A Pilot Study to Assess Lactate and Bicarbonate Detection Within Malignant Brain Tumors Using [1-13C]-Pyruvate DNP Magnetic Resonance Spectroscopy (MRS)
Phase: PHASE1
Conditions: Malignant Central Nervous System Neoplasm, Metastatic Malignant Neoplasm in the Central Nervous System
Sponsor: Daniel M. Spielman

 
 
 
 
A Pilot Study to Assess lactate and bicarbonate detection within Malignant Brain 
Tumors using [1-13C]-pyruvate DNP Magnetic Resonance Spectroscopy (MRS) 
Study Protocol and Statistical Analysis Plan 
NCT03565367 
May 15, 2019 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 1 of 18 
CONFIDENTIAL 
A Pilot Study to Assess lactate and bicarbonate detection within Malignant Brain 
Tumors using [1-13C]-pyruvate DNP Magnetic Resonance Spectroscopy (MRS) 
 
Protocol Director 
Lawrence Recht MD 
Phone: 650 725-8630 
Fax: 650 498-4686 
LRecht@stanford.edu 
 
Dept of Neurology, 
875 Blake Wilbur Drive, 
 
Palo Alto 94305 
 
 
Co-Investigators 
Daniel Spielman PhD 
Phone: 650 723-8697 
Fax: 650 723-9222 
spielman@stanford.edu 
 
 
Frederick T. Chin, Ph.D. 
Phone: 650 725-4182 
Fax: 650 618-0415 
chinf@stanford.edu 
 
 
Bin Shen Ph.D. 
Phone: 650-736-2844 
Fax: 650 618-0415 
binshen@stanford.edu 
 
 
Lucas Center for Medical Imaging, 
1201 Welch Rd, 
 
Stanford, CA  94305 
 
 
 
Biostatistician 
None 
 
Study Coordinator 
  Phone:  
 
  Phone: 
 
                                   
 Phone: 
 
 
800 Welch Road, 
 
Palo Alto, CA  94305 
 
Study Coordinators’ Fax:  
 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 2 of 18 
CONFIDENTIAL 
TABLE OF CONTENTS 
TABLE OF CONTENTS ............................................................................................................... 2 
SCHEMA ...................................................................................................................................... 4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ........................................................ 5 
1. 
OBJECTIVES ....................................................................................................................... 6 
1.1. 
Primary Objectives ........................................................................................................ 6 
2. 
BACKGROUND .................................................................................................................... 6 
2.1. 
Study Disease ............................................................................................................... 6 
2.2. 
Study Agent/Device/Imaging procedure ........................................................................ 6 
2.3. 
Clinicaltrials.gov ............................................................................................................ 7 
2.4. 
Rationale ....................................................................................................................... 8 
2.5. 
Preliminary results ......................................................................................................... 8 
2.6. 
Study Design ................................................................................................................. 8 
3. 
PARTICIPANT SELECTION AND ENROLLMENT PROCEDURES ................................... 8 
3.1. 
Inclusion Criteria............................................................................................................ 8 
3.2. 
Exclusion Criteria .......................................................................................................... 8 
3.3. 
Informed Consent Process ............................................................................................ 9 
3.4. 
Study Timeline............................................................................................................... 9 
4. 
IMAGING AGENT/DEVICE/PROCEDURE INFORMATION ................................................ 9 
4.1. 
Hyperpolarized [1-13C]-Pyruvate ................................................................................... 9 
4.2. 
Dissolution DNP MRSI .................................................................................................. 9 
4.3. 
Dose Modification .......................................................................................................... 9 
5. 
STUDY PROCEDURES ...................................................................................................... 10 
6. 
STUDY CALENDAR ........................................................................................................... 11 
7. 
ADVERSE EVENTS AND REPORTING PROCEDURES .................................................. 12 
7.1. 
Potential Adverse Events ............................................................................................ 12 
7.2. 
Adverse Event Collection ............................................................................................ 12 
7.3. 
Adverse Event Reporting ............................................................................................ 12 
8. 
REGULATORY CONSIDERATIONS ................................................................................. 13 
8.1. 
Institutional Review of Protocol ................................................................................... 13 
8.2. 
Data Management Plan ............................................................................................... 13 
8.3. 
Data and Safety Monitoring Plan ................................................................................. 13 
9. 
MEASUREMENTS .............................................................................................................. 14 
9.1. 
Primary and Secondary Outcome measures .............................................................. 14 
9.2. 
Measurement Methods ................................................................................................ 14 
9.3. 
Measurement Time Points .......................................................................................... 14 
9.4. 
Response Review ....................................................................................................... 14 
10. 
STATISTICAL CONSIDERATIONS ............................................................................... 14 
10.1. Statistical Design ......................................................................................................... 14 
10.2. Randomization ............................................................................................................ 14 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 3 of 18 
CONFIDENTIAL 
10.3. Interim analyses .......................................................................................................... 14 
10.4. Descriptive Statistics and Exploratory Data Analysis .................................................. 14 
10.5. Primary Analysis .......................................................................................................... 15 
10.6. Sample Size ................................................................................................................ 15 
10.7. Accrual estimates ........................................................................................................ 15 
10.8. Criteria for future studies ............................................................................................. 15 
11. 
REFERENCES ................................................................................................................ 16 
Appendix A:  Inclusion/Exclusion Criteria Checklist............................................................. 18 
 
