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Version Date: *06/01/2020* Abbreviated Title: Ph I/II TMZ+TG02 astrocytoma CC Protocol \#: 17C0009 F NCT Number: NCT02942264 Version Date: 06/19/2020 Title: Phase I Trial of Zotiraciclib (TG02) Plus Dose-Dense or Metronomic Temozolomide Followed by Randomized Phase II Trial of Zotiraciclib (TG02) plus Temozolomide versus Temozolomide alone in Adults with Recurrent Anaplastic Astrocytoma and Glioblastoma NCI Principal Investigator: Jing Wu, M.D., PhD
NOB, CCR, NCI
37 Convent Dr. Rm 1142A Bethesda, MD 20892 Tel: 240-760-6036 Email: jing.wu3@nih.gov Commercial Agents: Temozolomide  
| Investigational Agents:  Drug Name: Zotiraciclib (TG02) IND Number: 132001 Sponsor: CCR Manufacturer: Adastra Pharmaceuticals, Inc.   |
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Coordinating Center  
Institution: National Cancer Institute, Bethesda, MD Overall Study PI: Jing Wu, MD, PhD  
NOB, CCR, NCI  
37 Convent Dr. Rm 1142A Bethesda, MD 20892 Phone: 240-760-6036  
E-mail: jing.wu3@nih.gov
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precis background

Zotiraciclib (TG02) is a pyrimidine-based multi-kinase inhibitor that has been shown to have inhibitory effects on CDKs, Janus Kinase 2 (JAK2) and Fm-like tyrosine kinase 3 (Flt3). It is orally administered and penetrates blood brain barrier (BBB). There is clinical experience in using Zotiraciclib (TG02) as both a single agent and in combination with other chemotherapy agents for cancer treatment.  
Temozolomide (TMZ) is an oral alkylating agent that has proven efficacy in anaplastic glioma and glioblastoma. It was approved by the U.S. Food and Drug Administration (FDA) to treat anaplastic astrocytoma and glioblastoma in adults. Both a dose-dense (dd) schedule, 7 days on and 7 days off and a metronomic (mn) daily dosing schedule have been used to treat recurrent high-grade gliomas.  
Our preclinical data have demonstrated that Zotiraciclib (TG02) down-regulates CDK9 activity and its target proteins, such as anti-apoptotic protein Mcl-1, XIAP and survivin. A treatment with Zotiraciclib (TG02) and TMZ has synergistic anti-glioma effects in a variety of glioma models with different genetic background. This serves as the basis for this proposed clinical trial.
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objectives phase i

To determine the maximum tolerated dose (MTD) of Zotiraciclib (TG02) plus TMZ
using both the dd and mn TMZ schedules in adult patients with recurrent anaplastic astrocytoma or glioblastoma/gliosarcoma.
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phase ii

To determine the efficacy of Zotiraciclib (TG02) plus TMZ versus TMZ alone in patients with recurrent WHO grade III or IV astrocytoma as determined by progression free survival.
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eligibility

Documented pathology diagnosis of anaplastic astrocytoma [WHO grade III], or glioblastoma/gliosarcoma [WHO grade IV] with recurrent disease. If the pathology diagnosis is anaplastic glioma or anaplastic oligoastrocytoma, evidence of either intact 1p/19q chromosomes or molecular features suggesting astrocytic tumor must be present. (including, but not limited to *ATRX* and/or TP53 mutation)
No prior use of bevacizumab as a treatment for brain tumor.  
No more than two prior relapses for Phase I and no more than one prior relapse for Phase II.  
Patients must have recurrent disease, either histologically proven or with imaging suggestive of recurrent disease Tumor tissues available for review to confirm the histologic diagnosis.  
Tumor tissue blocks available for molecular profiling analysis.
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design

Phase I:
This portion of the study is conducted in two stages: The MTD finding and cohort extension. Two treatment arms and several dose levels are planned.  
In the MTD finding part, TMZ with two alternate schedules (dd and mn) in combination with Zotiraciclib (TG02) will be administered.  
A cohort extension of both arms will be performed at each MTD and the treatment arm with a better progression free survival at 4 months (PFS4) will be selected for the combination treatment arm for Phase II.  
Pharmacokinetic, pharmacogenetic studies and neutrophil analysis will be performed during the cohort extension of both arms.
A maximum of 72 patients will be enrolled to this component for the trial.
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phase ii

Patients will be randomized between two competing treatment arms: ("winner" of dd vs mn) TMZ + Zotiraciclib (TG02) versus dd/mn TMZ alone using a Bayesian clinical trial design. The dosage for the combination arm will be derived from the MTD determined in the Phase I component of the study.  
The treatment schedule will be identical to that described above in the phase I
component, with each cycle comprising 28 days.
Patients will continue treatment until tumor progression or unacceptable toxicity occurs. At progression, patients randomized to the control arm (Temozolomide [TMZ] alone)
will be offered the opportunity to continue TMZ and additional treatment with Zotiraciclib (TG02).  
3 Abbreviated Title: Ph I/II TMZ+ TG02 astrocytoma  
| Version Date: 06/01/2020                                                                                                                          |                                                                                                                                                     |
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| TABLE OF CONTENTS                                                                                                                                 |                                                                                                                                                     |
| TABLE OF CONTENTS .............................................................................................................................4  |                                                                                                                                                     |
| l                                                                                                                                                 | INTRODUCTION .                                                                                                                                      |
| 1.1                                                                                                                                               | Study Objectives                                                                                                                                    |
| 1.2                                                                                                                                               | Background and Rationale.................................................................................................................7          |
| ELIGIBILITY ASSESSMENT AND ENROLLMENT ............................................................30                                              |                                                                                                                                                     |
| 2                                                                                                                                                 |                                                                                                                                                     |
| 2.1                                                                                                                                               | Eligibility Criteria ............................................................................................................................30 |
| 2.2                                                                                                                                               | Screening Evaluation .                                                                                                                              |
| 2.3                                                                                                                                               | Participant Registration and Study Status Update Procedures ............................................34                                          |
| Baseline Evaluation ...........................................................................................................................35 |                                                                                                                                                     |
| 2.4                                                                                                                                               |                                                                                                                                                     |
| 3                                                                                                                                                 | STUDY IMPLEMENTATION ..........................................................................................................36                   |
| Study Design:                                                                                                                                     |                                                                                                                                                     |
| 3.1                                                                                                                                               | Drug administration ...........................................................................................................................39   |
| 3.2                                                                                                                                               |                                                                                                                                                     |
| 3.3                                                                                                                                               | Dose Modification/Delays: .....................................................................................................40                   |
| 3.4                                                                                                                                               | Questionnaires ..........................................................................................................................46         |
| 3.5                                                                                                                                               | Assessments while On Study Therapy .......................................................................................47                        |
| Assessments after completion of study therapy                                                                                                     |                                                                                                                                                     |
| 3.6                                                                                                                                               |                                                                                                                                                     |
| 3.7                                                                                                                                               | CR Self-Administered Study Drugs Policy .........................................................................48                                 |
| 3.8                                                                                                                                               | Study Calendar......................................................................................................................49              |
| 3.9                                                                                                                                               | SURGICAL GUIDELINES                                                                                                                                 |
| 3.10                                                                                                                                              | Criteria for Removal from Protocol Therapy and Off Study Criteria ...................................51                                             |
| 4                                                                                                                                                 | CONCOMITANT MEDICATIONS/MEASURES                                                                                                                    |
| Medications to Avoid or Use with Precaution (Zotiraciclib [TG02]) ...................................52                                           |                                                                                                                                                     |
| 4.1                                                                                                                                               |                                                                                                                                                     |
| 4.2                                                                                                                                               | Medications to Avoid or Use with Precaution (TMZ)                                                                                                   |
| 4.3                                                                                                                                               | G-CSF and erythropoietin administration: ..........................................................................................53               |
| 4.4                                                                                                                                               | Platelet support:                                                                                                                                   |
| Corticosteroids                                                                                                                                   |                                                                                                                                                     |
| 4.5                                                                                                                                               |                                                                                                                                                     |
| 4.6                                                                                                                                               | Febrile neutropenia                                                                                                                                 |
| 4.7                                                                                                                                               | Anti-emetics                                                                                                                                        |
| Other Anticancer or Experimental Therapies ...........................................................................................53          |                                                                                                                                                     |
| 4.8                                                                                                                                               |                                                                                                                                                     |
| S                                                                                                                                                 | BIOSPECIMEN COLLECTION                                                                                                                              |
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abbreviated title ph i/ii tmz+

| Version Date: 06/01/2020                                                                                                                                         |                                                                                                                                                                 |
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| 5.1                                                                                                                                                              | Correlative Studies for Research ..............................................................................................................54               |
| Sample Storage, Tracking and Disposition .......................................................................................58                               |                                                                                                                                                                 |
| 5.2                                                                                                                                                              |                                                                                                                                                                 |
| 6                                                                                                                                                                | DATA COLLECTION AND EVALUATION ............................................................................60                                                   |
| 6.1                                                                                                                                                              | DATA COLLECTION .............................................................................................................................................60 |
| 6.2                                                                                                                                                              | DATA SHARING Plans                                                                                                                                              |
| 6.3                                                                                                                                                              | Response Criteria                                                                                                                                               |
| 6.4                                                                                                                                                              | Toxicity Criteria                                                                                                                                               |
| 7                                                                                                                                                                | NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN....65                                                                                                |
| Definitions ..................................................................................................................................................65 |                                                                                                                                                                 |
| 7.1                                                                                                                                                              |                                                                                                                                                                 |
| 7.2                                                                                                                                                              | OHSRP OFFICE OF COMPLIANCE AND TRAINING/IRB Reporting..........................65                                                                               |
| 7.3                                                                                                                                                              | NCI Clinical Director Reporting ..................................................................................................66                            |
| NIH REQUIRED DATA AND SAFETY MONITORING PLAN .........................................66                                                                         |                                                                                                                                                                 |
| 7.4                                                                                                                                                              | SPONSOR SAFETY REPORTING                                                                                                                                        |
| 8                                                                                                                                                                |                                                                                                                                                                 |
| 8.1                                                                                                                                                              | Definitions                                                                                                                                                     |
| 8.2                                                                                                                                                              | Assessment of Safety Events ..........................................................................................................68                        |
| Reporting of Serious Adverse Events                                                                                                                              |                                                                                                                                                                 |
| 8.3                                                                                                                                                              |                                                                                                                                                                 |
| 8.4                                                                                                                                                              | Safety Reporting Criteria to the Pharmaceutical Collaborators ...........................................68                                                     |
| 8.5                                                                                                                                                              | Reporting Pregnancy .......................................................................................................................69                   |
| Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND .....................69                                                                       |                                                                                                                                                                 |
| 8.6                                                                                                                                                              |                                                                                                                                                                 |
| 9                                                                                                                                                                | Clinical monitoring plan ...........................................................................................................................69          |
| 10                                                                                                                                                               | STATISTICAL CONSIDERATIONS ..............................................................................................70                                     |
| Phase I component: ......................................................................................................................................70      |                                                                                                                                                                 |
| 10.1                                                                                                                                                             | Phase II component: ....................................................................................................................................74      |
| 10.2                                                                                                                                                             |                                                                                                                                                                 |
| 11                                                                                                                                                               | COLLABORATIVE AGREEMENTS ...................................................................................................76                                  |
| 1.1                                                                                                                                                              | A Agreement Type .......................................................................................................................................76      |
| 12                                                                                                                                                               | HUMAN SUBJECTS PROTECTIONS ................................................................................................76                                   |
| Rationale For Subject Selection                                                                                                                                  |                                                                                                                                                                 |
| 12.1                                                                                                                                                             |                                                                                                                                                                 |
| 12.2                                                                                                                                                             | Participation of Children                                                                                                                                       |
| 12.3                                                                                                                                                             | Participation Of Subjects Unable To Give Consent .................................................................76                                            |
| Evaluation of Benefits and Risks/Discomforts................................................................................77                                   |                                                                                                                                                                 |
| 12.4                                                                                                                                                             |                                                                                                                                                                 |
| 12.5                                                                                                                                                             | Risks/Benefits Analysis                                                                                                                                         |
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abbreviated title ph i/ii tmz+

| 12.6                                                                                                                             | Consent and Assent Process and Documentation ........................................................................78                                |
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| PHARMACEUTICAL INFORMATION ...................................................................................................79 |                                                                                                                                                        |
| 13                                                                                                                               |                                                                                                                                                        |
| 13.1                                                                                                                             | Temozolomide (Temodar®; Methazolastone)                                                                                                                |
| 13.2                                                                                                                             | Zotiraciclib (TG02)                                                                                                                                    |
| 14                                                                                                                               | REFERENCES .                                                                                                                                           |
| 15                                                                                                                               | APPENDICES .........................................................................................................................................90 |
| 15.1                                                                                                                             | Appendix: Performance Status Criteria ...................................................................................90                            |
| 15.2                                                                                                                             | Appendix: Pregnancy & contraception......................................................................................91                            |
| Appendix: CYP1A2 and CYP3A4 ..................................................................................................92 |                                                                                                                                                        |
| 15.3                                                                                                                             |                                                                                                                                                        |
| 15.4                                                                                                                             | Appendix: Study Flow Chart ..........................................................................................................93                |
| 15.5                                                                                                                             | Appendix: Rano ...............................................................................................................................95       |
| Appendix: Dosing schedule for both treatments arms for Phase I study............................96                               |                                                                                                                                                        |
| 15.6                                                                                                                             |                                                                                                                                                        |
| 15.7                                                                                                                             | Appendix: Pill diary..........................................................................................................................97       |
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1.1.1.1 phase i

To determine the maximum tolerated dose (MTD) of Zotiraciclib (TG02) plus TMZ using both the dd and mn TMZ schedules in adult patients with recurrent anaplastic astrocytoma or glioblastoma/gliosarcoma. 1.1.1.2 Phase II To determine the efficacy of Zotiraciclib (TG02) plus TMZ *versus* TMZ alone in patients with recurrent WHO grade III or IV astrocytoma as determined by progression free survival (PFS).
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1.1.2 secondary objectives

1.1.2.1 To select the treatment regimen with better PFS4 between Zotiraciclib (TG02) plus dd TMZ or mn TMZ at each of the MTDs following cohort expansion.  
1.1.2.2 To determine the objective response rate, PFS at 6 months (PFS6) and overall survival
(OS) in the treatment arms.  
1.1.2.3 To longitudinally evaluate patient reported outcome measures using self-reported symptom severity and interference with daily activities using the MDASI-BT
1.1.3 Exploratory Objectives 1.1.3.1 To obtain tumor tissues from initial occurrence and /or recurrence to detect TP53, PTEN, IDH mutation, MYC amplification status. The level of anti-apoptotic protein, such as Mcl-1 and XIAP, and caspase 3, cleaved PARP, c-Myc, CDK9 expression will be tested, if tissues are available at the time of recurrence.  
1.1.3.2 To evaluate the imaging features by magnetic resonance imaging, including perfusion, before and during the treatment every two months.  
1.1.3.3 To obtain blood samples after Zotiraciclib (TG02) dose on cycle 1, day -3 along with PK sample collection to analyze the effect of Zotiraciclib (TG02) on neutrophil count and functions.  
1.1.3.4 To perform pharmacokinetic and pharmacogenomic studies of Zotiraciclib (TG02) once the MTD is determined in each cohort.  
1.1.3.5 To explore the feasibility of Patient-Reported Outcomes Version of the Common Terminology Criteria for Adverse Events (PRO-CTCAE) in brain tumor patients receiving investigational cancer treatments.
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1.2 background and rationale 1.2.1

High grade astrocytomas include glioblastoma (GBM) [WHO grade IV] and anaplastic astrocytoma (AA), [WHO grade III]. Both are highly malignant and belong to gliomas. Gliomas represent 80% of malignant CNS tumors. GBM accounts for 54% of gliomas and AA accounts for 7%.[1]
Histologically, GBM displays a high mitotic index, cellular pleomorphism, and extensive vascular proliferation or necrosis. They are characterized by their intrinsic heterogeneity, high morbidity and mortality. Despite aggressive multimodality treatment approach in surgery, radiation therapy and cytotoxic chemotherapies, the prognosis of patients with GBM remains poor with a median survival rate of less than 18 months.[2] Recurrent GBM has an even more dismal prognosis with a median overall survival of approximately 25 weeks. [3] Anaplastic astrocytoma (AA) is classified by WHO as a grade III glioma, demonstrating a less aggressive behavior when comparing to GBM. AA frequently transforms to GBM. The treatment of recurrent AA is unsatisfying. Chemotherapy and re-operation for recurrent AA is of modest benefit and only bring short responses. Wong et al reported that a progression free survival rate at 6 months is 31% and the median progression free survival is only 13 weeks based on eight phase II clinical trials that included 150 patients with recurrent AA.[3] In the pivotal clinical trial that led to the accelerated FDA approval in 1999 for TMZ as the treatment of recurrent AA, the PFS6 and median overall survival were reported as 46% and 13.6 months respectively.[4] Most normal cells can activate the apoptotic pathways in response to DNA damage or abnormal proliferation. In contrast, tumor cells in AA developed mechanisms to increase cell proliferation and decreasing apoptosis. *TP53* is one of the notably known genes that are implicated in apoptotic pathways of high grade astrocytoma. The *TP53* gene that maps to chromosome 17p is frequently inactivated in astrocytoma and is found in one third of AAs.[5] P53 protein plays multiple important roles in a number of cellular processes including cell cycle arrest, response to DNA damage and apoptosis. As expected, the signaling pathways in gliomas including *TP53* inactivation are most commonly noted in secondary GBM, which evolves from AA, in contrast to the primary glioblastoma, which are featured by genetic alteration, including *EGFR* amplification and chromosome 10 loss with *PTEN* mutation.[6, 7]
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1.2.2 zotiraciclib (tg02) in human

