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Toxicity and Dose-Response Studies of 1 -Hydroxyvitamin D2 in a Retinoblastoma Xenograft Model
Richard J. Grostern, MD;
Paul J. Bryar, MD;
Michele L. Zimbric, BS;
Soesiawati R. Darjatmoko, MS;
Boaz J. Lissauer, MD;
Mary J. Lindstrom, PhD;
Janice M. Lokken;
Stephen A. Strugnell, PhD;
Daniel M. Albert, MD, MS
Arch Ophthalmol. 2002;120:607-612.
ABSTRACT
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Background Although calcitriol (1,25-dihydroxycholecalciferol) and vitamin D2 inhibit retinoblastoma growth in the athymic (nude) mouse xenograft
(Y-79 cell line) model of retinoblastoma, they can cause severe toxicity.
Objective To examine the toxicity of and dose-dependent response for the inhibition
of tumor growth for 1 -hydroxyvitamin D2 (1-OH-D2), an analogue with reduced systemic toxicity, in the athymic Y-79
mouse model.
Methods Mice were randomized into treatment and control groups for 5-week toxicity
and dose-response studies. Treatment was via oral gavage 5 times per week.
Dose-response studies measured tumor inhibition and drug serum levels. Tumor
size and body weight were measured weekly together with various criteria for
toxicity. Animals were euthanized at the end of the treatment period. Tumors
and kidneys were harvested, and serum was analyzed for calcium and drug levels.
Results Doses of 0.1 to 1.2 µg/d were selected on the basis of toxicity
studies for the dose-response trial. Tumor weight and volume in the 0.2-µg
and 0.3-µg doses were significantly lower than in controls. Mortality
rates and kidney calcification in mice treated with doses of 0.1 to 0.3 µg
were lower than those observed in studies of calcitriol and vitamin D2.
Conclusion A vitamin D analogue, 1-OH-D2, inhibits tumor growth
in this xenograft model of retinoblastoma with less toxicity than calcitriol
and vitamin D2.
INTRODUCTION
RETINOBLASTOMA IS the most common intraocular malignancy of childhood,
occurring once in 20 000 live births worldwide.1
In 1966, Frederick Verhoeff, MD, who had studied this tumor type for more
than 50 years, named it retinoblastoma,2 and
initiated radiation treatment,3-4
stressed that patients with regressed retinoblastoma had tumors that were
heavily calcified.5 He proposed using vitamin
D as an inhibitor of proliferating retinoblastoma tumors.5-6
In 1974, our laboratory established the first human retinoblastoma cell
line (Y-79 cell line) and subsequently confirmed the presence of high-affinity
vitamin D receptors in this cell line.7-8
We have demonstrated that calcitriol and vitamin D2 inhibit the
growth of Y-79 retinoblastoma cells in tissue culture, in a xenograft model
of a human Y-79 retinoblastoma cell line grown in athymic (nude) mice, and
in  -luteinizing hormone-Tag (LH-Tag) mice, a transgenic model
for hereditary retinoblastoma.6, 8-10
In contrast to Verhoeff's theory, this antineoplastic effect is unrelated
to the serum calcium level. However, marked toxicity in the form of high mortality
rates, hypercalcemia, and marked weight loss was seen in all animals treated
with calcitriol and vitamin D2at doses required to achieve a therapeutic
effect.
Vitamin D analogues were developed in the 1990s with comparable antineoplastic
activity but a reduced effect on calcium metabolism.11
The vitamin D analogue 1,25-dihydroxy-16-ene-23-yne-vitamin D3
(16,23-D3) was tested in our laboratory and found to be effective
against retinoblastoma tumors in LH-Tag mice at doses that produced
less toxicity than calcitriol and vitamin D2.12
This analogue has only recently been approved for experimental use in humans.
We are unaware of any available results in adult patients with tumors, a prerequisite
for human pediatric clinical trials.
In the current study, we report the toxicity and dose-dependent inhibition
of tumor growth in the athymic Y-79 retinoblastoma model with 1-hydroxyvitamin
D2(1-OH-D2) (BoneCare International Inc, Madison,
Wis).1 This vitamin D analogue was approved by the Food and Drug
Administration in 1999 for use in human tumor treatment. It is currently being
used in phase 1 human clinical trials of patients with prostate cancer (George
Wilding, MD, written communication, June 2001). Similar to 16,23-D3,
1-OH-D2 is known to induce a relatively low level of hypercalcemia
while producing effective systemic serum drug levels for tumor treatment.13-14
MATERIALS AND METHODS
All research using mouse models of retinoblastoma received institutional
review board approval by the Research Animal Resources Center of the University
of Wisconsin in Madison.
