a phase ib/ii study of
untitled h2

| Protocol Number:        | 11996                                                                                                                                                                                                                        |
|-------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Version:                | 6.0 (May 26, 2017)                                                                                                                                                                                                           |
| Study Drug:             | Eribulin                                                                                                                                                                                                                     |
| IND:                    | Exempt                                                                                                                                                                                                                       |
| Coordinating Center:    | University of California, San Francisco Helen Diller Family Comprehensive Cancer Center                                                                                                                                      |
| Principal Investigator: | Hope S. Rugo, M.D. UCSF Helen Diller Family Comprehensive Cancer Center                                                                                                                                                      |
| Statistician:           | Jimmy Hwang, PhD                                                                                                                                                                                                             |
| Revision History:       | Version 6.0 (May 26, 2017) Version 5.0 (May 31, 2013) Version 4.0 (May 29, 1012) Version 3.0 (July 1, 2011) Version 2.0 (June 27, 2011) Version 1.0 (June 13, 2011) Version 0.4 (April 18, 2011) Version 0.2 (April 1, 2011) |
a phase ib/ii study of
study synopsis

This is a phase Ib/II trial designed to determine the MTD and DLTs of the combination of eribulin and cyclophosphamide in solid tumors and make preliminary estimates regarding efficacy of this treatment in patients with advanced breast cancer. Phase 1b will be conducted at UCSF. Phase 2 will be conducted at multiple sites to be determined. UCSF will be the coordinating center.  
The study drug in this protocol is intravenous eribulin mesylate. Eribulin will be administered on day 1 and 8 in combination with cyclophosphamide 600 mg/m2 on day 1. Three weeks will constitute one cycle of treatment.  
The study includes a standard dose-confirmation schema (phase Ib portion) enrolling 3 to 6 patients/subjects, with any solid tumors, per cohort (3+3 design) with a total of 18 patients. The doseexpansion (phase II portion) will enroll 40 patients with advanced breast cancer to detect an effect size of 15% with a power of 80% with endpoints of safety, efficacy, and clinical benefit rate. A
maximum of 58 patients will be enrolled on the phase Ib and II portions of this trial combined and will be treated until disease progression or toxicity mandate treatment change.  
Eribulin is a non-taxane microtubule inhibitor that is FDA approved as monotherapy for the treatment of taxane and anthracycline resistant metastatic breast cancer. The combination of docetaxel and cyclophosphamide is a well-accepted adjuvant chemotherapy regimen that has become an increasingly common therapeutic choice for intermediate risk early stage breast cancer. Eribulin has a favorable toxicity profile compared to docetaxel with the most common adverse reactions (incidence
≤25%) including neutropenia, anemia, asthenia/fatigue, alopecia, peripheral neuropathy, nausea, and constipation. Eribulin appears to have activity in taxane resistant disease, making it an attractive partner with cyclophosphamide.  
Neuropathy can be a devastating complication from adjuvant chemotherapy and in the metastatic setting, may limit effective therapy and reduce quality of life. Understanding the host factors that predict risk for neuropathy is critical, as these patients may in particular benefit from the lower risk of neuropathy associated with eribulin therapy. In conjunction with this trial, we have included correlative studies to study the proposed pharmacogenomic factors associated with risk of neuropathy. In this way we will potentially be able to identify patients who could preferentially be treated with less neurotoxic microtubule inhibitors.  
| Table of Contents                                                                                                                                  |                                                                                                                                                                           |
|----------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| L List of Tables and Figures .                                                                                                                     |                                                                                                                                                                           |
| List of Abbreviations                                                                                                                              |                                                                                                                                                                           |
| BACKGROUND AND RATIONALE                                                                                                                           |                                                                                                                                                                           |
| 1.0                                                                                                                                                |                                                                                                                                                                           |
| 1.1                                                                                                                                                | Breast Cancer                                                                                                                                                             |
| 1.2                                                                                                                                                | Breast cancer treatment                                                                                                                                                   |
| 1.3                                                                                                                                                | Toxicity of taxanes ................................................................................................................................................... 9 |
| 1.4                                                                                                                                                | Toxicity of epothilones                                                                                                                                                   |
| 1.5                                                                                                                                                | Eribulin mesylate and Preclinical data ......................................................................................... 9                                        |
| 1.6                                                                                                                                                | Clinical data for eribulin mesylate                                                                                                                                       |
| 1.7                                                                                                                                                | In Vitro and Vivo Activity Studies .                                                                                                                                      |
| 1.7.1                                                                                                                                              | Nonclinical Pharmacokinetics .                                                                                                                                            |
| 1.7.2                                                                                                                                              | Clinical Pharmacokinetics                                                                                                                                                 |
| 1.7.3                                                                                                                                              | Toxicology .                                                                                                                                                              |
| 1.7.4                                                                                                                                              | Developmental/Reproductive Toxicity:                                                                                                                                      |
| 1.8                                                                                                                                                | Cyclophosphamide                                                                                                                                                          |
| 1.9                                                                                                                                                | RATIONALE                                                                                                                                                                 |
| 1.9.1                                                                                                                                              | Rationale for eribulin in combination with cyclophosphamide ..................................... 12                                                                      |
| 1.9.2                                                                                                                                              | Rationale for Dosing                                                                                                                                                      |
| 1.9.3                                                                                                                                              | Rationale for Starting Dose                                                                                                                                               |
| 1.10                                                                                                                                               | Correlative Studies Background                                                                                                                                            |
| 1.10.1                                                                                                                                             | Single-nucleotide polymorphisms                                                                                                                                           |
| 1.10.2                                                                                                                                             | Genome Analysis                                                                                                                                                           |
| 1.10.3                                                                                                                                             | CC                                                                                                                                                                        |
| 1.10.4                                                                                                                                             | Archived Tissue Analysis .                                                                                                                                                |
| 1.10.5                                                                                                                                             | Quality of life                                                                                                                                                           |
| 1.10.6                                                                                                                                             |                                                                                                                                                                           |
| 2.0                                                                                                                                                | OBJECTIVES .                                                                                                                                                              |
| 2.1                                                                                                                                                | Phase Ib .                                                                                                                                                                |
| 2.2                                                                                                                                                | Phase II .                                                                                                                                                                |
| 3.0                                                                                                                                                | PATIENT SELECTION                                                                                                                                                         |
| Subject Inclusion Criteria .                                                                                                                       |                                                                                                                                                                           |
| 3.1                                                                                                                                                |                                                                                                                                                                           |
| 3.2                                                                                                                                                | Subject Exclusion Criteria .                                                                                                                                              |
| Inclusion of Women and Minorities ............................................................................................................. 19 |                                                                                                                                                                           |
| 3.3                                                                                                                                                |                                                                                                                                                                           |
| 4.0                                                                                                                                                | SUBJECT REGISTRATION AND ENROLLMENT                                                                                                                                       |
| 4.1                                                                                                                                                | Subject Recruitment & Study Sites                                                                                                                                         |
| 4.2                                                                                                                                                | Registration Procedures                                                                                                                                                   |
| 4.2.1                                                                                                                                              | Institutional Registration .                                                                                                                                              |
| 4.2.2                                                                                                                                              | Lead Center Registration .                                                                                                                                                |
| 5.0                                                                                                                                                | STUDY DESIGN AND TREATMENT PLAN                                                                                                                                           |
| 5.1                                                                                                                                                | Dose and schedule                                                                                                                                                         |
| 5.2                                                                                                                                                | Dose-Limiting Toxicity Phase Ib                                                                                                                                           |
| 5.2.1                                                                                                                                              |                                                                                                                                                                           |
| 5.2.2                                                                                                                                              | Non-hematologic dose-limiting toxicity                                                                                                                                    |
| 5.3                                                                                                                                                | D Duration of Therapy                                                                                                                                                     |
| 5.4                                                                                                                                                | Conditions for Terminating the Study                                                                                                                                      |
| 6.0                                                                                                                                                | TOXICITY MANAGEMENT AÑD DOSE MODIFICATION                                                                                                                                 |
| 6.1                                                                                                                                                | Removal of patients from study:                                                                                                                                           |
| 6.2                                                                                                                                                | Concomitant Medications                                                                                                                                                   |
| Supportive Care Guidelines .                                                                                                                       |                                                                                                                                                                           |
| 6.3                                                                                                                                                |                                                                                                                                                                           |
| 7.0                                                                                                                                                | DRUG INFORMATION                                                                                                                                                          |
| Eribulin mesylate (Halaven™)                                                                                                                       |                                                                                                                                                                           |
| 7.1                                                                                                                                                |                                                                                                                                                                           |
| 7.2                                                                                                                                                | Cyclophosphamide (Cytoxan®)                                                                                                                                               |
| 8.0                                                                                                                                                | CORRELATIVE/SPECIAL STUDIES                                                                                                                                               |
| 8.1                                                                                                                                                | Pharmacokinetic (PK) Studies .                                                                                                                                            |
| 8.2                                                                                                                                                | CC                                                                                                                                                                        |  
| 8.3                                                                                                                                                             | Pharmacogenomic (PG studies) .                                                                                                                                     |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Archived Tissue Analysis .                                                                                                                                      |                                                                                                                                                                    |
| 8.4                                                                                                                                                             |                                                                                                                                                                    |
| 9.0                                                                                                                                                             | STUDY PROCEDURES .                                                                                                                                                 |
| 9.1                                                                                                                                                             | Screening assessments                                                                                                                                              |
| Assessments and Procedures During Treatment                                                                                                                     |                                                                                                                                                                    |
| 9.2                                                                                                                                                             | Study Termination                                                                                                                                                  |
