Phase II trial of cytarabine and mitoxantrone with devimistat in acute myeloid leukemia

Devimistat is a TCA cycle inhibitor. A previously completed phase I study of devimistat in combination with cytarabine and mitoxantrone in patients with relapsed or refractory AML showed promising response rates. Here we report the results of a single arm phase II study (NCT02484391). The primary outcome of feasibility of maintenance devimistat following induction and consolidation with devimistat in combination with high dose cytarabine and mitoxantrone was not met, as maintenance devimistat was only administered in 2 of 21 responders. The secondary outcomes of response (CR + CRi) and median survival were 44% (21/48) and 5.9 months respectively. There were no unexpected toxicities observed. An unplanned, post-hoc analysis of the phase I and II datasets suggests a trend of a dose response in older but not younger patients. RNA sequencing data from patient samples reveals an age-related decline in mitochondrial gene sets. Devimistat impairs ATP synthesis and we find a correlation between mitochondrial membrane potential and sensitivity to chemotherapy. Devimistat also induces mitochondrial reactive oxygen species and turnover consistent with mitophagy. We find that pharmacological or genetic inhibition of mitochondrial fission or autophagy sensitizes cells to devimistat. These findings suggest that an age related decline in mitochondrial quality and autophagy may be associated with response to devimistat however this needs to be confirmed in larger cohorts with proper trial design.

model derived from a 75 year old AML patient was injected into NSG mice which were then treated with vehicle, devimistat, metformin or the combination and followed for survival. For all viability assays a mean of three independent experiments each done in triplicate are shown. . *=p<0.05, **=p<0.01, ***=p<0.005, ****=p<0.001, ns=p>0.05. All error bars represent the standard deviation around the mean. Flow Cytometry Viable Cell Gating Strategy example:

Supplementary Note 1.
Complete Phase II Protocol

Proposed Mechanism of Action
CPI-613, the investigational drug, is a novel anti-tumor compound presumably with a novel mechanism of action that does not belong to any existing pharmacological class of anticancer agents currently used in the clinics. Specifically, CPI-613 is referred to as an Altered Energy Metabolism-Directed (AEMD) compound, and it is selective against tumor cells (but not normal cells) according to preclinical studies. The anti-tumor effects of CPI-613 are believed to be exerted by the inhibition of the pyruvate dehydrogenase complex (PDC).
In both normal and tumor cells, energy is derived from glucose, first by breaking down glucose to pyruvate via the glycolytic cycle in the cell cytoplasm. Once formed in mammalian cells, pyruvate is then converted by PDC to acetyl-CoA that is in turn metabolized via the tricarboxylic acid (TCA) cycle in the mitochondria.
Most tumor cells display profound perturbation of energy metabolism that tightly correlates with malignant transformation (Baggetto 1992). The PDC is regulated by the phosphorylation state of the E1 subunit. When phosphorylated by the PDC kinase the enzyme is inhibited and the conversion of pyruvate to acetyl-CoA is inhibited. Preclinical studies have shown that CPI-613 causes phosphorylation of the E1 subunit in tumor cells with little to no effect on normal cells. As a result of this phosphorylation ATP levels in tumors cells fall and cancer cell death ensues.
Consistent with the proposed mechanism is the fact that CPI-613 has been shown to have anti-tumor activity in cell culture and animal tumor models against diverse cancers independent of multiple drug resistance, cell cycle phase and activated signal transduction pathways. The novelty in the presumed mechanism of action for CPI-613 is further supported by the results from an ex vivo study demonstrating that CPI-613 is effective against various types of tumor cells excised from patients that were resistant to different anti-cancer drugs currently used in the clinics.
The significance of CPI-613 having a novel mechanism of action that is not shared by any existing pharmacological class of anti-cancer agents currently used in the clinics is that CPI-613 may be effective not only against naive tumors that have never been treated with any anti-cancer agents, but also effective against tumors that are resistant to anti-cancer agents currently used in the clinics. This is an important aspect because tumors frequently develop resistance to anti-cancer agents, rendering a shortage of treatment options. The availability of a novel anticancer agent would provide a new tool to treat cancer.

Background on Preclinical Studies
CPI-613 is being developed as an anti-tumor agent for the treatment of cancer. A number of preclinical anti-tumor efficacy studies; Administration, Distribution, Metabolism, and Elimination (ADME) studies; safety pharmacology studies; and toxicity studies of CPI-613 have been conducted. These studies are briefly described below, and the Investigator's Brochure provides detailed information regarding these preclinical studies.

Pharmacokinetic Studies
In rats, the toxicokinetic profile of CPI-613 administered 2x weekly for 3 weeks by IV administration at dose levels of 25, 30 or 35 mg/kg (i.e., 150, 180 or 210 mg/m 2 , respectively) was characterized by an apparent biphasic profilean initial distribution phase followed by an elimination phase. Systemic exposure was proportional to dose level, and there was no evidence of test article accumulation or sex differences in exposure. In minipigs, the toxicokinetic profile of CPI-613 administered 2x weekly for 3 weeks by IV administration at dose levels of 45, 42 and 46 mg/kg (i.e., 1575, 1470 and 1617 mg/m², respectively) was also characterized by an apparent biphasic profile.There was also no evidence of test article accumulation or sex differences in exposure.