Zotiraciclib (TG02) is a pyrimidine-based multi-kinase inhibitor that has been shown to have inhibitory effects on cyclin-dependent kinases (CDKs), Janus kinase 2 (JAK2) and Fm-like tyrosine kinase 3 (Flt3).[8] It most potently inhibits CDK1, 2, 3, 5 and 9 with an IC50 of 3-9 nM and IC50 of 37nM for CDK 7. The IC50 for JAK2 is 19 nM. It is also effective against Flt3 and its drug resistant mutant with an IC50 of 19-21 nM.  
Figure 1: Target kinase inhibition by Zotiraciclib (TG02) following oral dosing at 60mg/kg in  
 nude mice bearing MCV-11 AML xenografts.  
Among all mechanism of action, CDK9 inhibition is a one of the most appealing features of Zotiraciclib (TG02). CDK9 is a serine/threonine kinase that forms the catalytic core of the positive transcription elongation factor [9-11] and it is critical for stimulating transcription elongation through RNA polymerase II.[12] To assess target engagement by Zotiraciclib (TG02) in tumor tissues, tumor lysate were analyzed after dosing MV4-11 AML xenograft-bearing mice with a single dose of 60mg/kg Zotiraciclib (TG02). Level of RNA-polymerase II phosphorylation at serine 2 was used as a readout for CDK9 inhibition. The inhibition was observed starting from 0.5 hours and reached to a maximum at 6 hours as demonstrated in Figure 1 (Investigator's Brochure, Version 6). As a consequence of inhibiting CDK9 function, the short-lived proteins will be depleted due to their heightened requirement to be regularly refreshed.[13] Two such short-lived proteins that play an important role in cancer cell survival mechanisms are Myeloid Leukemia 1 (Mcl-1), a B-cell Lymphoma 2 (Bcl-2) family member and x-linked inhibitor of apoptosis (XIAP), an anti-apoptotic protein.[14] Both Mcl-1 and XIAP were found to be depleted following a treatment with TG02 in hematologic malignancy cell lines, including myeloma cell line, as demonstrated in Figure 2 (Investigator's Brochure, Version 6).  
Figure 2: Zotiraciclib (TG02) depletes anti-apoptotic proteins of the Bcl-2 and AP families in  
  
myeloma cell line.  
Since it was discovered in 1992, Mcl-1 has been shown to play an integral role in anti-apoptotic signaling pathways. Mcl-1 is highly expressed in a variety of human cancer cells, including CNS
cancers. [15] Its overexpression is one of the survival mechanisms that allow cancer cells to survive and to further develop resistance to various apoptotic-inducing therapies. To underscore this point, in high grade gliomas, Mcl-1 was found to be the dominant anti-apoptotic Bcl-2related protein.[16] XIAP expression has been noted in human glioma cell lines and apoptosis was blocked after XIAP gene transfer,[17] suggesting that XIAP is involved in apoptosis signaling in human gliomas. CDK9 activity is regulated though activators, such as c-Myc [18] and NFkB [19], both are heavily involved in pathogenesis of malignant glioma.[20, 21] In addition to their roles in transcription, CDKs can regulate other cellular process that were considered as the core function of cell cycle control mechanisms.[22] While not involved in cell cycle progression, CDK9 and its cyclin partner T1 have a role in cell differentiation.[23] CDK9 was also found to be crucial in MYC-driven tumors. Pharmacological or shRNA-mediated CDK9 inhibition led to anti-tumor effects that correlated with MYC expression level, suggesting CDK9 inhibition likely is a therapeutic approach for treating MYC-overexpression tumors.[24] In vitro studies have demonstrated that Zotiraciclib (TG02) depletes survival proteins, including Mcl-1 and XIAP, leading to apoptosis in human tumor cells.[25] An oral dosing at 30-60mg/kg of Zotiraciclib (TG02) in mice AML xenografts demonstrated an inhibition of several target kinases, suggesting that the agent inhibited CDK9, CDK2, JAK2 and FLT3.[8] 1.2.2.2 Preclinical and clinical experience of Zotiraciclib (TG02)
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preclinical experiences

Extensive preclinical experiments have been performed to demonstrate the characteristics of Zotiraciclib (TG02). A Good Laboratory Practice (GLP) safety pharmacology study was conducted in 12 beagle dogs (6 females and 6 male) to evaluate the cardiovascular and respiratory effects of Zotiraciclib (TG02) after a single oral administration at 30mg/kg and 60 mg/kg. A dose-related duration of emesis was observed. One female dog received Zotiraciclib (TG02) at 30mg/kg died due to circulatory failure secondary to diarrhea and dehydration caused by severe intestinal damage. Postmortem pathology exam demonstrated marked atrophy and hemorrhage of thymus in this individual dog. The surface of both small and large intestines was found to be covered by white mucosa layer. The histologic exam showed epithelial necrosis on the mucosa surface and in the lumen of crypts accompanied with marked hyperemia. All the other 11 dogs survived, and no changes were noted in skin and fur, eyes and mucous membranes, respiratory system, circulatory system, autonomic system and CNS. There were no Zotiraciclib (TG02) effects on cardiac function in dogs. The individual values for blood pressure, respiratory rate, body temperature and glucose levels were within physiological limits during the study in all groups. Neither dose- nor sex-dependent variations were observed. Serum chemistry examination performed during the study showed dose dependent escalation in LDH, AST, ALT and CK in interval 2 hours and 6 hours after the administration of Zotiraciclib (TG02) with a trend to recovery of LDH and AST at 24 and 48 hours after Zotiraciclib (TG02) treatment.  
Table 1: Pharmacokinetic parameters of Zotiraciclib **(TG02) in mice, rats and dogs**  
  
Pharmacokinetics (PK) analyses were conducted in female nude mice, male Wistar rats and male beagle dogs. Comparison of single intravenous (I.V.) and oral doses showed variable bioavailability following oral dosing. All three species showed a rapid absorption following oral dosing with Tmax ranging from 10 minutes to 60 minutes. The PK parameters of Zotiraciclib
(TG02) in these 3 species are summarized in Table 1 (Investigator's Brochure, Version 6). The plasma concentration versus time profile of Zotiraciclib (TG02) in female nude mice following a single dose of 5mg/kg intravenously and a single oral dose of 75mg/kg is demonstrated in Figure 3 (Investigator's Brochure, Version 6).  
Figure 3: Plasma concentrations vs. time profile of Zotiraciclib (TG02) in female nude mice  
 following a single IV dose of 5mg/kg and a single oral dose of 75 mg/kg (n=3)
The PK assessment following a single oral administration under fed and in unfed conditions in mice suggested no significant food-drug interaction. The percentage of plasma protein biding of Zotiraciclib (TG02) was measured by an equilibrium dialysis of Zotiraciclib (TG02) (1000ng/ml)
spiked into human, dog, and mouse plasma against phosphate-buffered saline (PBS) at 37oC for 4 hours. Results suggested that the percentage of plasma-bound Zotiraciclib (TG02) was 99.97%
±  in human and similar in all species studied. Limited tissue distribution of Zotiraciclib
(TG02) was assessed in female nude mice following a single oral dosing at 75mg/kg. The mean concentration of Zotiraciclib (TG02) in plasma and tissues were demonstrated in Figure 4 (Investigator's Brochure, Version 6).  
Figure 4: Mean Plasma and tissue concentrations of Zotiraciclib (TG02) determined at different  
 time points following a single oral administration of 75mg/kg to nude mice (n=3) Table 2: Mean Pharmaceutical Parameters of Zotiraciclib (TG02) Following Oral Dosing at 30  
 mg/kg and 60mg/kg in Nude Mice Bearing MV4-11 AML Xenografts Zotiraciclib (TG02) preferentially distributed to liver, lungs and kidney followed by heart and brain. The PK of Zotiraciclib (TG02) in plasma and tumor tissues in mice was also determined following a signal dosing of nude mice bearing subcutaneous xenografts of FLT-mutant AML MV4-11 at 30mg/kg and 60mg/kg. The PK parameters that are summarized in Table 2 (Investigator's Brochure, Version 6) suggested that the greater peak and aggregate drug exposure in the tumor than in the blood in this animal model. The tissue distribution after single dose of Zotiraciclib (TG02) in mice demonstrate that the AUC ratio of brain/plasma is 2.4, suggesting good blood brain barrier penetration, which is highly relevant to the potential use of Zotiraciclib (TG02) in brain tumor treatment (Table 3, data from Adastra Pharmaceutical). Figure 5 (Investigator's Brochure, Version 6) shows the mean plasma and tissue concentration profiles over time. The levels of Zotiraciclib (TG02) in plasma and tissue exceeded the biochemical IC50 on the kinases CDK1, 2, and 9 (<10 nM) and FLT3 and JAK2 (20 nM to 40 nM); and exceeded the cellular IC50 on primary AML blasts (40nM to 50nM) for at least 24 hours at both doses. In animals receiving an oral dose of 60mg/kg Zotiraciclib (TG02), intratumoral Zotiraciclib (TG02) concentrations peaked at 200-fold above these levels (~8 μM) and remained 10-fold over (~0.4 μM) after 24 hours. By contrast, Zotiraciclib (TG02) was cleared relatively rapidly from plasma, with blood levels dropping below the threshold for cellular activity within 3 to 4 hours after a 30mg/kg dose and 13 to 14 hours after a 60 mg/kg dose.  
Table 3: Limited Tissue distribution of Zotiraciclib (TG02) after a single PO administration at  
 75mg/kg Figure 5: Mean Plasma and Tumor Concentrations of Zotiraciclib (TG02) Following Oral Dosing  
 at 30mg/kg and 60mg/kg in Nude Mice Bearing MV4-11 AML Xenografts The *in vitro* metabolic T1/2 of Zotiraciclib (TG02) in human liver microsomes (LM; T1/2 = 48 minutes), indicates involvement of Phase 1 metabolism. Species differences for *in vitro* metabolic T1/2 were noted as demonstrated in Figure 6 (Investigator's Brochure, Version 6). The compound appears to be more stable in human and dog than in mouse and rat LMs. The major metabolites observed in all species were M1, N-de-methylation, and M2, resulting from oxidation of Zotiraciclib (TG02).  
  
To investigate the role of CYP450 isozymes in the metabolism of Zotiraciclib (TG02), CYP3A4, 2D6, 1A2, 2C9, and 2C19 Bactosomes (recombinant isoform expression systems) were incubated with 5 μM Zotiraciclib (TG02) at 37°C for 45 minutes in the presence of nicotinamide adenine dinucleotide phosphate-oxidase. In this assay system, the t1/2 of Zotiraciclib (TG02) was approximately 30 minutes for CYP1A2 and CYP3A4 Bactosomes but >45 minutes for the other isoforms. These results indicate that Zotiraciclib (TG02) is primarily metabolized by CYP1A2 and CYP3A4. The potential for Zotiraciclib (TG02) to inhibit CYP450 isozymes was assessed using pooled human LM. The capacity of Zotiraciclib (TG02) (0.05, 0.25, 0.5, 2.5, 5, and 25 μM) to inhibit various CYP450 isozymes was investigated by measuring the inhibition potential (IC50 and inhibition constant [Ki]) on prototype reactions using specific substrates. Zotiraciclib (TG02) inhibited the catalytic activity of CYP2D6 (IC50 = 0.95 μM ± 0.16 μM), but not the other isozymes tested. The potential of Zotiraciclib (TG02) to induce human CYP3A4 and CYP1A2 isozymes was investigated in cultured human hepatocytes from a single male human donor. Results suggested that Zotiraciclib (TG02) is a weak inducer of CYP1A2 in human hepatocytes when compared with positive control omeprazole. For CYP3A4, Zotiraciclib
(TG02) caused no significant induction at the concentration range tested. Trans-epithelial transport of Zotiraciclib (TG02) through Caco-2 monolayers in comparison with 2 control compounds indicates that Zotiraciclib (TG02) has high intestinal absorption. The efflux ratio value (~1) for Zotiraciclib (TG02) suggests no involvement of an active P-glycoprotein mediated transport mechanism. The toxicological profile of Zotiraciclib (TG02) was evaluated in mice and dogs following single and multiple oral doses for up to 28 days followed by a 28-day non-treatment recovery period. Genotoxicity (Ames test, *in vitro* chromosomal aberration assay, and *in vivo* mouse micronucleus test) was also conducted to provide an overall assessment of Zotiraciclib (TG02). The mean toxicokinetic parameters for Zotiraciclib (TG02) following a single oral administration to mice is summarized in Table 4 (Investigator's Brochure, Version 6) and those following 28 days of daily oral administration is summarized in Table 5 (Investigator's Brochure, version 6).  
Table 4: Mean Toxicokinetic Parameters for Zotiraciclib (TG02) Following a Single Oral  
 Administration to Mice  
  
In the GLP acute oral toxicity study in mice, groups of 10 mice/sex were administered 0 (vehicle), 25 mg/kg, 50 m/kg, or 100 mg/kg Zotiraciclib (TG02). Cohorts of mice were humanely terminated on Days 3 and 15 and given a full postmortem evaluation. In addition, a subgroup of mice was designated for neurobehavioral assessment through the use of a functional observational battery (FOB), and another subgroup of mice was designated for a full TK profile on Day 1. All animals survived until scheduled termination. Rough coat was observed on Day 3 in two males received 100 mg/kg and one male received 50 mg/kg. Body weight decreases were observed in the males and females in 100 mg/kg group on Days 2 and 3 and were considered the result of the test article administration. There were no changes in food consumption considered the result of the test article administration. The FOB suggests that a single oral dose of 50 mg/kg or 100 mg/kg Zotiraciclib (TG02) may induce acute increases in aggression and/or emotionality, consistent with minor increases in specific measures of spontaneous locomotor activity (e.g., rearing). Test article-related hematology changes included decreased reticulocytes on Day 3 in the 25 mg/kg males, and in both sexes of the 50 mg/kg and 100 mg/kg groups, which correlated with bone marrow atrophy/necrosis. Total WBC and differential WBC counts decreased in the 50 mg/kg males only and in both sexes of the 100 mg/kg groups. These were the result of test article administration and were consistent with thymic atrophy observed in the 100 mg/kg animals. Test article-related increases in platelet counts were noted in the 100 mg/kg females.  
There were no obvious changes in the serum chemistry parameters for any of the groups attributed to Zotiraciclib (TG02) citrate. Histopathologic changes due to Zotiraciclib (TG02) citrate were observed in the thymus, spleen, and bone marrow of mice examined on Day 3, and in the spleen of mice examined on Day 15. Zotiraciclib (TG02)-related atrophy was present in the thymus of all 100mg/kg mice examined at Day 3. Due to the thymic atrophy on Day 3, thymic weight parameters were reduced in both sexes of the 100 mg/kg group and in the 50 mg/kg males. Thymic atrophy was not evident in recovery mice examined on Day 15. Bone marrow atrophy and/or necrosis were present on Day 3 in most 100 mg/kg mice and in some 25 mg/kg and 50 mg/kg females and one (1) 50 mg/kg male. Bone marrow atrophy was not evident in recovery mice examined on Day 15. Hematopoiesis was reduced in the spleen of 100 mg/kg and 50 mg/kg mice and occasional 25 mg/kg females examined on Day 3 and was responsible for reduced splenic weight parameters in the 100 mg/kg males. Splenic hematopoiesis was not apparent in recovery group mice examined on Day 15. Based on the results of the single-dose study of Zotiraciclib (TG02) citrate in mice, a no observed adverse effect level was not determined. Test article-related findings on Day 3 included rough coat (50 mg/kg and 100 mg/kg); body weight decreases (100 mg/kg); decreased reticulocytes (25 mg/kg [males only], 50 mg/kg, and 100 mg/kg); decreased WBC counts (50 mg/kg [males only] and 100 mg/kg); increased platelet counts (100 mg/kg [females only]); and thymic, spleen, and bone marrow changes in organ weights and histopathology (25, 50, and 100 mg/kg). By Day 15, recovery was evident or complete for each of the parameters affected.  
Multiple dose toxicity was evaluated by a GLP oral 28-day toxicity study was conducted in CD1 mice to determine potential toxicity and TK of Zotiraciclib (TG02) citrate when administered by a daily oral gavage for up to 28 consecutive days and to monitor the persistence or reversibility of any adverse effects after a 28-day recovery period. Ten mice/sex/group were assigned to 1 of 4 dose groups at target dosage levels of 0 (control article), 10 mg/kg, 20 mg/kg, and 40 mg/kg Zotiraciclib (TG02). Endpoints used to evaluate the potential toxicity of Zotiraciclib (TG02) citrate were survival, clinical observations, body weights, food consumption, clinical pathology, necropsy, organ weights, and selected histopathology. Mice administered 10 mg/kg/day Zotiraciclib (TG02) did not display any adverse clinical signs or statistically significant differences in mean body weight, food consumption, clinical pathology or organ weight values when compared to respective control values. Preliminary data revealed significant decreases in mean body weight and food consumption values in the 40 mg/kg males and females with a noted decrease in survival in this group. Based on the inability to field recovery animals at 40 mg/kg (exceeded the MTD), animals within the core and recovery groups were reassigned so that recovery could also be assessed at the mid-dose level, 20 mg/kg. Mice administered 40 mg/kg displayed adverse clinical observations suggestive of a general debilitation in their health status; whereas only rough hair coat was noted in some mice administered 20 mg/kg. Administration of 40 mg/kg Zotiraciclib (TG02) was related to decreases in reticulocyte counts, red blood cell (RBC) counts, hemoglobin, and hematocrit in males and females, and decreases in total WBC, lymphocyte, neutrophil, and monocyte counts in the males on Day 29. Mice administered 20 mg/kg Zotiraciclib (TG02) displayed decreases in total WBC, lymphocyte, neutrophil, and monocyte counts on Day 29. On Day 57, the hematology results of the 20 mg/kg mice of both sexes and the 40 mg/kg females were similar to controls.  
Test article-related clinical chemistry changes on Day 29 in the 40 mg/kg group included increases in ALT, AST, CK, and LDH (males and females); increases in alkaline phosphate, BUN, sodium and chloride (males), and decreases in total bilirubin, total protein and ALB (males and females); decreases in serum cholesterol and blood glucose (males); and decreases in serum globulin and increases in the mean ALB/globulin ratio in the 40 mg/kg females. Zotiraciclib (TG02) related clinical chemistry changes on Day 29 in the 20 mg/kg males included increases in AST, LDH, BUN, and triglycerides and decreases in bilirubin; and in the females there were decreases in globulin and increases in the ALB/globulin ratio and BUN value. Zotiraciclib (TG02)-related changes were present in the thymus, spleen, bone marrow, stomach, liver, testes, uterus, and/or small intestines of 10 mg/kg, 20 mg/kg, and 40 mg/kg scheduled termination mice, and consisted of atrophy and or necrosis of lymphoid tissues, reduced or increased hematopoiesis in the spleen, atrophy of bone marrow elements, testicular degeneration, small uterus, and atrophy and necrosis and/or regeneration (increased mitoses) of enterocytes in the small intestines. Zotiraciclib (TG02)-related lesions were reversed at recovery (Day 57) except in the GI tract, where minimal residual lesions were present in the small intestine and lesions were mildly increased in the forestomach. Additionally, testicular degeneration was still present in the 20 mg/kg mice at recovery. In the confirmatory mutagenicity assay, no positive mutagenic response was observed. Zotiraciclib (TG02) citrate was evaluated for its genotoxic potential (clastogenicity/aneugenicity) as measured by its ability to increase the incidence of micronucleated polychromatic erythrocytes (MPCEs) in bone marrow of male and female mice. A significant increase in the incidence of MPCEs in bone marrow of male and female ICR mice received 100mg/kg Zotiraciclib (TG02), indicating the positivity in mouse micronucleus assay.
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clinical experiences

Two Phase 1 clinical studies with single agent Zotiraciclib (TG02) in hematological malignancy have been conducted (NCT01204164 and NCT01699152). The primary objective of these studies was to determine the MTD and DLT. Phase 1 study in combination with carfilzomib in multiple myeloma is ongoing. Additional study with Zotiraciclib (TG02) in combination with a hypomethylating agent is under development.  
  