 
 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 4 of 18 
CONFIDENTIAL 
SCHEMA 
 
 
 
 
 
 
 
 
 
Safety Monitoring until Day 2 (+2) 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 5 of 18 
CONFIDENTIAL 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
AE 
Adverse event 
ALT 
Alanine aminotransferase 
AST 
Asparagine aminotransferase 
BIC 
Bicarbonate 
CBV 
Cerebral blood volume 
CNS 
Central nervous system 
CRF 
Case report form 
DCA 
Dichloroacetate 
DLT 
Dose Limiting Toxicity 
DNP 
Dynamic nuclear polarization 
DSMC 
Data Safety Monitoring Committee 
ECG 
Electrocardiogram 
EPA 
Electron paramagnetic agent 
FDA 
Food and Drug Administration 
GBM 
Glioblastoma multiforme 
GCP 
Good clinical practices 
GFR 
Glomerular filtration rate 
GGT 
Gamma-glutamyltransferase 
GLY 
Glycolysis 
IRB 
Institutional Review Board 
IV 
Intravenous 
LAC 
Lactate 
MRI 
Magnetic resonance imaging 
MRSI 
Magnetic resonance spectroscopy imaging 
MRS 
Magnetic resonance spectroscopy 
OXPHOS 
Oxidative phosphorylation  
PD 
Protocol Director 
PYR 
pyruvate 
SAE 
Serious adverse event 
SNR 
Signal to noise ratio 
SRC 
Scientific Review Committee 
T1 
Time constant for magnetization decay 
ULN 
Upper limit of normal 
VS 
Vital signs 
 
 
 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 6 of 18 
CONFIDENTIAL 
1. OBJECTIVES 
 
1.1. Primary Objectives 
To assess the safety of IV injection of hyperpolarized [1-13C]-pyruvate for MRI. 
 
1.2. Exploratory Objectives 
To assess the frequency and sensitivity with which lactate and bicarbonate signals can be 
detected in malignant brain tumors after IV injection of hyperpolarized [1-13C]-pyruvate. 
 
2. BACKGROUND 
 
2.1. Study Disease 
Our ultimate goal is to develop a strategy to manage cancer as a chronic disease for which 
treatment can be calibrated according to the tumor’s metabolic state.  In particular, cancer cells, 
in addition to their high growth rate, universally demonstrate an abnormal metabolism 
characterized by an overproduction of lactate (via a process known as glycolysis [GLY]) relative 
to the more energy efficient conversion of glucose to carbon dioxide and water (oxidative 
phosphorylation [OXPHOS]).  Originally described over 80 years ago by Otto Warburg, this 
altered metabolism, more recently termed metabolic reprogramming [1], is now viewed as a 
hallmark of cancer and appears linked to the need for carbon skeletons to produce biomass and 
is thus intimately tied to the ability of cancer cells to proliferate [2].  The novel clinical implication 
of these observations suggest that a potential anti-tumor strategy lies in reversing the “Warburg 
Effect” by forcing cancer cell metabolism away from GLY towards OXPHOS.  Although this 
would not result in cell kill per se, it should result in stabilization of growth with minimal toxicity.  
Although a number of targets exist for which active drugs could be directed, a major obstacle 
towards taking this to the clinic has been the inability to measure cancer metabolism in vivo.   
 
Using a rat model, we have developed magnetic resonance spectroscopic imaging (MRSI) 
of hyperpolarized [1-13C]-pyruvate as a noninvasive measure of lactate (Lac) and bicarbonate 
(Bic) production reflecting GLY and OXPHOS respectively [3], [4].  We now wish to translate this 
imaging technology to the clinic and demonstrate that these critical aspects of glucose 
metabolism can be measured in patients with glioma.  A hyperpolarizer that can be used 
clinically has been purchased and is now ready to be utilized.  We have funds in which to test 
15 subjects and would like to perform a pilot test to confirm two necessary aspects of our 
hypothesis: (i) determining that our methods for polarizing [1-13C]-pyruvate are safe for patient 
use; and (ii) evaluating if and how frequently we can detect lactate and bicarbonate signaling 
within brain tumor tissue. 
 
2.2. Study Agent/Device/Imaging procedure 
Dynamic nuclear polarization (DNP) magnetic resonance spectroscopic imaging (MRSI) is 
based on the transfer of polarization from the electron spins of paramagnetic centers embedded 
in a glassy frozen solution to neighboring nuclear spins through dipolar interactions, thus driving 
nuclei temporarily into a significant redistribution of the ordinary populations of energy levels [5].  
These spin states are not stable in the sense that the induced massive spin polarization decays 
during a relatively short period but within this period allows one to follow a nucleus (usually 
carbon or nitrogen) as it is processed by a tissue.  The absence of ionizing radiation and 
convenient integration with standard MRI imaging of hyperpolarized nuclei therefore offers the 
prospect of monitoring tumor metabolism noninvasively, the potential value of which depends on 
three factors: (i) the T1 signal needs to be sufficiently long so that enough material is preserved 
after injection to measure; (ii) the metabolic pathway itself much be sufficiently rapid so that the 
fate of the tracer can be measured; and (iii) the process must be relevant to tumor biology [6].   