ADME Studies
A PK study in minipigs showed that the log scale of the half-life (t1/2) values tended to be directly and linearly related to the log of the dose. Specifically, t1/2 was ~1hour at 3 mg/kg (105 mg/m²), ~1.7 hours at 10 mg/kg (350 mg/m²), and 10 hours at 30 mg/kg (1050 mg/m²).
The tissue distribution pattern of radioactivity, after an intravenous (IV) administration of 14 C-labeled CPI-613 in rats showed that: 1. The liver might be involved in the metabolism of CPI-613 and/or 14 Ccontaining metabolites of CPI-613. 2. Both the GI tract and the kidneys might be involved in the elimination of CPI-613 and/or 14 C-containing metabolites of CPI-613. 3. CPI-613 and 14C-containing metabolites of CPI-613 were mostly present in the extracellular space in the circulation.
An in vitro human plasma protein binding study showed that CPI-613 exhibited 99.7% binding to human plasma proteins, a value that was similar to warfarin. These results are consistent with findings from tissue distribution studies (see above) which showed CPI-613 was mostly present in the extracellular space in the circulation, consistent with findings of extensive plasma protein binding.
An in vitro human liver S9 fraction hepatocyte study showed that CPI-613 was oxidized to form a sulfoxide metabolite rapidly, with a half-life of <19 minutes. CPI-613 was also glucuronidated rapidly at the terminal carboxyl group, with a half-life of <15 minutes. Due to oxidation and glucuronidation, potential metabolites are a sulfoxide, a metabolite with glucuronidation in the terminal carboxyl group; and/or a metabolite with combination of sulfoxidation and glucuronidation.

In Vitro and Animal Pharmacology Safety Studies
An in vitro study showed that CPI-613 did not inhibit Ether-a-Go-Go-Related Gene (hERG) channels. Since hERG channel inhibition is the predominant cause of QTc interval prolongation which has been linked to Torsades de Pointes conditions such as sudden cardiac death and ventricular arrhythmia, this study suggested that CPI-613 may not induce Torsades de Pointes conditions. This is consistent with the subsequent animal toxicology studies showing that CPI-613 did not induce abnormal ECG pattern.
The potential influence of air in the dead space of an IV catheter on the acute toxicity of CPI-613 in rats has been evaluated. The results suggested that standard drug administration procedures in the elimination of dead air space within indwelling IV catheters should be followed, since air in the catheter air space may enhance the acute toxicity of CPI-613 at supra-antitumor doses.
The acute toxicity of CPI-613 administered IV as a bolus vs. by infusion has been compared in a preliminary study in rats. The results suggested that CPI-613 at supra-antitumor doses might be less toxic when administered as a 1-hr IV infusion than as a 1-min IV bolus.

Animal Toxicology Studies
Single Dose: A toxicology study involving a single IV administration of CPI-613 in mice has been conducted. The results indicated that 0.3-3 mg/kg (0.9-9 mg/m²) of CPI-613 did not induce any toxicity, whereas 10-100 mg/kg (30-300 mg/m²) also did not induce toxicity, except inflammatory reactions at or around the site of injection with intensity that was dose-related.
Twice Weekly for 1 Week Dosing: Animal toxicology studies involving IV administrations of CPI-613 twice weekly for 1 week have been conducted in mice and minipigs. In mice, CPI-613 at 100 mg/kg (300 mg/m²) induced mortality in all female mice but not male mice. Also, CPI-613 at 30 and 75 mg/kg (90 and 215 mg/m², respectively) did not induce any toxicity, except inflammation around the site of injection and a small increase in the spleen weight in mice treated with 75 mg/kg (215 mg/m²) and in the surviving mice treated with 100 mg/kg (300 mg/m²) of CPI-613.
In minipigs, when given 2x weekly for 1 week, CPI-613 at doses up to 30 mg/kg (1,050 mg/m²), the highest dose investigated in this study, did not induce toxicity, other than local inflammatory reactions at and around the site of injection. These results are consistent with those observed with the mouse study (see above).
Twice Weekly for 3 Weeks Dosing: Animal toxicology studies involving IV administrations of CPI-613 twice weekly for 3 weeks have been conducted in rats and minipigs, as described below. Significant systemic inflammation was also observed in rats treated with CPI-613, resulting in dose-dependent incidence of mortality at the toxicological doses of 30-35 mg/kg (180-210 mg/m²) and might be due to an over enhancement of inflammatory responses induced by toxic doses of CPI-613, as supported by dose-related exaggeration in inflammatory effects according to gross necropsy and histopathology findings (see below).

Overall Summary of Preclinical Studies
Preclinical efficacy studies, ADME studies, tissue distribution studies, and toxicology studies on CPI-613 have been conducted. The efficacy studies demonstrated the anti-tumor effects of CPI-613, whereas the ADME studies provided PK, biodistribution, metabolism and plasma protein binding information on CPI-613 in the body. Finally, for the toxicology studies, CPI-613 was administered in a way that mimicked that in the proposed clinical study, which involved IV administration given twice weekly. The results of the animal toxicology showed that CPI-613 is sufficiently non-toxic.

Background on Clinical Studies
Six single-patient physician-sponsored clinical studies have been conducted for compassionate use. Three of these single-patient studies were conducted in the US, whereas the other 3 were conducted in Israel.
Additionally, a Phase I clinical study involving CPI-613 as a single agent in patients with solid tumors and lymphomas is ongoing. A phase I clinical study involving CPI-613 as a single agent in patients with advanced hematologic malignancies is ongoing. A phase I study of CPI-613 in combination with high dose cytarabine and mitoxantrone has recently been completed. This trial established the MTD of CPI-613 as 2,500 mg/m 2 . A Phase I/II clinical study involving CPI-613 in combination with Gemcitabine is ongoing.

Rationale
Chemotherapy resistance is a major cause of death in patients with relapsed or refractory AML. CPI-613 can increase the sensitivity of leukemia cells to chemotherapy in petri dishes and mice. As a single agent in a phase I clinical trial several relapsed/refractory AML patients had an objective response. Additionally, a phase I study of CPI-613 in combination with high dose cytarabine and mitoxantrone has recently been completed. This trial established the MTD ofCPI-613 as 2,500 mg/m 2 . Preliminary analysis of the study revealed an overall complete remission or complete remission with incomplete count recovery rate of 45%. This compares favorably with our historical experience of this regimen without CPI-613. In this trial patients with poor risk cytogenetics had a 46% response rate, far superior to the 19% response seen with historical data. This trial established that CPI-613 can be safely administered at 2,500 mg/m 2 in this combination and results in superior response rates. The novel mechanism of action, non-cross resistance with chemotherapeutic agents, lack of CPI-613related myelosuppression clinically and increase response rate make this regimen a suitable candidate for the proposed pilot study.

