A Phase I Trial of Ascorbic Acid and Gemcitabine for the Treatment ...

A Phase I Trial of Ascorbic Acid and Gemcitabine for the Treatment of
Metastatic Pancreatic Cancer


Principal Investigator:
Joseph J. Cullen, M.D.
Professor
Department of Surgery
University of Iowa Hospitals and Clinics
4622 JCP
353-8297
joseph-cullen@uiowa.edu


Co-Investigators:
Radiation Oncology— Garry R. Buettner, Ph.D.
Internal Medicine— Daniel J. Berg, M.D.
Internal Medicine — Nelson S. Yee, M.D., Ph.D.
Internal Medicine—Nicole Nisly, M.D.
Internal Medicine — Thorvardur R. Halfdanarson, M.D.
Pharmacy—D. J. Murry, PharmD.
Consultants:
Mark Levine, M.D.
Senior Investigator
NIH/NIDDK
markl@bdg8.niddk.nih.gov

Biostatistician:
Brian J. Smith, Ph.D.


Research Nurse:
Jane Hershberger, R.N.
Research Coordinator:
Kellie Bodeker-Goranson, B.Sc., CCRC


Protocol Editor:
Marilyn Rosenquist, MS


IRB Number:
200804753
Protocol Version Date:
2/20/2009

---

TABLE OF CONTENTS

Page
1. Background and Significance …………..……………………………… 2
2. Objectives………………………………….……………............................ 4
3. Drug Information………..……………………………………………..…..
4
4. Eligibility Criteria ………………………………………………….....….... 5
5. Registration Guidelines and Recruitment......................................... 7
6. Patient Evaluation................................................................................ 8
7. Method………………………………………………………………………. 9
8. Chemotherapy……………………………………………………….……… 11
9. Toxicities……………………………………………………………….…… 14
10. Criteria for Evaluation…………………………………………….…….. 16
11. Criteria for Response……………………………………… …………… 16
12. Toxicity Criteria……………………………………………… ………….. 19
13. Criteria for Removal from Study………………………… …………… 20
14. Data Submission Schedule……………………………… ……………. 20
15. Statistical Considerations……………………………………………… 20
16. Data Management, Quality Control and Data Security ………….... 22
18. References………………………………………………………………… 25
Appendix

Clinic schedules

---

SCHEMA





Screening physical, medical history and labs prior to study
R
E
initiation.
G
I
S
T
Ascorbic acid IV 2 days per week.
R
A
T
I
Gemcitabine IV weekly for 3 weeks and 1 week off.
O
N
Continue gemcitabine for 6 months or until disease progression
or unacceptable toxicity.
Maintenance ascorbic acid IV once weekly after 6 months if
disease is stable.

---

1.0
BACKGROUND
1.1 Ascorbate as an anti-tumor agent. Ascorbate (ascorbic acid, vitamin C, AscH) is one of the
early unorthodox therapies for cancer, based on two unsupported hypotheses. McCormick postulated
that ascorbate protects against cancer by increasing collagen synthesis1, 2, while Cameron hypothesized
that ascorbate could have anti-cancer action by inhibiting hyaluronidase and thereby prevent cancer
spread3. These hypotheses were subsequently promoted by Cameron and Pauling4, 5. Cameron and
Campbell initially published case reports of 50 patients; some seemed to have benefited from high dose
ascorbate6. Cameron and Pauling then published results of 100 patients with terminal cancer that were
given intravenous ascorbate7. The ascorbate-treated patients were compared to 1000 retrospective
controls with similar disease. Patients who received ascorbate survived 300 days longer than controls5,
7. A prospective study was then conducted that randomized patients to ascorbate treatment or
palliative therapy. Treated patients had a median survival of 343 d vs. 180 d for controls8. Smaller
studies have also reported benefits of ascorbate9, 10.
To test “definitively” whether ascorbate was effective, Moertel conducted two randomized placebo-
controlled studies randomized to oral ascorbate; neither study showed benefit11, 12. Subsequently,
ascorbate treatment was considered useless. However, it was not recognized until approximately 15
years later that oral and intravenous ascorbate have strikingly different pharmacokinetics13, 14. This
difference in the administration route is key. Cameron gave patients ascorbate intravenously as well
as orally, while Moertel’s patients received only oral ascorbate. Thus, the issue of ascorbate in cancer
treatment needs to be re-examined.