| 9.3                                                                                                                                                             |                                                                                                                                                                    |
| 9.4                                                                                                                                                             | Follow-up                                                                                                                                                          |
| 10.0 CRITERIA FOR EVALUATION .                                                                                                                                  |                                                                                                                                                                    |
| 10.1                                                                                                                                                            | Antitumor Effect - Solid Tumors                                                                                                                                    |
| 10.1.1                                                                                                                                                          | Response Definitions .                                                                                                                                             |
| 10.1.2                                                                                                                                                          | Disease Parameters                                                                                                                                                 |
| 10.1.3                                                                                                                                                          | Methods for evaluation of measurable disease .                                                                                                                     |
| 10.1.4                                                                                                                                                          | Response Criteria                                                                                                                                                  |
| 10.1.5                                                                                                                                                          | D Duration of Response .................................................................................................................41                         |
| 1.0 STATISTICAL CONSIDERATIONS ...............................................................................................................42                |                                                                                                                                                                    |
| Study Design/Endpoint Definitions .......................................................................................................42                     |                                                                                                                                                                    |
| 11.1                                                                                                                                                            |                                                                                                                                                                    |
| 11.2                                                                                                                                                            | Accrual Objectives ...................................................................................................................................42           |
| 11.2.1                                                                                                                                                          | Replacement of Patients: ...........................................................................................................................42             |
| 1.3                                                                                                                                                             | Stratification Factors .....................................................................................................................43                     |
| 1.4                                                                                                                                                             | Analysis of Secondary Endpoints                                                                                                                                    |
| Reporting and Exclusions .....................................................................................................................43                |                                                                                                                                                                    |
| 1.5                                                                                                                                                             |                                                                                                                                                                    |
| 11.5.1                                                                                                                                                          | Evaluation of toxicity.                                                                                                                                            |
| 11.5.2                                                                                                                                                          | Evaluation of response                                                                                                                                             |
| 12.0 MULTI-CENTER GUIDELINES .                                                                                                                                  |                                                                                                                                                                    |
| 12.1                                                                                                                                                            | Responsibilities of the coordinating/lead center                                                                                                                   |
| 12.1.1                                                                                                                                                          | OVersight                                                                                                                                                          |
| 12.1.2                                                                                                                                                          | Protocol Distribution                                                                                                                                              |
| 12.1.3                                                                                                                                                          | Documentation of Distribution                                                                                                                                      |
| 12.1.4                                                                                                                                                          | Documentation of Protocol Approvals                                                                                                                                |
| 12.2                                                                                                                                                            | Responsibilities of other participating sites .                                                                                                                    |
| 12.2.1                                                                                                                                                          | Study Initiation .                                                                                                                                                 |
| 12.2.2                                                                                                                                                          | Patient Enrollment .                                                                                                                                               |
| 12.2.3                                                                                                                                                          | Study Drug Supply and Accountability .                                                                                                                             |
| 12.2.4                                                                                                                                                          | Approval of Protocol and Amendments ...........................................................................................45                                  |
| 13.0  DATA MANAGEMENT                                                                                                                                           |                                                                                                                                                                    |
| 14.0  ETHICAL ASPECTS AND REGULATORY CONSIDERATIONS ......................................................46                                                    |                                                                                                                                                                    |
| 14.1                                                                                                                                                            | Conduct of the study ...........................................................................................................................................46 |
| 14.2                                                                                                                                                            | Scientific Review .......................................................................................................................................46        |
| 14.3                                                                                                                                                            | Institutional Review Board / Independent Ethics Committee ......................................................46                                                 |
| 14.4                                                                                                                                                            | Investigational New Drug Application (IND)                                                                                                                         |
| 14.5     Additional Reporting Requirements ..................................................................................................................46 |                                                                                                                                                                    |
| 15.1                                                                                                                                                            | Adverse Event Definitions ..................................................................................................................46                     |
| 15.1.1                                                                                                                                                          | Adverse Events .                                                                                                                                                   |
| 15.1.2                                                                                                                                                          | Serious Adverse Events .................................................................................................................47                         |
| 15.2                                                                                                                                                            | Adverse Event Grading .........................................................................................................................47                  |
| 15.3                                                                                                                                                            | Adverse Event Reporting Period                                                                                                                                     |
| 15.4                                                                                                                                                            | Adverse Event Reporting and Data Safety Monitoring ...........................................................48                                                   |
| 15.4.1                                                                                                                                                          | Oversight and Monitoring Plan .............................................................................................................48                      |
| 15.4.2                                                                                                                                                          | Monitoring and Reporting Guidelines ..........................................................................................48                                   |
| 15.4.3                                                                                                                                                          | Review and Oversight Requirements                                                                                                                                  |
| 16.0  REFERENCES .                                                                                                                                              |                                                                                                                                                                    |
| APPENDIX A .                                                                                                                                                    |                                                                                                                                                                    |
| APPENDIX B .                                                                                                                                                    |                                                                                                                                                                    |
| APPENDIX C .                                                                                                                                                    |                                                                                                                                                                    |
| APPENDIX D .                                                                                                                                                    |                                                                                                                                                                    |  
| APPENDIX E .   |
|----------------|  
| List of Tables and Figures Table 3-1. Adequate Organ Function Values 18 Table 5-1. Dose Escalation Schedule 20 Table 6-1. Toxicity management guidelines for eribulin and cyclophosphamide 26 Table 9-1 Study Calendar 35 Table 10-2. Evaluation of Patients with Measurable Disease (i.e., Target Disease) 41 Table 10-3. Evaluation of Patients with Non-Measurable Disease (i.e., Non-Target Disease) 41   |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| List of Abbreviations AC doxorubicin (adriamycin) and cyclophosphamide aCGH array comparative genomic hybridization AE adverse event ALDH aldehyde dehydrogenase ALT alanine transaminase ANC absolute neutrophil count AST aspartate transaminase AUC area under the curve BID twice a day BSA body surface area CAEPR Comprehensive Adverse Event and Potential  Risks CALGB Cancer and Leukemia Group B CBC complete blood count CBR clinical benefit rate CGH comparative genomic hybridization  CHR UCSF Committee on Human Research Cl plasma clearance CR complete response CRC Clinical Research Coordinator CRF case report form CT computerized tomography CTC circulating tumor cells CTCAE Common Terminology Criteria for Adverse  Events CTMS Clinical Trials Management System CV coefficient of variability CYP cytochrome P450 DLT dose limiting toxicity DFS disease free survival DNA deoxyribonucleic acid DSMB data safety monitoring board E7389 eribulin mesylate ECOG Eastern Cooperative Oncology Group EGFR epidermal growth factor receptor ECOG Eastern Cooperative Oncology Group EORTC European Organization for Research and  Treatment of Cancer FACS fluorescence-activated cell sorting FACT Functional Assessment of Cancer Therapy FDA Food and Drug Administration FNA Fine needle aspiration GCP Good Clinical Practice GLP Good Laboratory Practices GOG Gynecologic Oncology Group GWAS genome wide association study HER-2 human epidermal growth factor receptor 2 HIPPA The Health Insurance Portability and  Accountability Act HGB hemoglobin HR heart rate HV healthy volunteers   | IC50                                                 | 50% inhibitory concentration   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------|--------------------------------|
| IND                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | investigational new drug                             |                                |
| IEC                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Independent Ethics Committee                         |                                |
| IRB                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Institutional Review Board                           |                                |
| IB                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Investigator's Brochure                              |                                |
| IV                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | intravenous                                          |                                |
| KPS                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Karnofsky Performance Status                         |                                |
| MAD                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | maximum administered dose                            |                                |
| MBC                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | metastatic breast cancer                             |                                |
| MRI                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Magnetic Resonance Imaging                           |                                |
| MTD                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | maximum tolerated dose                               |                                |
| NCI                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | National Cancer Institute                            |                                |
| NOAEL                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | no observed adverse effect level                     |                                |
| NSABP                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | National Surgical Adjuvant Breast and Bowel  Project |                                |
| NYHA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | New York Heart Association                           |                                |
| OS                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | overall survival                                     |                                |
| PD                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | progressive disease                                  |                                |
| PFS                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | progression free survival                            |                                |
| Pgp                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | permeability glycoprotein                            |                                |
| PgR                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | progesterone receptor                                |                                |
| PHI                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | personal health information                          |                                |
| PI                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Principal Investigator                               |                                |
| PK                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | pharmacokinetic                                      |                                |
| PO                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | by mouth                                             |                                |
| PR                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | partial response                                     |                                |
| PRC                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | UCSF Protocol Review Committee                       |                                |
| QD                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | once daily                                           |                                |
| QID                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | four times a day                                     |                                |
| QOD                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | every other day                                      |                                |
| QOL                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Quality of life                                      |                                |
| QLQ                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Quality of life questionnaire                        |                                |
| QTc                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | QT corrected interval                                |                                |
| RECIST                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | Response Evaluation Criteria in Solid Tumors         |                                |
| RNA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | ribonucleic acid                                     |                                |
| RR                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | response rate                                        |                                |
| SAE                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | serious adverse event                                |                                |
| SC                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | subcutaneously                                       |                                |
| SD                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | stable disease                                       |                                |
| SiBP                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | sitting blood pressure                               |                                |
| t ½                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | elimination half-life                                |                                |
| TC                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | docetaxel and cyclophosphamide                       |                                |
| TPC                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | the physician's choice                               |                                |
| ULN                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | upper limit of normal                                |                                |
| Vdss                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | volume of distribution at steady state               |                                |
| WBC                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | white blood cell                                     |                                |
| WGA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | whole genome amplification                           |                                |
a phase ib/ii study of
1.0 background and rationale 1.1

