA methodology.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 346 347 348 Table of Contents 349 350 351 1. Background and Rationale ...... 22 352 353 1.1 Background ...... 22 354 355 1.1.1 Disease ...... 22 356 357 1.1.2 Study “Drug” ...... 23 358 359 1.2 Non-Clinical Experience ...... 23 360 361 1.2.1 Non-Clinical Pharmacology ...... 23 362 363 1.2.2 Non-Clinical Pharmacokinetics and Metabolism ...... 23 364 365 1.2.3 Non –Clinical Safety and Toxicology ...... 24 366 367 1.3 Previous Clinical Experience ...... 26 368 369 1.3.1 Safety ...... 27 370 1.3.1.1 Single Ascending Dose (SAD) Study ...... 27 371 372 1.3.1.2 Multiple Ascending Dose (MAD) Study ...... 27 373 374 1.3.1.3 Single Dose Alcohol Interaction Study ...... 28 375 376 1.3.1.4 Relative Bioavailability Studies ...... 28 377 378 1.3.1.5 Ketoconazole Interaction Study ...... 29 379 380 1.3.1.6 Phase 2a Study in Treatment-Resistant Depression ...... 30 381 382 1.3.1.7 Phase 2a Study in Fragile X Syndrome ...... 30 383 384 1.3.2 Pharmacokinetics ...... 30 385 1.3.3 Pharmacodynamics ...... 31 386 387 1.4 Rationale for the Study and Overall Risk-Benefit Assessment ...... 32 388 389 390 2. Objectives ...... 33 391 392 2.1 Primary Objective ...... 33 393 394 2.2 Secondary Objectives ...... 33 395 396 2.3 Exploratory Objectives ...... 33 397 398 2.3.1 Key Exploratory Objective ...... 33 399 400 2.3.2 Other Exploratory Objectives ...... 33 401 402 2.3.3 Pharmacokinetics ...... 34 403 2.3.4 Roche Clinical Repository (RCR), Mandatory Biomarker Samples (MBS) and Clinical Genotyping (CG) ...... 34 2.4 Roche Internal Criteria to Advance the Development of RO4917523 . 34

3. Study Design ...... 34

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 404 405 406 3.1 Overview of Study Design and Dosing Regimen ...... 34 407 408 3.1.1 Rationale for Study Design ...... 35 409 410 3.1.2 Rationale for Dose Selection ...... 36 411 412 3.1.3 End of Study ...... 37 413 414 3.2 Number of Patients/ Assignment to Treatment Groups ...... 37 415 416 3.3 Centers ...... 37

417 418 4. Study Population ...... 37 419 420 421 4.1 Overview ...... 37 422 423 4.2 Inclusion Criteria ...... 37 424 4.3 Exclusion Criteria ...... 39 425 426 4.4 Allowed and Prohibited Medication ...... 40 427 428 4.4.1 Ongoing Antidepressant Treatment ...... 40 429 430 4.4.2 Allowed Medications ...... 40 431 432 4.4.3 Prohibited Medications ...... 41 433 434 4.5 Criteria for Premature Withdrawal ...... 41 435 436 4.5.1 Withdrawal Of Patients From the Roche Clinical Repository (RCR) 42 437 4.6 Replacement Policy [Ensuring Adequate Numbers of Evaluable 438 439 Patients] ...... 43 440 441 4.6.1 For Patients ...... 43 442 443 4.6.2 For Centers ...... 43

444 445 5. Schedule of Assessments and Procedures ...... 43 446 447 448 5.1 Screening Period ...... 45 449 450 5.1.1 Informed Consent Form (ICF) ...... 45 451 5.1.2 Screening ...... 45 452 453 5.2 Procedures for Randomization of Eligible Patients ...... 45 454 455 5.3 Double-blind Treatment Period ...... 46 456 457 5.4 Follow-up Period ...... 46 458 459 5.5 Clinical Assessments and Procedures ...... 46 460 461 5.5.1 Rater Selection and Training ...... 46 462 463 5.5.2 Assessments for Eligibility ...... 47 464 465 5.5.2.1 Detailed psychiatric history ...... 47 466 5.5.2.2 Massachusetts General Hospital Antidepressant Treatment History Questionnaire (MGH ATRQ) ...... 47 5.5.2.3 Medical history and Physical examination ...... 47

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 467 468 469 5.5.2.4 Computer-Administered Diagnostic Interview ...... 47 470 471 5.5.2.5 Mini International Neuropsychiatric Inventory (MINI) ...... 48 472 473 5.5.3 Efficacy ...... 48 474 475 5.5.3.1 Montgomery Asberg Depression Rating Scale (MADRS) ...... 48 476 477 5.5.3.2 Clinical Global Impression- Severity (CGI-S) and Improvement 478 (CGI-I) ...... 49 479 480 5.5.3.3 Patient Global Impression of Improvement (PGI-I) ...... 49 481 482 5.5.3.4 Cambridge Neuropsychological Test Automated Battery 483 (CANTAB®) ...... 49 484 485 5.5.3.5 Quick Inventory of Depressive Symptomatology Self Report- 16 486 item version (QIDS-SR16) ...... 50 487 488 5.5.3.6 Quality of Life Enjoyment and Satisfaction Questionnaire- Short 489 Form (Q-LES-Q-SF) ...... 50 490 491 5.5.3.7 Sheehan Disability Scale (SDS) ...... 50 492 493 5.5.4 Safety ...... 50 494 495 5.5.4.1 Brief Psychiatric Rating Scale (BPRS) ...... 50 496 497 5.5.4.2 Young Mania Rating Scale (YMRS) ...... 50 498 499 5.5.4.3 Columbia-Suicide Severity Rating Scale (C-SSRS) ...... 51 500 501 5.5.4.4 Vital Signs and weight ...... 52 502 503 5.5.4.5 ECG ...... 52 504 505 5.6 Laboratory Assessments ...... 52 506 507 5.6.1 Pharmacokinetic [PK] Assessments ...... 53 508 509 5.7 Roche Clinical Repository Specimen(s) ...... 54 510 511 5.7.1 Specimen Types ...... 54 512 513 5.8 Mandatory Biomarker Sampling (MBS) ...... 55 514 515 5.9 Clinical Genotyping ...... 56 516 517 6. Investigational Medicinal Product ...... 56 518 519 6.1 Dose and Schedule of IMP ...... 56 520 521 6.1.1 Dose Modifications, Interruptions and Delays ...... 56 522 523 6.2 Formulation, Packaging and Labeling ...... 57 524 525 6.2.1 Packaging and Storage ...... 57 526 527 6.3 Blinding and Unblinding ...... 57 528 529 6.4 Accountability of IMP and Assessment of Compliance ...... 58 530 531 6.4.1 Accountability of IMP ...... 58

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 532 533 6.4.2 Assessment of Compliance ...... 59 6.5 Destruction of the IMP/Comparator ...... 59

7. Safety Instructions and Guidance ...... 60 7.1 Adverse Events (AEs) and Laboratory Abnormalities ...... 60 7.1.1 Clinical AEs ...... 60 7.1.1.1 Intensity ...... 60 7.1.1.2 Drug - Adverse event relationship ...... 60 7.1.1.3 Serious Adverse Events [Immediately Reportable to Roche] . . . . . 60 7.1.2 Treatment and Follow-up of AEs ...... 61 7.1.3 Laboratory Test Abnormalities ...... 61 7.1.3.1 Follow-up of Abnormal Laboratory Test Values ...... 61 7.2 Handling of Safety Parameters ...... 62 7.2.1 Reporting of AEs ...... 62 7.2.1.1 Recording of AEs related to Patient-Reported Outcomes ...... 62 7.2.2 Reporting of Serious Adverse Events [immediately reportable] . . . . 62 7.2.3 Abuse and Withdrawal Symptoms ...... 63 7.2.4 Pregnancy ...... 63 7.3 Warnings and Precautions ...... 63

8. Statistical Considerations and Analytical Plan ...... 63 8.1 Primary and Secondary Study Endpoints ...... 63 8.1.1 Primary Endpoints ...... 63 8.1.2 Secondary Endpoints ...... 63 8.1.3 Exploratory Endpoints ...... 64 8.1.4 Safety ...... 64 8.2 Statistical and Analytical Methods ...... 64 8.2.1 Analysis Population ...... 64 8.2.1.1 Intent-to-treat Population ...... 64 8.2.1.2 Per-protocol Population ...... 64 8.2.1.3 Safety Population ...... 65 8.2.2 Statistical Model ...... 65 8.2.3 Sample Size ...... 66 8.2.4 Hypothesis Testing ...... 66 8.2.5 Efficacy Analysis ...... 66

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 534 535 536 8.2.5.1 Interim Analysis ...... 66 537 538 8.2.5.2 Missing Data Handling ...... 67 539 540 8.2.6 Safety Data Analysis ...... 67 541 542 8.2.7 Other Analyses ...... 67 543 544 8.2.7.1 Pharmacokinetic Analysis ...... 67 545 546 8.2.7.2 Pharmacodynamic Analysis ...... 67 547 548 8.2.7.3 Subgroup Analysis ...... 67 549 550 9. Data Collection, Management and Quality Assurance ...... 68 551 552 553 9.1 Assignment of Preferred Terms and Original Terminology ...... 68 554 555 10. Study Committees ...... 68 556 557 558 11. References...... 69 559 560 561 12. Ethical Aspects ...... 71 562 563 12.1 Local Regulations/Declaration of Helsinki ...... 71 564 565 12.2 Informed Consent ...... 71 566 567 12.2.1 Main study Informed Consent ...... 71 568 569 12.2.2 RCR Informed Consent ...... 71 570 12.2.3 Death or Loss of Competence of Participant who has donated a 571 572 specimen(s) that is stored in the RCR ...... 72 573 12.3 Independent Ethics Committees/(IEC)Institutional Review Board(IRB) ...... 72 12.4 Role of the Science and Ethics Advisory Group (SEAG) ...... 73

13. Conditions for Modifying the Protocol...... 73

14. Conditions for Terminating the Study ...... 73

15. Study Documentation, CRFs and Record Keeping ...... 73 15.1 Investigator's Files / Retention of Documents ...... 73 15.2 Source Documents and Background Data ...... 74 15.3 Audits and Inspections ...... 74 15.4 Case Report Forms or Electronic Case Report Forms ...... 75 15.5 Financial Disclosure ...... 75

16. Monitoring the Study ...... 75

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 574 575 576 17. Confidentiality of Trial Documents and Patient Records ...... 76 577 578 579 18. Clinical Study Report (CSR) ...... 76 580 581 582 19. Publication of Data and Protection of Trade Secrets ...... 76

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 583 584 585 List of Tables 586 587 588 Table 1 Schedule of Assessments ...... 43 589 590 Table 2 Schedule of PK Sampling ...... 53 591 592 Table 3 IMP Packaging Configuration by Treatment Arm ...... 57

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 593 594 595 List of Figures 596 597 598 Figure 1 Study Flow ...... 35

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 599 600 601 List of Appendices 602 603 604 Appendix 1 AEs Categories for Determining Relationship to Test Drug . . . . . 78 605 606 Appendix 2 ICH Guidelines for Clinical Safety Data Management, 607 Definitions and Standards for Expedited Reporting, Topic E2 . 80 608 609 Appendix 3 Structured Interview Guide For The Montgomery And Asberg 610 Depression Rating Scale (Madrs-Sigma) ...... 82 611 612 Appendix 4 Clinical Global Impression (CGI) ...... 88 613 614 Appendix 5 Patient Global Impression of Improvement (PGI-I) ...... 89 615 616 Appendix 6 Quick Inventory of Depressive Symptomatology Self Report 617 (16-Item) (Self-Report) (QIDS-SR16) ...... 90 618 619 Appendix 7 Quality of Life Enjoyment and Satisfaction Questionnaire- Short 620 Form (Q-LES-Q-SF) ...... 94 621 622 Appendix 8 Sheehan Disability Scale (SDS) ...... 97 623 624 Appendix 9 Brief Psychiatric Rating Scale, Positive Symptom Subscale (4 625 items) ...... 98 626 627 Appendix 10 Young Mania Rating Scale (YMRS) (item 1) ...... 99

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 628 629 630 GLOSSARY OF ABBREVIATIONS 631 632 AE adverse event 633 634 ALP alkaline phosphatase 635 636 ALT [SGPT] alanine aminotransferase 637 638 AMPA α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate 639 640 ANCOVA analysis of covariance 641 642 AST [SGOT] aspartate aminotransferase 643 644 AUC area under the plasma concentration-time curve 645 646 BDNF brain-derived neurotrophic factor

647 b.i.d. Latin bis in die (twice a day)

648 BMI body mass index 649 650 BP blood pressure 651 652 BPRS Brief Psychiatric Rating Scale

653 cAMP cyclic adenosine monophosphate

654 CANTAB® Cambridge Neuropsychological Test Automated Battery 655 656 C-CASA Columbia Classification Algorithm of Suicidality Assessment 657 658 CDS core data sheet 659 660 CFF critical flicker fusion test 661 662 CFR Code of Federal Regulations, US

663 CG clinical genotyping

664 CGI-I Clinical Global Impression of Improvement 665 666 CGI-S Clinical Global Impression of Severity 667 668 CI confidence interval 669 670 CL plasma clearance 671 672 CNS central nervous system 673 674 Cmax maximum plasma concentration 675 676 CPK creatine phosphokinase 677 678 CRF case report form

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 679 680 681 GLOSSARY OF ABBREVIATIONS 682 683 CRO contract research organization 684 685 CRT choice reaction time 686 687 C-SSRS Columbia-Suicide Severity Rating Scale 688 689 CSR clinical study report 690 691 CYP cytochrome P 692 693 DCS data collection specification 694 695 DNA deoxyribonucleic acid 696 697 DSMB Data Safety Monitoring Board 698 699 DSM-IV-TR Diagnostic and Statistical Manual of Mental Disorders, Fourth 700 Edition, Text Revision 701 702 DSST digit symbol substitution test 703 704 DxV-MDD Diagnostic Validation-Major Depressive Disorder 705 706 EAF Eligibility Assessment Form 707 708 EC Ethics Committee 709 710 ECG electrocardiogram 711 712 eCRF electronic case report form(s)

713 EDC electronic data capture

714 EDTA Ethylenediaminetetraacetic acid 715 716 EEA European Economic Area

717 eform electronic form (page)

718 ES effect size 719 720 ESF eligibility screening form 721 722 FDA Food and Drug Administration

723 FSH follicle stimulating hormone

724 FTP file transfer protocol

725 GABA gamma-aminobutyric acid 726 727 GCP Good Clinical Practice

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 728 729 730 GLOSSARY OF ABBREVIATIONS 731 732 GGT gamma-glutamyltransferase 733 734 GI gastro intestinal 735 736 GLP Good Laboratory Practice 737 738 HDL high density lipoprotein 739 740 HIV human immunodeficiency virus 741 742 HPLC high performance liquid chromatography 743 744 HR heart rate 745 746 IB Investigator Brochure 747 748 IC50 inhibitory concentration, half maximal 749 750 ICD-10 The International Statistical Classification of Diseases and 751 Related Health Problems 10th Revision 752 753 ICF Informed Consent Form 754 755 ICH International Conference on Harmonization 756 757 ID identification 758 759 IEC Independent Ethics Committee

760 iGlu ionotropic glutamate

761 IMP investigational medicinal product

762 IND investigational new drug application

763 INR International Normalized Ratio

764 IP internet protocol 765 766 IR immediate release 767 768 IRB Institutional Review Board 769 770 ITT intent to treat 771 772 IUD intrauterine device

773 iv intravenous

774 IVRS interactive voice response system 775 776 IWRS interactive web response system

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 777 778 779 GLOSSARY OF ABBREVIATIONS 780 781 keo equilibration rate constant

782 kd dissociation rate constant

783 LC/MS/MS liquid chromatography/tandem mass spectrometry 784 785 LDL low density lipoprotein 786 787 LMT learning memory task 788 789 LOCF Last observation carried forward 790 791 MAD multiple ascending dose 792 793 MADRS Montgomery Asberg Depression Rating Scale 794 795 MBS Mandatory Biomarker Samples 796 797 MDD Major depressive disorder 798 799 MGH ATRQ Massachusetts General Hospital Antidepressant Treatment 800 Response Questionnaire

801 mGlu metabotropic glutamate

802 MINI Mini International Neuropsychiatric Inventory 803 804 MMRM mixed effect repeated measure analysis 805 806 MPEP 2-methyl-6-(phenylethynyl)-pyridine 807 808 MR modified release 809 810 MTD maximum tolerated dose 811 812 MTEP [(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine 813 814 NMDA N-methyl-D-aspartic acid 815 816 NOEL No observed effect level 817 818 NOAEL No observed adverse effect level 819 820 OCD Obsessive-Compulsive Disorder 821 822 OB olfactory bulbectomized 823 824 PCP Phencyclidine 825 826 PD Pharmacodynamic 827 828 PE Pharmacoeconomic

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 829 830 831 GLOSSARY OF ABBREVIATIONS 832 833 PGI-I Patient Global Impression of Improvement 834 835 PI principal investigator 836 837 PK pharmacokinetic 838 839 PP per protocol 840 841 PR pulse rate 842 843 PTSD Postraumatic Stress Disorder 844 845 QD Latin quaque die (every day) 846 847 QIDS-SR16 Quick Inventory of Depressive Symptomatology-Self Report, 848 16 item version 849 850 Q-LES-Q-SF Quality of Life Enjoyment and Satisfaction Questionnaire- 851 Short Form 852 853 RCR Roche Clinical Repository 854 855 REM rapid eye movement 856 857 RNA ribonucleic acid 858 859 RT PCR reverse transcriptase polymerase chain reaction 860 861 RVIP rapid visual information processing 862 863 SAD single ascending dose

864 SAE serious adverse event

865 SDS Sheehan Disability Scale

866 SEM saccadic eye movement

867 sFTP secure file transfer protocol 868 869 SMT study management team 870 871 SNRI serotonin and norepinephrine reuptake inhibitor 872 873 SPC Summary of Product Characteristics 874 875 SSRI selective serotonin reuptake inhibitor 876 877 SUSAR Suspected Unexpected Serious Adverse Reaction 878 879 T3 triiodothyronine 880 881 T4 thyroxine

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 882 883 884 GLOSSARY OF ABBREVIATIONS 885 886 t.b.d. to be determined 887 888 T1/2 terminal elimination half life

889 Tmax time of maximal concentration

890 TSH thyroid stimulating hormone 891 ULN Upper limit of normal

892 VSS volume of distribution at steady state 893 894 VAS Bond-Lader visual analog scale 895 896 YMRS Young Mania Rating Scale

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 897 898 899 PART I: STUDY DESIGN AND CONDUCT 900 901 1. BACKGROUND AND RATIONALE 902 903 1.1 Background 904 Glutamate is the main excitatory neurotransmitter that mediates its effects via ionotropic 905 glutamate (iGlu) receptors (NMDA, AMPA and kainate receptors) and metabotropic 906 glutamate (mGlu) receptors (G-protein coupled receptors). There are currently eight 907 known mGlu receptors that are classified into three clusters based on sequence 908 homology, preferred signal transduction pathway, and pharmacology. Group I includes 909 mGlu1 and mGlu5 receptors, which are coupled via Gq to phospholipase C. Group II 910 includes mGlu2 and mGlu3 receptors, and Group III includes mGlu 4, 6, 7 and 911 8 receptors, all of which are coupled to Gi and inhibit cAMP formation. 912 913 The mGlu5 receptor has emerged as an attractive target for the treatment of anxiety and 914 depressive disorders based on its expression pattern in the brain and the efficacy of 915 mGluR5 antagonists in various animal models of these diseases. mGlu5 receptors are 916 highly expressed in limbic areas of the brain including the limbic cortex, the 917 hippocampus, the amygdala and the basal ganglia. These areas are known to be involved 918 in emotional and motivational processes and play a critical role in affective disorders 919 including anxiety and depression. A general review of the relevance of the glutamatergic 920 system in the treatment of mood disorders is provided by Sanacora et al [1]. 921 922 There is evidence from nonclinical and clinical studies to suggest that glutamatergic 923 dysfunctions may be involved in the pathophysiology of depression, and that 924 interventions aimed to modify the glutamatergic neurotransmission may possess 925 antidepressant effects. In fact, the prototypical mGluR5 antagonists MPEP and MTEP 926 have been shown to induce positive results in the forced swim test and tail suspension 927 tests animal models of depression. Additional supportive evidence has been originated 928 from olfactory bulbectomized rats (a different animal model) on which known chronic 929 (but not acute) anti-depressant treatment selectively reduce passive avoidance deficits and 930 hyperactivity, also reversed by MPEP and MTEP administration. Recent clinical data 931 indicate that ketamine, an NMDA channel blocking agent, has a fast acting (within hours) 932 antidepressant-like effect in treatment-resistant patients [2]. Given the co-localization of 933 NMDA receptors and mGlu5 receptors in the limbic regions, and that the downstream 934 effects of mGlu5 receptor blockade are related to down-regulation of NMDA function, 935 there is a strong rationale for antidepressant-like activity for mGlu5 receptor antagonists. 936 937 1.1.1 Disease 938 Major Depressive Disorder (MDD) remains an area of considerable medical need despite 939 the many agents approved for treatment of this illness. Response rates for initial treatment 940 are estimated to be about 50%, while remission, considered to be the goal of treatment, 941 ranges from 15 to 40% [3]. The recently published results of the STAR*D trial have 942 provided sobering conclusions regarding the conceptualization of remission and its 943 implications for defining recovery, relapse, and recurrence [4]. The study showed that 944 only one-third of patients achieved remission with initial treatment, and remission rates 945 declined even further with each successive treatment attempt (36.8% after first, 30.6%

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 946 947 after second, 13.7% after third and 13% after fourth treatment). Moreover, various 948 switching strategies employed as second-line treatment did not yield very different 949 results [5]. 950 951 Failure to obtain remission, therefore, is a common clinical problem. Patients who are 952 treatment resistant use a disproportionately larger share of health care resources, have 953 significantly more claims for comorbid conditions, and cost employers more in lost 954 productivity compared with patients with major depression who respond to treatment. 955 Given the disabling impact of persistent depression and its medical consequences, one 956 can argue that new treatments addressing inadequate response to antidepressant therapy 957 in MDD would fulfill an important medical need. 958 959 1.1.2 Study “Drug” 960 RO4917523 will be provided in capsules for this study. Further information is provided 961 in Section 6, Investigational Medicinal Product. 962 963 1.2 Non-Clinical Experience 964 965 1.2.1 Non-Clinical Pharmacology

966 In vitro, RO4917523 has been shown to be a potent mGlu5 receptor antagonist (Kd of 967 0.8 nM for human mGlu5 receptors). When RO4917523 was tested for activity on other 968 relevant targets (over 100), it exhibited over 1000-fold lower activity at all targets 969 including receptors, enzymes and ion channels. In animal models of anxiety, RO4917523 970 elicits dose-dependent anxiolytic effects in the Stress-Induced Hyperthermia (SIH) model 971 in mice, in the Vogel Conflict Test in rats, the Fear Potentiated Startle response in rats, 972 and in the Conditioned Emotional Response (CER) tests in rats. Furthermore, 973 RO4917523 was also active in animal models used to detect antidepressant potential such 974 as the forced swim test and the stress-induced anhedonia test. In sleep studies, 975 RO4917523 induced an increase in non-REM:REM ratio supportive of an 976 anti-depressant-like profile. RO4917523 did not potentiate the effects of ethanol in mice 977 and had no cognitive impairing activity in the delayed match to position test in rats. 978 However, RO4917523 impaired performance in the Morris water maze in two different 979 protocols at doses above 0.1 mg/kg p.o. at 4 h post-administration. 980 981 1.2.2 Non-Clinical Pharmacokinetics and Metabolism

982 The estimated (single dose) volume of distribution at steady state (Vss) of RO4917523 983 was generally high in animals, 4 and 5 L/kg in rats and monkeys, respectively. The total 984 plasma clearance (CL) was low to moderate, approximately 6 and 9 mL/min/kg in rat and 985 monkeys, respectively. The terminal elimination half-life (T1/2) values were between 7.5 986 to 10 h in rat and monkey. 987 988 RO4917523 was well absorbed after oral administration and had a high oral 989 bioavailability in rodents and cynomolgus monkey. In monkeys, the bioavailability was 990 higher in fasted animals. 991 992 Plasma protein binding is expected to be high in man. The in vitro bound fraction was 993 approximately 95 to 98 %, respectively, in animal and human plasma. RO4917523 994 showed a moderate partitioning to blood cells with an in vitro blood/plasma concentration

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 995 996 ratio of 1.3 in rat blood and penetrated well into brain tissue. The brain to plasma 997 concentration ratio in the rat ranged from 1.7 to 2.9. 998 999 The results of in vitro drug metabolism studies suggest that RO4917523 is predominantly 1000 metabolized to hydroxy-metabolites by cytochrome P450 enzymes (CYP 1A1, 1A2, 2C9, 1001 3A4, 3A5). CYP 3A isoforms were responsible for > 90% human liver microsomal 1002 metabolism. 1003 1004 Mass balance studies in rats with [14C]-RO4917523 indicate complete excretion within 1005 120 h of administration. 20% and 80% of total drug radioactivity was recovered in urine 1006 and feces, respectively. Quantitative whole body autoradiography revealed elevated 1007 concentrations of radioactivity in the inner cortex of the kidney, the liver, thyroid gland 1008 and nasal mucosa at 6 h. In cynomolgus monkeys, approximately 82% of the total 1009 radioactivity was recovered after 168 h postdose, excreted mainly in the feces. 1010 1011 1.2.3 Non –Clinical Safety and Toxicology 1012 In repeat-dose toxicity studies, the main targets for RO4917523-related systemic toxicity 1013 were the CNS, which is the pharmacological target, and as an adaptive response to drug 1014 load, the liver. Other organs (pituitary, thyroid, adrenals) were most probably secondarily 1015 affected. Chronic toxicity studies in rats and monkeys are currently ongoing. 1016 1017 Based on the available non-clinical safety information, the overall NOAEL for general 1018 toxicology effects is considered to be 4 mg/kg/day in adult and 10 mg/kg/day in juvenile 1019 rats, and 10 mg/kg/day in monkeys. In rat fertility studies the NOAEL for male mating 1020 performance was 1 mg/kg/day and for female fertility 0.3 mg/kg/day. 1021 1022 CNS-related effects were transiently observed in rats in general toxicology studies on the 1023 first or first few study days. They included drowsiness and hypoactivity. In CNS safety 1024 pharmacology studies in rats, the effects also included gait abnormalities, changes in 1025 body posture and body tone, and a transient decrease in body temperature, and after the 1026 occurrence of these early effects, slightly increased exploratory behavior from 2 to 1027 6 hours post dose. There was no obvious dose-effect relationship.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1028 1029 1030 The NOEL for the CNS-effects is ≤ 0.1 mg/kg in rats (maximal effect reached at 1031 0.5-1 mg/kg without any further increase at higher doses. In rats, because of their low 1032 degree, transient nature with fast recovery, and occurrence on the first day or first few 1033 days of dosing only, and in monkeys, because of their low degree and inconsistent 1034 presence at doses ≤30 mg/kg, and because of ease of monitoring in humans, these effects 1035 are not considered ‘adverse’ from a safety perspective, but rather signs of exaggerated 1036 pharmacological activity. 1037 1038 The mechanism of these CNS effects is not known, but it is assumed that at the lower 1039 doses they are secondary effects of the mGlu5-antagonism of RO491723. Other 1040 mGlu5-antagonists have been reported to decrease locomotor activity, impair Rotarod 1041 performance and decrease body temperature. At the higher dose levels also off-target 1042 mechanisms may be involved. 1043 1044 Minor hepatic findings (minimal to slight liver cell hypertrophy), most likely related to 1045 the high drug load, were consistently only seen in rats (NOAEL 4 mg/kg/day). In clinical 1046 chemistry the most prominent change in rats was a 12-fold increase in GGT at 1047 100 mg/kg/day and dose-related, slightly higher levels in bilirubin, glucose, ALT, ALP, 1048 total cholesterol, LDL, and HDL at 25 and 100 mg/kg/day in the 4-week study and a 1049 slightly higher total cholesterol level at 28 mg/kg/day in the 13-week study. 1050 1051 In rats (males predominantly affected) there was a dose-related increase in incidence of 1052 hypertrophy of TSH-producing cells in the pituitary and an increase in incidence and 1053 severity of thyroid follicular cell hypertrophy starting at 25 mg/kg/day. These effects are 1054 considered secondary to the liver cell hypertrophy, which lead to increased metabolism of 1055 the thyroid hormone T4, a well known rat specific effect without relevance for humans. 1056 In the 13-week study this hypothesis was supported by the observations of (1) slightly 1057 reduced serum T4 levels (but not T3 and TSH) in mainly males, (2) slightly increased 1058 T4-glucuronidation activity in liver microsomes, and (3) induction of gene expression of 1059 UDP-glucuronyltransferase 2 in the liver. 1060 1061 Other effects in rats were a slight increase in adrenal weight in females at 25 and 1062 100 mg/kg/day and an increased incidence and degree of diffuse vacuolization in the 1063 adrenal cortex of males at 100 mg/kg/day. 1064 1065 In rats there were also some minor urinary findings which were considered of equivocal 1066 relevance, because of absence of adverse morphological changes in the 4-week study, an 1067 equivocal slight exacerbation only of ‘normal’ slight tubular basophilia in the kidney of 1068 males at the highest dose in the 13 week study, and absence of up-regulation of any of the 1069 known biomarker genes for kidney toxicity (in particular KIM-1). 1070 1071 All the findings in the multiple dose toxicity studies had fully recovered within the 1072 4-week recovery period, with the exception of liver and adrenal weights in female rats, 1073 which were still slightly higher than in concurrent controls.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1074 1075 In an oral toxicity study in juvenile rats (highest dose 10 mg/kg/day = MTD) RO4917523 1076 given from post-natal day 7 to 91 induced a decrease in male body weight gain at 1077 ≥0.5 mg/kg/day and an increase in female food intake at ≥ 2.5 mg/kg/day, behavioral 1078 changes of quantitative components of the functional observational battery (decreased 1079 forelimb grip strength and hindlimb splay) at ≥0.5 mg/kg/day, an increase in motor 1080 activity at 10 mg/kg/day, and reduced performance in the learning of path B in the 1081 Cincinnati water maze test (a learning and memory test) at ≥2.5 mg/kg/day. These 1082 effects, all considered target pharmacology-related, showed partial or full regression 1083 during the 4-week recovery period or the post recovery observation period. Sexual 1084 maturation and reproductive function were not adversely affected. In view of the 1085 pharmacology-related effects seen at all doses the NOEL was considered 1086 <0.5 mg/kg/day, and because of absence of adverse effects the NOAEL 10 mg/kg/day. 1087 1088 There were no signs of a genotoxic potential for RO4917523, and there was no 1089 experimental evidence of a direct systemic effect on the immune system. 1090 1091 RO4917523 was phototoxic in vitro (cultured murine fibroblasts), but not in vivo 1092 (hairless rat model), and therefore the risk for inducing a phototoxic skin reaction in man 1093 is considered very low. 1094 1095 In safety pharmacology studies, RO4917523 increased respiration rate in rats 1096 (NOAEL 0.2 mg/kg), increased heart rate (moderate, short lasting) in telemetered 1097 monkeys (NOAEL 10 mg/kg), and decreased gastric emptying and intestinal transit at 1098 ≥0.1 mg/kg. 1099 1100 In two rat models (Conditioned Place Preference and Intracranial Self Stimulation) for 1101 assessing the reinforcing potential of RO4917523 (doses of up to 3 mg/kg p.o. or i.p., 1102 respectively) there were no reinforcing effects. 1103 1104 RO4917523 was not teratogenic in the studies on embryo-fetal development in rats and 1105 rabbits. 1106 1107 In fertility studies in rats RO4917523 reduced the mating and fertility indices. These 1108 effects were fully reversible after a treatment-free recovery period of 9 weeks in males 1109 and 4 weeks in females. There were no morphological changes in sexual organs from 1110 both genders. The effect on the mating index was male-mediated (NOEL = 1 mg/kg/day), 1111 while the effect on fertility was female-mediated (NOEL = 0.3 mg/kg/day). In a 1112 combined fertility study (e.g. males and females treated with RO4917523), sperm count 1113 was slightly reduced at 25 mg/kg/day, but sperm motility was normal, whereas in the 1114 4-week general toxicology study with doses of up to 100 mg/kg no effect on either sperm 1115 count or sperm motility was seen. 1116 1117 1.3 Previous Clinical Experience 1118 RO4917523 has been dosed to 233 healthy volunteers in seven clinical studies (a single 1119 ascending dose [SAD] study, a multiple ascending dose [MAD] study, an alcohol 1120 interaction study, three relative bioavailability studies and a drug-drug interaction study) 1121 and to 86 patients in two clinical studies (one study in treatment-resistant depression and 1122 one in Fragile X Syndrome).