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 7 of 18 
CONFIDENTIAL 
 
[1-13C]-pyruvate is currently the tracer for which the most preclinical and clinical experience 
exists.  Pyruvate (Pyr) is situated at a hub of the glucose utilization pathway where it can be 
reduced to lactate, amidated to alanine or catalyzed to acetyl CoA and CO2.  Although an issue 
existed early in its development as to whether it could cross the blood brain barrier fast enough 
to allow imaging in brain tumors without disruption, unequivocal imaging of brain tumor 
metabolism of hyperpolarized [1-13C]-pyruvate in brain has been demonstrated [4]. 
 
Signal to noise (SNR) ratios are particularly strong for visualizing lactate (Lac), on which 
most studies to date have focused.  Using this technology, it has been possible to show lactate 
dehydrogenase catalyzed interconversion of label between Pyr and Lac in several tumor types 
including GBM and that changes occur in the rate of labeling with treatment [4], [7]-[9].  
Furthermore, it has also been demonstrated that an increased conversion of Pyr conversion to 
alanine is an early feature of experimental myc-driven hepatic tumors [10].   
 
Based on numerous previous reports, tracing Pyr fate into the mitochondria has been more 
challenging and there have been numerous reports noting that imaging both lactate and 
bicarbonate (Bic) is difficult, especially in brain tumor [11], [12], creating barriers for using this 
imaging modality to explore the question of whether forcing OXPHOS slows cancer growth.   
 
We recently overcame this hurdle through optimization of the acquisition protocol for spiral 
chemical shift imaging so as to be able to measure both lactate and bicarbonate in transplanted 
C6 glioma.  Furthermore, we were able to demonstrate a consistent and marked decrease in the 
ratio of Lac/Bic acutely after the administration of DCA [8], substantiating at least that change in 
Pyr flux is occurring so that more substrate is directed into the mitochondria (since that is where 
Bic is generated). 
 
This technology has been recently applied to clinical populations.  The first-in-man Phase I 
trial examined a cohort of prostate cancer patients.  It demonstrated the safety of administering 
250 mM hyperpolarized [1-13C]-pyruvate up to doses of 0.43 mL/kg body weight and that 
images could be acquired that demonstrated conversion of hyperpolarized pyruvate to lactate in 
regions of biopsy-proven tumor [13].  Since these experiments were reported from UCSF, this 
technology has become available for clinical study at Memorial Sloan Kettering Cancer Center, 
University of Texas at Southwestern (UTSW), Cambridge University, Oxford University, 
University of Toronto, and several other centers (including ours) are gearing up for study. 
 
2.3. Clinicaltrials.gov  
This study is submitted to IND-
 for the IV injection of [1-13C]-pyruvate, and will 
include submission of this protocol.  
 
For clinicaltrials.gov compliance:  This is a phase 0 (early phase 1), but relies in part on NIH 
funding so is required to register on clinicaltrials.gov and report results.  For Clinicaltrials.gov 
results reporting, the following outcome measures will be used: 
Primary Outcome Measure  
• 
Title: Safety of hyperpolarized [1-13C]-pyruvate injection 
• 
Time Frame: Up to Day 4 
• 
Description: Results will be reported as the number of Grade 2 or higher adverse 
events judged by the investigator to be clinically significant AND related/possibly-
related to hyperpolarized [1-13C]-pyruvate injection across all subjects administered 
study agent. 
 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 8 of 18 
CONFIDENTIAL 
Exploratory Outcome Measure  
• 
Title: Feasibility of Lactate and Bicarbonate signal detection 
• 
Time Frame: Up to 4 days 
• 
Description: Results will be reported as the percentage of subjects with CNS 
malignancy administered study agent and imaged who show 13C MR signal detection 
above background noise level, reported separately for lactate and bicarbonate 
signals. 
 
2.4. Rationale 
See Section 2.1 
 
2.5. Preliminary results 
The proposed study will be the first time at Stanford where this technology will be used in 
humans.  Prior use in cancer imaging, conducted at UCSF, is described in the Investigator’s 
Brochure. 
 
2.6. Study Design 
This is an open-label, two-armed study of 5 healthy volunteers and 10 patients with known 
CNS malignancy. 
• 
Arm 1: n = 5 healthy volunteers 
• 
Arm 2: n = 10 patients with known CNS malignancy 
 
3. PARTICIPANT SELECTION AND ENROLLMENT PROCEDURES 
 
3.1. Inclusion Criteria 
3.1.1. (Arm 2 patients with known CNS malignancy only) Patients with a known 
diagnosis of CNS malignancy, including metastases, with known enhancement on 
MR who are otherwise eligible to undergo MRI. 
3.1.2. At least 18 years old. 
3.1.3. eGFR > 30 ml/min. 
3.1.4. (Arm 2 patients with known CNS malignancy only) No allergy to gadolinium. 
3.1.5. Ability to understand and the willingness to sign a written informed consent 
document. 
 