The interim analysis of pharmacokinetic (PK) data from clinical studies has been evaluated for 55 acute leukemia patients, 18 multiple myeloma patients and 8 CLL patients. Mean exposures for Zotiraciclib (TG02) over twenty-four hours from Cycle 1 Day 1 (Figure 7) reveal dosedependent increases in exposure (AUC0-t), Cmax at approximately 2 hours and an estimated t1/2 of 10 to 15 hours. Drug-related nausea and vomiting occur most frequently around Cmax on dosing days.  
Table 6: Summary of Zotiraciclib (TG02) Clinical Studies  
  
Multiple consecutive daily doses resulted in Zotiraciclib (TG02) drug accumulation that was not predicted based on the nonclinical toxicokinetic studies. Drug accumulation was associated with higher incidence and frequency of exposure-related TEAEs, such as fatigue. Zotiraciclib (TG02) was administered three times per week (TIW) to multiple myeloma patients; TIW dosing reduced but did not alleviate drug accumulation. BIW dosing, twice per week, alleviated drug accumulation in all populations and reduced the incidence and severity of drug-related fatigue. Schedules of Zotiraciclib (TG02) clinical studies are summarized in Table 6 (Investigator's Brochure, Version 6). The preclinical pharmacodynamics data demonstrate that daily administration is not required to achieve anticipated therapeutic drug levels. Base on PK profiles, Zotiraciclib (TG02) will be administered no more than twice weekly (BIW). In a Phase 1 study of Zotiraciclib (TG02) in multiple myeloma patients, a single agent Zotiraciclib (TG02) was administered to 18 multiple myeloma patients on three different schedules of a 28-day cycle: 6 patients administered Zotiraciclib (TG02) daily (3 patients at 50 mg and 70 mg each); 9 patients received Zotiraciclib (TG02) TIW on days 1, 3, 5, 8, 10, 12, 15, 17, 19 followed by a 9-day rest (3 patients at 100 mg and 6 at 150 mg); 3 patients administered Zotiraciclib (TG02) BIW on days 1, 4, 8, 11, 15, 18 followed by a 10-day rest (all 3 patients at 200 mg). For the BIW schedule, all 3 patients successfully completed Cycle 1. No patient experienced a dose-limiting toxicity (DLT). At this time, it was decided to amend the protocol and pursue Zotiraciclib (TG02) in combination with carfilzomib. No further dose escalation was evaluated for Zotiraciclib (TG02) as a single agent and no MTD was identified. An open label phase Ib study was developed with the primary objectives to determine the MTD of Zotiraciclib (TG02) in combination with carfilzomib (American Society for Hematology 2015 Abstract \#3052). Zotiraciclib (TG02) was administered once daily on days 1, 4, 8, 11, 15, 18 of a 28-day schedule. The starting dose of Zotiraciclib (TG02) was 150 mg with dose escalation in 50 mg increments up to 300 mg. Carfilzomib was administered per the *Full Prescribing Information* or in accordance with institutional guidelines at the time of the study (i.e., dosing days 1, 2, 8, 9, 15 and 16 at 20 mg/m2 for Cycle 1 and 27 mg/m2 for Cycle 2 and subsequent cycles). Fourteen patients were enrolled for dose escalation and 10 patients were enrolled for the MTD cohort expansion. The MTD for Zotiraciclib (TG02) in combination with carfilzomib was established at 250 mg Zotiraciclib (TG02) administered twice a week for 3 weeks (Days 1, 4, 8, 11, 15, and 18) on a 28days cycle. Two DLTs were observed, grade (Gr) 4 sepsis and neutropenia, both in 300 mg cohort. The most common drug-related adverse events were diarrhea (Gr 1-2: 4% and Gr3: 17%), nausea (Gr1-2: 79%), vomiting (Gr1-2: 50%), fatigue (Gr1-2: 38%, Gr3: 4%), anorexia (Gr: 21%), anemia (Gr1-2: 4%; Gr3:17%) and thrombocytopenia (Gr3: 8%, Gr4:13%). Zotiraciclib (TG02) inhibited the hERG channel, suggesting a risk factor for torsade de pointes (QTc prolongation) in human. In the clinic, electrocardiographic QTc interval results for 73 patients enrolled in Parts 1 and 2 demonstrated that there were no clinically meaningful changes from baseline in resting 12-lead QTc intervals observed following administration of Zotiraciclib (TG02) on Cycle 1 Day 1 at 10 to 200 mg doses. Additionally, no clinically meaningful changes from baseline in resting 12-lead QTc intervals were observed following multiple doses of Zotiraciclib (TG02) at 10 to 200 mg doses through Cycle 1, regardless of schedule. Patients with prolonged QTc intervals (males: >450 ms; females: >470 ms as calculated by Fridericia's correction formula) at screening/ baseline (based on the average QTc of triplicate ECGs), should be assessed for the following: normal electrolyte balance and concomitant medications known to prolong QTc. A patient can be reassessed for QTc interval after electrolytes are balanced and/or concomitant medications are stopped or replaced with an alternate medication. Patients, who continue to have prolonged QTc despite normal electrolyte balance and discontinuation of medications known to prolong QTc, will be excluded from the study. Use of concomitant medications that prolong QTc interval should be avoided if possible, during this study. Zotiraciclib (TG02) may carry a risk of damage to chromosomes and should not be given to pregnant women or WOCBP not using an effective form of birth control. Men receiving Zotiraciclib (TG02) with female sexual partners of childbearing potential should use an effective form of birth control. Zotiraciclib (TG02) may suppress bone marrow and the lymphoid system. Based on the preliminary observation from the first 16 patients on the phase I portion of this study, moderate to severe neutropenia has been observed. 1.2.3 Rationale for using Zotiraciclib (TG02) in glioma treatment High grade glioma is a common primary CNS tumor with a poor prognosis. Despite aggressive treatment, the disease eventually progresses, and the treatment options are often limited at the time of recurrence. There is a great unmet need to develop novel therapies in patients with recurrent high-grade glioma. TMZ is an oral alkylating agent that has proven efficacy in AA and GBM. It has been proposed that the cytotoxic effect of TMZ is associated with induction of cell death or cell cycle arrest.[26] Mcl-1 was found to be essential for TMZ induced cell death.[27] Importantly, apoptosis was not found to be enhanced by ABT-737, an inhibitor of Bcl-2, but not Mcl-1. However, reducing expression of Mcl-1 increased TMZ-induced apoptosis, suggesting that targeting Mcl-1 enhances TMZ-induced cell death and therefore may improve response to TMZ treatment, even in patients with TMZ–refractory disease. p53 is a transcription factor and has an important role in regulating cell cycle and apoptosis in response to a variety of cellular stress events. *TP53* mutation is a common genetic alteration in glioma. The incidence of *TP53* mutations is high in malignant gliomas and is reported to be higher in AA than GBM.[28] The mutant *TP53* has complex effects. There is not only the lack of normal p53 protein function, but there is also other mutation related gain-of-functions,[29]
such as the ability to disrupt mechanisms that maintain cellular genome integrity and antiapoptosis. This confers a selective survival advantage on cells within a competitive microenvironment. The ability to modulate Mcl-1 makes the rational to test Zotiraciclib (TG02) in an effort to enhance the apoptosis in glioma cells. Furthermore, MYC gene amplification and MYC over-expression have been found in human glioblastoma.[30] MYC expression has an important role in cell growth and apoptosis. However, attempts to inhibit MYC haven't been successful. Therefore, there may be indirect mechanisms, through inhibition of CDK9 dependent transcription, to intervene in MYC oncogenic signaling pathway. PTEN is a tumor suppressor that is frequently inactivated in brain tumors and inactivating *PTEN* mutations were found in 36% of glioblastoma based on a TCGA study.[31] Importantly, this was only found in primary but not secondary glioblastoma.[32] *PTEN* mutation is known to convey resistance to apoptosis-inducing treatments such as radiation and TMZ treatment, by upregulating anti-apoptotic proteins, including Mcl-1.[33] It was also shown that lack of PTEN can fuel the cell cycle by increasing the nuclear availability of cyclin D and thus promotes the transition from G1-phase to S phase of the cell cycle.[34]
Figure 8: The cell viability of patient-derived glioma cell lines is determined using Beckman  
 Coulter Vi-CELL TM XR cell viability analyzer with ViCELL Quad Pak Kit. U251, U87, LN229, LN18 were obtained from ATCC and the sphere-forming GSC 923, GSC827 were developed from NOB laboratory. All cells were treated with different concentrations of Zotiraciclib (TG02) at 24-hour after the cell seeding and cell viability is detected following 72 hours culture.  
The preliminary results of in vitro studies from our NOB laboratory and other laboratories have demonstrated that a subset of patient-derived glioblastoma cells is highly sensitive to Zotiraciclib
(TG02). The IC50 of Zotiraciclib (TG02) are in the scale of nanomolar in the glioma cell lines that were studied (Figure 8).  
Figure 9: Western blot of several key molecular signals, reflecting the different genetic  
 background of the cell lines that were used to study the effects of Zotiraciclib (TG02) treatments. Figure 10: Western blot of cleaved Caspase 3, PARP and phospo-H2AX in GSC923 following  
 treatment with Zotiraciclib (TG02) at 8, 16, 24, and 48 hours.  
The cells that were tested have a wide range of genetic alterations as demonstrated in Figure 9. The high sensitivity to a variety of glioblastoma cell lines suggested that the board spectrum of effects of Zotiraciclib (TG02) in treating high grade glioma, which is known for inter-tumoral heterogeneity. This provides the opportunity to allow enrichment of molecular markers through carefully designed clinical trials. GSC923, a patient derived stem-like cell line developed in NOB laboratory was selected as a cell model to investigate the apoptotic effect of Zotiraciclib (TG02) in glioma. GSC923 has MGMT expression, which makes it insensitive to TMZ. It is also featured by *TP53* mutation and *PTEN* mutation. High c-Myc expression was also detected in this cell line. The genetic makeup makes GSC923 an appropriate *in vitro* model to study the biology of high grade astrocytoma. Caspase 3 activation was detected by both flow cytometry and Western blot in GSC923 following Zotiraciclib (TG02) treatment. Along with the presence of cleaved Poly (ADP-ribose) polymerase (PARP) and phosphorylated histone H2AX, the data suggested that Zotiraciclib (TG02) can induce apoptosis in glioma cells (Figure 10). As early as 8 hours after treatment with Zotiraciclib (TG02) at 50 nM, CDK9 expression was shown to be decreased and further decreased expression was detected at 48 hours following Zotiraciclib (TG02) treatment (Figure 11). The expression of anti-apoptotic proteins including survivin, XIAP, Mcl-1decreased along with the CDK9 inhibition following Zotiraciclib (TG02) treatment. The cell cycle analysis in GSC923 demonstrated the G2 arrest. In summary, the potential to inhibit CDK9 activity leading to a subsequent apoptotic effect and modulation of cell cycle and other tyrosine kinases in cancer signaling pathways makes Zotiraciclib (TG02) a logical therapeutic agent to be tested in treatment of high grade astrocytoma. As described above, high-grade gliomas are known to harbor multiple genetic alterations, such as TP53 mutation, MYC amplification and PTEN that can lead to resistance to apoptosis and treatment failure. Zotiraciclib (TG02) is orally administered and penetrates BBB with good concentration in the brain. The pharmacological inhibition of CDKs with optimized pharmacokinetic features of Zotiraciclib (TG02) may provide a new therapeutic option for high grade gliomas.  
Figure 11: Western blot of surviving, XIAP Mcl-1 and c-MyC in GSC923 following treatment  
 with Zotiraciclib (TG02) at 8, 16, 24, 48 hours.
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1.2.4 rationale for zotiraciclib (tg02)

The starting dose of Zotiraciclib (TG02) was decided based on the results of an open label phase Ib study that was developed with the primary objectives to determine the MTD of Zotiraciclib (TG02) in combination with carfilzomib (American Society for Hematology 2015 Abstract \#3052). In that study, Zotiraciclib (TG02) was administered once daily on days 1, 4, 8, 11, 15, 18 of a 28-day schedule. The starting dose of Zotiraciclib (TG02) was 150mg with dose escalation in 50 m increments up to 300mg. Carfilzomib was administered according to the Full Prescribing Information or in accordance with institutional guidelines at the time of the study
(i.e., dosing days 1, 2, 8, 9, 15 and 16 at 20mg/m2 for Cycle 1 and 27mg/m2 for Cycle 2 and subsequent cycles). Fourteen patients were enrolled for dose escalation and 10 patients were enrolled for the MTD cohort expansion. The MTD for Zotiraciclib (TG02) in combination with carfilzomib was established at 250mg Zotiraciclib (TG02) administered twice a week for 3 weeks (Days 1, 4, 8, 11, 15, and 18) on a 28-days cycle. Carfilzomib is a selective proteasome inhibitor being evaluated for the treatment of relapsed and refractory multiple myeloma. In an open-label, single-arm phase 2 study (PX-171-003- A1 \#NCT00511238), patients received single-agent carfilzomib 20 mg/m2 intravenously twice weekly for 3 of 4 weeks in cycle 1, then 27 mg/m2 for ≤ 12 cycles. Adverse events (AEs) were found to be manageable without cumulative toxicities.[35] Common AEs were fatigue (49%), anemia (46%), nausea (45%), and thrombocytopenia (39%). Thirty-three patients (12.4%) experienced peripheral neuropathy, primarily grades 1 or 2. Based on the *Full Prescribing Information* of temozolomide, more severe and frequent hematologic toxicities were described. In anticipation of more toxicities in patients receiving combination treatment with Zotiraciclib (TG02) and TMZ comparing to those receive Zotiraciclib (TG02) and carfilzomib, we decided to set the starting dose of Zotiraciclib (TG02) relatively lower and dosing less frequently followed by careful dose escalation, if the treatment is proved to be well tolerated. The starting dose of Zotiraciclib (TG02) is at 200 mg/day orally on days 1, 12, 15, and 26 on a 28-days cycle in both treatment arms, except for that the first dose of Zotiraciclib (TG02) will be given 3 days prior to Day1 in cycle 1. TMZ is administered at 125mg/m2 daily on days 1-7 and 15-21 orally in dd TMZ schedule and 50mg/m2 daily in mn TMZ schedule. The dosing schedule was also designed with the intention to avoid potential overlapping toxicity from both drugs. The proposed dosing schedule will allow patients have less chance to have to take both medications on the same day. Based on our preclinical data that Zotiraciclib (TG02) reduces expression of anti-apoptotic protein and better anti-glioma effects with pretreatment of Zotiraciclib (TG02) and followed by combination treatment, one dose of Zotiraciclib (TG02) will be administered 3 days prior to first dose of TMZ in the first cycle. In the subsequent cycles, the doses that patients receive on day 26 would be the exactly 3 days prior to TMZ administration on day 1 of the following cycles.
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1.2.5 rationale for combination treatment