The evidence for use of ascorbate in cancer treatment falls into two categories: clinical data on
dose concentration relationships and laboratory data describing potential cell toxicity with high
concentrations of ascorbate in vitro. Clinical data show that when ascorbate is given orally, fasting
plasma concentrations are tightly controlled at < 100 μM15. As doses exceed 200 mg, absorption
decreases, urine excretion increases and ascorbate bioavailability is reduced13, 15. In contrast, when
1.25 grams of ascorbate are administered intravenously, concentrations as high as 1 mM are achieved.
Some clinicians have infused more than 10 grams of ascorbate in cancer patients and achieved plasma
concentrations of 1 to 5 mM16. Thus, it is clear that intravenous administration of ascorbate can yield
very high plasma levels, while oral treatment does not.
Pharmacologic ascorbate concentrations have been shown to selectively kill some cancer cell
types. Chen et al. measured cell death in 10 cancer and 4 normal cell types using 1-hour exposures to
pharmacological ascorbate17. Normal cells were unaffected by 20 mM ascorbate whereas 5 cancer cell
lines had EC50 values of < 4 mM, a concentration achievable by intravenous administration. In addition,
cell death was independent of metal chelators, but dependent on formation of H
18
2O2 . H2O2 generation
was dependent on ascorbate concentration, incubation time; [H2O2] displayed a linear increase with
[AscH] and it increased as a quadratic function of ascorbate radical, ascorbate being an electron donor
to O2 to form H2O2.
When ascorbate is infused intravenously the resulting pharmacologic concentration will distribute
rapidly into the extracellular water space19. In vivo, Chen and colleagues demonstrated that
intravenous injection of ascorbate (0.25-0.5 mg/g body weight) increased baseline concentrations of
ascorbate in blood and extracellular fluid to > 8 mM and increased formation of H2O2 (human study)20.
More recent studies have demonstrated that intraperitoneal doses of 4 g/kg ascorbate resulted in blood
concentrations of 40 mM; tumor extracellular fluid increased to peaks of 20 mM for up to 3 hours15. Our
preliminary data demonstrate that pharmacologic doses of ascorbate are cytotoxic to pancreatic cancer
cells and that intraperitoneal administration of high dose ascorbate inhibits pancreatic tumor growth in
mice. Combined, these studies provide a foundation for pursuing pharmacologic ascorbate as a
prooxidant agent in cancer therapy.

Ascorbate-mediated cell death has been shown to be due to H2O2 generation, via ascorbate
radical formation, with ascorbate as the electron donor17, 18, 20. When ascorbate is infused intravenously
the resulting pharmacologic concentration distributes rapidly in the extracellular water space20. Thus,
pharmacologic ascorbate concentrations in media, as a surrogate for extracellular fluid, should
generate ascorbate radical and H2O2. In contrast, the same pharmacologic ascorbate concentrations in
2

---

whole blood generate little detectable ascorbate radical and no detectable H
12
2O2 . This can be
accounted for by efficient and redundant H2O2 catabolic pathways in whole blood relative to those in
media or extracellular fluid. Thus, ascorbic acid administered intravenously in pharmacologic
concentrations may serve as a pro-drug for H2O2 delivery to the extracellular milieu, but without H2O2
accumulation in blood.

In a recent Phase I trial of intravenous ascorbic acid in patients with advanced cancers, adverse
events and toxicity were minimal at all dose levels. The high-dose intravenous ascorbic acid was well
tolerated when administered to patients. Ascorbic acid concentrations reached up to 25 mmol/L in
patients who received ascorbic acid of 1.5 g/kg. Of the 24 patients in the study, only 4 recorded minor
adverse events including headache, dizziness and diarrhea 24.