Justification of the Dose of CPI-613
WFUCCC 22112, a phase I study of CPI-613 in combination with high dose cytarabine and mitoxantrone given exactly as proposed in the current study established the MTD of CPI-613 to be 2,500 mg/m 2 . A detailed analysis of the response data indicated that similar response rates were seen at the 2,000 mg/m 2 dose with less toxicity. Further studies have indicated a dose of 1500 mg/m 2 has a similar efficacy profile as 2,000 mg/m 2 but with even less toxicity. The rationale behind using a dose of 500mg/m 2 is supported by three lines of evidence: 1) preclinical evidence in mice suggesting lower doses may be more efficacious overtime than higher doses, 2) a traditional dose response effect of CPI-613 is not observed in AML patients thus far, and 3) a previous study in pancreatic cancer that showed remarkable synergy between CPI-613 at 500mg/m 2 in combination with standard chemotherapy.

Primary Objective
To determine the feasibility of CPI-613 when administered with high dose cytarabine, and mitoxantrone in all three phases of salvage therapy (induction, and maintenance). The regimen will be considered feasible if ≥50% of patients eligible for maintenance therapy complete at least 3 cycles.

Secondary Objectives
To observe the response rate (CR, and CRi) of CPI-613 in combination with high dose cytarabine and mitoxantrone.
To observe the overall survival of patients treated with CPI-613 in combination with high dose cytarabine and mitoxantrone in induction, consolidation and maintenance.
To monitor toxicities experienced by patients treated with CPI-613 in combination with high dose cytarabine and mitoxantrone in induction, consolidation and maintenance.

Patient Selection
This clinical trial can fulfill its objective only if patients appropriate for this trial are enrolled. All relevant medical and other considerations should be taken into account when deciding whether this protocol is appropriate for a particular patient. Physicians should consider the risks and benefits of any therapy, and therefore only enroll patients for whom this treatment is appropriate.

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

Registration Procedures
All patients entered on any CCCWFU trial, whether treatment, companion, or cancer control trial, must be linked to the study in EPIC within 24 hours of Informed Consent. Patients must be registered prior to the initiation of treatment.
You must perform the following steps in order to ensure prompt registration of your patient: To complete the registration process, the Registrar will:  assign a patient study number  assign the patient a dose  register the patient on the study

Treatment Plan
Patients recruited at 500 mg/m² will receive this dose for all treatment phases.
Salvage Induction Cycle 1: The dose of CPI-613 will be 500 mg/m²/day or 1,500 mg/m²/day given over 2 hours on days 1-5. Immediately following CPI-613 dosing 50 ml of D5W will be run through the central line. Cytarabine will be given following CPI-613 at 3gm/m2 for age <60 or 1.5 gm/m2 for age >60 in 500 mL NS over 3 hours every 12 hours for 5 doses starting on day 3. Mitoxantrone will be given at 6mg/m2 daily for 3 doses given in 50 mL NS over 15 minutes after 1st, 3rd and 5th doses of cytarabine.
Dose modifications for cytarabine and mitoxantrone are as follows (Please see section 6 for CPI-613 dose modifications): Hold Cytarabine and notify physician if:  Bilirubin 1.5-3: Consider Decrease dose by 25%.  Bilirubin greater than 3: Consider Decrease dose by 50%.  Cr Cl less than 60 mL/min: Consider dose reduction.  Hold mitoxantrone and notify physician if:  Bilirubin greater than 3: Consider Decrease dose by 25%.
All dose modifications are at the discretion of the treating physician.
Salvage Induction Cycle 2 (Optional, at the discretion of the treating physician): A repeat of the initial cycle as outlined above or at the discretion of the treating physician an abbreviated second cycle can be given. In the abbreviated course CPI-613 is given as above on days 1-3, cytarabine given as above for 3 doses starting on day 2, mitoxantrone given as above after the first and third cytarabine doses.
Salvage Consolidation: All responding patients are eligible for consolidation therapy with up to 2 cycles of the abbreviated course of CPI-613 at 500 mg/m²/day or 1,500 mg/m²/day, high dose cytarabine and mitoxantrone. Responding patients can be removed from trial at any time to receive a stem cell transplant at the discretion of the treating physician. Patients can move to maintenance following 1, 2 or no cycles of consolidation at the discretion of the treating physician.
Maintenance Therapy: Any patient that has completed all planned consolidation therapy (i.e. 0, 1 or 2 cycles) and refuses or is not eligible for a stem cell transplant may receive maintenance therapy with CPI-613. The dose for the maintenance phase of treatment in this study is 2,500 mg/m 2 except for patients who received 500 mg/m 2 at induction. Patients receiving 500 mg/m 2 at induction will continue with this dose during maintenance therapy. Patients may continue on maintenance therapy until evidence of disease progression, availability of stem cell transplant or occurrence of intolerable side effects. Creatinine, BUN  10  10  10  10  10  10  10  10 Anti-Tumor Efficacy (i.e., imaging for granulocytic sarcoma, bone marrow biopsy for AML) 3.7 Obtained within 2 weeks prior to the first dose.