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1123 1124 1.3.1 Safety 1125 1126 1.3.1.1 Single Ascending Dose (SAD) Study 1127 In the SAD study, single oral doses of 0.25 to 2 mg RO4917523 were administered. 1128 Single doses of 0.25 mg and 0.5 mg were well tolerated. Most of the adverse events 1129 occurred within 0.5 to 3 hours after administration and were mostly related to CNS 1130 (mainly dizziness and somnolence). The number of adverse events (AEs) increased with 1131 the dose, particularly at doses above 1 mg (1 AE at 0.25 mg to 29 AEs at 2 mg). The AEs 1132 included hypoesthesia/paresthesia, myoclonus, vasovagal malaise (one with, one without 1133 loss of consciousness), disturbance in attention and amnesia, gastro-intestinal disorders 1134 (nausea, dry mouth), eye disorders (blurred vision, mydriasis), and psychiatric disorders 1135 (euphoric mood). Most of the AEs were of mild to moderate intensity but 5 AEs were 1136 reported as of severe intensity at the doses of 1.5 and 2 mg (4 cases of dizziness, 1 case of 1137 syncope). The dose escalation was stopped at 2 mg due to the occurrence of adverse 1138 events of moderate to severe intensity in the majority of the subjects. 1139 1140 1.3.1.2 Multiple Ascending Dose (MAD) Study 1141 During the MAD study, fixed or up-titrated doses of 0.1 mg to 2 mg were administered 1142 b.i.d. in the fasted or fed state during 14 days (with the exception of the 0.1 mg b.i.d. 1143 cohort in fasted condition that was terminated at Day 8 due to the occurrence of an acute 1144 psychotic state, rated as an SAE, and an acute state of anxiety). The most frequently 1145 reported AEs affected the ‘central nervous system’, ‘gastro-intestinal system’, and 1146 ‘general disorders system’. Dizziness was reported with a higher frequency in the active 1147 drug groups (1 to 3 per group of 6 to 9 subjects) than in the placebo group (1 in 1148 15 subjects). Although the exposure levels in this study exceeded those reached in the 1149 SAD study, there were no reports of severe dizziness and no clear relationship emerged 1150 between the dose and the incidence or intensity of dizziness, suggesting development of 1151 tolerance upon repeated dosing. No dose-response relationship emerged either for 1152 headache or drowsiness. However, when the drug was given in the fed state, a dose 1153 response pattern was suggested for the following neuropsychiatric events: mood 1154 (dysphoria, euphoria), cognition (attention and memory, including the cognitive battery 1155 results), perception and ideation (hallucinations, paranoid ideation) and sleep (insomnia, 1156 nightmares, and hypnagogic hallucinations in up to 38% of the subjects); these events 1157 occurred at doses > 1 mg. Such a pattern of dose response was not observed in the fasted 1158 state (0.1 and 0.25 mg b.i.d.). All effects resolved within a few days after discontinuation. 1159 There were no reports of withdrawal syndrome as assessed by the Rickels withdrawal 1160 questionnaire applied from Day 14 until Day 24. In most subjects there were only some 1161 single occurrences of abnormalities in the laboratory tests without clinical significance, 1162 except one instance of markedly increased ALT whereas Alkaline phosphatase, GGT, 1163 total bilirubin and CPK levels were at all times normal leading to the subject’s 1164 withdrawal. There were no indication for trends in the vital signs and ECGs compared to 1165 placebo. 1166 1167 Interestingly, RO4917523 at 0.25 mg b.i.d was better tolerated when administered in the 1168 fed state compared to the fasted state, in particular with respect to psychiatric or CNS 1169 adverse events. This was possibly due to the lower exposure to the study drug, measured 1170 in this group compared to the corresponding fasted cohort. Indeed Cmax and AUC0-τ on

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1171 1172 Day 14 were respectively 1.8 and 1.6 fold higher in the subjects having received the drug 1173 in the fasted state. 1174 1175 1.3.1.3 Single Dose Alcohol Interaction Study 1176 During the Single Dose Alcohol Interaction study, most AEs occurred during the 1177 combined treatment with 0.5 mg RO4917523 and 0.5 g/kg alcohol (36 AEs in 19 1178 subjects) followed by treatment with 0.5 g/kg alcohol alone (30 AEs in 14 subjects) and 1179 then treatment with 0.5 mg RO4917523 alone (21 AEs in 12 subjects). CNS AEs were 1180 more commonly reported under alcohol alone (17), followed by the combined treatment 1181 (13) and then RO4917523 alone (7). Gastrointestinal disorders (dry mouth, nausea) were 1182 more frequently reported with the combination (8), followed by RO4917523 alone (5). 1183 The intensity of these AEs was mostly mild, 6 AEs were recorded as moderate (3 with 1184 combined treatment with RO4917523 and alcohol, 2 with RO4917523 alone), 1 with 1185 alcohol alone, and none were severe. The 3 AEs of moderate intensity after combined 1186 treatment with RO4917523 and alcohol were dizziness, nausea and vomiting. These AEs 1187 were resolved without sequelae within 5 minutes. The 3 other AEs of moderate intensity 1188 were gastroenteritis, joint dislocation (RO4917523 alone) and headache (alcohol alone) 1189 and were considered as unrelated to treatment. 1190 1191 1192 1193 1.3.1.6 Phase 2a Study in Treatment-Resistant Depression 1194 NP22022 was a phase 2a, adaptive fixed dose, randomized, double-blind, parallel-group, 1195 placebo-controlled study of the safety and therapeutic effects of RO4917523 in patients 1196 with treatment-resistant depression. A total of 46 patients received either placebo, 1197 RO4917523 0.1 mg, 0.5 mg, 1.0 mg, or 1.5 mg given once daily with food for 10 days. 1198 The unblinded safety data from this study reveal that the treatments were well tolerated. 1199 ‘Non-serious’ adverse events and changes in vital signs were not clinically significant, 1200 and any changes in laboratory parameters were either not clinically significant or were 1201 reversible. Two serious adverse events (suicidal ideation and a suicide attempt) occurred 1202 in one patient taking 0.5 mg/day, whose medical history contained similar events. 1203 1204 Based upon some neuropsychiatric events observed in Phase I studies, close attention was 1205 given to the reporting of all psychotomimetic adverse events in NP22022. Importantly, no 1206 clinically significant changes were reported in a subscale of the BPRS score (including 1207 the four items Conceptual Disorganization, Hallucinatory Behavior, Suspiciousness, 1208 Unusual Thought Content), confirming the absence of psychotic related events. In 1209 addition, no clinically significant changes were reported in the YMRS ‘elevated mood’ 1210 item score, confirming that events related to elevation of mood or euphoria have not been 1211 observed. Only insomnia, abnormal dreams and nightmares were reported as psychiatric 1212 adverse events, allowing us to conclude that the compound has been generally well 1213 tolerated in this population. 1214 1215 1.3.1.7 Phase 2a Study in Fragile X Syndrome 1216 NP22578 is an ongoing study in adult patients with Fragile X Syndrome. 40 patients have 1217 completed 42 days of treatment with RO4917523 in an outpatient setting (IND 103,917, 1218 S-000). The compound was overall well tolerated and safe. One case of auditory 1219 hallucinations and one case of catatonia were reported (in the 0.5 mg/day dose cohort); 1220 the symptoms were completely resolved after discontinuation without sequelae, and no 1221 other psychotomimetic adverse events were observed in the higher dose cohorts of 1.0

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1222 and 1.5 mg/day. 1223 1224 1.3.2 Pharmacokinetics 1225 RO4917523 exhibits linear and dose proportional pharmacokinetics after single 1226 administration within the dose range of 0.5-2 mg, in fasted condition. The linearity was 1227 confirmed in the MAD study where, on the first day, in the fed state, exposure after 1228 0.5 mg b.i.d. was approximately double to that after 0.25 mg b.i.d. When the immediate 1229 release formulation is administered maximum plasma concentrations are reached in 1230 approximately 1 h (fasted state) to 2-3 h (fed state), and thereafter the concentrations 1231 decline in a triphasic manner. The estimate of the terminal elimination half-life by non 1232 compartmental analysis is approximately 30-70 hours after single dose and 50-100 hours 1233 after reaching steady-state. The compartmental analysis used in the population PK

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1234 1235 analysis provides a terminal half-life value of 86 hours in woman and of 62 hours in man 1236 in healthy volunteers and depressed patients and an effective half-life value of 69 hours in 1237 woman and to 45 hours in man in the same population. 1238 1239 Administration with food decreases peak concentration (Cmax) and increases time to peak 1240 concentration (Tmax) compared to administration in the fasted state. Food had no 1241 significant effect on exposure (AUC) calculated after single dose, however higher 1242 exposure was noted after multiple b.i.d. administration in the fasted state as compared to 1243 fed state (1.7-fold higher AUC0-τ on Day 14). 1244 1245 Concomitant alcohol intake had no relevant effect on the exposure of RO4917523, and 1246 vice versa. 1247 1248 There is a gender effect on RO4917523 pharmacokinetics; in general exposure is higher 1249 in women. In the alcohol interaction study there was 2.4- and 1.4-fold higher AUClast and 1250 Cmax values respectively in females compared to males, following a single 0.5 mg 1251 RO4917523 dose. There was a trend for higher exposure and longer half-life in females 1252 than in males in the multiple dose study. In the relative bioavailability study comparing 1253 an optimized formulation of matrix tablet to the reference capsule formulation, AUClast 1254 was estimated as 3.45 times higher in a small sample of 6 females and 15 males. 1255 However, considering the entire population of female and male subjects, the population 1256 PK analysis estimates the gender effect as AUC female = 1.55 x AUC male. 1257 1258 The strong CYP3A4 inhibitor ketoconazole (single 200 mg dose) had no significant 1259 effect on the exposure (Cmax or AUC) to a single 0.15 or 0.25 mg dose of RO4917523. In 1260 a dedicated drug-drug interaction study ketoconazole 400 mg once daily was given four 1261 days prior to a single dose of 0.25 mg RO4917523 and during 14 subsequent days. The 1262 exposure ratio was 130% on AUC0-312h (90% CI = 122-139) and 102% on Cmax 1263 (90% CI = 94-111). It can be concluded that ketoconazole at steady-state and 1264 administered during a large part of RO4917523 elimination phase, had a modest 1265 inhibitory effect on RO4917523 clearance. 1266 1267 1.3.3 Pharmacodynamics 1268 The effects of RO4917523 on psychomotor and cognitive functions were assessed in 1269 healthy volunteers, using computerized psychometric tests batteries, Bond Lader visual 1270 analogue scale (VAS), of mood, alertness, and attention (computerized version), and 1271 ARCI-49 questionnaire. 1272 1273 • RO4917523 appeared to induce some impairment in the psychomotor and 1274 cognitive tests especially in the learning memory test (LMT), body sway, and tests 1275 of frontal/executive functions such as choice reaction time (CRT), rapid visual 1276 information processing (RVIP), and digit symbol substitution test (DSST). The 1277 degree was dose proportional and relatively modest at doses below 1 mg. 1278 • Impairment in cognitive performances noted after single administration of 0.5 mg 1279 were not present when this dose was administered repeatedly, indicating the 1280 development of tolerance to the effects of RO4917523.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1281 1282 1283 • In the MAD study, tests indicative of CNS mediated sedation, such as critical 1284 flicker fusion test (CFF) and saccadic eye movement (SEM) did not produce any 1285 statistically significant changes compared to placebo except for SEM at the 1286 highest tested dose (2 mg b.i.d.) only. 1287 • Overall, there was no potentiation of the effects when a 0.5 mg single dose was 1288 co-administered with alcohol (0.5g/kg). Additive effects were noted on some 1289 CNS parameters, principally assessing sedation and information processing. 1290 1291 There was no evidence of an analgesic effect in the intradermal capsaicin test performed 1292 in the MAD study. The low number of control placebo subjects limited the interpretation 1293 of the finding. 1294 1295 The effects of RO4917523 on postural stability were assessed in healthy volunteers using 1296 body sway tests. In the SAD study, the body sway test revealed an ataxic effect, which 1297 was only significant at doses higher than 1 mg and only when the tests were performed 1298 with the eyes closed, a condition which amplifies postural imbalance. There was no 1299 evidence of an effect of formulation (IR vs. MR) on postural stability. 1300 1301 1.4 Rationale for the Study and Overall Risk-Benefit 1302 Assessment 1303 Evidence of the potential benefit of RO4917523 in the treatment of depressive episodes 1304 has been substantiated based on: (1) the effect of the compound in preclinical models of 1305 depression, (2) the clinically reported antidepressant effect of several agents that 1306 modulate the glutamatergic neurotransmitter system (ketamine, riluzole, lamotrigine), 1307 and (3) the phenomenological analogy between the side effect profile observed during the 1308 phase I studies with RO4917523 and the side effect profile observed with ketamine, 1309 particularly on the psychotomimetic events of ideation and perception. The study design 1310 rationale can be found in the sections 3.1.1 and 3.1.2. Taking these factors in 1311 consideration, the safety of RO4917523 was recently investigated in a study of patients 1312 with treatment resistant depression (1.3.1.6) which showed that the medication was 1313 overall safe and well tolerated (see study NP22022 in Investigator’s Brochure for 1314 additional details). 1315 1316 As previously mentioned, there is a clear unmet medical need to provide more effective 1317 therapeutics to patients that have failed to respond to antidepressant treatments which are 1318 currently available. In fact, it has been shown that response and remission rates continue 1319 to decline with each successive antidepressant treatment in depressed patients [4]. Would 1320 it be acceptable to evaluate RO4917523 tolerability in the context of providing a possible 1321 resolution of symptoms that is often elusive with conventional therapeutics? Based on the 1322 accumulated clinical experience with RO4917523 (up to 1.5 mg/day in patients), Roche 1323 considers that the expected benefit for improving the efficacy of treatments in this 1324 population outweigh the potential risk associated with this experimental intervention, 1325 provided that sufficient safety monitoring is maintained during the study. That is to say, 1326 there is a positive benefit-risk assessment to studying the efficacy of RO4917523 in 1327 depressed outpatients who have failed to respond to ongoing antidepressant therapy.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1328 1329 2. OBJECTIVES 1330 1331 2.1 Primary Objective 1332 To evaluate the efficacy of two fixed doses of RO4917523 compared to placebo in a 1333 confirmatory manner over 6 weeks as adjunctive therapy in patients with MDD with 1334 inadequate response to ongoing antidepressant treatment, based on mean change in the 1335 Montgomery Asberg Depression Rating Scale (MADRS) total score from baseline to end 1336 of treatment. 1337 1338 2.2 Secondary Objectives 1339 The secondary objectives are to evaluate change after 6 weeks of treatment with 1340 RO4917523 versus placebo as adjunctive therapy on the following: 1341 1342 • Clinical Global Impression Scores: Severity (CGI-S) from baseline to end of 1343 treatment, and Improvement (CGI-I) at end of treatment 1344 • Safety and tolerability of RO4917523 1345 • Proportion of patients exhibiting remission (a MADRS score of less than or equal 1346 to 10) 1347 • Proportion of patients exhibiting response (reduction in MADRS score equal to or 1348 greater than 50% of the baseline score) 1349 • Quick Inventory of Depressive Symptomatology Self Report (QIDS-SR16) 1350 • Patient Global Impression of Improvement (PGI-I) score at end of treatment 1351 1352 1353 2.3 Exploratory Objectives 1354 1355 2.3.1 Key Exploratory Objective 1356 To evaluate the effect size (ES) of two fixed doses of RO4917523 compared to placebo 1357 over 6 weeks as adjunctive therapy in patients with MDD with inadequate response to 1358 ongoing antidepressant treatment, based on mean change in the Montgomery Asberg 1359 Depression Rating Scale (MADRS) total score from baseline to end of treatment. 1360 1361 2.3.2 Other Exploratory Objectives 1362 The other exploratory objectives include the investigation of the differential effect of 1363 RO4917523 vs. placebo in: 1364 1365 • The proportion of patients who meet MADRS responder criteria plus CGI-I score 1366 of 1) “very much improved” or 2) “much improved” 1367 • The proportion of patients who meet MADRS remission criteria plus CGI-I score 1368 of 1) “very much improved” or 2) “much improved” 1369 • The speed of onset of antidepressant effect based on change over time in any of 1370 the depression symptom scales 1371 • CANTAB Cognitive Test Battery subset 1372 • Quality of Life Enjoyment and Satisfaction Questionnaire-Short Form (Q-LES-Q- 1373 SF) 1374 • Sheehan Disability Scale (SDS)

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1375 1376 2.3.3 Pharmacokinetics 1377 • Pharmacokinetics of RO4917523 in the target population with the objective of 1378 performing a population pharmacokinetic analysis with non-linear mixed effect 1379 modeling. 1380 1381 1382 2.3.4 Roche Clinical Repository (RCR), Mandatory Biomarker Samples 1383 (MBS) and Clinical Genotyping (CG) 1384 Specimens collected for the Roche Clinical Repository (RCR), Mandatory Biomarker 1385 Samples (MBS) and Clinical Genotyping (CG) will be used to possibly: 1386 1387 • Identify biomarkers that are predictive of response to RO4917523 treatment 1388 • Increase our knowledge and understanding of the pathogenesis, course and 1389 outcome of depression and related diseases 1390 • Develop biomarker or diagnostic assays, and establish the performance 1391 characteristics of these assay 1392 1393 See sections 5.7, 5.8 and 5.9 for further information on RCR, MBS and CG sampling. 1394 1395 2.4 Roche Internal Criteria to Advance the Development of 1396 RO4917523 1397 For the purpose of internal decision making, Roche may consider to advance the 1398 development of RO4917523 if the observed ES is equal to or larger than 0.35 utilizing 1399 the LOCF method. 1400 1401 3. STUDY DESIGN 1402 1403 3.1 Overview of Study Design and Dosing Regimen 1404 This is a phase 2, randomized, double-blind, placebo-controlled, parallel-group, 1405 multi-center study that will evaluate two fixed doses of RO4917523 versus placebo as 1406 adjunctive treatment for six weeks in patients with major depressive disorder with 1407 inadequate response to ongoing antidepressant therapy. Patients must have had at least 1408 one but no more than three treatment failures (of adequate dose and duration according to 1409 the Massachusetts General Hospital Antidepressant Treatment History Questionnaire 1410 [MGH ATRQ]); failure to ongoing antidepressant treatment in the current episode is 1411 counted as one treatment failure. Further details are provided in section 4.2 Inclusion 1412 Criteria. 1413 1414 Following screening procedures to confirm eligibility, patients on ongoing antidepressant 1415 therapy will be randomized to one of the three treatment arms: a) RO4917523 0.5 mg, 1416 b) RO4917523 1.5 mg, or c) placebo, once daily. 1417 1418 The total duration of the study for each patient will be approximately 2 and a half 1419 months, divided as follows: 1420 1421 • Screening Period: up to 14 days 1422 • 6-Week Double-blind Treatment Period 1423 • Follow-up period: 21 days

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1424 1425 Figure 1 Study Flow 1426 1427 1428 1429 1430 1431 Ongoing Antidepressant Treatment (SSRI/SNRI) 1432 1433 1434 1435 1442 RO4917523RO4917523 0.5 mgmg 1436 1443 1437 1 to 3 1444

1438 Historical 1445 RO4917523 1.5 mg 1439 Failure1 to 3

1440 Historis cal RO4917523 1.5 mg 1446 1441 Failure 1447 PLACEBO 1448 1449 PLACEB 1450 O 1451 1452 1453 1454 1455 1459 1463 1456 1460 1464 1457 14 Day 1461 6 Week Double-blind 21 Day Follow-up 14651458 Screening 1462 Treatment 1466 1467 1468 1469 1470 3.1.1 Rationale for Study Design 1471 This study will include outpatients 18 to 70 years of age with a primary diagnosis of 1472 major depressive disorder (MDD) without psychotic features as defined by DSM-IV-TR 1473 criteria, and having inadequate response to ongoing antidepressant therapy. To establish a 1474 basal level of treatment resistance, the patients must have had at least one (of adequate 1475 dose and duration according to the MGH ATRQ) but no more than three treatment 1476 failures. 1477 1478 In order to enhance the effect of antidepressant treatments for patients with 1479 inadequate response, several clinical strategies are utilized. Note that these 1480 strategies may or may not be approved as safe and efficacious by health authorities 1481 and should be checked with local regulations before its utilization. They include 1482 among others, the addition of a new antidepressant from a different class or 1483 mechanism of action, the addition of new generation antipsychotics, potentiation 1484 with thyroid hormones, or potentiation with lithium. In this context, this study will 1485 help us to understand if RO4917523 added to the ongoing antidepressant 1486 medication (SSRI/SNRI only) is efficacious in the treatment of depressive symptoms 1487 and if it is safe and well tolerated. 1488 1489 As mentioned, the safety of RO4917523 has been recently investigated in a study of 1490 patients with treatment resistant depression (1.3.1.6) showing that the medication was 1491 overall safe and well tolerated (see study NP22022 in Investigator’s Brochure for details). 1492 As there is a clear unmet medical need to provide improved therapeutics to these patients, 1493 this study aims to investigate the effects RO4917523 in addition to the medication on 1494 which the patients have failed to adequately respond. This study will employ a placebo 1495 controlled design, in order to minimize the effect that the expectation of receiving an 1496 efficacious treatment may have in caregivers and patients alike. Moreover, the adjunctive 1497 efficacy of RO4917523 on SSRI or SNRI antidepressant treatment will also be CONFIDENTIAL Roche Protocol NP25620 C (RO4917523) - Page34