1.2 PRELIMINARY DATA

1.2.1 Differential susceptibility of pancreatic ductal epithelial cells vs. pancreatic cancer cells
to ascorbate-induced cytotoxicity and oxidative stress: Figure 1 demonstrates the differential
susceptibility of pancreatic ductal epithelial cell line (H6c7) vs. pancreatic cancer cells. The effects of
pharmacologic ascorbate concentrations were determined on pancreatic cancer and pancreatic ductal
epithelial cells. All cells were treated with ascorbate (0, 5, 10 mM) for one hour. Cell viability was
determined by MTT assay. MIA PaCa-2, AsPC-1, BxPC-3 are pancreatic cancer cell lines.
Immortalized pancreatic ductal epithelial cell line, H6c7 and its derivatives, H6c7er-Kras (H6c7 cells
expressing K-ras oncogene), and H6c7eR-KrasT (tumorigenic H6c7 cells expressing K-ras oncogene),
also received ascorbate (0, 5, 10 mM) for one hour.

Figure 1. Pancreatic cancer cells and tumorigenic H6c7
1
e
)
cells expressing K-ras oncogene are more susceptible


c
e
l
l

l
i
n
than normal pancreatic ductal epithelial cells to
0.75
0 mM
5 mM
pharmacological ascorbate-induced cytotoxicity. Cell
r

each
10 mM
l
i
t
y
f
o
viability was determined using the MTT assay. Cell lines
M
abi

m
0 0.5
were treated with ascorbate (0, 5, or 10 mM for one hour and
Vi
to
*
*
MTT assay performed. Cell-viability was decreased in all
a
l
i
zed
pancreatic cancer cell lines and in H6c7 cells expressing K-ras
r
m
o 0.25
(
n
*
*
oncogene. Cell-viability was unchanged in the pancreatic
*
* *
*
*
*
ductal epithelial cell line H6c7. Means, n = 3.*P < 0.05 vs. 0 mM
0
for each cell line.
MiaPaCa-2
AsPC-1
BxPC-3
H6c7
Kras
KrasT

*P<0.05 vs 0 mM for each cell line
These results support the hypothesis that pancreatic cancer cells are more susceptible than normal
cells to ascorbate-induced cytotoxicity.

1.2.2 Treatment of established pancreatic tumors with ascorbate inhibits in vivo growth—We
wanted to determine if treatment of established pancreatic tumors in a xenograft model with ascorbate
would inhibit growth. MIA PaCa-2 tumor cells (2 x 106) were delivered subcutaneously into the flank
region of nude mice and allowed to grow until they reached 3 mm in greatest dimension (~ 10 days), at
which time they were randomly assigned to a treatment group. This was defined as day 1 of the
experiment. The animals were randomized to receive either ascorbate (4 g/kg) or osmotically
equivalent i.p. saline as a control (1 M) given to mice i.p. every day for two weeks. Data from Dr. Mark
Levine’s laboratory have demonstrated that 4 g/kg i.p. ascorbate resulted in blood concentration from
baseline of 40 μM to peaks of 40 mM while tumor extracellular fluid increased to peaks of 20 mM for up
to 3 hours [5 and personal communication]. The primary outcomes of interest were tumor growth over
time. Tumor size (mm3) was periodically measured throughout the experiments, resulting in repeated
measurements across time for each mouse. Linear mixed effects regression models were used to
estimate and compare the group-specific tumor growth curves. The observed tumor volumes for all
mice are plotted over time in Figure 2.

3

---

1000
saline
Ascorbate
)3 800
m


(
m
e
Ascorbate (4 g/kg
Figure 2. Ascorbate decreased tumor growth in
m
600
I.P.every day for 14
l
u
nude mice. The group of animals that received
o
days). Hypertonic saline
r

v
(g/kg I.P. every day for
ascorbate (4 g/kg, i.p.) had significantly slower rate of
o
m
14 days).
400
u
tumor growth when compared to the controls receiving
T
osmotically equivalent saline (P < 0.0001, n = 5-
200
8/group). Ascorbate (4 g/kg, i.p.) was given every day
for 14 days beginning on day 1 of the experiment. On
0
day 15 there was a 3.5-fold decrease in tumor size in
1
5
8
12
15
animals receiving ascorbate when compared to controls.
Days