Breast cancer is the most common female cancer in the United States and is estimated to affect 207,090 women in the United States in 2010, with over 40,000 deaths expected (Jemal, Siegel et al. 2010). As many as 20% of patients will develop metastatic disease and most will have received prior adjuvant and/or neoadjuvant chemotherapy. Despite advances in therapies, the overall long-term prognosis of patients with metastatic breast cancer remains poor, with time to progression ranging from 6 to 9 months in the first-line setting and median survival overall of approximately 3 years.
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1.2 breast cancer treatment

For breast cancer, anthracyclines are one of the most commonly administered intravenous chemotherapy agents and are known to cause cardiac toxicity and rare cases of acute leukemia
(Patt, Duan et al. 2007) (Du, Xia et al. 2009). The taxanes were introduced in 1990 and quickly became an integral part of chemotherapy regimens. Significant side effects include bone marrow suppression, peripheral neuropathy, nail changes, epiphora, and, less commonly, permanent alopecia (Sparano, Wang et al. 2008). Patients with metastatic disease develop cumulative toxicity from multiple lines of chemotherapy as well as chemotherapy resistance which limits efficacy. Selection of less toxic regimens with improved efficacy is important to achieve the goal of improved outcome without worsening quality of life. Development of agents targeted to the biology that drives tumor growth are a critical step in this process, however, chemotherapy remains the backbone of breast cancer treatment. New agents and new combinations may not only improve response and duration of response, but also could provide an improved backbone for the addition of targeted agents. The combination of doxorubicin and cyclophosphamide (AC) is a standard contemporary adjuvant chemotherapy regimen based on data from NSABP-15, NSABP-23 and other studies (Fisher, Brown et al. 1990; Fisher, Anderson et al. 2001). Jones and colleagues studied the combination of docetaxel and cyclophosphamide (TC) in patients with early stage breast cancer in order to evaluate a nonanthracycline containing regimen that might avoid the risk of long-term toxicity. 1800 women with stage I and II breast cancer were randomized to receive TC every three weeks for 4 cycles or AC on the same schedule. At a median follow-up of five years, patients treated with TC had a significantly improved disease free survival (DFS) of 86% versus 80% p=0.015, compared to those receiving AC (Jones, Savin et al. 2006). Subsequently, 7 year follow-up data demonstrated a significant improvement in overall survival (OS) favoring TC, 87% versus 82% p=0.032 (Jones, Holmes et al.  
2009). Grade 3-4 toxicities were evaluated by age group and anemia was highest in the age group ≥ 65 occurring in 5% of patients who received AC. Febrile neutropenia was 5% with TC and 2.5% with AC. Patients treated with TC also experienced more peripheral edema (1% vs. <1%), diarrhea (5%
vs. 1%), myalgia (2% vs. 1%). The AC group had more asthenia (9% vs. 6%), infections (10% vs. 7%), nausea (7% vs. 3%) and vomiting (6% vs. 1%). Four late deaths without breast cancer relapse and probably related to treatment occurred in the AC group: a 45-year-old woman died of cardiomyopathy and congestive heart failure, and two older women died of complications related to myelodysplasia and myelofibrosis. One more patient died of acute leukemia 10 years after AC. No cardiac toxicities or leukemia were observed in the group receiving TC.  
Based on this data, TC has become a generally accepted adjuvant chemotherapy regimen for the treatment of lower risk early stage disease. Ongoing or planned studies have incorporated TC as a chemotherapy backbone, including two NSABP trials. NSABP B-46 is a three-arm trial and randomizes women with node-positive or high-risk node-negative breast cancer to 6 cycles of TAC
(docetaxel, doxorubicin, and cyclophosphamide), TC, or TC combined with bevacizumab. NSABP B47 is studying the impact of trastuzumab in patients with HER2 normal disease when given with either TC or AC followed by paclitaxel.
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1.3 toxicity of taxanes

Despite the success of taxanes in the treatment of early stage breast cancer, toxicity remains an issue. Mild to moderate peripheral neuropathy is seen in up to 88% of women, with 3% having severe neuropathy, and some suffering from long-term neuropathy (Rowinsky and Donehower 1995). In ECOG 1199, a randomized trial of doxorubicin and cyclophosphamide followed by various schedules of taxane therapy, 27% of patients who received weekly paclitaxel had Grade 2 or greater neuropathy(Sparano, Wang et al. 2008). Other toxicities, such as epiphora, edema, and delayed hair growth, among others, limit time to recovery from adjuvant therapy.
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1.4 toxicity of epothilones

Epothilones also target microtubules and stabilize their polymerization, but differ from taxanes in that they demonstrate less susceptibility to Pgp-mediated efflux and retain antitumor activity even in the setting of tubulin mutations (Rivera and Gomez 2010). Phase II studies for the mostly widely studied epothilone, ixabepilone, when given as monotherapy, has shown overall response rates (ORR) for metastatic breast cancer patients not previously treated with taxanes of 41.5 and 57% and in taxanepretreated patients ORR of 11.5 to 22% (Denduluri, Low et al. 2007) (Low, Wedam et al. 2005)
(Perez, Lerzo et al. 2007) (Roche, Yelle et al. 2007) (Thomas, Gomez et al. 2007). In a phase II study with 113 patients, grade 3/4 adverse events included 14% of patients with peripheral sensory neuropathy, 54% with neutropenia, 14% with fatigue and grade 3 myalgia/arthralgia in 14% (Perez, Lerzo et al. 2007). Based on this toxicity profile, newer, less toxic agents are still needed.
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1.5 eribulin mesylate and preclinical

Eribulin mesylate (E7389) is a nontaxane, microtubule dynamic inhibitor with a novel mechanism of action. Eribulin suppresses polymerization, has no effect on microtubule depolymerization, and sequesters tubulin into nonfunctional aggregates (Kuznetsov, Towle et al. 2004; Jordan, Kamath et al. 2005; Okouneva, Azarenko et al. 2008) (Towle, Salvato et al. 2001).  
Results of in vitro studies demonstrate that eribulin inhibits cell growth with sub- to low-nmol/L halfmaximal inhibitory concentration (IC50) values in a wide range of established human cancer cell lines, including breast, colon, prostate, ovarian, small cell lung, and non-small cell lung cancers, histiocytic lymphoma, promyelocytic leukemia, head and neck carcinoma, melanoma, and uterine sarcoma. Eribulin exerts its anticancer effects via a tubulin-based antimitotic mechanism, leading to G2/M (GAP 2/mitosis stages of cell cycle) cell cycle blocks, disruption of mitotic spindles, and ultimately apoptotic cell death after prolonged mitotic blockage. Among tubulin-targeted agents, eribulin is a mechanistically unique inhibitor of microtubule dynamics, leading to inhibition of microtubule growth in the absence of effects on microtubule shortening, and formation of nonproductive tubulin aggregates. This unique pattern of inhibitory effects on microtubule dynamics is not shared by other known tubulin-targeted agents. Preclinical studies have also demonstrated antitumor activity in cell lines that are taxane resistant as a result of β-tubulin mutations (Cortes and Lorca 2011) (Kuznetsov et al. abstract ESMO 2007). In addition, eribulin appears to have less neurotoxicity than other microtubule antagonists. A murine model compared the effect on nerve conduction of paclitaxel and ixabepilone to eribulin. This data, presented at the annual European Society for Medical Oncology found that treatment with paclitaxel and ixabepilone resulted in a reduction in caudal nerve conduction velocity as well as caudal and digital amplitude, but eribulin had no deleterious effect on these endpoints. Eribulin also caused less severe morphological changes in dorsal root ganglion and sciatic nerves on pathological assessment
(Wozniak, Lapidus et al. 2010).
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1.6 clinical data for eribulin

In two phase II studies, eribulin has demonstrated efficacy in patients with heavily pre-treated metastatic breast cancer. E7389-A001-201 evaluated the safety and efficacy of eribulin in subjects with locally advanced or metastatic breast cancer who had been previously treated with an anthracycline and taxane and had documented progression during or within 6 months following the last dose of chemotherapy. 103 patients with a median of 4 prior chemotherapy regimens for advanced disease enrolled (Vahdat, Pruitt et al. 2009). 70 subjects received 1.4 mg/m2 eribulin administered as an IV bolus on days 1, 8, and 15 of a 28-day cycle; another 33 subjects received eribulin administered as an IV bolus on days 1 and 8 of a 21-day cycle. The 21-day cycle cohort was added because 63% of subjects in the 28-day cycle cohort experienced dose delays, reductions, or omissions due to neutropenia; in most cases the day 15 dose was being omitted. The response rate
(RR) was 11.5% (all PR), the clinical benefit rate (CBR) was 17.2%, and progression free survival (PFS) was 2.6 months. The second phase II trial, E7389-A001-211, enrolled 299 patients with locally advanced or metastatic breast cancer who had received anthracycline, taxane, and capecitabine as prior therapy, and were refractory to their last chemotherapy regimen as documented by progression on or within 6 months of therapy (Cortes, Vahdat et al. 2010). All patients received 1.4 mg/ m2 on day 1 and 8 every 21 days. Results were similar by independent review, with RR of 9.3%, CBR of 17.1%
and PFS of 2.6 months. Between the two trials, OS ranged from 9 to 10.4 months. The most common drug-related grades 3/4 toxicities were neutropenia (54-64%), fatigue (5-10%), peripheral neuropathy (5-6.9%), and febrile neutropenia (4-5.5%).  
The results of a phase III trial comparing eribulin to treatment of physician's choice (TPC) in patients with anthracycline and taxane pre-treated MBC and at least two prior chemotherapy regimens for advanced disease recently led to FDA approval of this novel chemotherapy agent (Twelves, Cortes et al. 2010). The EMBRACE trial randomized 508 women to single agent eribulin and 254 women to TPC; the primary endpoint of OS was 13.12 months in the eribulin arm versus 10.65 months in the TPC arm (HR 0.81, p=0.041). The RR was also greater in the eribulin arm (12.2 versus 4.7%,
p=0.002), and although PFS was significantly longer by investigator assessment, by central review this was not significantly different, likely due to the inclusion of patients with evaluable rather than solely measurable disease (3.7 versus 2.2 months, HR 0.87, p=0.14). Subset analysis suggested benefit from eribulin across identified risk groups. 144 patients were identified with ER/PgR/HER2 negative disease, determined locally, and in this group the PFS for eribulin was 2.23 months, compared to 1.9 months for TPC. In the overall study, grade 3/4 toxicities that were higher in the eribulin arm included higher rates of neutropenia (21% versus 14%) and leukopenia (11.7% versus 5%) as well as febrile neutropenia (3% versus 0.8%). However, anemia (1.8 vs 3.2%), asthenia/fatigue (8.2 vs 10.1%), nausea (1.2 vs 2.4%), mucositis (1.4 vs 2%) and hand-foot syndrome
(0.4 vs 3.6%) were more prevalent in the TPC arm.
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1.7 in vitro and vivo