Downloaded From: https://jamanetwork.com/ on 09/29/2021 1498 1499 investigated with this design. It has been determined that patients who have failed to 1500 respond after six weeks of antidepressant treatment on SSRIs will achieve remission 1501 levels after twelve weeks with the sole continuation of the same antidepressant; in fact, 1502 up to 41% of the patients that were initially non-responders and 48% of those who were 1503 partial-responders at week six will achieve remission at week twelve [6]. 1504 1505 Experimental glutamatergic medications (NMDA antagonists) [2, 7] have been reported 1506 to produce a rapid antidepressant effect (e.g., during the first week of treatment) of 1507 adequate magnitude (comparable to the mean response reached after six weeks of 1508 treatment with available antidepressants) in a population of depressed patients who have 1509 previously failed to respond to adequate treatments. We are investigating how quickly 1510 and robust an antidepressant effect can be detected or observed once RO4917523 has 1511 been initiated within the 42 days timeframe of blinded treatment. This timeframe 1512 1) would allow a determination of the clinical relevance in the efficacy outcome 1513 measures, and 2) would also allow the continued characterization of the safety and 1514 tolerability profile of RO4917523 in this population. 1515 1516 A 21 day post treatment follow-up period would allow for the duration and persistence of 1517 the therapeutic effect to be tracked, as well as to monitor residual adverse events or newly 1518 emerging adverse events. This period exceeds five times the effective half-life of 1519 RO4917523 (76 hr in woman and 45 hr in man on average, respectively) and even five 1520 times the terminal half-life of RO4917523 (93 hr in woman and 63 hr in man on average, 1521 respectively) within which 97% of the compound is eliminated following discontinuation. 1522 1523 3.1.2 Rationale for Dose Selection 1524 The proposed doses for this study have been selected based on the safety and tolerability 1525 observations from the study NP22022 in treatment resistant depression and from the 1526 study NP22578 in Fragile X Syndrome, in which doses of RO4917523 ranging from 1527 0.1 mg/day to 1.5 mg/day were studied. 1528 1529 Close attention was given to the reporting of all psychotomimetic adverse events in the 1530 NP22022 study, in which 46 patients received either RO4917523 or placebo in an 1531 inpatient setting for 10 days. Very importantly, hallucinations, effects on mood 1532 (e.g., dysphoria or euphoria), and delusions (e.g., paranoid ideation), were not reported 1533 during the study. Only insomnia, abnormal dreams and nightmares were reported as 1534 psychiatric adverse events, allowing us to conclude that the compound has been generally 1535 well tolerated in this population. Furthermore, no cases of increased psychotic or manic 1536 symptoms were reported or detected. 1537 1538 In the NP22578 study in Fragile X Syndrome, 38 patients completed 42 days of treatment 1539 with RO4917523 in an outpatient setting. The compound has been overall well tolerated 1540 and safe. One case of auditory hallucinations and one case of catatonia were reported in 1541 the 0.5 mg/day dose arm; the symptoms were completely resolved without sequelae after 1542 discontinuation of the study medication.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1543 1544 Based on the aforementioned clinical experience, and provided that sufficient safety 1545 monitoring is maintained, the present study will investigate RO4917523 0.5 mg and 1546 1.5 mg once daily taken in the morning immediately after breakfast as the dosing 1547 schedule and regimen. 1548 1549 3.1.3 End of Study 1550 The end of the study will be considered to be the date of the last visit (including the last 1551 follow-up visit) of the last patient in the study. 1552 1553 3.2 Number of Patients/ Assignment to Treatment Groups 1554 Approximately 300 patients (100 per treatment arm) will be recruited. 1555 1556 3.3 Centers 1557 The study will be conducted in approximately 60 centers worldwide. 1558 1559 4. STUDY POPULATION 1560 Under no circumstances are patients who enroll in this study permitted to be 1561 re-randomized to this study and enrolled for a second course of treatment. 1562 1563 4.1 Overview 1564 The study will include male and female outpatients from 18 to 70 years of age with a 1565 primary diagnosis of major depressive disorder (MDD) without psychotic features as 1566 defined by DSM-IV-TR criteria, and having inadequate response to ongoing 1567 antidepressant therapy. Patients must have had at least one but no more than three 1568 treatment failures (of adequate dose and duration according to the MGH ATRQ). Failure 1569 to ongoing antidepressant treatment in the current episode is counted as one treatment 1570 failure. 1571 1572 4.2 Inclusion Criteria 1573 A patient may be included if the answer to all of the following statements is “yes” at 1574 screening, unless otherwise indicated. 1575 1576 General: 1577 1578 1. Male or female, legally adult (minimum 18) up to 70 years of age at time of informed 1579 consent 1580 2. Body mass index (BMI) between 18 and 38 kg/m2 inclusive 1581 3. Patients with reproductive potential must agree to use contraceptive protection from 1582 screening until 21 days after the last dose of study medication as follows: 1583 a. Males with partners of childbearing potential must use a barrier method of 1584 contraception (e.g. condom, diaphragm, or spermicide) or remain sexually 1585 abstinent. 1586 b. Non-lactating, non-pregnant females of child-bearing potential must choose one 1587 of the following contraceptive options: 1588 i. Any one of the following methods: an IUD which was implanted at least 2 1589 months prior to screening, a depot form of medroxyprogesterone, tubal 1590 ligation, or male partner surgical sterilization.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1591 1592 ii. Any two of the following methods: oral hormonal contraception, condom, 1593 diaphragm or cervical cap plus spermicide, or vaginal sponge. 1594 iii. Remain sexually abstinent. 1595 4. Able and willing to sign a written consent prior to any study related procedures 1596 1597 Neuropsychiatric: 1598 1599 5. A primary diagnosis of major depressive disorder (MDD) without psychotic features 1600 as defined by DSM-IV-TR criteria, on the basis of a structured interview (Mini 1601 International Neuropsychiatric Inventory [MINI]). The diagnosis of MDD will be 1602 confirmed via a central vendor specialized in MDD diagnosis (hereafter referred to as 1603 the central vendor) via a computer-administered diagnostic interview (DxV-MDD), 1604 when available1. 1605 6. Having inadequate response to an ongoing antidepressant treatment: 1606 a. Inadequate response defined as having at screening a rater-administered 1607 MADRS score of 25 or greater and a CGI-S score of 4 or greater (moderately 1608 ill or worse). When available 1, the computer-administered MADRS score 1609 must be 23 or greater. If the above conditions are met, there must also be no 1610 more than a 7 point difference between the rater and computer MADRS scores. 1611 b. Antidepressant treatment defined as pharmacotherapy with a selective 1612 serotonin reuptake inhibitor (SSRI) or serotonin and norepinephrine reuptake 1613 inhibitor (SNRI) of at least 6 weeks duration at the minimum acceptable dose 1614 per the MGH ATRQ. 1615 7. Dose and duration of ongoing antidepressant treatment must be verified during the 1616 screening period by written documentation which must include one of the following: 1617 a. Medical records 1618 b. Pharmacy records 1619 c. Referral form from treating physician indicating medication, dose, and dates 1620 of treatment 1621 8. Having at least one but no more than three antidepressant treatment failures within the 1622 current depressive episode. Treatment failure is defined as having inadequate 1623 response to an antidepressant treatment of at least 6 weeks duration at the minimum 1624 acceptable dose per the MGH ATRQ. The ongoing antidepressant therapy is counted 1625 as one treatment failure. 1626 9. Existing medication regimens for other medical conditions should be stable for 1627 6 weeks prior to Baseline, with the intent to remain stable throughout the study 1628 (see [4.4] Concomitant Medication below for restrictions). 1629 1630 1631 1632 1633 1634 1635 1636 1637 1638 1639 1640 1641 1642 1 And deemed appropriate according to local medical practices.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1643 1644 1645 4.3 Exclusion Criteria 1646 A patient will be excluded if the answer to any of the following statements is “yes” at 1647 screening, unless otherwise indicated. 1648 1649 Treatment history: 1650 1651 1. Currently receiving treatment with a combination of antidepressants (two or more), or 1652 on adjunctive or potentiating treatment including antipsychotic agents (typical or 1653 atypical), mood stabilizers (anticonvulsants), lithium, triiodothyronine, or stimulants. 1654 2. Previously received RO4917523 1655 3. Enrollment/participation in a clinical trial or intake of an investigational drug within 1656 90 days of screening or 5 times the half life of the investigational drug (whichever is 1657 longer) 1658 4. History of failure, or utilization during the current episode of Electroconvulsive 1659 Therapy (ECT) or repetitive Transcranial Magnetic Stimulation (rTMS) 1660 5. History of use at any time of Vagus Nerve Stimulation (VNS) or Deep Brain 1661 Stimulation (DBS) 1662 6. Planning to begin individual or group psychotherapy during the study. Patients 1663 undergoing regular psychotherapy (i.e., at least 90 days duration at the time of 1664 Screening) are eligible to participate in the study 1665 1666 Diagnosis and psychiatric history: 1667 1668 7. Other current DSM-IV-TR axis I diagnosis. Comorbid anxiety which dominates the 1669 clinical presentation or troubles the patient the most is not accepted: Obsessive- 1670 Compulsive Disorder (OCD) and Posttraumatic Stress Disorder (PTSD) are 1671 specifically not allowed under any circumstances. 1672 8. Current or past history of psychotic symptoms. 1673 9. Current or past history of Bipolar Disorder (e.g., manic, hypomanic, or mixed 1674 episodes). 1675 10. Mood disorder due to medical condition or substance use/abuse/dependence. 1676 11. Personality disorder which might interfere with compliance or increase suicidal risk. 1677 12. Alcohol and/or substance abuse/dependence during the last 6 months. Additionally, 1678 patients should be advised not to consume alcohol during the duration of the trial. 1679 13. Significant risk of suicide or suicidal behavior, including a history of previous 1680 suicide attempts during the current episode, or to develop such a risk during the study. 1681 1682 Past and current other medical history: 1683 1684 14. Other significant or unstable medical condition that could interfere with, or for which 1685 the treatment of might interfere with the conduct of the study, or that would, in the 1686 opinion of the investigator, pose an unacceptable risk to the patient in this study, 1687 including among others: 1688 a. Significant or unstable neurological disorder 1689 b. Unstable or uncontrolled hypertension

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1690 1691 c. Hypomagnesemia 1692 d. Abnormal thyroid function. Note that patients under treatment may be allowed 1693 to participate in the study if currently euthyroid and have not had a change in 1694 treatment regime within the last 8 weeks 1695 15. Serology positive for HIV, Hepatitis B or C 1696 16. Clinically significant abnormality on 12-lead electrocardiogram (ECG) at Screening, 1697 including a QTcF equal to or greater than 450 milliseconds 1698 17. Clinically significant laboratory abnormality (note that re-testing is allowed to rule 1699 out potential laboratory errors) 1700 18. For females, pregnancy test positive at screening, breast feeding, or intend to become 1701 pregnant during the course of the trial 1702 19. Positive result for urine drugs of abuse test at screening 1703 20. Hypersensitivity to the excipients of the study drug 1704 1705 Other: 1706 1707 21. Individuals whose occupation is to drive or operate mass transportation (i.e., buses, 1708 trains), large vehicles (i.e., trucks), or heavy machinery. 1709 1710 1711 4.4 Allowed and Prohibited Medication 1712 1713 4.4.1 Ongoing Antidepressant Treatment 1714 Ongoing treatment with a single antidepressant of the SSRI or SNRI class (see inclusion 1715 criteria number 7b and 8) must be continued for the duration of the double-blind 1716 treatment period without modification of the dosing schedule. 1717 1718 4.4.2 Allowed Medications 1719 • Medications used for the treatment of stable medical conditions other than 1720 depression. Existing medication regimens (which may include benzodiazepines) 1721 should be stable for 6 weeks prior to baseline, and with the intent to remain stable 1722 throughout the study. 1723 • Anticonvulsants being utilized for the treatment of epilepsy only of non- 1724 glutamatergic or non-GABAergic mechanism of action (e.g., phenytoin, valproic 1725 acid, carbamazepine). 1726 • For acute anxiety and/or insomnia, a benzodiazepine (up to 2 mg/day lorazepam, 1727 or equivalent), or a non-benzodiazepine hypnotic (immediate release zolpidem up 1728 to 10 mg, controlled release zolpidem up to 12.5 mg, or zaleplon up to 10 mg, at 1729 bedtime) can be utilized. Use of any one of these agents should be prescribed on 1730 an as needed basis only, and limited to no more than three days per week, and not 1731 within 8 hours of any visit. Use of alternative agents may be allowed after 1732 consultation with the Sponsor/Medical Monitor.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1733 1734 1735 4.4.3 Prohibited Medications 1736 All other psychotropic medications (with the exception of those allowed in 4.4.1) are 1737 prohibited starting two weeks before randomization (or a period equivalent to 5 half lives 1738 for that medication, which ever is longer). These include: 1739 1740 • Fluvoxamine 1741 • Non-SSRI or non-SNRI antidepressants (including St. John’s Wort) 1742 • Antipsychotics 1743 • Psychostimulants 1744 • Lithium 1745 • Thyroid hormone used for antidepressant potentiation 1746 • Opioid analgesics 1747 • Anticonvulsant medications utilized as mood stabilizers, adjunctive, or 1748 potentiating antidepressant treatment 1749 • GABAergic drugs including: tiagabine, vigabatrin, baclofen, pregabalin and 1750 gabapentin 1751 • Glutamatergic drugs including: riluzole, topiramate, memantine, lamotrigine 1752 • Herbal supplements 1753 • Omega-3 fatty acids (i.e. fish oil and flaxseed oil) 1754 • Magnesium supplements 1755 • Dextromethorphan 1756 • All other psychotropic drugs 1757 1758 1759 4.5 Criteria for Premature Withdrawal 1760 Patients have the right to withdraw from the study at any time for any reason. 1761 1762 In the case that the patient decides to prematurely discontinue study treatment [“refuses 1763 treatment”], he/she should be asked if he/she can still be contacted for further 1764 information. The outcome of that discussion should be documented in both the medical 1765 records and in the CRF. If lost to follow-up, the investigator should contact the patient 1766 or a responsible relative by telephone followed by registered mail or through a personal 1767 visit to establish as completely as possible the reason for the withdrawal. A complete 1768 final evaluation at the time of the patient’s withdrawal should be made with an 1769 explanation of why the patient is withdrawing from the study. 1770 1771 When applicable, patients should be informed of circumstances under which their 1772 participation may be terminated by the investigator without the patient’s consent. The 1773 investigator may withdraw patients from the study in the event of intercurrent illness, 1774 adverse events, treatment failure after a prescribed procedure, lack of compliance with 1775 the study and/or study procedures (e.g., dosing instructions, study visits), or any reason 1776 where it is felt by the investigator that it is in the best interest of the patient to be 1777 terminated from the study. In particular, the investigator should consider 1778 withdrawing a patient due to ‘lack of response with persistence of unpleasant 1779 symptoms’ if such symptoms are deemed unsafe or intolerable by the investigator or 1780 the patient. Any administrative or other reasons for withdrawal must be documented and 1781 explained to the patient.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1782 1783 The investigator may also withdraw a patient in case of evidence of suicidal ideation, 1784 behavior or risk revealed by the Columbia-Suicide Severity Rating Scale (C-SSRS) at 1785 any time during the study. A confirmed positive finding on the C-SSRS, as defined in 1786 section 5.5.4.3 (e.g., a “yes” to question 4, indicating suicidal ideation with intention 1787 to act), will warrant discontinuation of the patient from the study. The reasons for 1788 withdrawal must be documented and explained to the patient. 1789 1790 In addition, as an additional safeguard, according to the FDA’s “Guidance for 1791 Industry – Drug-Induced Liver Injury: Premarketing Clinical Evaluation” (June 1792 2009), discontinuation of treatment should be considered if patients exceed the 1793 following liver function test threshold values: 1794 1795 o ALT or AST >8xULN 1796 o ALT or AST >5xULN for more than 2 weeks 1797 o ALT or AST >3xULN and (Serum Total Bilirubin >2xULN or INR 1798 >1.5)*. 1799 o ALT or AST >3xULN with the appearance of fatigue, nausea, vomiting, 1800 right upper quadrant pain or tenderness, fever, rash, and/or eosinophilia 1801 (>5%)*. 1802 1803 *Note that if ALT or AST >3xULN, the investigator should perform INR testing via local lab to 1804 ensure that the threshold condition is comprehensively assessed. 1805 1806 Whether the decision to discontinue is made by the patient or by the investigator, an 1807 accurate and detailed description of the reason for discontinuation should be captured in 1808 the eCRF. If the reason for removal of a patient from the study is an Adverse Event, the 1809 principal specific event will be recorded on the eCRF. The patient should be followed 1810 until the Adverse Event has resolved, if possible. 1811 1812 An excessive rate of withdrawals can render the study non-interpretable; therefore, 1813 unnecessary withdrawal of patients should be avoided. Should a patient decide to 1814 withdraw, all efforts will be made to complete and report the observations prior to 1815 withdrawal as thoroughly as possible. If a patient withdraws from the study early, an 1816 attempt should be made to complete all study assessments listed under End-of-treatment 1817 in the Schedule of Assessments Section 5, on the last day of study medication dosing or 1818 as soon as feasible after that. 1819 1820 4.5.1 Withdrawal Of Patients From the Roche Clinical Repository (RCR) 1821 Patients who gave consent to provide RCR specimens have the right to withdraw their 1822 specimen from the RCR at any time for any reason. If a patient wishes to withdraw 1823 his/her consent to the testing of his/her specimen(s), the investigator must inform the 1824 Roche monitor in writing of the patient’s wishes using the RCR Subject Withdrawal 1825 Form and enter the date of withdrawal in the patient’s Case Report Form (CRF). A 1826 patient withdrawal from the main trial does not, by itself, constitute withdrawal of the 1827 specimen from the RCR, likewise a patient withdrawal from the RCR does not constitute 1828 a withdrawal from the main trial.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1829 1830 4.6 Replacement Policy [Ensuring Adequate Numbers of 1831 Evaluable Patients] 1832 1833 4.6.1 For Patients 1834 Patients discontinued from the study after randomization for any reason will not be 1835 replaced. 1836 1837 4.6.2 For Centers 1838 A center may be replaced for the following administrative reasons: 1839 1840 – Excessively slow recruitment 1841 1842 – Poor protocol adherence 1843 1844 – Non-compliance with GCP or any significant audit findings 1845 1846 – Administrative decision made by the sponsor. 1847 1848 New centers may also be added during the course of the study. 1849 1850 5. SCHEDULE OF ASSESSMENTS AND PROCEDURES 1851 1852 Table 1 Schedule of Assessments 1853 1 7 Visit Name/Study Screening Day 1 Day 3 Day 7 Day 11 Day 14 Day 21 Day 28 Day 35 Day 42 Day 63 Week (baseline) (Wk 1) telephone (Wk 2) (Wk 3) (Wk 4) (Wk 5) (End-of- (Follow- call only treatment) up) Visit Window (days) < 14 days N/A +1 day +/- 1 +/-1 +/- 2 +/- 2 +/- 2 +/- 2 +/- 2 21+/- 3 prior to days after baseline ‘end of treatment’ Informed Consent X Inclusion/Exclusion X criteria ATRQ X 2 DxV-MDD X Medical and psychiatric X history (incl. MINI) 3 Physical Examination X X 4,5 12 lead ECG X X X Weight X X Vital Signs4 (BP, HR, X X X X X X X X X X Temp) Blood Chemistry X X X X Hematology X X X X Urinalysis/menstrual X X X X status Urine drug screen X Virology X Pregnancy Test X X X X PK Samples X9,10 X9 X9 X9,10 X11 Optional RCR X sampling- inherited Optional RCR X X sampling- non inherited Mandatory biomarker X X sampling

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1854 1855 1 7 Visit Name/Study Screening Day 1 Day 3 Day 7 Day 11 Day 14 Day 21 Day 28 Day 35 Day 42 Day 63 Week (baseline) (Wk 1) telephone (Wk 2) (Wk 3) (Wk 4) (Wk 5) (End-of- (Follow- call only treatment) up) Visit Window (days) < 14 days N/A +1 day +/- 1 +/-1 +/- 2 +/- 2 +/- 2 +/- 2 +/- 2 21+/- 3 prior to days after baseline ‘end of treatment’ Clinical Genotyping X sampling CANTAB X12 X X 6 MADRS X X X X X X X X X X CGI-S X X X X X X X X X X CGI-I X X X X X X X X PGI-I X X X X X X X X 8 C-SSRS X X X X X X X X X X QIDS-SR16 X X X X X X X X X X Q-LES-Q-SF X X X SDS X X X Prior and Concomitant X X X X X X X X X X Treatments 13 Adverse Events X X X X X X X X X X X BPRS (sub-scale) Completed ONLY as required to follow-up on AEs as described in section 5.5.4 YMRS (item 1) Completed ONLY as required to follow-up on AEs as described in section 5.5.4 Drug Dispensation and X X X X X X X X Return 1856 1). All baseline assessments must be conducted within 24 hours and prior to administration of the first 1857 dose of study medication. 1858 2). Patients will complete the DxV-MDD computer-administered diagnostic interview prior to the MINI, 1859 when available. 1860 3). A complete physical examination will be performed at screening and week 6 (end-of-treatment). 1861 Height will be collected only at screening. 1862 4). 30 minutes should elapse between the last venipuncture and the measurement of vital signs and ECG. 1863 ECG is conducted after remaining in the semi supine position for at least 10 minutes. 1864 5). All ECGs will consist of three consecutive interpretable ECG recordings (as close as possible, and not 1865 more than 2 minutes apart). 1866 6). In addition to the rater-administered MADRS, the MADRS assessment will also be completed by the 1867 patient via a computer supplied by a central vendor, when available. The central vendor will be 1868 blinded to study treatment. The rater’s MADRS score will be entered onto the central vendor’s 1869 computer system. 1870 7). If double-blind treatment is discontinued early, the ‘end-of-treatment visit’ should be performed as 1871 soon as possible (“early discontinuation visit”). One PK sample will be taken at any time during the 1872 “early discontinuation visit.” A ‘follow-up visit’ should be conducted 21 days afterwards and no PK 1873 sample taken. 1874 8). The ‘C-SSRS baseline’ will be administered at screening. The ‘C-SSRS since last visit’ will be 1875 administered at subsequent visits (including the baseline). 1876 9). At these visits, PK sampling performed at 0 (pre-dose) and 4 hours (according to patient availability). 1877 10). At these visits, an additional PK sample performed at 6 hours (according to patient availability).

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1878 1879 Table 1 Schedule of Assessments (Cont.) 1880 1881 11). PK sample drawn anytime. 1882 12). The CANTAB at screening is administered to patients with confirmed eligibility (pending only 1883 laboratory results). 1884 13). After informed consent, but prior to initiation of study medication (i.e. during screening), only SAEs 1885 caused by a protocol-mandated intervention will be collected (e.g., SAEs related to invasive 1886 procedures). 1887 1888 5.1 Screening Period 1889 1890 5.1.1 Informed Consent Form (ICF) 1891 All patients must sign and date the most current IRB/IEC-approved written informed 1892 consent before any study specific assessments or procedures are performed. 1893 Documentation of informed consent must be kept in the patients’ study medical records. 1894 The original informed consent document will be retained by the investigator. 1895 1896 5.1.2 Screening 1897 The study center will access an Interactive Voice Response System/Interactive Web 1898 Response System (IVRS) to register the patient into screening. Screening procedures 1899 should be performed within a period of time not exceeding 14 days (see required 1900 assessments in Table 1). The screening period may be extended by the PI in consultation 1901 with the Sponsor/Medical Monitor. 1902 1903 Patients who fail to meet eligibility criteria may be screened again at a later date after 1904 consultation with the Sponsor/Medical Monitor. 1905 1906 Once all inclusion and exclusion criteria are met, an Eligibility Screening 1907 Form/Eligibility Assessment Form (ESF/EAF) is completed by the investigator and sent 1908 to the Sponsor/Medical Monitor for approval of the patient to participate in the study. 1909 The Sponsor/Medical Monitor will confirm the patient’s eligibility in IVRS and notify 1910 the site. 1911 1912 A screen failure log must be maintained by the investigator. 1913 1914 5.2 Procedures for Randomization of Eligible Patients 1915 In order to be enrolled into the double-blind treatment period, patients must have: 1916 1917 – No significant risk of suicidal behavior (e.g., consider the Suicidal Ideation section of 1918 the C-SSRS “Since Last Visit” for this evaluation) 1919 1920 – No significant change in medical or psychiatric condition, or change in medications 1921 since screening (as agreed with the Sponsor/Medical Monitor when appropriate) 1922 1923 – Negative result on the Baseline pregnancy test (if applicable) 1924 1925 – No change in ongoing antidepressant therapy, and the ability to continue for the 1926 duration of the double-blind treatment period without modification to the dosing 1927 schedule 1928 1929 – An EAF approved by the Sponsor/Medical Monitor

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1930 1931 The Double-blind Treatment Period begins with the investigational site call into IVRS 1932 confirming the patient’s eligibility as per the ESF/EAF. The patient will be randomized 1933 and receive their first dose of study medication on Day 1. 1934 1935 The patient randomization numbers will be generated by Roche or its designee. The 1936 treatment allocation will be stratified by geographical region/country, as follows: 1937 Europe, United States, Latin America, Asia (excluding Japan) and Japan. 1938 1939 The patient randomization numbers are to be allocated sequentially in the order in which 1940 the patients are enrolled according to the specification document agreed with the IVRS 1941 vendor. A patient Enrollment and Identification Code List must be maintained by the 1942 investigator. 1943 1944 5.3 Double-blind Treatment Period 1945 Dose 1 of the blinded study medication is to be administered in the clinic immediately 1946 after a meal and before 12 pm (noon), or soon thereafter upon consultation with the 1947 Sponsor/Medical Monitor, once all baseline procedures and assessments (Table 1) are 1948 completed. Patients will remain at the clinic for 6 hours after the first dose for safety 1949 monitoring and for the PK samples (according to clinical observation and patient 1950 availability). Subsequently, dosing will be once daily in the morning immediately after 1951 breakfast. 1952 1953 As with any experimental drug at this stage of development, it is advisable for patients 1954 not to drive or operate dangerous machinery until known side effects (e.g., dizziness and 1955 somnolence) can be adequately assessed on an individual basis during the trial. 1956 1957 At Day 11, telephone contact with the patient by a clinician is required to monitor 1958 for the emergence of any clinically significant adverse events. 1959 1960 Patients will arrive at each study visit without having taken their daily dose of study 1961 medication, and site staff will record the time of their last dose. Following collection of 1962 the pre-dose PK blood sample (if applicable) and a meal, patients will take their next dose 1963 of study medication before 12 pm (noon) or soon thereafter upon consultation with the 1964 Sponsor/Medical Monitor. The last dose of study medication will be administered on Day 1965 42 (End-of-Treatment visit). 1966 1967 5.4 Follow-up Period 1968 A single Follow-up visit will be performed 21 days after the End-of-Treatment visit 1969 (see Table 1). During the follow-up period, adjustments to antidepressant treatments may 1970 be initiated if deemed necessary by the investigator. 1971 1972 5.5 Clinical Assessments and Procedures 1973 1974 5.5.1 Rater Selection and Training 1975 To be considered as a rater in this study, a member of the site staff must be a physician, a 1976 nurse with certifiable psychiatric practice, or a clinical psychologist. Qualified nurses and 1977 clinical psychologists must have a minimum of two years experience administering 1978 standardized rating scales. (participation of raters with different qualifications should be 1979 agreed upon with the Sponsor/Medical Monitor on a case by case basis).

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 1980 1981 Raters may be trained and certified on the clinician-administered scales and a quality 1982 monitoring program may be implemented to ensure rating consistency during the trial. 1983 1984 5.5.2 Assessments for Eligibility 1985 1986 5.5.2.1 Detailed psychiatric history 1987 A detailed psychiatric history will be taken during the screening period. This will include 1988 documentation of previous treatment for major depression and medication regimens and 1989 changes. Previous psychiatric hospitalizations and out-patient treatment should also be 1990 noted. 1991 1992 5.5.2.2 Massachusetts General Hospital Antidepressant Treatment 1993 History Questionnaire (MGH ATRQ) 1994 The MGH ATRQ is a patient-completed instrument used to determine treatment 1995 resistance in major depressive disorder [8, 9]. A clinician may assist the patient to 1996 complete the instrument. The MGH ATRQ defines 6 weeks on a minimally acceptable 1997 dose of antidepressant medication as an adequate treatment duration. As noted in 1998 Inclusion criterion number 7, information regarding the ongoing antidepressant treatment 1999 must be verified by way of written documentation 2000 2001 5.5.2.3 Medical history and Physical examination 2002 The patient’s medical history will be taken during the screening period. A complete 2003 physical exam will be performed at screening and Week 6. The patient’s height will be 2004 recorded at screening only. 2005 2006 For pre-menopausal females, menstrual status will be recorded at each visit when a urine 2007 sample is collected. 2008 2009 5.5.2.4 Computer-Administered Diagnostic Interview 2010 Patients will be instructed to complete a computer-administered diagnostic interview 2011 (DxV-MDD) at the screening visit, when available. The DxV-MDD will collect data 2012 about the subjects’ history relative to lifetime history of MDD. Each site will be 2013 provided with a laptop computer that will record the subject’s responses to the 2014 computerized questionnaire. Each site will receive training on the use of the laptop and 2015 perform a secure data transmission test with the central vendor. 2016 2017 After the screening visit, the site will transfer the information collected by the laptop 2018 through a secure, encrypted sFTP connection. The subject’s responses will be evaluated 2019 by independent clinical experts at the central vendor. The details of the subject’s history 2020 will be evaluated by an independent MDD expert based on the responses to the 2021 computerized interview. To establish confidence in the diagnosis, any diagnostic 2022 uncertainty raised by the patient’s responses to the diagnostic interview must be resolved 2023 by the Investigator, in collaboration with a central vendor clinician. Patients for whom 2024 diagnostic agreement between the Investigator and the central vendor cannot be reached 2025 may not be considered appropriate for study participation2. 2026 2027 2028 2029 2030 2 If deemed appropriate according to local medical practices.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2031 2032 5.5.2.5 Mini International Neuropsychiatric Inventory (MINI) 2033 In order to verify a diagnosis of major depressive disorder (MDD) without psychotic 2034 features for inclusion in this study (and other related inclusion criteria), the patient will be 2035 evaluated by use of the MINI [10] during the screening period. The MINI is a short, 2036 structured diagnostic interview designed to provide psychiatric diagnoses according to 2037 the DSM-IV and ICD-10 criteria. Thus it was intended to serve as a tool that could be 2038 applied internationally in such settings as multicenter clinical trials. 2039 2040 5.5.3 Efficacy 2041 The collection of efficacy assessments is detailed in the Schedule of Assessments 2042 [Table 1]. 2043 2044 5.5.3.1 Montgomery Asberg Depression Rating Scale (MADRS) 2045 The MADRS [11] was designed to assess the overall severity of depressive symptoms in 2046 patients with major depressive disorder. The scale is also intended to be sensitive to 2047 treatment effects and to discriminate between medication versus placebo. The MADRS is 2048 a 10-item instrument. Items are rated on a scale of 0 to 6 with anchors at 2-point 2049 intervals; hence the total score ranges from 0 to 60. To improve the quality and 2050 consistency of the MADRS ratings across study centers, the MADRS-SIGMA (an 2051 interview guide which provides structured probes) will be utilized [12]. See Appendix 2052 [Appendix 3] for details. 2053 2054 This study will utilize a rater quality control system conducted by a central vendor, 2055 blinded to study treatments. The rater quality control system allows the study team to 2056 monitor the primary outcome measure at treatment phase assessments in the study and 2057 give ongoing feedback and remediation to the rater in the study, when necessary. 2058 2059 Each study site will be provided with a laptop computer containing the quality control 2060 system software, receive training on use of the laptop, and perform a secure server 2061 connection test with the central vendor. The rater will administer the MADRS to the 2062 subject per protocol procedures. The rater’s MADRS scores will be entered onto the 2063 laptop computer. Afterwards, the subject will complete an interactive interview on the 2064 computer (i.e., the computer-administered MADRS). The interview involves a series of 2065 probe and follow up questions with multiple choice response options (mean time for 2066 completion is < 10 minutes). Subjects will not be required to type any responses. 2067 2068 The subject responds to computer prompts presented. The laptop is not connected to the 2069 Internet during the interactive interview. The quality control system software restricts the 2070 user’s access to only the subject-user interface. The quality control system software 2071 places an unalterable time and date on each report which cannot be modified (meets 2072 requirement 21 CFR Part 11). The study assigned subject ID number is the only 2073 identifying information that is stored into the laptop computer and transmitted to the 2074 vendor; therefore, the vendor will be blinded to the study treatment. 2075 2076 After the subject’s visit, the site will transfer the information collected by the laptop 2077 through a secure, encrypted FTP connection (meets requirement 21 CFR Part 11). The 2078 transfer will be acknowledged to the site on the laptop screen. The information is stored 2079 on a password protected server that restricts access to registered users or IP addresses.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2080 2081 After the transfer, the information is analyzed by the vendor’s quality control system 2082 software. The vendor examines the subject’s interview responses and quality control 2083 system software ratings in parallel with the rater’s outcome measure scores and compares 2084 the two for consistency. If an individual report falls outside the study thresholds, the 2085 central vendor may schedule a structured review with the site rater to determine probable 2086 sources of the discordance. If a review is necessary, the central vendor will contact the 2087 site within five business days. The rater’s assigned scores are not altered by the review or 2088 any exchange with the central vendor. The central vendor will apply the same 2089 methodology for review at each study visit. 2090 2091 The rater quality control system maintains an ongoing performance profile for each rater. 2092 Quality issues are evaluated and corrected continuously throughout the study. Reports 2093 are updated continuously and available to the study team. 2094 2095 5.5.3.2 Clinical Global Impression- Severity (CGI-S) and Improvement 2096 (CGI-I) 2097 The CGI rating scales are tools used to evaluate both the severity of illness and change 2098 from baseline [13]. The CGI-S reflects the clinician’s impression of the patient’s current 2099 illness state on a 7-point scale ranging from no symptoms (1) to very severe (7). The 2100 CGI-I is used to assess the clinical change as compared to symptoms at baseline using a 2101 7-point scale, ranging from very much improved (1) to very much worse (7). In this 2102 study, overall clinical status will be evaluated with the CGI-S and CGI-I. See Appendix 4 2103 for details. 2104 2105 5.5.3.3 Patient Global Impression of Improvement (PGI-I) 2106 The PGI-I is a self reported instrument to record the patient’s own assessment of 2107 improvement since baseline. The patient is asked to rate their condition as compared to 2108 before they began study medication using a 7-point scale, ranging from very much better 2109 (1) to very much worse (7). The PGI-I is essentially a patient-reported version of the 2110 CGI-I. See Appendix 5 for details. 2111 2112 5.5.3.4 Cambridge Neuropsychological Test Automated Battery 2113 (CANTAB®) 2114 Cognitive performance will be assessed using the CANTAB® computerized battery 2115 provided by Cambridge Cognition™. The main cognitive domains known to be impaired 2116 in depression include memory, attention, executive function and speed of processing. A 2117 subset of the CANTAB® tests will be used to assess function in these domains: Motor 2118 Control Task (MOT), Rapid Visual Processing (RVP), Delayed Match to Sample (DMS), 2119 Emotional Recognition Task (ERT), -10 min break-, Paired Associates Learning (PAL), 2120 the Stockings of Cambridge (SOC, at screening) or the One-Touch Stockings of 2121 Cambridge (OTS, at subsequent visits), and Attention Shifting Test (AST). 2122 2123 During the screening period, the CANTAB battery will be administered to patients with 2124 confirmed eligibility (pending only laboratory results).