Table of Study-Related Interventions
Obtained on day 14 of cycle 1.
Optional blood, urine and plasma samples, as well as bone marrow via biopsy 9 for possible testing of biomarkers, predictors of biological responses, toxicity, genotype vs. drug response relationship, etc.
Obtained within 2-4 weeks prior to the first dose.
Blood sample to be obtained prior to and following the dose of CPI-613 at 2, 4, and 6 hours from end of CPI-613 infusion on day 1 and bone marrow sample to be obtained on day 14 of Cycle 1. Bone marrow samples may be collected +/-1-2 days if falls on a weekend or holiday.
Optional urine samples will be collected from the first void after the first infusion of CPI-613. Details of the collection will be recorded on Appendix G. 1 Height, weight are also determined during pre-enrollment screening. 2 Renal function will be assessed utilizing the Cockcroft-Gault formula. 3 The Response Criteria will be assessed by standard criteria (Blood. 2010;115:453-474). 4 Vital signs will be performed immediately after CPI-613 administration and the patient only re-examined if clinically indicated. 5 These tests are performed within 24 hrs prior to dosing. Only the results of the creatinine are needed before CPI-613 is dosed. 6 For Day 1 of Treatment Cycle 1, the pre-enrollment test results can be used for all labs except creatinine which must be rechecked 24 hours prior to dosing. 7 The frequency of assessment beyond Cycle 1 is: -For medical history, physical exam and vital signs, ECOG PS, evaluation of symptoms and medications, they are assessed within 5 days of the next cycle doses.
-Clinical chemistry and hematology are assessed on day 5 of each cycle (or day 3 if short dosing cycle is given). 9 Bone Marrow via biopsy will only be collected if deemed appropriate by physician 10 Creatinine and BUN to be checked within 24 hours of every dose and at 24, 48 and 72 hours post last dose of CPI-613, mitoxantrone, and cytarabine (i.e., days 6, 7, and 8) 11 Coagulation (PT/PTT) to be completed Day 1 and weekly during Cycle 1. Obtained upon completion of all planned consolidation therapy.

Assessments
Optional blood and plasma samples, as well as bone marrow via biopsy 9 for possible testing of biomarkers, predictors of biological responses, toxicity, genotype vs. drug response relationship, etc.
Blood sample to be obtained prior to and following the dose of CPI-613 at 2, 4, and 6 hours from end of CPI-613 infusion on day 1 and bone marrow sample to be obtained at the end of planned consolidation therapy. Bone marrow samples may be collected +/-1-2 days if falls on a weekend or holiday. 1 Height, weight are determined during pre-enrollment screening. 2 Renal function will be assessed utilizing the Cockcroft-Gault formula. 3 The Response Criteria will be assessed by standard criteria (Blood. 2010;115:453-474). 4 Vital signs will be performed immediately after CPI-613 administration and the patient only re-examined if clinically indicated. 5 These tests are performed within 24 hrs prior to dosing. Only the results of the creatinine are needed before CPI-613 is dosed. 7 The frequency of assessment is: -For medical history, physical exam and vital signs, ECOG PS, evaluation of symptoms and medications, they are assessed within 5 days of the next cycle doses. 9 Bone Marrow via biopsy will only be collected if deemed appropriate by physician 10 Creatinine and BUN to be checked within 24 hours of every dose of CPI-613 11 3.7 Obtained upon completion of every fourth cycle.
Optional blood and plasma samples, as well as bone marrow via biopsy 9 for possible testing of biomarkers, predictors of biological responses, toxicity, genotype vs. drug response relationship, etc.
Blood sample to be obtained prior to and immediately following the dose of CPI-613 on day 1and bone marrow sample to be obtained at the end of planned maintenance therapy. Bone marrow samples may be collected +/-1-2 days if falls on a weekend or holiday. 1 Height, weight are determined during pre-enrollment screening. 2 Renal function will be assessed utilizing the Cockcroft-Gault formula. 3 The Response Criteria will be assessed by standard criteria (Blood. 2010;115:453-474). 4 Vital signs will be performed immediately after CPI-613 administration and the patient only re-examined if clinically indicated. 5 These tests are performed within 24 hrs prior to dosing. Only the results of the creatinine are needed before CPI-613 is dosed. 7 The frequency of assessment for cycles after cycle 1 is: -For medical history, physical exam and vital signs, ECOG PS, evaluation of symptoms and medications, they are assessed within 7 days of the next cycle doses. 9 Bone Marrow via biopsy will only be collected if deemed appropriate by physician. 10 Creatinine and BUN to be checked within 24 hours of every dose of CPI-613.

Pre-Enrollment Medical Screening
Informed consent must be obtained prior to pre-enrollment medical screening. Investigators who are listed on US "Food and Drug Administration (FDA) Form 1572" or Canadian "Clinical Trial Site Information Form" are authorized to obtain informed consent. Pre-enrollment medical screening is used to determine the eligibility of each candidate. All enrollment evaluations must be performed within the time frame listed below, prior to CPI-613 treatment and include: Within 2 weeks: -Bone marrow biopsy or cytologic evidence of relapsed or refractory AML or granulocytic sarcoma -Optional blood, plasma and bone marrow biopsy sample for biomarker evaluation -a complete medical history -physical exam, including vital signs, height, and weight -ECOG PS -baseline evaluation of symptoms and medications -clinical chemistry (including renal function), hematology and coagulation (see Section 5.6 for specifics) Within 1 week: -a pregnancy test for women of child-bearing potential (performed within 1 week prior to CPI-613 treatment)

CPI-613 Dosing
This is a Pilot trial to determine the feasibility of CPI-613 given with high dose cytarabine and mitoxantrone in salvage induction, consolidation and maintenance. This is an open-label study, and investigators and subjects are not blinded to the treatment. The reason this is an open-label study is because this is a feasibility trial, and the investigators need to determine whether this regimen is feasible to be tested in larger clinical trials. The assignment of patients will not be randomized, since this is a feasibility trial. The doses of CPI-613 for the induction and consolidation phases of treatment in this study are 500 mg/m 2 or 1,500 mg/m 2 with 20 patients in each cohort for both 500 and 1,500 the study has already accrued 17 patients at 2,000 mg/m 2 cohort; the dose for the maintenance phase of treatment in this study is 2,500 mg/m 2 except for patients who received 500mg/m 2 at induction. Patients receiving 500mg/m 2 at induction will continue with this dose for all phases of therapy including maintenance.
CPI-613 must be diluted from 50 mg/mL to 12.5 mg/mL with D5W (i.e., 1 portion of CPI-613 diluted with 3 portions of D5W) and must be administered IV at a rate of ~0.5 mL/min via a central venous catheter with D5W running at a rate of about 125-150 mL/hr. Please refer to Section 5.4 for methods of administering CPI-613.

