Cantex Clinical Study Protocol DSF-Cu CAN-201

CAN-201 A Phase II, Multicenter, Open-Label, Single Arm Study to Evaluate the Safety, Tolerability, and Efficacy of Disulfiram and Copper Gluconate in Recurrent Glioblastoma

IND #: 131305 Initial Draft Version Date: July 6, 2016 Final Version Date: January 5, 2017

SPONSOR Cantex Pharmaceuticals 1792 Bell Tower Lane Weston, FL 33326

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SPONSOR CONTACT Stephen Marcus, MD Telephone: (954) 315-3660 Fax: (954) 315-3661 E-mail: [email protected]

MEDICAL MONITOR Cristina Damatarca, MD Telephone: (760) 658-5903 Fax: (760) 520-0608 E-mail: [email protected]

STUDY CHAIRMAN Jiayi Huang, MD Washington University School of Medicine in St. Louis Office: (314) 362-8567 Fax: (314) 362-8521 [email protected]

Confidentiality Statement The document contains confidential and proprietary information and is the property of Cantex Pharmaceuticals. The information may be provided only to investigators, appropriate trial personnel, members of the Institutional Review Board, and personnel of the authorities to whom it is submitted. This protocol may not be photocopied or discussed with third parties without prior written authorization from Cantex Pharmaceuticals.

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Cantex Clinical Study Protocol DSF-Cu CAN-201 CAN-201 A Phase II, Multicenter, Open-Label, Single-Arm Study to Evaluate the Safety, Tolerability, and Efficacy of Disulfiram and Copper Gluconate in Recurrent Glioblastoma

Investigator’s Agreement

I have read the CAN-201 protocol for which Cantex Pharmaceuticals is the sponsor. I agree to conduct the study as outlined in the protocol and to comply with all the terms and conditions set out therein. I agree to maintain the confidentiality of all information received or developed in connection with this protocol. I confirm that I will conduct the study in accordance with ICH GCP Guidelines and applicable local regulations. I will also ensure that sub-investigator(s) and other relevant members of my staff have access to copies of this protocol and the ICH GCP Guidelines to enable them to work in accordance with the provisions of these documents.

Investigator Signature

Printed Name of Investigator

Date

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Table of Contents 1.0 BACKGROUND AND RATIONALE ...... 9 1.1 Recurrent Glioblastoma...... 9 1.2 Re-sensitizing Glioblastoma to Temozolomide ...... 9 1.3 Preclinical Data of Disulfiram for Temozolomide Sensitization ...... 10 1.4 Clinical Trial of Repurposing Disulfiram for GBM...... 13 1.5 Drug Metabolism and Pharmacokinetics ...... 13 1.6 Toxicology and Safety ...... 15 1.7 Disulfiram and Copper Administration ...... 16 1.8 TID Dosing of Disulfiram and Copper ...... 17 1.9 IDH Mutation in GBM ...... 17 1.10 Study Rationale ...... 17 2.0 STUDY OBJECTIVES ...... 18 2.1 Primary Objective ...... 18 2.2 Secondary Objectives ...... 18 3.0 PATIENT SELECTION ...... 18 3.1 Inclusion Criteria ...... 18 3.2 Exclusion Criteria ...... 19 3.3 Inclusion of Women and Minorities...... 20 4.0 STUDY DESIGN...... 20 5.0 INVESTIGATIONAL PLAN ...... 20 5.1 Pretreatment Evaluation (Screening/Baseline)...... 20 5.2 Disulfiram and Copper Dosing (Investigational Product) ...... 21 5.3 Temozolomide Dosing (Standard of Care) ...... 21 5.4 Study Evaluations ...... 21 5.5 Optional Pharmacokinetics Study ...... 22 5.6 General Concomitant Medication and Supportive Care Guidelines ...... 23 5.7 Men and Women of Childbearing Potential ...... 25 5.8 Duration of Therapy ...... 25 5.9 Duration of Follow-up ...... 26 6.0 RESPONSE ASSESSMENT ...... 26 6.1 Evaluation of Best Overall Response ...... 28 6.2 Duration of Response ...... 29 6.3 Neurological Exam and Performance Status ...... 29 6.4 Progression-Free Survival ...... 30 7.0 DOSE MODIFICATIONS ...... 30 7.1 Temozolomide Dose Modifications ...... 30 7.2 Disulfiram Dose Modifications ...... 30 8.0 REGULATORY AND REPORTING REQUIREMENTS ...... 31 8.1 Adverse Events (AEs) ...... 31 8.2 Unanticipated Problems (UAP) ...... 33 8.3 Noncompliance...... 33 8.4 Serious Noncompliance...... 34

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8.5 Protocol Exceptions/Deviations ...... 34 8.6 Reporting to the Institutional Review Board (IRB) ...... 34 8.7 Reporting to the FDA and Other Investigational Sites ...... 34 9.0 INVESTIGATIONAL PRODUCT INFORMATION ...... 35 9.1 Disulfiram...... 35 9.2 Treatment Compliance, Accountability and Return ...... 36 10.0 CORRELATIVE STUDIES ...... 37 10.1 Collection of Specimens for Optional Pharmacokinetics Study ...... 37 11.0 STUDY CALENDAR ...... 38 12.0 STATISTICAL CONSIDERATIONS...... 39 12.1 Definition of Primary Endpoints and Analytical Plan ...... 39 12.2 Definition of Secondary Endpoints and Analytical Plans ...... 39 13.0 ADMINISTRATIVE PROCEDURES AND CONSIDERATIONS ...... 40 13.1 IRB Approval ...... 40 13.2 Ethical Conduct ...... 40 13.3 Informed Consent ...... 40 13.4 Subject Confidentiality ...... 41 13.5 Study Monitoring ...... 41 13.6 Case Report Forms and Study Records/Data ...... 42 13.7 Criteria for Termination of the Study ...... 43 13.8 Investigator Final Report(s) ...... 43 13.9 Financial Disclosure ...... 43 13.10 Publication and Disclosure ...... 43 14.0 REFERENCES ...... 44 APPENDIX A: KPS and ECOG Scales...... 48 APPENDIX B: Sample Concurrent Disulfiram and Copper Medication Diary ...... 49 APPENDIX C: Sample Concurrent Temozolomide Medication Diary ...... 50

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Glossary of Abbreviations

AA Anaplastic Astrocytoma AE Adverse Event ALDH Aldehyde Dehydrogenase ALT Alanine aminotransferase AST Aspartate aminotransferase BMP Basic Metabolic Panel BVZ Bevacizumab CBC Complete Blood Count CCNU Lomustine CR Complete Response CRF Case Report Form Cu Copper Gluconate DDTC Diethyldithiocarbamate DSF Disulfiram ECOG Eastern Cooperative Oncology Group Status EDC Electronic Data Capture FDA Food and Drug Administration FLAIR Fluid-attenuated inversion recovery GBM Glioblastoma GCP Good Clinical Practice GSC Glioma-like stem cells H & P History and Physical ICF Informed Consent Form ICH International Conference on Harmonisation IDH Isocitrate dehydrogenase INR International normalized ratio LFTs Liver Function Tests Me-DDTC Methyl ester of diethyldithiocarbamate MGMT O6-methylguanine-DNA-methyltransferase KPS Karnofsky Performance Status MRI Magnetic resonance imaging OBG O6-benzylguanine ORR Objective response rate OS Overall survival PD Progressive Disease PFS Progression-free survival PFS6 Progression-free survival at six months PR Partial response RANO Response Assessment in Neuro- Oncology Criteria RT Radiation therapy

Final Version: January 5, 2017 7 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 SAE Serious Adverse Event SD Stable Disease SOP Standard Operating Procedure TMZ Temozolomide UAP Unanticipated Problem ULN Upper limit of normal WHO World Health Organization

SCHEMA

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1.1 Recurrent Glioblastoma

Glioblastoma (GBM, World Health Organization/WHO grade IV) is the most common malignant primary brain tumor and one of the most devastating cancers (Johnson and O'Neill 2012). Between 2004 and 2007, there were 37,690 patients newly diagnosed with GBM, with an estimated incidence rate of 3 cases per 100,000 people in the United States (Ostrom, et al. 2014). The current standard of care for GBM includes maximal safe resection followed by radiotherapy (RT) and temozolomide (TMZ). However, despite optimal multi-modality therapy, median progression-free survival (PFS) is less than 7 months, and median overall survival (OS) is less than 15 months (Stupp, et al. 2005). Effective treatment for recurrent GBM is even more dismal. A meta-analysis of 8 phase II studies of different cytotoxic drugs or cytostatic agents on recurrent GBM showed objective response rate (ORR, including both complete response and partial response) of 6%, median progression-free survival (PFS) of 9 weeks, and PFS at 6 months (PFS6) of 15%. Based on remarkable radiological response from two single-arm, non-comparative phase II studies, bevacizumab (BVZ), an anti-angiogenesis antibody, was approved by the Food and Drug Administration (FDA) for the treatment of recurrent GBM (Friedman, et al. 2009; Kreisl, et al. 2009). However, the duration of response is relatively short with median PFS of 3-4 months and OS of 7-9 month (Piccioni, et al. 2014). A recent randomized study (EORTC 26101) showed that the addition of BVZ to lomustine (CCNU) improved PFS but failed to improve OS as compared to CCNU alone (Wilk, et al. 2015). Previously, two large randomized studies also showed that the addition of BVZ to standard RT and TMZ failed to improve OS (Chinot, et al. 2014; Gilbert, et al. 2014). Altogether, these data confirm that the radiological response from BVZ does not accurately reflect its anti-tumor effect and challenges the current prevalent approach of testing BVZ-based combination therapies in clinical studies for recurrent GBM. Therefore, a novel agent is desperately needed for recurrent GBM and should be ideally tested without BVZ in future trials.

1.2 Re-sensitizing Glioblastoma to Temozolomide

Currently, TMZ represents the only systemic therapy that has been shown to improve OS for GBM (Stupp, et al. 2005). If GBM recurs after at least a 2 month interval from discontinuing TMZ, re-challenging with TMZ has demonstrated promising results (Perry, et al. 2010). Another phase II study attempted to re-sensitize recurrent TMZ- resistant GBM (those who would recur while receiving TMZ or within 2 months of the last treatment of TMZ) using an O6-methylguanine-DNA methyltransferase (MGMT) inhibitor called O6-benzylguanine (OBG) (Quinn, et al. 2009). Since MGMT is an enzyme that repairs DNA damage by alkylating agents such as TMZ, an MGMT inhibitor is thought to be a rational agent to re-sensitize GBM to TMZ. Unfortunately, combination of OBG and TMZ did not show any evidence of significant re-sensitization: ORR of 3% (95% CI: 0-15%), PFS6 of 9% (95% CI: 2-21%), and median PFS of 7.5 weeks (95% CI: 4.2-7.9 weeks) (Quinn, et al. 2009).

Final Version: January 5, 2017 9 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 1.3 Preclinical Data of Disulfiram for Temozolomide Sensitization

Disulfiram (DSF) is a FDA-approved oral medication that has been used for treating alcoholism since 1951. It inhibits aldehyde dehydrogenase (ALDH), which leads to accumulation of acetaldehyde in the blood after ingestion of alcohol. It has well-known safety profile for up to 3000 mg per day in the absence of alcohol consumption and has been shown to readily cross the blood-brain barrier (Faiman, et al. 1978; Suh, et al. 2006). Multiple preclinical in vitro studies have demonstrated promising activity against GBM cells (Hothi, et al. 2012; Liu, et al. 2012; Lun, et al. 2016; Triscott, et al. 2012). Specifically, two independent high-throughput drug screening studies have identified DSF with potent and selective activity against large panels of patient-derived glioma stem-like cells (GSCs, a subset of GBM tumor cells that have been shown to be more resistant to RT and chemotherapy) (Hothi, et al. 2012; Lun, et al. 2016).