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2125 2126 5.5.3.5 Quick Inventory of Depressive Symptomatology Self Report- 16 2127 item version (QIDS-SR16) 2128 The QIDS-SR16 [14, 15] was designed to assess the severity of depressive symptoms in a 2129 self-rated format. The scale assesses all of the symptom domains selected by DSM-IV to 2130 diagnose a major depressive episode. Each of the 16 items is scored on a 4-point 2131 anchored scale, representing least severe (0) to most severe (3). Specific instructions for 2132 calculating a total score are included in the scale. See Appendix 6 for details. 2133 2134 5.5.3.6 Quality of Life Enjoyment and Satisfaction Questionnaire- Short 2135 Form (Q-LES-Q-SF) 2136 The Q-LES-Q-SF [16] was developed to measure the degree of enjoyment and 2137 satisfaction experienced in various areas of daily life in a self-rated format. The 2138 instrument is intended to detect differences among groups of patients, as well as 2139 individual patients over time. The full version of the Q-LES-Q consists of 60 items, 2140 however the 16-item short form version will be utilized for this study. See Appendix 7 for 2141 details. 2142 2143 5.5.3.7 Sheehan Disability Scale (SDS) 2144 The SDS [17] is a combination of three self-rated items designed to measure the extent to 2145 which three major areas of a patient’s life are impaired by panic, phobic, anxiety, or 2146 depressive symptoms. The scale was developed as an outcome measure that would be 2147 sensitive to change and to differences over time when evaluating medication treatment 2148 versus placebo. The patient rates the extent to which his or her 1) work or school, 2149 2) social life or leisure activities, and 3) home life or family responsibilities have been 2150 impaired by symptoms, using a 10-point visual analog scale. See Appendix 8 for details. 2151 2152 5.5.4 Safety 2153 The safety and tolerability of RO4917523 will be assessed by monitoring vital signs, 2154 laboratory tests, ECGs, the emergence of symptoms/adverse events of interest and other 2155 adverse events as detailed in the Schedule of Assessments [Table 1]. For additional safety 2156 instructions and guidance, including Adverse Events (AEs) & Laboratory Abnormalities 2157 and Handling of Safety Parameters, refer to Section 7. 2158 2159 5.5.4.1 Brief Psychiatric Rating Scale (BPRS) 2160 A subscale (4 items) of the BPRS [18] will be completed only to follow up on treatment 2161 emergent psychotic-related and mania-related (mania or hypomania) adverse events. The 2162 symptom constructs included are conceptual disorganization, suspiciousness, 2163 hallucinatory behavior, and unusual thought content. See Appendix 9 for details. 2164 2165 5.5.4.2 Young Mania Rating Scale (YMRS) 2166 The YMRS [19], item 1 only for mood elevation will be completed only to follow up on 2167 treatment emergent psychotic-related and mania-related (mania or hypomania) adverse 2168 events. See Appendix 10 for details.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2169 2170 5.5.4.3 Columbia-Suicide Severity Rating Scale (C-SSRS) 2171 The C-SSRS is a tool used to assess the lifetime suicidality of a patient (C-SSRS 2172 baseline) as well as any new instances of suicidality (C-SSRS since last visit). The 2173 C-SSRS incorporates a structured interview to prompt recollection of suicidal ideation, 2174 including the intensity of the ideation, behavior and attempts with actual/potential 2175 lethality. A validated, self rated version of the C-SSRS, the eC-SSRS [20] will be used to 2176 capture these data via an interactive voice response telephone system. The eC-SSRS 2177 baseline version will be administered at screening and the C-SSRS since last visit version 2178 will be administered at subsequent visits, including the baseline visit (see [Table 1]). 2179 Training will be provided on the administration of the C-SSRS rating scale, and on the 2180 eC-SSRS system for patient use. 2181 2182 eC-SSRS Administration: During a visit, patients will be directed to a private, quiet place 2183 with a telephone to complete the assessment. This assessment should be conducted early 2184 in the visit to provide sufficient time for the report to be received at the study site prior to 2185 patient departure. At the conclusion of each assessment, the site will receive an eC-SSRS 2186 Findings Report via e-mail or fax. The report presents the findings for suicidal ideation, 2187 intensity of ideation, suicidal behavior, and lethality/medical damage (for actual suicide 2188 attempts only). 2189 2190 Positive reports are generated for ANY of the following findings: 2191 2192 • Suicidal ideation with intention to act 2193 • Suicidal ideation with specific plan and intent 2194 • Made suicide attempt 2195 • Interrupted suicide attempt 2196 • Aborted suicide attempt 2197 • Preparatory behaviors for making a suicide attempt 2198 2199 Negative suicidality indication reports are generated when there are NO indications of the 2200 above. 2201 2202 Positive Findings: Should the system report that the patient has a positive suicidal 2203 indication, the site will be immediately notified by a telephone call from the vendor, as 2204 well as by fax / email. The site staff should take appropriate steps to interview the 2205 patient, e.g. complete a clinician administered C-SSRS. Depending on the results of the 2206 interview, the patient may be referred to a psychiatrist for a follow-up evaluation. The 2207 patient should not be released from the evaluation site until it is confirmed that the call is 2208 complete, the report is reviewed and the patient is not considered to be at risk. Ultimately, 2209 the determination of suicidality and risk is up to the evaluation site’s clinical judgment. 2210 2211 Suicidality Monitoring Data: The eC-SSRS Findings Report will remain at the evaluation 2212 site as source data and the data will be sent by the vendor electronically for inclusion in 2213 the database. Any clinician administered C-SSRS will be kept at the site as source data 2214 and entered into the eCRF.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2215 2216 5.5.4.4 Vital Signs and weight 2217 Vital signs including BP and pulse will be recorded in the supine position after resting 2218 3 – 5 minutes, and in the standing position after 2 minutes. The same arm should be used 2219 for all BP measurements. Vitals should be measured either prior to or no less than 2220 30 minutes after the last blood draw. 2221 2222 Weight will be measured at the time points specified in [Table 1]. 2223 2224 5.5.4.5 ECG 2225 Twelve lead ECG measurements (indicated in [Table 1]) will be performed in the supine 2226 position and will consist of three interpretable recordings (performed as close as possible, 2227 not more than two minutes apart). ECGs will be interpreted by cardiologists using a 2228 centralized ECG vendor. Procedures are described separately in the ECG manual. The 2229 investigator or designated qualified clinician must review, sign, and date the reports. All 2230 ECG results will be electronically loaded into the study database. 2231 2232 ECG should be measured either prior to or no less than 30 minutes after the last blood 2233 draw. 2234 2235 5.6 Laboratory Assessments 2236 Laboratory assessments (blood and urine samples) will be collected at the time points 2237 specified in [Table 1] and analyzed by a central laboratory. Parameters to be analyzed are 2238 shown below: 2239 2240 • Chemistry: magnesium, sodium, potassium, bicarbonate, chloride, urea 2241 (BUN), serum creatinine, fasting glucose, calcium, total protein, 2242 serum albumin, total bilirubin, AST (SGOT), ALT (SGPT), 2243 alkaline phosphatase, total cholesterol, triglycerides, Free T4 and 2244 TSH. FSH and estradiol will be measured in females if clinically 2245 appropriate to confirm post-menopausal state. 2246 2247 • Hematology: hemoglobin, hematocrit, erythrocytes (RBC), leukocytes (WBC), 2248 platelets, differential (counts): neutrophils, eosinophils, and 2249 lymphocytes 2250 2251 • Virology: Hepatitis B, Hepatitis C and HIV (screening only) 2252 2253 • Urinalysis: Midstream urine sample collected and analyzed by dipstick for 2254 pH, glucose, blood, and protein. If there is clinically significant 2255 positive result, urine will be sent to the central laboratory vendor 2256 for microscopy and culture. 2257 2258 • Pregnancy test: Female patients of childbearing potential will undergo urine 2259 pregnancy tests (analyzed by dipstick). 2260 2261 • Drugs of Abuse Urine samples will be analyzed for the presence of the following 2262 drugs: amphetamine, cannabinoids, opiates, cocaine, barbiturates, 2263 PCP.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2264 2265 The total volume of blood loss for laboratory assessments will be approximately 139 mL. 2266 (including volume collected for PK sampling, MBS, CG and optional RCR samples). 2267 2268 The samples for this study should be classified, packed and shipped as UN3373 2269 Biological Substance, Category B. 2270 2271 5.6.1 Pharmacokinetic [PK] Assessments 2272 Blood samples for population pharmacokinetic determinations of RO4917523 will be 2273 collected, based on patient availability, as specified in the Schedule of Assessments and 2274 Schedule of PK Sampling (Table 1 and Table 2).

2275 Plasma concentrations will be measured by a specific and validated LC/MS/MS method.

2276 Up to 11 samples (2 mL each) for each patient will be collected during the study. The 2277 procedures for the collection, handling and shipping of laboratory samples will be 2278 provided in the Sample Collection, Handling and Logistics Manual. 2279 2280 Table 2 Schedule of PK Sampling 2281 Visit Pre-dose 4h Post-dose 6h Post-dose Any time Day 1 √ √ √ Day 14 √ √ Day 28 √ √ Day 42 (end-of-treatment) √ √ √ Day 63 (follow-up/early √ withdrawal) 2282 2283 Patients will take their medication at the study centre on all study visit days and in 2284 particular on PK visit days. 2285 2286 For all patients who withdraw before completion of the double-blind treatment, one PK 2287 sample will be taken at any time during the early discontinuation visit. The date and time 2288 of the last dose of study medication should be recorded in the eCRF. 2289 2290 To ensure that evaluable data are collected for each patient, PK samples must be taken at 2291 the time points stated in Table 2 or as close to these time points as possible. On the day of 2292 PK assessment, the following will be recorded onto the eCRF: 2293 2294 • the actual date and time of dosing on the PK day 2295 • the actual date and time of dosing on the two preceding days 2296 • the actual time of meal in relation to dosing on PK day 2297 • the actual date and time of PK sampling. 2298 2299 Reasons for missed PK samples or those taken late in error must be provided and the 2300 actual sampling time recorded in the eCRF.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2301 2302 5.7 Roche Clinical Repository Specimen(s) 2303 Specimens for dynamic (A. non inherited) biomarker discovery and validation will be 2304 collected from consenting patients and where permissible with local regulations. These 2305 specimens will be used for research purposes to identify dynamic biomarkers that are 2306 predictive of response to RO4917523 treatment (in terms of efficacy-dose, safety and 2307 tolerability) and will help to better understand the pathogenesis, course and outcome of 2308 depression and related diseases. To these ends analysis may include determination of 2309 markers of synaptic plasticity and glutamate signaling. Specimens for dynamic biomarker 2310 discovery will be single coded like any other clinical sample (labeled and tracked using 2311 the patient’s study identification number- see Section 17). 2312 2313 Specimens for genetic biomarker (B. inherited) discovery and validation will also be 2314 collected from consenting patients and where permissible with local regulations. The 2315 pharmacogenetic information gathered through the analysis of specimens in the Roche 2316 Clinical Repository (RCR) is hoped to improve patient outcome by predicting which 2317 patients are more likely to respond to specific drug therapies, predicting which patients 2318 are susceptible to developing adverse side effects and/or predicting which patients are 2319 likely to progress to more severe disease states. Such genetic samples collected for 2320 analysis of heritable DNA variations will be double coded: a new independent code will 2321 be added to the first code to increase confidentiality and data protection (see Section 17). 2322 2323 The results of specimen analysis from the RCR will facilitate the rational design of new 2324 pharmaceutical agents and the development of diagnostic tests, which may allow for 2325 individualized drug therapy for patients in the future. 2326 2327 All RCR specimens will be destroyed no later than 15 years after the final freeze of the 2328 respective clinical database unless regulatory authorities require that specimens be 2329 maintained for a longer period. The specimens in the RCR will be made available for 2330 future biomarker research towards further understanding of treatment with RO4917523, 2331 of depression, related diseases and adverse events, and for the development of potential 2332 associated diagnostic assays. The implementation and use of the RCR specimens is 2333 governed by the Roche Clinical Repository policy to ensure the appropriate use of the 2334 RCR specimens. 2335 2336 5.7.1 Specimen Types 2337 The biomarker sampling below is optional. Samples will be stored for exploratory 2338 analysis after the end of the study. 2339 2340 A. Non-inherited: 1. Plasma and serum at baseline and end of treatment = 4 samples 2341 2342 2. RNA at baseline and end of treatment = 2 samples 2343 2344 3. DNA at baseline and end of treatment for epigenetics = 2 samples 2345 2346 B. Inherited: 4. DNA at baseline for genetics = 1 sample

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2347 2348 1. Plasma / Serum assays 2349 Blood samples for plasma and serum isolation will be obtained at baseline and the end of 2350 treatment as shown in Table 1. A total of 4 samples (2x6 mL for plasma and 2x6 mL for 2351 serum; 24 mL total) for each patient will be collected during the study. These samples 2352 will be used for biomarker assays for candidate depression biomarkers. For sampling 2353 procedures, storage conditions and shipment instructions see study Sample Collection, 2354 Handling and Logistics Manual. 2355 2356 2. Blood for RNA expression profiling 2357 Blood samples (2 x approximately 2.5 mL collected in PAXgene vacutainers) for RNA 2358 isolation will be collected at baseline and the end of treatment as shown in Table 1 2359 (10 mL total). The samples may be tested using techniques such as a micro array 2360 profiling system and / or RT PCR to study the expression profile of genes known to be 2361 involved with depression and any other differentially expressed genes relative to 2362 treatment response, dose response or re-treatment. For sampling procedures, storage 2363 conditions and shipment instructions see study Sample Collection, Handling and 2364 Logistics Manual. 2365 2366 3. Blood sample for epigenetic analysis 2367 Blood samples (approximately 6 mL in K3 EDTA) for DNA isolation will be collected at 2368 baseline and the end of treatment as shown in Table 1 (12 mL total). The sample may be 2369 processed using techniques to identify epigenetic modifications such as DNA methylation 2370 microarray profiling. See study Sample Collection, Handling and Logistics Manual for 2371 more details. 2372 2373 4. Blood sample for genetic analysis 2374 A single blood sample (approximately 6 mL in K3 EDTA) for DNA isolation will be 2375 collected as indicated in Table 1. If, however, the RCR genetic blood sample is not 2376 collected during the scheduled visit, it may be collected at any time (after randomization) 2377 during the conduct of the clinical study. The sample may be processed using techniques 2378 such as sequencing or microarray profiling. See study Sample Collection, Handling and 2379 Logistics Manual for more details. 2380 2381 For all samples, dates of consent and specimen collection should be recorded on the 2382 associated RCR page of the case report form (CRF) and/or in the clinical database. 2383 2384 5.8 Mandatory Biomarker Sampling (MBS) 2385 Blood samples for serum and plasma protein biomarker discovery and validation (such 2386 as, but not limited to, neurotrophic factors or cytokines) will be collected from all patients 2387 where permissible with local regulations at baseline and the end of treatment as indicated 2388 in Table 1. A total of 4 samples (2x6 mL for serum and 2x6 mL for plasma; 24 mL total) 2389 for each patient will be collected during the study. Specimens will be used for research 2390 purposes to identify or validate dynamic protein biomarkers that may be predictive of 2391 response to RO4917523 treatment (in terms of dose, safety and tolerability) or contribute 2392 to the pathogenesis, course and outcome of depression and related diseases. These 2393 specimens will be destroyed no later than 5 years after the end of the study. See study 2394 Sample Collection, Handling and Logistics Manual for more details.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2395 2396 5.9 Clinical Genotyping 2397 A 3 mL whole blood sample will be taken for DNA extraction from all patients where 2398 permissible with local regulations at baseline as indicated in Table 1. The DNA will be 2399 used to explore whether genetic variants are related to the pharmacokinetic / 2400 pharmacodynamic behavior, or observations on the safety and efficacy of RO4917523 or 2401 the ongoing antidepressant medication for one or more of the following specific genes: 2402 the cytochrome P450 variant 1A2 gene (CYP1A2), the metabotropic glutamate receptor 5 2403 (GRM5), the serotonin 2A receptor (HTR2A), the serotonin transporter (SLC6A4), and 2404 brain derived neurotropic factor (BDNF). Data arising from this analysis will be subject 2405 to standard confidentiality and data protection. This specimen will be destroyed 2406 immediately after the analysis has been completed. See study Sample Collection, 2407 Handling and Logistics Manual for more details. 2408 2409 6. INVESTIGATIONAL MEDICINAL PRODUCT 2410 For the purposes of this study, the Investigational Medicinal Product (IMP) is defined as 2411 the supplied dosage form of RO4917523 or matching placebo provided during the 2412 randomized treatment period. Ongoing treatment with antidepressant medication of the 2413 SSRI or SNRI class is not considered IMP and will not be supplied. 2414 2415 6.1 Dose and Schedule of IMP 2416 The first dose of study medication (RO4917523 0.5 mg, RO4917523 1.5 mg, or placebo) 2417 will be on Day 1, after all baseline assessments and pre-dose PK sampling have been 2418 conducted, and after a meal. Study medication will be administered once a day in the 2419 morning immediately after breakfast. Each dose will consist of two capsules. 2420 2421 6.1.1 Dose Modifications, Interruptions and Delays 2422 If the patient accidentally or by intention consumes more of the study medication than 2423 directed they should be instructed to notify their PI immediately, who will in turn 2424 evaluate and implement the appropriate clinical care strategy necessary. 2425 2426 If dose schedule needs modification, prior approval of the sponsor/Medical Monitor 2427 should be obtained. This should be appropriately documented and every effort taken to 2428 assure the time of dosing be as consistent as possible for the duration of the study. 2429 2430 If patient accidentally forgets to take the medication, they should be instructed to inform 2431 the site PI who in turn may recommend the following schedule: 2432 2433 – If before 12 pm (noon) of the same day of the missing dose, instruct the patient to 2434 take the medication with food. 2435 2436 – If after 12 pm (noon) the same day of the missing dose, instruct the patient not to take 2437 the medication that day. The treatment will be reinitiated the next day according to 2438 schedule. Explicitly instruct the patient not to take the double of the dose the 2439 following day to prevent the patient from attempting to make up the skipped dose. 2440 2441 – If more than 24 hours have elapsed since last dose, study PI should contact the 2442 sponsor/Medical Monitor.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2443 2444 If the patient is unable to tolerate the study medication due to intolerable adverse events, 2445 they should be withdrawn from the study and appropriate (end of study) procedures 2446 should be followed as shown in Table 1. 2447 2448 6.2 Formulation, Packaging and Labeling 2449 Study medication consists of RO4917523 pellets in hard gelatin capsules in strengths of 2450 0.5 mg (Ro 491-7523/F18) and 1.0 mg (Ro 491-7523/F19). The capsules are identical in 2451 size (no. 1). A matching placebo capsule (Ro 491-7523/F21) is also available. 2452 2453 Study drug packaging will be overseen by the Roche clinical trial supplies department 2454 and bear a label with the identification required by local law, the protocol number, drug 2455 identification and dosage. 2456 2457 6.2.1 Packaging and Storage 2458 Daily doses will be packaged as single blister strips containing 2 capsules as shown 2459 below in Table 3: 2460 2461 Table 3 IMP Packaging Configuration by Treatment Arm 2462

Number and Type of Capsules Treatment Arm Placebo 0.5 mg 1.0 mg Ro 491-7523/F21 Ro 491-7523/F18 Ro 491-7523/F19

Placebo 2 0 0

RO4917523 1 1 0 0.5 mg RO4917523 0 1 1 1.5 mg 2463 2464 Medication will be packed in blister packs which contain a one week supply. The drug 2465 must not be stored at temperatures exceeding 25°C (77°F). 2466 2467 Upon arrival of investigational products at the site, site personnel should check them for 2468 damage and verify proper identity, quantity, integrity of seals and temperature conditions, 2469 and report any deviations or product complaints to the monitor upon discovery. 2470 2471 6.3 Blinding and Unblinding 2472 The Randomization List will not be available at the study center, to the Sponsor’s 2473 monitors, project Statisticians, the CRO, or to the project team at Roche (except as noted 2474 in section 9 for the analysis of pharmacokinetic samples). Unblinding should not occur 2475 except in the case of emergency situations. Any request from the investigator for 2476 information about the treatment administered to study patients for another purpose must 2477 be discussed with Roche. Unblinding will be performed by means of the Interactive 2478 Voice Response System/Interactive Web Response System (IVRS).

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2479 2480 As per regulatory reporting requirement, Roche will unblind the identity of the study 2481 medication for all unexpected serious adverse events that are considered by the 2482 investigator to be related to study drug as per safety reference document(s), e.g., IB, 2483 CDS, SPC. Details of patients who are unblinded during the study will be included in the 2484 Clinical Study Report. 2485 2486 Unblinding for independent pharmacological analysis of biological samples, or ongoing 2487 safety monitoring by a Drug Safety Monitoring Board [DSMB], will be performed 2488 according to the sponsor’s internal procedures to ensure integrity of the data. 2489 2490 All other individuals directly involved in this study will remain blinded until the final 2491 analysis of the primary parameter. 2492 2493 The password-protected and/or encrypted electronic Master Randomization List is kept 2494 by the IVRS vendor in their secure system and is only accessible to the Randomization 2495 List Managers. No open key to the code will be available at the study center, to the 2496 Sponsor’s monitors, project statisticians, or to the project team at Roche or the CRO. 2497 2498 6.4 Accountability of IMP and Assessment of Compliance 2499 2500 6.4.1 Accountability of IMP 2501 The investigator is responsible for the control of drugs under investigation. Adequate 2502 records for the receipt (e.g. Drug Receipt Record) and disposition (e.g. Drug Dispensing 2503 Log) of the study drug must be maintained. Accountability will be assessed by maintaining 2504 adequate drug dispensing and return records. 2505 2506 Accurate records must be kept for each study drug provided by the sponsor. These 2507 records must contain the following: 2508 2509 • Documentation of drug shipments received from the sponsor (date received and 2510 quantity) 2511 • Disposition of unused study drug not dispensed to patient 2512 2513 A Drug Dispensing Log must be kept current and should contain the following 2514 information: 2515 2516 • the identification of the patient to whom the study medication was dispensed 2517 • the date[s], quantity of the study medication dispensed to the patient 2518 • the date[s] and quantity of the study medication returned by the patient 2519 2520 All records and drug supplies must be available for inspection by the Monitor at every 2521 monitoring visit. 2522 2523 Patients will be asked to return all used and unused drug supply containers at the end of 2524 the treatment as a measure of compliance.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2525 2526 When the study is terminated, the investigator will return any used and unused study drug 2527 (i.e. empty, partially used, and unused containers) to the Monitor. The completed Drug 2528 Dispensing Log and Drug Return Record(s) will be returned to Roche, unless alternate 2529 destruction has been authorized by Roche, or required by local or institutional 2530 regulations. (Section 6.5). The investigator's copy of the Drug Return Record(s) must 2531 accurately document the return of all study drug supplies to Roche. 2532 2533 6.4.2 Assessment of Compliance 2534 The investigator is responsible for ensuring that dosing is administered in compliance 2535 with the protocol. Delegation of this task must be clearly documented and approved by 2536 the investigator. 2537 2538 Accountability and patient compliance will be assessed by maintaining adequate drug 2539 dispensing and return records. Patients will be asked to return all used and unused drug 2540 supply containers at each study visit as a measure of compliance. 2541 2542 A Drug Dispensing Log must be kept current and should contain the following 2543 information: 2544 2545 • the identification of the patient to whom the study medication was dispensed 2546 • the date[s], quantity of the study medication dispensed to the patient 2547 • the date[s] and quantity of the study medication returned by the patient 2548 2549 This inventory must be available for inspection by the monitor. All supplies, including 2550 partially used or empty containers and the dispensing logs, must be returned to the 2551 monitor at the end of the study. 2552 2553 6.5 Destruction of the IMP/Comparator 2554 Local or institutional regulations may require immediate destruction of used 2555 investigational medicinal product (IMP) for safety reasons. In these cases, it may be 2556 acceptable for investigational site staff to destroy dispensed IMP before a monitoring 2557 inspection provided that source document verification is performed on the remaining 2558 inventory and reconciled against the documentation of quantity shipped, dispensed, 2559 returned and destroyed. Written authorization must be obtained from the sponsor at study 2560 start up before destruction. 2561 2562 If there are any issues with the drug it should be returned to the appropriate Roche 2563 clinical trial supplies department for long-term storage and not destroyed. 2564 2565 Written documentation of destruction must contain the following: 2566 2567 – Identity [batch numbers or subject numbers]of IMP[s] destroyed 2568 2569 – Quantity of IMP[s]destroyed 2570 2571 – Date of destruction 2572 2573 – Method of destruction

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2574 2575 – Name and signature of responsible person (or company) who destroyed 2576 investigational products[s] 2577 2578 7. SAFETY INSTRUCTIONS AND GUIDANCE 2579 2580 7.1 Adverse Events (AEs) and Laboratory Abnormalities 2581 2582 7.1.1 Clinical AEs 2583 According to the International Conference of Harmonization [ICH], an AE is any 2584 untoward medical occurrence in a patient or clinical investigation subject administered a 2585 pharmaceutical product and which does not necessarily have a causal relationship with 2586 this treatment. An AE can therefore be any unfavorable and unintended sign [including 2587 an abnormal laboratory finding], symptom, or disease temporally associated with the use 2588 of a medicinal [investigational] product, whether or not considered related to the 2589 medicinal [investigational] product. Pre-existing conditions which worsen during a study 2590 are to be reported as AEs. 2591 2592 7.1.1.1 Intensity 2593 All clinical AEs encountered during the clinical study will be reported on the AE form of 2594 the CRF. Intensity of AEs will be graded on a three-point scale [mild, moderate, or 2595 severe] and reported in detail on the CRF. 2596 Mild discomfort noticed but no disruption of normal daily activity. Moderate discomfort sufficient to reduce or affect daily activity. Severe inability to work or perform normal daily activity

2597 2598 2599 7.1.1.2 Drug - Adverse event relationship 2600 Relationship of the AE to the treatment should always be assessed by the investigator. 2601 2602 7.1.1.3 Serious Adverse Events [Immediately Reportable to Roche] 2603 A Serious Adverse Event is any experience that suggests a significant hazard, 2604 contraindication, side effect or precaution. It is any AE that at any dose fulfils at least 2605 one of the following criteria: 2606 2607 – is fatal; [results in death**; NOTE: death is an outcome, not an event] 2608 2609 – is Life-Threatening [NOTE: the term "Life-Threatening" refers to an event in which 2610 the subject was at immediate risk of death at the time of the event; it does not refer to 2611 an event which could hypothetically have caused a death had it been more severe] 2612 2613 – requires in-patient hospitalization or prolongation of existing hospitalization 2614 2615 – results in persistent or significant disability/incapacity 2616 2617 – is a congenital anomaly/birth defect