The pharmacokinetics (PK) of eribulin were examined after single IV administrations in male mice (0.5 or 2 mg/kg), male rats (0.5 or 1 mg/kg), and male dogs (0.08 mg/kg). The PK of eribulin can be characterized by an extensive volume of distribution (7.45 - 12.2 l/kg in mice, 36.3 - 44.1 l/kg in rats, and 18.8 - 20.4 l/kg in dogs), a moderate clearance (2.03 - 2.79 l/hr/kg in mice, 1.61 - 2.04 l/hr/kg in rats, and 0.688 - 1.06 l/hr/kg in dogs), and a relatively slow elimination (t½, 3.55 - 6.88 hours in mice, 15.9 - 27.9 hours in rats, 21.9 - 28.2 hours in dogs). In multiple dose PK studies in which eribulin was administered every four days for three courses no accumulation was observed in both rats and dogs.  
The protein binding of eribulin in mouse, rat, dog and human plasma in vitro was investigated. Between 100 and 1000 ng/mL, eribulin was not strongly bound to plasma protein in vitro: mouse
(28.47%-35.94%), rat (23.01%-34.09%), dog (15.41%-26.37%), and human plasma (48.92%- 65.07%). Cytochrome 3A4 (CYP3A4) appears to be the major enzyme responsible for the human hepatic metabolism of eribulin, and forms mainly isomeric monohydroxylates in vitro.  
No significant inhibition of CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1 was detected with eribulin at concentrations up to 5 μmol/L. Eribulin inhibited CYP3A4-mediated R-warfarin 10hydroxylation, testosterone 6-β hydroxylation, nifedipine dehydration (Ki: 3-17 μmol/L), and exhibited weak inhibitions on the metabolism of carbamazepine, diazepam, paclitaxel, tamoxifen, midazolam, and terfenadine. Eribulin did not alter the activities or the protein expressions of CYP1A, CYP3A,
CYP2C9, and CYP2C19 in the primary cultures of human hepatocytes (up to 5 or 10 μmol/L). It is unlikely that eribulin would cause metabolic inhibition of concurrent drugs based on anticipated low therapeutic dose in humans. However, caution will be taken for patients who are taking drugs that are metabolized by CYP3A4 since CYP3A4 was substrate dependant and appears to be the major enzyme responsible for human hepatic metabolism of eribulin in vitro.
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1.7.2 clinical pharmacokinetics

The pharmacokinetics of eribulin in human subjects is characterized by a rapid distribution phase, with a prolonged elimination phase after intravenous infusion. Plasma concentrations of drug are sufficiently low during the elimination phase to preclude significant accumulation at the dosing intervals being tested. The disposition of eribulin follows linear kinetics over the dose range studied, as shown by consistent dose-independent pharmacokinetic parameters (t½, CI, Vdss) and similar dose-normalized parameters (Cmax/Dose, AUC0-t/Dose and AUC0-∞/Dose) between eribulin doses ranging from 0.25 to 1.4 mg/ m2(E7389-A001-101) and from 0.25 to 4.0 mg/ m2(E7389-A001-102).
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1.7.3 toxicology

The toxicity of eribulin has been evaluated in rats and dogs with the drug administered in 5% ethanol in 0.9% sodium chloride (NaCl) as a slow IV bolus to rats or as a 1-hour IV infusion to dogs once a day on days 1, 5, 9. In range-finding studies, single doses of 0.75 mg/kg/day (4.5 mg/ m2/day) were lethal to rats and two doses of 0.075 mg/kg/day (1.5 mg/ m2/day) were lethal to dogs. Bone marrow toxicity appeared to be dose-limiting in both rats and dogs. In the Investigational New Drug Application (IND)-directed, Good Laboratory Practices (GLP) toxicity studies, doses of 0.08 mg/
m2/day produced no toxicity in dogs or rats, while doses of 0.6 to 0.8 mg/ m2/day produced reversible bone marrow toxicity in both species. Other toxicities that were considered to be drug-related occurred in the lymphoid tissue, testes, and skeletal muscle. All observed toxicities (except testicular toxicity) were reversible in both dogs and rats. Based on these studies, the recommended starting dose for phase I clinical trials is 0.12 mg/ m2/day, as dose less than 1/10th the maximum tolerated dose (MTD) in rats (1.2-1.5 mg/ m2/day) and 1/6th the Toxic Dose Low (0.4 mg/ m2/day) in dogs.  
In *vitro* bone marrow assays did not demonstrate significant species difference between human, dog, and mouse CFU-G/M cell sensitivity to eribulin. Based on the ratios of human to murine and human to canine IC90S and in vivo MTDs, the calculated human MTD was projected to be 0.9 mg/ m2/day
(based on canine data) and 3.1 mg/ m2/day (based on murine data.) Similar bone marrow effects were also confirmed by in vitro assays using multipotential stem cells (CFC-GEMM) from mouse, dog and human. The murine CFC-GEMM cells appeared to be less sensitive to the toxic effects, whereas human and canine cells appeared to be equally sensitive. In *in vitro* genotoxicity studies, eribulin was negative in the Ames test with or without S9, but was weakly positive in the L5178Y/TK+/- mouse lymphoma mutagenesis assay.
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1.7.4 developmental/reproductive toxicity

Developmental and reproductive toxicity studies of eribulin have not been performed thus far. Eribulin was not tested in pregnant or breast-feeding women. Women of child-bearing potential and men participating in clinical studies of eribulin must use appropriate contraception, including abstinence and double-barrier methods, throughout eribulin therapy. In preclinical mutagenicity studies, eribulin was neither genotoxic or mutagenic.
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1.8 cyclophosphamide

Cyclophosphamide (Endoxan, Cytoxan, Neosar, Procytox, Revimmune) is a prodrug nitrogen mustard alkylating agent, from the oxazophorines group. It is converted by oxidase enzymes in the liver to active toxic metabolites, 4-hydroxycyclophosphamide and aldophosphamide. Most of the aldophosphamide is oxidized by the enzyme aldehyde dehydrogenase (ALDH) to make carboxyphosphamide. A small proportion of aldophosphamide is converted into phosphoramide mustard and acrolein. Phosphoramide mustard forms DNA crosslinks between (interstrand crosslinkages) and within (intrastrand crosslinkages) DNA strands at guanine N-7 positions. This is irreversible and leads to cell death(Cohen and Jao 1970) (Brock 1989) (Brock 1996).  
Phosphoramide mustard is only formed in cells that have low levels of ALDH. Cyclophosphamide has relatively little typical chemotherapy toxicity as ALDH is present in relatively large concentrations in bone marrow stem cells, liver and intestinal epithelium. ALDH protects these actively proliferating tissues against toxic effects phosphoramide mustard and acrolein by converting aldophosphamide to carboxyphosphamide that does not give rise to the toxic metabolites (phosphoramide mustard and acrolein).  
Cyclophosphamide has activity in a broad range of human cancer types and has emerged as front line therapy for many advanced cancers including breast cancer. Phase III clinical trials have shown that cyclophosphamide in combination with adriamycin prolongs survival in patients with breast cancer (Fisher, Brown et al. 1990). In early stage breast cancer patients, the regimen of cyclophosphamide and docetaxel has recently been adopted since it is a less toxic regimen and data shows that it improves DFS and OS (Jones, Holmes et al. 2009).  
The most common toxicities associated with cyclophosphamide include bone marrow suppression, nausea, vomiting, anorexia, diarrhea, abdominal discomfort, alopecia, mucositis, infertility; and rare cases of hemorrhagic cystitis due to the metabolite acrolein, hemorrhagic colitis, Stevens-Johnson syndrome or interstitial pneumonitis.
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1.9 rationale 1.9.1 rationale for

Docetaxel or paclitaxel are well-established active and effective treatments for advanced breast cancer in both the front-line and pre-treated setting. *In vitro* data with taxane resistant cell lines offers a biological basis for the efficacy of eribulin in this setting. Due to the favorable toxicity profile and efficacy seen with the use of eribulin as a single agent, it is feasible to study combinations of eribulin with standard chemotherapeutic agents given in breast cancer. The success of docetaxel combined with cyclophosphamide in the adjuvant setting has led to considerable interest in anthracycline-free chemotherapy options. Given the advantages of eribulin in both toxicity and efficacy, the combination of eribulin and cyclosphosphamide is intriguing. If both efficacy and safety is demonstrated in the metastatic setting, this could be an important combination therapy to test as adjuvant therapy for early stage breast cancer.
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1.9.2 rationale for dosing

On November 15, 2010, the U. S. Food and Drug Administration granted approval for eribulin mesylate for the treatment of patients with metastatic breast cancer who have previously received an anthracycline and a taxane in either the adjuvant or metastatic setting, and at least two chemotherapeutic regimens for the treatment of metastatic disease. In the phase III trial which led to this approval, eribulin was administered as an intravenous dose of 1.4 mg/ m2 on days 1 and 8 of a 21-day cycle, with dose delays and reductions for pre-specified toxicities.
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1.9.3 rationale for starting dose

We anticipate the possibility of increased toxicity, including bone marrow suppression, when eribulin is combined with standard dose cyclophosphamide (600 mg/m2 on day 1 of a 21-day cycle.)
Therefore we plan to test this combination initially with a reduced dose of eribulin (1.1 mg/m2 days 1 and 8 of a 21-day cycle).
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1.10 correlative studies background 1.10.1

Chemotherapy-associated toxicity is highly variable. This may be largely due to host factors such as differences in absorption, distribution, and particularly metabolism and clearance. Sources of variability may be compounded by the presence of vomiting and diarrhea, poor nutritional status, concomitant medications, prior therapies, and liver metastases or effusions (Ratain 1992). Response to chemotherapy is also highly variable and may be influenced by both host and tumor specific factors. Variability in hepatic metabolism and membrane transport has been the focus of many studies addressing the sources of interindividual differences in response to medications, particularly chemotherapy. Several CALGB studies have included correlative pharmacogenetics studies to investigate genetic contributions to toxicity and response. The most mature of these studies in breast cancer, CALGB 40101, has recently identified novel genetic predictors of paclitaxel-induced peripheral neuropathy. Surprisingly, polymorphisms in known candidate genes for metabolism and transport did not contribute significantly to toxicity. Results from a genome-wide scan of over 500,000 single nucleotide polymorphisms (SNPs) identified three novel candidate genes, FZD3, FGD4 and EPHA5.  
FZD3 encodes a Wnt receptor with a known role in neurite outgrowth (Endo, Beauchamp et al. 2008), suggesting that altered function in individuals carrying the variant allele of this gene may protect against paclitaxel-induced peripheral nerve injury. A COX model was also used to identify genetic predictors of time to developing peripheral neuropathy. Although not reaching genome-wide significance, the two top hits were in genes of biological interest. One SNP was in EPHA5 (p = 9.6 x 10-7; HR = 1.63) and another in FGD4 (p = 2.6 x 10-6; HR = 1.57). EPHA5 encodes an ephrin receptor involved in axon guidance. Studies in mice have shown that EPHA5 is important for neuronal regeneration following injury (Barrette, Calvo et al. 2010). Patients carrying a reduced function EPHA5 allele may have an attenuated repair response following paclitaxel-induced injury. FGD4 encodes a Rho GTPase that is critical for proper myelination of peripheral nerves. Rare mutations in FGD4 are associated with the congenital peripheral neuropathy Charcot-Marie-Tooth disease
(Stendel, Roos et al. 2007). The more common FGD4 polymorphisms identified in CALGB 40101 may be associated with demyelination of peripheral nerves following paclitaxel treatment. The molecular bases for each of these three genes in paclitaxel-induced peripheral neuropathy are currently under study at Dr. Kroetz' laboratory at USCF. Therefore, in addition to specific hypothesis testing for the above candidate genes, this study will also provide the framework for hypothesis generation investigations of genotype and/or haplotype in additional candidate genes of putative importance to response and toxicity of the agents being evaluated in this study.  
For patients who consent, whole blood will be collected prior to the start of treatment. DNA extraction will occur at UCSF Genomics Core Facility, the Cancer Center Genome Analysis Core, the Cancer Center Tissue Core or within the laboratory of Dr. Kroetz. Exploratory SNP analysis will then be performed in Dr. Kroetz laboratory. Data from this trial will be combined with other SNP data for separate larger analysis of pooled patients.
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1.10.2 genome analysis