1.3.1 In Vitro Data of Disulfiram for Temozolomide Sensitization

In addition to promising cytotoxicity against GBM cells, DSF has also appeared to synergize with TMZ. Triscott et al. showed that DSF is active against TMZ-resistant GBM cell lines and provided synergistic activity when combined with TMZ. As seen in Figure 1, DSF remarkably sensitized GBM cells that were relatively resistant to TMZ treatment (Triscott, et al. 2012).

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Lun et al. recently demonstrated that DSF-Cu not only could increase the sensitivity TMZ- naïve GSCs to TMZ (Figure 2A) but also recurrent TMZ-resistant GSCs (Figure 2B).

1.3.2 In Vivo Data of Disulfiram for Temozolomide Sensitization

Lun et al. further verified the synergistic effect of DSF-Cu using orthotopic GBM models that were produced from the GSCs. As seen in Figure 3A and 3B, DSF-Cu alone had limited effect on mice implanted with TMZ-sensitive GSCs. However, when given with

Final Version: January 5, 2017 11 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 TMZ, the combination significantly improved survival of mice with orthotopic tumors as compared to TMZ alone. Furthermore, as seen in Figure 3C, the combination of DSF-Cu and TMZ also significantly improved survival of mice implanted with TMZ-resistant BT73R cells as compared to TMZ alone or DSF-Cu alone (either treatment alone was ineffective) (Lun, et al. 2016). Altogether, these experiments provided proof of concept that DSF-Cu can re-sensitize TMZ-resistant GBM tumors to TMZ and the combination of both would be required to observe the maximal anti-tumor effect.

1.3.3 Mechanism of DSF’s Anticancer Activity

Final Version: January 5, 2017 12 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 The mechanism of DSF’s anti-cancer property is not definitively understood. Multiple studies have suggested that it may be due to the inhibition of chymotrypsin-like proteasome activity (Chen, et al. 2006; Cvek, et al. 2008; Hothi, et al. 2012). Others have showed that DSF-Cu complex can induce intracellular reactive oxygen species to trigger intrinsic apoptosis of GBM cells through activation of c-Jun amino-terminal kinases (JNK) and p38 pathways (Liu, et al. 2012). Paranjpe et al. showed that DSF increased the sensitivity of GBM cells to TMZ through inhibition of MGMT and demonstrated that DSF preferentially inhibited tumor MGMT in GBM xenografts as compared to MGMT in the normal tissue (Paranjpe, et al. 2014). Lun et al. demonstrated that proteasome inhibition was not responsible for all the cytotoxicity of DSF-Cu. Complete proteasome inhibition using proteasome inhibitor bortezomib did not produce the same level of cell death as DSF-Cu, while very low concentration of DSF-Cu with minimal proteasome inhibition was able to produce significant cytotoxicity. Lun et al. also showed that DSF-Cu inhibited DNA repair pathways and thus enhanced the effects of DNA alkylating agents and radiation to increase DNA damage (Lun, et al. 2016). These data suggest that the mechanism of DSF against GBM may be multi-factorial and may be more likely to overcome TMZ-resistance than a pure MGMT inhibitor such as OBG.

1.4 Clinical Trial of Repurposing Disulfiram for GBM

A phase I, dose-escalation pharmacodynamic study of DSF in combination with adjuvant TMZ for newly diagnosed GBM after standard RT and concurrent TMZ determined that the maximum tolerated dose (MTD) of DSF with adjuvant TMZ is 500 mg per day. Dose- limiting toxicities of delirium and ataxia occurred at dose of 1000 mg per day within the first month of administration. The combination of 500 mg of DSF with adjuvant TMZ had an acceptable safety profile but was associated with reversible neurological toxicities of delirium, ataxia, and neuropathy after prolonged use. The median PFS with RT plus concurrent TMZ followed by 500 mg of DSF and adjuvant TMZ was 8.1 months (Huang, et al. 2016). In comparison, previous randomized studies showed only a median PFS of 5.5 to 7.3 months with adjuvant TMZ alone after chemoradiotherapy (Gilbert, et al. 2014; Gilbert, et al. 2013; Stupp, et al. 2005). However, in the previous phase I study, the relative contributions of RT and TMZ could not be distinguished, so the single-agent efficacy of DSF could not be clearly defined (Huang et al, 2016). A logical next step would be to test DSF and Cu against recurrent TMZ-resistant GBM to elucidate their single-agent efficacy and their potential for TMZ re-sensitization.

1.5 Drug Metabolism and Pharmacokinetics

After oral ingestion, DSF is partially reduced to diethyldithiocarbamate (DDTC) in the acidic stomach, which in turn forms a complex with Cu to form Cu(DDTC)2. Both the parent DSF and Cu(DDTC)2 are absorbed through the gastrointestinal tract. Generally, more than 80% of an oral dose is absorbed. Enteric formulation and oil may enhance absorption. After absorption, DSF is again reduced to DDTC and then Cu(DDTC)2. Downstream metabolites also include diethylamine, carbon disulfide, diethyldithiomethylcarbamate (Me-DDTC), and glucuronic acid of DDTC. Me-DDTC is biotransformed into active inhibitors of ALDH (Johansson 1992). Experiments with

Final Version: January 5, 2017 13 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 radiolabeled DSF have shown distribution in the blood, liver, kidney, heart, adrenal gland, thyroid, pancreas, testes, spleen, marrow, muscles, and, most importantly, brain (Faiman, et al. 1978).

Previous pharmacokinetic studies were done on alcoholic patients after either single dose of 250 mg daily or repeated doses for 12 consecutive days (Figure 4).

Apparent half-lives of DSF, DDTC, and Me-DDTC were 7.3 ± 1.5, 15.5 ± 4.5, and 22.1 ± 3.8 hours, respectively. Average time to reach maximal plasma concentration was 8-10 hours for DSF and its metabolites. The mean peak plasma concentrations of DSF and Me- DDTC were 1.3 nM and 4.7 nM, respectively. However, there was marked inter-subject variability in the plasma concentrations (Faiman, et al. 1984; Jensen, et al. 1982). Doses as low as 100 mg of DSF could produce detectable plasma concentrations of Me- DDTC and complete blockade of ALDH activity in erythrocytes (Johansson, et al. 1991). As seen in Figure 5, three daily doses of DSF will allow for DSF and its metabolites to reach steady state.

The metabolites of DSF are mainly excreted via kidneys, feces, and the lungs. Up to 20% of an oral dose may not be absorbed and thus excreted in the feces. About 65% is eliminated through the

Final Version: January 5, 2017 14 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 kidneys, mostly as the glucuronide of DDTC and inorganic sulfates. The metabolite carbon disulfide is mostly eliminated via the lungs (Johansson 1992).

In the blood, both DSF and Me-DDTC are mostly bound to albumin, with average binding percentages of 96 and 80% over the ranges 200-800 and 345-2756 nM, respectively. The average number of binding sites was approximately one for both substances, suggesting a single binding site for both. The average association constants were 7.1 x 104 and 6.1 x 103 M-1, respectively. Therefore, patients with impaired protein synthesis and decreased albumin levels may have considerably different plasma concentration of DSF and its metabolites. Both DSF and Me-DDTC are also very lipophilic, with Log P (octanol-water partition coefficient) of 2.81 and 1.85, respectively (Johansson 1990), which support their ability to cross the blood brain barrier.

1.6 Toxicology and Safety

DSF has been used clinically for more than 50 years, so its safety profile is well known. Early toxicology studies done in mice, rats, dogs, and rabbits have shown that DSF has very low toxicity, with LD50 between 1.8-10g/kg when administered orally. At those extreme doses, demyelination of brain and spinal cord was observed on histopathology (Child and Crump 1952). Interestingly, long-term administration of high doses of DSF to rats did not induce any laboratory or histological signs of liver damage (Milandri, et al. 1980). High doses of DSF (up to 6 g daily) are relatively nontoxic in humans. Symptoms of overdose include vomiting, headache, apathy, ataxia, motor restlessness, irritability, hallucinations, psychosis, loss of consciousness and convulsions. Death occurs by respiratory arrest, preceded by ascending paralysis, and pathological lesions are seen in the liver, spleen, kidney and CNS, with congestion in the adrenal gland and edema in the heart muscle.

The current FDA-recommended dose of DSF is 250-500 mg daily for alcohol abstinence. In early clinical practice, a much higher dose of 1000-3000 mg per day was used (Fuller and Gordis 2004). At high dosages, the DSF-ethanol reaction may be severe and even fatal, but such high dosage in the absence of alcohol is well tolerated. In the early 1950s, 4 patients (out of an estimated 11,000 patients prescribed high doses of DSF) died of sudden respiratory or cardiovascular causes likely related to the DSF-ethanol reaction (Jacobsen 1952). At such high dosages, there were also case reports of psychosis in the absence of alcohol ingestion (Guild and Epstein 1951; Martensen-Larsen 1951). A phase I study administered a single dose of oral DSF prior to cisplatin every 3 weeks and encountered dose-limiting reversible confusion at 3000 mg/m2 (approximately 4800 mg) (Stewart, et al. 1987). High doses of DSF may inhibit cerebrospinal dopamine B-hydroxylase (Nilsson, et al. 1987), and people with very low activity of dopamine hydroxylase may be prone to transient psychosis with such inhibition (Major, et al. 1979). In another phase I study of non-metastatic recurrent prostate cancer patients treated with 250-500 mg of DSF alone, grade 3 toxicities included double vision, hearing loss, liver function test (LFT) abnormality, diarrhea, constipation, and ataxia (Schweizer, et al. 2013).

Final Version: January 5, 2017 15 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 In the previous phase I study of 500-1000 mg DSF in combination with adjuvant TMZ in newly diagnosed GBM patients after chemoradiotherapy, grade 2-3 toxicities that were attributed to DSF included delirium, ataxia, dizziness, neuropathy, and fatigue (Table 1). Higher dose of 1000 mg DSF per day could not be tolerated due to relatively fast onset of delirium and ataxia within the first month of therapy. Therefore, it was determined that 500 mg per day of DSF is the MTD in combination with adjuvant TMZ. However, even at the MTD of 500 mg per day, 2 of 7 patients eventually stopped DSF due to possibly DSF- related toxicity. One patient developed grade 3 delirium after 55 days of DSF; another patient developed grade 3 motor neuropathy (lower extremity weakness and foot drop). Both toxicities resolved after discontinuing DSF (Huang, et al. 2016).

Table 1: Possible DSF-related toxicity during Adjuvant TMZ Toxicities* DSF 500 mg (n=7) DSF 1000 mg (n=5) Grade 2 Grade 3 Grade 2 Grade 3 Ataxia 1 (14%) 0 2 (40%) 1 (20%)† Delirium 0 1 (14%)† 2 (40%) 2 (40%)§ Dizziness 1 (14%) 0 1 (20%) 0 Fatigue 3 (43%) 0 1 (20%) 0 Peripheral motor neuropathy 0 1 (14%)† 1 (20%) 0 Peripheral sensory neuropathy 2 (29%) 0 1 (20%) 0 *Grade 2-3 adverse events according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v. 4.0 (2009) or higher that were possibly or probably related to DSF and that were not present at baseline. †Not dose-limiting toxicity as occurred after the first month of DSF. §Dose-limiting toxicity as occurred within the first month of DSF.