3.2. Exclusion Criteria 
3.2.1. Refusal to have an IV placed for injection. 
3.2.2. Acute major illness (e.g., unstable angina, etc.) or other condition that makes 
participation unsafe, per the investigator’s judgement. 
3.2.3. Total Bilirubin > 1.5 x ULN; AST/ALT > 2.5 x ULN; GGT > 2.5 x ULN. 
3.2.4. Pregnant or breast-feeding.   
3.2.5. Cardiovascular risk, including: 
o 
Poorly controlled hypertension, defined as either systolic > 170 or 
diastolic > 110. 
o 
Congestive heart failure 
o 
Myocardial infarction within the past year 
o 
QT prolongation, defined as pretreatment QTc > 440 msec in males or 
> 460 msec in females 
3.2.6. (Arm 1 Healthy volunteers only) Undergoing active treatment for malignancy 
 
 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 9 of 18 
CONFIDENTIAL 
3.3. Informed Consent Process 
All participants must be provided a consent form describing the study with sufficient 
information for participants to make an informed decision regarding their participation.  
Participants must sign the IRB approved informed consent prior to participation in any study 
specific procedure.  The participant must receive a copy of the signed and dated consent 
document.  The original signed copy of the consent document must be retained in the medical 
record or research file.   
 
 
3.4. Study Timeline 
3.4.1. Primary Completion 
The study will reach primary completion once the last subject undergoes Day 2 safety 
evaluations. 
 
3.4.2. Study Completion 
We estimate that this study will take one year from first subject enrollment to complete. 
 
4. IMAGING AGENT/DEVICE/PROCEDURE INFORMATION 
 
4.1. Hyperpolarized [1-13C]-Pyruvate 
13C is a stable non-radioactive isotope of carbon.  [1-13C]-pyruvate has exactly the same 
chemical characteristics as pyruvate.  In [1-13C]-pyruvate, the C-1 carbonyl has been enriched 
with 13C-nuclei, which has a magnetic moment and can be hyperpolarized by DNP in the 
presence of an electron paramagnetic agent (EPA), i.e., AH111501 sodium salt (a stable trityl 
radical).  Although [1-13C]-pyruvate has the same chemical characteristics as unlabeled 
pyruvate and is metabolized the same way, the fact that it can be hyperpolarized means that 
MRSI can be used to rapidly detect the hyperpolarized 13C-label in [1-13C]-pyruvate and its 
metabolic products. 
 
Hyperpolarized [1-13C]-pyruvate will be prepared by qualified staff at the Lucas Center under 
aseptic GMP/GLP conditions using a DNP polarizer (SpinLab, GE Healthcare).  The imaging 
agent will be administered by IV injection at a rate of 5 ml/sec followed by a 20 ml saline flush at 
5 ml/sec (on average 30-40 seconds).  The formulation procedures are detailed in the current 
Investigator’s Brochure (IB).   
 
4.2. Dissolution DNP MRSI 
Hyperpolarized [1-13C]-pyruvate will be injected at a dosage of 0.43 ± 0.05 mL/kg body 
weight (concentration of 250 mM), the maximum safely administered dose attained in the Phase 
I study [13].  After hyperpolarization in the SpinLab polarizer, a syringe is automatically filled.  
The quality control module of the SpinLab measures the pyruvate concentration, volume, 
temperature, pH, polarization, and removal of the EPA in the syringe.  A quality control 
assessment is then performed to ensure all parameters are reached, after which the syringe is 
released by the protocol director and immediately passed to a nurse or radiology technician for 
initiation of injection.   
 
The overall imaging protocol will consist of 1) standard non-contrast clinical collection setup 
for brain imaging (~45 min), 2) hyperpolarized [1-13C]-pyruvate MRSI (single- or multi-slice 
acquisition, ~3-min acquisition), and 3) standard clinical brain gadolinium-enhanced MRI 
(~10min). 
 
4.3. Dose Modification 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 10 of 18 
CONFIDENTIAL 
The starting dose of [1-13C]-pyruvate will be 0.43 ± 0.05 mL/kg body weight (250 mM 
[1-13C]-pyruvate).  If we encounter a DLT at this dose (which seems unlikely since it has been 
well tolerated in safety studies elsewhere), we will decrease dose to 0.28 mL/kg. 
 
The choice whether or not to use the [1-13C]-pyruvate and/or gadolinium MRI contrast agent 
is made at Protocol Director’s discretion.  If the investigator choses to forego gadolinium 
contrast (due to concerns of gadolinium accumulation or other concerns), the second MRI brain 
scan may be skipped. 
 
 
 
5. STUDY PROCEDURES  
Healthy volunteers (Arm 1 of the study) will be recruited via an IRB-approved e-mail that 
refers them to the clinical research coordinator or research team. Eligible patients for arm 2 will 
be invited to participate at any time during their course of CNS malignancy treatment.  
Generally, arm 2 patients will be recruited during a regular clinic visit. 
 