PRELIMINARY TOXICITY TRIAL
To assess the relative toxicity of 1-OH-D2, a preliminary
toxicity trial was conducted. Forty-eight athymic hybrid mice were divided
into 6 treatment groups of 7 animals each, with a corresponding control group
of 6 animals. The doses given were 0.1 µg, 0.3 µg, 0.6 µg,
0.9 µg, 1.2 µg, 1.8 µg, and 2.4 µg in 0.1 mL of solution.
1-OH-D2 was prepared as described below, and animals' baseline
body weights were measured. The mice were treated via oral gavage 5 times
per week for 5 weeks. Toxicity was assessed according to mortality rates,
weight loss, serum calcium levels, and kidney calcification.
DOSE-DEPENDENT RESPONSE FOR INHIBITION OF TUMOR GROWTH
A total of 292 athymic (nude) mice at ages ranging from 8 to 10 weeks
were given dorsal subcutaneous injections of 1 x 107 human
retinoblastoma cells from a cultured Y-79 cell line. Details of culture methods
have been described previously.15 Because of
limitations in available housing for the large number of animals needed, this
study was done in several layers of treated and control animals until group
sizes of at least 30 animals per group were reached. The tumors were allowed
to grow for 5 days prior to the start of treatment. Pretreatment body weight
was recorded. For 2 weeks prior to treatment with 1-OH-D2
and throughout the treatment period, the animals were fed a vitamin D- and
calcium-deficient diet (Vitamin D/Calcium Deficient PD; Purina Mills Inc,
St Louis, Mo) to reduce hypercalcemia.
The mice were randomized into 5 1-OH-D2 treatment
groups and a corresponding control group (37 animals in the 0.1 µg dose
group, 60 animals in the 0.2 µg group, 55 animals in the 0.3 µg
group, 45 animals in the 0.6 µg group, and 40 animals in the 1.2 µg
group; 55 animals were in the control group). 1-OH-D2 was
provided by BoneCare International Inc in a crystalline form, which was then
dissolved with 100% ethanol for a stock solution of 2.98 mg/mL. This solution
was diluted in coconut oil to concentrations corresponding to drug doses of
0.1 µg, 0.2 µg, 0.3 µg, 0.6 µg, and 1.2 µg per
0.1 mL. Spectrophotometric analysis was used to confirm the drug concentrations.
The mice in the control group were given 0.1 mL of coconut oil. Stock solutions
of the drug were prepared weekly and stored in amber glass bottles at -40°C
to protect the compound from degradation due to temperature or UV light.
Animals were treated 5 times per week for 5 weeks by oral gavage using
a 1-in, 1.25-mm-ball diameter steel gavage needle (22GX; Becton Dickinson,
Franklin Lakes, NJ) attached to a 1-mL syringe. Each animal was weighed twice
a week during treatment. Approximately 2 hours after the last dose at the
fifth week, animals were euthanized by cervical dislocation using isofluorane
anesthesia. Immediately prior to euthanasia, serum was collected for the determination
of calcium and vitamin D metabolite levels. The subcutaneous tumors were harvested,
measured, and weighed. Tumor volume was also measured using water displacement.
The kidneys were removed from representative animals in each treatment group
and histologically examined for calcification. Toxicity was assessed according
to mortality rate, decreased body weight, increased lethargy, and kidney calcification.
Animal care followed standards set by the Research Animal Resources
Center at the University of Wisconsin and previously published guidelines
for mice treated with vitamin D compounds.8
Animals were monitored daily for signs of toxicity, such as lethargy and weight
loss, by lab personnel and by veterinarians responsible for the Animal Care
Unit at the Clinical Science Center at the University of Wisconsin. Treatment
was withheld for animals that were severely lethargic for a maximum of 2 consecutive
days, allowing them to regain weight and recover normal physical activity.
Animals that experienced marked weight loss or were extremely ill were euthanized
prior to treatment completion on the advice of the attending veterinarians.
ANALYSIS OF TUMOR SIZE
Tumors were measured externally 3 times per week using calipers calibrated
in 1-mm increments, according to methods previously described in our laboratory.15 A single investigator performed all tumor measurements.