TMZ is an alkylating agent that has been used in malignant gliomas of all grades. It produces O6-methylguanine in DNA, which mispairs with thymine during the next cycle of DNA replication. Subsequent futile cycles of DNA mismatch repair lead to a p53-associated apoptotic cell death. A seminal study from 2005 provided level 1 evidence that the addition of TMZ to radiotherapy for newly diagnosed glioblastoma resulted in a clinically meaningful and statistically significant survival benefit.[2] Since then, TMZ has become part of standard treatment for glioblastoma. Not only used in newly diagnosed glioblastoma, TMZ has been also frequently used in grade II and III gliomas, in both upfront and recurrent settings. Clinical studies in recurrent high grade gliomas also demonstrated objective response to TMZ.[4, 36] However, the treatment effects are always limited by the resistance mechanisms, including not only MGMT expression, but also p53 and p21 status when the mismatch repair mechanism is intact.[37] In gliomas with intact p53 function, TMZ can cause prolonged cell cycle arrest and apoptosis. However, in gliomas with *TP53* mutation, TMZ induced cell cycle arrest is not sustained.[38] Zotiraciclib (TG02) not only promotes apoptosis by decreasing expression of survival proteins, such as Mcl-1, but also by regulating the cell cycle by inhibiting CDKs. This broad spectrum of mechanisms of action makes Zotiraciclib (TG02) a strong candidate to overcome resistance to TMZ treatment and subsequently lead to a prolonged response to treatment.  
Figure 12: Cell viability analysis of A. GDC923; and B. U251 by using cell counting following  
 treatment with Zotiraciclib (TG02) and TMZ alone or in combination with different sequence: Zotiraciclib (TG02) treatment for 6 hours followed by TMZ; TMZ treatment for 6 hours followed by Zotiraciclib (TG02).  
The preclinical study of cell proliferation assay from Neuro-Oncology Branch (NOB) laboratory using human-derived glioblastoma cell lines have demonstrated a significant additive effect of inhibiting tumor cell growth with combination treatment of Zotiraciclib (TG02) and TMZ. This additive effect was observed in both GSC and non-GSC cell lines (Figure 12). In *in vitro* study, GSC923 was served as a model of high grade glioma with *MGMT* promotor unmethylation and U251 was served as a model of glioma with methylated *MGMT* promotor. Cell lines with different MGMT promotor methylation status respond to combination treatment differently, suggesting the role of MGMT methylation as a stratification factor in the randomization of the clinical trials that is designed to test clinical efficacy of combination treatment of Zotiraciclib (TG02) and TMZ in high grade gliomas. Our cell-based assay also suggests that the pretreatment with Zotiraciclib (TG02) followed by a combination treatment with TMZ cause better antiglioma effects comparing to the opposite treatment sequence. This is consistent with the hypothesis that Zotiraciclib (TG02) enhances the apoptotic effects of TMZ by suppressing the expression of anti-apoptotic proteins, which is supported by the data demonstrated in Figure 13. In Figure 13, more decreased expression of survivin, XIAP and Mcl-1 is demonstrated after combination treatment with Zotiraciclib (TG02) and TMZ, comparing to the single treatment with either Zotiraciclib (TG02) or TMZ. These anti-apoptotic proteins seem to slightly induce by TMZ treatment, which potentially contributes to the treatment resistance.  
Figure 13: Western blot Western blot of survivin, XIAP and Mcl-1 in GSC923 following  
 treatment with Zotiraciclib (TG02), TMZ alone and in combination at 8, 16, 24, and 48 hours.  
1.2.6 Rationale to test Zotiraciclib (TG02) in combination with TMZ with two different dosing schedules Once patients with glioblastoma have recurrent disease after standard treatment defined as concurrent radiation plus daily TMZ followed by 6-12 cycles of adjuvant TMZ (150-200 mg/m2 days 1-5 of a 28-day cycle), the disease is typically less responsive to TMZ. This resistance has been attributed to an increase in the tumor intracellular concentration of the enzyme O6methlyguanine DNA methyltransferase (MGMT). It has been demonstrated that if the MGMT can be depleted, sensitivity to alkylating agents such as TMZ can be restored. A study by Tolcher and his colleagues demonstrated that there was a more pronounced depletion of MGMT in peripheral blood mononuclear cells (PBMCs) after dose-intense dosing schedule of TMZ.[39] Building on this data, many studies explored the use of alternative schedules. The most notable evaluated the 7-day on - 7-day off and a metronomic schedule that used daily continuous dosing of 50 mg/m2 of TMZ. Dose-dense schedule: In a prospective study, ninety adult patients with recurrent glioma were treated with TMZ at 150mg/m2/day on days 1-7 and 15-21.[40] PFS6 was reported to be 43.8% and one-year survival rate was 23%. In contrast, another study in a similar patient population reported a PFS6 of 10% and median overall survival was 21.6 weeks.[41] Both studies demonstrated good tolerance of the treatment. Metronomic dosing: A study of recurrent glioma patients who received metronomic TMZ at 50mg/m2 yielded a PFS6 of 23.9% and a 1-year survival rate of 27.3% in recurrent glioblastoma and a PFS6 of 35.7% and a 1-year survival of 60.7% in recurrent anaplastic astrocytoma patients.[42] TMZ with both the dose-dense (dd) and metronomic (mn) schedules showed promising responses in recurrent high grade glioma patients. However, the superiority of either dosing schedule hasn't been established and comparison within the same clinical trial setting is therefore warranted. 1.2.7 Rationale for pharmacokinetic, pharmacogenetic and neutrophil function assay Since the clinical trial 17C0009 was open to accrual, an unexpected phenomenon has been observed on multiple occasions- neutropenia with quick onset and quick recovery. Per current protocol, the hematologic toxicities have been mainly attributed to TMZ which is known for the bone marrow toxicities, including thrombocytopenia, leukopenia, neutropenia and lymphopenia. The complete blood count (CBC) of more than one patient happened to be tested based on clinical needs at several occasions. In those tests, absolute neutrophil count (ANC) in some patients was found to be decreased significantly. Interestingly, the low ANC can be found to be recovered within 24-48 hours in most cases. In one case, patient was found to have grade 4 neutropenia within 24 hours after one dose Zotiraciclib (TG02) but recovered to grade 1 neutropenia 24 hours later. The quick onset and quick recovery suggested that the bone marrow dysfunction is unlikely to be the etiology for the neutropenia that was observed in these cases. In addition, in most cases where this transient neutropenia occurred, there were no decreases in lymphocytes, platelets and hemoglobin. Although there were no serious consequences, such as infection in these cases, study drug had to be discontinued. Without knowing the pathophysiology of this type of neutropenia, the study drug can be discontinued prematurely. On the other hand, patients will be at risk if the follow up lab tests are not scheduled appropriately. It would be important to understand the effect of Zotiraciclib (TG02) on neutrophil biology. To analyze the neutrophil function after Zotiraciclib (TG02) administration, information from pharmacokinetic study is crucial. To further understand why this neutropenia only happened in a subgroup of patients but not all patients receiving Zotiraciclib (TG02), we will add pharmacogenetic study by analyzing the genomic DNA and assess genotype of the most relevant drug metabolizing enzymes and transporters (DMET).
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1.2.8 rationale for the patient-reported

Precedence for measuring "non-therapeutic" endpoints exists in oncology research. For example, Gemcitabine was approved by the FDA partially as a consequence of the decrease in pain reported in pancreatic patients who were treated, not on the basis of survival improvement, which was modest, at best. There have been efforts in neuro-oncology to evaluate secondary endpoints using validated instruments as an additional indicator of benefit.  
The M.D. Anderson Symptom Inventory-Brain Tumor Module (MDASI-BT) allow the selfreporting of symptom severity and interference with daily activities. The MDASI-BT have demonstrated reliability and validity in the adult primary brain tumor patient population[43, 44] This tool represents a modification of the widely used and validated M.D. Anderson Symptom Inventory, with particular attention to symptoms common in patients with brain tumors. The availability of validated instruments provides an opportunity to prospectively assess the impact of treatment, both positive and negative, on patients. The evaluation of symptom burden in this study will assist in finding the best possible treatment with the least toxicity.
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1.2.9 rationale for utilizing pro-ctcae

PRO-CTCAE is a patient-reported outcome measurement system developed by the NIH to capture the symptomatic adverse events in patients on cancer clinical trials. It was designed as a companion to the Common Terminology Criteria for Adverse Event (CTCAE), which is often used per medical professionals' evaluation. This instrument will allow us to select, but not be limited to the symptoms that are anticipated based on the previous experiences. It can be a flexible tool for descriptive reporting system, which patients can complete it at their own convenience. Since the timing for assessments are within 7 days, it certainly avoids the recall biases. It also can be used in conjunction with the CTCAE to provide a better understanding of the toxicities of the clinical trial treatments.
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2 eligibility assessment and enrollment

Inclusion criteria are same in both Phase I and Phase II parts, except for the number of prior disease relapses in **2.1.1.5**.  
2.1.1.1 Patients must have pathologic diagnosis of anaplastic astrocytoma (defined as WHO
grade III, or glioblastoma/gliosarcoma, WHO grade IV, which are confirmed by NCI Laboratory of Pathology. If the pathology diagnosis is anaplastic glioma or anaplastic oligoastrocytoma, evidence of either intact 1p/19q chromosomes or molecular features suggesting astrocytic tumor must be present. (including, but not limited to ATRX, TP53).  
2.1.1.2 Patients must have recurrent disease, histologically proven or imaging suggestive of recurrent disease as determined by PI. Prior implantation of Gliadel wafers is acceptable, if tumor recurrence is confirmed by histologic examination of the recurrent tumor.  
2.1.1.3 Patients must have the ability to understand and the willingness to sign a written informed consent document.  
2.1.1.4 Patients must be >18 years old. 2.1.1.5 No more than two prior disease relapses to be eligible for the phase I portion of the study and no more than one prior relapse to be eligible for phase II.  
2.1.1.6 Patients must have undergone prior standard therapy for their primary disease. For patients with glioblastoma, this would include surgical resection or biopsy, if safe resection was not permitted due to the tumor location, radiation and adjuvant temozolomide. For patients with anaplastic astrocytoma, this would include surgical resection, radiation and adjuvant chemotherapy (PCV or temozolomide).  
2.1.1.7 Tumor tissue must be available for review to confirm histological diagnosis. 2.1.1.8 Tumor block or unstained slides must be available for molecular profiling. 2.1.1.9 Karnofsky > 60%, (see 15.1.)
2.1.1.10 Patients must have adequate bone marrow function (ANC > 1,500/mm3, platelet count of > 100,000/mm3), adequate liver function (ALT and AST< 3 times upper limit normal and alkaline phosphatase < 2 times upper limit normal, total bilirubin < 1.5mg/dl), and adequate renal function (BUN < 1.5 times institutional normal and serum creatinine < 1.5 mg/dl) prior to registration. These tests must be performed within 14 days prior to registration. Total bilirubin: patients with Gilbert's Syndrome are eligible for the study. (Total bilirubin level can be exempted from the eligibility criterion.)
2.1.1.11 Patients must have recovered from the toxic effects of prior therapy to < grade 2 toxicity per CTC version 4 (except deep vein thrombosis)
2.1.1.12 At the time of registration, subject must be removed from prior therapy as follows:
≥ 28 days from any investigational agent,
≥ 4 weeks (28 days) from prior cytotoxic therapy,
≥ 2 weeks (14 days) from vincristine,
≥ 6 weeks (42 days) from nitrosoureas,
≥ 3 weeks (21 days) from procarbazine administration, ≥ 1 week (7 days) for non-cytotoxic agents, e.g., interferon, tamoxifen, thalidomide, cis-retinoic acid, etc. (radiosensitizer does not count).  
2.1.1.13 Patients having undergone recent resection of recurrent or progressive tumor will be eligible given all of the following conditions apply:
A. At least 2 weeks (14 days) have elapsed from the date of surgery and the patients have recovered from the effects of surgery. B. Evaluable or measurable disease following resection of recurrent malignant glioma is not mandated for eligibility into the study. C. To best assess the extent of residual disease post-operatively, an MRI should be done no later than 96 hours in the immediate post-operative period or at least within 4 weeks postoperatively, within 14 days prior to registration. If the 96-hour scan is more than 14 days before registration, the scan needs to be repeated. The patient must have been on a stable steroid dose for at least 5 days prior to the baseline MRI. Steroids may be initiated as clinically indicated once baseline imaging has been completed with a goal of titrating steroids as soon as clinically warranted.  
2.1.1.14 Patients must have received prior radiation therapy and must have an interval of greater than or equal to 12 weeks (84 days) from the completion of radiation therapy to study entry except if there is unequivocal evidence for tumor recurrence (such as histological confirmation or advanced imaging data such as PET scan) in which case the principal investigator's discretion may determine appropriate timepoint at which study therapy may begin.  
2.1.1.15 Women of childbearing potential must have a negative -HCG pregnancy test documented within 14 days prior to registration. The effects of Zotiraciclib (TG02) on the developing human fetus are unknown. For this reason, women of childbearing potential must not be pregnant, must not be breast-feeding, and must practice adequate contraception (see **15.2** for details and definitions) for the duration of the study, and for 30 days after the last dose of study medication.  
2.1.1.16 Male patients on treatment with Zotiraciclib (TG02) must agree to use an adequate method of contraception for the duration of the study, and for 30 days after the last dose of study medication as the effects of Zotiraciclib (TG02) on the developing human fetus are unknown (see **15.2** for definition of adequate methods of contraception.  
2.1.1.17 Patients must agree to enroll on the NOB Natural History protocol to allow the assessment of molecular tumor markers.  
2.1.2 Exclusion Criteria 2.1.2.1 Patients who are receiving any other investigational agents. However, prior enrollment on a study using investigational agents is acceptable as per section **2.1.1.12**.  
2.1.2.2 Patients with prior bevacizumab use for tumor treatment. Patients who received bevacizumab for symptom management, including but not limited to cerebral edema, pseudo progression can be included in the study. (To date, there have been no effective regimens developed for recurrent malignant gliomas that are refractory to bevacizumab. Inclusion of this patient population may impact the ability to determine the efficacy of Zotiraciclib [TG02] with TMZ.)
2.1.2.3 Any serious medical condition, laboratory abnormality, or psychiatric illness that would prevent the subject from providing informed consent.  
2.1.2.4 Any condition, including the presence of clinically significant laboratory abnormalities, which places the patient at unacceptable risk if he/she were to participate in the study or confounds the ability to interpret data from the study. These would include:
2.1.2.4.1 Active infection (including persistent fever) including known history of HIV or Hepatitis C infection, because these patients are at increased risk of lethal infections when treated with marrow-suppressive therapy.  
2.1.2.4.2 Diseases or conditions that obscure toxicity or dangerously alter drug metabolism. 2.1.2.4.3 Serious concurrent medical illness (e.g. symptomatic congestive heart failure).  
2.1.2.5 History of allergic reactions attributed to compounds of similar chemical or biologic composition to temozolomide and/or Zotiraciclib (TG02).  
2.1.2.6 Patients with a history of any other cancer (except non-melanoma skin cancer; or melanoma in-situ following curative surgical resection; or carcinoma in-situ of the cervix or bladder), unless in complete remission and off all therapy for that disease for a minimum of 3 years, are ineligible.  
2.1.2.7 Zotiraciclib (TG02) is primarily metabolized by CYP1A2 and CYP3A4. Patients receiving any medications or substances that are strong inhibitors or inducers of CYP1A2 and/or CYP3A4 are ineligible. Lists including medications and substances known or with the potential to interact with the CYP1A2 and CYP3A4 isoenzymes are provided in **15.3.**
2.1.2.8 Patients, who continue to have prolonged QTc (males: >450ms; females: >470ms as calculated by Fridericia's correction formula) despite normal electrolyte balance and discontinuation of medications known to prolong QTc, will be excluded from the study.
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2.1.3 recruitment strategies

This protocol may be abstracted into a plain language announcement posted on NIH websites and on NIH social media platforms.
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2.2 screening evaluation

2.2.1 Screening activities performed prior to obtaining informed consent Minimal risk activities that may be performed before the subject has signed a consent include the following:
Email, written, in person or telephone communications with prospective subjects Review of existing medical records to include H&P, laboratory studies, etc.  
Review of existing MRI, x-ray, or CT images Review of existing photographs or videos Review of existing pathology specimens/reports from a specimen obtained for diagnostic purposes A waiver of consent for these activities has been requested in section **12.6.1**.
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2.2.2 screening activities performed after

The following activities will be performed only after the subject has signed the study consent OR the consent for study 01-C-0129 (provided the procedure is permitted on that study) on which screening activities may also be performed. Assessments performed at outside facilities or on another NIH protocol within the timeframes below may also be used to determine eligibility once a patient has signed the consent. To be completed within 14 days of study registration, unless otherwise indicated 2.2.3 Unstained paraffin tissue from surgical samples will be obtained. A representative paraffin tissue block (at least 5 mm x 5 mm) or unstained slides will be obtained from previous surgery to confirm the histological diagnosis and molecular markers before entry into the clinical trial.  
2.2.4 All patients with recurrent anaplastic astrocytoma or glioblastoma/gliosarcoma will need to have confirmation of pathology diagnosis on initial disease or recurrent disease, by a Neuropathologist at the NIH. The recurrent status of the disease must be confirmed by either by pathology diagnosis on tissue from recurrent disease or by imaging review by the PI. Confirmation of pathology diagnosis may occur any time prior to study entry.  
2.2.5 A Gd-DPTA enhanced MRI, with DWI, ADC, SWI, FLAIR, perfusion, dynamic contrast enhanced, and dynamic susceptibility contrast sequences is needed within 14 days of entry into the study in all patients without surgical resection to establish the diagnosis of recurrent disease. The treating physician will review the Gd-DPTA MRI scan done prior to study entry documenting progression, if no tumor tissue available for pathology diagnosis.  
2.2.6 Complete history and physical exam completed by principal or associate investigator, including thorough documentation of signs and symptoms caused by the tumor, determination of KPS, neurological function and mental status.  
2.2.7 Screening laboratory tests will include CBC, differential, platelets, PT, PTT, INR, total protein, albumin, calcium, phosphorus, magnesium, glucose, BUN, creatinine, sodium, potassium, total bilirubin, alkaline phosphatase, LDH, SGPT (ALT), SGOT (AST), 12lead EKG and a pregnancy test for women of child bearing potential.
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2.3 participant registration and study

Registration and status updates (e.g. when a participant is taken off protocol therapy and when a participant is taken off-study) will take place per CCR SOP ADCR-2, CCR Participant Registration & Status Updates found here.
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patients must initiate study treatment

2.3.1 Treatment Assignment and/or Randomization/ Stratification Procedures*  
| Number   | Name     | Description                                                                                     |
|----------|----------|-------------------------------------------------------------------------------------------------|
| 1        | Cohort 1 | Patients with recurrent anaplastic astrocytoma or  glioblastoma/gliosarcoma assigned to Phase 1 |  
Cohorts  
2 Cohort 2 Patients with recurrent anaplastic astrocytoma or  
glioblastoma/gliosarcoma assigned to Phase 2
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arms stratifications

In Phase I, study patients will be assigned to one of two arms to receive Zotiraciclib (TG02) plus dd TMZ or Zotiraciclib (TG02) plus mn TMZ in 1:1 ratio*. Once a phase II dose/schedule is determined, patients will be randomized to the following arms in 1:1 ratio - Zotiraciclib (TG02) plus TMZ (dose and schedule selected from phase 1 component) or Control Arm - TMZ (dosing schedule will match combination arm). Patients' KPS, MGMT promoter methylation status of the tumors and the presence of measurable disease at study entry will be used as stratification factors. *In Phase I, Post-interim analysis patients will be registered to cohort 1 and assigned to ARM 1, at a TG02 dose level of 200mg. Patients will be enrolled consecutively to cohort 1. Details are described in Section **3.2.1.3**.  
| Number   | Name           | Description                         |
|----------|----------------|-------------------------------------|
| 1        | Phase I Arm I  | dose dense TMZ+ Zotiraciclib (TG02) |
| 2        | Phase I Arm 2  | metronomic TMZ+ Zotiraciclib (TG02) |
| 3        | Phase II Arm 1 | TMZ + Zotiraciclib (TG02)           |
| 4        | Phase II Arm 2 | TMZ (Control Arm)                   |  
| Name                                    | Distinct Options   |
|-----------------------------------------|--------------------|
| KPS                                     | Yes No             |
| MGMT promoter methylation status of the | Yes                |
| tumor                                   | No                 |
| Presence of Measurable Disease          | Yes No             |
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2.4 baseline evaluation