1.7 Disulfiram and Copper Administration

Although the MTD of DSF in combination with adjuvant TMZ was established at 500 mg once per day, continuous administration of this dose over time was associated with reversible delirium or neuropathy that prompted discontinuation of therapy in some patients. Furthermore, the pharmacodynamic study showed that 500 mg DSF per day (without concurrent Cu) produced limited proteasome inhibition on peripheral blood cells, one of the presumed mechanisms against GBM cells (Huang et al 2016). Given that Cu has been shown to be essential for DSF’s anti-cancer effects, co-administration of Cu with DSF may be necessary to maximize its efficacy (Hothi, et al. 2012; Lun, et al. 2016). A previous phase I study for patients with advanced liver metastasis showed that 6 mg of elemental Cu in the form of copper gluconate is well tolerated when given with 250 mg of DSF daily. In this study, Cu was given in the morning half hour before breakfast, and DSF was given with the evening meals to avoid gastro-intestinal irritation from complexation of DSF and Cu in the stomach (Grossmann, et al. 2011). Currently, Dr. Jiayi Huang at Washington University, is conducting a phase I expansion cohort study in which patients are taking 500 mg of DSF every morning along with 2mg of Cu three times a day (TID). The combination of DSF and Cu has been well tolerated to date (personal communications).

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A recent double-blinded, randomized phase II study compared chemotherapy with and without DSF for metastatic non-small cell lung cancer. Patients who received concurrent DSF had significantly better PFS and OS than those who received chemotherapy alone (5.9 vs. 4.9 months, and 10.0 vs. 7.1 months, respectively) (Nechushtan, et al. 2015). In that study, DSF was given at 40 mg TID. In contrast to the phase I study by Huang et al., Nechushtan et al. did not report any significant neurological toxicity except fatigue. Given the half-life of DSF is approximately 7 hours, TID administration may be superior to improve its bioavailability and tolerability. Based on simulated modeling, TID administration of DSF may permit a more consistent systemic dose. Combining the promising results of the randomized phase II study for metastatic non-small cell lung cancer (Nechushtan et al, 2015) and the rationales above, we would like to investigate further on the TID formulation of DSF with concurrent Cu to treat GBM. However, as the previous pharmacokinetic study of a single dose or repeated doses of DSF was performed with once daily dosing (Figure 4), there is uncertainty whether TID dosing would cause accumulation. Thus, although the previous phase I showed that the MTD of DSF with concurrent TMZ is 500 mg per day, this phase II study will administer DSF at 80 mg TID (total of 240mg per day) together with 1.5 mg of Cu TID, to keep the total daily dose below the established MTD. Selective pharmacokinetic measurements will also be performed to check for drug accumulation.

1.9 IDH Mutation in GBM

Isocitrate dehydrogenase (IDH) mutation was first reported in a minority of GBM patients and have been shown to characterize a distinct entity known as secondary GBM, which represent approximately 10% of all GBMs and has much more favorable prognosis compared to the more common primary IDH-wildtype GBM. IDH mutation status has been shown to improve classification of gliomas and has been incorporated into the new 2016 CNS WHO Classification (Eckel-Passow, et al. 2015; Louis, et al. 2016). According to the 2016 WHO classification, GBM and anaplastic astrocytoma (AA, WHO grade III) should be characterized as either IDH-wildtype or IDH-mutant. IDH-wildtype AA has similar outcome as IDH-wildtype GBM and is currently treated similarly with RT with concurrent TMZ followed by adjuvant TMZ. Given such molecular classification provides better classification of glioma than grading, it has been advocated that future clinical trials should be stratified by IDH mutation status and that a clinical trial should ideally not combine IDH-mutant GBM with IDH-wildtype GBM (Reuss, et al. 2015).

1.10 Study Rationale

Recurrent TMZ-resistant GBM currently lacks any effective treatment. Preclinical studies have identified DSF-Cu as having promising activity against GBM cells and can potentially re-sensitize TMZ-resistant cells and orthoptic tumors to TMZ again. A recent phase I study has demonstrated the feasibility and safety of combining once daily DSF with adjuvant TMZ in newly diagnosed GBM patients after chemoradiotherapy (Huang, et al. 2016). Another randomized phase II study has shown promising results by combining TID DSF

Final Version: January 5, 2017 17 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 with concurrent chemotherapy for metastatic non-small cell lung cancer (Nechushtan et al, 2015). This clinical trial of TMZ with the addition of TID DSF-Cu for TMZ-resistant GBM will provide valuable single-agent efficacy data regarding DSF-Cu and could prospectively validate its potential to re-sensitize GBM to TMZ.

2.0 STUDY OBJECTIVES

2.1 Primary Objective

To determine evidence of an antitumor effect in patients who have TMZ-resistant GBM treated concurrently with DSF-Cu and TMZ.

2.2 Secondary Objectives

1. To evaluate the safety and tolerability of DSF-Cu administered TID when given concurrently with TMZ. 2. To determine PFS and OS of TMZ-resistant GBM patients treated with DSF-Cu in combination with TMZ.

3.0 PATIENT SELECTION

Approximately 20 patients will be enrolled and receive treatment with study drug.

3.1 Inclusion Criteria

1. At least 18 years of age. 2. Diagnosis of histologically confirmed GBM (WHO grade IV). Patients with previous identified, that is, known IDH mutant GBM are not eligible. Patients with primary GBM with IDH status that is unknown are eligible. Patients with secondary GBM (GBM with antecedent astrocytoma or oligodendroglioma) are eligible upon confirmation of transformation to IDH wild-type GBM. 3. The subject must have completed RT with concurrent TMZ at least 12 weeks prior to the planned start of treatment on this study UNLESS there is pathological verification of recurrent tumor and at least 4 weeks have elapsed since the end of RT with concurrent TMZ. 4. Experienced first unequivocal progression of tumor by magnetic resonance imaging (MRI) [as assessed via Radiologic Assessment in Neuro-Oncology (RANO) criteria (See Section 6.0)] within 3 months from the last dose of TMZ. 5. Recent resection of recurrent or progressive tumor will be allowed as long as at least 2 weeks have elapsed from the date of surgery and the subject has recovered from the effects of surgery. Evaluable or measurable disease following resection of recurrent tumor is mandated for eligibility into the study. 6. Karnofsky performance status (KPS) of at least 60% (see Appendix A). 7. Willing to remain abstinent from consuming alcohol while on DSF. 8. Recovered from the toxic effects of prior therapy to < grade 2 toxicity per NCI CTCAE version 4.0 (2009) or higher prior to study registration (except lymphopenia). The

Final Version: January 5, 2017 18 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 minimum duration required between prior therapy and initiation of study drug treatment is as follows: a. 12 weeks from completion of radiation therapy UNLESS there is histological confirmation of tumor recurrence in which case at least 4 weeks from completion of RT will suffice b. 4 weeks from prior cytotoxic therapy c. 4 weeks from prior experimental drug d. 6 weeks from nitrosoureas e. 3 weeks from procarbazine f. 1 week for non-cytotoxic agents, such as interferon, tamoxifen, and cis-retinoic acid 9. Meets the following laboratory criteria: a. Absolute neutrophil count ≥ 1,000/µL b. Platelets ≥ 75,000/µL c. Hemoglobin > 10.0 g/dL (transfusion and/or ESA allowed) d. Total bilirubin and alkaline phosphatase ≤ 2x institutional upper limit of normal (ULN) e. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) < 3 x ULN f. Blood urea nitrogen (BUN) and creatinine < 1.5 x ULN 10. Able to take oral medication. 11. Females of childbearing potential (defined as a female who is not menopausal or surgically sterilized) must be willing to use an acceptable method of birth control (i.e., intra-uterine device, diaphragm with spermicide, condom with spermicide, or abstinence) for the duration of the study. Should a woman become pregnant or suspect she is pregnant while participating in this study, she must inform her treating physician immediately. 12. Able to understand and willing to sign an institutional review board (IRB)-approved written informed consent document (legally authorized representative permitted).

3.2 Exclusion Criteria

1. Radiographic evidence of leptomeningeal dissemination, gliomatosis cerebri, infratentorial tumor, or disease at sites remote from the supratentorial brain. 2. Enrolled in another clinical trial testing a novel therapy or drug within the past 4 weeks. 3. Received more than one course of radiation therapy or more than a total dose of 75 Gy. Subjects may have received radiosurgery as part of the initial therapy (i.e., in addition to one course of radiation therapy); however, the dose used for the radiosurgery counts against the total dose limit listed above. 4. History of allergic reaction/hypersensitivity to temozolomide, dacarbazine, DSF or Cu. 5. Treatment with the following medications are contraindicated with DSF: metronidazole, isoniazid, dronabinol, carbocisteine, lopinavir, paraldehyde, ritonavir, sertraline, tindazole, tizanidine, atazanavir. (Note: the following medications are not contraindicated but should be cautioned if taking concurrently with DSF: warfarin, phenytoin, theophylline, chlorzoxazone, chlordiazepoxide, or diazepam. If the patient is taking warfarin, INR should be monitored closely. If the patient has to remain on phenytoin, its serum concentration and response should be monitored closely.) 6. Fever ≥ 38.5°C within 3 days prior to study enrollment.

Final Version: January 5, 2017 19 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 7. Active or severe hepatic or renal disease. 8. Grade 2 or higher peripheral neuropathy or ataxia per NCI CTCAE version 4.0 (2009) or higher. 9. History of idiopathic seizure disorder schizophrenia, or psychosis unrelated to glioblastoma, corticosteroid, or anti-epileptic medications. 10. History of Wilson’s disease. 11. History of hemochromatosis. 12. Pregnant or breastfeeding. Refusal or inability to use highly effective means of contraception in female participants of child‐bearing potential.

3.3 Inclusion of Women and Minorities

Both men and women and members of all races and ethnic groups are eligible for this trial.

4.0 STUDY DESIGN

This multi-center study is a single arm, open-label, phase II study of DSF-Cu in combination with TMZ for recurrent TMZ-resistant GBM patients.

SCHEMA

5.0 INVESTIGATIONAL PLAN

5.1 Pretreatment Evaluation (Screening/Baseline)

Prior to dosing, patients must have a complete medical history, physical examination including neurological exam, evaluation of performance status using the KPS and the ECOG scale (Appendix A), assessment of corticosteroid usage (mg per day), baseline laboratory studies (CBC, BMP, and LFTs), and a baseline brain MRI. The baseline MRI should be within 6 weeks prior to dosing. Women of childbearing potential must have a negative serum pregnancy test within 14 days of initiation of treatment.

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5.2 Disulfiram and Copper Dosing (Investigational Product)

Two study drugs will be provided: DSF in 40 mg capsules and Copper Gluconate in 1.5 mg capsules. DSF will be given at 80 mg (two capsules) three times daily approximately 4-8 hours apart. To avoid the potential effect of food on absorption, patients should take DSF with approximately 8 ounces of water at least one hour before food. Copper gluconate at a dose of 1.5 mg (1 capsule) should be taken with meals TID and not within one hour of DSF.A previous phase I study determined that the MTD is 500 mg per day in combination with TMZ (Huang et al, 2016). However, in this protocol, DSF-Cu will be administered TID as discussed in section 1.8. The DSF dose will be a total of 240 mg per day. If a patient misses a dose, s/he should be instructed not to make up that dose but should instead resume dosing with the next scheduled dose. Patients will be instructed to bring all unused drug and the completed medication diary (sample provided in Appendix B) to each study visit for assessment of compliance.