2.0 OBJECTIVES

Adenocarcinoma of the pancreas is the fourth leading cause of cancer death in the United
States and is increasing in incidence; the prognosis remains dismal. We propose to investigate an
entirely new approach, using pharmacological ascorbate, combined with Gemcitabine, to treat this
cancer. Intravenous ascorbate (i.e., ascorbic acid, vitamin C), but not oral ascorbate, produces high
plasma concentrations, which are in the range that can be cytotoxic to tumor cells. Though ascorbate
has been utilized in cancer therapy, few studies have investigated intravenous deliver of ascorbate.
Preliminary studies from our group have demonstrated that ascorbate induces oxidative stress and
cytotoxicity in pancreatic cancer cells; this cytotoxicity appears to be greater in tumor vs. normal cells.
We hypothesize that production of H2O2 mediates the increased susceptibility of pancreatic cancer cells
to ascorbate-induced metabolic oxidative stress. Gemcitabine is the standard chemotherapy drug used
to treat pancreatic cancer21. As a first step to test this hypothesis, we will determine the maximum
tolerable dose of ascorbic acid in combination with Gemcitabine in patients with metastatic pancreatic
cancer.

2.1 Primary Objective
Determine the safety, acute toxicity, and maximum tolerated dose (MTD) of ascorbic acid
when given twice/week with standard Gemcitabine chemotherapy for patients with advanced
pancreatic cancer.

2.2 Secondary Objective
Determine the effect of escalating doses of ascorbic acid on ascorbic acid levels in
pancreatic cancer patients.

3.0
DRUG INFORMATION

3.1
Ascorbic Acid (Vitamin C) (cost covered by The Susan L. Bader Foundation of Hope Pancreatic Cancer Fund)
Ascorbic Acid (vitamin C) is a water-soluble vitamin. It occurs as a white or slightly
yellow crystal or powder with a slight acidic taste. On exposure to air and light it gradually darkens. In
the dry state it is reasonably stable in air, but in solution it rapidly oxidizes. Ascorbic Acid is freely
soluble in water; sparingly soluble in alcohol; insoluble in chloroform, ether, and benzene.
The chemical name of Ascorbic Acid is L-ascorbic acid. The molecular formula is C6H8O6 and the
molecular weight is 176.13.
Ascorbic Acid Injection is a clear, colorless to slightly yellow sterile solution of Ascorbic Acid in Water
for Injection, for intravenous, intramuscular or subcutaneous use. Each mL contains Ascorbic Acid 500
mg, Edetate Disodium 0.025%, Water for Injection q.s. pH (range 5.5-7.0) adjusted with Sodium
Bicarbonate.
Side effects associated with Ascorbic Acid (vitamin C) are transient mild soreness may occur at the site
of intramuscular or subcutaneous injection. Too-rapid intravenous administration of the solution may
cause temporary faintness or dizziness.
4

---

3.2
Gemcitabine
Gemzar® (gemcitabine HCl) is a nucleoside analogue that exhibits antitumor activity. Gemcitabine HCl
is 2′-deoxy-′, 2′-difluorocytidine monohydrochloride (b-isomer).
The empirical formula for gemcitabine HCl is C9H11F2N3O4· HCl. It has a molecular weight of 299.66.
Gemcitabine HCl is a white to off-white solid. It is soluble in water, slightly soluble in methanol, and
practically insoluble in ethanol and polar organic solvents.
The clinical formulation is supplied in a sterile form for intravenous use only. Vials of Gemzar contain
either 200 mg or 1 g of gemcitabine HCl (expressed as free base) formulated with mannitol (200 mg or
1 g, respectively) and sodium acetate (12.5 mg or 62.5 mg, respectively) as a sterile lyophilized
powder. Hydrochloric acid and/or sodium hydroxide may have been added for pH adjustment.
Gemzar is indicated as first-line treatment for patients with locally advanced (nonresectable
Stage II or Stage III) or metastatic (Stage IV) adenocarcinoma of the pancreas. Gemzar is
indicated for patients previously treated with 5-FU.
Gemzar has been used in a wide variety of malignancies, both as a single-agent and in
combination with other cytotoxic drugs. In Single-agent use, Myelosuppression is the principal
dose-limiting toxicity.
In Single-Agent Use for Pancreatic Cancer, Gemzar is administered by intravenous infusion at a dose
of 1000 mg/m2 over 30 minutes for 3 weeks. Each cycle consist of infusions once weekly for 3
consecutive weeks out of every 4 weeks.
Gemzar is commercially available in an injection form.