If the patient signs the informed consent form and agrees to participate, the following 
screening procedures will be performed within 14 days prior to Day 1: 
• 
Collect demographics 
• 
Measure vital signs (heart rate, temperature, blood pressure), height and weight 
• 
Serum chemistry blood test 
• 
Serum pregnancy test, if woman of childbearing potential 
• 
Electrocardiogram (ECG) 
 
On Day 1, MRI and MRSI will be performed in the Richard M. Lucas Center for Imaging at 
Stanford University.  Production of hyperpolarized [1-13C]-pyruvate will be coordinated with 
imaging schedule such that MRSI begins within 1 minute of hyperpolarized [1-13C]-pyruvate 
administration.  Each subject will undergo the following procedures: 
• 
Measure vital signs 
• 
MRI scan of brain, 45 min acquisition time using standard non-contrast clinical brain 
collection setup 
• 
Administration of hyperpolarized [1-13C]-pyruvate (250 mM, 0.43 ± 0.05 mL/kg body 
weight) IV injection at a rate of 5 mL/sec followed by a 20 mL saline flush at 5 
mL/sec (on average 30-40 seconds) 
• 
MRSI scan of brain, 3 min acquisition on a 3T MRI scanner equipped with both 1HF 
and 13C RF coils. 
• 
MRI scan of brain, 10 min acquisition time using standard Gadolinium-enhanced 
contrast clinical brain collection. Gadolinium contrast agent may or may not be 
administered at the discretion of the Protocol Director. Gadolinium will not be used 
for Arm 1 Healthy Volunteers.  
• 
Within 10 min after the conclusion of image collection: 
o Measure vital signs 
o Collect any adverse events and monitor injection site 
• 
At least once more  20-45 minutes after the conclusion of image collection: 
o Measure vital signs 
o Collect any adverse events and monitor injection site 
• 
1 to 5 hours post pyruvate administration: 
o Measure vital signs 
o ECG 
o Collect any adverse events and monitor injection site 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 11 of 18 
CONFIDENTIAL 
 
Between Day 2 and 4, subjects will be contacted by phone or at an in-person clinic visit to 
collect any adverse events. 
 
Once initial imaging is completed, patients will be followed as expected by standard of care.  
Patients are free to enroll in other studies.   
 
 
 
6. STUDY CALENDAR 
 
 
Pre-Study 
(Within 14 Days of 
Day 1) 
Day 1 
Day 2 
(+2 days) 
Informed consent 
 
X 
 
 
 
 
Demographics 
 
X 
 
 
 
 
Vital signs 
 
X 
Xf 
 
Height 
 
X 
 
 
 
 
Weight 
 
X 
 
 
Serum chemistry a 
 
X 
 
 
ECG 
 
X 
 
X e 
 
 
Pregnancy test 
 
X b 
 
 
 
 
Non-contrast enhanced MRI c 
 
 
 
X 
 
 
[1-13C]-pyruvate IV injection 
  
X  
 
 
MRSI 
 
 
 
X 
 
 
Contrast enhanced MRI d 
  
 
X 
 
AE evaluation 
 
 
 
X 
 
X 
 
a: GFR., Total bilirubin, SGOT[AST], SGPT[ALT], GGT. 
b: 
Women of childbearing potential only 
c: 
Before [1-13C]-pyruvate IV injection and MRSI 
d:    After [1-13C]-pyruvate IV injection and MRSI. Arm 1 Healthy Volunteers will not receive contrast 
e:  Within 1-5 hours post [1-13C]-pyruvate IV injection 
f:     Collect on Day 1: at baseline prior to non-contrast MRI;  within 10 min after the conclusion of the image 
collection, 20-45 min after the conclusion of image collection; and 1-5 hours post [1-13C]-pyruvate IV 
injection 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 12 of 18 
CONFIDENTIAL 
7. ADVERSE EVENTS AND REPORTING PROCEDURES 
 
7.1. Potential Adverse Events 
In the original Phase I studies with [1-13C]-pyruvate, no dose-limiting toxicities or toxicities 
deemed to be clinically significant were noted [13].  We will consider any Grade 2 or higher 
toxicity judged related or possibly-related to [1-13C]-pyruvate as significant (excluding 
asymptomatic lab abnormalities).   
7.1.1. Stopping Rules 
The study will be stopped if infection attributable to any component of the pyruvate infusion 
is observed.  The study may resume after the necessary chemistry, manufacturing and controls 
(CMC) changes are made and the IND amendment is submitted to FDA and approved by IRB. 
In addition, the study will pause enrollment pending a safety review if two dose-limiting toxicity 
events (any Grade 2 or higher toxicity judged related or possibly-related to [1-13C]-pyruvate, 
excluding asymptomatic lab abnormalities) are observed. 
 
7.2. Adverse Event Collection 
Adverse events will be graded according to CTCAE v5.0.  Both Serious and Non-Serious 
Adverse Events will be clearly noted in source documentation and listed on study specific Case 
Report Forms (CRFs).  The Protocol Director (PD) or designee will assess each Adverse Event 
(AE) to determine whether it is unexpected according to the Informed Consent, Protocol 
Document, or Investigator’s Brochures, and related to the investigation.   
 
All AEs, regardless of seriousness or relationship to investigational agent, will be collected 
from Day 1 through safety follow-up contact.  From signing of informed consent to prior to 
Day 1, only AEs deemed related to study procedures will be collected.  All Serious Adverse 
Events (SAEs) will be tracked until resolution or 4 days after the last dose of the study 
treatment.  After the safety follow-up contact, any SAEs spontaneously reported to investigator 
and deemed related to investigational agent should be reported to the IND Sponsor-Investigator 
(Daniel Spielman, PhD). 
 