5.3 Temozolomide Dosing (Standard of Care)

TMZ should be continued according to the standard adjuvant dose. TMZ will not be provided by the study sponsor. Dosage will be 150 mg/m2 PO per day on days 1-5 of every 28-day cycle, and no dose escalation will be allowed after the first month. If the patient was on 200 mg/m2 per day previously, he/she should receive 150 mg/m2 of TMZ with concurrent DSF/Cu on this study. TMZ should be taken at bedtime on an empty stomach and should not be taken within one hour of DSF and Cu administration. Patients will be instructed to bring their completed medication diary (sample provided in Appendix C) to the study visit for assessment of compliance.

5.4 Study Evaluations

Consistent with standard of care, patients will be seen approximately every 4 weeks (± 10 days) and typically within 10 days before the start of the next cycle. At each visit, a Karnofsky Performance Status (KPS) evaluation and corticosteroid usage (mg per day) will be recorded. Brain MRI will be obtained approximately every 8 weeks (± 2 weeks) to assess for radiological response.

Patients are evaluated for adverse events from the time of consent to 30 days after the last treatment of DSF-Cu, or until death, whichever occurs first. Patients removed from study for adverse events will be followed until resolution or stabilization of the adverse event.

Table 2: Potential Adverse Events Related to DSF MedDRA Term Frequency: Likely: greater than 10% Less Likely: 1-10% Rare: less than 1% Neoplasms Rare: Tumor necrosis

Final Version: January 5, 2017 21 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 MedDRA Term Frequency: Likely: greater than 10% Less Likely: 1-10% Rare: less than 1% Blood and lymphatic Rare: Neutropenia, anemia, leukopenia, system disorders thrombocytopenia, lymphopenia (likely due to TMZ) Immune system Rare: Hypersensitivity disorders Metabolism and Likely: Alcohol intolerance, metallic or garlic-like nutrition disorders aftertaste Psychiatric disorders Less Likely: psychosis, delirium (need to rule out tumor progression) Nervous system Likely: Drowsiness, headache, confusion disorders Less Likely: ataxia, gait disturbance, peripheral neuropathy Rare: Extrapyramidal symptoms Eye disorders Rare: Optic neuritis Cardiac disorders Less Likely: Tachycardia, hypotension (need to rule out DSF-alcohol reaction) Respiratory, thoracic, Less Likely: Dyspnea (need to rule out DSF-alcohol and mediastinal reaction) disorders Gastrointestinal Likely: Nausea, vomiting, diarrhea disorders Less Likely: constipation (likely due to temozolomide) Hepatobiliary disorders Rare: Hepatitis Skin and subcutaneous Less Likely: Allergic dermatitis tissue disorders Musculoskeletal Rare: Arthralgia, myalgia Renal and urinary Rare: Dysuria, hematuria disorders Reproductive system Less likely: impotence General disorders Likely: Fatigue

5.5 Optional Pharmacokinetics Study

As discussed in section 1.8, previous single-dose and multiple-dose pharmacokinetic measurements of DSF were performed with 250 mg once daily and without concomitant Cu. To ensure TID administration of DSF with Cu in this protocol would not lead to drug accumulation or any unanticipated changes in pharmacokinetics, approximately 5 patients at Washington University will be enrolled on an optional informed consent to participate in pharmacokinetics measurements. This will be a pilot exploratory study to guide future larger formal pharmacokinetic analyses. The selected participants will take a single dose of 80 mg of DSF on the day prior to the start of protocol treatment (day 0). Approximately 10 mL of peripheral blood (into a green top tube containing sodium heparin) will be drawn at the following times: hour 0 (before taking DSF), 1, 2, 4, 8, and 24 (before the start of protocol treatment). After starting the protocol treatment on day 1, additional blood will

Final Version: January 5, 2017 22 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 also be collected approximately 4 hours after the morning dose of DSF (but before the afternoon dose) on the following days during the treatment: day 3, 7, and 10 (see Schema). To facilitate blood collection, the participants of the pharmacokinetics study should take the single dose of DSF on a Monday, so they will start DSF with TMZ on a Tuesday.

5.6 General Concomitant Medication and Supportive Care Guidelines

The following medications and procedures are prohibited during the study:  Any antineoplastic therapy other than TMZ and DSF  Any investigational therapy other than DSF or Cu

The following medications are contraindicated with DSF and are prohibited during the study: metronidazole, isoniazid, dronabinol, carbocisteine, lopinavir, paraldehyde, ritonavir, sertraline, tindazole, tizanidine, atazanavir.

The use of alcohol-containing medications and products are to be avoided during the study, such as cough and cold syrups.

The following medications are allowed but should be cautioned if patient has to remain on them while taking DSF: warfarin, phenytoin, theophylline, chlorzoxazone, chlordiazepoxide, or diazepam. If the patient is taking warfarin, INR should be monitored closely. If the patient has to remain on phenytoin, its serum concentration and seizure control should be monitored closely.

The use of the following medications/supplements are to be avoided at study entry and during the study as they may effect Cu levels in the body:  Birth control medications  Estrogen  Penicillamine  Allopurinol  Cimetidine  Nifedipine  Zinc

The use of valproic acid is to be avoided as it was shown to decrease the oral clearance of temozolomide.

All other medical conditions or manifestations of the patient’s malignancy should be treated at the discretion of the investigator in accordance with local community standards of medical care.

Patients should not drive, operate dangerous tools or machinery, or engage in any other potentially hazardous activity that requires full alertness and coordination if they experience sedation while enrolled in this study.

Patients are to be instructed to abstain from alcohol while enrolled in this study.

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5.6.1 Nausea and Vomiting

Prophylactic antiemetic therapy may be used in this study at the discretion of treating physician. Because of the potential of benzodiazepines to interact with DSF, the use of benzodiazepines for antiemetic prophylaxis should be reserved for patients who cannot be satisfactorily managed otherwise.

5.6.2 Diarrhea

Antidiarrheal medications will not be used prophylactically; however, patients will be instructed to take loperamide, 4 mg, at the occurrence of the first loose stool and then 2 mg every 2 hours until they are diarrhea-free for at least 12 hours. During the night, patients may take 4 mg of loperamide every 4 hours. Fluid intake should be maintained to avoid dehydration.

5.6.3 Central Nervous System Effects

Doses of 500-1000 mg of DSF per day with adjuvant TMZ produced neurological toxicities in some patients, including delirium/psychosis, ataxia, and peripheral neuropathy, especially at the 1000 mg dose. Patients should be carefully monitored for early signs of these symptoms. Once other causes such as tumor progression are ruled out, dose reduction of DSF to 40 mg (one capsule) three times daily should be considered if the toxicity is grade 2 or greater (refer to Section 6.2). If symptoms are not improving with dose reduction, DSF should be discontinued. Patients whose symptoms are not considered immediately life-threatening should be carefully monitored. Each patient may be approached individually with a systematic assessment to rule out other causes. Appropriate tests may include vital signs measurement, computerized tomography, MRI scans, or other appropriate medical assessment; the investigator may consult with the Medical Monitor or the Sponsor if needed.

If the patient’s level of consciousness is considered to be life-threatening, the patient should be hospitalized and necessary measures should be instituted to secure the airway, ventilation, and intravenous access.

5.6.4 Management of Disulfiram-Alcohol Reaction

In severe reactions caused by the patient's excessive ingestion of alcohol, supportive measures to restore blood pressure and treat shock should be instituted. Other recommendations include: oxygen, carbogen (95% oxygen and 5% carbon dioxide), vitamin C intravenously in massive doses (1 g), and ephedrine sulfate. Antihistamines have also been used intravenously. Potassium levels should be monitored, particularly in patients on digitalis, since hypokalemia has been reported.

Final Version: January 5, 2017 24 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 5.7 Men and Women of Childbearing Potential

Temozolomide may cause birth defects. Male and female patients who take temozolomide should use effective birth control. Female patients and female partners of male patients should avoid becoming pregnant while taking temozolomide.

Women of childbearing potential (defined as women with regular menses, women with amenorrhea, women with irregular cycles, women using a contraceptive method that precludes withdrawal bleeding, or women who have had a tubal ligation) are required to have a negative serum pregnancy test within 14 days prior to the first dose of the study treatment.

Female patients are required to use a method of acceptable contraception during participation in the study until the last dose of study treatment.

For male patients, barrier methods of contraception, such as condoms, are recommended.

If a patient is suspected to be pregnant, study treatment should be immediately discontinued. In addition, a positive urine test must be confirmed by a serum pregnancy test. If it is confirmed that the patient is not pregnant, the patient may resume treatment.

If a female patient or female partner of a male study patient becomes pregnant during therapy, the investigator must be notified in order to facilitate outcome follow-up; study drug must be discontinued and the patient must be withdrawn from the study. Pregnancies will be followed through completion/termination of the pregnancy. If the outcome of the pregnancy meets the criteria for a serious adverse event, it must be reported following the procedures for reporting serious adverse events.

5.8 Duration of Therapy

Patients can receive investigational drug treatment during multiple temozolomide cycles over the course of several months, depending on their response (estimated to be approximately 6 months).

If at any time the constraints of this protocol are considered to be detrimental to the patient’s health and/or the patient no longer wishes to continue protocol therapy, the protocol therapy should be discontinued and the reason(s) for discontinuation documented in the case report form.

In the absence of treatment delays due to adverse events, DSF-Cu treatment may continue until:

 Documented and confirmed disease progression (see Section 6.0)  Death  Adverse event(s) that, in the judgment of the investigator, may cause severe or permanent harm or which rule out continuation of study drug

Final Version: January 5, 2017 25 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201  Patient develops unacceptable toxicity deemed possibly, probably, or definitely related to drug that does not resolve to grade 1 or to the patient’s baseline status  Patient requires more than 1 dose reduction  General or specific changes in the patient’s condition render the patient unacceptable for further treatment in the judgment of the investigator  Suspected or confirmed pregnancy  Serious noncompliance with the study protocol  Lost to follow-up  Patient withdraws consent at any time for any reason  Investigator removes the patient from study  The sponsor (Cantex), the investigator, or the investigator’s institution decides to close the study

In the case of tumor progression, DSF-Cu should be discontinued, and the patient may be considered for second-line therapy.

If TMZ is temporarily withheld due to toxicity, DSF-Cu should be continued until TMZ may be resumed again.

If TMZ is discontinued, DSF-Cu may be continued as per the discretion of the treating physician.

If DSF-Cu is discontinued due to toxicity, TMZ may be continued as per the discretion of the treating physician.

5.9 Duration of Follow-up

Patients are evaluated for adverse events for 30 days after the last dose of DSF-Cu or until death, whichever occurs first. Patients removed from study for adverse events will be followed until resolution or stabilization of the adverse event. Follow-up after the conclusion of the study participation will be per routine clinical care (approximately 6 months). Two years after the patient goes off study drug (two years after the last dose), the patient’s chart will be reviewed to collect data on progression and survival.