Tumor volume on day 1 of treatment was used as a baseline measurement. After
the animals were euthanized, the tumors were dissected from the subcutaneous
location and measured using calipers in a similar manner. These measurements
were identified as end-of-study tumor volume values and are referred to as tumor size. Tumor weight was recorded to the nearest 0.01
mg. Tumor volume was measured by suspending the tumors in water and measuring
displacement. All tumors were fixed in 10% buffered formalin, processed histologically,
embedded in paraffin, sectioned, and stained with hematoxylin-eosin. The slides
were examined using light microscopy to confirm the presence of retinoblastoma.
ANALYSIS OF TOXICITY TO THE KIDNEYS
Kidneys were harvested immediately following euthanasia from 6 mice
in the 0.1 µg and 0.2 µg groups, 12 mice in the 0.3 µg group,
9 mice in the 0.6 µg group, and 3 mice in the 1.2 µg group. Each
kidney was assigned a unique identifying number, fixed in 10% buffered formalin,
and processed for light microscopy examination. Three hematoxylin-eosin–stained
sections of each kidney were examined. Renal calcification was assessed by
a single masked reviewer and rated as none, mild, moderate, or severe.
ANALYSIS OF BLOOD FOR HYPERCALCEMIA
Prior to euthanasia, serum was collected from the axillary veins of
all study mice and sent for calcium measurement by an independent commercial
laboratory (Marshfield Laboratories Inc, Marshfield, Wis).
SERUM VITAMIN D METABOLITE ANALYSIS
Prior to euthanasia, serum was collected from the axillary veins of
all study mice and sent for quantitation of serum concentrations of 1,25(OH)2D2, the active metabolite of 1-OH-D2.
This analysis was performed at BoneCare International using solid-phase extraction16 followed by high-performance liquid chromatography
purification and radioimmunoassay. The detection limit
for 1,25(OH)2D2 in this assay was 17 pg/mL (44.2 pmol/L). A minimum of
0.5 mL of serum was required to perform serum vitamin D metabolite analysis,
which necessitated the pooling of samples within dose groups.
STATISTICAL ANALYSIS
Tumor size, weight, and volume as well as animal weight were analyzed
using a 1-way analysis of variance (ANOVA) test to detect statistical differences
in tumor size among the groups. Pairwise comparisons were then performed to
detect statistical differences between particular dose groups. Tumor size,
weight, and volume were transformed to the log scale before analysis to obtain
approximately normal distributed residuals. Differences were considered significant
at P<.05.
RESULTS
PRELIMINARY TOXICITY STUDY
Mortality data for the preliminary toxicity study are shown in Figure 1. From
these results, we selected doses of 0.1 µg, 0.2 µg, 0.3 µg,
0.6 µg, and 1.2 µg per day for the dose-response efficacy study.
DOSE-RESPONSE EFFICACY STUDY
All animals in the 5 treatment groups and control group had visible
tumors at the start of treatment that remained observable until the end of
the study. There was no significant difference in tumor size between the groups
on day 1, when treatment was initiated. Mortality rates for all dose groups
appear in Figure 2. As expected
from the preliminary toxicity study, high mortality rates were observed in
the 0.6 µg and 1.2 µg dose groups. The 1.2 µg dose group
had only 3 surviving animals at the end of the treatment period. Because of
the small number of surviving animals (3 of 12), the 1.2 µg dose group
is not included in the data analyses.
At the end of the study, all tumors were harvested and confirmed to
be retinoblastomas using light microscopic examination. Tumor size showed
a strong inverse relationship to dose groups of 1-OH-D2
with statistically significant differences in tumor size between controls
and each drug group (P<.03) (Table 1). Tumor weight and volume showed similar strong relationships
with dose. Statistically significant differences in weight and volume existed
between controls and 0.2 µg and 0.3 µg dose groups (P<.004 and P<.003, respectively) (Table 1 and Figure 3). A decrease in tumor weight and volume was also seen in
the 0.6 µg dose group, but statistical significance was not reached,
possibly owing to the low survival rate of the animals in this group.
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Dose-Response Efficacy Study*
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Animal weight in the 0.2 µg and 0.3 µg dose groups at the
end of the study was found to be significantly different from that in controls
(Table 1). When compared with
baseline animal weights at the start of treatment, the control animals had
an average weight gain of 12.43%, the 0.1 µg group had an average gain
of 9.68%, the 0.2 µg group had an average gain of 2.43%, the 0.3 µg
group had an average weight loss of 5.70%, and the 0.6 µg group had
an average loss of 4.99%. Of note, tumor size, tumor volume, and tumor weight
were not significantly different between the 0.2 µg and 0.3 µg
groups (P = .41, P = .44,
and P = .41, respectively), whereas animal weight
was different between those groups at P = .002.