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2618 2619 – is medically significant or requires intervention to prevent one or other of the 2620 outcomes listed above. 2621 2622 **The term sudden death should be used only when the cause is of a cardiac origin as per standard 2623 definition. The terms death and sudden death are clearly distinct and must not be used 2624 interchangeably. 2625 2626 The study will comply with all local regulatory requirements and adhere to the full 2627 requirements of the ICH Guideline for Clinical Safety Data Management, Definitions 2628 and Standards for Expedited Reporting, Topic E2. 2629 2630 7.1.2 Treatment and Follow-up of AEs 2631 AEs, especially those for which the relationship to study medication(s) is not “unrelated”, 2632 should be followed up until they have returned to baseline status or stabilized. If after 2633 follow-up, return to baseline status or stabilization cannot be established an explanation 2634 should be recorded on the CRF. 2635 2636 7.1.3 Laboratory Test Abnormalities 2637 Laboratory test results will be provided to the sites as faxed reports from the central 2638 laboratory vendor. 2639 2640 Any laboratory result abnormality fulfilling the criteria for a serious adverse event [SAE] 2641 should be reported as such, in addition to being recorded as an AE in the CRF. 2642 2643 Any treatment-emergent abnormal laboratory result which is clinically significant, 2644 i.e., meeting one or more of the following conditions, should be recorded as a single 2645 diagnosis on the AE form in the CRF: 2646 2647 • Accompanied by clinical symptoms 2648 • Leading to a change in study medication [e.g. dose modification, interruption or 2649 permanent discontinuation] 2650 • Requiring a change in concomitant therapy [e.g. addition of, interruption of, 2651 discontinuation of, or any other change in a concomitant medication, therapy or 2652 treatment]. 2653 This applies to any protocol and non-protocol specified safety and efficacy laboratory 2654 result from tests performed after the first dose of study medication, which falls outside 2655 the laboratory reference range and meets the clinical significance criteria. 2656 2657 This does not apply to any abnormal laboratory result which falls outside the laboratory 2658 reference range but which does not meet the clinical significance criteria [these will be 2659 analyzed and reported as laboratory abnormalities]; those which are considered AEs of 2660 the type explicitly exempted by the protocol; or those which are a result of an AE which 2661 has already been reported. 2662 2663 7.1.3.1 Follow-up of Abnormal Laboratory Test Values 2664 In the event of medically significant unexplained abnormal laboratory test values, the 2665 tests should be repeated and followed up until they have returned to the normal range 2666 and/or an adequate explanation of the abnormality is found. If a clear explanation is 2667 established it should be recorded on the CRF.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2668 2669 7.2 Handling of Safety Parameters 2670 2671 7.2.1 Reporting of AEs 2672 Adverse events will be recorded from randomization until 21 days following treatment 2673 discontinuation. As with all assessments, eCRF completion begins when the patient is 2674 randomized in the study. For screen failure patients, the adverse events will only be 2675 recorded in source documents. 2676 2677 7.2.1.1 Recording of AEs related to Patient-Reported Outcomes 2678 AE reports will not be derived from PRO data. If during site review of the completed 2679 PRO questionnaires a possible AE is identified, site staff will alert the investigator to 2680 determine if the criteria for an AE has been met (as described in section 7.1). 2681 2682 7.2.2 Reporting of Serious Adverse Events [immediately reportable] 2683 Any clinical AE or abnormal laboratory test value that is serious [as defined in 2684 Section 7.1.1.3 above] and which occurs during the course of the study, regardless of the 2685 treatment arm, occurring from the enrollment visit (start of study screening procedures), 2686 must be reported to Roche within one working day of the investigator becoming aware 2687 of the event [expedited reporting]. The investigator must complete the SAE Reporting 2688 Form [gcp_for000031] and forward it to the SAE Responsible. 2689 2690 Additionally, after the informed consent has been signed, but prior to initiation of study 2691 medications, only SAEs caused by a protocol-mandated intervention will be collected 2692 (e.g., SAEs related to invasive procedures or medication washout). After first study 2693 medication, all SAEs must be reported. 2694 2695 Related Serious Adverse Events MUST be collected and reported regardless of the time 2696 elapsed from the last study drug administration, even if the study has been closed. 2697 Suspected Unexpected Serious Adverse Reactions (SUSARs) are reported to 2698 investigators at each site and associated IRB/IEC when the following conditions occur: 2699 2700 • The event must be a SAE. 2701 • There must be a certain degree of probability that the event is an adverse reaction 2702 from the administered drug. 2703 • The adverse reaction must be unexpected, that is to say, not foreseen in the SPC text 2704 (Summary of Product Characteristics (for an authorized medicinal product) or the 2705 Investigator’s Brochure (for an unauthorized medicinal product). 2706 2707 When all patients at a particular site are off treatment as defined by the protocol: 2708 2709 • only individual SUSAR reports originating in that particular trial will be forwarded to 2710 the site and associated IRB/IEC on an expedited basis; 2711 • individual SUSARs considered to be a significant safety issue and/or which result in 2712 Roche recommending a change to the Informed Consent Form (ICF), will be reported 2713 in an expedited manner to all investigators and IRBs/IECs; 2714 • SUSAR reports originating from other trials using the same IMP will be provided as 2715 six monthly SUSAR Reports (SSRs) to investigators and IRBs/IECs where long-term 2716 follow-up studies are carried out, unless they are considered significant.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2717 2718 Unrelated Serious Adverse Events must be collected and reported during the study and 2719 for up to 21 days after the last dose of study medication. 2720 2721 This study adheres to the definition and reporting requirements of ICH Guideline for 2722 Clinical Safety Data Management, Definitions and Standards for Expedited 2723 Reporting, Topic E2. Complete information can be found in Appendix 2. 2724 2725 7.2.3 Abuse and Withdrawal Symptoms 2726 An abuse potential monitoring strategy will ensure close monitoring for potential 2727 diversion and/or abuse of study drug as well as adverse events which may be consistent 2728 with abuse or withdrawal. The strategy is as follows: 2729 2730 – The PI/site staff will regularly check for patterns of suspected hoarding of study 2731 medication (e.g., based on recurring instances of missing medication). Such patterns 2732 should be brought to the attention of the Medical Monitor for appropriate follow up. 2733 2734 – The Sponsor/Medical monitor and PI will regularly review of all AEs related to abuse 2735 potential 2736 2737 Any findings will be documented in a note to file. 2738 2739 7.2.4 Pregnancy 2740 A female patient must be instructed to stop taking the study medication and immediately 2741 inform the investigator if she becomes pregnant during the study. 2742 2743 The investigator should report all pregnancies within 24 hours to the sponsor. The 2744 investigator should counsel the patient, discuss the risks of continuing with the pregnancy 2745 and the possible effects on the fetus. Monitoring of the patient should continue until 2746 conclusion of the pregnancy. The outcome of the pregnancy must be reported to the 2747 sponsor with a follow-up report. Pregnancies occurring up to 90 days after the completion 2748 of the study medication must also be reported to the investigator. 2749 2750 7.3 Warnings and Precautions 2751 No evidence available at the time of the approval of this study protocol indicated that 2752 special warnings or precautions were appropriate, other than those noted in the 2753 Investigators Brochure. 2754 2755 8. STATISTICAL CONSIDERATIONS AND ANALYTICAL PLAN 2756 2757 8.1 Primary and Secondary Study Endpoints 2758 2759 8.1.1 Primary Endpoints 2760 Change in the Montgomery Asberg Depression Rating Scale (MADRS) total score from 2761 baseline to end of treatment. 2762 2763 8.1.2 Secondary Endpoints 2764 The secondary endpoints are: 2765 2766 • Change in Clinical Global Scores Severity (CGI-S) from baseline to end of 2767 treatment and Improvement (CGI-I) at end of treatment

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2768 2769 2770 • Frequency of patients exhibiting remission (a MADRS score of less than or equal 2771 to 10) at end of treatment 2772 • Frequency of patients exhibiting response (reduction in MADRS score equal to or 2773 greater than 50% of the baseline score) at end of treatment 2774 • Quick Inventory of Depressive Symptomatology Self Report (QIDS-SR16) 2775 change from baseline to end of treatment 2776 • Patient Global Impression of Improvement (PGI-I) score at end of treatment 2777 2778 2779 8.1.3 Exploratory Endpoints 2780 The key exploratory endpoint is: 2781 2782 • Change in the Montgomery Asberg Depression Rating Scale (MADRS) total 2783 score from baseline to end of treatment for exploratory evaluation. 2784 2785 The other exploratory endpoints are: 2786 2787 • The proportion of patients who meet MADRS responder criteria plus CGI-I score 2788 of 1) “very much improved” or 2) “much improved” at end of treatment 2789 • The proportion of patients who meet MADRS remission criteria plus CGI-I score 2790 of 1) “very much improved” or 2) “much improved” at end of treatment 2791 • The speed of onset of antidepressant effect based on change over time in any of 2792 the depression symptom scales 2793 • CANTAB Cognitive Test Battery subset 2794 • Quality of Life Enjoyment and Satisfaction Questionnaire-Short Form (Q-LES-Q- 2795 SF) 2796 • Sheehan Disability Scale (SDS) 2797 2798 2799 8.1.4 Safety 2800 Safety and tolerability of the treatment will be evaluated by AEs, laboratory tests, vital 2801 signs, electrocardiogram, the C-SSRS, BPRS, and YMRS (if appropriate). 2802 2803 All patients who belong to the safety population will be included in the safety evaluation. 2804 2805 8.2 Statistical and Analytical Methods 2806 2807 8.2.1 Analysis Population 2808 2809 8.2.1.1 Intent-to-treat Population 2810 The intent-to-treat (ITT) population will include all patients who were randomized and 2811 had a baseline and at least one post-baseline assessment of MADRS (i.e., valid total 2812 score). The ITT analysis will be done by randomized treatment. The ITT population will 2813 be the primary population for all analyses of primary and secondary clinical efficacy 2814 variables. 2815 2816 8.2.1.2 Per-protocol Population 2817 The per-protocol (PP) population will be defined as the subset of the intent-to-treat 2818 population. Definition of the PP population will be finalized before the database closure 2819 and will be documented in the analysis plan. The definition may include sufficient

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2820 2821 treatment duration of study medication, and lack of any major protocol violations that 2822 affect efficacy evaluation. 2823 2824 For the PP population analysis, data will be analyzed according to the treatment actually 2825 taken. 2826 2827 8.2.1.3 Safety Population 2828 The safety population will consist of all patients who have received at least one dose of 2829 study medication, whether withdrawn prematurely or not. All safety data will be analyzed 2830 for the safety population according to the medication actually dispensed. 2831 2832 8.2.2 Statistical Model 2833 All efficacy data will be analyzed using intent-to-treat population. PP analysis will be 2834 conducted for the primary and selected secondary efficacy variables only if more than 2835 10% of the ITT population are not included in the PP population (defined in 8.2.1.2). For 2836 all efficacy variables, the baseline value will be defined as the last value taken prior to the 2837 start of double-blind study medication. 2838 2839 The efficacy parameters of the primary endpoint and of the continuous secondary 2840 endpoints will be analyzed using a mixed effects covariance pattern model to utilize all 2841 the data collected over time with consideration of the variance-covariance matrix of the 2842 repeated measures (MMRM). 2843 2844 The model will include independent variables of the fixed, categorical effects of 2845 treatment, geographical region/countries, assessment weeks relative to the first dose of 2846 study medication (i.e., time), and treatment-by-time interaction, along with the 2847 continuous effect of baseline. The geographical region/countries will use as categories 2848 the strata of the randomization: Europe, United States, Latin America, Asia 2849 (excluding Japan) and Japan. 2850 2851 An unstructured variance-covariance matrix will be applied to model the within-patient 2852 errors. A treatment-by-time interaction contrast will be used to estimate the difference 2853 between each of the RO4917523 doses and placebo in the mean change from baseline to 2854 day 42 of randomized treatment. 2855 2856 As additional sensitivity analysis, the primary efficacy variable will also be assessed at 2857 day 42 by ANCOVA using the LOCF method as exploratory analysis to assess the 2858 robustness of the results. The model will include the baseline measure as covariate, and 2859 the categorical variable treatment. The effect size of each dose compared to placebo and 2860 its 90% confidence interval will be calculated using estimates from the treatment 2861 contrasts. The results will be used for internal decisions on the continuation of the 2862 development of the project. 2863 2864 Ordered categorical data, like CGI-S, CGI-I or PGI-I, will be analyzed using Wilcoxon 2865 rank test. Binary data, such as responders or remissions, will be analyzed using Fisher’s 2866 exact test. 2867 2868 All exploratory variables and safety and tolerability will be summarized descriptively.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2869 2870 8.2.3 Sample Size 2871 A sample size of approximately 100 patients per group (approximately 300 for 3 groups) 2872 has been chosen to obtain a power of approximately 80% at two-sided maximum 2873 familywise error rate of 0.05 in a closed testing procedure. 2874 2875 The power calculation was based on simulations of the MMRM analysis planned for the 2876 primary efficacy variable. The following assumptions were included: 2877 2878 • 7 post-baseline assessment visits 2879 • Overall dropout rate of 15%, incremental rates over the 6 week period 2880 • An effect size of 0.46 for one dose group at day 42 assuming increasing magnitude 2881 of treatment difference over the period and an effect size of 0.0 for the other dose 2882 group 2883 • A moderately decreasing correlation structure between assessments that are further 2884 apart 2885 2886 2887 8.2.4 Hypothesis Testing 2888 For the primary endpoint the following hypotheses will be tested: 2889 2890 H0: The mean reduction in total MADRS score at end of treatment in both dose groups 2891 are same as in the Placebo group µRO 1 = µPlacebo = µRO 2 2892 2893 vs. 2894 2895 H1: At least in one dose group the mean reduction in total MADRS score at end of 2896 treatment is different from the Placebo group µRO 1 ≠ µPlacebo or µRO 2 ≠ µPlacebo 2897 2898 For each dose group i=1,2 the following hypotheses will be tested: 2899 2900 H0i: The mean reduction in total MADRS score at end of treatment in dose group i is the 2901 same as in the Placebo group µRO i = µPlacebo 2902 2903 vs. 2904 2905 H1i: The mean reduction in total MADRS score at end of treatment in dose group i is 2906 different from the Placebo group µRO i ≠ µPlacebo 2907 2908 A closed testing procedure will be utilized: The overall hypothesis H0 will be tested at the 2909 two-sided significance level α = 0.05 using the Dunnett’s test within the MMRM 2910 analysis. If H0 will be rejected, the elementary hypothesis H0i will be tested at the 2911 two-sided significance level α = 0.05 utilizing t- test within MMRM, separately for i=1, 2912 2. The closed testing procedure has a two-sided maximum family-wise error rates of 0.05. 2913 Therefore no further adjustment for multiplicity is required. 2914 2915 8.2.5 Efficacy Analysis 2916 2917 8.2.5.1 Interim Analysis 2918 No interim analyses are planned.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2919 2920 8.2.5.2 Missing Data Handling 2921 The main analysis of the primary and continuous secondary efficacy variables will be 2922 done using a mixed effects model, and no imputation for missing data will be applied. 2923 2924 To understand the pattern of missing data observed during the study and thus the missing 2925 data mechanism, the following data will be reviewed: 2926 2927 • Timing of discontinuations by treatment group 2928 • Reasons for discontinuation by treatment group and time 2929 • Mean of the primary efficacy variable of those who dropped out vs. those who 2930 remained at each scheduled assessment week 2931 2932 A sensitivity analysis for deviation from the assumption for the randomness of 2933 missing data as required for the MMRM analysis (Missing at Random) will be 2934 performed. Details will be described in the statistical analysis plan prior to database 2935 lock and unblinding. 2936 2937 8.2.6 Safety Data Analysis 2938 All safety variables (e.g., adverse events, lab tests, ECG, vital signs, BPRS, YMRS) will 2939 be summarized for each assessment time (including follow-up) using descriptive statistics. 2940 The items of the C-SSRS will be presented by individual listings and the outcomes from 2941 this scale will be classified using the C-CASA methodology. Incidence of AEs will be 2942 summarized based on body systems and preferred terms. Incidence of marked abnormal 2943 lab test results will be summarized based on the Roche COG 3007 definition. 2944 2945 8.2.7 Other Analyses 2946 2947 8.2.7.1 Pharmacokinetic Analysis 2948 Nonlinear mixed effects modeling (with software NONMEM [20]) will be used to 2949 analyze the sparse sampling dose-concentration-time data of RO4917523. Population and 2950 individual pharmacokinetic parameters (e.g. Cl/F and Vss/F) will be estimated and the 2951 influence of various covariates on these parameters will be investigated. The data 2952 collected in this study may be pooled with data collected in previous phase I and phase 2953 IIa studies as appropriate to build pharmacokinetic model. Secondary PK parameters such 2954 as AUC and Cmax will be derived from the individual post-hoc predictions. 2955 2956 The results will be reported in a document separate from the clinical study report. 2957 2958 8.2.7.2 Pharmacodynamic Analysis 2959 Exploratory analyses or modeling techniques (if possible) will be used to investigate the 2960 relationship between RO4917523 exposure and response (e.g. MADRS, CGI-I for 2961 efficacy and occurrence of psychiatric AEs for safety). The results will be reported in a 2962 document separate from the clinical study report. 2963 2964 8.2.7.3 Subgroup Analysis 2965 To assess whether safety, efficacy and pharmacokinetic results of the patients enrolled in 2966 different geographical regions/countries are similar, selected key data may be analyzed 2967 for each. The comparability will be assessed descriptively.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 2968 2969 9. DATA COLLECTION, MANAGEMENT AND QUALITY ASSURANCE 2970 The overall procedures for quality assurance of clinical study data are described in the 2971 Contract Research Organization (CRO) Standard Operational Procedures. 2972 2973 Data for this study will be recorded via an Electronic Data Capture system EDC using 2974 electronic Case Report Forms (eCRF). It will be transcribed by the site from the paper 2975 source documents into the eCRF. In no case is the eCRF to be considered as source data 2976 for this trial. 2977 2978 Accurate and reliable data collection will be assured by verification and cross–check of 2979 the eCRFs against the investigator’s records by the study monitor (source document 2980 verification), and the maintenance of a drug–dispensing log by the investigator. 2981 2982 A comprehensive validation check program will verify the data. Discrepancies will be 2983 generated automatically in the system at the point of entry or added manually for 2984 resolution by the Investigator. 2985 2986 In order to facilitate analysis of the pharmacokinetic samples collected in this study, the 2987 treatment code will be released to the responsible analyst when the samples have been 2988 received at the analytical site and are ready for assay. The result of the analysis must not 2989 be released with individual identification of the patient until the database is closed. 2990 2991 9.1 Assignment of Preferred Terms and Original Terminology 2992 For classification purposes, preferred terms will be assigned by the Sponsor to the 2993 original terms entered on the eCRF, using the most up-to-date version of the Medical 2994 Dictionary for Regulatory Activities (MedDRA) terminology for adverse events and 2995 diseases and the International Non-proprietary Name (INN) Drug Terms and Procedures 2996 Dictionary for treatments and surgical and medical procedures. 2997 2998 10. STUDY COMMITTEES 2999 An independent Data Safety Monitoring Board (DSMB) will review potential safety 3000 signals of concern. The DSMB will be composed of non-sponsor members who are not 3001 involved in the conduct of this study and with no other ongoing financial relationship 3002 with the sponsor. The DSMB will review available safety data (and potentially efficacy 3003 data, only if deemed necessary to better assess safety) from this trial at regularly 3004 scheduled intervals as specified in the DSMB Charter. Two DSMB review cycles are 3005 planned. The first review cycle is planned to occur when approximately 40 to 60 patients 3006 have completed the study. The second will occur when approximately 140 to 160 patients 3007 have completed. The DSMB has the option to change the data review schedule as deemed 3008 necessary. 3009 3010 Following each data review, the DSMB will make recommendations regarding the 3011 conduct of this study, including continuing the study without modifications, or to modify 3012 the study design in any other way. Details of the DSMB’s responsibilities and logistics 3013 will be outlined in the DSMB Charter.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3014 3015 11. REFERENCES 3016 1. Sanacora G, Zarate CA, Krystall JH, Manji HK. Targeting the glutamatergic 3017 system to develop novel, improved therapeutics for mood disorders. Nature 3018 Drug Discovery 2008;7:426-437. 3019 3020 2. Zarate CA, Singh JB, Carlson PJ, et al. A randomized trial of an N-methyl-D- 3021 aspartate antagonist in treatment-resistant major depression. Arch Gen 3022 Psychiatry 2006;63:856-864. 3023 3024 3. Nemeroff CB. Prevalence and management of treatment-resistant depression. 3025 J Clin Psychiatry 2007;68 [suppl 8]:17-25. 3026 3027 4. Nelson JC. The STAR*D study. Am J Psychiatry 2006;163:1864-1866. 3028 3029 5. Rush AJ, Trivedi MH, Wisniewski SR, et al. STAR*D Study Team. 3030 Bupropion-SR, sertraline, or venlafaxine-XR after failure of SSRIs for 3031 depression. N Engl J Med 2006;354:1231-1242. 3032 3033 6. Quitkin FM, Petkova E, McGrath PJ, et al. When should a trial of fluoxetine 3034 for major depression be declared failed? Am J Psychiatry 2003;160: 734-740. 3035 3036 7. Preskorn SH, Baker B, Kolluri S, et al. An innovative design to establish 3037 proof of concept of the antidepressant effects of the NR2B subunit selective 3038 N-Methyl-D-Aspartate antagonist, CP-101606, in patients with treatment- 3039 refractory major depressive disorder. J Clin Psychpharmacol 2008;28:631- 3040 637. 3041 3042 8. Fava M. Diagnosis and definition of treatment-resistant depression. Biol 3043 Psychiatry 2003;53:649-659. 3044 3045 9. Chandler GM, Iosifescu DV, Pollack MH, et al. Validation of the 3046 Massachusetts General Hospital Antidepressant History Questionnaire 3047 (ATRQ). CNS Neuroscience & Therapeutics 2009;16:322-325. 3048 3049 10. Sheehan DV, Lecrubier Y, Harnett-Sheehan K, et al. The Mini International 3050 Neuropsychiatric Interview (M.I.N.I.): the development and validation of a 3051 structured diagnostic psychiatric interview for DSM-IV and ICD-10. J Clin 3052 Psychiatry 1998;59(suppl 20):22-33. 3053 3054 11. Montgomery SA and Asberg M. A new depression scale designed to be 3055 sensitive to change. Br. J. Psychiatry 1979;134:382-389. 3056 3057 12. Williams JB and Kobak KA. Development and reliability of a structured 3058 interview guide for the Mongomery-Asberg Depression Rating Scale 3059 (SIGMA). Br. J. Psychiatry 2008;192:52-58. 3060 3061 13. Guy W, ed. Clinical global impressions (028-CGI). In: ECDEU Assessment 3062 Manual for Psychopharmacology. Revised, 1976. NIMH, U.S. Department of 3063 Health, Education, and Welfare, Pub No (ADM) 76 -338, 1976:215-222.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3064 3065 14. Rush AJ, Trivedi MH, Ibrahim HM, et al. The 16-item Quick Inventory of 3066 Depressive Symptomotology (QIDS), Clinician Rating (QIDS-C), and Self- 3067 Report (QIDS-SR): a psychometric evaluation in patients with chronic major 3068 depression. Biol Psychiatry 2003;54:573-583. 3069 3070 15. Trivedi MH, Rush AJ, Ibrahim HM, et al. The Inventory of Depressive 3071 Symptomotology, Clinician Rating (IDS-C) and Self-Report (IDS-SR), and 3072 the Quick Inventory of Depressive Symptomotology, Clinician Rating 3073 (QIDS-C) and Self-Report (QIDS-SR) in public sector patients with mood 3074 disorders: a psychometric evaluation. Psychol Med 2004;34:73-82. 3075 3076 16. Endicott J, Nee J, Harrison W, et al. Quality of Life Enjoyment and 3077 Satisfaction Questionnaire: a new scale. Psychopharmocol Bull 1993;29:321- 3078 326. 3079 3080 17. Sheehan DV. The Anxiety Disease. New York, Scribners, 1983. 3081 3082 18. Overall JE, Gorham DR. The Brief Psychiatric Rating Scale (BPRS): recent 3083 developments in ascertainment and scaling. Psychopharmocol Bull 1988;24: 3084 97-99. 3085 3086 19. Young RC, Biggs JT, Ziegler VE, et al. A rating scale for mania: reliability, 3087 validity, and sensitivity. British Journal of Psychiatry 1978;133: 429-435. 3088 3089 20. Mundt JC, Greist JH, Gelenberg AJ, et al. Feasibility and validation of a 3090 computer-automated Columbia-Suicide severity rating scale using interactive 3091 voice response technology. J Psychiatric Research. 2010 May 27 [Epub ahead 3092 of print]. 3093 3094 21. Beal S and Sheiner L (Eds.). NONMEM Users Guides, NONMEM Project 3095 Group, University of California at San Francisco, San Francisco 1998.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3096 3097 PART II: ETHICS AND GENERAL STUDY ADMINISTRATION 3098 3099 12. ETHICAL ASPECTS 3100 3101 12.1 Local Regulations/Declaration of Helsinki 3102 The investigator will ensure that this study is conducted in full conformance with the 3103 principles of the “Declaration of Helsinki” or with the laws and regulations of the country 3104 in which the research is conducted, whichever affords the greater protection to the 3105 individual. The study must fully adhere to the principles outlined in “Guideline for Good 3106 Clinical Practice” ICH Tripartite Guideline or with local law if it affords greater 3107 protection to the subject. For studies conducted in the EU/EEA countries, the 3108 investigator will ensure compliance with the EU Clinical Trial Directive [2001/20/EC]. 3109 For studies conducted in the USA or under US IND, the investigator will additionally 3110 ensure adherence to the basic principles of “Good Clinical Practice” as outlined in the 3111 current version of 21 CFR, subchapter D, part 312, “Responsibilities of Sponsors and 3112 Investigators”, part 50, “Protection of Human Subjects”, and part 56, “Institutional 3113 Review Boards”. 3114 3115 In other countries where a “Guideline for Good Clinical Practice” exists, Roche and the 3116 investigators will strictly ensure adherence to the stated provisions. 3117 3118 12.2 Informed Consent 3119 Written Informed Consent from Patients: 3120 3121 12.2.1 Main study Informed Consent 3122 It is the responsibility of the investigator, or a person designated by the investigator [if 3123 acceptable by local regulations], to obtain signed informed consent from each patient 3124 prior to participating in this study after adequate explanation of the aims, methods, 3125 anticipated benefits, and potential hazards of the study. 3126 3127 The investigator or designee must also explain that the patients are completely free to 3128 refuse to enter the study or to withdraw from it at any time, for any reason. 3129 3130 The Case Report Forms (CRFs) for this study contain a section for documenting patient 3131 informed consent, and this must be completed appropriately. If new safety information 3132 results in significant changes in the risk/benefit assessment, the consent form should be 3133 reviewed and updated if necessary. All patients (including those already being treated) 3134 should be informed of the new information, given a copy of the revised form and give 3135 their consent to continue in the study. 3136 3137 12.2.2 RCR Informed Consent 3138 It is the responsibility of the investigator, or a person designated by the investigator (if 3139 acceptable under local regulations), to obtain written informed consent from each 3140 individual who has consented to RCR sampling after adequate explanation of the aims, 3141 methods, objectives and potential hazards. Patients must receive an explanation that they 3142 are completely free to refuse to provide the RCR specimen(s) and may withdraw his/ her 3143 sample at any time and for any reason during the 15 year storage period of the 3144 specimen(s). The Informed Consent for an optional specimen donation will be