Baseline tests do not need to be repeated if the same tests were done within 14 days of treatment initiation.  
2.4.1 A complete history and physical (including baseline blood pressure measurement) and neurological examination (include documentation of the patients' height, weight and Karnofsky Performance Status), as well as documentation of measurable and/or evaluable disease shall be performed on all patients.  
2.4.2 A baseline Gd-DPTA enhanced MRI, with DWI, ADC, SWI, FLAIR, perfusion, dynamic contrast enhanced and dynamic susceptibility contrast sequences should be performed. The baseline on-study MRI should be performed on a steroid dosage that has been stable or decreasing for at least 5 days. If the steroid dose was increased between the date of imaging and the initiation of therapy (or at that time), a new baseline MRI is required. MRI study needs to be done within 14 days of the initiation of the study treatment.  
2.4.3 Pre-treatment laboratory tests will include CBC, differential, platelets, PT, PTT, INR,
total protein, albumin, calcium, phosphorus, magnesium, glucose, BUN, creatinine, sodium, potassium, total bilirubin, alkaline phosphatase, LDH, SGPT (ALT), SGOT (AST), 12-lead EKG and a pregnancy test for women of child bearing potential. Blood tests must be performed within 14 days (+ 3 working days) of registration, with day 0 = registration date. Pregnancy test must be obtained 14 days (+ 3 working days) before treatment starts.  
2.4.4 Patients will complete a baseline MD Anderson Symptom Inventory-Brain Tumor Module (MDASI-BT) (**Error! Reference source not found.**). The MDASI-BT will only be completed by the patient, unless changes in vision or weakness make this difficult. If this occurs, then the caregiver or research assistant may read the questions to the patient or assist with marking the severity number or score as described by the patient. A patient caregiver may complete the questionnaires as a patient-preference proxy if the patient's deficits preclude self-report. If the MDASI-BT is completed by the patient for Natural History study, 16C0151 within (+/-) 14 days from the initiation of the treatment, then it can be used as the baseline.  
2.4.5 Rapid clinical deterioration from the time of study entry to initiation of treatment will mandate a full re-evaluation including physical examination, KPS, MRI, and laboratory studies including CBC and blood chemistries and MDASI as outlined in Sections 2.5.1 - 2.4.4.
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3 study implementation 3.1 study

The phase I component is designed to find the MTD of Zotiraciclib (TG02) in combination with 2 schedules of TMZ - dose dense and metronomic. Patients will be enrolled and equally assigned into the two dosing schedule arms for MTD finding. In each arm, the MTD will be determined independently using the Bayesian Optimal Interval (BOIN) design, fully described in Section 8 and recently published [45]. Once the MTDs of the two regimens (dd TMZ + Zotiraciclib (TG02) and mn TMZ + Zotiraciclib [TG02]) have been established, cohort expansion will be carried out at the each of the MTDs. Patients will be equally randomized into to the two cohort expansions. A post interim analysis cohort will be carried out in cohort 1 arm 1 to confirm MTD. The MTD that has higher progression-free survival rate at 4 months (PFS4) will be selected and used in phase II.
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3.1.2 phase ii

The two-arm randomized phase II will determine if the addition of Zotiraciclib (TG02) to the dd or mn TMZ schedule (determined by the expanded phase I) improves outcome in patients with recurrent malignant glioma using PFS as the primary outcomes measure. Patients will be equally randomized into the combination Arm (Zotiraciclib [TG02] plus TMZ selected from phase I) and control arm (TMZ). The randomization will stratify by the presence of measurable disease at the study entry, KPS and MGMT promoter methylation status. Additional outcomes such as PFS6, overall survival from study entry and symptom burden (using the MDASI-BT instrument in Appendix **Error! Reference source not found.**) will also be collected. The study schema is illustrated in Study Flow Chart in Appendix **15.4.**
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3.1.3 definition of dose limiting

All patients in the MTD finding phase of the phase I part will be evaluable for assessment of toxicity to define DLTs. The DLT-defining period is the first 4 weeks' treatment in the Phase I
study. Toxicities will be graded per the Common Terminology Criteria for Adverse events (CTCAE) Version 4.0. If multiple toxicities are seen, the presence of DLT should be based on the most severe toxicity experienced. DLT is defined as any of the following events occurring in the first cycle of treatment (4 weeks) and attributable to the study drugs:
Any of the following hematological toxicities:
Grade 4 neutropenia lasting 5 days or more. Febrile neutropenia defined as grade 3-4 neutropenia with fever ≥38.5ºC and/or infection requiring antibiotic or antifungal treatment.  
Grade 4 thrombocytopenia lasting more than 2 days.  
Grade 3 thrombocytopenia with bleeding of any duration
Grade 3 or 4 anemia lasting more than 2 days.  
Any non-hematologic grade 3 or 4 toxicity, except the following nausea or vomiting that responds to symptomatic therapy and lasts ≤7 days.  
Diarrhea that responds to symptomatic therapy within 2 days.
fatigue that responds to symptomatic therapy and lasts ≤7 days.  
weight gain (in patients on steroids)
venous thromboembolic disease (rationale for not including this event as a DLT
described below)
If there is a dose reduction of either or both study medications or if a dose is held or discontinued in cycle 1, this constitutes a DLT, per PI discretion.  
Grade 4 lymphopenia is not considered as a DLT for this study. All patients for this study have treated with alkylating agents and radiation treatment. Lymphopenia can be highly associated with the previous treatment or have a low threshold to have severe lymphopenia because of the previous treatments. In case of isolated laboratory abnormalities that may not reach clinical significance, a determination will be made by PI or in consultation with the IRB on a case-by-case basis to determine if DLT needs to be declared.  
Failure to recover from treatment related toxicities to be eligible for re-treatment within 4 weeks of the last dose of the drugs.
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3.1.4 dose escalation

We will employ the Bayesian optimal interval (BOIN) design [46] to guide dose escalation and find the MTD. The details of the design are provided in Section 8. There are two treatment arms in the Phase I study: the combination treatment with Zotiraciclib (TG02) and dd TMZ; and the combination treatment of Zotiraciclib (TG02) with mn TMZ. Dose escalation will be conducted independently in these two arms. Several dose levels of Zotiraciclib (TG02) are planned as outlined in the Dose Escalation Table below.  
| Arm 1: Dose-dense temozolomide   |                                       |                                                  |
|----------------------------------|---------------------------------------|--------------------------------------------------|
| Level                            | TMZ (mg/m2  x 7 days on/7 days off)** | TG 02 (mg/day on 1, 12,15,26  per 28 day cycle)* |
| -1                               | 125                                   | 150                                              |
| 0 (starting dose)                | 125                                   | 200                                              |
| I                                | 125                                   | 250                                              |
| II                               | 125                                   | 300***                                           |  
| Arm 2: Metronomic temozolomide   |                      |                                                 |
|----------------------------------|----------------------|-------------------------------------------------|
| Level                            | TMZ (mg/m2  daily)** | TG 02 (mg/day on 1, 12,15,26 per 28 day cycle)* |
| -1                               | 50                   | 150                                             |
| 0 (starting dose)                | 50                   | 200                                             |
| I                                | 50                   | 250                                             |
| II                               | 50                   | 300***                                          |  
* The first dose of Zotiraciclib (TG02) will be given 3 days prior to Day1 of cycle 1. The subsequent doses for cycle 1 will be given on days 1, 12, 15, and 26. Zotiraciclib (TG02) will be given on days 1, 12, 15, and 26 every 28-days cycle in all the cycles starting cycle 2.  
**BSA (body surface area) for Temozolomide dosing calculation will be capped at 2.2m2 to avoid excessive risk of myelosuppression. *** One patient in MN arm developed severe toxicities which lead to ICU admission after one dose of Zotiraciclib (TG02) at 300mg. The etiology was not completely clear. However, due to the patient's safety concern, does level II of Zotiraciclib (TG02) was determined to be eliminated in both study arms by the investigator and pharmaceutical manufacturer.
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3.2 drug administration 3.2.1 phase

There are two treatment arms in this component: the combination treatment of Zotiraciclib (TG02) and dd TMZ; and the combination treatment of Zotiraciclib (TG02) with mn TMZ. In both treatment arms, several dose levels are planned as outlined in section **3.1.4.** The starting dose of Zotiraciclib (TG02) is 200 mg/day orally on days 1, 12, 15, and 26 on a 28-days cycle in both treatment arms, except for that the first dose of Zotiraciclib (TG02) will be given 3 days prior to Day1 in cycle 1. The dosing schedule for both treatment arms are illustrated in the Appendix **15.6**. TMZ is administered at 125mg/m2 daily on days 1-7 and 15-21 orally in dd TMZ schedule and 50mg/m2 daily in mn TMZ schedule. The dosing schedule was designed with the intention to avoid potential overlapping toxicity from both drugs. The proposed dosing schedule will allow patients have less chance to have to take both medications on the same day. Based on our preclinical data that Zotiraciclib (TG02) reduces expression of anti-apoptotic protein and better anti-glioma effects with pretreatment of Zotiraciclib (TG02) and followed by combination treatment, one dose of Zotiraciclib (TG02) will be administered 3 days prior to first dose of TMZ in the first cycle. In the subsequent cycles, the doses that patients receive on day 26 would be the exactly 3 days prior to TMZ administration on day 1 of the following cycles. We will employ the Bayesian optimal interval (BOIN) design [46] to guide dose escalation and find the MTD. The details of the design are provided in Section 8.
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3.2.1.2 cohort expansion

After identifying the MTD for each of the TMZ dosing schedules in combination with Zotiraciclib (TG02) (i.e., 125mg/m2 TMZ on days 1-7 and 15-21 per cycle, and 50 mg/m2 TMZ daily), cohort expansion will be conducted independently at each of the MTDs until the total number of patients treated at each of the MTDs reaches 18 patients (including the patients who have been treated in the dose-finding part). The dosing schedule with a higher PFS4 will be selected and used in phase II.
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3.2.1.3 post interim analysis cohort

Patients will be enrolled to Arm 1 only to receive Zotiraciclib (at Dose level 0 and in combination of dd TMZ). TG02 dose is 200 mg/day orally on days 1, 12, 15, and 26 on a 28days cycle, except for that the first dose of Zotiraciclib (TG02) will be given 3 days prior to Day1 in cycle 1. TMZ is administered at 125mg/m2 daily on days 1-7 and 15-21 orally on a 28days cycle.
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3.2.2 phase ii treatment

The combination treatment selected from phase I (with better PFS4) will be tested against the TMZ alone with the same dosing schedule as it is in the combination arm. Patients will be randomized on a 1:1 basis between two competing treatment arms: TMZ + Zotiraciclib (TG02) versus TMZ alone using a Bayesian phase II trial design. The details of the design are provided in Section 8. The dosage for the combination arm is derived from the MTD of the combination determined in the phase I component of the study. The treatment schedule will be identical to that described above in the phase I component, with each cycle comprising 28 days. Patients will continue treatment until tumor progression or unacceptable toxicity occurs or completion of 12 cycles of treatment. At progression, patients randomized to the control arm (TMZ alone) will be offered the opportunity to continue TMZ and the additional treatment with Zotiraciclib (TG02). After administration of 12 cycles of treatment, patients may stay on the study treatment, if there is evidence of clinical benefit based on treating physician's discretion. If patient vomits Zotiraciclib (TG02), do not re-administer.
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3.3 dose modification/delays

If an adverse event occurs wherein both TMZ and Zotiraciclib (TG02) would require dose deescalation, the Principal Investigator shall have the discretion of first dose reducing one of the 2 study drugs with the consideration of previously known drug toxicities/side effects in an effort to further define attribution.  
| Table 8: Start of Each Cycle for both TMZ and Zotiraciclib (TG02) Temozolomide and Zotiraciclib (TG02) Hematologic* ANC ≥ 1.5 x 109 /L  and PLT ≥ 100 x  109 /L No dose modification ANC< 1.5 x 109 /L  and/or Platelet count <  100 x 109 /L Delay up to 4 weeks until ANC ≥ 1.5 x  109 /L and PLT ≥ 100 x 109 /L. If  unresolved after 4 weeks, then stop. If  resolved, dose delay/reductions based on  Table 9.  NonHematologic Grade 1 or less No dose modification Grade 2 Zotiraciclib (TG02): Delay up to 4  weeks until ≤ Grade 1. If unresolved after  4 weeks, then dose reduce by 1 dose level. Temozolomide: dose delay/reductions  based on Table 9, if adverse event  attributed to Temozolomide. Grade 3 Zotiraciclib (TG02): Delay up to 4 weeks  until ≤ Grade 1. If unresolved after 4  weeks, then stop. If resolved to </=  Grade 1, then dose reduce by 1 dose level. Temozolomide: dose delay/reductions  based on Table 9, if adverse event  attributed to Temozolomide. Grade 4 Discontinue treatment *With the exception of lymphopenia, grade 1-4. Patient may start a cycle with G2 WBC, as long   |
|---|  
*With the exception of lymphopenia, grade 1-4. Patient may start a cycle with G2 WBC, as long  
as the ANC ≥ 1.5 x 109/L.
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3.3.1 temozolomide (tmz) dose modifications

Dose modification(s) are based on adverse event occurrence and may be implemented during any treatment cycle (Table 11) or prior to initiation of each new cycle per Table 8. If multiple AEs are seen, the dose administered should be based on the dose reduction required for the most severe grade of any single AE. If TMZ is delayed or discontinued in combination therapy, the Zotiraciclib (TG02) component of treatment may continue, unless there is clear reason to discontinue Zotiraciclib (TG02) simultaneously.  
| DD TMZ Dose,   | MN TMZ Dose,   |    |
|----------------|----------------|----|
| mg/m2 /day     | mg/m2 /day     |    |
| Dose Level -2  | 80             | 32 |
| -1             | 100            | 40 |
| 0              | 125            | 50 |
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3.3.1.1 delay

For treatment on day 1 of each cycle (within the prior 72 hours), ANC ≥ 1.5 x 109/L, platelet count ≥ 100 x 109/L and all grade 2 -3 non-hematologic AEs (except nausea and vomiting) must have resolved (to grade ≤ 1). For grade 4, discontinue treatment. For the purposes of this study, any clinically insignificant Grade 2 or 3 electrolyte abnormalities with attribution of unlikely related to or unrelated to study medications, will not require holding or dose-reducing study medications. If AEs persist, treatment may be delayed by 1 week for up to 4 consecutive weeks. If, after 4 weeks of delay, all AEs have still not resolved to grade 1 or less: then any further treatment with temozolomide should be stopped, as summarized in Table 8 and Table 11. 3.3.1.2 Dose reductions (Summarized in Table 11) If any non-hematologic AE observed was grade 3 (except nausea and vomiting) and/or if platelets < 50 x 109/L and/or ANC < 1 x 109/L, then the dose should be reduced by one dose level. For patients who would require dose reductions to a dose level < -2 as defined in table 10, temozolomide will be stopped. Also, if any of the same non-hematologic grade 3 AE recurs (except, nausea and vomiting,) after reduction for that AE, then temozolomide will be stopped.  
If any treatment-related non-hematologic AE observed was grade 4 (except nausea and vomiting)
then adjuvant temozolomide treatment should be stopped. Any dose reductions of temozolomide will be determined according to (1) non-hematologic AE, as well as (2) the lowest/worst ANC and platelet counts observed. No dose escalation should be attempted. If the dose reduction occurs in the middle of the cycle, the reduced dose should be applied as soon as the treatment resumes (i.e. next dose).
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3.3.2 zotiraciclib (tg02) dose modifications