6.0 RESPONSE ASSESSMENT

For the purposes of this study, patients should be evaluated for response every 8 weeks.

Response and progression will be evaluated in this study using the updated RANO response assessment criteria for high-grade gliomas (Wen, et al. 2010). Patients who have measurable disease as defined by RANO criteria at baseline and have received at least one dose of DSF/Cu with concurrent TMZ will be considered evaluable for response.

Final Version: January 5, 2017 26 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 Criteria for Determining First Progression Depending on Time from Initial Chemoradiotherapy First Progression Definition Progression can only be defined using diagnostic imaging if there is new enhancement outside of the radiation field (beyond the high-dose region or 80% isodose line) or if there is unequivocal evidence of viable tumor on histopathologic sampling (e.g., solid tumor areas [i.e., > 70% tumor cell Progressive disease < 12 nuclei in areas], high or progressive increase in MIB-1 proliferation index weeks after completion of compared with prior biopsy, or evidence for histologic progression or chemoradiotherapy increased anaplasia in tumor). Note: Given the difficulty of differentiating true progression from pseudoprogression, clinical decline alone, in the absence of radiographic or histologic confirmation of progression, will not be sufficient for definition of progressive disease in the first 12 weeks after completion of concurrent chemoradiotherapy. 1. New contrast-enhancing lesion outside of radiation field on decreasing, stable, or increasing doses of corticosteroids. 2. Increase by ≥ 25% in the sum of the products of perpendicular diameters between the first post-radiotherapy scan, or a subsequent scan with smaller tumor size, and the scan at 12 weeks or later on stable or increasing doses of corticosteroids. 3. Clinical deterioration not attributable to concurrent medication or Progressive disease ≥ 12 comorbid conditions is sufficient to declare progression on current weeks after treatment but not for entry onto a clinical trial for recurrence. chemoradiotherapy 4. For patients receiving antiangiogenic therapy, significant increase completion in T2/FLAIR non-enhancing lesion may also be considered progressive disease. The increased T2/FLAIR must have occurred with the patient on stable or increasing doses of corticosteroids compared with baseline scan or best response after initiation of therapy and not be a result of comorbid events (e.g., effects of radiation therapy, demyelination, ischemic injury, infection, seizures, postoperative changes, or other treatment effects).

Criteria for Response Assessment Incorporating MRI and Clinical Factors (Adapted from (Wen, et al. 2010). Response Criteria  Requires all of the following: complete disappearance of all enhancing measurable and non-measurable disease sustained for at least 4 weeks.  No new lesions; stable or improved non-enhancing (T2/FLAIR) lesions. Complete response  Patients must be off corticosteroids (or on physiologic replacement doses only) and stable or improved clinically. Note: Patients with non-measurable disease only cannot have a complete response; the best response possible is stable disease. Requires all of the following:  ≥ 50% decrease compared with baseline in the sum of products of Partial response perpendicular diameters of all measurable enhancing lesions sustained for at least 4 weeks.  No progression of non-measurable disease.

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Response Criteria  Stable or improved non-enhancing (T2/FLAIR) lesions on same or lower dose of corticosteroids compared with baseline scan; the corticosteroid dose at the time of the scan evaluation should be no greater than the dose at time of baseline scan.  Stable or improved clinically. Note: Patients with non-measurable disease only cannot have a partial response; the best response possible is stable disease. Requires all of the following:  Does not qualify for complete response, partial response, or progression.  Stable non-enhancing (T2/FLAIR) lesions on same or lower dose of corticosteroids compared with baseline scan. In the event that the corticosteroid Stable disease dose was increased for new symptoms and signs without confirmation of disease progression on neuroimaging, and subsequent follow-up imaging shows that this increase in corticosteroids was required because of disease progression, the last scan considered to show stable disease will be the scan obtained when the corticosteroid dose was equivalent to the baseline dose. Defined by any of the following:  ≥ 25% increase in sum of the products of perpendicular diameters of enhancing lesions compared with the smallest tumor measurement obtained either at baseline (if no decrease) or best response, on stable or increasing doses of corticosteroids*. The absolute increase in any dimension must be at least 5mm when calculating the products.  Significant increase in T2/FLAIR non-enhancing lesion on stable or increasing doses of corticosteroids compared with baseline scan or best response after Progression initiation of therapy* not caused by comorbid events (e.g. radiation therapy, demyelination, ischemic injury, infection, seizures, postoperative changes, or other treatment effects).  Any new measureable lesion.  Clear clinical deterioration not attributable to other causes apart from the tumor (e.g. seizures, medication adverse effects, complications of therapy, cerebrovascular events, infection, and so on) or changes in corticosteroid dose.  Failure to return for evaluation as a result of death or deteriorating condition; or clear progression of non-measurable disease.  NOTE. All measurable and non-measurable lesions must be assessed using the same techniques as at baseline.  Abbreviations: MRI, magnetic resonance imaging; FLAIR, fluid-attenuated inversion recovery.  Stable doses of corticosteroids include patients not on corticosteroids.

6.1 Evaluation of Best Overall Response

The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence. The patient's best response assignment will depend on the achievement of both measurement and confirmation criteria.

Final Version: January 5, 2017 28 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 Summary of the RANO Response Criteria (Adapted from (Wen, et al. 2010)). Criterion CR PR SD PD T1 gadolinium < 50% ↓ but < 25% None ≥ 50% ↓ ≥ 25% ↑* enhancing disease ↑ Stable or Stable or T2/FLAIR Stable or ↓ ↑* ↓ ↓ New lesion None None None Present* Stable or Corticosteroids None Stable or ↓ NA† ↓ Stable or Stable or Clinical status Stable or ↑ ↓* ↑ ↑ Requirement for All All All Any* response Abbreviations: RANO, Response Assessment in Neuro-Oncology; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; FLAIR, fluid-attenuated inversion recovery; NA, not applicable. * Progression occurs when this criterion is present. † Increase in corticosteroids alone will not be taken into account in determining progression in the absence of persistent clinical deterioration. NOTE: Patients may continue on treatment and remain under close observation and evaluation at 4-8 week intervals if there is uncertainty regarding whether pseudoprogression may be present as determined by the investigators. If subsequent radiographic or clinical assessments suggest that the patient is in fact experiencing progression, then the date of progression should be the time point at which this issue was first raised. Similarly, stable disease may be assigned in cases of presumed “pseudoprogression” associated with decreased steroid use.

6.2 Duration of Response

Duration of overall response: The duration of overall response is measured from the time measurement criteria are met for complete response (CR) or partial response (PR) (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started).

The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented.

Duration of stable disease: Stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements.

6.3 Neurological Exam and Performance Status

Patients will be graded using the KPS scale and their neurological function evaluated as improved, stable or deteriorated in addition to objective measurement of tumor size. These parameters will be used to determine the overall response assessment.

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6.4 Progression-Free Survival

PFS is defined as the duration of time from start of treatment to time of progression or death, whichever occurs first.

7.0 DOSE MODIFICATIONS

7.1 Temozolomide Dose Modifications

TMZ dosage modification is at the discretion of treating physician as per routine clinical care.

7.2 Disulfiram Dose Modifications

If a patient experiences any grade 2 or higher non-hematologic toxicity considered by the investigator to be possibly, probably, or definitely related to DSF, the dose of DSF may be decreased by 50% (i.e., from 80 mg TID to 40 mg TID) once. Special attention should be paid to neurological symptoms such as delirium/psychosis, gait disturbance/ataxia, and peripheral neuropathy. A grade 2 toxicity of the above neurological symptoms should prompt a consideration for further work-up and dose reduction. No second dose reduction is allowed—if a second dose reduction is required, DSF and Cu will be discontinued and the patient will be taken off study. Of note, hematologic toxicity is uncommon for DSF and is likely related to TMZ.

7.2.1 Administration of DSF to Patients with Abnormal Hepatic Function

DSF-Cu should only be administered if hepatic function is within the parameters established in the eligibility criteria. Hepatic toxicity from DSF-Cu is uncommon but may occur. Therefore, hepatic dysfunction that occurs while the patient is on study should prompt an evaluation to determine the cause, including the possibility of hepatotoxicity from concurrent medications.

7.2.2 Hypersensitivity Reactions

Hypersensitivity reactions rarely occur. If they do occur, minor symptoms such as flushing, skin reactions, dyspnea, lower back pain, hypotension, or tachycardia may require no intervention; however, severe reactions, such as hypotension requiring treatment, dyspnea requiring bronchodilators, angioedema, or generalized urticaria require immediate discontinuation of study drug administration and aggressive symptomatic therapy. Patients who experience a severe hypersensitivity reaction to DSF should discontinue DSF immediately and not be re-challenged.

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8.1 Adverse Events (AEs)

Definition (as per 21 CFR 312.32): An AE is any untoward medical occurrence associated with the use of a drug in humans, whether or not considered drug related.

An AE therefore can be any unfavorable and unintended sign, symptom, or disease temporally associated with the use of a medicinal product whether or not considered related to the medicinal product.

The investigator or designee will monitor each patient for adverse events on a routine basis during the study, starting after the patient signs consent and continuing until 30 days after the last dose of study treatment. The investigator or designee will assess and record any AE in the source documents and in the case report form (CRF), including seriousness, the date of onset, description, severity, duration and relationship to study drug, action taken and outcome.

Conditions that started before signing of consent and for which no symptoms or treatment are present at the time of consent are considered medical history.

Conditions that started before signing consent and for which symptoms are present after signing consent are considered part of medical history if the condition did not worsen (change in frequency or severity).

Conditions that start or worsen after signing consent will be captured as AEs.

Surgical or diagnostic procedures in and of themselves are not AEs. The condition leading to the procedure or the resulting diagnoses would be considered an AE.

Abnormal laboratory test results will be considered AEs if they are considered clinically significant. If the abnormality is part of a diagnosis, only the diagnosis would be considered an AE.

Pre-planned procedures that were scheduled prior to signing consent will not be considered AEs. However, if the pre-planned procedure is performed earlier due to a worsening of a pre-existing condition, the worsening condition would be considered an AE.

Elective procedures performed where there is no change in the subject’s medical condition should not be recorded as AEs but should be documented in the source documents.

All AEs will be followed until resolution or stabilization of the event, completion of the patient’s participation or study termination, or upon agreement between the investigator and sponsor that no further follow-up in necessary whichever occurs first.

Final Version: January 5, 2017 31 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 All AEs will be treated appropriately. Such treatment may include changes in study drug treatment as outlined in section 7.0 of this protocol.

Serious adverse events (as defined in 21 CFR 312.32) A serious adverse event (SAE) is defined as any AE occurring during an investigational study that, in the opinion of the investigator or sponsor, results in any of the following outcomes:

 Death  A life-threatening AE  Inpatient hospitalization or prolongation of existing hospitalization  A persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions  Congenital anomaly/birth defect

Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered serious when, based on appropriate medical judgment, they jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition.

SAEs must be reported to the sponsor (or designee) within 24 hours from the time when the site personnel first learn about the event. Notification may be made via email or fax using the report form provided by the sponsor (paper or electronic data capture (EDC) forms will be acceptable):

Email: [email protected] Fax number: 760-683-6549

If the subject is hospitalized because of or during the course of an SAE, the investigator should attempt to obtain a copy of the hospital discharge summary and any pertinent laboratory or diagnostic reports and provide them to the sponsor (or designee) as soon as possible.

New information about the event as it becomes available to the investigator must be reported to the sponsor within 1 working day of receipt by the investigator and site personnel via the same methods as described above.

All SAEs will be followed until resolution or stabilization of the event, or upon agreement between the investigator and sponsor that no further follow-up is necessary.

Severity Grading: the descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 (2009) or higher will be utilized for all toxicity reporting. A copy of the CTCAE can be downloaded from the CTEP website: http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm.

Final Version: January 5, 2017 32 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 Expectedness: AEs will be assessed by the sponsor as to whether they were expected to occur or unexpected. An unexpected AE is one that is not listed in the investigator brochure or that is not listed at the specificity or severity that has been previously observed; or, if an investigator brochure is not required or available, is not consistent with the risk information described in the general investigational plan or elsewhere in the current application, as amended.

“Unexpected,” as used in this definition, also refers to adverse events or suspected adverse reactions that are mentioned in the investigator brochure as occurring with a class of drugs or as anticipated from the pharmacological properties of the drug, but are not specifically mentioned as occurring with the particular drug under investigation.

Causality assessments: A causal relationship between all AES and SAEs to study drug (investigational product) will be assessed by the investigator and the sponsor.

8.2 Unanticipated Problems (UAP)

Definition (as per Department of Health and Human Services’ Office for Human Research Protections [ORHP] Guidance):

 unexpected (in terms of nature, severity, or frequency) given (a) the research procedures that are described in the protocol-related documents, such as the IRB- approved research protocol and informed consent document; and (b) the characteristics of the subject population being studied;  related or possibly related to participation in the research (in this guidance document, possibly related means there is a reasonable possibility that the incident, experience, or outcome may have been caused by the procedures involved in the research); and  suggests that the research places subjects or others at a greater risk of harm (including physical, psychological, economic, or social harm) than was previously known or recognized.

UAPs will reported by the investigator to regulatory authorities and the IRB as required per 45 CFR part 46 and as per the local IRB requirements.

The ORHP guidance can be found at: http://www.hhs.gov/ohrp/regulations-and-policy/guidance/reviewing-unanticipated- problems/index.html

8.3 Noncompliance

Definition: failure to follow any applicable regulation or institutional policies that govern human subjects research or failure to follow the determinations of the IRB. Noncompliance may occur due to lack of knowledge or due to deliberate choice to ignore regulations, institutional policies, or determinations of the IRB.

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8.4 Serious Noncompliance

Definition: noncompliance that materially increases risks, that results in substantial harm to subjects or others, or that materially compromises the rights or welfare of participants.

8.5 Protocol Exceptions/Deviations

Definition: A planned deviation from the approved protocol that is under the research team’s control. Exceptions apply only to a single participant or a singular situation. Deviations are any intentional or unintentional changes from an IRB-approved protocol that is not approved by the IRB prior to initiation of the change. Major protocol deviations are deviations that result in increased risk to subjects, affect the rights, safety, or welfare of the subjects or affect the integrity of the study (impacts the quality of the data or the outcome of the study). Examples include (but are not limited to) deviations from inclusion/exclusion criteria, informed consent deviations, and concomitant medication restriction deviations. Major protocol violations are to be reported to the IRB.

Protocol exceptions are not expected or encouraged, and should be discussed first with the Study Sponsor.

8.6 Reporting to the Institutional Review Board (IRB)

The PI is required to promptly notify the IRB of the following events according to the local IRB requirements:

 Any unanticipated problems involving risks to participants or others which occur at the institution, or that impacts participants or the conduct of the study.  Noncompliance with federal regulations or the requirements or determinations of the IRB.  Receipt of new information that may impact the willingness of participants to participate or continue participation in the research study.

These events must be reported to the IRB within 10 working days of the occurrence of the event or notification to the PI of the event or as otherwise required by the local IRB. The death of a research participant that qualifies as a reportable event should be reported within 1 working day of the occurrence of the event or notification to the PI of the event, or as otherwise required by the local IRB.

8.7 Reporting to the FDA and Other Investigational Sites

The conduct of the study will comply with all FDA safety reporting requirements. It is the responsibility of the investigator to report any SAEs to the Sponsor (or the Sponsor’s designee) (within 24 hours) and the Sponsor (or designee) will report events to FDA and all investigational sites as per applicable regulations.

Final Version: January 5, 2017 34 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 9.0 INVESTIGATIONAL PRODUCT INFORMATION

9.1 Disulfiram

9.1.1 Disulfiram Description

DSF is an alcohol antagonist drug approved by the FDA for the treatment of alcoholism. Its powder is white, odorless, and almost tasteless. It is soluble in water to the extent of about 20mg in 100mL, and in alcohol to the extent of about 3.8 g in 100 mL.

Molecular formula: C10H20N2S4 Chemical name: bis(diethylthiocarbamoyl) disulfide. Molecular weight: 296.54.

9.1.2 Clinical Pharmacology

DSF is mostly known as an irreversible inhibitor of aldehyde dehydrogenase, which affects alcohol metabolism and causes accumulation of acetaldehyde. However, increasing preclinical studies have shown that DSF is also a proteasome inhibitor, specifically the chymotrypsin-like activity. GBM cells and its stem-like subpopulation (also referred to as CSC) may be more susceptible to the effect of proteasomal inhibition than normal brain cells. DSF is very lipophilic and readily crosses the blood-brain barrier.

9.1.3 Supplier

DSF will be manufactured by IriSys, LLC in San Diego, California in accordance with current Good Manufacturing Practices (cGMP) and provided by Cantex Pharmaceuticals for the study participants. Label text will be compliant with FDA requirements.

9.1.4 Dosage Form

DSF is supplied as 40 mg capsules in bottles containing 60 capsules.

9.1.5 Storage and Stability

DSF is dispensed in a tight, light-resistant container. It should be stored at the study site at controlled room temperature (20° to 25°C or 68° to 77°F) in its original container to protect from bright light. A broader range is acceptable for subject home storage (15° to 30°C or 59° to 86°F).

Final Version: January 5, 2017 35 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 9.1.6 Disulfiram Administration

DSF is taken by mouth three times daily approximately 4-8 hours apart. It should be taken at least one hour before food with approximately 8 ounces of water. It should not be taken together with Cu. It should not be taken within one hour of TMZ. Patients should not have consumed any alcohol at least 12 hours prior to the first dose or at any time during the study. In the rare event of a severe hypersensitivity reaction, discontinue DSF immediately.

9.1.7 Copper Gluconate Description

Cu gluconate is a dietary food supplement generally recognized as safe (GRAS) under §21CFR184.1260. Cu is an important nutrient in the human body and plays an essential role by participating in numerous metabolic reactions in the body (Stern, et al. 2007). Exposure to Cu usually occurs from consumption of food and drinking water. Several studies have concluded that chronic dietary intakes of Cu less than 10 mg/day pose no significant health risk.

9.1.8 Supplier

Cu gluconate will be manufactured by IriSys, LLC in San Diego, California in accordance with current Good Manufacturing Practices (cGMP) and provided by Cantex Pharmaceuticals for the study participants. Label text will be compliant with FDA requirements.

9.1.9 Dosage Form

Cu gluconate is supplied as 1.5 mg capsules in bottles of 60 capsules.

9.1.10 Storage and Stability

Cu gluconate is dispensed in a tight, light-resistant container. It should be stored at the study site at controlled room temperature (20° to 25°C or 68° to 77°F) in its original container to protect from bright light. A broader range is acceptable for subject home storage (15° to 30°C or 59° to 86°F).

9.1.11 Copper Gluconate Administration

Cu gluconate is taken by mouth three times daily with meals at least one hour after DSF administration.

9.2 Treatment Compliance, Accountability and Return

Study medications will be dispensed to patients at the investigation site by authorized site personnel. The investigator or designee must maintain accurate records of all study treatments, including dates, lot numbers and quantities of study drug received at the site as

Final Version: January 5, 2017 36 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 well as dates, lot numbers and quantities dispensed to each patient as well as the amount returned at each visit.

The investigator will instruct patients to return empty bottles and unused product back to the site for each visit. New bottles will be dispensed at each visit.

Upon completion of the study, all unused and/or partially used study drug must be returned to the Sponsor or authorized designee along with the clinical supplies return documentation. Study drug may only be returned to the Sponsor or designee after accountability has been completed and documented, and verified by the study monitor. Any discrepancies must be identified and explained. Drug accountability records, study drug supply receipt and returns must be maintained by the investigator.

10.0 CORRELATIVE STUDIES

The correlative studies to measure single-dose and multiple-dose pharmacokinetics of DSF will be performed in approximately 5 patients on an optional informed consent as described in section 5.5.

If the tumor recurs during the treatment period and the patient has surgery, we will request a portion of the tissue removed during surgery along with blood for future correlative studies.

10.1 Collection of Specimens for Optional Pharmacokinetics Study

Approximately 10 mL of peripheral blood (into a green top tube containing sodium heparin) will be drawn on day 0 (hour 0, 1, 2, 4, 8, and 24), day 3 (hour 4), day 7 (hour 4), and day 10 (hour 4) as described in section 5.6. Blood will be submitted to a laboratory at Washington University in St. Louis for pharmacokinetics analysis.

Final Version: January 5, 2017 37 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 11.0 STUDY CALENDAR

Screening/baseline evaluations are to be conducted within 4 weeks prior to start of protocol therapy. MRIs must be done no more than 6 weeks prior to the start of the protocol therapy. Each treatment cycle is 28 days. The number of cycles will vary per patient but are estimated to be up to 6 cycles.

Screening / Within 10 End of Every End of Follow- Baseline Days Even- Treatment Up1 Before Numbered Each Cycle Cycle (Excluding Cycle 1) Informed consent X Medical history X Physical incl. neurologic X X X exam, KPS, ECOG Evaluation of X X corticosteroid usage CBC X X BMP X X LFTs X X β-hCG2 X Brain MRI X X3 X TMZ Days 1-5 of each cycle4 DSF Days 1-28 of each cycle5 Cu gluconate Days 1-28 of each cycle6 AE assessment X ------X7 Optional blood collection Day 0 (hour 0, 1, 2, 4, 8, 24) for pharmacokinetics Day 3 (hour 4) analysis8 Day 7 (hour 4) Day 10 (hour 4) 1. Follow-up will be as clinically indicated. A chart review will be done at 2 years after the last dose of DSF to look for progression and survival. 2. Women of childbearing potential only. 3. A confirmatory MRI should be considered in 4-8 weeks after documentation of complete response (CR) or partial response (PR) if consistent with the institutional standard of care. 4. To be taken at bedtime on an empty stomach. Must not be taken within an hour of DSF and Cu. 5. To be taken three times a day at least one hour before each meal, with water, approximately 4-8 hours apart. 6. To be taken three times a day with each meal. 7. AEs will be tracked for 30 days after the last dose of DSF. 8. Will be performed on approximately 5 volunteer participants at Washington University, and the participants will take a single dose of 80 mg of DSF on the day prior to the protocol treatment (day 0), and day 1 will be the first day of TMZ and DSF. Hour 4 will be 4 hours after the morning dose of DSF on day 3, 7, and 10. Each blood collection will be 10 mL.

Final Version: January 5, 2017 38 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 12.0 STATISTICAL CONSIDERATIONS

12.1 Definition of Primary Endpoints and Analytical Plan

The primary endpoints will be ORR. ORR will be defined as the percentage of patients with CR or PR according to the RANO criteria as described in Section 6.0. For recurrent TMZ-resistant GBM, continuing TMZ should yield ORR of less than 5% (Quinn, et al. 2009). Patients who have measurable disease as defined by RANO criteria at baseline and have received at least one dose of DSF/Cu with concurrent TMZ will be considered evaluable for response.