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3145 3146 incorporated as a specific section into the main Clinical Trial [or Experimental Research 3147 study] Informed Consent Form (ICF). A second, separate, specific signature consenting 3148 to specimen donation will be required to document the study participant’s agreement to 3149 provide an optional specimen; if the participant declines, he/ she will check a “no” box in 3150 the appropriate section and not provide a second signature. 3151 3152 The CRF for the associated clinical study contains a page for documenting patient 3153 informed consent to the RCR, and this must be completed appropriately. 3154 3155 12.2.3 Death or Loss of Competence of Participant who has donated a 3156 specimen(s) that is stored in the RCR 3157 In case the Informed Consent Form and/or the Study Protocol do not provide any specific 3158 provisions for death or loss of competence, specimen and data will continue to be used as 3159 part of RCR research. 3160 3161 In the event of the death of a participant of a Roche Clinical Trial or Experimental 3162 Medicine Research study or if a participant is legally incompetent at the time of the 3163 specimen and data procurement, or becomes legally incompetent thereafter, applicable 3164 provisions as stated for such situations in the respective Informed Consent Form and/or 3165 the Study Protocol shall become effective and be followed accordingly. 3166 3167 Additional procurement of assent from legally incompetent persons and minors shall take 3168 place according to local laws and international best practice, as it applies to the specific 3169 case. 3170 3171 12.3 Independent Ethics Committees/(IEC)Institutional Review 3172 Board(IRB) 3173 The protocol, informed consent and any accompanying material provided to the patient in 3174 the U.S. will be submitted by the investigator to an IRBfor review. For EEA member 3175 states, the sponsor will submit to the Competent Authority and IEC, the protocol and any 3176 accompanying material provided to the patient. In both the US and EEA member states, 3177 the accompanying material may include patient information sheets, descriptions of the 3178 study used to obtain informed consent and terms of any compensation given to the patient 3179 as well as advertisements for the trial. 3180 3181 An approval letter or certificate (specifying the protocol number and title) from the 3182 IEC/IRB must be obtained before study initiation by the investigator specifying the date 3183 on which the committee met and granted the approval. This applies whenever subsequent 3184 amendments/modifications are made to the protocol. 3185 3186 Any modifications made to the protocol, informed consent or material provided to the 3187 patient after receipt of the IEC/IRBapproval must also be submitted by the investigator in 3188 the U.S. and by the Sponsor in the EEA member states in accordance with local 3189 procedures and regulatory requirements. 3190 3191 When no local review board exists, the investigator is expected to submit the protocol to 3192 a regional committee. If no regional committee exists, Roche will assist the investigator 3193 in submitting the protocol to the European Ethics Review Committee.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3194 3195 Sampling for the RCR is contingent on review and approval for the exploratory 3196 biomarker assessments and written informed consent by an appropriate regulatory body 3197 (depending on the country where the study is performed) and a site’s Institutional Review 3198 Board (IRB) / Ethics Committee (EC). If a regulatory or site’s IRB/ EC does not approve 3199 the sampling for the exploratory assessments the section on biomarker sampling will not 3200 be applicable. 3201 3202 Roche shall also submit an Annual Safety Report once a year to the IEC and Competent 3203 Authorities (CAs) according to local regulatory requirements and timelines of each 3204 country participating in the study. In the U.S. Roche submits an IND Annual Report to 3205 the FDA according to local regulatory requirements and timelines. 3206 3207 12.4 Role of the Science and Ethics Advisory Group (SEAG) 3208 A Science and Ethics Advisory Group consisting of experts in the fields of biology, 3209 ethics, sociology and law will advise Roche regarding the use of specimens stored in the 3210 RCR and on the scientific and ethical aspects of handling genetic information. The SEAG 3211 is independent of Roche. 3212 3213 13. CONDITIONS FOR MODIFYING THE PROTOCOL 3214 Requests from investigators to modify the protocol to ongoing studies will be considered 3215 only by consultation between an appropriate representative of the sponsor and the 3216 investigator [investigator representative[s] in the case of a multicenter trial]. Protocol 3217 modifications must be prepared by a representative of the sponsor and initially reviewed 3218 and approved by the Clinical Science Leader and Biostatistician. 3219 3220 All protocol modifications must be submitted to the appropriate Independent Ethics 3221 Committee or Institutional Review Board for information and approval in accordance with 3222 local requirements, and to Regulatory Agencies if required. Approval must be obtained 3223 before any changes can be implemented, except for changes necessary to eliminate an 3224 immediate hazard to trial patients, or when the change[s] involves only logistical or 3225 administrative aspects of the trial [e.g. change in monitor[s], change of telephone 3226 number[s]]. 3227 3228 14. CONDITIONS FOR TERMINATING THE STUDY 3229 Both the sponsor and the investigator reserve the right to terminate the study at any time. 3230 Should this be necessary, both parties will arrange the procedures on an individual study 3231 basis after review and consultation. In terminating the study, Roche and the investigator 3232 will assure that adequate consideration is given to the protection of the patients’ interests. 3233 The appropriate IRB/EC and Regulatory Agencies should be informed accordingly. 3234 3235 15. STUDY DOCUMENTATION, CRFS AND RECORD KEEPING 3236 3237 15.1 Investigator's Files / Retention of Documents 3238 The Investigator must maintain adequate and accurate records to enable the conduct of 3239 the study to be fully documented and the study data to be subsequently verified. These 3240 documents should be classified into two different separate categories [1] Investigator's 3241 Study File, and [2] patient clinical source documents.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3242 3243 The Investigator's Study File will contain the protocol/amendments, CRF/DCS and 3244 schedule of assessments, Independent Ethics Committee/Institutional Review Board and 3245 governmental approval with correspondence, sample informed consent, drug records, 3246 staff curriculum vitae and authorization forms and other appropriate 3247 documents/correspondence, etc. In addition at the end of the study the investigator will 3248 receive the patient data, which includes an audit trail containing a complete record of all 3249 changes to data, query resolution correspondence and reasons for changes, in human 3250 readable format on CD which also has to be kept with the Investigator’s Study File. 3251 3252 Patient clinical source documents [usually defined by the project in advance to record key 3253 efficacy/safety parameters independent of the CRFs] would include patient hospital/clinic 3254 records, physician's and nurse's notes, appointment book, original laboratory reports, 3255 ECG, EEG, X-ray, pathology and special assessment reports, signed informed consent 3256 forms, consultant letters, and patient screening and enrollment logs. The Investigator 3257 must keep the two categories of documents as described above (including the archival 3258 CD) on file for at least 15 years after completion or discontinuation of the study. After 3259 that period of time the documents may be destroyed, subject to local regulations. 3260 3261 Should the Investigator wish to assign the study records to another party or move them to 3262 another location, Roche must be notified in advance. 3263 3264 If the Investigator can not guarantee this archiving requirement at the investigational site 3265 for any or all of the documents, special arrangements must be made between the 3266 Investigator and Roche to store these in a sealed container[s] outside of the site so that 3267 they can be returned sealed to the Investigator in case of a regulatory audit. Where source 3268 documents are required for the continued care of the patient, appropriate copies should be 3269 made for storing outside of the site. 3270 3271 ICH GCP guidelines require that Investigators maintain information in the study patient’s 3272 records which corroborate data collected on the CRF(s). Completed CRF will be 3273 forwarded to Roche. 3274 3275 15.2 Source Documents and Background Data 3276 The investigator shall supply the sponsor on request with any required background data 3277 from the study documentation or clinic records. This is particularly important when errors 3278 in data transcription are suspected. In case of special problems and/or governmental 3279 queries or requests for audit inspections, it is also necessary to have access to the 3280 complete study records, provided that patient confidentiality is protected. 3281 3282 15.3 Audits and Inspections 3283 The investigator should understand that source documents for this trial should be made 3284 available to appropriately qualified personnel from the Roche Pharma Development 3285 Quality Assurance Unit or its designees, or to health authority inspectors after appropriate 3286 notification. The verification of the CRF data must be by direct inspection of source 3287 documents.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3288 3289 15.4 Case Report Forms or Electronic Case Report Forms 3290 Data for this study will be captured via an online Electronic Data Capture (EDC) system. 3291 The data collected in the source documents is entered onto the study eCRF. An audit trail 3292 will maintain a record of initial entries and changes made; reasons for change; time and 3293 date of entry; and user name of person authorizing entry or change. 3294 3295 For each patient randomized, an eCRF must be completed and electronically signed by 3296 the principal investigator or authorized delegate from the study staff. This also applies to 3297 records for those patients who fail to complete the study. If a patient withdraws from the 3298 study, the reason must be noted on the eCRF. If a patient is withdrawn from the study 3299 because of a treatment-limiting AE, thorough efforts should be made to clearly document 3300 the outcome. 3301 3302 The investigator should ensure the accuracy, completeness and timeliness of the data 3303 reported to the sponsor in the eCRFs and in all required reports. 3304 3305 15.5 Financial Disclosure 3306 The investigator(s) will provide the Sponsor with sufficient accurate financial 3307 information (PD35) to allow the Sponsor to submit complete and accurate financial 3308 certification or disclosure statements to the appropriate regulatory authorities. The 3309 investigator is responsible to promptly update any information provided to the Sponsor if 3310 relevant changes occur in the course of the investigation and for 1 year following the 3311 completion of the study (last patient, last visit). 3312 3313 16. MONITORING THE STUDY 3314 It is understood that the responsible Roche monitor [or designee] will contact and visit 3315 the investigator regularly and will be allowed, on request, to inspect the various records 3316 of the trial [CRFs and other pertinent data] provided that patient confidentiality is 3317 maintained in accord with local requirements. 3318 3319 It will be the monitor's responsibility to inspect the CRFs at regular intervals throughout 3320 the study, to verify the adherence to the protocol and the completeness, consistency and 3321 accuracy of the data being entered on them. The monitor must verify that the patient 3322 received the study drug assigned by the randomization center (by controlling the written 3323 confirmation of the randomization by IVRS). The monitor should have access to 3324 laboratory test reports and other patient records needed to verify the entries on the CRF. 3325 The investigator [or deputy] agrees to cooperate with the monitor to ensure that any 3326 problems detected in the course of these monitoring visits are resolved. 3327 3328 Roche Clinical Repository specimens will at all times be tracked in a manner consistent 3329 with Good Clinical Practice, by a quality controlled, auditable and validated Laboratory 3330 Information Management System, to ensure compliance with data confidentiality as well 3331 as adherence to authorized use of specimens as specified in the study protocol and ICF, 3332 respectively. Roche monitors and auditors will have direct access to appropriate parts of 3333 records relating to patients participating in this study for the purposes of verifying the 3334 data provided to Roche. The site will permit monitoring, audits, Institutional Review 3335 Board/Independent Ethics Committee (IRB/IEC) review, and regulatory inspections by

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3336 3337 providing direct access to source data and documents related to the RCR Research 3338 Project. 3339 3340 17. CONFIDENTIALITY OF TRIAL DOCUMENTS AND PATIENT RECORDS 3341 The investigator must assure that patients’ anonymity will be maintained and that their 3342 identities are protected from unauthorized parties. On CRFs or other documents 3343 submitted to the sponsor, patients should not be identified by their names, but by an 3344 identification code. The investigator should keep a patient enrollment log showing codes, 3345 names and addresses. The investigator should maintain documents not for submission to 3346 Roche, e.g., patients' written consent forms, in strict confidence. 3347 3348 Roche already maintains rigorous confidentiality standards for clinical studies by 3349 “coding” (i.e. assigning a unique patient ID number at the investigator site) all patients 3350 enrolled in Roche clinical studies. This means that patient names are not included in 3351 data sets that are transmitted to any Roche location. Given the sensitive nature of genetic 3352 data, Roche has implemented a number of additional processes to assure patient 3353 confidentiality. All specimens taken for inherited genetic research that will be stored in 3354 the RCR (see 5.6.1) undergo a second level of “coding”. At Roche, the specimen is 3355 transferred to a new tube and labeled with a new random number. This is referred to as 3356 “Double Coding (De-Identification)”. Data generated following the use of these 3357 specimens and all clinical data transferred from the clinical study database and 3358 considered relevant, will also be labeled with this same code. The “linking key” between 3359 the participant’s identification number and this new independent code will be stored in a 3360 secure database system. Access to the table linking the participant identification number 3361 to the specimen code will be strictly limited and monitored by audit trail. Legitimate 3362 operational reasons for accessing the “linking key” will be documented in a standard 3363 operating procedure. Access to the “linking key” for any other reason will require written 3364 approval from the Governance Committee responsible for the specimen(s). 3365 3366 18. CLINICAL STUDY REPORT (CSR) 3367 A clinical study report will be written and distributed to Health Authorities as required by 3368 applicable regulatory requirements. 3369 3370 To fulfill the requirement of the EU Directive No 75/318/EEC the CSR will be 3371 signed by a coordinating investigator who will be designated at a later stage. 3372 3373 Note: EU Regulation (EC) No.1901/2006, states: For pediatric studies the CSR must be 3374 distributed to the applicable Health Authorities within six months of completion of the 3375 study. 3376 3377 19. PUBLICATION OF DATA AND PROTECTION OF TRADE SECRETS 3378 Roche will comply with the requirements for publication of study results. 3379 3380 The results of this study may be published or presented at scientific meetings. If this is 3381 foreseen, the investigator agrees to submit all manuscripts or abstracts to Roche prior to 3382 submission. This allows the sponsor to protect proprietary information and to provide 3383 comments based on information from other studies that may not yet be available to the 3384 investigator.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3385 3386 In accordance with standard editorial and ethical practice, Roche will generally support 3387 publication of multicenter trials only in their entirety and not as individual center data. In 3388 this case, a coordinating investigator will be designated by mutual agreement. 3389 3390 Authorship will be determined by mutual agreement and in line with International 3391 Committee of Medical Journal Editors (ICMJE) authorship requirements.. Any formal 3392 publication of the study in which contribution of Roche personnel exceeded that of 3393 conventional monitoring will be considered as a joint publication by the investigator and 3394 the appropriate Roche personnel. 3395 3396 Data derived from RCR specimen analysis on individual patients will not be provided to 3397 study investigators, except where explicitly stipulated in a study protocol (e.g. if the 3398 result is an enrollment criterion). Exceptions may be granted (e.g. if biomarker data 3399 would be linked to safety issues). The aggregate results of any research conducted using 3400 RCR specimens will be available in accordance with the effective Roche policy on study 3401 data publication. 3402 3403 Any inventions and resulting patents, improvements and / or know- how originating from 3404 the use of the RCR will become and remain the exclusive and unburdened property of 3405 Roche, except where agreed otherwise.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3406 3407 Appendix 1 AEs Categories for Determining Relationship to Test Drug 3408 PROBABLE [must have first three] 3409 3410 This category applies to those AEs which are considered, with a high degree of certainty, to 3411 be related to the test drug. An AE may be considered probable, if: 3412 3413 1. It follows a reasonable temporal sequence from administration of the drug. 3414 2. It cannot be reasonably explained by the known characteristics of the patient’s 3415 clinical state, environmental or toxic factors, or other modes of therapy administered 3416 to the patient. 3417 3. It disappears or decreases on cessation or reduction in dose. [There are important 3418 exceptions when an AE does not disappear upon discontinuation of the drug, yet 3419 drug-relatedness clearly exists; e.g., [1] bone marrow depression, [2] tardive 3420 dyskinesias.] 3421 4. It follows a known pattern of response to the suspected medication. 3422 5. It reappears upon rechallenge.

3423 POSSIBLE [must have first two]

3424 This category applies to those AEs in which the connection with the test drug 3425 administration appears unlikely but cannot be ruled out with certainty. An AE may be 3426 considered possible if, or when: 3427 3428 1. It follows a reasonable temporal sequence from administration of the drug. 3429 2. It may have been produced by the patient’s clinical state, environmental or toxic 3430 factors, or other modes of therapy administered to the patient. 3431 3. It follows a known pattern of response to the suspected medication.

3432 REMOTE [must have first two]

3433 In general, this category is applicable to an AE which meets the following criteria: 3434 3435 1. It does not follow a reasonable temporal sequence from administration of the drug. 3436 2. It may readily have been produced by the patient’s clinical state, environmental or 3437 toxic factors, or other modes of therapy administered to the patient. 3438 3. It does not follow a known pattern of response to the suspected medication. 3439 4. It does not reappear or worsen when the drug is readministered.

3440 UNRELATED

3441 This category is applicable to those AEs which are judged to be clearly and 3442 incontrovertibly due only to extraneous causes [disease, environment, etc.] and do not 3443 meet the criteria for drug relationship listed under remote, possible, or probable.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3444 3445 Appendix 1 AEs Categories for Determining Relationship to Test Drug 3446 (Cont.) 3447 Probable Possible Remote Unrelated Clearly due to extraneous causes – – – +

Reasonable temporal association with drug + + – – administration

May be produced by patient clinical state, – + + + etc.

Known response pattern to suspected drug + + – –

Disappears or decreases on cessation or + – – – reduction in dose

Reappears on rechallenge + – – –

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3448 3449 Appendix 2 ICH Guidelines for Clinical Safety Data Management, 3450 Definitions and Standards for Expedited Reporting, Topic E2 3451 A serious adverse event is any experience that suggests a significant hazard, 3452 contraindication, side effect or precaution. It is any AE that at any dose fulfills at least 3453 one of the following criteria: 3454 3455 – is fatal; [results in death] [NOTE: death is an outcome, not an event] 3456 3457 – is Life-Threatening [NOTE: the term "Life-Threatening" refers to an event in which 3458 the patient was at immediate risk of death at the time of the event; it does not refer to 3459 an event which could hypothetically have caused a death had it been more severe] 3460 3461 – requires in-patient hospitalization or prolongation of existing hospitalization 3462 3463 – results in persistent or significant disability/incapacity 3464 3465 – is a congenital anomaly/birth defect 3466 3467 – is medically significant or requires intervention to prevent one or other of the 3468 outcomes listed above 3469 3470 Medical and scientific judgment should be exercised in deciding whether expedited 3471 reporting to the sponsor is appropriate in other situations, such as important medical 3472 events that may not be immediately life-threatening or result in death or hospitalization 3473 but may jeopardize the patient or may require intervention to prevent one of the outcomes 3474 listed in the definitions above. These situations should also usually be considered serious. 3475 3476 Examples of such events are intensive treatment in an emergency room or at home for 3477 allergic bronchospasm; blood dyscrasias or convulsions that do not result in hospitalization; 3478 or development of drug dependency or drug abuse. 3479 3480 An unexpected AE is one in which the nature or severity is not consistent with the 3481 applicable product information. 3482 3483 Causality is initially assessed by the investigator. For Serious Adverse Events, possible 3484 causes of the event is indicated by selecting one or more options. (Check all that apply) 3485 3486 – Pre-existing/Underlying disease – specify 3487 3488 – Study treatment – specify the drug(s) related to the event 3489 3490 – Other treatment (concomitant or previous) – specify 3491 3492 – Protocol-related procedure 3493 3494 – Other (e.g. accident, new or intercurrent illness) – specify 3495 3496 The term severe is a measure of intensity, thus a severe AE is not necessarily serious. For 3497 example, nausea of several hours' duration may be rated as severe, but may not be 3498 clinically serious.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3499 3500 Appendix 2 ICH Guidelines for Clinical Safety Data Management, 3501 Definitions and Standards for Expedited Reporting, Topic E2 3502 [Cont.] 3503 A serious adverse event occurring during the study or which comes to the attention of the 3504 investigator within 15 days after stopping the treatment or during the protocol-defined 3505 follow-up period, if this is longer, whether considered treatment-related or not, must be 3506 reported. In addition, a serious adverse event that occurs after this time, if considered 3507 related to test “drug”, should be reported. 3508 3509 Such preliminary reports will be followed by detailed descriptions later which will 3510 include copies of hospital case reports, autopsy reports and other documents when 3511 requested and applicable. 3512 3513 For serious adverse events, the following must be assessed and recorded on the AEs 3514 eform of the CRF: intensity, relationship to test substance, action taken, and outcome to 3515 date. 3516 3517 The investigator must notify the Ethics Review Committee/Institutional Review Board of 3518 a serious adverse event in writing as soon as is practical and in accordance with 3519 international and local laws and regulations. 3520 3521 ROCHE LOCAL COUNTRY CONTACT for SAEs: Local Monitor: 3522 3523 See attached Protocol Administrative and Contact Information & List of Investigators 3524 Form,[ gcp_for000227], for details of administrative and contact information. 3525 3526 ROCHE HEADQUARTERS CONTACT for SAEs and other medical emergencies: 3527 Clinical Operations/Clinical Science: 3528 3529 See attached Protocol Administrative and Contact Information & List of Investigators 3530 form,[gcp_for000227], for details of administrative and contact information. 3531 3532 24 HOUR MEDICAL COVERAGE: 3533 3534 Identification of a contact for 24 Hour Medical Coverage is mandatory to be compliant 3535 with worldwide regulatory agencies and to ensure the safety of study patients. 3536 3537 An Emergency Medical Call Center Help Desk will access the Roche Medical 3538 Emergency List, escalate emergency medical calls, provide medical translation service (if 3539 necessary), connect the investigator with the Roche medical contact for this study and 3540 track all calls. The Emergency Medical Call Center Help Desk will be manned 24 hours 3541 7 days a week. Toll free numbers will be distributed to all investigators running Roche 3542 Pharma Development clinical trials. The Help Desk will be used for medical 3543 emergencies outside regular business hours, or when the regular Clinical Science 3544 Leader/Clinical Pharmacology Leader cannot be reached. 3545 3546 See the attached Protocol Administrative and Contact Information & List of Investigators 3547 form [gcp_for000227], for details of administrative, contact information, and Emergency 3548 Medical Call Center Help Desk toll-free numbers.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3549 3550 Appendix 3 Structured Interview Guide For The Montgomery And 3551 Asberg Depression Rating Scale (Madrs-Sigma) 3552 Source [12] 3553 3554 INTERVIEWER: The questions in bold for each item should be asked exactly as written. 3555 Often these questions will elicit enough information about the severity and frequency of a 3556 symptom for you to rate the item with confidence. Follow-up questions are provided, 3557 however, for use when further exploration or additional clarification of symptoms is 3558 necessary. The specified questions should be asked until you have enough information to 3559 rate the item confidently. In some cases, you may also have to add your own follow-up 3560 questions to obtain necessary information. Note that questions in parentheses are 3561 optional, for use, for example, if information is unknown. 3562 3563 NOTES:

3564 Time period. The ratings should be based on the patient's condition in the past week.

3565 Change from baseline. In general, a symptom is rated as present only when it reflects a 3566 change from before the depression began (baseline). The interviewer must identify a 2- 3567 month period of non-depressed functioning and use this as a reference point. In some 3568 cases, such as when the patient has dysthymia the referent should be to the last time the 3569 person felt all right (i.e. not depressed or high) for at least a few weeks. 3570 3571 This interview guide is based on the Montgomery-Asberg Depression Rating Scale 3572 (MADRS) (Montgomery SA, Asberg M: A new depression scale designed to be sensitive 3573 to change. Br J Psychiatry ;1979 134: 382-9). The scale itself has been retained in its 3574 original form, except for reversing the order of the first two items. This guide adds 3575 interview questions to aid in the assessment and application of the MADRS. Previous 3576 versions of this guide appeared in 1988, 1992, 1996, and 2005. 3577 3578 ©2008 The Royal College of Psychiatrists. The SIGMA may be copied by individual 3579 researchers or clinicians for their own use without seeking permission from the 3580 publishers. The scale must be copied in full and all copies must acknowledge the 3581 following source: Williams JBW, Kobak KA. Development and reliability of a structured 3582 interview guide for the Montgomery-Asberg Depression Rating Scale (SIGMA). Br J 3583 Psychiatry 2008; 192: 52-58. Written permission must be obtained from the Royal 3584 College of Psychiatrists for copying and distribution to others or for replication ( in print, 3585 online or by any other medium). Correspondence should be addressed to Dr. J Williams, 3586 MedAvante, Inc., 100 American Metro Blvd., Suite 106, Hamilton, NJ 08619, USA; 3587 email: [email protected] To inform an ongoing survey, researchers and clinicians are 3588 asked to notify Dr Williams of their intention to use the SIGMA. 3589 3590 3591 *NOTE: Scale removed due to copyright protection.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3592 3593 Appendix 4 Clinical Global Impression (CGI) 3594 Source [13] 3595 3596 Clinical Global Impression – Severity of Illness (CGI-S) 3597 3598 Considering your total clinical experience with this particular population, how mentally 3599 ill is the patient at this time? 3600 3601 1 = normal, not at all ill 3602 3603 2 = borderline mentally ill 3604 3605 3 = mildly ill 3606 3607 4 = moderately ill 3608 3609 5 = markedly ill 3610 3611 6 = severely ill 3612 3613 7 = among the most extremely ill patients 3614 3615 Clinical Global Impression – Improvement (CGI-I) 3616 3617 Rate total improvement whether or not in your judgment it is due entirely to drug 3618 treatment. Compared to his/her condition at admission to the study, how much has he/she 3619 changed? 3620 3621 1 = very much improved 3622 3623 2 = much improved 3624 3625 3 = minimally improved 3626 3627 4 = no change 3628 3629 5 = minimally worse 3630 3631 6 = much worse 3632 3633 7 = very much worse 3634 3635 Guy W, ed. ECDEU Assessment Manual for Psychopharmacology. Revised, 1976. NIMH, U.S. 3636 Department of Health, Education, and Welfare, Pub No (ADM) 76-338, 1976:218.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3637 3638 Appendix 5 Patient Global Impression of Improvement (PGI-I) 3639 Check the one number that best describes how your depression is now, compared with 3640 how it was before you began taking medication in this study. 3641 3642 1 = very much better 3643 3644 2 = much better 3645 3646 3 = a little better 3647 3648 4 = no change 3649 3650 5 = a little worse 3651 3652 6 = much worse 3653 3654 7 = very much worse

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3655 3656 Appendix 6 Quick Inventory of Depressive Symptomatology Self Report

3657 (16-Item) (Self-Report) (QIDS-SR16) 3658 Source [14, 15] 3659 3660 *NOTE: Scale removed due to copyright protection. Please see www.ids-qids.org/ for more 3661 information. 3662

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3663 3664 3665 3666 3667 3668 Appendix 7 Quality of Life Enjoyment and Satisfaction Questionnaire- Short Form (Q-LES-Q-SF) 3669 Source [16] 3670 3671 QUALITY OF LIFE ENJOYMENT AND SATISFACTION QUESTIONNAIRE – SHORT FORM* 3672 3673 (Q-LES-Q-SF) 3674 CONFIDENTIAL 3675 Jean Endicott, Ph.D** 3676 3677 This questionnaire is designed to help assess the degree of enjoyment and satisfaction experienced during the past week.

3678 Name ID# _ Date: / / oh rtclN260C R4153 -Pg 94 Page - (RO4917523) C NP25620 Roche Protocol 3679 (3-10)+ (11-16)+ 3680 3681 Sex: 1 - Male, 2 - Female Age: 3682 3683 (17)+ (18-19)+ 3684 3685 Study # 3687 Group: 36883686 3689 (20-21)+ (22-24)+ (79-80 = DA+) 3690 3691 3696 6/13/95-R 3692 3693 * The Short Form of the Q-LES-Q has the same content as the general activities section of the regular Q-LES-Q. 3694 ** Developed with the assistance of Wilma Harrison, M.D. and Dianne Schechter, Ph.D. (11/29/90) 36973695 Available from Jean Endicott, Ph.D., Department of Research Assessment and Training, Unit 123, 1051 Riverside Drive, New York, NY 10032. (Under 3698 Copyright) 3699 3700 NOTE: Scale removed due to copyright protection.

Downloaded From: https://jamanetwork.com/ on 09/29/2021 3701 Appendix 8 Sheehan Disability Scale (SDS) 3702 Source [17] 3703 *NOTE: Scale removed due to copyright protection. 3704 3705 CONFIDENTIAL CONFIDENTIAL Roche Protocol NP25620 C (RO4917523) - Page Page 95 - (RO4917523) C NP25620 Roche Protocol

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3706 Appendix 9 Brief Psychiatric Rating Scale, Positive Symptom Subscale 3707 (4 items) 3708 Source [18] 3709 3710 BRIEF PSYCHIATRIC RATING SCALE (BPRS) 3711 3712 Please enter the score for the term which best describes the patient’s condition 3713 3714 0 = not assessed, 1 = not present, 2 = very mild, 3 = mild, 4 = moderate, 5 = moderately 3715 severe, 6 = severe, 7 = extremely severe 3716 3717 Item 4. Conceptual Disorganization 3718 3719 Degree to which the thought processes are confused, disconnected, or disorganized, Rate 3720 on the basis of integration of the verbal products of the patient. 3721 3722 Do not rate on the basis of patient’s subjective impression of his own level of functioning. 3723 3724 Score 3725 3726 Item 11. Suspiciousness 3727 3728 Belief (delusional or otherwise) that others have now, or have had in the past, malicious 3729 or discriminatory intent toward the patient. On the basis of verbal report, rate only those 3730 suspicions which are currently held whether they concern past or present circumstances. 3731 3732 Score 3733 3734 Item 12. Hallucinatory Behavior 3735 3736 Perceptions without normal external stimulus correspondence. Rate only those 3737 experiences which are reported to have occurred within the last week and which are 3738 described as distinctly different from the thought and imagery processes of normal people. 3739 3740 Score 3741 3742 Item 15. Unusual Thought Content 3743 3744 Unusual, odd, strange or bizarre thought content. Rate here the degree of unusualness, not 3745 the degree of disorganization of thought processes. 3746 3747 Score 3748 3749 Adapted from Overall JE, Gorham DR: „The Brief Psychiatric Rating Scale. 3750 „Psychological Reports 10:799-812. 1962

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3751 3752 Appendix 10 Young Mania Rating Scale (YMRS) (item 1) 3753 Source [19] 3754 3755 *NOTE: Scale removed due to copyright protection. 3756 3757 © 1978 The Royal College of Psychiatrists

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3758 STATISTICAL ANALYSIS PLAN

TITLE: A randomized, double-blind, parallel-group study of the safety and efficacy of RO4917523 versus placebo, as adjunctive therapy in patients with major depressive disorder with inadequate response to ongoing antidepressant treatment. PROTOCOL NUMBER: NP25620

IND NUMBER: 103001

EUDRACT NUMBER: 2011-001436-33

SPONSOR: F. Hoffmann-La Roche Ltd 3759 3760 3761 SAP APPROVAL 3762 3763 3764 Plan Number / Version: 1 3765 Date: See last date in electronic signature manifestation 3766 below. 3767 Plan approved by: See electronic signature manifestation 3768 below. 3769 3770 3771 3772 3773 3774 3775 Name Reason for Signing Date and Time 3776 (UTC) 3777 Beyer,Ulrich 3779 Project Statistician 3781 03-Jul-2013 09:33:10 3778 3780

F. Hoffmann-La Roche Ltd 1 NP25620 Statistical Analysis Plan

Downloaded From: https://jamanetwork.com/ on 09/29/2021 3782 TABLE OF CONTENTS 3783 3784 1. BACKGROUND ...... 4 3785 3786 2. STUDY DESIGN ...... 4 3787 2.1 Protocol Synopsis ...... 4 2.2 Outcome Measures ...... 4 2.3 Determination of sample size ...... 4 2.4 Analysis Timing ...... 5 2.4.1 Data to Be Analyzed ...... 5 2.4.2 Measures to Minimize Bias ...... 5

3. STUD Y CONDUCT ...... 5

3.1 Randomization...... 5 3.2 Data Monitoring ...... 6 3788 3789 4. STATISTICAL METHODS ...... 6 3790 3791 4.1 Analysis Populations ...... 6 3792 3793 4.1.1 Intent-to-Treat Analysis Population...... 6 3794 3795 4.1.2 Per Protocol Population ...... 6 3796 3797 4.1.3 Completer Analysis Population ...... 7 3798 3799 4.1.4 Safety Analysis Population ...... 7 3800 3801 4.2 Analysis of study conduct ...... 8 3802 3803 4.2.1 Study Enrollment ...... 8 3804 3805 4.2.2 Protocol Violations ...... 8 3806 3807 4.2.3 Patient Disposition ...... 8 3808 3809 4.3 Analysis of treatment group comparability ...... 8 3810 3811 4.3.1 Demographics ...... 8 3812 3813 4.3.2 Baseline Disease Characteristics ...... 8 3814 3815 4.3.3 History of Major Depressive Disorder ...... 8 3816 3817 4.3.4 Other Diseases Previous and Current at Baseline ...... 9 3818 3819 4.3.5 Previous and Concomitant Medications (Other 3820 than Study Treatment) ...... 9 3821 3822 4.4 Efficacy Analysis...... 10 3823 3824 4.4.1 Primary Efficacy Endpoint...... 11 3825 3826 4.4.2 Secondary Efficacy Endpoints ...... 13