If multiple AEs are seen, the dose administered should be based on the dose reduction required for the most severe grade of any single AE. Prior studies of Zotiraciclib (TG02) indicate that non-hematologic toxicities have been the dose-limiting toxicities. Therefore, in the combination regimens, attribution of treatment-related toxicities will be made based on best available information. As Zotiraciclib (TG02) has not been reported with high rates of prolonged myelotoxicity, these toxicities will likely be attributed to temozolomide and dose modifications made as outlined in Section **3.3.1** above. Conversely, non-hematologic adverse events such as nausea and vomiting, intractable diarrhea, fatigue and prolongation of the QTc interval on ECG
will be attributed to Zotiraciclib (TG02). Liver toxicities are a known toxicity of TMZ, which has been tested extensively in patients with high grade glioma. Zotiraciclib (TG02) has also been reported to cause elevated liver enzymes. To better understand the attribution of this toxicity, in the event of elevated ALT/ AST of grade 2 or above when a patient has received the combined treatment, dose modification will be made on one drug at a time. Should the need for dose modification arise again due to the same toxicity, dose of the second drug will be reduced. If a dose reduction occurs in the middle of the cycle, the reduced dose should be applied as soon as the treatment resumes. Details for dose modification, delay at beginning of each cycle and during the cycle are summarized in Table 8 and Table 11 respectively. The dose of Zotiraciclib (TG02) will be initiated at the phase I dose for patients enrolled on the dose-finding component or at the MTD for both the cohort expansion of the phase I and the phase II component of the trial. The same dose will be continued unless > or = grade 3 drug related toxicity occurs; treatment will be held until toxicity resolves to grade 1 or less. At that time, the dose will be reduced to one level below the dose that caused the grade 3 toxicity. Only two dose reductions will be allowed for Zotiraciclib (TG02). Patients enrolled prior to determination of the MTD will have stepwise reductions as necessary based on the Table 10 below. Please note, if the MTD is 150 mg/day, then no dose reduction will be allowed. Additionally, if the patient is treated at dose Level 0, only one dose reduction (to 150 mg/day) will be allowed.  
| Dose Level   | TG 02 (mg/day on days 1, 12, 15, 26  per 28 day cycle)*   |
|--------------|-----------------------------------------------------------|
| -1           | 150                                                       |
| 0            | 200                                                       |
| 1            | 250                                                       |
| 2            | 300                                                       |  
Summary of Dose Modification or Discontinuation  
| Table 11: Dose Modifications and Delay*   Zotiraciclib (TG02)   | TMZ           |                 |               |                 |
|-----------------------------------------------------------------|---------------|-----------------|---------------|-----------------|
| Grade                                                           | Hematologic** | Non-hematologic | Hematologic** | Non-hematologic |
| 0-1                                                             | No dose       | No dose         | No dose       | No dose         |
| modification                                                    | modification  | modification    | modification  |                 |  
| Table 11: Dose Modifications and Delay*   Zotiraciclib (TG02)                                            | TMZ                                                                                 |                                                                                                                                          |                                                                             |                                                                                                                    |
|----------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------|
| 2                                                                                                        | Hold until resolved  to ≤ G1; if persists  beyond 4 weeks,  dose reduce one  level. | Hold until  resolved to ≤ grade 1; if > 4  weeks then dose  reduce 1 level.                                                              | Hold until resolved  to ≤ grade 1; if > 4  weeks then dose  reduce 1 level. | No dose  modifications for  non-hematologic  AEs (G 0-2),  unless the  adverse event  attributed to  Temozolomide. |
| 3                                                                                                        | If not recovered to  ≤ G2 within 3  days, STOP.                                     | Delay up to 4  weeks until ≤ Grade 1. If  unresolved after 4  weeks, then stop.  If resolved to ≤ grade 1; then dose  reduce by 1 level. | Hold until resolved  to ≤ grade 1; if > 4  weeks, then stop.                | Hold until  resolved to ≤ grade 2; if  greater than 4  weeks then stop.  If less than 4  weeks, reduce by  one dose level. If  the same nonhematologic  grade 3 AE  recurs after  reduction for that  AE, then stop.                                                                                                                    |
| If recovered to ≤ G2 within 3 days,  re-start @ one dose  level lower once  recovered to G1 or  less.*** |                                                                                     |                                                                                                                                          |                                                                             |                                                                                                                    |
| 4                                                                                                        | If not recovered to  ≤ G2 within 3  days, STOP.                                     | Stop treatment.                                                                                                                          | Stop treatment.                                                             | Stop treatment.                                                                                                    |
| If recovered to ≤ G2 within 3 days,  re-start @ one dose  level lower once  recovered to G1 or  less     |                                                                                     |                                                                                                                                          |                                                                             |                                                                                                                    |
| * If the patient develops a lower extremity deep venous thrombosis and requires anticoagulation or       |                                                                                     |                                                                                                                                          |                                                                             |                                                                                                                    |  
* If the patient develops a lower extremity deep venous thrombosis and requires anticoagulation or intervention (grade 2 or 3 toxicity), treatment may be restarted after a 2-week rest period if the patient is stably anticoagulated and is judged medically stable to begin chemotherapy. (Please note: DVT is graded in the CTC version 4.0 as grade 1= superficial thrombosis, grade 2= DVT, medical intervention indicated, grade 3= uncomplicated pulmonary embolism, medical intervention indicated, grade 4=life-threatening pulmonary embolism, cerebrovascular event, arterial insufficiency, urgent intervention indicated).  
** With the exception of lymphopenia, grade 1-4.  
***If G3 neutropenia recovers within 72 hours, the Principal Investigator may consider no dose reduction of Zotiraciclib (TG02).  
If leukopenia is grade 2 or above while ANC is grade 1 or less, the principal investigator may not consider it for dose modification and delay.
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3.3.3 pre-medications 3.3.3.1 zotiraciclib (tg02)

Patients must receive vigorous prophylactic treatment for nausea and vomiting. Prophylactic use of moderately anti-emetogenic treatments (e.g., 5-HT3 antagonist) has been more effective at controlling nausea and vomiting than lightly anti-emetogenic treatments or treatment after onset. 3.3.3.1.1 Prophylaxis for Nausea/Vomiting Zotiraciclib (TG02) is potentially highly emetogenic. Vigorous anti-emetic prophylaxis is required prior to administration of Zotiraciclib (TG02) and should continue at least 24 hours after Zotiraciclib (TG02) administration or until nausea/vomiting returns to Grade 0 or 1. Anti-emetic prophylaxis is also highly recommended to be started on the day prior to Zotiraciclib (TG02) administration. Some recommended prophylaxis regimens include but is not limited to the following:
5-HT3 antagonist (e.g., ondansetron, granisetron, palonosetron, etc.)
2.5 mg olanzapine or  
other moderate anti-emetic regimens tailored to the individual patient at the Investigator's discretion
3.3.3.1.2 Prophylaxis for Diarrhea Due to frequent reports of diarrhea with Zotiraciclib (TG02) administration, vigorous antidiarrheal prophylaxis (e.g., loperamide or equivalent) is required prior to administration of Zotiraciclib (TG02) and should continue at least 24 hours after Zotiraciclib (TG02) administration or until diarrhea returns to Grade 0 or 1.  
The recommended prophylaxis includes but is not limited to the following:  
a. Loperamide the day prior to and the day of Zotiraciclib (TG02). Suggest that if patient had diarrhea on day of Zotiraciclib (TG02) that Loperamide 2-4mg is administered following first loose stool on day after Zotiraciclib (TG02) and after each stool if an hour as elapsed since last dose, as needed thereafter.  
b. Once diarrhea has occurred, Loperamide 4mg after first loose stool, and Loperamide 4mg after the second and third stool respectively, with patient to call the study team practitioner after third loose stool.  
c. Consider Lomotil and Tincture of Opium, if diarrhea continues. A nutrition consult may also be beneficial for potential diet modifications.
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3.4 questionnaires 3.4.1 md anderson

The MDASI-BT will be administered at baseline and at the time of imaging analysis as outlined in the protocol. Of note, every attempt should be made to administer the MDASI-BT prior to informing the patients about the results of the imaging study. The MDASI-BT will be completed only by the patient, unless changes in vision or weakness make this difficult. If this occurs, then the caregiver or research assistant may read the questions to the patient or assist with marking the severity number or score as described by the patient. A caregiver may complete the questionnaires as a patient proxy if the patient's deficits preclude self-report; however, this must be done at every assessment from baseline to end of treatment. In addition, information regarding demographics and treatment history will be collected as part of the larger study and used in this analysis. The MDASI-BT consist of symptoms rated on an 11-point scale (0 to 10) to indicate the presence and severity of the symptom, with 0 being "not present" and 10 being "as bad as you can imagine." Each symptom is rated at its worst in the last 24 hours. Symptoms included on the instrument include those commonly associated with cancer therapies, and those associated with the underlying disease. The questionnaire also includes ratings of how much symptoms interfered with different aspects of a patient's life in the last 24 hours. These interference items include: general activity, mood, work (includes both work outside the home and housework), relations with other people, walking, and enjoyment of life. The interference items are also measured on 0 - 10 scales. The average time to complete this instrument is 5 minutes. The MDASI-BT has been translated into multiple languages, but the English language version will be used for this study[44].
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3.4.2 pro-ctcae

Patients will complete a baseline PRO-CTCAE any time after enrollment but before starting treatment, and again on days 2, 8, 14, 21, and 28 in each treatment cycle, if the patient is able. There are 13 questions in total. The questions are focused on 12 symptoms that are commonly reported by the patients taking Zotiraciclib (TG02). It takes 5-10 minutes to complete the questionnaire. It will only be completed by the patient, unless changes in vision or weakness make this difficult. If this occurs, then the caregiver may read the questions to the patient or assist with marking the answers as described by the patient. A patient caregiver may complete the questionnaires as a patient-preference proxy if the patient's deficits preclude self-report. The questionnaires are allowed to be completed within +/-2 days form the scheduled dates. The PROCTCAE is only going to be conducted in the cohort expansion part of the study.  
Patients will complete at least 2 questionnaires per cycle. *The PRO-CTCAE will not be performed in the Post Interim Analysis cohort.
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3.5 assessments while on study

During the 2nd **week** of treatment (i.e. 28-day cycle), the patient will have a complete blood count (CBC) with differential and platelet count. In phase I study, if grade 3 neutropenia is identified, repeat CBC with differential every 3 days until it improves to ≤ grade 2. Patient may have CBC drawn at NIH lab or outside community lab.  
During the 4th **week** of treatment (i.e. 28-day cycle), the patient will have the following assessments: On days 2, 8, 14, 21, and 28, patients will complete PRO-CTCAE form. (+/- 2 days). The PRO-CTCAE will not be performed in the Post Interim Analysis cohort.
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3.5.2 cycle 2 assessments

During the 2nd **week** of treatment, the patient will have a complete blood count (CBC) with differential and platelet count. In phase I study, if grade 3 neutropenia is identified, repeat CBC with differential every 3 days until it improves to ≤ grade 2. Patient may have CBC drawn at NIH lab or outside community lab.  
During the 4th **week** of treatment (i.e. 28-day cycle), the patient will have the following assessments:
Physical exam with vital signs, neurological exam, neurological function score and neurological examination (including documentation of the patient's Karnofsky Performance Status), mental status score. Weight is also included.  
A Gd-DPTA enhanced MRI, with DWI, ADC, SWI, FLAIR, perfusion, dynamic contrast enhanced and dynamic susceptibility contrast sequences should be performed.  
MD Anderson Symptom Inventory-Brain Tumor Module (MDASI-BT). If the MDASIBT is completed by the patient for Natural History study within 14 days from the MRI
assessment, it can be used but only if it was administered prior to informing patient of tumor imaging results.  
Toxicity monitoring Physical exam with vital signs, neurological exam, neurological function score and neurological examination (including documentation of the patient's Karnofsky Performance Status), mental status score. Weight is also included. Toxicity monitoring.  
A complete blood count (CBC) with differential and platelet count. In phase I study, if grade 3 neutropenia is identified, repeat CBC with differential every 3 days until it improves to ≤ grade 2.  
PT, PTT, INR, total protein, albumin, calcium, phosphorus, magnesium, glucose, BUN,
creatinine, sodium, potassium, total bilirubin, alkaline phosphatase, LDH, SGPT (ALT), SGOT (AST) and a pregnancy test for women of childbearing potential 12-lead EKG.  
A complete blood count (CBC) with differential and platelet count every two weeks. In phase I study, if grade 3 neutropenia is identified, repeat CBC with differential every 3 days until it improves to ≤ grade 2.  
PT, PTT, INR, total protein, albumin, calcium, phosphorus, magnesium, glucose, BUN,
creatinine, sodium, potassium, total bilirubin, alkaline phosphatase, LDH, SGPT (ALT), SGOT (AST) and a pregnancy test for women of childbearing potential 12-lead EKG.  
Response evaluation.
3.5.3 Subsequent cycles For all subsequent odd-numbered cycles (i.e. cycle 3, 5, 7, 9, 11), patients will repeat assessments outlined in section **3.5.1**
For all subsequent **even-numbered cycles (i.e. cycle 4, 6, 8, 10, 12)**, patients will repeat assessments outlined section **3.5.2**.
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3.6 assessments after completion of

Follow-Up Phase - Survival/Post-Disease Progression Once a subject completes Phase II of study therapy or starts a new anti-cancer therapy, the subject moves into the survival follow up phase and should be contacted approximately within every 6 months to assess for survival status until death, withdrawal of consent, or the end of the study, whichever occurs first.
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3.7 ccr self-administered study drugs

All oral self-administered investigational agents will be properly accounted for, handled, and disposed in accordance with existing federal regulations and principles of Good Clinical Practice. All oral study drugs will be recorded in the patient diaries found in Appendix **15.7.** This will be used as a memory aide for subjects. A clinical research team maintains the primary source record.  
| 3.8                       | STUDY CALENDAR   | Cycle 1   | Cycle 2   | Subsequent  Odd  numbered  cycles   | Subsequent Even  numbered  cycles   | Offstudy visit8    |    |    |    |    |    |                                      |              |
|---------------------------|------------------|-----------|-----------|-------------------------------------|-------------------------------------|----|----|----|----|----|----|--------------------------------------|--------------|
| Screening/ Baseline       |                  |           |           |                                     |                                     |    |    |    |    |    |    |                                      |              |
| Procedure                 | Wk               | Wk        | Wk        | Wk                                  | Wk                                  | Wk | Wk | Wk | Wk | Wk | Wk | Wk                                   | 30 days (+/- |
| 1                         | 2*               | 3         | 4*        | 1                                   | 2*                                  | 3  | 4* | 2* | 4* | 2* | 4* | 15) after  last dose of  treatment 8 |              |
| Informed Consent          | X                |           |           |                                     |                                     |    |    |    |    |    |    |                                      |              |
| Medical History           | X                |           |           |                                     |                                     |    |    |    |    |    |    |                                      |              |
| Physical Exam1            | X                | X1        | X1        | X1                                  | X1                                  | X1 |    |    |    |    |    |                                      |              |
| MDASI-BT                  | X                | X         | X7        |                                     |                                     |    |    |    |    |    |    |                                      |              |
| ProCTCAE***               | X                | X         | X         | X                                   | X                                   | X  | X  | X  | X  | X  | X  | X                                    | X            |
| GD-DPTA                   | X                | X         | X6        |                                     |                                     |    |    |    |    |    |    |                                      |              |
| MRI of brain  Hematology2 | X                | X         | X         | X                                   | X                                   | X  | X  | X  | X  | X  |    |                                      |              |
| Chemistry3                | X                | X         | X         | X                                   | X                                   | X  |    |    |    |    |    |                                      |              |
| PT/PTT/INR                | X                | X         | X         | X                                   | X                                   |    |    |    |    |    |    |                                      |              |
| 12-lead EKG               | X                | X         | X         | X                                   |                                     |    |    |    |    |    |    |                                      |              |
| Pregnancy Test 4          | X                | X         | X         | X                                   | X                                   | X  |    |    |    |    |    |                                      |              |
| Tumor Pathology           | X                |           |           |                                     |                                     |    |    |    |    |    |    |                                      |              |
| Paraffin Tissue block 5   | X                |           |           |                                     |                                     |    |    |    |    |    |    |                                      |              |
| Response evaluation       | X                | X         |           |                                     |                                     |    |    |    |    |    |    |                                      |              |
| Research samples (PK,     | X9,10            |           |           |                                     |                                     |    |    |    |    |    |    |                                      |              |
| PG, neutrophil)9,10       |                  |           |           |                                     |                                     |    |    |    |    |    |    |                                      |              |  
1. Physical examination includes vital signs, neurological exam, neurological function score, Karnofsky performance score, mental status score. Height and weight are also included. **Height measured only** at baseline.  
2. Includes CBC with differential and platelets. In phase I study, once grade 3 neutropenia is identified, CBC with differential will be tested every 3 days until it improves to ≤ grade 2.  
3. Includes: Total protein, albumin, Ca, Mg, P, glucose, BUN, creatinine, Na, K, total bilirubin, alkaline phosphatase, LDH, SGPT (ALT), SGOT (AST).  
4. Only for women of childbearing potential as defined in section **15.2.** To be performed before each new cycle of therapy.  
5. If a tissue block is unavailable, unstained slides are acceptable. 6. Brain MRI w/wo contrast needs to be done every two cycles of treatment. 7. MDASI-BT questionnaire needs to be completed at imaging cycles within +/- 1week. (prior to imaging results having been discussed with the patient). If the MDASI-BT is completed by the patient for Natural History study (CC 16C0151) within 14 days from the MRI assessment, it can be used but only if it was administered prior to informing patient of tumor imaging results.  
8. If patient does not return for 30-day safety visit, patient will be taken off study for reason listed in section 3.10.3 If patient does not return, physical exam and blood work is not mandatory.  
9. On cycle 1 day -3, collect following samples from patients in the **Phase 1 cohort expansion only**: PK
studies (1 6mL green sodium heparin tube at following timepoints-pre-dose, 1hr, 2hr, 4hr, 12hr, 24hr, 48hr, and 72hr post-oral dose); PG studies (1 purple tube EDTA, 6mL, pre-dose) and neutrophil analysis (2 additional purple top EDTA, 6mL each for CBC/diff and neutrophil marker; and 1 additional heparinized green top tube, 6 mL for plasma isolation for monitoring neutrophil activation markers) at following timepoints- pre-dose, 1hr, 2hr, 4hr, 12hr, 24hr, 48hr, and 72hr post-oral dose). All effort will be made to collect samples at prescribed time points; missed timepoints or collections outside the window will be documented but not constitute a deviation.  
10. Research sample will be collected for PK/PG study from patients enrolled to the Post-Interim Analysis cohort at the following timepoints: pre-dose, 1hr, 2hr, 4hr, 24hr, and 48hr post-oral dose. This will avoid the requirement for inpatient admission. The neutrophil sample will not be collected.  
*+/- 3 business days ** Allowance for scheduling changes: Brief interruption and delay in the 28-day cycle may occasionally be required due to travel delays, airport closure, inclement weather, family responsibilities, security alerts and government holidays, etc. This can also extend to complications of disease not attributable to disease progression or protocol therapy. These delays will not be considered protocol deviation. However, any interruption more than 14 calendar days will not be permitted.  
*** on days 2, 8, 14, 21, and 28 (+/- 2 days). The ProCTCAE data will not be collected from patients enrolled to the post-interim analysis cohort.
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3.9 surgical guidelines

If neurosurgical management is required for reasons not due to tumor progression, these procedures must be documented, including the indications for surgery, the surgical operative note and pathology report. Study treatment may be resumed per discretion of the PI.
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3.10 criteria for removal from

3.10.1 Criteria for Removal from Protocol Therapy  
After administration of 12 cycles of treatment, unless there is clear evidence of clinical benefit to justify continuation of treatment based on treating physician's opinion.  
Progressive disease if patient not eligible for or refuses crossover or progressive disease * after crossover to TMZ+ Zotiraciclib (TG02)
The patient may withdraw from the study at any time for any reason
Unacceptable Toxicity as defined in section 3.3 Investigator discretion  
Treatment related adverse events not resolved within a 4-weeks rest period or requiring more than two dose de-escalations of any drug whichever is earlier.  
Pregnancy
* Delayed imaging responses have been observed in some patients who received protocol treatment. If a patient is clinically stable or clinically improved, even while MRI showed >25% increase in tumor size, the patient may continue on study treatment per P.I. discretion for one additional cycle, at which time a repeat scan will be done. If tumor size continues to increase, disease progression will be dated back to the date of prior MRI.
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3.10.2 off-study visit

Prior to removal from study, effort must be made to have all subjects complete a safety visit approximately 30 days following the last dose of study therapy. If patient does not return for off study follow up safety visit, patient will be taken off study per appropriate reason listed in section **3.10.3.** During the follow up safety visit, the following study tasks will be performed:  
A complete physical (including baseline blood pressure measurement) and neurological examination (include documentation of the patients' Karnofsky Performance Status).  
MD Anderson Symptom Inventory-Brain Tumor Module (MDASI-BT) **(Error!**
Reference source not found.)
Laboratory tests will include CBC, differential, platelets, total protein, albumin, calcium, phosphorus, magnesium, glucose, BUN, creatinine, sodium, potassium, total bilirubin, alkaline phosphatase, LDH, SGPT (ALT), SGOT (AST) and a pregnancy test for women of childbearing potential.  
Take patient off study (section **Error! Reference source not found.**)
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3.10.3 off-study criteria

Participant requests to be withdrawn from study  
Patients lost to or refuse to follow up Death Screen failure PI discretion based on clinical indications Patient to start alternative therapy Patient completed follow up period Phase II patient completed follow up until death
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4 concomitant medications/measures 4.1 medications

Use of certain medications that prolong QTc interval should be avoided throughout the trial. These medications include but are not limited to: azithromycin, clarithromycin, erythromycin, roxithromycin, metronidazole (with alcohol), moxifloxacin, fluconazole (in cirrhosis), ketoconazole, nelfinavir, chloroquine, mefloquine, halothane, disopyramide, procainamide, quinidine, amiodarone, sotalol, amitriptyline, clomipramine, impipramine, dothiepin, doxepin, risperidone, fluphenazine, haloperidol, clozapine, thioridiazine, ziprasidone, pimozide, droperidol, terfenadine, astemizole, probucol, dolasetron mesylate, and cisapride. However, it is understood that patients with malignancy treated with potentially myelotoxic cytotoxic chemotherapy may require certain classes of medications such as azole antifungal agents and certain antibiotics that have been reported to prolong QTc. If patients require these medications every effort should be made to maintain a normal electrolyte balance with special attention to K+ and Mg++ levels. An ECG in triplicate should be obtained after beginning such a medication to determine any change to the QTc duration based on the average QTc of triplicate ECGs.  
In the event of QT/QTc prolongation, electrolytes will be corrected to keep the potassium and magnesium within normal limits and the ECG in triplicate will be repeated. Patients may be monitored on telemetry at the Investigator's discretion. During the trial, patients who show a QTc interval prolongation as per CTCAE (based on the average QTc of triplicate ECGs) as calculated by Fridericia's correction formula, should be tested for the electrolytes noted above. If the electrolytes are at normal levels but QTc interval remains prolonged, study drug should be held until QTc interval is no longer prolonged.
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4.1.2 cyp450 drug interactions (related

Strong inhibitors/inducers of CYP3A4 and CYP1A2 and drugs that are known substrates for CYP1A2 and CYP2D6 with a narrow therapeutic index should be used with caution in this study. Known inhibitors of CYP1A2 include acyclovir and fluvoxamine, and known inhibitors of CYP3A4 include atazanavir, clarithromycin, indinavir, itraconazole, ketoconazole, nefazodone, nelfinavir, ritonavir, saquinavir, and telithromycin. Known inducers of CYP1A2 include omeprazole, insulin, and cigarette smoking, and known inducers of CYP3A4 include rifampin and carbamazepine.  
Sensitive CYP1A2 substrates with narrow therapeutic indices include theophylline and tizanidine, and sensitive CYP2D6 substrates with narrow therapeutic indices include thioridazine and tamoxifen. Exceptions are allowed per PI discretion, if clinically indicated.
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4.2 medications to avoid or

Administration of valproic acid decreases oral clearance of temozolomide by about 5%.
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4.3 g-csf and erythropoietin administration

Routine prophylactic use is not permitted. However, therapeutic use in patients with complications (severe neutropenia with fever or anemia), may be considered at the investigator's discretion.
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4.4 platelet support

Prophylactic use of platelet is only used when Platelet count < 20 x 109/L. Platelet transfusion is provided for active bleeding patients with thrombocytopenia to keep platelet count above 50 x 109/L.
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4.5 corticosteroids

Should be used in the smallest dose to control symptoms of cerebral edema and mass effect and discontinued if possible.
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4.6 febrile neutropenia

It may be managed according to the local institution's Infectious Disease guidelines. Measures may include appropriate laboratory testing, including blood and urine cultures and the institution of broad-spectrum antibiotics. If a source for the fever is not identified or the fever resolves when the neutrophil count recovers, antibiotics may be discontinued, and the patient observed.
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4.7 anti-emetics

Prophylactic and therapeutic use is allowed. Other concomitant medications and the therapies considered necessary for the well-being of the patient may be given at the discretion of the treating physician. All concomitant medications must be recorded.
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4.8 other anticancer or experimental

No other anticancer therapy (including chemotherapy, radiation, hormonal treatment or immunotherapy) of any kind is permitted during the study period. No other antitumor drugs under investigation may be used concomitantly with the study drug.
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5 biospecimen collection

| 5.1                     | CORRELATIVE STUDIES FOR RESEARCH   |                                                                                                                                                                         |                                                                                                                                                                          |                |
|-------------------------|------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------|
| Test/assay              | Volume  blood  (approx.)           | Type                                                                                                                                                                    | Collection point (+/-                                                                                                                                                    | Location of    |
| of tube                 | 48hrs)                             | specimen analysis                                                                                                                                                       |                                                                                                                                                                          |                |
| Target  DNA  Sequencing | Tumor                              | -                                                                                                                                                                       | Initial and/or Recurrence                                                                                                                                                | Laboratory of  |
| tissue block            | Pathology                          |                                                                                                                                                                         |                                                                                                                                                                          |                |
| Western  Blot and  PCR  | Fresh  Frozen  Tumor               | -                                                                                                                                                                       | Surgery at Disease                                                                                                                                                       | NOB Laboratory |
| recurrence              |                                    |                                                                                                                                                                         |                                                                                                                                                                          |                |
| PK and                  | Dr. Figg's Lab                     |                                                                                                                                                                         |                                                                                                                                                                          |                |
| PG*                     | 6mL                                | Sodium  Heparin  (green  top)                                                                                                                                           | Cycle 1 day -3 at the  following time points:  pre-dose, 1hr, 2hr (peak),  4hr, 12hr-, 24hr, 48hr, and  72hr post oral dose. For  patients in the cohort  expansion only |                |
| Up to 6mL               | EDTA  (purple  top)                | Cycle 1 day - 3 For  patients in the cohort  expansion only                                                                                                             | Dr Figg's Lab                                                                                                                                                            |                |
| Neutrophil              | 12mL                               | EDTA                                                                                                                                                                    |                                                                                                                                                                          |                |
| analysis**              | (purple  top)                      | Cycle 1 day -3 at the  following time points:  pre-dose, 1hr, 2hr (peak),  4hr, 12hr, 24hr, 48hr, and  72hr post oral dose. For  patients in the cohort  expansion only | (1 tube)  Processed by NOB  Laboratory and sent  to Dept of Laboratory  Medicine (1 tube) Processed by NOB  Laboratory and sent  to Neutrophil  Monitoring Lab  (NML)    |                |  
| Test/assay   | Volume  blood  (approx.)                                                                                                                         | Type                                                                        | Collection point (+/-   | Location of   |
|--------------|--------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------|-------------------------|---------------|
| of tube      | 48hrs)                                                                                                                                           | specimen analysis                                                           |                         |               |
| 6mL          | (green                                                                                                                                           | Cycle 1 day -3 at the                                                       |                         |               |
| top)         | following time points:  pre-dose, 1hr, 2hr (peak),  4hr, 12hr, 24hr, 48hr, and  72hr post oral dose. For  patients in the cohort  expansion only | Processed by NOB  Laboratory and sent  to Neutrophil  Monitoring Lab  (NML) |                         |               |  
*PK/PG analysis: For patients in Post-Interim Analysis cohort, samples will be collected in Cycle 1 day -3 at the following time points only: pre-dose, 1hr, 2hr (peak), 4hr, 24hr, and 48hr post oral dose. **Neutrophil analysis will not be collected and performed in patients in Post-Interim Analysis cohort.  
5.1.1 The brain tumor tissue block from initial and /or recurrence will be collected to detect MGMT methylation, *TP53, PTEN, IDH* mutation, MYC amplification status by performing targeted DNA sequencing at the laboratory of Pathology, CCR, NIH (if not included in the Natural History Study [CC 16C0151]).  
5.1.1.1 Tissue blocks can be shipped to Department of Pathology at NCI at the following address:
Laboratory of Pathology Center for Cancer Research National Cancer Institute Building 10, Room 2S235 Bethesda, MD 20892-1500 Ph: 301-480-5010 5.1.1.2 Procedures for storage of tissue specimens in the Laboratory of Pathology Tissues designated for clinical diagnostics are transported to the Laboratory of Pathology (LP) where they are examined grossly, and relevant portions are fixed, embedded in paraffin and sectioned and stained for diagnostic interpretation. Unutilized excess tissues are stored for up to three months, in accordance with College of American Pathologists/Joint Commission on Accreditation of Healthcare Organizations (CAP/JCAHO) guidelines, and then discarded. Following completion of the diagnostic workup, the slides and tissue blocks are stored indefinitely in the LP's clinical archives. All specimens are catalogued and retrieved utilizing the clinical laboratory information systems, in accordance with CAP/JCAHO regulations. The use of any stored specimens for research purposes is only allowed when the appropriate IRB approval has been obtained. In some cases, this approval has been obtained via the original protocol on which the patient was enrolled. 5.1.2 Fresh frozen brain tumor tissue will be obtained if patient undergoes surgery at the time of disease progression. Pro-survival proteins, including Mcl-1, XIAP and survivin will be tested at protein by Western blot and at mRNA level by PCR in the NOB laboratory.  
5.1.2.1 The samples should be shipped to the following address ON DRY ICE in appropriately labeled package in accordance with local and federal regulations for biological material:
Wei Zhang MD, PhD. Neuro-Oncology Branch, NCI, NIH Bldg. 37, Room 1142 Bethesda, MD 20892 Office: 240-760-6835 or 301-435-3136 Cell: 240-274-0469 Pager: 102-12209 5.1.2.2 If the surgery is performed at Neurosurgery Department at NINDS, NIH, the following personnel will be contacted for tissue pick up.  
Wei Zhang MD, PhD. Neuro-Oncology Branch, NCI, NIH Bldg. 37, Room 1142 Bethesda, MD 20892 Office: 240-760-6835 or 301-435-3136 Cell: 240-274-0469 Pager: 102-12209 5.1.3 Pharmacokinetic (PK) and Pharmacogenetic (PG)Studies 5.1.3.1 Pharmacokinetic Studies PK studies will be done in all patients that are enrolled in the cohort expansion part and the PostInterim Analysis cohort. Blood samples for the determination of plasma levels of Zotiraciclib
(TG02) will be obtained from each patient (see section **5.1.3.3** for schedule and tube types). Bioanalytical measurements for Zotiraciclib (TG02) will be conducted on an ultra HPLC-MSMS system using an assay developed and validated by the Clinical Pharmacology Program. This data will be used to monitor plasma concentrations of Zotiraciclib (TG02) to correlate to pharmacodynamic endpoints, clinical response, toxicity, and pharmacogenetic analyses.
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5.1.3.2 pharmacogenetic studies (pg)

PG studies will be done in all patients that are enrolled in the cohort expansion part and the PostInterim Analysis cohort. One blood sample per patient (6mL) will be collected (see section 5.1.3.3 for schedule) to analyze the genomic DNA and assess genotype of the most relevant drug metabolizing enzymes and transporters (DMET). DNA will be analyzed on a DMET Plus (Affymatrix) genotyping platform that has the capacity to test for 1,936 genetic variations in 225 drug disposition genes, including 47 CYP (phase I metabolism) genes, 13 non-CYP (phase I metabolism) genes, 78 phase II metabolizing genes (including UGTs), 63 transporters, 4 genes involved in facilitation of drug transporters, 9 genes involved in global regulation of drug metabolizing/transporting proteins, 4 drug binding proteins. Of specific interest to Zotiraciclib (TG02) are polymorphisms in CYP3A4, CYP1A2, and ABCB1 (P-glycoprotein), all of which are included in the DMET analysis. The samples will be placed at room temperature. The date and exact time of each blood draw should be recorded on the sample tube and the PK/PG sheet. Contact Dr. Figg's Clinical Pharmacology Program (Blood Processing Core) in Bldg. 10/5A09 at NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the Friday before is preferred).
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5.1.3.3 sample collection

PKs: Blood samples for the determination of plasma levels of Zotiraciclib (TG02) will be obtained from each patient via 6mL green sodium heparin tube collected on the first daily dose on cycle 1 day -3 at the following time points: pre-dose, 1hr, 2hr (peak), 4hr, 12hr, 24hr, 48hr, and 72hr post oral dose. For patients in the post-interim analysis cohort, blood will be collected, pre-dose, 1hr, 2hr, 4hr, 24hr, and 48hr post oral dose. PGs: 6 mL of blood will be collected in an EDTA (purple top) tube on day -3 of cycle 1. 5.1.3.4 Sample processing The samples will be placed immediately on wet ice and refrigerated. The date and exact time of each blood draw should be recorded on the sample tube and the PK sheet. Please e-mail NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the Friday before is preferred). For sample pickup, page 102-11964. For immediate help, call 240-760-6180 (main blood processing core number) or, if no answer, 240-760-6190 (main clinical pharmacology lab number). For questions regarding sample processing, contact NCIBloodcore@mail.nih.gov. Upon arrival in the Clinical Pharmacology Program, samples will be centrifuged, and the plasma transferred into cryovials for storage at -80 C until the time of analysis. In addition, samples will be barcoded.
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5.1.4 neutrophil analysis

Neutrophil analysis studies will be done in all patients that are enrolled in the cohort expansion part. Three tubes of blood will be collected at all PK time points for the following studies: WBC and differential analysis, neutrophil morphology studies (photographs), neutrophil function assays, surface marker analysis and for spinning out cells and saving plasma to send to Dr. Douglas Kuhns for monitoring activation markers released into the plasma such as specific granule marker. (See section **5.1.4.1** for tube types and collection information. Note: The pediatric size tubes can be used as alternatives for blood sample collection, if it is necessary.) WBC with differential and imaging analysis will be done at Department of Laboratory Medicine. Plasma and neutrophil isolation will be done by NOB laboratory and analysis will be done in Neutrophil Monitoring Laboratory (NML).  
5.1.4.1 Collection  
a. CBC with differential (a tube of 6 cc blood collected in purple top tube) will be done on cycle 1 day -3 at the following time points*: pre-dose, 1hr, 2hr, 4hr, 12hr, 24hr, 48hr, and 72hr post oral Zotiraciclib (TG02). Neutrophil morphology will be studies by using Cellavision program.  
b. Collect blood sample (6 cc of blood in heparinized green top tube) at the time of PK sample collection for plasma isolation for monitoring neutrophil activation markers.  
c. Collect (6 cc of blood in purple top tube with EDTA), fix and stain neutrophils at the same time points of the PK sample collection. Samples will be used for neutrophil cell surface markers including but not limited to CD11a, CD11b, CD11c, CD32, CD18, CD67, CD64, CD18, CD45, CD62L, CD63, 7D5.
* All effort will be made to collect samples at prescribed time points; missed timepoints or collections outside the window (+/- 30 minutes from the scheduled time) will be documented but not constitute a deviation. 5.1.4.2 Contact of NOB laboratory Ying Pang, MD., PhD. Neuro-Oncology Branch, NCI, CCR Bldg. 37, Room 1142 Bethesda, MD 20892 Office: 240-760-6216 5.1.4.3 Contact of NML Douglas Kuhns, PhD. Building 310, Rm 204 Frederick, MD 21702 Office: 301-846-6378 5.1.5 Labeling of Samples Labels on all samples will at least include the following information:
Patient initials, ID number Protocol number Collection date
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5.2 sample storage tracking and

Samples will be ordered in CRIS and tracked through a Clinical Trial Data Management system. Should a CRIS screen not be available, the CRIS downtime procedures will be followed. Samples will not be sent outside NIH without appropriate approvals and/or agreements, if required.
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5.2.1 clinical pharmacology program

All samples sent to the Clinical Pharmacology Program (CPP) will be barcoded, with data entered and stored in Labmatrix utilized by the CPP. This is a secure program, with access to Labmatrix limited to defined Figg lab personnel, who are issued individual user accounts. Installation of Labmatrix is limited to computers specified by Dr. Figg. These computers all have a password restricted login screen. Labmatrix creates a unique barcode ID for every sample and sample box, which cannot be traced back to patients without Labmatrix access. The data recorded for each sample includes the patient ID, name, trial name/protocol number, time drawn, cycle time point, dose, material type, as well as box and freezer location. Patient demographics associated with the clinical center patient number are provided in the system. For each sample, there are notes associated with the processing method (delay in sample processing, storage conditions on the ward, etc.).Barcoded samples are stored in barcoded boxes in a locked freezer at either -20 or -80C according to stability requirements. These freezers are located onsite in the CPP and offsite at NCI Frederick Central Repository Services in Frederick, MD. Visitors to the laboratory are required to be accompanied by laboratory staff at all times. Access to stored clinical samples is restricted. Samples will be stored until requested by a researcher named on the protocol. All requests are monitored and tracked in Labmatrix. All researchers are required to sign a form stating that the samples are only to be used for research purposes associated with this trial (as per the IRB approved protocol) and that any unused samples must be returned to the CPP. It is the responsibility of the NCI Principal Investigator to ensure that the samples requested are being used in a manner consistent with IRB approval. Following completion of this study, samples will remain in storage as detailed above. Access to these samples will only be granted following OHSRP/IRB approval of an additional protocol, granting the rights to use the material or if the use is not considered to be human subjects research.
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5.2.2 neutrophil monitoring laboratory (nml)