4.0
ELIGIBILITY CRITERIA
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. The exclusion criteria
of this protocol will be strictly followed by the investigators:

• Psychiatric illness that would prevent the patient from giving informed consent.
• Patient is not deemed a candidate for gemcitabine based on overall condition and co-
morbidities.
• Patients who have a medical condition such as active/uncontrolled infection, which would make
this protocol unreasonably hazardous for the patient in the opinion of the treating physician.
• Life
expectancy
≤ 12 weeks.
• Participants in this study must use adequate contraception for the duration of treatment and for
up to 3 months after the completion of protocol therapy.

4.1
Inclusion Criteria
Each of the criteria must be met in order for a patient to be considered eligible for inclusion:
4.1.1. G6PD status normal. Cost covered by The Susan L. Bader Foundation of Hope Pancreatic
Cancer Fund
4.1.2. Patients must have histologically or cytologically diagnosed pancreatic adenocarcinoma.

Documentation of disease extent by CT scan is required. Radiologically measurable disease is
not required.
4.1.3. The patient must screened for eligibility and have care approved by treating oncologist; the
oncology care is to be dictated by the oncology team and patient.
4.1.4. Patients must be of ambulatory status without evidence of spinal cord compression.
5

---

4.1.5. Prior Therapy
4.1.6. No prior chemotherapy for metastatic disease.
4.1.7. Metastatic disease, failed curative therapy, and patient ineligible for definitive curative therapy.
4.1.8. If the patient received adjuvant therapy, it must have been completed at least 4 weeks prior to
enrollment on this study. The patient must have recovered from all treatment related toxicities
and must have evidence of disease progression following adjuvant treatment.
4.1.9. Prior radiation therapy. With or without a radiosensitizing dose of fluoropyrimidines, is allowed
provided the patient has disease outside of the radiation port. At least 4 weeks must have
elapsed from completion of the radiation therapy and all signs of toxicity must have resolved.
4.1.10. Patients with a “currently active” second malignancy other than non-melanoma skin cancers are
not to be registered. Patients are not considered to have a “currently active” malignancy if they
have completed therapy and considered by their physician to be a less than 30% risk of
relapse.
4.1.11. Women must be non-pregnant and non-breast feeding.
4.1.12. ECOG Performance Status 0, 1 or 2.
4.1.13. Required Initial Laboratory Data:
Granulocytes
≥ 1,500 / ul
Platelet
count
≥ 100,000 / ul
Creatinine
≤ 1.5 mg /dL or creatinine clearance ≥ 60 mL / min
Total
Bilirubin
≤ 2 x upper limit of normal

SGOT (AST) ALT
3 x upper limit of normal if no liver metastases; < 5 x if liver metastases
are
present
PT
INR ≤ 1.5, unless patient is on full dose warfarin
4.1.14. Patients who have no language barrier, are cooperative, and can give informed consent before entering
the study after being informed of the medications and procedures to be used in this study may
participate.
4.1.15. Age≥18 with diagnosis of pancreatic cancer.
Children will be excluded from this study because most people who develop pancreatic cancer are
older adults. The average age of patients is 71 years old.


4.2 Exclusion
Criteria

4.2.1. Patients with evidence of a significant psychiatric disorder by history/examination that would prevent
completion of the study will not be allowed to participate.
4.2.2. ECOG Performance Status of 4.
4.2.3. Co-morbid condition that would affect survival: end stage congestive heart failure, unstable angina,
myocardial infarction within 6 weeks of study, uncontrolled blood sugars ≥ 300 mg/dL, patients with
known chronic active hepatitis or cirrhosis.
4.2.4. Patients who consume an excess of alcohol or abuse drugs (an excess of alcohol is defined as more
than four of any one of the following per day: 30 mL distilled spirits, 340 mL beer, or 120 mL wine) will
not be allowed.
4.2.5 Patients taking over-the-counter anti-oxidants (dietary supplements like oral vitamin C and grape
seeds). These should be discontinued prior to starting therapy.