F. Hoffmann-La Roche Ltd 2 NP25620 Statistical Analysis Plan

Downloaded From: https://jamanetwork.com/ on 09/29/2021 3827 TABLE OF CONTENTS (contd.) 3828 4.4.3 Exploratory Efficacy Endpoints ...... 14 4.4.4 Sensitivity Analyses ...... 16 4.4.5 Subgroup Analyses ...... 17 4.5 Pharmacokinetic and Pharmacodynamic Analyses ...... 18 4.5.1 Population Pharmacokinetic Analysis ...... 18 4.6 Biomarker Analysis ...... 18 4.7 Safety Analyses ...... 18 4.7.1 Exposure to Study Drug...... 19 4.7.2 Adverse Events ...... 19 4.7.3 Suicidality Assessment ...... 21 4.7.4 Laboratory Data ...... 21 4.7.5 Vital Signs and Weight...... 21 4.7.6 Physical Examinations...... 22 4.7.7 ECGs ...... 22 4.8 Missing Data ...... 22 4.9 Interim Analyses ...... 23

5. REF ERENCES ...... 23 3829 TABLE OF TABLES 3830 3831 Table 1 Classification of Treatment Period for Medications ...... 10 3832 Table 2 Adult Normal Ranges for ECG Listings and Summary Tables .... 22 3833 3834 3835 3836 TABLE OF APPENDICES 3837 3838 Appendix 1 Protocol Synopsis ...... 24

F. Hoffmann-La Roche Ltd 3 NP25620 Statistical Analysis Plan

Downloaded From: https://jamanetwork.com/ on 09/29/2021 3839 1. BACKGROUND 3840 3841 This Statistical Analysis Plan (SAP) documents the statistical methods for summarizing 3842 and analyzing the efficacy and safety data from study NP25620 to be collected from 3843 patients with major depressive disorder with an inadequate response to ongoing 3844 antidepressant treatment who will be administered RO4917523. The main purpose of 3845 this SAP is to describe the data handling rules, derivation rules, and statistical analysis 3846 methods. 3847 3848 2. STUDY DESIGN 3849 3850 Study NP25620 is a randomized, double-blind, placebo-controlled, parallel-group, 3851 multicenter study that will evaluate two fixed doses of RO4917523 versus placebo as 3852 adjunctive treatment for 6 weeks in patients with major depressive disorder with 3853 inadequate response to ongoing antidepressant therapy. 3854 3855 Following screening procedures to confirm eligibility, patients on ongoing antidepressant 3856 therapy will be randomized to one of the three treatment arms: a) RO4917523 0.5 mg 3857 once daily, b) RO4917523 1.5 mg once daily, or c) placebo once daily. 3858 3859 This outpatient trial consists of the following periods: 3860 3861 • Screening period: up to 14 days 3862 3863 • Double-blind treatment period: 6 weeks 3864 3865 • Follow-up period: 21 days 3866 3867 2.1 PROTOCOL SYNOPSIS 3868 3869 The protocol synopsis is in Appendix 1. 3870 3871 2.2 OUTCOME MEASURES 3872 3873 See the protocol synopsis in Appendix 1 for the efficacy, safety, and pharmacokinetic 3874 (PK) outcome measures. 3875 3876 2.3 DETERMINATION OF SAMPLE SIZE 3877 3878 A sample size of approximately 100 patients per group (approximately 300 patients for 3879 the three groups) has been chosen to obtain a power of approximately 80% at two-sided 3880 maximum family-wise error rate of 0.05 in a closed testing procedure. The power 3881 calculation was based on simulations of the mixed-effect model repeat measures 3882 (MMRM) analysis planned for the primary efficacy variable. The following assumptions 3883 were included: 3884 • Seven post-baseline assessment visits 3885 3886 • An overall dropout rate of 15%, incremental rates over the 6-week double-blind 3887 treatment period

F. Hoffmann-La Roche Ltd 4 NP25620 Statistical Analysis Plan

Downloaded From: https://jamanetwork.com/ on 09/29/2021 3888 • An effect size of 0.46 for one dose group at Day 42, assuming increasing magnitude 3889 of treatment difference over the period and an effect size of 0.0 for the other dose 3890 group 3891 3892 • A moderately decreasing correlation structure between assessments that are further 3893 apart 3894 3895 2.4 ANALYSIS TIMING 3896 3897 The primary efficacy analysis will be performed at the end of the study, after the last 3898 patient has completed the last follow-up visit. 3899 3900 2.4.1 Data to Be Analyzed 3901 3902 In the case that (safety) follow-up data and patient data from the additional Japanese 3903 patients will continue after the primary and secondary efficacy analysis, the 3904 efficacy analysis will be carried out by an independent group consisting of the Sponsor 3905 and the contract research organization as described in Section 2.4.2. In this case, the 3906 unblinded Sponsor’s Neuroscience Therapeutic Area Head and/or designees will have 3907 no contact with the study management team regarding the conduct of the study and data 3908 cleaning activities until final database lock. 3909 3910 2.4.2 Measures to Minimize Bias 3911 3912 The randomization list will only be provided to the independent group. The data will be 3913 stored in an access-restricted area to which only the independent group will have access. 3914 Unblinded summary data will be reviewed only by the Neuroscience Therapeutic Area 3915 Head and/or designees who will have no contact with the study management team 3916 regarding the conduct of the study and data cleaning activities until database lock. The 3917 results will be presented to representatives from upper management who will be 3918 involved directly in the decision on the planning of future research for this project. 3919 Dissemination of the results will be limited, and, particularly, the details will not be 3920 distributed to those who are directly involved the conduct of the study, and the data 3921 cleaning activities. 3922 3923 An audit trail will be maintained to track any changes made to efficacy data points 3924 between the efficacy analysis and final database lock. If any change in the variables 3925 used for the analysis occurs, the respective analyses will be repeated and both results 3926 will be reported in the CSR. 3927 3928 3. STUDY CONDUCT 3929 3930 3.1 RANDOMIZATION 3931 3932 Randomization will be performed centrally by telephone or web using an interactive 3933 response system (IxRS). After being screened, those patients who meet all eligibility 3934 criteria will be randomly assigned to one of the three treatment groups: placebo, 3935 RO4917523 0.5 mg, or RO4917523 1.5 mg, in the ratio of 1:1:1.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3936 The patient numbers will be allocated sequentially in the order in which the patients are 3937 enrolled according to the specification document agreed with the IxRS vendor. The 3938 treatment allocation will be stratified by geographical region/country, as follows: 3939 • Europe, United States, Latin America, and Japan 3940 3941 3942 The study is planned in a double-blind manner to minimize potential bias. The Sponsor 3943 will be blinded to the study treatments (those patients are randomized to and those 3944 actually given). 3945 3946 3.2 DATA MONITORING 3947 3948 An independent Data Monitoring Committee (iDMC) will review unblinded safety data at 3949 scheduled time intervals and on an ad-hoc basis as needed. The safety data package 3950 will be prepared by a team external to the Sponsor. Details are described in the iDMC 3951 Charter. 3952 3953 4. STATISTICAL METHODS 3954 3955 4.1 ANALYSIS POPULATIONS 3956 3957 The following analysis populations will be defined: intent to treat (ITT), per-protocol (PP), 3958 completer (CP) and safety. 3959 3960 4.1.1 Intent-to-Treat Analysis Population 3961 3962 The ITT analysis population will include all randomized patients who received at least 3963 one dose of the randomized study drug. Patients who receive study drug different from 3964 that to which they were randomized will be included in the group to which they were 3965 randomized. 3966 3967 The ITT analysis population will be the primary analysis population for all analyses of 3968 primary and secondary clinical efficacy data. 3969 3970 4.1.2 Per Protocol Population 3971 3972 The PP analysis population will be defined as the subset of the ITT population that 3973 excludes those patients who have major protocol violations affecting efficacy 3974 assessments. Patients in the PP population should meet all the following criteria: 3975 • Meet all inclusion criteria with the exception of the body mass index (BMI) 3976 requirement, contraceptive protections, and stability of existing medication regimens 3977 for other medical conditions for at least 6 weeks prior to baseline 3978 3979 • Show overall compliance of 80% − 120% with study drug during the individual 3980 treatment period (Day 42/end of treatment [EOT]) (see Section 4.7.1 for calculation 3981 of compliance) 3982 3983 • Have assessment of Montgomery Asberg Depression Rating Scale (MADRS) 3984 conducted by certified site raters

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 3985 • Have valid primary efficacy variable values (rater MADRS total score) at baseline 3986 and at least one post-baseline treatment assessment 3987 3988 • Have none of the following protocol violation codes: pregnancy positive, ongoing 3989 antidepressant medication modified or non-SSRI/SNRI, prohibited medication 3990 3991 3992 If, during the active phase of treatment the patients receive a drug other than the one to 3993 which they were randomized and if overall compliance is within 80% − 120%, the efficacy 3994 variables will be analyzed for this population on the basis of the actual treatment 3995 received. 3996 3997 PP analyses will be performed on the primary efficacy endpoint if a > 10% difference 3998 exists between the ITT and PP populations in the number of patients qualified to be 3999 assessed for consistency of the results between the two populations. 4000 4001 4.1.3 Completer Analysis Population 4002 4003 The CP analysis population will be defined as the subset of the PP population that 4004 excludes those patients with an incomplete treatment period. Patients in the CP 4005 population should meet all the following criteria: 4006 • Completed 6 weeks of double blind treatment (i.e. duration of treatment ≥ 40 days) 4007 4008 • Have valid primary efficacy variable values (MADRS total score) at baseline and 4009 Day 42 4010 4011 If, during the active phase of treatment the patients receive a drug other than the one to 4012 which they were randomized and if overall compliance is within 80% − 120%, the efficacy 4013 variables will be analyzed for this population on the basis of the actual treatment 4014 received. 4015 4016 CP analyses will be performed on the primary efficacy endpoint if a > 10% difference 4017 exists between the CP and ITT populations in the number of patients qualified to be 4018 assessed for consistency of the results between the two populations. 4019 4020 4.1.4 Safety Analysis Population 4021 4022 The safety analysis population will consist of all patients who have received at least one 4023 dose of randomized study medication, regardless of whether they withdrew prematurely 4024 or not. Patients whose first randomized study medication received differed from the 4025 medication to which they were randomized will be included in the group according to the 4026 first randomized study medication actually taken. 4027 4028 All safety parameters will be summarized and presented in tables based on this safety 4029 population.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4030 4.2 ANALYSIS OF STUDY CONDUCT 4031 4.2.1 Study Enrollment 4032 • The number of patients for each of the ITT, PP, and safety populations will be 4033 summarized by treatment group. 4034 4035 o The number of patients excluded from each of the populations will be 4036 summarized by reason for exclusion 4037 4038 • The number of patients enrolled at each site will be summarized by treatment group. 4039 4040 4.2.2 Protocol Violations 4041 4042 The major protocol violations will be identified according to the Management of 4043 Violations to Protocol Specifications document and recorded on the Roche PD 99V Form 4044 before database lock. The number and percentage of patients with major protocol 4045 violations will be summarized by treatment group and protocol violation criterion. 4046 4047 4.2.3 Patient Disposition 4048 4049 The number and percentage of patients in each treatment group will be summarized by 4050 duration (based on the last dosing day and the date of last contact day, respectively) and 4051 reason for withdrawal during the treatment period and follow-up period, respectively. 4052 4053 4.3 ANALYSIS OF TREATMENT GROUP COMPARABILITY 4054 4055 Summary tables of demographics and baseline disease characteristics will be produced 4056 for the ITT and safety populations. No formal hypothesis tests will be performed. 4057 4058 4.3.1 Demographics 4059 4060 Summary statistics will be presented for each treatment group for the following 4061 demographics and baseline characteristics: sex, age, race, ethnicity, geographic 4062 region/country (Europe, United States, Latin America, and Japan), female reproductive 4063 status, weight, height, BMI, smoking status/history, and years of education. 4064 4065 4.3.2 Baseline Disease Characteristics 4066 4067 Summary statistics will be presented for each treatment group for the following baseline 4068 disease characteristics: MADRS total rater score, Clinical Global Impression of Severity 4069 (CGI-S), and others if appropriate. 4070 4071 4.3.3 History of Major Depressive Disorder 4072 4073 Summary statistics and listings will be generated for each treatment group for patient’s 4074 history of major depressive disorder. The key variables from the electronic Case Report 4075 Form (eCRF) pages for the psychiatric history and evaluation of depression will be 4076 summarized.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4077 4.3.4 Other Diseases Previous and Current at Baseline 4078 4079 For all diseases (other than depression), the term entered by the investigator to describe 4080 the disease (the “verbatim term”) will be assigned to a standardized term 4081 (the “preferred term”) and system organ class based on the Medical Dictionary for 4082 Regulatory Activities (MedDRA). All analyses will be performed using these preferred 4083 terms and body systems. Diagnoses will be categorized as “not active,” or “active” at the 4084 screening visit, using the end date or “ongoing” tick box responses on the Medical 4085 History page of the eCRF. 4086 4087 The number and percentage of patients with previous diseases (i.e., those marked “not 4088 active”) will be summarized by treatment group. Multiple occurrences of a same disease 4089 (i.e., same coded term) in the same patient will be counted only once. Diseases 4090 concurrent at baseline (i.e., those marked “active”) will be summarized similarly in a 4091 separate table. 4092 4093 4.3.5 Previous and Concomitant Medications (Other than Study 4094 Treatment) 4095 4096 For all medications, the term entered by the investigator to describe the medication 4097 (the “verbatim term”) will be assigned to a standardized term (the “preferred term”) and 4098 drug class based on the International Non-proprietary Name (INN) Drug Terms and 4099 Procedures Dictionary. All analyses will be performed using these preferred terms and 4100 medication classes. 4101 4102 The number and percentage of patients taking each medication will be presented for each 4103 treatment group. Previous and concomitant medications will be summarized separately 4104 by the purpose of the treatment (e.g., for depression treatment, for adverse events [AEs], 4105 etc.). Previous medications completed 6 months prior to screening will not be 4106 summarized. Multiple occurrences of a same medication taken by same patient 4107 (i.e., same coded term) will be counted only once. All summary tables will be sorted by 4108 medication class (in decreasing order of overall incidence), then by preferred term 4109 (in decreasing order of overall incidence). 4110 4111 The rules shown in Table 1 will be applied in classifying the medications into previous, 4112 concomitant, or previous and concomitant.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4113 Table 1 Classification of Treatment Period for Medications 4114 CRF page Medication Start Medication End Assigned Treatment Period where reported Date Date Previous and Missing, or before Before start date of PREVIOUS concomitant start date of first first dose of study treatments dose of study medication and medication ongoing box not checked Missing, or before On or after start PREVIOUS_CONCOMITANT start date of first date of first dose of dose of study study medication or medication ongoing box checked

Before start date of Missing and PREVIOUS_CONCOMITANT first dose of study ongoing box not medication checked

On or after start CONCOMITANT date of first dose of study medication

Missing Missing PREVIOUS_CONCOMITANT

4115 4116 4117 4.4 EFFICACY ANALYSIS 4118 4119 All efficacy data collected will be grouped by actual assessment visit day relative to the 4120 first day (i.e., Day 1) or last day of randomized study drug. 4121 4122 For the screening assessments, the scheduled visit will be used regardless of the actual 4123 assessment days relative to the first day of study drug. 4124 4125 In general, the baseline value will be defined as the last non-missing value recorded 4126 prior to or on the first day of the study drug, using the latter if both are available.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4127 Other assessments during the double-blind period and follow-up period will have the 4128 following time windows: 4129 Visit Start Day End Day Target Day Day 3 2 4 3 Day 7 5 9 7 Day 11 10 12 11 Day 14 13 17 14 Day 21 18 24 21 Day 28 25 31 28 Day 35 32 38 35 Day 42 39 44 42 Follow-up Date of last dose + 18 Date of last dose + 24 Date of last dose + 21 4130 4131 Note that the above defined time windows (day intervals) will be used for all parameters, 4132 including those that were not scheduled to be collected at all visits for the sake of 4133 consistency. Data from assessments performed during a visit unscheduled for the 4134 parameter will not be included in summary tables or hypothesis testing. 4135 4136 If multiple valid (non-missing) values for a variable are recorded at the same time 4137 window, one record will be selected for summary of the data by the following priority: 4138 1. The originally scheduled visit assessment 4139 4140 2. The assessment closest to the target assessment day 4141 4142 3. The last assessment within the time window 4143 4144 4.4.1 Primary Efficacy Endpoint 4145 4.4.1.1 Definition of Primary Efficacy Endpoint 4146 The primary efficacy endpoint of the study is the change from baseline in the MADRS 4147 total score at Day 42, as assessed by the site raters. 4148 4149 MADRS comprises 10 items; each item is rated on a 0–6 scale with anchors at 2-point 4150 intervals 4151 4152 MADRS total score = sum of all 10 items 4153 4154 If any item score is missing, then the MADRS total score will be set to missing.

4155 A higher MADRS total score indicates a greater pathology.

4156 The change from baseline of MADRS total score at Day 42 will be calculated as the total 4157 score at Day 42 minus the total score at baseline. A negative change from baseline in 4158 MADRS total score indicates improvement.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4159 4.4.1.2 Hypothesis Testing 4160 For the primary endpoint the following hypotheses will be tested: 4161 4162 H0: The mean reduction in total MADRS score at Day 42 in both dose groups are

4163 same as in the placebo group μRO 1 = μPlacebo = μRO 2 4164 4165 versus 4166 4167 H1: At least in one dose group the mean reduction in total MADRS score at Day 42

4168 is different from the placebo group μRO 1 ≠ μPlacebo or μRO 2 ≠ μPlacebo 4169 4170 For each dose group i=1,2 the following hypotheses will be tested: 4171 4172 H0i: The mean reduction in total MADRS score at Day 42 in dose group i is the same

4173 as in the placebo group μRO i = μPlacebo 4174 4175 versus 4176 4177 H1i: The mean reduction in total MADRS score at Day 42 in dose group i is different

4178 from the placebo group μRO i ≠ μPlacebo 4179 4180 A closed testing procedure will be utilized: The overall hypothesis H0 will be tested at the 4181 two-sided significance level α = 0.05 using the Dunnett test within the MMRM analysis. If

4182 H0 will be rejected, the elementary hypothesis H0i will be tested at the two-sided 4183 significance level α = 0.05 utilizing t- test within MMRM, separately for i=1, 2. The 4184 closed testing procedure has a two-sided maximum family-wise error rates of 0.05. 4185 Therefore no further adjustment for multiplicity is required. 4186 4187 4.4.1.3 Statistical Analysis Method 4188 For the assessment of differences between each RO4917523 group and placebo with 4189 regard to the mean change from baseline in MADRS total score at Day 42, an MMRM 4190 analysis incorporating data from up to Day 42 of treatment will be used for all the data 4191 collected over time with consideration of the variance-covariance matrix of the repeated 4192 measures. This method allows a general unstructured variance-covariance matrix and 4193 will include data from patients with incomplete data from some scheduled time points. 4194 4195 The model 4196 4197 yijk = μ + πk + τi + (π τ)ik + γij + β* Mij0 + εijk, i=1,2,3, j=1,…,ni, k=1,7,..,35,42 4198 4199 will include the change from baseline in MADRS total score yijk of subject j in the 4200 treatment group i at visit day k as the dependent variable. The fixed effects in the model 4201 will include the independent variables the overall mean μ, of the fixed categorical effects

4202 of treatment τi, assessment day relative to the first dose of randomized study drug

4203 (i.e., time) πk, treatment-by-time interaction (π τ)ik, geographical region γij, along with the

4204 baseline MADRS total score as a covariate β* Mij0. Time will be treated as a repeated

4205 variable within a patient. The individual within-patients residual vectors εij = (εij1,…, εij,42) 4206 are assumed to be independently normal distributed with identical unstructured 4207 variance-covariance matrix. Patient, treatment, and time will be treated as factor

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4208 variables. The Restricted Maximum Likelihood (REML) method will be used for 4209 estimating the variance components. Denominator degrees of freedom will be estimated 4210 using Satterthwaite’s approximation. 4211 4212 A treatment-by-time interaction contrast (i.e., pairwise treatment group contrast at 4213 Day 42) will be constructed to estimate the difference between each of the RO4917523 4214 doses versus placebo in the mean change from baseline to Day 42. Based on the 4215 analysis above, least square means, standard errors, the 95% confidence intervals (CIs) 4216 of the treatment difference, and p-values of t-test and Dunnett test will be reported for 4217 each comparison. 4218 4219 The MADRS total score and the change from baseline in MADRS total score at each 4220 scheduled visit will be summarized using descriptive statistics for each treatment group. 4221 4222 4.4.1.4 Adjustment for Multiple Testing 4223 The adjustment for multiple testing is incorporated into the hypothesis testing in 4224 Section 4.4.1.2. 4225 4226 4.4.2 Secondary Efficacy Endpoints 4227 4.4.2.1 Definition of Secondary Efficacy Endpoints 4228 The secondary efficacy variables include the following: 4229 4230 • Change from baseline in the CGI-S rating score at Day 42. 4231 4232 The CGI-S is rated on a 1–7 scale. Ratings are based on degree of severity. A 4233 CGI-S score of 1 refers to “normal” and a score of 7 refers to “most severely ill.” 4234 • Distribution of the Clinical Global Impression of Improvement (CGI-I) rating score at 4235 Day 42. 4236 4237 The CGI-I is rated on a 1–7 scale. Ratings are based on the degree of improvement. 4238 A CGI-C score of 1 refers to “very much improved” and a score of 7 refers to “very 4239 much worse.” 4240 4241 • Frequency of patients exhibiting remission, as defined by a MADRS total score 4242 of ≤ 10 at Day 42. 4243 4244 • Frequency of patients exhibiting response, as defined by a MADRS total score at 4245 Day 42 ≤ 50% of the baseline score. 4246 4247 The percentage of the score at Day 42 relative to the baseline score is calculated as 4248 (MADRS total score at Day 42) / (MADRS total score at baseline) *100% 4249 4250 • Quick Inventory of Depressive Symptomatology Self Report (QIDS-SR16) change 4251 from baseline to Day 42. 4252 4253 QIDS-SR16 is comprised of 16 items. Each item is scored on a 4-point anchored 4254 scale, representing least severe (0) to most severe (3).

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4255 • Patient Global Impression of Improvement (PGI-I) score at Day 42. 4256 4257 The PGI-I is rated on a 1–7 scale. Ratings are based on the degree of improvement. 4258 A PGI-I score of 1 refers to “very much improved” and a score of 7 refers to “very 4259 much worse.” 4260 4261 4.4.2.2 Analysis Methods of the Secondary Efficacy Endpoints 4262 There will be no formal statistical analysis for secondary efficacy endpoints. 4263 4264 Continuous Variables 4265 4266 The secondary efficacy variable QIDS will be analyzed using the MMRM method 4267 described in Section 4.4.1.3 for the primary efficacy variable and the analysis of 4268 covariance (ANCOVA) described in Section 4.4.3 for the key exploratory endpoint. 4269 4270 Categorical Variables 4271 4272 Ordered categorical variables, such as change in CGI-S, CGI-I, and PGI-I, all at Day 42, 4273 will be analyzed using Wilcoxon’s rank test. For CGI-I, PG-I, and change in CGI-S, the 4274 MMRM method described in Section 4.4.1.3 will be performed as an additional, 4275 approximative analysis. For CGI-I, no baseline variable will be used as a covariate. 4276 4277 Binary data, such as remissions and responders, will be analyzed using Fisher’s exact 4278 test. 4279 4280 The values, frequencies, and changes from baseline in the secondary efficacy variables, 4281 if applicable, at each scheduled visit will be summarized using descriptive statistics for 4282 each treatment group. 4283 4284 Given the exploratory nature of the secondary efficacy variables, adjustment for multiple 4285 comparisons will not be applied to any of the secondary efficacy variables. 4286 4287 The analysis for categorical data will be applied to the observed data and last 4288 observation carried forwarded values (Section 4.8), except for the MMRM analyses. 4289 4290 4.4.3 Exploratory Efficacy Endpoints 4291 4292 The key exploratory endpoint is: 4293 4294 • Change in the MADRS total score from baseline to Day 42 for exploratory 4295 evaluation. 4296 4297 4298 The other exploratory endpoints are: 4299 4300 • Frequency of patients who meet MADRS responder criteria (see Section 4.4.2.1) 4301 plus CGI-I score of 1) “very much improved” or 2) “much improved” at Day 42 4302 4303 • Frequency of patients who meet MADRS remission criteria(see Section 4.4.2.1) 4304 plus CGI-I score of 1) “very much improved” or 2) “much improved” at Day 42

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4305 • Cambridge Neuropsychiatric Test Automated Battery (CANTAB®) Cognitive Test 4306 Battery subset 4307 A subset of the CANTAB computerized battery provided by Cambridge Cognition™ 4308 will be used to assess function in the domains memory, attention, executive function 4309 and speed of processing: Motor Control Task (MOT), Rapid Visual Processing 4310 (RVP), Delayed Match to Sample (DMS), Emotional Recognition Task (ERT), Paired 4311 Associates Learning (PAL), the Stockings of Cambridge (SOC, at screening) or the 4312 One-Touch Stockings of Cambridge (OTS, at subsequent visits), and Attention 4313 Shifting Test (AST). 4314 4315 • Quality of Life Enjoyment and Satisfaction Questionnaire-Short Form (Q-LES-Q-SF). 4316 4317 Q-LES-Q-SF is a 16-item short form of a 60-item full version. Each item is scored 4318 from 1 for “Very Dissatisfied” to 5 for “Very Satisfied.” 4319 4320 • Sheehan Disability Scale (SDS) 4321 4322 SDS is a combination of three self-rated items using 10-points scales for impairment 4323 at 1) work or school, 2) social life or leisure activities, and 3) home life or family 4324 responsibilities. 4325 4326 • Change in the MADRS total score from baseline to Days 3, 7, 11, 14, 21, 28, and 35. 4327 4328 4329 The key exploratory endpoint will be assessed at Day 42 by ANCOVA using the last 4330 observation carried forward (LOCF) method as exploratory analysis. The model will 4331 include the baseline measure as covariate, and the categorical variable treatment. The 4332 effect size of each dose compared to placebo and its 90% CI will be calculated using 4333 estimates from the treatment contrasts. The results will be used for internal decisions 4334 regarding future research in the project. 4335 4336 Change in MADRS score from baseline to different visits during the treatment period 4337 except Day 42 will be analyzed using the model described above for the key exploratory 4338 variable. The LOCF method will be used. 4339 4340 The exploratory variables Q-LES-Q-SF and SDS will be analyzed using the MMRM 4341 method described in Section 4.4.1.3 for the primary efficacy variable.