To justify future drug development of DSF-Cu for GBM, we hypothesized that the addition of DSF-Cu to TMZ would improve ORR to 20%. The sample size is calculated based on the above hypotheses with one-sided exact test at an approximate α level of 0.10 and 80% statistical power. At these type I and II error rates, 20 patients will detect a proportion of patients who have ORR significantly higher than 5% Of note, previous phase II study testing addition of OBG (a MGMT inhibitor) to TMZ for TMZ-resistant GBM used similar hypothesis and observed a disappointing ORR of 3% (Quinn, et al. 2009). This negative phase II study has detailed information on ORR, PFS, and OS as well as associated estimated 95% CIs and will serve as a historical comparison for this study.

12.2 Definition of Secondary Endpoints and Analytical Plans

The secondary endpoints will be toxicity, PFS, OS, and pharmacokinetics.

1. Adverse events will be coded using a standardized medical dictionary (MedDRA) and categorized by preferred term and System Organ Class (SOC). Toxicities (adverse events) that are possibly/probably/definitely related to DSF will be graded by NCI CTCAE version 4.0 (2009) or higher and will be tabulated by type and grade. Patients who received at least one dose of DSF or Cu will be considered evaluable for toxicity. 2. PFS and OS will be measured from the date of the first dose of DSF-Cu to the date of death or, otherwise, the last follow-up date on which the patient was reported alive. PFS6 will be defined as proportion of patients that are free from progressive disease (PD) as per RANO criteria or death at 6 months from the date of the first dose of DSF-Cu. PFS6 and median PFS will be estimated using the Kaplan-Meier product-limit method. Median OS and OS12 (proportion of patients that are alive at 12 months) will also be estimated using the Kaplan-Meier product-limit method. 3. Plasma concentration of DSF will be measured after single dose and multiple doses for approximately 5 patients. An exploratory analysis will be performed to correlate response and toxicity with the plasma concentration of DSF.

Final Version: January 5, 2017 39 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 13.0 ADMINISTRATIVE PROCEDURES AND CONSIDERATIONS

13.1 IRB Approval

The protocol and informed consent form (ICF) will be reviewed and approved by the Institutional Review Board (IRB). The investigator must submit and obtain (when applicable) approval from the IRB for all subsequent protocol amendments. If requested, the investigator will permit audits by the IRB and regulatory inspections by providing direct access to source data/documents.

The investigator will provide the IRB with progress reports at required intervals (not to exceed one year) and a Study Progress Report following completion, termination, or discontinuation of the investigator’s participation in the study.

13.2 Ethical Conduct

The study will be conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki, and that are consistent with the ICH consolidated Guideline E6 for GCP, and the applicable regulatory requirements and laws.

13.3 Informed Consent

Written informed consent will be obtained from the patient prior to any study related procedures are conducted. Information about the study will be provided to the subject both verbally and in writing and will include an explanation of the objectives of the study, the risks, and the benefits. The patient will be permitted time and opportunity to inquire about details of the study and to decide whether or not to participate and will be informed that participation is voluntary and that he/she can withdraw consent at any time without penalty or loss of benefits to which the patient is otherwise entitled. The investigator/designee will answer any questions regarding the study, prior to obtaining consent.

The investigator must be satisfied that the patient has read and understood the information provided before written consent is obtained. If there is any doubt as to whether the patient has understood the written and verbal information, the patient should not participate in the study.

If the patient agrees to participate in the study, the patient will be asked to sign and date the study ICF which will be retained by the investigator. The investigator/designee must sign and date the ICF before the patient can participate in the study. A copy of the signed and dated ICF will be given to the patient. The informed consent process will be in accordance with all applicable regulatory requirements and must be documented in the patient’s source documents. The original ICF must be retained by the investigator and made available for inspection by the study monitor.

If the ICF is amended during the study, the investigator must follow all applicable regulatory requirements pertaining to approval of the amended ICF by the IRB and use of

Final Version: January 5, 2017 40 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 an amended form (including for ongoing patients). The patients will be required to sign an updated ICF if there is any information that affects the conduct of the study or the subject’s willingness to participate in the study.

13.4 Subject Confidentiality

Subject confidentiality will be strictly held in trust by the participating investigators, their staff, the sponsor and their authorized representatives. This confidentiality is extended to cover testing of biological samples in addition to the clinical information relating to participating patients.

No information that would permit the identification of a specific individual will be provided for entry into the study database or study report. Study documentation submitted to the sponsor will identify patients by study code numbers and initials. The investigator will keep a separate confidential enrollment log that matches identifying study codes with the patients’ names and residences.

The study protocol, documentation, data, and all other information generated will be held in strict confidence. No information concerning the study or data will be released to any unauthorized third party, without prior written approval of the Sponsor.

Authorized representatives of the Sponsor, the designated contract research organization (if applicable), the study monitor, employees of the government authorities such as the US FDA or other government authorities, and members of the IRB may inspect all documents and records required to be maintained by the investigator, including but not limited to, medical records (office, clinic, or hospital) and pharmacy records for the patients in the study. The clinical study site will permit access to such records.

13.5 Study Monitoring

The Sponsor or its designee are responsible for monitoring the study in accordance with the requirements of ICH/GCP, and in accordance with written standard operating procedures (“SOPs”) and monitoring plans. The study will be monitored by the Sponsor or designee at all stages of study conduct from inception to completion in accordance with ICH/GCP. The investigator will allocate adequate time for such monitoring activities. This monitoring will be in the form of site visits and other communication and will include review of original source documents, case report forms (CRFs), facilities and equipment, recruiting, record-keeping, protocol adherence, data collection, AE reporting, and other factors. The frequency of these visits will depend upon the progress of the study.

The investigator will ensure that the monitor or other compliance or quality assurance reviewers are given access to all the above noted study-related documents and study-related facilities (e.g., pharmacy, diagnostic laboratory, etc.) and has adequate space to conduct the monitoring visit.

Final Version: January 5, 2017 41 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 13.6 Case Report Forms and Study Records/Data

Source documents are defined as original documents, data, and records. This may include hospital records, clinical and office charts, laboratory data/information, subjects’ diaries or evaluation checklists, pharmacy dispensing and other records, recorded data from automated instruments, microfiches, photographic negatives, microfilm or magnetic media, and/or x-rays. Data collected during this study must be recorded on the appropriate source documents. All source documents should be completed in a neat, legible manner to ensure accurate interpretation of data. The management and recording of data will be consistent with applicable ICH/GCP guidelines.

Case report forms (CRFs) will be electronic and will be used to collect data during the study. CRFs will be patient-specific. CRFs will be completed as soon as data are available in the source documents as some monitoring procedures may be done in real-time. Sites will be trained on the appropriate completion of the CRFs and training records will be maintained. A CRF will be completed for each patient who signs an ICF. Data collected in the CRF may be queried by the sponsor. All queries must be resolved in a timely manner.

The investigator will sign and date where indicated in the CRF which will indicate that the investigator has reviewed all data collected, queries, or other clarifications and agrees that the content is accurate.

The investigator is responsible for the accuracy, completeness, and timeliness of the data reported in the CRF. Study data management, monitoring, statistical analysis, and reporting will be performed by the sponsor or designee according to SOPs.

Completed datasets and associated files are the property of the sponsor and should not be made available in any form to third parties, except for authorized business representatives or appropriate government health or regulatory authorities, without the written permission of the sponsor.

The investigator/institution will maintain all CRFs and all source documents that support the data collected from each subject, and all study documents as specified in ICH/GCP and as specified by applicable regulatory requirements. The investigator/institution will take measures to prevent accidental or premature destruction of documents. Essential documents should be retained for at least 2 years after the last approval of a marketing application in an ICH region or at least 2 years have elapsed since the formal discontinuation of clinical development of the study drug (investigational product). These documents should be retained longer if required by the applicable regulatory requirements or by an agreement with the Sponsor. It is the responsibility of the Sponsor to inform the investigator/institution as to when these documents no longer need to be retained.

The Sponsor must be notified if the investigator retires, relocates or otherwise will no longer work on the study and record custody and retention must be transferred to a person who will accept the responsibility. The investigator must not relocate study documents or dispose of any study documents before obtaining written approval from the Sponsor.

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13.7 Criteria for Termination of the Study

If the Sponsor or designee, the investigator or regulatory authority discovers any condition arising during the study that indicates that the study or study site should be terminated, this action may be taken after appropriate consultation between the Sponsor or its designee and the investigator. The Sponsor or its designee has the right to terminate the participation of either an individual site or the study at any time, for any reason, which may include the following:

 The incidence and severity of AEs in this or other studies indicates a potential health hazard to patients  Patient enrollment is unsatisfactory  Data recording is inaccurate or incomplete  Investigator(s) do(es) not adhere to the protocol or applicable regulatory guidelines in conducting this study  Submission of knowingly false information from the study site to the Sponsor or its designee or regulatory authorities.

13.8 Investigator Final Report(s)

Upon completion of the trial, the investigator should, where required by the applicable regulatory requirements, inform the institution, and the investigator/institution should provide the Sponsor with all required reports, the IRB with a summary of the trial’s outcome, and the regulatory authority(ies) with any report(s) they require of the investigator/institution.

13.9 Financial Disclosure

The principal and sub-investigators are required to provide certification of the following:

 no financial arrangements with the Sponsor exist where study outcome could affect compensation  the investigator does not have significant equity interest in the sponsor  the investigator has not received payments of other sorts.

The principal and sub-investigator must inform the sponsor if the above circumstances change during the course of the study or within 1 year of the end of his/her participation in the study.

13.10 Publication and Disclosure

All unpublished information that the Sponsor gives to the investigator shall be kept confidential and shall not be published or disclosed to a third party without the prior written consent of the Sponsor.

Final Version: January 5, 2017 43 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 When the Sponsor generates reports for presentation to regulatory agencies, one or more of the investigators who has/have contributed significantly to the study may be asked to endorse the final report.

The investigator shall not make a patent application based on the results of this study and shall not assist any third party in making such an application without the written authorization of the Sponsor unless otherwise specified in a clinical study agreement.

14.0 REFERENCES

Chen, D., et al. 2006 Disulfiram, a clinically used anti-alcoholism drug and copper-binding agent, induces apoptotic cell death in breast cancer cultures and xenografts via inhibition of the proteasome activity. Cancer Res 66(21):10425-33. Child, G. P., and M. Crump 1952 The toxicity of tetraethylthiuram disulphide (antabuse) to mouse, rat, rabbit and dog. Acta Pharmacol Toxicol (Copenh) 8(3):305-14. Chinot, O. L., et al. 2014 Bevacizumab plus radiotherapy-temozolomide for newly diagnosed glioblastoma. N Engl J Med 370(8):709-22. Cvek, B., et al. 2008 Ni(II), Cu(II), and Zn(II) diethyldithiocarbamate complexes show various activities against the proteasome in breast cancer cells. J Med Chem 51(20):6256-8. Eckel-Passow, J. E., et al. 2015 Glioma Groups Based on 1p/19q, IDH, and TERT Promoter Mutations in Tumors. N Engl J Med 372(26):2499-508. Faiman, M. D., D. E. Dodd, and R. E. Hanzlik 1978 Distribution of S35 disulfiram and metabolites in mice, and metabolism of S35 disulfiram in the dog. Res Commun Chem Pathol Pharmacol 21(3):543-67. Faiman, M. D., J. C. Jensen, and R. B. Lacoursiere 1984 Elimination kinetics of disulfiram in alcoholics after single and repeated doses. Clin Pharmacol Ther 36(4):520-6. Friedman, H. S., et al. 2009 Bevacizumab alone and in combination with irinotecan in recurrent glioblastoma. J Clin Oncol 27(28):4733-40. Fuller, R. K., and E. Gordis 2004 Does disulfiram have a role in alcoholism treatment today? Addiction 99(1):21-4. Gilbert, M. R., et al. 2014 A randomized trial of bevacizumab for newly diagnosed glioblastoma. N Engl J Med 370(8):699-708. Gilbert, M. R., et al. 2013 Dose-dense temozolomide for newly diagnosed glioblastoma: a randomized phase III clinical trial. J Clin Oncol 31(32):4085-91. Grossmann, K.F. , et al.