7.3. Adverse Event Reporting 
Non serious adverse events will be reported annually to FDA via an Annual Report 
(
) and to IRB via Continuing Review. 
 
The PD (aka Principle Investigator) must immediately report within 24 hours of knowledge of 
event to the IND Sponsor-Investigator (Daniel Spielman, PhD), any serious adverse event, 
whether or not considered related to study drug.  SAEs of all grades will be reported to the 
Sponsor-Investigator using the Stanford Cancer Institute SAE CRF.   
 
Adverse events deemed serious, unexpected (i.e. not described in the protocol, 
Investigator’s Brochure or informed consent documents) and related to investigational agent 
must be reported to the IND Sponsor-Investigator by email or fax using the FDA MedWatch 
form 3500a within 24 hours of knowledge of event.  The IND Sponsor-Investigator is responsible 
for deciding whether the event meets IND Safety Reporting criteria. 
 
Events will be submitted to Sponsor-Investigator Dr. Daniel Spielman, at: 
Daniel Spielman, PhD  
Professor of Radiology and Electrical Engineering (by courtesy) 
1201 Welch Rd 
Lucas Center, 
 
Stanford, CA  94305-5488 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 13 of 18 
CONFIDENTIAL 
Phone: 650-723-8697 
Fax: 650-723-9222 
spielman@stanford.edu 
 
It will be the responsibility of the Sponsor-Investigator to provide to the FDA all information 
concerning significant hazards, contraindications, side effects or precautions felt significant to 
the safety of the investigational agent being studied.  The PD is responsible for accurate and 
timely communication to the Sponsor-Investigator of any events reportable to FDA.   
 
SAEs CTCAE Grade 2 and above, and all subsequent follow-up reports will be reported to 
the Stanford Cancer Institute Data and Safety Monitoring Committee (DSMC) using the study 
specific CRF regardless of the event’s relatedness to the investigation.  Following review by the 
DSMC, events meeting the IRB definition of ‘Unanticipated Problem’ will be reported to the IRB 
using eProtocol within 10 working days of DSMC review, or within 5 working days for deaths or 
life-threatening experiences. 
 
8. REGULATORY CONSIDERATIONS 
 
8.1. Institutional Review of Protocol 
The protocol, the proposed informed consent and all forms of participant information related 
to the study (e.g.  advertisements used to recruit participants) will be reviewed and approved by 
the Stanford IRB and Scientific Review Committee (SRC).  Any changes made to the protocol 
will be submitted as a modification and will be approved by the IRB, and SRC if applicable, prior 
to implementation.  The Protocol Director will disseminate the protocol amendment information 
to all participating investigators. 
 
Protocol amendments will be submitted to the FDA under IND 
in accordance to the 
regulations. 
 
8.2. Data Management Plan 
The Protocol Director, or his/her designee, will prepare and maintain adequate and accurate 
participant case histories with observations and data pertinent to the study.  Study specific Case 
Report Forms (CRFs) will document treatment outcomes for data analysis.  Case report forms 
will be developed using the Oncore database system and will be maintained by CCTO.  CRFs 
will be kept in a locked office, only accessible to the research team.” 
 
8.3. Data and Safety Monitoring Plan 
During the clinical investigation, the Protocol Director will evaluate the progress of the trial, 
including periodic assessments of data quality and timeliness, participant recruitment, accrual 
and retention, participant risk versus benefit, performance of trial sites, and other factors that 
can affect study outcome.  Monitoring of the trial will occur every 8 weeks and a record of 
monitoring activities will be maintained by the study team.   
 
The Stanford Cancer Institute Data and Safety Monitoring Committee (DSMC) will audit 
study related activities at least annually in accordance with the DSMC SOP to determine 
whether the study has been conducted in accordance with the protocol, local standard operating 
procedures, FDA regulations, and Good Clinical Practice (GCP).  This may include review of 
regulatory binders, case report forms, eligibility checklists, and source documents.  In addition, 
the DSMC will regularly review serious adverse events and protocol deviations associated with 
the research to ensure the protection of human subjects.  Results of DSMC audits will be 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 14 of 18 
CONFIDENTIAL 
communicated to the IRB and the appropriate regulatory authorities at the time of continuing 
review, or in an expedited fashion, as needed. 
 
9. MEASUREMENTS 
 
9.1. Primary and Exploratory Outcome measures 
Primary Outcome Measure: This is a pilot study which will allow us to develop a novel MRS 
technique for clinical application.  Our primary measurement will be safety of [1-13C]-pyruvate IV 
injection defined as the absence of Grade 2 toxicity (except for asymptomatic lab increases).   
 
Exploratory Outcome Measure: Detection of lactate and bicarbonate production in tumor 
and normal brain tissue, defined as the frequency of 13C MR signal detection above background 
noise level of both lactate and bicarbonate in all tumors screened. 
 
9.2. Measurement Methods 
Images will be analyzed for several conventional MR parameters, including tumor volume 
and CBV.  Pyruvate, bicarbonate and lactate signals will be assessed by peak integration of the 
corresponding spectral peaks.   
 