4342 CANTAB and speed of onset will be summarized descriptively.

4343 Binary data, such as remissions + CGI and responders + CGI, will be analyzed using 4344 Fisher’s exact test. 4345 4346 The values, frequencies, and changes from baseline in the secondary efficacy variables 4347 will be summarized using descriptive statistics for each treatment group.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4348 4.4.4 Sensitivity Analyses 4349 4.4.4.1 Effects on Covariates 4350 To assess effects of relevant covariates (baseline MADRS score and geographic 4351 region/country) on the outcome of the primary efficacy variable, analyses of interaction 4352 effects will be performed. 4353 4354 The interaction effects of treatment-by-baseline score and treatment-by-geographic 4355 region/country of the study centers will be assessed at Day 42 using ANCOVA. A model 4356 including main effects of treatment, the covariate, and the treatment-by-covariate 4357 interaction term will be used. The statistical significance of the interaction terms will be 4358 assessed at the 0.10 α level for the homogeneity of the treatment effect across the 4359 baseline value or the geographic region/country of the study centers. If a significant 4360 interaction effect is noted, the nature of the interaction (e.g., quantitative or qualitative) will 4361 be examined using descriptive summaries of the data by various levels of the baseline 4362 value or by geographic region/country. 4363 4364 4.4.4.2 Missing Data Assumptions 4365 The analysis on the assumptions for missing data will only be performed for the primary 4366 efficacy endpoint. The primary analysis assumes a missing at random (MAR) 4367 missing-data mechanism (i.e., the probability that missing data are dependent on other 4368 observed variables, but not on the missing data itself. The assumption of MAR will be 4369 assessed by the following data review. However, it will not be possible to completely 4370 rule out the “missing not at random.” 4371 • Comparison between treatment groups regarding dropout rates and time 4372 of dropouts 4373 4374 • Comparison between subsequent dropouts versus those who remained in the study 4375 regarding outcome of the primary efficacy variable at each assessment week 4376 4377 4378 The random-effects pattern-mixture model will be applied as a sensitivity analysis to 4379 explore the robustness of the MMRM results of the primary efficacy variable [1]. 4380 4381 In the random-effects pattern-mixture model, the effects of missing data patterns will be 4382 included as explanatory variables in the MMRM analysis to assess whether treatment 4383 effects vary by dropout status. For example, patients will be classified into separate 4384 patterns based on dropout status, such as dropouts, potential split into sub-groups 4385 based on the timing of the dropout, or by type of dropout, and completers. This 4386 patient-level variable and interaction terms with this variable will then be included in the 4387 primary analysis model of MMRM to assess their influence on the longitudinally observed 4388 outcome of the primary efficacy variable. The MMRM model incorporating the pattern- 4389 mixture approach will include fixed terms of status of dropout and its two-way interaction 4390 terms (i.e., dropout-by-treatment, dropout-by-assessment time), and the 4391 three-way interaction term (dropout-by-treatment-by-assessment time). The MMRM 4392 model will enable the evaluation of whether study treatment effects are consistent across

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4393 different patterns of missing data. The overall estimate of treatment effects will be 4394 compared with the estimates from the main model not including pattern mixture. 4395 4396 In the random random-effects pattern-mixture analysis, the statistical significance of the 4397 interaction terms will be assessed at the 0.10 significance level. If a significant 4398 interaction is noted the following sensitivity analyses are performed including a 4399 multiple-imputation analysis to evaluate the sensitivity of not MAR scenarios. 4400 4401 For the multiple-imputation analysis, it will be assumed that majority of missing data will 4402 be monotone due to early withdrawals (observations for all visit after a missing 4403 observation are missing). PROC MI in SAS with the Markov chain Monte Carlo (MCMC) 4404 method will be used for multiple imputations using a pattern mixture model as follows: All 4405 change from baseline MADRS data at all visits under treatment will be used. Non 4406 monotone missing data (missing observation[s] followed by non-missing observation at a 4407 later visit) will be imputed assuming that they follow the same model as other patients in 4408 the same treatment group. Monotone missing data will be imputed under the 4409 assumption that the patients who discontinued their treatment will follow the same 4410 pattern of disease evolution as patients in the placebo group. The imputation will be 4411 performed in a stepwise process for each visit: All placebo patients and those who drop 4412 out at a given visit will be included to impute the missing values of those who 4413 discontinued. 4414 4415 A completer analysis will be performed including only patients who have completed 4416 Day 42. 4417 4418 4.4.5 Subgroup Analyses 4419 4420 Subgroup analyses of the primary efficacy variable will be performed for subgroups 4421 defined by stratification factors at randomization and clinically relevant factors at 4422 baseline as follows: 4423 • Sex: male versus female 4424 4425 • Geographic region: Europe versus United Stated versus Latin America versus 4426 Japan; and Japan versus non-Japan 4427 4428 • Class of antidepressant co-medication (SSRI vs. SNRI) 4429 4430 • Number of prior treatment failures (i.e., 1, 2, or 3) 4431 4432 • Single episode versus recurrent major depressive disorder 4433 4434 • Length of current episode (≤ 1 year vs. > 1 year) 4435 4436 4437 The MMRM model described in Section 4.4.1.3 will be modified by adding the 4438 categorical effect subgroup (if not already in the model) and replacing the treatment 4439 effect by treatment nested in subgroup, both as effect on its own and as part of the 4440 treatment-time interaction.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4441 For the comparison of Japan versus non-Japan, the MMRM analysis will be performed 4442 separately for each of the two subgroups. 4443 4444 4.5 PHARMACOKINETIC AND PHARMACODYNAMIC ANALYSES 4445 4.5.1 Population Pharmacokinetic Analysis 4446 4447 All patients with at least one adequately documented PK sample will be included in the 4448 population PK analysis. 4449 4450 Nonlinear mixed-effects modeling (with software NONMEM®) will be used to analyze the 4451 dose-concentration–time data of RO4917523. Population and individual PK parameters 4452 (e.g., apparent plasma clearance [CL/F] and apparent volume of distribution at steady

4453 state [Vss/F]) and their variability will be estimated. The influence of various covariates 4454 (e.g., age, sex, body weight, BMI, race) on these PK parameters will be investigated. 4455 4456 The data collected in this study may be pooled with data from other studies with 4457 RO4917423 PK measurements, as appropriate. Secondary PK parameters e.g., area

4458 under the plasma concentration − time curve [AUC], steady-state concentration [Css]) will 4459 be derived from the individual post-hoc predictions. Details of the analyses will be 4460 described in the Modeling and Simulation Analysis Plan before the database lock. 4461 4462 The results will be reported in a document separate from the CSR. 4463 4464 4.6 BIOMARKER ANALYSIS 4465 4466 For each of the single nucleotide polymorphisms (SNPs) cytochrome P450 variant 1A2 4467 gene (CYP1A2), serotonin 2A receptor (HTR2a), serotonin transporter (SLC6A4), 4468 metabotropic glutamate receptor 5 (GRM5), and brain derived neurotropic factor (BDNF), 4469 an MMRM analysis will be performed using the models described in Section 4.4.5, 4470 where the SNP allelic results are used as subgroups. Race will be added as fixed 4471 categorical covariable. The genotypic model will be based on additive inheritance where 4472 genotype will be treated as a three-level categorical variable (-1, 1, 0). For the rs6265 4473 SNP in BDNF, corresponding to Val66Met, the patients will be classified according to 4474 genotype distribution under a dominant model of inheritance and thus comparing Val/Val 4475 homozygotes versus Met carriers (Val/Met and Met/Met group). 4476 4477 4.7 SAFETY ANALYSES 4478 4479 For all safety variables collected by visit, the definitions of the baseline and time window 4480 will be the same as those for efficacy variables. 4481 4482 All safety variables will be summarized using descriptive statistics by treatment group for 4483 the safety population. 4484 4485 Safety variables include: 4486 4487 • Exposure to study drug

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4488 • AEs 4489 4490 • Columbia-Suicide Severity Rating Scale (C-SSRS) assessments 4491 4492 • Clinical laboratory tests 4493 4494 • Vital sign and body weight assessments 4495 4496 • Physical examinations 4497 4498 • ECG assessments 4499 4500 • Brief Psychiatric Rating Scale (BPRS) assessments 4501 4502 • Young Mania Rating Scale (YMRS) assessments 4503 4504 4.7.1 Exposure to Study Drug 4505 4506 The following extent of exposure to study drug will be summarized by treatment group: 4507 4508 • Duration of treatment, which will be calculated from the first day of the double-blind 4509 study medication to the last day of study treatment (i.e., the date of the last dose 4510 minus the date of the first dose plus 1). 4511 4512 • Extent of compliance to the prescribed treatment. The percentage of compliance for 4513 each patient will be calculated as “sum of total number of capsules taken” divided by 4514 “sum of total number of capsules expected to be taken,” then multiplied by 100. The 4515 “total number of capsules expected to be taken” will be calculated as the duration of 4516 treatment multiplied by 2 (due to two capsule prescribed per day). 4517 4518 4.7.2 Adverse Events 4519 4520 For each AE recorded, the term entered by the investigator describing the event (the 4521 “verbatim term”) will be assigned to a standardized term (the “preferred term”) based on 4522 the MedDRA. All data displays of AEs will be performed using the system organ class 4523 (also referred to as Body System) and preferred terms. All AEs with all occurrences will 4524 be listed by patient and will contain preferred terms and comments for 4525 each event. For summaries of AE incidences, patients who experienced the same event 4526 on more than one occasion will be counted once in the calculation of the event 4527 frequency at the highest intensity reported. Each table will also present the overall 4528 number of patients experiencing at least one AE and the total number of AEs reported. 4529 In summary tables, the AEs will be sorted by body system (in decreasing order of overall 4530 incidence), then by preferred term (in decreasing order of overall incidence). A glossary 4531 of superclass terms, preferred terms, and verbatim terms will be prepared. 4532 4533 All AEs will be summarized by treatment group for the following analysis periods 4534 separately: 4535 • Screening period: includes the serious AEs (SAEs; caused by protocol-mandated 4536 procedures) for which the onset date is before the day of the start of the study 4537 medication. Pre-existing conditions and AEs with start date prior to study drug 4538 administration will be considered baseline signs and symptoms (medical history).

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4539 • Treatment period: includes (a) the AEs for which the onset date is on or after the 4540 first day of the study drug and on or prior to the last dose day; OR (b) the AEs for 4541 which the onset date is prior to the first dose day with the end date on or after the 4542 first dose day or is unresolved and the most extreme intensity started during the 4543 treatment period. 4544 4545 • Follow-up period: includes the AEs of which onset date is after the last dose day. 4546 4547 4548 The following rules will be applied for AEs with missing onset and/or end dates: 4549 4550 • Events that are missing both onset and end dates will be considered treatment 4551 emergent, given that a patient had at least one dose of study drug. 4552 4553 • If the onset date is missing and the end date is on or after the first dosing date or 4554 unresolved or missing, then the event will be considered treatment emergent. 4555 4556 • If the end date is missing and the onset date is on or after the first dosing date, then 4557 the event will be considered treatment emergent. 4558 4559 • If the end date is missing and the extreme intensity is worse than the initial intensity 4560 and the onset date is prior to the first dosing date, then the event will be considered 4561 treatment emergent. 4562 4563 • The duration will be set to missing. 4564 4565 4566 Summaries will also be done by intensity and relationship to study drug as assigned by 4567 the investigator. 4568 4569 In the summary table of AEs by intensity, if a patient has more than one occurrence of 4570 an event, the event with the most severe intensity will be counted. If the intensity of an 4571 AE is missing, then the AE will be included only in the total number of events column, 4572 and not in the count of patients with the event by intensity. 4573 4574 In the summary table of AEs by relationship to study treatment, if a patient has more 4575 than one occurrence of an event, the most closely related event will be counted. If the 4576 relationship of an AE is missing, then the AE will be included only in the total number of 4577 events column, and not in the count of patients with the event by relationship. 4578 4579 All SAEs, AEs that led to death, and AEs that led to withdrawal of study treatment will 4580 also be summarized by treatment group. 4581 4582 The listing of patients who prematurely withdrew due to AEs will include all AEs 4583 (not only the events leading to withdrawal) that were reported by the patient. 4584 4585 In addition, for suicidality and severe liver injury Standardized MedDRA Query (SMQ) 4586 will be used to identify the events that may not be reported as AEs of special interest by 4587 the investigator. The AEs of suicidality will be identified by the MedDRA basket 4588 suicide/self-injury (SMQ 20000037 Narrow). The AEs of severe liver injury will be 4589 identified by a MedDRA basket to be defined.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4590 Further analysis of AEs (e.g., temporal relationship between duration of treatment and 4591 the AE) may be conducted in case some AEs that require special attention are observed. 4592 4593 For any SAE with the outcome of death, patient listings will be generated containing all 4594 details, including autopsy findings, recorded on the patient AE or Additional 4595 Observations eform. 4596 4597 4.7.3 Suicidality Assessment 4598 4599 The items of the C-SSRS will be summarized and presented in a data listing. The 4600 number and percentage of patients will be summarized for each suicidality ideation or 4601 behavior (i.e., those marked “yes”) by study treatment and scheduled assessment week 4602 starting at randomization. 4603 4604 4.7.4 Laboratory Data 4605 4606 Results of all laboratory tests collected will be summarized for each assessment week 4607 using descriptive statistics for the actual and change from baseline values. 4608 4609 Incidence of marked abnormal laboratory test results will be summarized by treatment 4610 group based on the Roche COG 3007 definition. 4611 4612 4.7.5 Vital Signs and Weight 4613 4614 Vital signs (blood pressure and pulse) and weight are measured throughout the study. 4615 Summaries for vital signs will be presented at each scheduled assessment visit using 4616 descriptive statistics. 4617 4618 The number and percentage of patients with abnormal changes in the vital signs 4619 measures will be summarized at each scheduled assessment visit and any time during 4620 the treatment period and follow-up period, respectively, based on the following criteria: 4621 4622 Vital Signs Parameter Abnormality Criteria 4623 4626 4624 Blood pressure 4627 Percent increase ≥ 20% from baseline 4625 4628 Pulse rate Percent decrease ≥ 20% from baseline 4629 4632 4630 Weight 4633 Percent increase ≥ 7% from baseline 4631 4634 Percent decrease ≥ 7% from baseline 4635 Percent increase ≥ 14% from baseline 4636 Percent decrease ≥ 14% from baseline 4637 4638 4639 In addition, orthostatic changes in blood pressure or pulse rate will be summarized. 4640 Orthostatic changes in blood pressure or heart rate are defined as the occasions when 4641 the measurements, upon moving from supine to standing position, fulfilled the following 4642 criteria based on the American Academy of Neurology’s consensus statement (1996). 4643 • Decrease in diastolic blood pressure ≥ 10 mmHg, or 4644 4645 • Decrease in systolic blood pressure ≥ 20 mmHg, or

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4646 • Increase in heart rate ≥ 20 beats/minute 4647 4648 4649 Summary of number and percentage of patients meeting the criteria will be presented for 4650 each assessment visit and any time during the treatment period and follow-up period, 4651 respectively. A listing of patients with orthostatic changes will be presented. 4652 4653 4.7.6 Physical Examinations 4654 4655 Physical examination findings (i.e., normal/abnormal) will be summarized for all patients 4656 who have a baseline and Day 42 (end-of-treatment) assessment using descriptive 4657 statistics. 4658 4659 4.7.7 ECGs 4660 4661 Actual values and change from baseline values of each parameter will be summarized 4662 using descriptive statistics for each scheduled assessment visit. 4663 4664 The number and percentage of patients with abnormal PR or QRS intervals, QT interval 4665 corrected using Bazett’s formula (QTcB), or QT interval corrected using Fridericia’s 4666 formula (QTcF) will be summarized for each scheduled assessment visit and at any time 4667 during the study (including follow-up) based on criteria in Table 2 . 4668 4669 Table 2 Adult Normal Ranges for ECG Listings and Summary Tables 4670 Low High Heart Rate ECG (bpm) 40 100 PR (msec) 120 200 QT (msec) 200 500 QRS (msec) 80 120 RR (msec) 600 1500 > 450 > 480

QTcB (msec) 300 > 500 Increase of > 30 from baseline Increase of > 60 from baseline > 450 > 480 QTcF (msec) 300 > 500 Increase of > 30 from baseline Increase of > 60 from baseline 4671 QTcB = QT interval corrected using Bazett’s formula; QTcF = QT interval corrected using 4672 Fridericia’s formula. 4673 4674 4.8 MISSING DATA 4675 4676 For all rating scales, if any item score contributing to the total/factor/subscale score is 4677 missing, then the total/factor/subscale will be set to missing.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4678 The main analysis of the primary and secondary continuous efficacy variables will be 4679 performed using a mixed-effects model, and no imputation for missing data will be 4680 applied. All observed assessments up to Day 42 will be included in the MMRM analyses. 4681 4682 For the supportive efficacy analysis using the ANCOVA model and the categorical data 4683 analysis, as an exploratory analysis to assess the robustness of the results, analyses of 4684 the ITT population will be repeated using the LOCF replacement for missing values. The 4685 LOCF dataset includes data recorded at a given visit or, if no observation was recorded 4686 at that visit, data carried forward from the previous post-baseline visit. To perform an 4687 efficacy analysis at Day 42, the last observed post-baseline value up to 6 days after the 4688 last dose day of patients who dropped out of the study before Week 6, whether 4689 scheduled or unscheduled, will be carried forward to Week 6. Baseline data will not be 4690 carried forward or averaged with post-treatment data to impute missing values for the 4691 LOCF dataset. 4692 4693 To understand the pattern of missing data observed during the study and, thus, the 4694 missing data mechanism, the following data will be summarized: 4695 • Timing of discontinuations by treatment group 4696 4697 • Reasons for discontinuation by treatment group and time 4698 4699 • Mean of the primary efficacy variable of those who dropped out versus those who 4700 remained at each scheduled assessment week. The dropout cohort at each 4701 assessment week will include the patients who had their last primary efficacy 4702 assessment in the corresponding week interval. 4703 4704 4705 Sensitivity analyses for the influence of the missing-data mechanism are described in 4706 Section 4.4.4.2. 4707 4708 No imputation will be applied for missing data of safety variables. 4709 4710 4.9 INTERIM ANALYSES 4711 4712 No interim analyses are planned for this study. 4713 4714 5. REFERENCES 4715 4716 1. Hedeker D, Gibbons RD. Application of random-effects pattern-mixture models for 4717 missing data in longitudinal studies. Psychological Methods 1997;2:64–78. 4718 4719 2. The Consensus Committee of the American Autonomic Society and the American 4720 Academy of Neurology. Consensus statement on the definition of orthostatic 4721 hypotension, pure autonomic failure, and multiple system atrophy. Neurology 4722 1996;46:1470.

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 4723 Appendix 1 4724 Protocol Synopsis 4725 4726 SYNOPSIS OF PROTOCOL NUMBER NP25620 4727 4728 4729 TITLE • A randomized, double-blind, parallel-group study of the safety 4730 and efficacy of RO4917523 versus placebo, as adjunctive 4731 therapy in patients with major depressive disorder with 4732 inadequate response to ongoing antidepressant treatment. 4733 4734 SPONSOR F. Hoffmann-La Roche Ltd CLINICAL 4737 IIb 4735 PHASE 47384736 INDICATION Major Depressive Disorder (MDD) 4739 OBJECTIVES Primary 4740 • To evaluate the efficacy of two fixed doses of RO4917523 4741 compared to placebo in a confirmatory manner over 6 weeks as 4742 adjunctive therapy in patients with MDD with inadequate 4743 response to ongoing antidepressant treatment, based on mean 4744 change in the Montgomery Asberg Depression Rating Scale 4745 (MADRS) total score from baseline to end of treatment. 4746 4747 Secondary 4748 • The secondary objectives are to evaluate change after 6 weeks 4749 of treatment with RO4917523 versus placebo as adjunctive 4750 therapy on the following: 4751 • Clinical Global Impression Scores: Severity (CGI-S) from 4752 baseline to end of treatment, and Improvement (CGI-I) at end 4753 of treatment 4754 • Safety and tolerability of RO4917523 4755 • Proportion of patients exhibiting remission (a MADRS score 4756 of less than or equal to 10) 4757 • Proportion of patients exhibiting response (reduction in 4758 MADRS score equal to or greater than 50% of the baseline 4759 score) 4760 • Quick Inventory of Depressive Symptomatology Self Report 4761 (QIDS-SR16) 4762 • Patient Global Impression of Improvement (PGI-I) score at 4763 end of treatment 4764 4765 Exploratory 4766 • Key Exploratory Objective 4767 • To evaluate the effect size (ES) of two fixed doses of RO4917523 4768 compared to placebo over 6 weeks as adjunctive therapy in 4769 patients with MDD with inadequate response to ongoing 4770 antidepressant treatment, based on mean change in the 4771 Montgomery Asberg Depression Rating Scale (MADRS) total 4772 score from baseline to end of treatment. 4773 4774 • Other Exploratory Objectives 4775 To investigate the differential effect of RO4917523 vs. placebo in: 4776 • Proportion of patients who meet MADRS responder criteria 4777 plus CGI-I score of 1) “very much improved” or 2) “much

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4778 improved” 4779 • Proportion of patients who meet MADRS remission criteria 4780 plus CGI-I score of 1) “very much improved” or 2) “much 4781 improved 4782 • Speed of onset of antidepressant effect based on change 4783 over time in any of the depression symptom scales 4784 • CANTAB Cognitive Test Battery subset 4785 • Quality of Life Enjoyment and Satisfaction Questionnaire- 4786 Short Form (Q-LES-Q-SF) 4787 • Sheehan Disability Scale (SDS) 4788 4789 • Pharmacokinetics 4790 • Pharmacokinetics of RO4917523 in the target population with 4791 the objective of performing a population pharmacokinetic 4792 analysis with non-linear mixed effect model 4793 • Biomarkers 4794 • Identify biomarkers that are predictive of response to 4795 RO4917523 treatment 4796 • Increase our knowledge and understanding of the 4797 pathogenesis, course and outcome of depression and related 4798 diseases 4799 • Develop biomarker or diagnostic assays, and establish the 4800 performance characteristics of these assays 4801 TRIAL DESIGN • This is an outpatient trial consisting of three consecutive periods: 4802 screening (up to 14 days), 6-week double-blind treatment, and a 4803 21 day follow-up period. 4804 4805 NUMBER OF 4808 Approximately 300 patients, randomized in equal proportions to the 4806 SUBJECTS 4809 three treatment arms. 4810 48114807 4812 TARGET 4815 • The study will include male and female outpatients from 18 to 4813 POPULATION 4816 70 years of age with a primary diagnosis of major depressive 4814 4817 disorder (MDD) without psychotic features as defined by DSM- 4818 IV-TR criteria, and having inadequate response to ongoing 4819 antidepressant therapy. Patients must have had at least one but 4820 no more than three treatment failures (of adequate dose and 4821 duration according to the Massachusetts General Hospital 4822 Antidepressant Treatment History Questionnaire [MGH ATRQ]). 4823 Failure to ongoing antidepressant treatment in the current 4824 episode is counted as one treatment failure. 4825 4826 LENGTH OF STUDY • 14 day screening period 4827 • 6 week double-blind treatment 4828 • 21 day follow-up 4829 END OF STUDY The date of the last visit (including the follow-up period) of the last 4830 patient in the study 4831 INVESTIGATIONAL4837 • Doses of RO4917523 0.5 mg or 1.5 mg supplied as pellets in 4832 MEDICAL 4838 capsules, in combinations of Ro 491-7523/F18 0.5 mg, 4833 PRODUCT(S) 4839 Ro 491-7523/F19 1.0 mg, taken orally once a day 4834 DOSE/ ROUTE/ 4835 REGIMEN 4836 F. Hoffmann-La Roche Ltd 25 NP25620 Statistical Analysis Plan

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4840 4841 4842 COMPARATOR “DRUG”4845 Matching capsules of placebo Ro 491-7523/F21, taken orally once a 4843 DOSE/ ROUTE/ REGIMEN4846 day 48474844 4848 ASSESSMENTS OF: 4849 -EFFICACY • MADRS total score 4850 • Remission (a MADRS score less than or equal to 10) 4851 • Response (reduction in MADRS score equal to or greater than 4852 50% from baseline) 4853 • CGI-S 4854 • CGI-I 4855 • PGI-I 4856 • QIDS-SR16 4857 • Q-LES-Q-SF 4858 • SDS 4859 • CANTAB 4860 4861 -SAFETY • Adverse events (AEs) and concomitant medications will be 4862 monitored throughout the entire study (screening through follow- 4863 up). Intensity of AEs will be graded on a 3 point scale (mild, 4864 moderate, or severe) 4865 • A subscale (4 items) of the BPRS will be completed only to 4866 follow up on treatment emergent psychotic-related and mania- 4867 related (mania or hypomania) adverse events. The symptom 4868 constructs included are conceptual disorganization, 4869 suspiciousness, hallucinatory behavior, and unusual thought 4870 content 4871 • The YMRS (item 1 only for mood elevation) will be completed 4872 only to follow up on treatment emergent psychotic-related and 4873 mania-related (mania or hypomania) adverse events 4874 • Columbia-Suicide Severity Rating Scale (C-SSRS) 4875 • Physical examination 4876 • Vital signs (pulse, blood pressure) 4877 • 12-lead ECG 4878 • Laboratory parameters 4879 • Chemistry panel 4880 • Hematology panel with differential 4881 • Free T4 and TSH 4882 • Viral Serology 4883 • Urinalysis (dipstick) 4884 • Urine drug screen 4885 • Pregnancy test in females 4886 • Data Safety Monitoring Board (DSMB) 4887 • An independent Data Safety Monitoring Board (DSMB) will 4888 review potential safety signals of concern. The DSMB will be 4889 composed of non-sponsor members who are not involved in 4890 the conduct of this study. The DSMB will review available 4891 safety data from this trial at regularly scheduled intervals as

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4892 4893 4926 specified in the DSMB Charter 4894 4927 • Statistical Interim Analyses 4895 No statistical interim analyses are planned 4896 4928 • 4897 - 4929 PK sampling will be obtained from all patients according to the 4898 PHARMACOKINETIC4930 Schedule of Assessments. 4899 S 4931 4900 -EXPLORATORY - 4932 Specimens for the Roche Clinical Repository (RCR) will be collected 4901 BIOMARKERS (non-4933 for dynamic (non-inherited) biomarker discovery and validation. The 4902 inherited) 4934 RCR sampling is optional. Blood specimens will be collected as per 4903 4935 Schedule of Assessments, with details as follows: 4904 4936 • Plasma and Serum assays: Blood samples for plasma and 4905 4906 4937 serum isolation will be obtained at baseline and the end of 4907 4938 treatment. A total of 4 samples (2x6 mL for serum and 2x6 mL 4908 4939 for plasma; 24 mL total) for each patient will be collected during 4909 4940 the study. These samples will be used for biomarker assays for 4910 4941 candidate depression biomarkers. 4911 4942 • Blood for RNA expression profiling: Blood samples for RNA 4912 4913 4943 isolation will be obtained at baseline and the end of treatment 4914 4944 (5 mL each; 10 mL total). 4915 4945 • Blood sample for epigenetic analysis: Blood samples for DNA 4916 4946 isolation will be collected at baseline and the end of treatment 4917 4947 (6 mL each; 12 mL total). 4918 4919 4948 4920 4949 These specimens will be stored for up to 15 years after the end of 4921 4950 the study. 4922 -EXPLORATORY 4951 Additional specimens for the Roche Clinical Repository (RCR) will be 4923 BIOMARKERS 4952 collected from consenting patients for genetic biomarker (inherited) 4924 (inherited) 4953 discovery and validation. The RCR sampling (inherited) is optional. 4925 4954 A single blood specimen will be collected from consenting patients, 4955 with details as follows: 4956 • Blood sample for genetic analysis: A blood sample (approx. 4957 6 mL) for DNA isolation will be collected at a single study visit as 4958 per Schedule of Assessments. If however, the RCR genetic 4959 blood sample is not collected during the scheduled visit, it may 4960 be collected at any time (after randomization) during the conduct 4961 of the clinical study The sample may be processed using 4962 techniques such as sequencing or microarray profiling. 4963 4964 These specimen(s) will be stored for up to 15 yearsafter the end of 4965 4977 4966 4978 the study. 4967 -MANDATORY 4979 Blood samples for serum and plasma protein biomarker discovery 4968 BIOMARKERS (MBS)4980 and validation will be collected from all patients where permissible 4969 4981 with local regulations at baseline and the end of treatment. A total of 4970 4982 4 samples (2x6 mL for serum and 2x6 mL for plasma; 24 mL total) 4971 for each patient will be collected during the study. These specimens 4972 4983 4973 4984 will be destroyed no later than 5 years after the end of the study. 4974 - CLINICAL 4985 A 3 mL whole blood sample will be taken for DNA extraction from all 4975 GENOTYPING (CG)4986 patients where permissible with local regulations at baseline. The 4976 4987 DNA will be tested for one or more of the following specific genes: 4988 the cytochrome P450 variant 1A2 gene (CYP1A2), the metabotropic 4989 glutamate receptor 5 (GRM5), the serotonin 2A receptor (HTR2A), 4990 the serotonin transporter (SLC6A4), and brain derived neurotropic 4991 factor (BDNF). This specimen will be destroyed immediately after the 4992 analysis has been completed.

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4993 4994 PROCEDURES (summary): Screening Period (up to 14 days) 4995 During the screening period, informed consent will be obtained, and the investigator will 4996 determine whether the candidate meets all inclusion criteria and does not meet any 4997 exclusion criteria. 4998 4999 6-Week Double-blind Treatment Period 5000 In order to be randomized into the double-blind treatment period, patients must have at 5001 baseline: 5002 • No significant risk of suicidal behavior (e.g., consider the Suicidal Ideation section of 5003 the C-SSRS “Since Last Visit” for this evaluation) 5004 5005 • No significant change in medical or psychiatric condition, or change in medications 5006 since screening (unless agreed with the Sponsor/Medical Monitor) 5007 5008 • Negative result on the Baseline pregnancy test (if applicable) 5009 5010 • No change in ongoing antidepressant therapy, and ability to continue for the 5011 duration of the double-blind treatment period without modification to the dosing 5012 schedule 5013 5014 • An ESF/EAF approved by the Sponsor/Medical Monitor 5015 5016 5017 The double-blind treatment period begins with the investigational site call into IVRS 5018 confirming the patient’s eligibility. The patient will be randomized and receive their first 5019 dose of study medication on Day 1. 5020 5021 Dose 1 of the blinded study medication is to be administered in the clinic immediately 5022 after a meal and before 12 pm (noon), or soon thereafter upon consultation with the 5023 Sponsor/Medical Monitor, once all baseline procedures and assessments are 5024 completed. Patients will remain at the clinic for 6 hours after the first dose for safety 5025 monitoring and for the PK samples (according to clinical observation and patient 5026 availability). Subsequently, dosing will be once daily in the morning immediately after 5027 breakfast. 5028 5029 As with any experimental drug at this stage of development, it is advisable for patients 5030 not to drive or operate dangerous machinery until known side effects (e.g., dizziness and 5031 somnolence) can be adequately assessed on an individual basis during the trial. 5032 5033 Patients will arrive at each study visit without having taken their daily dose of study 5034 medication, and site staff will record the time of their last dose. Following collection of the 5035 pre-dose PK blood sample (if applicable) and a meal, patients will take their next dose of 5036 study medication before 12 pm (noon) or soon thereafter upon consultation with the 5037 Sponsor/Medical Monitor. The last dose of study medication will be administered on Day 5038 42 (End-of-Treatment visit).

F. Hoffmann-La Roche Ltd 28 NP25620 Statistical Analysis Plan

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5039 Early Discontinuers will be instructed to return as soon as possible for the End-of- 5040 Treatment visit, and 21 days later for the Follow-up visit. 5041 5042 Follow-up Period (21 days) 5043 A Follow-up Visit will take place 21 days after the End-of-Treatment visit. During the 5044 follow-up period, adjustments to antidepressant treatments may be initiated if deemed 5045 necessary by the investigator. 5046 5047 STATISTICAL ANALYSES: 5048 Main efficacy analysis will be performed in a confirmatory manner based on ITT 5049 population, using a mixed effects covariance pattern model (MMRM) to utilize all the 5050 data collected over time. A closed testing procedure will be used to take multiple 5051 comparisons into account. As supporting analysis, the analysis may be repeated on the 5052 per-protocol population. Another supportive analysis will use an ANCOVA with LOCF 5053 imputation for missing data. The primary efficacy variable is change in the Montgomery 5054 Asberg Depression Rating Scale (MADRS) total score from baseline to end of treatment. 5055 5056 All safety variables (e.g., adverse events, lab tests, ECG, vital signs, BPRS, YMRS, 5057 ASEX) will be summarized for each assessment time (including follow-up) using 5058 descriptive statistics. The items of the C-SSRS will be presented by individual listings 5059 and the outcomes from this scale will be classified using the C-CASA methodology. 5060

F. Hoffmann-La Roche Ltd 29 NP25620 Statistical Analysis Plan

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