In genetic epidemiology, a genome-wide association study (GWA study, or GWAS), also known as whole genome association study (WGA study, or WGAS), is an examination of all or most of the genes (the genome) of different individuals of a particular species to see how much the genes vary from individual to individual. Different variations can then be associated with different traits, for example diseases or toxicity from treatment. One challenge for successful GWAS in the future will be to apply the findings in a way that accelerates drug and diagnostics development, including better integration of genetic studies into the drug-development process and a focus on the role of genetic variation. One such success was found relating genetic variant with response to anti-hepatitis C virus treatment. For genotype 1 hepatitis C treated with Pegylated interferon-alpha-2a or Pegylated interferon-alpha-2b (brand names Pegasys or PEG-Intron) combined with ribavirin, a GWAS study has shown that genetic polymorphisms near the human IL28B gene, encoding interferon lambda 3, are associated with significant differences in response to the treatment. A later report demonstrated that the same genetic variants are also associated with the natural clearance of the genotype 1 hepatitis C virus (Ge, Fellay et al. 2009).  
For purposes of hypothesis generation, we plan to collect whole blood on patients prior to the start of treatment and perform a genome scan in the Genome Analysis Core or the UCSF Genomics Core. Data from this trial will be combined with other genome scan data from UCSF for a separate larger analysis of pooled patients. These samples will not be sent to the National Institutes of Health (NIH) and will not be part of the NIH GWAS database.
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1.10.3 circulating tumor cells

The development of techniques to detect circulating tumor cells (CTCs) or micrometastases in the blood of cancer patients provides a new approach to monitoring cancer activity and response to therapy. Numerous studies have demonstrated the utility of using CTC enumeration as a prognostic marker in advanced breast cancer (Cristofanilli, Budd et al. 2004; Cristofanilli, Hayes et al. 2005). More recently, considerable attention has turned to the development of more sophisticated assays to better understand the biology and molecular nature of CTCs. Current models suggest that CTCs may represent tumor cells with metastatic potential, and therefore gene-expression profiling of CTCs may be a rich resource for the identification of biomarkers with therapeutic relevance to micrometastatic disease. In addition, as molecular targeted therapies are being developed, it is imperative that we have strategies to assess target levels in patients, as well as changes in target status during treatment. CTCs offer an ideal focus for such strategies, as they are readily obtainable and can be subjected to further molecular analysis.  
Molecular profiling of CTCs is challenging because CTCs are a rare cell population. Isolating CTCs and handling picogram levels of RNA and DNA is extremely difficult and can be wrought with technical complications (Racila, Euhus et al. 1998). Efforts to detect CTCs have involved the use of antibody-based capture methods which greatly enrich the tumor cell component relative to hematopoetic cells, however these current methods still retain a considerable fraction of leukocytes which results in a heterogeneous sample that prevents accurate characterization of isolated CTCs.  
Dr. John Park's laboratory at UCSF has developed a novel approach using sequential immunomagnetic enrichment and FACS (Fluorescence-activated cell sorting) for CTC
isolation. Using this protocol, the Park laboratory has successfully isolated small pools of highly purified CTCs from breast cancer patients. Genomic DNA from these CTCs were subjected to whole genome amplification (Howgate, Gamie et al.) followed by array comparative genomic hybridization
(aCGH ) for copy number analysis. Their results demonstrate that CTCs can be isolated from hematopoietic cells with high enough purity to perform molecular profiling.  
We plan to utilize this novel isolation strategy to perform the following exploratory CTC analyses: 1)
enumeration of CTCs prior to starting therapy and if ≥5 CTC/mL, a second sample at cycle 2 day 1; 2)
whole genome amplification (Howgate, Gamie et al.) followed by aCGH of isolated CTCs 3) sequencing analysis for beta tubulin mutations as indicated; and 4) gene expression using a 64 gene panel including beta 3 tubulin (\#2, 3, will be performed only if an adequate number of CTCs are available.)
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1.10.4 archived tissue analysis

Over the past several years, advances in molecular technology have allowed scientists to perform genome-wide queries using oligonucleotide arrays to identify genes that are differentially expressed in tumor metastases, as well as in pre- and post-treatment tissue samples (Ramaswamy and Perou 2003; Weigelt, Glas et al. 2003).  
Archived tumor tissue will be collected for the analysis of biomarkers predictive of clinical response to eribulin. This may include, but is not limited to, proteins, phosphoproteins, and RNA expression related to the activity of eribulin may be assessed in this tissue. DNA will be extracted from tissue specimens in order to sequence and genotype the samples, to evaluate the prevalence of genetic polymorphisms of all membrane transporters as well as all other genes of interest which could potentially be interacting with the membrane transporters or having a effect on the expression levels of the membrane transporter genes. Sequencing and genotyping of the tissue samples will be carried out using traditional sequencing methods, or using the genomewide association array chip (Illumina, Affymetrix or others) and/or using the high-throughput DNA sequencing technologies (Illumina Genome Analyzer, 454 technologies and SOLiD) mRNA levels of all membrane transporters and other potential genes of interest will be measured by quantitative PCR, microarray analysis or other cutting edge technologies such as high-throughput RNA sequencing technologies (Illumina Genome Analyzer, 454 technologies and SOLiD). The protein levels of the interested genes in the tissue specimens will be determined by immunohistochemistry and Western Blot analyses using targeted antibodies. RNA/protein levels will be associated with various genotypes to identify genetic variants that have an impact on RNA/protein concentration. Other analysis of tumor tissue may include, but is not limited to, markers of response to treatment or potential for metastasis including, but not limited to, proteins, phosphoproteins, and other biological markers.
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1.10.5 quality of life

We plan to evaluate quality of life endpoints using the standard assessment tool EORTC QLQ-C30. QLQ-30 is a copyrighted instrument, which has been translated and validated into 81 languages and is used in more than 3,000 studies worldwide (Groenvold, Klee et al. 1997; Taenzer, Speca et al. 1997; Apolone, Filiberti et al. 1998; McLachlan, Devins et al. 1998).  
This questionnaire will be administered at baseline (within 7 days of cycle 1 day 1), and thereafter at 6 weeks, 3 months, 6 months, 12 months, 18 months and 24 months after the start of treatment, before drug administration and before any tumor assessments are communicated to the patient. Questionnaires should continue to be completed at the scheduled time-points (including beyond study termination), until the patient has progressive disease or starts a different course of anti-tumor treatment, whichever comes first. One person in each center will be designated to take responsibility for the administration, collection, and verification of the QLQ forms. Further details regarding QLQ administration are provided separately to on-site staff. An online questionnaire may also be available.
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1.10.6 neuropathy measurements

Cellular functions that rely on tubulin, such as axonal transport and cytoskeletal support of transmembrane proteins, may also be affected when cells are treated with microtubule inhibitors. In paclitaxel induced neuropathy, these disruptions lead to axonal atrophy of large diameter peripheral nerve axons (Sahenk, Barohn et al. 1994; Persohn, Canta et al. 2005). Patients with taxane-induced neuropathy present with neuropathic pain, dyesthesias, parathesias in a stocking-and-glove pattern, decreased deep tendon reflexes, increased vibration thresholds, decreased sensory nerve action potential amplitudes, and distal muscle weakness (Lipton, Apfel et al. 1989; Chaudhry, Rowinsky et al. 1994; New, Jackson et al. 1996; Forsyth, Balmaceda et al. 1997).  
Investigators at UCSF have developed a 10-point Modified Total Neuropathy Score (mTNS) and validated the measure in a group of 40 patients (Wampler, Miaskowski et al. 2006). It is inexpensive, noninvasive, and can be performed by a health care provider in less than ten minutes. The Functional Assessment of Cancer Therapy/Gynecologic Oncology Group-Neurotoxicity (FACT/GOG-Ntx) questionnaire is an eleven-item subscale (Ntx subscale) that evaluates symptoms and concerns associated specifically with chemotherapy-induced neuropathy. It assesses levels of patient neuropathy, severity of toxicity, and patient quality of life and has been validated (Calhoun, Welshman et al. 2003).  
The mTNS and FACT/GOG-NTX assessments will take place by a health care provider at baseline (within 7 days of cycle 1 day 1), and at the start of each cycle and study termination. It will be performed before drug administration and before any tumor assessments are communicated to the patient. Assessments will take place until the patient has progressive disease, starts a different course of anti-tumor treatment, or until 12 months from time of enrollment on study, whichever comes first. One person in each center will be designated to take responsibility for the collection and verification of the mTNS and FACT/GOG-Ntx forms. Further details regarding mTNS and FACT/GOG-Ntx administration are provided separately to on-site staff.
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2.0 objectives 2.1 phase ib

1. Determine the maximum tolerated dose (MTD) of eribulin in combination with cyclophosphamide in patients with any solid tumor.
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secondary objectives