KIDNEY CALCIFICATION
Kidneys were harvested and histologically processed from 15 mice in
the control group, 6 mice each in the 0.1 µg and 0.2 µg groups,
12 mice in the 0.3 µg group, 9 mice in the 0.6 µg group, and 3
mice in the 1.2 µg group. One masked reviewer (R.J.G.) graded calcification
as none, mild, moderate, or severe. The calcification of all kidneys was rated
as none to mild with the exception of 1 kidney in the 0.3 µg dose group
(moderate), 2 kidneys in the 0.6 µg dose group (moderate), and 1 kidney
in the 1.2 µg dose group (moderate). No kidneys were rated as having
severe calcification.
SERUM VITAMIN D METABOLITE ANALYSIS
Serum obtained from the mice prior to euthanasia was pooled and analyzed
at BoneCare International for levels of 1,25(OH)2D2.
There was a strong relationship between dose and serum level of the drug (Figure 4). However, the available volume
of serum for analysis was limited, so statistical analysis could not be performed
(N = 8).
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Figure 4. Serum levels of 1,25-dihydroxyvitamin
D2 , the active metabolite of 1-OH-D2, measured
by BoneCare International, Madison, Wis. To convert serum values to SI units
(pmol/L), multiply by 2.6.
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COMMENT
The need for improved treatment alternatives for retinoblastoma is generally
acknowledged.17 The most widely used current
treatment, external beam radiation, is associated with a 35% or higher risk
of secondary cancers in patients with bilateral retinoblastoma studied during
a 30-year period.18-20
Radiation therapy also increases the risks of adverse visual consequences
as well as cosmetic and functional abnormalities. Large retinoblastoma tumors
and those with vitreous seeding respond poorly to radiation therapy and often
require enucleation. Chemotherapy, particularly triple therapy using carboplatin
with vincristine sulfate and teniposide, is increasingly being used for the
chemoreduction of tumors but can be mutagenic and is linked to an increased
risk of secondary cancers in treated patients.1, 20-21
In our study, we tested a vitamin D analogue, 1-OH-D2,
that was recently approved for human use against a human cell line of retinoblastoma
(Y-79) and that was subcutaneously injected into athymic mice. Previous studies
with this compound have focused primarily on the characterization and metabolic
action of the drug,13-14,22
phase 1 clinical trials for prostate cancer (George Wilding, MD, written communication,
June 2001), and its efficacy in the treatment of secondary hyperparathyroidism.23 This is the first study that describes the antineoplastic
properties of 1-OH-D2 in an animal model of retinoblastoma.
Our results indicate that 1-OH-D2, a compound not known
to be a mutagen, can inhibit tumor growth in a nontoxic dose range.
Using the data generated from the preliminary toxicity experiment, we
designed a study to examine combined dose-response and toxicity characteristics
in this mouse model. Animals receiving doses of 0.1 to 0.3 µg of 1-OH-D2 had similar survival percentages (68%, 65%, and 62%, respectively,
in each dose group) (Figure 2). With 1-OH-D2, the mortality rate in the control group was
15% and ranged from 32% to 38% in the groups receiving lower effective treatment
doses. Comparing this with treatment of similar mice with vitamin D2, the latter compound resulted in a 0% mortality rate in the controls
and a 46% mortality rate in the low-dose (2.8 mg/kg) group.6
This dose of vitamin D2 had a comparable tumor effect with 0.1
to 0.3 µg of 1-OH-D2. Looking at previous results
with calcitriol in athymic mice, we found a control mortality rate of 10%
in the control group and 60% for the therapeutic dose that was comparable
with 0.1 to 0.3 µg of 1-OH-D2.10
On the basis of mortality rate, 1-OH-D2 appears less toxic
than vitamin D2 or calcitriol.
We suggest that the mortality rates in this study can be attributed
to 3 factors in addition to the calcemic effect of the drug: (1) the learning
curve of the personnel treating the animals (for 1-OH-D2,
gastric tubes were used to deliver the drug as opposed to intraperitoneal
injection for vitamin D2 and calcitriol); (2) the sensitivity of
the immunocompromised athymic mouse model; and (3) the stress of the 0.1%
calcium diet (a full-calcium diet had previously been used with vitamin D2 and calcitriol). Current studies in our laboratory using similar doses
of 1-OH-D2 in mice with intact immune systems have resulted
in lower mortality rates (D. M. A., unpublished data, August 2001).
Tumor size, weight, and volume were observed to be reduced the most
significantly in the 0.3 µg dose group when compared with controls.