Final Version: January 5, 2017 44 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 2011 A phase I clinical study investigating disulfiram and copper gluconate in patients with advanced treatment-refractory solid tumors involving the liver. Cancer Res 71(Suppl 8):[Abstract] nr 1308. Guild, J., and N. B. Epstein 1951 Psychosis during the treatment of alcoholism with tetraethylthiuram disulfide. Q J Stud Alcohol 12(3):360-6. Hochreiter, J., et al. 2012 Disulfiram metabolite S-methyl-N,N-diethylthiocarbamate quantitation in human plasma with reverse phase ultra performance liquid chromatography and mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 897:80-4.Hothi, P., et al. 2012 High-throughput chemical screens identify disulfiram as an inhibitor of human glioblastoma stem cells. Oncotarget 3(10):1124-36. Huang, J., et al. 2016 A phase I study to repurpose disulfiram in combination with temozolomide to treat newly diagnosed glioblastoma after chemoradiotherapy. J Neurooncol. 2016 Jun;128(2):259-66 Jacobsen, E. 1952 Deaths of alcoholic patients treated with disulfiram (tetraethylthiuram disulfide) in Denmark. Q J Stud Alcohol 13(1):16-26. Jensen, J. C., M. D. Faiman, and A. Hurwitz 1982 Elimination characteristics of disulfiram over time in five alcoholic volunteers: a preliminary study. Am J Psychiatry 139(12):1596-8. Johansson, B. 1990 Plasma protein binding of disulfiram and its metabolite diethylthiocarbamic acid methyl ester. J Pharm Pharmacol 42(11):806-7. 1992 A review of the pharmacokinetics and pharmacodynamics of disulfiram and its metabolites. Acta Psychiatr Scand Suppl 369:15-26. Johansson, B., et al. 1991 Dose-effect relationship of disulfiram in human volunteers. II: A study of the relation between the disulfiram-alcohol reaction and plasma concentrations of acetaldehyde, diethyldithiocarbamic acid methyl ester, and erythrocyte aldehyde dehydrogenase activity. Pharmacol Toxicol 68(3):166-70. Johnson, D. R., and B. P. O'Neill 2012 Glioblastoma survival in the United States before and during the temozolomide era. J Neurooncol 107(2):359-64. Kreisl, T. N., et al. 2009 Phase II trial of single-agent bevacizumab followed by bevacizumab plus irinotecan at tumor progression in recurrent glioblastoma. J Clin Oncol 27(5):740-5. Liu, P., et al. 2012 Cytotoxic effect of disulfiram/copper on human glioblastoma cell lines and ALDH-positive cancer-stem-like cells. Br J Cancer 107(9):1488-97. Louis, D. N., et al. 2016 The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. Acta Neuropathol 131(6):803-20. Lun, X., et al.

Final Version: January 5, 2017 45 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 2016 Disulfiram when combined with copper enhances the therapeutic effects of temozolomide for the treatment of Glioblastoma. Clin Cancer Res. 2016 Aug 1;22(15):3860-75 Major, L. F., et al. 1979 Dopamine-beta-hydroxylase in the cerebrospinal fluid: relationship to disulfiram- induced psychosis. Biol Psychiatry 14(2):337-44. Martensen-Larsen, O. 1951 Psychotic phenomena provoked by tetraethylthiuram disulfide. Q J Stud Alcohol 12(2):206-16. Milandri, M., et al. 1980 Effect of long-term disulfiram administration on rat liver. Pharmacology 21(1):76-80. Nechushtan H, et al. 2015 A phase IIb trial assessing the addition of disulfiram to chemotherapy for the treatment of metastatic non-small cell lung cancer. Oncologist. 20(4):366-7 Nilsson, G. E., O. Tottmar, and G. Wahlstrom 1987 Effects of aldehyde dehydrogenase inhibitors on hexobarbital sensitivity and neuroamine metabolism in rat brain. Brain Res 409(2):265-74. Ostrom, Q. T., et al. 2014 CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2007-2011. Neuro Oncol 16 Suppl 4:iv1-63. Paranjpe, A., et al. 2014 Disulfiram is a direct and potent inhibitor of human O6-methylguanine-DNA methyltransferase (MGMT) in brain tumor cells and mouse brain and markedly increases the alkylating DNA damage. Carcinogenesis 35(3):692-702. Perry, J. R., et al. 2010 Phase II trial of continuous dose-intense temozolomide in recurrent malignant glioma: RESCUE study. J Clin Oncol 28(12):2051-7. Piccioni, D. E., et al. 2014 Deferred use of bevacizumab for recurrent glioblastoma is not associated with diminished efficacy. Neuro Oncol 16(6):815-22. Quinn, J. A., et al. 2009 Phase II trial of temozolomide plus o6-benzylguanine in adults with recurrent, temozolomide-resistant malignant glioma. J Clin Oncol 27(8):1262-7. Reuss, D. E., et al. 2015 Adult IDH wild type astrocytomas biologically and clinically resolve into other tumor entities. Acta Neuropathol 130(3):407-17. Schweizer, M. T., et al. 2013 Pharmacodynamic study of disulfiram in men with non-metastatic recurrent prostate cancer. Prostate Cancer Prostatic Dis 16(4):357-61. Stern, B. R., et al. 2007 Copper and human health: biochemistry, genetics, and strategies for modeling dose-response relationships. J Toxicol Environ Health B Crit Rev 10(3):157-222. Stewart, D. J., S. Verma, and J. A. Maroun 1987 Phase I study of the combination of disulfiram with cisplatin. Am J Clin Oncol 10(6):517-9.

Final Version: January 5, 2017 46 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201 Stupp, R., et al. 2005 Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352(10):987-96. Suh, J. J., et al. 2006 The status of disulfiram: a half of a century later. J Clin Psychopharmacol 26(3):290-302. Triscott, J., et al. 2012 Disulfiram, a drug widely used to control alcoholism, suppresses the self-renewal of glioblastoma and over-rides resistance to temozolomide. Oncotarget 3(10):1112-23. Wen, P. Y., et al. 2010 Updated response assessment criteria for high-grade gliomas: response assessment in neuro-oncology working group. J Clin Oncol 28(11):1963-72. Wilk, W., et al. 2015 Phase III trial exploring the combination of bevacizumab and lomustine in patients with first recurrence of a glioblastoma: the EORTC 26101 trial. Neuro Oncol 17(suppl 5):v1 [abstract: LB-05]. Wilmotte, R., et al. 2005 B7-homolog 1 expression by human glioma: a new mechanism of immune evasion. Neuroreport 16(10):1081-5. Zhang, L., et al. 2013 A novel UPLC-ESI-MS/MS method for the quantitation of disulfiram, its role in stabilized plasma and its application. J Chromatogr B Analyt Technol Biomed Life Sci 937:54-9. Zhou, Q., A. Xing, and K. Zhao 2014 Simultaneous determination of nickel, cobalt and mercury ions in water samples by solid phase extraction using multiwalled carbon nanotubes as adsorbent after chelating with sodium diethyldithiocarbamate prior to high performance liquid chromatography. J Chromatogr A 1360:76-81.

Final Version: January 5, 2017 47 of 50 Cantex Clinical Study Protocol DSF-Cu CAN-201

APPENDIX A: KPS and ECOG Scales

KPS

100 Normal to no complaints; no evidence of disease 90 Able to carry on normal activity; minor signs or symptoms of disease 80 Normal activity with effort; some signs or symptoms of disease 70 Cares for self; unable to carry on normal activity or do active work 60 Requires occasional assistance but is able to care for most personal needs 50 Requires considerable assistance and frequent medical care 40 Disabled; requires special care and assistance 30 Severely disabled; hospital admission is indicated although death not imminent 20 Very sick; hospital admission necessary; active supportive treatment necessary 10 Moribund; fatal processes progressing rapidly 0 Dead

ECOG 0 Asymptomatic (Fully active, able to carry on all pre-disease activities without restriction 1 Symptomatic but completely ambulatory (Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature. For example, light housework, office work) 2 Symptomatic, <50% in bed during the day (Ambulatory and capable of all self-care but unable to carry out any work activities. Up and about more than 50% of waking hours) 3 Symptomatic, >50% in bed, but not bedbound (Capable of only limited self-care, confined to bed or chair 50% or more of waking hours) 4 Bedbound (Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair) 5 Death

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APPENDIX B: Sample Concurrent Disulfiram and Copper Medication Diary

Today’s Date: Agent: Disulfiram/Copper Month: ____

Patient Name:

INSTRUCTIONS TO THE PATIENT: 1. Complete one form for each month. Take __ mg ( ___capsules) of disulfiram with 8 ounces of water three times a day at approximately 4-8 hours apart, at least one hour before food. Please also take 1.5 mg of copper gluconate supplement three times a day with each meal but not within one hour of disulfiram. Do not take disulfiram or copper within one hour of your dose of temozolomide. 2. Record the date, and how many times you took disulfiram and copper on that date. 3. If you have any questions or notice any side effects, please record them in the comments section. Record the time if you should vomit. 4. Please return the forms to your physician or your study coordinator when you go to your next appointment. Please bring your unused study medications and/or empty bottles with you to each clinic visit so that a pill count can be done. 5. Avoid consuming alcohol before and throughout the entire study.

Day Date Check each time you take your Check each time you take your Comments disulfiram dose copper dose Morning Afternoon Evening Morning Afternoon Evening 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

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APPENDIX C: Sample Concurrent Temozolomide Medication Diary

Today’s Date: Agent: Temozolomide Month: ____

Patient Name:

INSTRUCTIONS TO THE PATIENT: 1. Complete this form. Take _____mg ( ___capsules) of temozolomide daily as instructed by your oncologist. Temozolomide should be taken at bedtime on an empty stomach and should not be taken within one hour of your disulfiram or copper supplement. 2. Record the date, the number of capsules taken, and when you took them. 3. If you forget to take temozolomide before midnight, then do not take a dose that day. Restart it the next day. 4. If you have any questions or notice any side effects, please record them in the comments section. Record the time if you should vomit. 5. Please return the forms to your physician or your study coordinator when you go to your next appointment. Please bring your unused study medications and/or empty bottles with you to each clinic visit so that a pill count can be done. 6. Avoid consuming alcohol before and throughout the entire study.

Day Date What time was # of tablets Comments dose taken? taken 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Final Version: January 5, 2017 50 of 50