1. Evaluate the safety of treatment with the combination of eribulin and cyclophosphamide in patients with any solid tumor.  
2. Determine the dose-limiting toxicities (DLTs) of eribulin in combination with cyclophosphamide in this cohort.  
3. Characterize the pharmacokinetics (Bettmann, Dake et al.), including evaluating the potential for drug-drug interaction of eribulin and cyclophosphamide.  
4. Evaluate quality of life endpoints using assessment tools EORTC QLQ-C30, FACT/GOG-Ntx and 10-point Modified Neuropathy Score.  
5. Explore the relationships between the combination of eribulin/cyclophosphamide pharmacodynamic, pharmacogenomic and response prediction biomarkers. a. Explore single-nucleotide polymorphism (SNP) variations evaluating potential toxicities including peripheral neuropathy and bone marrow suppression.  
b. Perform circulating tumor cell (CTC) analysis to enumerate and sort CTCs, and perform exploratory analyses evaluating WGA and β-tubulin expression in CTCs.  
c. Evaluate archival tumor specimens for exploratory markers of target validation and response, including, but not limited to tubulin mutations and comparative analysis with CTC aCGH analysis.
a phase ib/ii study of
2.2 phase ii primary objective

1. Estimate the clinical benefit rate (complete response, partial response, and stable disease) of the combination of eribulin and cyclophosphamide in patients with advanced breast cancer.
a phase ib/ii study of
secondary objectives

1. Make a preliminary assessment of efficacy of this combination in patients with advanced breast cancer including response rate, duration of response and time to progression.  
2. Evaluate the safety of this combination in an expanded cohort of patients.  
3. Evaluate quality of life endpoints using assessment tools EORTC QLQ-C30, FACT/GOG-Ntx and 10-point Modified Neuropathy Score.  
4. Explore the relationships between the combination of eribulin/cyclophosphamide pharmacodynamic, pharmacogenomic and response prediction biomarkers. a. Explore single-nucleotide polymorphism (SNP) variations evaluating potential toxicities including peripheral neuropathy and bone marrow suppression.  
b. Perform circulating tumor cell (CTC) analysis to enumerate and sort CTCs, and perform exploratory analyses evaluating WGA and β-tubulin expression in CTCs.  
c. Evaluate archival tumor specimens for markers of target validation and response, including, but not limited to tubulin mutations and comparative analysis with CTC aCGH analysis.
a phase ib/ii study of
3.0 patient selection

3.1 Subject Inclusion Criteria 1. Phase Ib: Patient must have histologically or cytologically documented solid tumor malignancies. Phase II: Patients must have histologically or cytologically confirmed locally advanced, unresectable or metastatic carcinoma of the breast.  
2. Patient is male or female and ≥18 years of age on the day of signing informed consent.  
3. Patient must have performance status of 0-2 on the ECOG Performance Scale and life expectancy > 3 months.  
4. Patients with ≤ grade 1 peripheral neuropathy are eligible for this trial using the CTCAE v4.0, regardless of use of therapy for neuropathy including gabapentin.  
5. Patient must have evaluable disease. Measureable disease is not required 6. Patient must have adequate organ function as indicated by the following laboratory values:  
| Table 3-1. Adequate Organ Function Values     |                                                                                           |
|-----------------------------------------------|-------------------------------------------------------------------------------------------|
| System                                        | Laboratory Value                                                                          |
| Hematological Absolute neutrophil count (ANC6 | ≥1,000/μL                                                                                 |
| Platelets                                     | ≥100,000/μL                                                                               |
| Hemoglobin                                    | ≥9 g/dL                                                                                   |
| Renal Serum creatinine                        | ≤1.5 x upper limit of normal (ULN) OR                                                     |
| or calculated creatinine clearance†           | ≥60 mL/min for patients with creatinine levels >1.5 x  institutional ULN                  |
| Hepatic Serum total bilirubin                 | ≤1.5 x ULN OR direct bilirubin ≤ ULN for patients with  total bilirubin levels >1.5 x ULN |
| AST (SGOT) and ALT (SGPT)                     | ≤3 x ULN or ≤5 x ULN in patients with known liver  metastasis                             |
| Metabolic Potassium§                          | in normal range                                                                           |
| †                                             | Creatinine clearance should be calculated per institutional standard.                     |
| §                                             | Patients with hypokalemia at screening must be corrected prior to initiating treatment.   |  
7. Female patient of childbearing potential must have a negative serum or urine pregnancy test β-hCG within 72 hours prior to receiving the first dose of study medication and agree to the use of effective methods of contraception while on study.  
8. Any number of prior lines of chemotherapy in the metastatic setting is allowed. 9. Concomitant use of bisphosphonates is allowed.  
10. Patients with stable and clinically insignificant CNS disease are allowed. Patients must be off steroids with no new CNS symptoms or findings on radiographic imaging for 1 month.  
11. Patients willing and able to complete the questionnaires. 12. Patients willing and able to comply with the study protocol for the duration of the study. 13. Written informed consent prior to any study-specific screening procedures with the understanding that the patient may withdraw consent at any time without prejudice.  
3.2 Subject Exclusion Criteria
A patient meeting any of the following criteria is not eligible to participate in this study:  
1. Patients who have had chemotherapy or radiotherapy within two weeks, 4 weeks for nitrosoureas, mitomycin C, pegylated-doxorubicin and one half-life for bevacizumab, hormone therapy within one week, trastuzumab within 2 weeks or lapatinib within one week of study Day 1.  
2. If the patient has residual toxicity from prior treatment, toxicity must be ≤ Grade 1.  
3. Patients with non-healing surgical wounds. Patients must be at least two weeks from a major surgical procedure, and surgical wounds must be completely healed.  
4. Patients with known active CNS metastases and/or carcinomatous meningitis. However, patients with CNS metastases who have completed a course of therapy would be eligible for the study provided they are clinically stable for at least 1 month prior to entry as defined as: a. no evidence of new or enlarging CNS metastasis b. off steroids that are used to minimize surrounding brain edema. Patients with clinically insignificant brain metastases that do not require treatment are eligible.  
5. Patients with known hypersensitivity to the components of study drug or its analogs.  
6. Significant cardiovascular impairment:
a. Congestive heart failure, Clinically significant cardiac arrhythmia, history or current evidence of a myocardial infarction during the last 6 months, and/or a current ECG tracing that is abnormal in the opinion of the treating Investigator, or unstable angina b. QTc prolongation >480 msec (Bazett's Formula) or congenitally long QT syndrome
7. Severe/uncontrolled concurrent illness/infection  
8. Patients with other active, current primary malignancies, other than carcinoma in situ of the cervix or non-melanoma skin cancer 9. Patients with a hypersensitivity to halichondrin B and/or halichondrin B chemical derivative 10. Patient is pregnant or breastfeeding, or expecting to conceive or father children within the projected duration of the study.  
11. Patients with other significant disease or disorders that, in the Investigator's opinion, would exclude the patient from the study
Both men and women and members of all races and ethnic groups are eligible for this trial.  
3.3 Inclusion of Women and Minorities
a phase ib/ii study of
4.0 subject registration and enrollment

Patients will be screened by the medical oncologists in the oncology practice clinics at participating sites. Patients to be screened will include those currently followed in these practices, as well as those referred from outside providers.
a phase ib/ii study of
4.2 registration procedures

After eligibility screening, patients selected to participate will be registered with the local study site/institution first, then with the lead/coordinating center at University of San Francisco-California Helen Diller Family Comprehensive Cancer Center.
a phase ib/ii study of
4.2.1 institutional registration

Patient registration at each study site/institution will be conducted according to the institution's established policies. Prior to registration, patients will be asked to sign and date an Institutional Review Board (IRB)-approved consent form and a research authorization form/Heath Insurance Portability and Accountability Act (HIPAA) authorization form. Patients must be registered with the institution before beginning any treatment or study activities.
a phase ib/ii study of
4.2.2 lead center registration

To initiate lead center registration, local study sites/institutions should forward copies of the signed informed consent, research authorization/HIPAA forms, and the completed and signed eligibility checklist and any supporting source documentation to clinical research coordinator (CRC) at the lead center by fax to the number on Page 1. The CRC will check the forms for completeness and contact the site regarding any discrepancies The lead center will then assign a unique patient study identification number, and send a confirmation of patient registration to the local CRC via electronic mail. A copy of this confirmation will be retained by both the lead center and local institution. All future study documentation related to that patient should include the assigned patient ID number.  
A patient cannot be treated until confirmation of enrollment is received.
a phase ib/ii study of
5.0 study design and treatment