However, reduction in animal weight was also highly significant in the 0.3
µg dose group vs the control group. Tumor size, tumor volume, and tumor
weight were not significantly different between the 0.2 µg and 0.3 µg
groups, whereas animal weight was significantly different from controls in
those groups (Table 1). This may
indicate that between the doses of 0.2 µg and 0.3 µg in this model,
there is a relatively large difference in the level of toxicity but a small
difference in tumor inhibition.
Inhibition of tumor growth with corresponding low toxicity is an ideal
outcome when searching for alternatives for cancer treatments. As noted previously,
other vitamin D compounds and analogues have been studied in our laboratory
and are inhibitors of retinoblastoma in studies of Y-79 cell lines and mouse
models.6, 9-10 We
demonstrated that, although vitamin D2 and calcitriol (1,25-dihydroxycholecalciferol)
were effective in inhibiting retinoblastoma tumors in athymic and transgenic
mice, they were highly toxic.6, 10
All groups treated with these agents experienced hypercalcemia, weight loss,
and death. A synthetic analogue of calcitriol, 16,23-D3 (1,25-dihydroxy-16-ene-23-yne-vitamin
D3), has shown an impressive tumor-inhibiting effect in previous
experiments.12, 15, 24
In our studies with 16,23-D3 in the athymic Y-79 retinoblastoma
model and a transgenic mouse model, toxicity was a limiting factor in dose
selection.15, 24 In comparing the
present study with a similar study of 16,23-D3 in the same mouse
model,15 1-OH-D2 appears
to be similar in tumor reduction capability at 0.3 µg/d in comparison
with 0.5 µg/d of 16,23-D3 (ie, an approximately 44%-45% reduction
in mean tumor size when compared with controls) while having a similar survival
rate to animals treated with 16,23-D3.
The mechanism of action of 1-OH-D2 and other synthetic
analogues and vitamin D compounds in limiting tumor growth is not fully understood.
Calcitriol and its analogues inhibit cellular proliferation in several malignant
cell types including retinoblastoma and breast, colon, renal, and lung carcinomas.25-28 It
is hypothesized that the antiproliferative effects of these compounds are
mediated by a vitamin D receptor–linked mechanism, although exceptions
have been reported.29-32
Considerable evidence indicates that the antineoplastic and differentiating
effects of vitamin D compounds result from alterations in the fundamental
cellular processes of proliferation, differentiation, and apoptosis. The resulting
key biochemical events are related to activation of cyclin-dependent kinase
inhibitors such as p21.33-36
Calcitriol and vitamin D analogues can induce apoptosis in leukemic
(HL60) cells as well as human breast cancer and colon cancer cell lines.37-39 Recent studies have
shown that human retinoblastoma and retinoblastoma cell lines are extremely
susceptible to p53-mediated apoptosis,40 and
preliminary studies in our laboratory have shown that Y-79 xenografts in athymic
mice treated with 16,23-D3 result in tumor growth attenuation through
increased apoptotic cell death (Robert W. Nickells, PhD, and Daniel M. Albert,
MD, unpublished data, November 1999). We hypothesize that 1-OH-D2 has a similar mechanism of action against retinoblastoma tumors in
the athymic mouse, and studies are planned to determine if this is correct.
We believe that 1-OH-D2 has potential value in the treatment
of human retinoblastoma; a phase 1 clinical trial is being developed.
AUTHOR INFORMATION
Submitted for publication December 13, 2001; final revision received
February 1, 2002; accepted February 6, 2002.
This study was supported by grant RO1 EYO1917 from the National Eye
Institute, National Institutes of Health, Bethesda, Md (Dr Albert), and Research
to Prevent Blindness, New York, NY.
We acknowledge BoneCare International Inc for the generous gift of 1-OH-D2, additional expenses for animal care costs, and expert serum metabolite
analysis by John Banach, BSMT, and Leon W. Levan, PhD.
Corresponding author and reprints: Daniel M. Albert, MD, MS, F4/338
Clinical Science Center, 600 Highland Ave, Madison, WI 53792-3220 (e-mail: dalbert{at}facstaff.wisc.edu).
From the Departments of Ophthalmology, Rush University (Dr Grostern)
and Northwestern University (Dr Bryar) Chicago, Ill, the Department of Ophthalmology
& Visual Sciences, School of Medicine (Drs Lissauer and Albert and Mss
Zimbric, Darjatmoko, and Lokken), and Department of Biostatistics (Dr Lindstrom),
University of Wisconsin, Madison, and BoneCare International Inc, Madison
(Dr Strugnell).
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