4.3
Pregnant and/or Nursing
Pregnant and nursing women are excluded from this study.




4.4
Inclusion of Women and Minorities
6

---

Although there is no evidence to suggest that the outcome will differ by gender or ethnicity and there is
insufficient power to detect small or moderate effects, we will, in a secondary analysis, report the results by
gender and ethnicity.


4.5 Sample
Size

This study will aim to accrue 10 subjects with histologically or cytologically diagnosed pancreatic
adenocarcinoma. The number of subjects was chosen based on the estimated ability to provide meaningful
information on pancreatic cancer. For discussion of statistics and power analyses, please see Section 15.

5.0
Registration Guidelines and Recruitment
5.0.1. At the time of registration, the study subjects must have completed the Registration Form.
5.0.2. Patients will not be registered if the IRB approval is not provided or is greater than 1 year prior to the
date of registration. The patient must have informed consent.
5.0.3. Study subjects will be recruited from the community from referrals. Posters, advertisements, and
media announcements will attract patients as well. Patients with cancer, in numbers that reflect
national surveys, currently ask to have complementary and alternative therapies added to their
chemotherapeutic regimens.
5.0.4. It is anticipated that the patient age range will be over 50 years of age. The patient population is
derived from the state of Iowa and surrounding states and represents a racially and economically
diverse population. We will make all efforts to enroll minority study subjects to this protocol.

5.1 Informed
Consent
Signed informed consent for enrollment in this protocol will be obtained from eligible subjects by a member of
the research team before the start of the study. Subjects will be fully informed of the purpose and potential risks
and benefits of participating in the study. Subjects have the opportunity to have questions answered to their
satisfaction before signing the consent.
7

---

6.0 PATIENT EVALUATION/STUDY CALENDAR
Study follow-up mirrors standard visits for pancreatic cancer management.

Study
Follow-
Screena
1Cycle=28 days=4 Weeksb EOS
Procedure
up
Day of cycle

1
5
8 12 15 19 22 26 D1C2


History/Physical X X X X x X X

Vital
signs
X
X X X X X X X X X
X

Height
X


Weight, BSA c
X X X X X X X

Performance
X X
X X

Status
Drug Toxicity

X X X X X X

Assessmentd
CBC
with
diff

X X X X X X X

Chemistry testse X


Creatininef
X X X X X
X
CA 19.9g
X X

Blood for
ascorbic acid
X
X X X X X X X X X


levelsh
Chest, Abd.,
X


Pelvis CT Scani
Pregnancy
Test X


Survival j











X
Ascorbic acid

X X X X X X X X X


infusion
Gemcitabine

X X X X


infusion
a. Screening labs and history/physical must be obtained < 14 days from initiation of study treatment, and CT scan, IVG
and EKG obtained < 28 days.
b. Subjects will be seen in clinic weekly for first 2 cycles and cycle day 1 and 15 for subsequent cycles.
c. Weight should be obtained prior to each cycle, and BSA recalculated if weight has changed >10%. At EOS, obtain
weight only; BSA does not require recalculation.
d. Drug assessment q weekly for first 2 cycles then day 1 and 15 for subsequent cycles.
e. Once a month: BUN, AST, ALT, LDH, ALP, T.bilirubin, T.protein, albumin, calcium, Na/K/Cl and CO2.
f. After screening, only creatinine need be checked for ascorbate toxicity at clinic visits. If the renal function declines, the
ascorbic acid dose may need to be adjusted per PI discretion. CBCs done weekly for first 2 cycles and then days 1+15.
g. CA 19.9 checked monthly.
h. Draw blood specimens before infusion and immediately after completing ascorbic acid infusion.
i. Scans after every 2 cycles after screening. If received Chest CT, do not need to receive Chest x-ray.
j. Patient survival is assessed every 3 months.

8

---