5.1 Dose and schedule  
| Table 5-1. Dose Escalation Schedule Dose*                                                          |                   |                  |                    |
|----------------------------------------------------------------------------------------------------|-------------------|------------------|--------------------|
| Dose Level                                                                                         | Eribulin mesylate | Cyclophosphamide | Number of patients |
| (mg/m2 )                                                                                           | (mg/ m2 )         |                  |                    |
| Phase Ib Level -1                                                                                  | 0.7 day 1, 8      | 600 day 1        | 3-6                |
| Level 0 (start)                                                                                    | 1.1 day 1, 8      | 600 day 1        | 3-6                |
| Level 1                                                                                            | 1.4 day 1, 8      | 600 day 1        | 3-6                |
| Phase II                                                                                           | MTD determined in | 600 day 1        | 40                 |
| phase I                                                                                            |                   |                  |                    |
| *Doses are stated as exact dose in units (e.g., mg/m2 , mcg/kg, etc.) rather than as a percentage. |                   |                  |                    |  
Phase Ib: The study will follow a standard dose-confirmation schema (phase Ib portion) with 3 to 6 patients per cohort (3+3 design) for a total of 9-18 patients with any solid tumor malignancy. The starting dose level will consist of eribulin mesylate 1.1 mg/ m2 on days 1 and 8 followed by cyclophosphamide 600 mg/ m2 on day 1 of a 21-day cycle.  
If no DLTs are observed in the first three patients, the dose of eribulin will be escalated to the next dose level for the subsequent patients.  
If one of three patients experiences a DLT, three additional subjects will be enrolled in that expanded cohort for a total of no more than 6 patients in each cohort.  
If no additional DLT is observed at the expanded dose level, i.e. 5 of 6 do not experience DLT
during the first cycle, the dose of eribulin will be escalated to the next level.  
If 2 or more dose-limiting toxicities (DLTs) during the first cycle of therapy at any dose level occur, escalation will cease and cohort expansion will continue at the next lower dose level.  
All patients at a given dose level will be followed on treatment for at least 3 weeks (one cycle)
before accrual to the next cohort can commence.  
There will be no intra-patient dose escalation allowed.  
For any dose cohort, if a patient is removed from study for reasons that are clearly not treatment-related, then an additional patient will be accrued to that dose level.  
For the purposes of Phase Ib dose escalation, DLTs will be defined as any treatment-related toxicity occurring within the first 21 days of combination therapy as grade 3 or 4 clinically evident non-hematologic toxicity; grade 4 neutropenia or thrombocytopenia lasting > 7 days or febrile neutropenia; or any clinically significant toxicity grade 2 or higher that requires more than 14 days to resolve.  
The highest dose level at which no more than one of six subjects experience DLT defines the MTD. Once the MTD has been defined, enrollment of subjects into the dose-expansion (Phase II) study in advanced breast cancer patients will commence.  
Phase 1b will be conducted at UCSF.
Phase II: The dose-expansion (phase II portion) will enroll 40 patients with advanced breast cancer to detect a clinically meaningful benefit rate (efficacy and response) of at least 25% in the study cohort. Phase 2 will be conducted at multiple sites, to be determined. Using a two-stage Simon's minimax design, the null and alternative hypothesis will be H0: p0 < 10% versus Ha: p1 > 25% for proportion of patients with complete or partial response by RECIST criteria. Based on defined p0=10% and p1=25% under the type I error of 5% and the type II error rate of 20%, the sample size for the first stage will be 22 patients. If 2 or fewer responses are observed in the first 22 patients (after all patients in the first stage have completed at least 2 months of treatment), the study will be terminated in the first stage; otherwise, the study will be expanded by additional 18 patients. If there are 7 or fewer responses are observed by the end of the second stage, no further investigation of the drug is warranted. If the clinical benefit rate is less than 10%, the probability of ending the study during the first stage is 62%. If the clinical benefit rate is greater than 25%, the probability that the study will be stopped in the first stage is 6%.
a phase ib/ii study of
5.2 dose-limiting toxicity phase ib

Toxicities will be graded in severity according to the guidelines outlined in the NCI-CTCAE version 4.0, published date 28-May-2009. Dose-limiting hematologic and non-hematologic toxicities will be defined differently, and will be based on events occurring during the first cycle of study drug administration (weeks 1-3). To be considered a dose-limiting toxicity (DLT), an adverse experience must be related to study drug, and must not be related to disease progression or intercurrent illnesses. Patients must receive ≥1 of 2 weekly treatments of eribulin during the first cycle in order to be evaluable for DLT. If therapy is delayed >14 days during the first cycle attributable to study drug, this is considered a DLT and the patient will not be replaced. If therapy is delayed due to another reason, the patient will be replaced.  
5.2.1 Hematologic dose-limiting toxicity  
Grade 4 neutropenia lasting for ≥7 days in duration despite growth factor support. GCSF
(Filgrastim) or Pegylated-GCSF (Neulasta) may be administered. Filgrastim may be used for ANC < 1000 at any time, or as prophylaxis in patients at risk for neutropenia. GCSF may not be administered within 24 hours of a chemotherapy infusion. When administered, this does not constitute a DLT.  
Grade 4 neutropenia with fever >38.5° C and/or infection requiring antibiotic or anti-fungal treatment Grade 4 thrombocytopenia (≤25.0 x 109/L)
Grade 3 thrombocytopenia complicated by bleeding and/or requiring platelet or blood transfusion 5.2.2 Non-hematologic dose-limiting toxicity
This will be defined as any Grade ≥3 non-hematologic toxicity, with the specific exception of:  
Grade 3 nausea or Grade 3 vomiting that in the opinion of the investigator/sponsor occurs in the setting of inadequate compliance with supportive care measures and lasts for less than 48 hours.  
Grade 3 diarrhea that in the opinion of the investigator/sponsor occurs in the setting of inadequate compliance with supportive care measures and lasts for less than 48 hours.  
Grade 3 dehydration that in the opinion of the investigator/sponsor occurs in the setting of inadequate compliance with supportive care measures and lasts for less than 48 hours.  
Alopecia Asthenia Inadequately treated (in the opinion of the investigator/sponsor) hypersensitivity reactions Grade 3 elevated transaminases of ≤1 week in duration. In patients with known liver metastases and abnormal LFTs at baseline, grade 3 elevated transaminases of ≤1 week in duration must be ≥2x baseline to meet criteria for DLT.  
Grade 3 total hyperbilirubinemia if <35% is direct component Hyperglycemia: isolated non-fasting Grade 3 glucose values or Grade 3 hyperglycemia that in the opinion of the investigator/sponsor occurs in the setting of inadequate compliance with supportive care measures.
The following will be considered DLT:  
≥ Grade 3 electrolyte (Na, K, Ca, Mg, Cl, phosphate, and bicarbonate) abnormalities due to glucose intolerance and not attributable to another cause Symptomatic bradycardia Persistent increases in QTc interval (>60 milliseconds from baseline and/or >500ms) Treatment delay of greater than 14 days Failure to administer ≥75% of the planned study drugs during cycle 1 as a result ≥ Grade 2 treatment-related toxicity
a phase ib/ii study of
5.3 duration of therapy 6.0

Subjects who fail to complete the first cycle due to reasons other than toxicity will be classified as not evaluable for toxicity, and will be replaced as in Section 5.1. No dose reductions can occur within the DLT window, see Table 6-1.  
In the absence of treatment delays due to adverse events, treatment may continue until one of the following criteria applies:
Disease progression Need for exclusionary concurrent treatment Unacceptable toxicity Patient decides to withdraw from the study Protocol non-compliance Study is completed or terminated The reason for study removal and the date the patient was removed must be documented in the data management system.  
5.4 Conditions for Terminating the Study The Principal Investigator may terminate the study for any of the following reasons:
Significant toxicities The study treatment is less effective than standard treatment Completion of all study data.  
Toxicities will be graded for management according to NCI-CTCAE version 4.0. Eribulin and cyclophosphamide dose-modification guidelines are outlined in Table 6-1. Both agents should be held for all instances of febrile neutropenia (ANC < 1000/μL). GCSF
(Filgrastim) or Pegylated-GCSF (Neulasta) growth factor support is encouraged for ANC < 1500/ μL and may be used at the discretion of the treating physician in order to maintain adequate blood counts. Filgrastim may be used for ANC < 1000 at any time, or as prophylaxis in patients at risk for neutropenia. Growth factor support is not permitted within 24 hours prior to or following any infusion.  
How to administer eribulin and cyclophosphamide when a delay is required: the goal is to administer the agents on the same day whenever possible. See table 6-1 for full details.
a phase ib/ii study of
6.1 removal of patients from

If toxicity occurs in a patient at the 0.7 mg dose of eribulin, no further reduction will occur and the patient will be discontinued from the study. The dose of cyclophosphamide will not be reduced.  
If any treatment is held for >3 weeksdue to delayed toxicity recovery from any of the therapies, the patient will be removed from the study.  
After the first cycle, patients are allowed to miss 1 out of every 2 weeks of treatment due to hematologic or non-hematologic toxicity. Treatment delays are made at the discretion of the treating physician. Treatment or visit delays due to public holidays, weather conditions, or natural disasters, do not constitute protocol violations. The following treatment delays are allowable for patient coordination:
Cycle 1, Day 8 - delay eribulin administration up to 1 day Cycle 2+, Day 1 - delay treatment up to 14 days Cycle 2+, Day 8 - delay treatment up to 3 days If a delay of >3 weeks in subsequent cycles (after Cycle 1) is required, the patient must discontinue permanently from the study protocol.
a phase ib/ii study of
6.2 concomitant medications

Concomitant treatments permitted: anti-emetics, anti-diarrheals, anti-allergic medications including antihistamines and steroids, granulocyte colony-stimulating factor/granulocyte-macrophage colonystimulating factor/erythropoietin is allowed in response to cytopenias, palliative radiotherapy, bisphosphonate treatment, weak inhibitors of CYP3A4.  
For patients receiving study treatment, any medication (including over-the-counter medications) will be recorded on the CRF (case report form).  
Concomitant treatments not permitted: other investigational drugs; anti-tumor therapies including chemotherapy, hormone therapy, radiation therapy (other than required for palliation), gene therapy, biologics, or immunotherapy.  
Use of strong CYP3A4 inhibitors should be avoided: ketoconazole, itraconazole, clarithromycin, nefazadone, saquinavir, telithromycin, ritonavir, indinavir, nefinavir. See Appendix B. Grapefruit juice may also increase plasma concentrations of eribulin and should be avoided.  
Caution should be used when administering mild or moderate CYP3A4 inhibitors during treatment, and alternative therapeutic agents that do not inhibit CYP3A4 should be considered. Patients receiving CYP3A4 inhibitors during treatment should be monitored closely for acute toxicities (e.g blood counts in between cycles.) See Appendix B.  
Caution should be used for CYP3A4 substrates with a narrow therapeutic index.See Appendix B.
a phase ib/ii study of
6.3 supportive care guidelines