CPI-613 Administration Guidelines
Since information on the use of CPI-613 in humans is limited, investigators should be aware of potential AEs and toxicities observed in preclinical studies, as described below.
According to animal toxicology studies, IV administration of CPI-613 can induce reactions at and around the site of administration. These reactions are consistent with local inflammation, which include red/purple/black color, swelling, necrosis, warm to touch, etc. These local reactions occurred more severely when there was accidental leakage of CPI-613 into the perivascular space during IV administration. This is possibly because of low perfusion in the perivascular areas, causing prolonged exposure of local tissues to CPI-613. Therefore, care must be taken to avoid perivascular leakage of CP-613.
CPI-613 must be administered IV by infusion (not bolus), via a central venous catheter that is free flowing, with D5W running at a rate of about 125-150 mL/hr, and free of air in the dead space of the IV catheter. This is to minimize vascular irritation, inflammation and acute toxicity of CPI-613. Accidental coadministration of extra air that is present in the dead space of an IV catheter has been shown to induce excessive acute toxicity of CPI-613 according to animal studies (Study NCL-049).
CPI-613 must be diluted prior to administration, as described in Section 5.3. Dilution of CPI-613 prior to administration reduces the rates of delivery of CPI-613 to the body, thus minimizing the acute toxicity of CPI-613.
Accordingly, the following procedures in administering CPI-613 should be taken to minimize local reactions and acute toxicity of CPI-613: A. Confirmation of the placement of the IV line to ensure that there is a lack of leakage of CPI-613 into the perivascular space. B. Confirmation that the IV line is free flowing and with D5W running at a rate of about 125-150 mL/hr. C. Confirmation that the IV line is free of air in the dead space. D. If necessary, rotate the site of administration for different doses of CPI-613. E. Dilute CPI-613 drug product with D5W, as instructed in the study protocol.
F. Administer CPI-613 by infusion, not bolus, as instructed in the study protocol. G. Upon completion of administering CPI-613, "slowly" flush the IV line with ~10 mL of D5W to remove residual CPI-613.
Investigators should be aware of the following toxicity observed in animals treated with toxic doses of CPI-613: 1. CPI-613 at the toxicological doses of 30-35 mg/kg (180-210 mg/m²) induced dose-related incidence of mortality in rats. The cause of death in most rats was due to significant systemic inflammation, and might be related to an over enhancement in the inflammatory responses induced by CPI-613. Deaths due to significant systemic inflammation were also observed in minipigs treated with the minipig toxicological dose of 46.2 mg/kg (i.e., 1617 mg/m²) of CPI-613. Additionally, CPI-613 at 30 and 35 mg/kg (i.e., 180 and 210 mg/m², respectively) in minipigs increased reticulocyte levels, which appeared to be associated with hematopoietic cell proliferation of the spleen, and sternal bone marrow hyperplasia that were secondary to inflammation initiated at the administration site or port. Therefore, investigators should be aware of significant increases in reticulocyte levels, which can be used as an indication of severe inflammation.

General Concomitant Medication and Supportive Care Guidelines
Patients will receive standard prophylactic treatment for drug-related symptoms. Supportive treatments may include anti-emetic, anti-diarrhea, anti-allergic, antihypertensive medications, analgesics, antibiotics, allopurinol, and others such as blood products and bone marrow growth factors. The treating physician may utilize marrow growth factors, or blood or platelet transfusions at their discretion. For patients with an ANC ≥ 500/mm3 at the time of CPI-613 administration loperamide can be administered as a premedication at a dose of 2 to 4 mg PO 30 minutes prior to CPI-613.

Study Procedures -Assessment of Safety, Plasma Concentration, and Possible Anti-Tumor Efficacy
Safety Assessment: The safety of CPI-613 will be assessed based on: -physical exams -vital signs -clinical pathology • clinical chemistry • renal function • hematology For physical exams and vital signs, they are to be performed during screening (performed within 2 weeks prior to treatment with CPI-613) and after CPI-613 administration in Cycle 1. Beyond Cycle 1, physical exam and vital signs will be assessed within 5 days of the next cycle doses.
For clinical pathology (i.e., clinical chemistry, renal function, hematology and coagulation), they are to be performed during screening (performed within 2 weeks prior to treatment with CPI-613), and within 24 hrs prior to dosing. Creatinine results within the last 24 hours must be reviewed prior to CPI-613 dosing. For Day 1 of Treatment Cycle 1, pre-dose assessments of clinical pathology are not needed because the pre-enrollment results can be used. Beyond cycle 1, laboratory assessments will be assessed on at least day 1 and 5 of each cycle rather than prior to beginning each cycle.
For left ventricular ejection fraction assessments, they are evaluated during screening and prior to consolidation therapy. Additional assessments can be made at the discretion of the treating physician.

Anti-Tumor Efficacy -A Secondary Endpoint:
Baseline anti-tumor efficacy (i.e., bone marrow biopsy) will be obtained within 2 weeks prior to the first dose of treatment cycle 1. Follow-up anti-tumor efficacy will be obtained on day 14 of cycle 1. In patients treated with additional treatment cycles beyond Cycle 1, anti-tumor efficacy will be assessed upon count recovery (defined as ANC>1000, freedom from RBC transfusions and platelets>100k) or day 42 from the first day of the cycle whichever comes first. Anti-tumor efficacy assessment will also be based on investigator and institutional routine monitoring of the disease progression of the patients, together with baseline evaluations performed prior to treatment with CPI-613. During maintenance therapy anti-tumor efficacy will be assessed after every 4 th cycle.
Optional Sampling and "Banking" of Blood, Plasma, urine and Bone Marrow Biopsy Samples: Optional blood samples (5 mL), urine (5-10 ml) as well as bone marrow via biopsy (if deemed appropriate by physician), will be obtained and "banked" for possible testing of biomarkers, predictors of biological responses, toxicity, relationship between genotype and drug responses, etc. These samples will be obtained prior to treatment initiation, as well as prior to and following administration of CPI-613 during salvage Cycle 1. The specific procedures are outlined below.
Whole Blood Sample • One (5 mL) Lavendar Top K EDTA Vacutainer Tube will be used. • Do not centrifuge or freeze sample.
• Samples will be shipped on ice pack to the address shown at the bottom of this section. Fresh Bone Marrow Biopsy for Banking of Tumor Cells • Samples will be collected into a green top lithium heparin tube. Harvested fresh tumor specimens are placed on ice and shipped to the address below.
(Note the tumor bank will Ficoll separate mononuclear cells and freeze aliquots in RPMI media containing 10% DMSO.) Urine Samples Urine samples will be collected from the first void after the first infusion of CPI-613 in the first induction cycle only. Details of the collection will be recorded on Appendix G.  Approximately 5-10 ml of urine will be collected in a 15 mL conical tube  The tube will placed at 4°C until placed into the Tumor Tissue Core Facility (see below) All optional samples and specimens should be shipped to:

ECOG PS:
The ECOG performance status (see Table below) will be used to classify patient's functional impairment. The higher the score, the worse the survival for most serious illnesses including AML.

ECOG Performance Scale (EPS) Score Definition 0
Fully active, able to carry on all pre-disease performance without restriction 1 Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature, e.g., light house work, office work. 2 Ambulatory and capable of all self-care but unable to carry out any work activities. Up and about more than 50% of waking hours.

3
Capable of only limited self-care, confined to bed or chair more than 50% of waking hours. 4 Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair. 5 Dead.
Lab Studies: Clinical chemistry assessed includes the following parameters. Renal function will be assessed utilizing the Cockcroft-Gault formula.

Future Research
Optional blood samples will be collected before and at various times after the 1st dose of CPI-613. Provisions should be made to record and report the actual clock times when samples are drawn. Whenever possible samples should be taken 5 minutes before and at completion of the infusion of CPI-613 on day 1 and at 2, 4 and 6 hours after the completion of the infusion. Blood (5 mL for each sample) should be obtained from an IV catheter that is not used for administration of CPI-613. The blood samples should be collected in 5-mL lavender top collection tubes containing K2-or K3-EDTA anticoagulant. Immediately after blood collection, gently invert tube 3 times to ensure proper mixing of blood and anticoagulant. Blood should then be immediately placed at 4 degrees Celsius. Additionally, several milliliters of urine from the first void following completion of the CPI-613 infusion on day one will be collected in consenting subjects. Once collected 5-10 ml of urine should be placed in a 15 ml conical tube and placed at 4 degrees Celsius until it can be sent to the tumor tissue core facility (see below).

Criteria for Removal from Study
Listed below are criteria for the discontinuation of the study. However, patients who fail to return for the follow-up visits will be contacted and queried as to the reason they have failed to complete the study with special attention to health status.
A. Patients have the right to withdraw from the study at any time for any reason.
B. The investigator has the right to withdraw patients from the study according to his/her discretion, if the investigator determines that continued participation is not in the patient's best interest. As an excessive rate of withdrawals can render the study not interpretable, unnecessary withdrawal of patients should be avoided. When a patient discontinues investigational treatment, the investigator should make every effort to contact the patient and to perform a final evaluation. The reason(s) for withdrawal must be recorded. Criteria for terminating subject's participation in the study are listed below: -Protocol defined disease progression significantly greater than expected -Unacceptable toxicity of the investigational product -Patient withdrawal of consent -Investigator's discretion -Undercurrent illness: a condition, injury, or disease unrelated to the intended disease for which the study is investigating, that renders continuing the treatment unsafe or regular follow-up impossible -General or specific changes in the patient's condition that renders the patient ineligible for further investigational treatment -Non-compliance with investigational treatment, protocol-required evaluations or follow-up visits -Termination of the clinical trial by the sponsor

Dosing Delays/Dose Modifications for CPI-613
The occurrence of Grade 1 toxicity does not generally require dose modification for subsequent doses for that patient. However, if Grade 2 non-hematologic, non-infectious toxicity develops attributed as at least probably related to CPI-613, treatment can resume only after the Grade 2 toxicity has been reduced to Grade 1 or below, and the dose level for subsequent doses for that patient will be reduced by 25% of the dose at which such Grade 2 toxicity occur. If Grade 3 or 4 non-hematologic, non-infectious toxicity develops, dosing of that patient will be withheld and the patient shall be monitored for recovery from, and reversibility of, such Grade 3 or 4 toxicity. To resume treatment for a patient who has had Grade 3 or 4 toxicity, the Grade 3 or 4 toxicity must be reduced to Grade 1 or below, and the dose level for subsequent doses for that patient will be reduced to 50% of the dose at which such Grade 3 or 4 toxicity occur. If the reduced dose of CPI-613 results in no toxicity, patients may be dose escalated to the prior dose at the discretion of the treating physician.
Dose adjustments during maintenance therapy will be done as described above with the addition that should patients experience hematologic toxicity ≥ Grade 2 attributed to CPI-613 the next dose of maintenance therapy must be held until the Grade 2 toxicity has been reduced to Grade 1 or below, and the dose level for subsequent doses for that patient will be reduced by 25% of the dose at which such Grade 2 toxicity occurred. If Grade 3 or 4 hematologic toxicity develops, dosing of that patient will be withheld and the patient shall be monitored for recovery from, and reversibility of, such Grade 3 or 4 hematologic toxicity. To resume treatment for a patient who has had Grade 3 or 4 hematologic toxicity, the Grade 3 or 4 hematologic toxicity must be reduced to Grade 1 or below, and the dose level for subsequent doses for that patient will be reduced to 50% of the dose at which such Grade 3 or 4 hematologic toxicity occurred. If the reduced dose of CPI-613 results in no toxicity, patients may be dose escalated to the prior dose at the discretion of the treating physician.

Adverse Events List and Reporting Requirements
Given the severe nature of the disease and treatment modalities associated with this patient population, any adverse event lower than grade 3 during a patient's treatment AE evaluation period will not be part of data analysis or subject to expedited reporting.

Adverse Event Characteristics
 CTCAE term (AE description) and grade: The CTEP Active Version of the NCI Common Terminology Criteria for Adverse Events (CTCAE 4.0) will be utilized for AE reporting. The CTEP Active Version of the CTCAE is identified and located on the CTEP website at (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. All appropriate treatment areas should have access to a copy of the CTEP Active Version of CTCAE.  'Expectedness': AEs can be 'Unexpected' or 'Expected' (see Section 7.1 above) for expedited reporting purposes only.
 Attribution of the AE: -Definite -The AE is clearly related to the study treatment.
-Probable -The AE is likely related to the study treatment.
-Possible -The AE may be related to the study treatment.
-Unlikely -The AE is doubtfully related to the study treatment.
-Unrelated -The AE is clearly NOT related to the study treatment.

STRC SAE Reporting Requirements
The Safety and Toxicity Review Committee (STRC) is responsible for reviewing SAEs for CCCWFU Institutional studies as outlined in Appendix B. STRC currently requires that all unexpected grade 4 and all grade 5 SAE's on these trials be reported to them for review. This procedure is a part of the CCCWFU Data Safety Monitoring Plan that our institution has on file at the NCI. All CRM staff members assisting a PI in investigating, documenting and reporting an SAE qualifying for STRC reporting are responsible for informing a clinical member of the STRC committee as well as the entire committee via the email notification procedure of the occurrence of an SAE.

WFUHS IRB AE Reporting Requirements
Any unanticipated problems involving risks to subjects or others and adverse events shall be promptly reported to the IRB, according to institutional policy.
Reporting to the IRB is required regardless of the funding source, study sponsor, or whether the event involves an investigational or marketed drug, biologic or device. Reportable events are not limited to physical injury, but include psychological, economic and social harm. Reportable events may arise as a result of drugs, biological agents, devices, procedures or other interventions, or as a result of questionnaires, surveys, observations or other interactions with research subjects.
All members of the research team are responsible for the appropriate reporting to the IRB and other applicable parties of unanticipated problems involving risk to subjects or others. The Principal Investigator, however, is ultimately responsible for ensuring the prompt reporting of unanticipated problems involving risk to subjects or others to the IRB. The Principal Investigator is also responsible for ensuring that all reported unanticipated risks to subjects and others which they receive are reviewed to determine whether the report represents a change in the risks and/or benefits to study participants, and whether any changes in the informed consent, protocol or other study-related documents are required.
Any unanticipated problems involving risks to subjects or others occurring at a site where the study has been approved by the WFUHS IRB (internal events) must be reported to the WFUHS IRB within 7 calendar days of the investigator or other members of the study team becoming aware of the event.
Any unanticipated problems involving risks to subjects or others occurring at another site conducting the same study that has been approved by the WFUHS IRB (external events) must be reported to the WFUHS IRB within 7 calendar days of the investigator or other members of the study team becoming aware of the event.
Any event, incident, experience, or outcome that alters the risk versus potential benefit of the research and as a result warrants a substantive change in the research protocol or informed consent process/document in order to insure the safety, rights or welfare of research subjects.

Pharmaceutical Accountability
Drug accountability logs will be maintained for all investigative agents used under this protocol. These logs shall record quantities of study drug received and quantities dispensed to patients, including lot number, date dispensed, patient identifier number, patient initials, protocol number, dose, quantity returned, balance remaining, and the initials of the person dispensing the medication.

Description of CPI-613 Drug Product
CPI-613 is provided in 10-mL amber glass vials. Each 10-mL vial contains 10 mL of CPI-613 at a concentration of 50 mg/mL (in 1M triethanolamine [TEA, also called trolamine]), which is equivalent to 500 mg of CPI-613. The drug product of CPI-613 is a clear and colorless solution that is free of any particulate matter.

Handling of CPI-613
CPI-613 is an investigational drug and the toxicity in humans is not fully understood. All necessary precautions in handling potentially toxic chemicals must be strictly adhered to. Gloves and protective clothing must be worn when handling CPI-613. Avoid contact by all modes of exposure. If the solution contacts the skin, it must be washed immediately and thoroughly with soap and water. If the solution comes in contact with mucous membranes, the membranes must be flushed thoroughly with water. Spills should be picked up with absorbent material and the area must be washed at least 3 times with ethyl alcohol followed by water.
CPI-613 drug product is slightly photosensitive (Study# PHO-001). Therefore, after removal of CPI-613 drug product from the amber vials, CPI-613 drug product should be protected from excessive light before administration to patients.
If CPI-613 is to be transferred from one storage area to another, or is to be prepared for dosing, care must be taken to maintain appropriate product temperature.
UNDER NO CIRCUMSTANCES SHOULD CPI-613 BE FROZEN.

Reconstitution and Administration of CPI-613
CPI-613 must be diluted from 50 mg/mL to 12.5 mg/mL with 5% Dextrose Water or D5W (i.e., 1 portion of CPI-613 diluted with 3 portions of D5W) prior to administration. The diluted drug product should be visually inspected for clarity.
If haziness or precipitate is observed, do not use the diluted drug product for dosing. After dilution with sterile D5W, the solution is clear and has a pH of 8.4-8.8. The diluted CPI-613 drug product has been found to be stable for 24 hrs at room temperature and refrigeration temperature (Studies STA-010).
CPI-613 must be administered IV, via a central venous catheter that is free flowing, free of air in the dead space, and with D5W running at a rate of about 125-150 mL/hr. This is to minimize vascular irritation, inflammation and acute toxicity of CPI-613 (Study NCL-049). Accidental co-administration of extra air in the dead space of IV catheters during administration of CPI-613 has demonstrated the potential to induce acute toxicity of CPI-613 according to animal studies (Study NCL-049). Also, accidental leakage of CPI-613 into the perivascular space during IV administration, which prolongs exposure of perivascular tissue to CPI-613, can induce significant local inflammation according to animal studies (Studies NCL-027 and NCL-030).
CPI-613 must not be administered as a bolus, but by infusion via a central venous catheter, over a total of 2 hours. All doses of CPI-613 will be administered over 2 hours to simplify PK/PD analysis. The infusion will also minimize potential acute toxicity of CPI-613, according to animal studies (Study NCL-049). The following precautions must be taken when administering CPI-613: A. Confirmation of the placement of the IV line to ensure a lack of leakage of CPI-613 into the perivascular space. B. Confirmation that the IV line is free flowing, and with D5W running at a rate of about 125-150 mL/hr. C. Confirmation that the IV line is free of dead air space. D. Rotate the site of administration for different doses of CPI-613. E. Dilute CPI-613 drug product with D5W, as instructed in the study protocol. F. Administer CPI-613 by infusion, not as a bolus. G. After administration of CPI-613, "slowly" flush the IV line with ~10 mL of D5W to remove residual CPI-613.

Request for CPI-613
CPI-613 must be requested from Rafael by the Principal Investigator (or authorized designees). CPI-613 may not be used outside the scope of this protocol, nor can it be transferred or licensed to any party not participating in this clinical study. Rafael policy requires that CPI-613 be shipped directly to the institution where the patient is to be treated. Rafael does not permit the transfer of CPI-613 between institutions (unless with prior written approval from Rafael). Requests must be submitted to Rafael by fax or email to the following address: The following information must be provided in the request of CPI-613 from Rafael: -Names of the principal investigator and the requestor (if different) -Name of the study site -Name of the pharmacist responsible for receiving and storing CPI-613 -Name of the person and address where CPI-613 is to be shipped to -Amount (# vials) requested -Date of request -Date shipment expected -Study Protocol (title and protocol#) for which the requested CPI-613 is to be used

Procurement of Investigational Drug
Relevant regulations require investigators to establish a record of the receipt, use and disposition of all investigational products. Investigators may delegate responsibility of drug ordering, storage, accountability and preparation to their designees.
The investigator, or the designee, will be responsible for dispensing and accounting of CPI-613 provided by Rafael and for exercising accepted medical and pharmacy practices.
Records of inventory, dispensation and disposition (vials received, source and dates) must be maintained. In addition, all doses dispensed should be accounted for by recording the date, study number and name, patient identification, patient initials, patient medical record number and balance forward. These records must be maintained and kept at the study site, and will be reviewed by Rafael, or its designee, during periodic monitoring visits.

Disposal of CPI-613
The following procedures are to be taken in disposal of CPI-613: -During the study, store the used CPI-613 vials (which must be separate from the unused CPI-613 vials) at room temperature in an access-limited area. Alternatively, destroy the used CPI-613 vials according to institutional policy after documentation of the number of used CPI-613 vials and remaining volume in each used vial.

Antitumor Effect
Although response is not the primary endpoint of this trial, patients with measurable disease will be assessed by standard criteria for AML (Blood. 2010;115:453-474). Peripheral blood (PB) cell counts and samples of bone marrow aspirates will be assessed for changes in disease status. Hematologic improvement (HI) requires improvement in one of the three hematopoietic lineages compared with baseline measurements, and stable disease (SD) requires the absence of objective disease progression for at least 2 months. Relevant responses have to be confirmed by a repeat measurement at least 1 wk later. Assessments will be done as described above.

Definitions
Evaluable for toxicity. All patients will be evaluable for toxicity from the time of their first treatment with CPI-613. Evaluable for objective response. Only those patients who have measurable disease present at baseline, have received at least one cycle of therapy, and have had their disease re-evaluated will be considered evaluable for response. These patients will have their response classified according to the definitions stated above. (Note: Patients who exhibit objective disease progression prior to the end of cycle 1 will also be considered evaluable.) Overall Survival (OS): Time from enrollment on trial to death from any cause.

Statistical Considerations
This is a pilot trial investigating three doses of CPI-613 (500, 1500, and 2,000 mg/m 2 . We plan to accrue between 10 and 15 patients per year for a total of 67 patients (20 at 500, 1,500 mg/m 2 and 3 additional patients at 2,000 mg/m 2 ). Of note 17 patients at 2,000 mg/m 2 and 7 patients at 2,500 mg/m 2 were already accrued.
Analysis Plan: The primary objective of determining the feasibility of administering CPI-613 in combination with high dose cytarabine and mitoxantrone during induction, consolidation and maintenance therapies will be completed by determining the percentage of patients eligible for maintenance therapy who complete at least 3 cycles. If ≥50% of eligible patients complete 3 cycles of maintenance therapy we will consider this regimen feasible for future study. Given an estimated response rate of 50% and a 25% attrition to stem cell transplant, if we enroll 60 patients we should be able to assess feasibility of maintenance in approximately 22 patients. With 22 evaluable patients, a two-sided 95.0% confidence interval using the large sample normal approximation will extend 22% in either direction from the observed value when the expected value is 50%. Within a dose we should be able to estimate feasibility in 7 patients with a 95% confidence interval extending 36% in either direction.
Participants will also be followed and analyzed for secondary outcomes of response rate and overall survival both overall and by dose. Participants will be monitored for survival through routine follow-up visits. Confidence intervals will be calculated around the estimates of the response rate (CR and CRi). Assuming a response rate of 0.5, with 60 participants, we would be able to create 95 percent confidence intervals with a 0.13 margin of error (0.37, 0.63). We will use Kaplan-Meier estimation to analyze overall survival. The frequency of toxicities experienced by the participants will be presented by type and grade in an effort to monitor and report safety of the treatment.
In exploratory analysis we plan to compare both complete response and early mortality (death within 60 days of beginning of treatment) to the observed rates in a historical cohort of subjects (CCCWFU 22111), both overall and by dose. In the historical cohort the response rate was estimated to be 41%. Using a onesided chi-square test, a 0.05 significance level, null hypothesis of 41%, and assuming 70% power, we would be able to show significant improvement in response if we are able to achieve a response rate of 56% or higher. Early mortality in the historical cohort was estimated to be 29% at 60 days. We will use a one-sided exact test for a single proportion, a 0.05 significance level, a null hypothesis of 29%, and 70% power. To show a significant decrease in early mortality in this study we would need to observe a 60 day mortality rate of 16.8% or lower.