9.3. Measurement Time Points 
Assessments will be made only at one time point.   
 
9.4. Response Review 
No response review is planned. 
 
10. STATISTICAL CONSIDERATIONS 
 
10.1. 
Statistical Design 
This pilot study seeks to demonstrate safety of the [1-13C]-pyruvate IV injection as well as 
the ability to detect lactate and bicarbonate spectra.  Safety will be defined as the absence of 
Grade 2 toxicity (except for asymptomatic laboratory abnormalities). 
 
Another goal is an assessment of whether lactate and bicarbonate spectra can be detected 
in tumor.  Three answers are anticipated.  Either there will be bicarbonate spectra detected in 
all, some or none.  Our experimental data suggests that we should be able to see it in all 
enhancing areas, although the technical differences between experimental and clinical 
machines does not guarantee this outcome. 
 
5 healthy volunteers and 10 patients with CNS malignancies will be assessed, which is the 
maximum that our budget will allow.  These results will then be used to properly formulate a 
more formal experiment that will subsequently be submitted. 
 
10.2. 
Randomization 
No randomization. 
 
10.3. 
Interim analyses 
No interim analysis is planned.   
 
10.4. 
Descriptive Statistics and Exploratory Data Analysis 
N/A 
 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 15 of 18 
CONFIDENTIAL 
10.5. 
Primary Analysis  
N/A 
 
10.6. 
Sample Size 
Our budget allows 5 healthy volunteers and 10 patients with CNS malignancies for a total of 
15 subjects.  This number will allow us to accrue enough data to better understand lactate and 
bicarbonate detectability and safety.   
 
10.7. 
Accrual estimates 
15 subjects will be assessed over one year. 
 
10.8. 
Criteria for future studies 
 
The data obtained from the current study will be instrumental in helping us craft the first in 
human study assessing [1-13C]-pyruvate DNP MRS in GBM patients receiving bevacizumab. 
 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 16 of 18 
CONFIDENTIAL 
11. REFERENCES 
 
[1] 
E.  M.  Palsson-McDermott and L.  A.  J.  O'Neill, “The Warburg effect then and now: 
From cancer to inflammatory diseases,” BioEssays, vol.  35, no.  11, pp.  965–973, Sep.  
2013. 
[2] 
M.  G.  Vander Heiden, L.  C.  Cantley, and C.  B.  Thompson, “Understanding the 
Warburg Effect: The Metabolic Requirements of Cell Proliferation,” Science, vol.  324, 
no.  5930, pp.  1029–1033, May 2009. 
[3] 
J.  M.  Park, S.  Josan, T.  Grafendorfer, Y.-F.  Yen, R.  E.  Hurd, D.  M.  Spielman, and 
D.  Mayer, “Measuring mitochondrial metabolism in rat brain in vivo using MR 
Spectroscopy of hyperpolarized [2-¹³C]pyruvate.,” NMR Biomed., vol.  26, no.  10, pp.  
1197–1203, Oct.  2013. 
[4] 
J.  M.  Park, S.  Josan, T.  Jang, M.  Merchant, R.  Hurd, D.  Mayer, L.  Recht, and D.  
Spielman, “Metabolic Changes in a Rat Glioma Model After Anti-Angiogenic Treatment 
Measured by MR Spectroscopic Imaging of Hyperpolarized [1-13C]Pyruvate,” 
presented at the Scientific Meeting of the ISMRM, Milan, Itraly. 
[5] 
J.  H.  Ardenkjær-Larsen, A.  M.  Leach, N.  Clarke, J.  Urbahn, D.  Anderson, and T.  
W.  Skloss, “Dynamic nuclear polarization polarizer for sterile use intent,” NMR 
Biomed., vol.  24, no.  8, pp.  927–932, Mar.  2011. 
[6] 
R.  E.  Hurd, Y.-F.  Yen, A.  Chen, and J.  H.  Ardenkjaer-Larsen, “Hyperpolarized 13C 
metabolic imaging using dissolution dynamic nuclear polarization,” J.  Magn.  Reson.  
Imaging, vol.  36, no.  6, pp.  1314–1328, Nov.  2012. 
[7] 
I.  Park, R.  Bok, T.  Ozawa, J.  J.  Phillips, C.  D.  James, D.  B.  Vigneron, S.  M.  
Ronen, and S.  J.  Nelson, “Detection of early response to temozolomide treatment in 
brain tumors using hyperpolarized 13C MR metabolic imaging,” J.  Magn.  Reson.  
Imaging, vol.  33, no.  6, pp.  1284–1290, May 2011. 
[8] 
J.  M.  Park, L.  D.  Recht, S.  Josan, M.  Merchant, T.  Jang, Y.  F.  Yen, R.  E.  Hurd, 
D.  M.  Spielman, and D.  Mayer, “Metabolic response of glioma to dichloroacetate 
measured in vivo by hyperpolarized 13C magnetic resonance spectroscopic imaging,” 
Neuro-Oncology, vol.  15, no.  4, pp.  433–441, Mar.  2013. 
[9] 
M.  J.  Albers, R.  Bok, A.  P.  Chen, C.  H.  Cunningham, M.  L.  Zierhut, V.  Y.  Zhang, 
S.  J.  Kohler, J.  Tropp, R.  E.  Hurd, Y.-F.  Yen, S.  J.  Nelson, D.  B.  Vigneron, and J.  
Kurhanewicz, “Hyperpolarized 13C lactate, pyruvate, and alanine: noninvasive 
biomarkers for prostate cancer detection and grading.,” Cancer Research, vol.  68, no.  
20, pp.  8607–8615, Oct.  2008. 
[10] 
M.  M.  Darpolor, Y.-F.  Yen, M.-S.  Chua, L.  Xing, R.  H.  Clarke-Katzenberg, W.  Shi, 
D.  Mayer, S.  Josan, R.  E.  Hurd, A.  Pfefferbaum, L.  Senadheera, S.  So, L.  V.  
Hofmann, G.  M.  Glazer, and D.  M.  Spielman, “In vivo MRSI of hyperpolarized 
[1-13C]pyruvate metabolism in rat hepatocellular carcinoma,” NMR Biomed., vol.  24, 
no.  5, pp.  506–513, Dec.  2010. 
[11] 
M.  M.  Chaumeil, T.  Ozawa, I.  Park, K.  Scott, C.  D.  James, S.  J.  Nelson, and S.  
M.  Ronen, “Hyperpolarized 13C MR spectroscopic imaging can be used to monitor 
Everolimus treatment in vivo in an orthotopic rodent model of glioblastoma,” 
NeuroImage, vol.  59, no.  1, pp.  193–201, Jan.  2012. 
[12] 
S.  E.  Day, M.  I.  Kettunen, M.  K.  Cherukuri, J.  B.  Mitchell, M.  J.  Lizak, H.  D.  
Morris, S.  Matsumoto, A.  P.  Koretsky, and K.  M.  Brindle, “Detecting response of rat 
C6 glioma tumors to radiotherapy using hyperpolarized [1-13C]pyruvate and 13C 
magnetic resonance spectroscopic imaging,” Magnetic Resonance in  Medicine, vol.  
65, no.  2, pp.  557–563, Nov.  2010. 
[13] 
S.  J.  Nelson, J.  Kurhanewicz, D.  B.  Vigneron, P.  E.  Z.  Larson, A.  L.  Harzstark, M.  
Ferrone, M.  van Criekinge, J.  W.  Chang, R.  Bok, I.  Park, G.  Reed, L.  Carvajal, E.  