Plasma and neutrophil isolation will be done by NOB laboratory and analysis will be done in Neutrophil Monitoring Laboratory (NML) in Frederick. Samples will be sent to the NOB laboratory and sent to Frederick laboratory will be barcoded with data entered and stored in the Labmatrix system utilized by the CCR, NCI. This is a secure system with access limited to defined personnel. All such personnel with access to patient information annually complete the NIH online Protection of Human Subjects course. Labmatrix creates a unique barcode ID for every sample which cannot be traced back to patients without Labmatrix access. The data recorded for each sample includes the patient ID, name, trial name/protocol number, date/time drawn, as well as box and freezer location. Patient demographics associated with the Clinical Center patient number are provided in the system. For each sample, there are notes associated with the processing method (delay in sample processing, storage conditions on the ward, etc.).
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5.2.3 department of laboratory medicine

Samples will be processed by the NOB laboratory and then sent to Department of Laboratory Medicine will be barcoded with data entered and stored in the Labmatrix system utilized by the CCR, NCI. This is a secure system with access limited to defined personnel. All such personnel with access to patient information annually complete the NIH online Protection of Human Subjects course. 5.2.4 Protocol Completion/Sample Destruction All specimens obtained in the protocol are used as defined in the protocol. Any specimens that are remaining at the completion of the protocol will be stored in the conditions described below. The study will remain open so long as sample or data analysis continues. Samples from consenting subjects will be stored until they are no longer of scientific value or if a subject withdraws consent for their continued use, at which time they will be destroyed. If the patient withdraws consent the participant's data will be excluded from future distributions, but data that have already been distributed for approved research use will not be able to be retrieved. The PI will record any loss or unanticipated destruction of samples as a deviation. Reporting will be per the requirements of section 7.2.
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6 data collection and evaluation

The PI will be responsible for overseeing entry of data into an in-house password protected electronic system (C3D) and ensuring data accuracy, consistency and timeliness. The MDASIBT responses will be entered by study subjects directly into Scribe/Labmatrix.  
The principal investigator, associate investigators/research nurses and/or a contracted data manager will assist with the data management efforts. All data obtained during the conduct of the protocol will be kept in secure network drives or in approved alternative sites that comply with NIH security standards. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant. All adverse events, including clinically significant abnormal findings on laboratory evaluations, regardless of severity, will be followed until return to baseline or stabilization of event. Document AEs from the first study intervention, Study Day 1, through 30 days after the last administration of investigational agent/intervention. Adverse events that are serious need to be recorded through 30 days after the last administration of investigational agent/intervention. Beyond 30 days after the last intervention, only adverse events which are serious and related to the study intervention need to be recorded. An abnormal laboratory value will be considered an AE if the laboratory abnormality is characterized by any of the following:
Results in discontinuation from the study  
Is associated with clinical signs or symptoms Requires treatment or any other therapeutic intervention Is associated with death or another serious adverse event, including hospitalization. Is judged by the Investigator to be of significant clinical impact If any abnormal laboratory result is considered clinically significant, the investigator will provide details about the action taken with respect to the test drug and about the patient's outcome.
End of study procedures: Data will be stored according to HHS, FDA regulations and NIH Intramural Records Retention Schedule as applicable. Loss or destruction of data: Should we become aware that a major breach in our plan to protect subject confidentiality and trial data has occurred, this will be reported expeditiously per requirements in section **7.2.1**. 6.1.1 Non-Relevant Toxicities for Brain Tumor Protocols Lymphopenia is a common finding among patients with primary brain tumors and is directly attributable to concurrent use of corticosteroids. To date, even Grade III lymphopenia has not been associated with a worsening of clinical outcome. Therefore, we will not consider this parameter in the determination of optimal dosing of drugs. Weight gain is a common finding among patients with primary brain tumors. This too is directly attributed to the concurrent use of corticosteroids. Doses of steroids or study agents will not be modified as a consequence of weight gain. Corticosteroid dosage will be based on maintenance of control of edema in the brain and the standard clinical practice to use the minimal effective dose of corticosteroids will be employed. Alopecia is a common occurrence in patients with brain tumors as a consequence of cranial radiotherapy. Alopecia will not be recorded or graded as toxicity. Venous thromboembolic disease is a common complication occurring in up to 30% of patients with malignant gliomas.[47, 48] In this study, patients who develop deep vein thrombosis may receive anticoagulation and resume therapy once they are stable on anticoagulation. Grade III deep vein thrombosis will not be considered a DLT for the phase I portion. The routine use of filgrastim or other white cell growth factors is not permitted during cycle 1 of the Phase I portion unless clinically indicated, but it may be used beyond this period or once the assessment of DLT has been completed.
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6.2 data sharing plans 6.2.1

I will share human data generated in this research for future research as follows:
Coded, linked data in an NIH-funded or approved public repository.  
Coded, linked data in BTRIS (automatic for activities in the Clinical Center) Coded, linked or identified data with approved outside collaborators under appropriate agreements.
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how and where will the

Data will be shared through:
BTRIS (automatic for activities in the Clinical Center)  
Approved outside collaborators under appropriate individual agreements.  
Publication and/or public presentations.
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when will the data be

Before publication.  
At the time of publication or shortly thereafter.  
6.2.2 Genomic Data Sharing
Unlinked genomic data will be deposited in public genomic databases such as dbGaP in compliance with the NIH Genomic Data Sharing Policy.
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6.3 response criteria 6.3.1 general

The primary efficacy endpoint for this study is progression free survival (PFS) from patient registration. However, objective response status should be measured and recorded.
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6.3.2 definitions

Evaluable for maximum tolerated dose: All subjects enrolled in the Phase I dose escalation cohorts from the time of their first treatment with Zotiraciclib (TG02) +TMZ to the end of cycle 1. Patients who develop a DLT during cycle 1 and thus do not complete treatment for the cycle should be considered evaluable for MTD. Evaluable for toxicity: All eligible patients who complete at least one cycle of protocol therapy or who have a DLT after the protocol therapy is started are evaluable for toxicity from the time of their first treatment.  
*If the patients miss doses of Zotiraciclib (TG02) or TMZ in the first cycle of treatment due to reasons that are unrelated to toxicity, it will be the PI's decision about whether they are evaluable for toxicity in the dose finding stage.  
Evaluable for objective response: Only those patients who have measurable disease present at baseline, have received at least one cycle of therapy at MTD and have had their disease reevaluated will be considered evaluable for response. These patients will have their response classified according to the definitions stated below. (Note: Patients who exhibit objective disease progression prior to the end of cycle 1 will also be considered evaluable.) Phase I patients removed from study treatment within 28 days for reasons other than toxicity may be replaced. 6.3.3 Disease Parameters Measurable Disease: Bi-dimensionally measurable lesions with clearly defined margins by GDDPTA enhanced MRI scan.  
Evaluable Disease: Uni-dimensionally measurable lesions, masses with margins not clearly defined. Patients with only this kind of imaging will not be allowed to enter this study unless they have recently undergone surgery and have histologically proven recurrent disease. Non-Evaluable Disease: Not Applicable for response evaluation. Objective-Status, To Be Recorded at Each Evaluation: If there are too many measurable lesions to measure at each evaluation, choose the largest two to be followed before a patient is entered on study. The remaining lesions will be considered evaluable for the purpose of objective status determination. Unless progression is observed, objective status can only be determined when ALL measurable and evaluable sites and lesions are assessed.
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6.3.4 response criteria

Response and progression will be evaluated in this study using the new proposed criteria Response Assessment in Neuro-Oncology Criteria (RANO) (see Appendix **15.5** ).[49] Complete response: Requires all of the following: Partial response: Requires all of following:
≥ 50 % decrease compared to baseline (prior to treatment) in the sum of the products of perpendicular diameters of all measurable enhancing lesions sustained for at least 4 weeks; No new lesions and stable or improved non-enhancing lesions (T2/FLAIR).  
Patients off steroids or on physiologic replacement only.  
Complete disappearance of all enhancing measurable and non-measurable disease sustained for at least 4 weeks.  
No new lesions and stable or improved non-enhancing lesions (T2/FLAIR)
Patient off steroids or on physiologic replacement only.  
Stable or improved clinically.
Stable or improved clinically.  
Stable disease: Requires all of the following:
Not complete response, partial response or progression.  
Stable non-enhancing lesions (T2/FLAIR).  
Patient on same or lower dose of steroids compared to baseline scan.  
Progression: Requires any of the following:
≥ 25 % increase compared to baseline in the sum of the products of perpendicular diameters of enhancing lesions compared with the smallest measurement obtained either at baseline or best response with the patient on stable or increasing doses of steroids.  
Significant increase in T2/FLAIR non-enhancing lesions with the patient on stable or increasing doses of steroids (not caused by comorbid events e.g. Radiation therapy, demyelination, ischemic injury, infection, seizures, medication adverse events or changes in corticosteroid dose).
Any new lesions. Failure to return for evaluation as a result of death or deteriorating condition or clear progression of non-measurable disease.  
6.3.5 Patients will undergo response evaluations every 2 months.  
RANO criteria will be used to determine response and progression (includes CR, PR, SD
and PD) **15.5.**
Neurological exam.  
Performance status by Karnofsky Performance Status.  
6.3.6 Evaluation of Best Overall Response This will be calculated from the sequence of objective statuses. For patients with all disease sites assessed every evaluation period, the best response will be defined as the best objective status as measured according to Section **6.3.4**. If the response does not persist at the next regular scheduled MRI, the response will still be recorded based on the prior scan but will be designated as a non-sustained response. If the response is sustained, e. g. still present on the subsequent MRI, it will be recorded as a sustained response, lasting until the time of tumor progression. Best response is unknown if the patient does not qualify for a best response or increasing disease and if all objective status determinations before progression are unknown.
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6.3.7 duration of response

Duration of overall response: The duration of overall response is measured from the time measurement criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented.  
Duration of stable disease: Stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements.  
6.3.8 Progression-Free Survival PFS is defined as the duration of time from start of registration to time of progression or death, whichever occurs first. 6.3.9 Time to Death: From date of registration to date of death due to any cause. 6.3.10 Neurological Exam: Although not used for determining response, it is useful to evaluate improvement in the neurologic exam, (as compared to the baseline assessment), that should coincide with objective measurement of tumor size. The criteria are as follows: 0= No neurologic symptoms, fully active at home/work without assistance  
1= Minor neurologic symptoms, fully active at home/work without assistance 2= Moderate neurologic symptoms, fully active at home/work but requires assistance 3= Moderate neurologic symptoms, less than fully active at home/work and requires assistance
4= Severe neurologic symptoms, totally inactive requiring complete assistance at home or institution, unable to work
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6.4 toxicity criteria

The following adverse event management guidelines are intended to ensure the safety of each patient while on the study. The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 4.0. A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm\#ctc_40).
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7 nih reporting requirements/data and

Please refer to definitions provided in Policy 801: Reporting Research Events found here.
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7.2 ohsrp office of compliance

Please refer to the reporting requirements in Policy 801: Reporting Research Events and Policy 802 Non-Compliance Human Subjects Research found here. Only IND Safety Reports that meet the definition of an unanticipated problem will need to be reported per these policies.  
7.2.2 IRB Requirements for PI Reporting at Continuing Review Please refer to the reporting requirements in Policy 801: Reporting Research Events found here.
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7.3 nci clinical director reporting

Problems expeditiously reported to the OHSRP in iRIS will also be reported to the NCI Clinical Director. A separate submission is not necessary as reports in iRIS will be available to the Clinical Director. In addition to those reports, all deaths that occur within 30 days after receiving a research intervention should be reported via email to the Clinical Director unless they are due to progressive disease. To report these deaths, please send an email describing the circumstances of the death to Dr. Dahut at NCICCRQA@mail.nih.gov within one business day of learning of the death.
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7.4 nih required data and

The clinical research team will meet on a regular basis when patients are being actively treated on the trial to discuss each patient. Decisions about dose level enrollment and dose escalation if applicable will be made based on the toxicity data from prior patients. All data will be collected in a timely manner and reviewed by the principal investigator or a lead associate investigator. Events meeting requirements for expedited reporting as described in section **7.2.1** will be submitted within the appropriate timelines. The principal investigator will review adverse event and response data on each patient to ensure safety and data accuracy. The principal investigator will personally conduct or supervise the investigation and provide appropriate delegation of responsibilities to other members of the research staff.
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8 sponsor safety reporting 8.1

Any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product and which does not necessarily have a causal relationship with this treatment. An adverse event (AE) can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal (investigational) product, whether or not related to the medicinal (investigational) product (ICH E6 (R2))
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8.1.2 serious adverse event (sae)

An adverse event or suspected adverse reaction is considered serious if in the view of the investigator or the sponsor, it results in any of the following:
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death

A life-threatening adverse event (see section **8.1.3**) Inpatient hospitalization or prolongation of existing hospitalization  
o A hospitalization/admission that is pre-planned (i.e., elective or scheduled surgery arranged prior to the start of the study), a planned hospitalization for pre-existing condition, or a procedure required by the protocol, without a serious deterioration in health, is not considered a serious adverse event.  
o A hospitalization/admission that is solely driven by non-medical reasons (e.g.,
hospitalization for patient convenience) is not considered a serious adverse event.  
o Emergency room visits or stays in observation units that do not result in admission to the hospital would not be considered a serious adverse event. The reason for seeking medical care should be evaluated for meeting one of the other serious criteria.  
Persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions A congenital anomaly/birth defect. Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a serious adverse drug experience when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition.
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8.1.3 life-threatening

An adverse event or suspected adverse reaction is considered "life-threatening" if, in the view of either the investigator or sponsor, its occurrence places the patient or subject at immediate risk of death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a more severe form, might have caused death. (21CFR312.32)
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8.1.4 severity

The severity of each Adverse Event will be assessed utilizing the CTCAE version 4.0.  
8.1.5 Relationship to Study Product All AEs will have their relationship to study product assessed using the terms: related or not related.  
Related - There is a reasonable possibility that the study product caused the adverse event. Reasonable possibility means that there is evidence to suggest a causal relationship between the study product and the adverse event.  
Not Related - There is not a reasonable possibility that the administration of the study product caused the event.
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8.2 assessment of safety events

AE information collected will include event description, date of onset, assessment of severity and relationship to study product and alternate etiology (if not related to study product), date of resolution of the event, seriousness and outcome. The assessment of severity and relationship to the study product will be done only by those with the training and authority to make a diagnosis and listed on the Form FDA 1572 as the site principal investigator or sub-investigator. AEs occurring during the collection and reporting period will be documented appropriately regardless of relationship. AEs will be followed through resolution. SAEs will be:  
Assessed for severity and relationship to study product and alternate etiology (if not related to study product) by a licensed study physician listed on the Form FDA 1572 as the site principal investigator or sub-investigator.  
Recorded on the appropriate SAE report form, the medical record and captured in the clinical database.  
Followed through resolution by a licensed study physician listed on the Form FDA 1572 as the site principal investigator or sub-investigator.
For timeframe of recording adverse events, please refer to section 6.1. All serious adverse events recorded from the time of first investigational product administration must be reported to the sponsor.
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8.3 reporting of serious adverse

Any AE that meets protocol-defined serious criteria or meets the definition of Adverse Event of Special Interest that require expedited reporting must be submitted immediately (within 24 hours of awareness) to OSRO Safety using the CCR SAE report form. All SAE reporting must include the elements described in section 8.2. SAE reports will be submitted to the Center for Cancer Research (CCR) at: OSROSafety@mail.nih.gov and to the CCR PI and study coordinator. CCR SAE report form and instructions can be found at: https://ccrod.cancer.gov/confluence/pages/viewpage.action?pageId=157942842 Following the assessment of the SAE by OSRO, other supporting documentation of the event may be requested by the OSRO Safety and should be provided as soon as possible.
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8.4 safety reporting criteria to

All events listed below must be reported in the defined timelines to OSROSafety@mail.nih.gov. The CCR Office of Regulatory Affairs will send all reports to the manufacturer as described below: Safety reports (subject to redaction, if required) sent to the FDA will be concurrently sent to the manufacturer:
Jillian Chapas-Reed Vice President, Clinical Operations Adastra Pharmaceuticals, Inc. Telephone: 410-920-2475 jchapas-reed@adastrarx.com www.adastrarx.com
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8.5 reporting pregnancy 8.5.1 maternal

If a patient becomes pregnant during the course of the study, the study treatment should be discontinued immediately, and the pregnancy reported to the Sponsor no later than 24 hours of when the Investigator becomes aware of it. The Investigator should notify the Sponsor no later than 24 hours of when the outcome of the Pregnancy become known, Pregnancy itself is not regarded as an SAE. However, congenital abnormalities or birth defects and spontaneous miscarriages that meet serious criteria (section **8.1.2**) should be reported as SAEs. The outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or congenital abnormality) should be followed up and documented.
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8.5.2 paternal exposure

Male patients should refrain from fathering a child or donating sperm during the study and for 30 days after the last dose of Zotiraciclib (TG02) Pregnancy of the patient's partner is not considered to be an AE. However, the outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or congenital abnormality) occurring from the date of the first dose until 30 days after the last dose should, if possible, be followed up and documented.
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8.6 regulatory reporting for studies

Following notification from the investigator, CCR, the IND sponsor, will report any suspected adverse reaction that is both serious and unexpected. CCR will report an AE as a suspected adverse reaction only if there is evidence to suggest a causal relationship between the study product and the adverse event. CCR will notify FDA and all participating investigators (i.e., all investigators to whom the sponsor is providing drug under its INDs or under any investigator's IND) in an IND safety report of potential serious risks from clinical trials or any other source, as soon as possible, in accordance to 21 CFR Part 312.32. All serious events will be reported to the FDA at least annually in a summary format.
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9 clinical monitoring plan

As a sponsor for clinical trials, FDA regulations require the CCR to maintain a monitoring program. The CCR's program allows for confirmation of: study data, specifically data that could affect the interpretation of primary and secondary study endpoints; adherence to the protocol, regulations, ICH E6, and SOPs; and human subjects protection. This is done through independent verification of study data with source documentation focusing on:
Informed consent process Eligibility confirmation Drug administration and accountability Adverse events monitoring Response assessment.  
The monitoring program also extends to multi-site research when the CCR is the coordinating center. This trial will be monitored by personnel employed by a CCR contractor. Monitors are qualified by training and experience to monitor the progress of clinical trials. Personnel monitoring this study will not be affiliated in any way with the trial conduct.