Patients should be treated with supportive care measures at the discretion of the treating physician according to standard practices guidelines.  
Diarrhea: Anti-diarrheal agents should not be taken prophylactically. Patients should be instructed to begin taking anti-diarrhea medication at the first sign of poorly formed or loose stool, occurrence of more bowel movements than usual in one day, or unusually high volume of stool. Anti-diarrheal agents should be deferred if blood or mucus is present in the stool or if diarrhea is accompanied with fever. In this setting, an infectious etiology must be considered and excluded. Rash: Skin rash and can be treated with standard topical steroids and/or oral antibiotics, antihistamines, sulfadiazine, or even a short course of oral steroids (less than 7 days) if clinically indicated.  
Nausea/Vomiting: Anti-emetic therapy beyond routine premedication is not necessary, but symptomatic patients should be treated with standard anti-emetic medications including a 5HT3 receptor antagonist.  
Anemia: Transfusions and/or erythropoietin may be utilized as clinically indicated for the treatment of anemia after the first cycle.  
Neutropenia: GCSF (Filgrastim) or Pegylated-GCSF (Neulasta) may be administered to treat ANC < 1000 at any time, or as prophylaxis in patients at risk for neutropenia. GCSF may not be administered within 24 hours of a chemotherapy infusion.  
Hypersensitivity/Allergic reaction: Hypersensitivity reactions will be managed by the treating physician per standard protocols. Whether to rechallenge the patient or discontinue the offending agent will be decided by the treating physician. Dose reductions will not be made for hypersensitivity reactions.  
| Table 6-1. Toxicity management guidelines for eribulin and cyclophosphamide   |                                             |                          |                            |                         |                     |
|-------------------------------------------------------------------------------|---------------------------------------------|--------------------------|----------------------------|-------------------------|---------------------|
| Toxicity                                                                      | NCI-CTC grade,  unless otherwise  specified | management of            | eribulin dose              | management of           | cyclophosphamide    |
| eribulin                                                                      | upon resumption                             | cyclophosphamide         | dose upon  resumption      |                         |                     |
| Neutropenia (ANC <1000)                                                       | 1st episode                                 | hold until afebrile and  | no change and              | hold until afebrile and | no change and add   |
| +/- fever (T>38)                                                              | ANC ≥ 1000                                  | add filgrastim           | ANC ≥ 1000                 | filgrastim              |                     |
| 2nd episode                                                                   | hold until afebrile and                     | no change                | hold until afebrile and    | reduce to 500 mg/m2     |                     |
| ANC ≥ 1000                                                                    | ANC ≥ 1000                                  | and continue  filgrastim |                            |                         |                     |
| 3rd episode                                                                   | hold until afebrile and                     | reduce by one            | hold until afebrile and    | continue 500 mg/m2 ;    |                     |
| ANC ≥ 1000                                                                    | dose level                                  | ANC ≥ 1000               | continue filgrastim        |                         |                     |
| 4th episode                                                                   | remove from study                           | remove from              | remove from study          | remove from study       |                     |
| study                                                                         |                                             |                          |                            |                         |                     |
| Thrombocytopenia                                                              | Grade 2 or                                  | hold until ≥ 75K         | no change                  | hold until ≥ 75K        | reduce to 500 mg/m2 |
| greater (<75K) Recurrent grade 2                                              | hold until ≥75K                             | reduce by one            | hold until ≥ 75K           | no change               |                     |
| or greater                                                                    | dose level                                  |                          |                            |                         |                     |
| 3rd occurrence                                                                | hold until ≥ 75K                            | no change                | hold until ≥ 75K           | reduce to 400 mg/m2     |                     |
| grade 2 or greater 4th occurrence                                             | remove from study                           | remove from              | remove from study          | remove from study       |                     |
| grade 2 or greater                                                            | study                                       |                          |                            |                         |                     |
| Rash                                                                          | 1 or 2                                      | continue treatment;      | no change**                | continue treatment      | no change           |
| supportive care                                                               |                                             |                          |                            |                         |                     |
| 3                                                                             | Hold until grade 1 or                       | no change**              | Hold until grade 1 or less | no change               |                     |
| less; supportive care                                                         |                                             |                          |                            |                         |                     |
| Recurrent grade 3                                                             | Hold until grade1 or  less; supportive care | reduce by one            | Hold until grade 1 or less | no change               |                     |
| dose level                                                                    |                                             |                          |                            |                         |                     |
| 3 rd occurrence                                                               | remove from study;                          | remove from              | remove from study          | remove from study       |                     |
| grade 3                                                                       | supportive care                             | study                    |                            |                         |                     |  
| Toxicity                     | NCI-CTC grade,  unless otherwise  specified   | management of        | eribulin dose              | management of              | cyclophosphamide   |
|------------------------------|-----------------------------------------------|----------------------|----------------------------|----------------------------|--------------------|
| eribulin                     | upon resumption                               | cyclophosphamide     | dose upon  resumption      |                            |                    |
| GI toxicity: diarrhea,       | 1, 2                                          | Continue treatment   | no change;                 |                            |                    |
| nausea, vomiting             | maximize  supportive  treatment               | no change; maximize  | no change;                 |                            |                    |
| supportive treatment         | maximize  supportive  treatment               |                      |                            |                            |                    |
| 3                            | Hold until grade 1                            | no change            | hold until grade 1 or less | no change                  |                    |
| or less                      |                                               |                      |                            |                            |                    |
| recurrent 3                  | Hold until grade 1                            | no change            | hold until grade 1 or less | reduce to 500 mg/          |                    |
| or less                      | m2                                            |                      |                            |                            |                    |
| 3 rd occurrence              | Hold until grade 1                            | reduce by one        | hold until grade 1 or less | no change                  |                    |
| or less                      | dose level                                    |                      |                            |                            |                    |
| 4 th occurrence              | remove from study                             | remove from          | remove from study          | remove from study          |                    |
| grade 3 or 4                 | study                                         |                      |                            |                            |                    |
| Liver function abnormalities | 1, 2                                          | Continue treatment   | no change                  | Continue treatment         | no change          |
| (ALT, AST, Alk Phos only)    | 3 and > 2x baseline                           | Hold until less than | no change                  | Hold until less than grade | Reduce to 500 mg/  |
| grade 3                      | 3                                             | m2                   |                            |                            |                    |
| recurrent 3 and >            | Hold until less than                          | reduce by one        | Hold until less than grade | no change                  |                    |
| 2x baseline                  | grade 3                                       | dose level           | 3                          |                            |                    |
| 4 th occurrence              | remove from study                             | remove from          | remove from study          | remove from study          |                    |
| grade 3 or 4                 | study                                         |                      |                            |                            |                    |
| Neuropathy (motor and        | 1, 2                                          | continue treatment   | no change                  | continue treatment         | no change          |
| sensory)                     | 3                                             | Hold until less than | no change                  | Hold until less than grade | no change          |
| grade 3                      | 3                                             |                      |                            |                            |                    |
| Recurrent 3                  | Hold until less than                          | reduce by one        | Hold until less than grade | no change                  |                    |
| grade 3                      | dose level                                    | 3                    |                            |                            |                    |
| 3rd occurrence 3             | remove from study                             | remove from          | remove from study          | remove from study          |                    |
| study                        |                                               |                      |                            |                            |                    |  
* A cycle is a 3-week course. If treatment needs to be held for >48 hours, the dose should be held for that week and resumed the following week. If doses are held for more than 3 weeks, the patients must be removed from study. ** If Grade 2 rash develops but resolves to grade ≤ 1 within 7 days allowing the patient to resume treatment, the patient should be restarted at the same dose.
a phase ib/ii study of
7.0 drug information 7.1 eribulin

(Source: FDA-approved product insert, dated 11/2010)
Classification: microtubule inhibitor Mode of Action: Eribulin inhibits the growth phase of microtubules without affecting the shortening phase and sequesters tubulin into nonproductive aggregates. Eribulin exerts its effects via a tubulinbased antimitotic mechanism leading to G2/M cell-cycle block, disruption of mitotic spindles, and, ultimately, apoptotic cell death after prolonged mitotic blockage.  
Metabolism: Eribulin is eliminated primarily in feces unchanged. After administration of 14C-eribulin to patients, approximately 82% of the dose was eliminated in feces and 9% in urine. Unchanged eribulin accounted for approximately 88% and 91% of the dose in feces and urine, respectively.  
Storage and Stability: Store at 25°C (77°F); excursions permitted to 15° - 30° C (59° -86° F). Do not freeze. Store the vials in their original cartons. Store undiluted eribulin in the syringe for up to 4 hours at room temperature or for up to 24 hours under refrigeration (40°F or/ 4°C). Store diluted solutions of eribulin for up to 4 hours at room temperature or up to 24 hours under refrigeration.  
Discard unused portions of the vial. Preparation and administration: Eribulin is a clear, colorless, sterile solution for intravenous administration. Each vial contains 1 mg of eribulin mesylate as a 0.5 mg/mL solution in ethanol: water
(5:95).  
Aseptically withdraw the required amount of eribulin from the single-use vial and administer undiluted or diluted in 100 mL of 0.9% Sodium Chloride Injection, USP as IVP over 2-5 minutes.  
Patient care implications: Do not dilute in or administer through an intravenous line containing solutions with dextrose. Do not administer in the same intravenous line concurrent with the other medicinal products. Incompatibilities/Contraindication: No drug-drug interactions are expected P-gp inhibitors.  
Cytochrome P450 3A4 (CYP3A4) negligibly metabolizes eribulin *in vitro*. Eribulin inhibits CYP3A4 activity in human liver microsomes, but it is unlikely that eribulin will substantially increase the plasma levels of CYP3A4 substrates. However, strong CYP3A4 inhibitors and inducers will be prohibited in this study, see Appendix B. Eribulin does not inhibit CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 or CYP3A4 enzymes or induce CYP1A2, CYP2C9, CYP2C19 or CYP3A4 enzymes at relevant clinical concentrations. Eribulin is not expected to alter the plasma concentrations of drugs that are substrates of these enzymes There are no known contraindications.
a phase ib/ii study of
side effects

| Common side effects: Neutropenia Anemia asthenia/fatigue alopecia   | peripheral neuropathy nausea constipation   |
|---------------------------------------------------------------------|---------------------------------------------|  
Page 28  Protocol Version: 6.0 (05/26/2017)  
| Less common side effects: lacrimation dyspepsia abdominal pain stomatitis dry mouth  peripheral edema   | upper respiratory tract infection hypokalemia  muscle spasms, muscular weakness  dysgeusia, dizziness  insomnia, depression  rash liver function test changes   |
|---------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------|  
Cardiac toxicity: QT prolongation has rarely been observed. ECG and electrolyte (potassium and magnesium)
monitoring is recommended in patients with history of heart failure or bradyarrhythmias and in patients using drugs known to prolong the QT interval. Eribulin should be avoided in patients with congenital long QT syndrome. Availability: Eribulin mesylate injection, 1 mg/2 mL, in a single-use vial. One vial per carton. Drug will be provided by Eisai, Inc and will investigationally labeled, but is identical in formulation to the commercial product.  
Accountability: Drug accountability records will be maintained by the investigational pharmacy as per standard pharmacy practice. Eisai will request drug accountability confirmation on a periodic basis. Standard information such as drug supply utilized and supply remaining will be requested.  
Unused or expired drug will be destroyed by USCF per standard operating procedures and a certificate of destruction (including product lot and expiry, amount destroyed and method of destruction) will be provided to Eisai.