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 17 of 18 
CONFIDENTIAL 
J.  Small, P.  Munster, V.  K.  Weinberg, J.  H.  Ardenkjaer-Larsen, A.  P.  Chen, R.  E.  
Hurd, L.  I.  Odegardstuen, F.  J.  Robb, J.  Tropp, and J.  A.  Murray, “Metabolic 
Imaging of Patients with Prostate Cancer Using Hyperpolarized [1-13C]Pyruvate,” Sci 
Transl Med, vol.  5, no.  198, pp.  198ra108–198ra108, Aug.  2013. 
 
 

Study IRB-39845 / BRN0036 
Version Date 15 May 2019 
Page 18 of 18 
CONFIDENTIAL 
Appendix A:  Inclusion/Exclusion Criteria Checklist 
*All subject files must include supporting documentation to confirm subject eligibility.  The 
method of confirmation can include, but is not limited to, laboratory test results, radiology test 
results, subject self-report, and medical record review.   
 
Inclusion Criteria 
(From IRB approved protocol) 
Yes 
No 
Not 
Applicable 
Supporting Documentation* 
1. (Arm 2 patients with known CNS 
malignancy only) Patients with a known 
diagnosis of CNS malignancy, including 
metastases, with known enhancement on 
MR who are otherwise eligible to undergo 
MRI 
 
 
 
      
2. At least 18 years old 
 
 
 
      
3. eGFR > 30 ml/min 
 
 
 
      
4. (Arm 2 patients with known CNS 
malignancy only) No allergy to gadolinium 
 
 
 
      
5. Ability to understand and willingness to 
sign a written informed consent document 
 
 
 
      
Exclusion Criteria 
(From IRB approved protocol) 
 
1. Refusal to have an IV placed for injection 
 
 
 
      
2. Acute major illness (e.g., unstable angina, 
etc.) or other condition that makes 
participation unsafe, per the investigator’s 
judgement 
 
 
 
      
3. Total Bilirubin > 1.5 x ULN;  
AST/ALT > 2.5 x ULN;  
GGT > 2.5 x ULN 
 
 
 
      
4. Pregnant or breast-feeding 
 
 
 
      
5. Cardiovascular risk, including: 
o 
Poorly controlled hypertension, defined as 
either systolic > 170 or diastolic > 110. 
o 
Congestive heart failure 
o 
Myocardial infarction within the past year 
o 
QT prolongation, defined as pretreatment 
QTc > 440 msec in males or > 460 msec 
in females 
 
 
 
      
6. (Arm 1 Healthy volunteers only) 
Undergoing active treatment for 
malignancy 
 
 
 
      
 
IV.  Statement of Eligibility 
This subject is [ 
 eligible / 
 ineligible ] for participation in the study. 
Signature: 
Date: 
Printed Name: