Veterinary Therapeutics • Vol. 4, No. 1, Spring 2003
Preclinical Tolerance and Pharmacokinetic
Assessment of MU-Gold, a Novel
Chemotherapeutic Agent, in Laboratory Dogs*
Mary Lynn Higginbotham, DVMa
Carolyn J. Henry, DVM, MSa
Kattesh V. Katti, MS, PhD, FRSCb
Stan W. Casteel, DVM, PhDc
Patricia M. Dowling, DVM, MSd
Nagavarakishore Pillarsetty, MSb
aDepartment
of Veterinary Medicine and Surgery
for Radiological Research
cVeterinary Medical Diagnostic Laboratory
University of Missouri-Columbia
Columbia, MO 65211
dVeterinary
■ ABSTRACT
MU-Gold, tetrakis (trishydroxymethyl)
phosphine gold(I) chloride, a novel gold compound, has cytotoxic effects against human
androgen-dependent and -independent prostatic,
gastric, and colonic carcinoma in cell culture
and against malignant lymphoma in rodent
models. A pilot study was conducted to evaluate
the tolerance and pharmacokinetic properties
of MU-Gold in normal dogs in anticipation of
clinical trials in cancer-bearing dogs. MUGold (10 mg/kg) was administered by IV
injection to three purpose-bred dogs. Serum
was collected from all dogs for measurement of
gold levels via atomic absorption spectrometry.
In addition, complete blood counts and biochemical profiles were monitored for Dogs 2
and 3 every 7 days for 30 days. A twocompartment IV bolus model with first-order
kinetics, mean elimination half-life of approximately 40 hours, and mean volume of distribution of 0.6 L/kg was established. Serum gold
concentrations ranging from 10 to 50 µg/ml
were sustained for 2 to 3 days with no clinically significant toxicities observed. Based on in
vitro results in earlier studies and preliminary
pharmacokinetic data collected in the present
study, Phase I clinical trials should be conducted to define the optimal dosage, dose-limiting
toxicities, and other characteristics of MUGold that will be used to design Phase II
clinical trials.
bCenter
*Funding for this study was provided by the University of Missouri-Columbia, Department of
Veterinary Medicine and Surgery Committee on
Research, Columbia, MO.
76
Physiologic Sciences
University of Saskatchewan
Western College of Veterinary Medicine
52 Campus Drive
Saskatchewan, Saskatoon, Canada S7N 5B4
■ INTRODUCTION
The role of chemotherapy has been defined
as induction therapy, or therapy used as the
primary treatment for disease in which no
adjunctive therapies exist, adjuvant therapy for
local disease, primary treatment for local disease,
and site-specific therapy for sanctuary sites of
particular cancers.1 Unfortunately, the ability
to completely eradicate disease is limited both
M. L. Higginbotham, C. J. Henry, K. V. Katti, S. W. Casteel, P. M. Dowling, and N. Pillarsetty
P(CH2OH)3
Au
+
P(CH2OH)3
(HOH2C)3P
P(CH2OH)3
Figure 1. MU-Gold chemical structure.
by toxicity to normal tissues, such as bone
marrow and the gastrointestinal system, and
development of resistance of the neoplastic
cells to chemotherapeutic agents.1 Although
great strides have been made in the ability to
treat cancer, there is still a need to develop new
compounds with lower levels of toxicity and
with known mechanisms of action that can be
used in combination treatment protocols. In
particular, the development of drugs that are
not affected by multidrug resistance will improve
the ability to treat neoplastic disease.
Gold compounds are a class of drugs that are
most commonly used in the treatment of
rheumatoid arthritis. Although it is not precisely
known how these gold agents work, they appear
to reduce inflammation and occasionally may
bring about remission of the disease. These
agents have also been used for the treatment of
other rheumatic diseases and various inflammatory skin disorders.2,3 In vitro and in vivo antitumor activity have also been described for gold
compounds.3–5 Although the mechanism of action is not completely understood, one possibility is a preferential inhibition of DNA synthesis,
similar to that exhibited by the platinum anticancer drug cisplatin.3 The oral gold compound
auranofin showed in vivo activity
against leukemia cells but was inactive against various solid tumor
models.4,6 Various digold complexes, including [Au(dppe)2]Cl2 were
later found to have antitumor activity but did not enter clinical trials
due to cardiotoxicity.7 In fact, to the
authors’ knowledge, no clinical trials
CI –
investigating gold compounds for
use as anticancer agents have been
completed to date.8,9 Gold agents
that have been used clinically and
experimentally for other applications have produced toxicities including cardiotoxicity,10 nephrotoxicity, thrombocytopenia,11 skin disorders, and
possible hepatotoxicity.12
In an effort to develop new radionuclides for
imaging and therapy, researchers at the University
of Missouri-Columbia investigated a nonradioactive compound, MU-Gold (Figure 1), for
comparison with radioactive gold compounds
in tissue distribution studies.13 Using radiolabeling
techniques, they found that this compound
remained in the peripheral circulation too long
to be of benefit for radiopharmaceutical applications. However, slow clearance may be beneficial
for a compound intended for use as a
chemotherapeutic agent in terms of achieving
and maintaining adequate drug concentration.
Further collaborative investigation with Dr.
Hideo Kameia of Aichi-Gaikun University,
Japan, demonstrated that MU-Gold has anticancer activity in vitro against several human
tumor cell lines, including androgen-dependent
and -independent prostatic carcinoma, gastric
carcinoma, and colonic carcinoma. The compound slows cell growth in both tissue culture
and rodent models of cancer, and has been
shown to elongate the G1 phase (for gap 1) of
the cell cycle.14 The G1 phase is considered the
aDeceased.
77
Veterinary Therapeutics • Vol. 4, No. 1, Spring 2003
initial phase of the cell cycle in which cellular
modifications occur in preparation for entry
into the synthesis phase (S phase) of the cell cycle.15 In vitro and in vivo studies of MU-Gold
demonstrated considerable tumor suppression
in cell culture and remarkable increases in survival time of sarcoma-bearing mice. This agent
was less toxic than cisplatin given at similar
dosages in rodent models.14 It was determined,
therefore, that MU-Gold may have potential as
a new cytotoxic agent.14
Despite the absence of adverse side effects in
rodent trials, specific toxicity testing of MUGold has not yet been performed for any
species. Likewise, pharmacokinetic and pharmacodynamic studies of this agent are lacking. The
currently marketed parenteral gold compounds,
gold sodium thiomalate and aurothioglucose,
are administered by IM injection due to poor
water solubility. The drugs accumulate in the
reticuloendothelial cells of the lymph nodes,
kidneys, liver, spleen, and bone marrow.2 MUGold is unique among the gold compounds in
that it is a water-soluble compound,13,16 thus
permitting IV administration (Figure 1).
The purpose of this preliminary pilot study
was to evaluate the pharmacokinetics and tolerance of MU-Gold following administration
of a single dose at 10 mg/kg IV to three
healthy, purpose-bred dogs. The study presented
here is considered the first step of preclinical
development of this novel cytotoxic agent.
Data collected in this preliminary investigation
will be used to establish the model for pivotal
toxicity and pharmacokinetic studies in dogs.
■ MATERIALS AND METHODS
Three adult male, mixed-breed, purpose-bred
dogs weighing 25.2 to 29.7 kg each were obtained for this study. This study was approved by
and conformed to guidelines of the University of
Missouri Animal Care and Use Committee.
Baseline evaluations included a physical ex-
78
amination, complete blood count (CBC),
serum biochemical profile, complete urinalysis, fecal flotation for parasite ova, heartworm
status determination by Knott and ELISA
tests, and Rickettsia rickettsia, Ehrlichia canis,
and Ehrlichia platys titers by immunofluorescent assay. Components of the serum biochemical profile included glucose, urea nitrogen
(BUN), creatinine, sodium, potassium, chloride, total carbon dioxide, anion gap, albumin,
total protein, globulin, calcium, phosphorus,
cholesterol, total bilirubin, alanine aminotransferase (ALT), alkaline phosphatase, and amylase. Dogs with azotemia (BUN greater than
28 mg/dl, creatinine greater than 1.8 g/dl),
isosthenuria (urine specific gravity 1.008 to
1.012), increased ALT activity (greater than
122 U/L), neutropenia (absolute number of
cells less than 3,000 per dl), thrombocytopenia
(absolute number of cells less than 200,000 per
dl), positive rickettsial or ehrlichial titers
(greater than 1:80), or positive heartworm tests
were excluded from the study.
Seventy-two hours before drug administration,
6-Fr jugular catheters were placed for the
administration of MU-Gold and for blood
sampling. Sedation of the dogs for jugular
catheter placement was achieved using morphine (0.4 mg/kg), xylazine (0.4 mg/kg), and
atropine (0.04 mg/kg) administered by IM injection. After jugular catheter placement,
yohimbine (one-quarter to one-half of the volume of xylazine) was administered IM to reverse
the effects of xylazine. Heart rate, respiratory rate,
and capillary refill time were monitored
throughout the sedation period. Jugular catheters
were kept patent by flushing with heparinized
saline between placement and sampling times.
Because MU-Gold had not previously been
administered to dogs, this study was performed
in two phases. Dog 1 received a single dose of
MU-Gold (10 mg/ml concentration) by IV
injection (Table 1). Subsequently, it was deter-
M. L. Higginbotham, C. J. Henry, K. V. Katti, S. W. Casteel, P. M. Dowling, and N. Pillarsetty
TABLE 1. Dosing Information for MU-Gold Administered by IV Injection to Healthy Dogs
Dog
Weight (kg)
Dosage (mg/kg)
MU-Gold
Concentration (mg/ml)
Dose (mg)
Total Volume
(ml)
1
2
3
29.7
26.5
25.2
10
10
10
10
100
100
297
265
252
29.7
2.65
2.52
mined that this concentration was unsuitable because of the length of time required to inject this
volume and the inability to obtain Time 0 (designated to be the moment the injection of material
was complete) samples. Therefore, MU-Gold was
reformulated at a concentration of 100 mg/ml for
Dogs 2 and 3. MU-Gold was given to these two
dogs as a single dose at 10 mg/kg IV (Table 1).
Serum was collected for gold measurement
at 0, 3, 6, 9, 15, 25, 40, 60, 90 minutes and 2,
3, 4, 5, 6, 7, 8, 12, 18, 24, 48, and 72 hours
after administration. Serum was separated within
30 minutes after collection and stored at –20˚C
until analysis. Storage of serum has been shown
to have no significant effects on the quantitative
measurement of gold;17 therefore, storage of samples for batch analysis was considered appropriate.
Elemental gold concentrations in serum were
quantified using atomic absorption spectrometry,
which has previously been established as an
effective means for serum gold quantification.17–19 Gold concentrations were measured
using a cathode adjusted to give maximum
sensitivity at 242.8 nm, with the cathode lamp
carrying a current of 14 to 15 mAmp. Gold
concentrations were calculated from the height
of the recorded peaks.17 Serum biochemical
profiles and CBCs were evaluated every 7 days
for 30 days after injection (Dogs 2 and 3) for a
preliminary assessment of acute toxicity related
to drug administration.
The pharmacokinetic profile was established
using computer software for compartmental
modeling and kinetic analysis as well as for
calculating noncompartmental analysis parameters (WinNonlin, Pharsight Corporation).
Mean values for pharmacokinetic parameters
were calculated.
■ RESULTS
Physical examinations, serum biochemical
profiles, and CBCs performed before jugular
catheter placement and MU-Gold administration were considered within normal limits for
all three dogs. Dog 2 exhibited a stress leukogram with mild elevations in both neutrophils
and monocytes; however, these findings were
considered inconsequential, and the dog was a
suitable candidate for treatment and evaluation.
Tests for heartworm and gastrointestinal parasites
were all negative, and titers for R. rickettsia, E.
canis, and E. platys were all less than 1:80.
MU-Gold, as it was administered to Dog 1
was formulated at 10 mg/ml; however, the formulation was subsequently modified to 100
mg/ml for Dogs 2 and 3 because of difficulties
with administering the large volume (approximately 30 ml) of solution in a given time frame
to Dog 1 (Table 1).
Clinical signs of toxicity were not seen immediately following administration of MUGold or within the 30 days the animals were
evaluated. There were no apparent signs of toxicity in any serum biochemistry or CBC variables;
however, slight increases in BUN values, not exceeding the normal range, were noted for Dogs
2 and 3 while the creatinine values remained relatively unchanged (Figure 2).
79
Veterinary Therapeutics • Vol. 4, No. 1, Spring 2003
MU-Gold at 10 mg/kg in preparation for the design of similar large30
scale studies. The data collected in
the present study can be used to
25
design treatment protocols for fu20
ture in vitro and in vivo studies of
15
this compound. Novel chemotherapeutic agents for treatment of
10
Dog 2
cancers with decreased toxicity are
5
Dog 3
needed to improve our ability to
0
treat malignancies. Because the dog
0
7
14
21
28
offers a model of spontaneously ocTime after Treatment (days)
A
curring cancers that, in some cases,
translate to human disease,20,21 evaluation of the tolerance and phar1
macokinetic properties was the first
0.9
0.8
step toward further evaluation of
0.7
MU-Gold for animal use.
0.6
Treatment of one of the three
0.5
dogs with MU-Gold formulated at
0.4
10 mg/ml demonstrated that the
0.3
time required for infusion of this
0.2
Dog 2
formulation was prolonged, and
0.1
Dog 3
the formulation was subsequently
0
0
7
14
21
28
modified to 100 mg/ml before
Time after Treatment (days)
B
treating Dogs 2 and 3. This more
concentrated formulation was satFigure 2. Serum (A) urea nitrogen and (B) creatinine levels measured
at weekly intervals after IV administration of MU-Gold to healthy dogs. isfactory for administration of the
gold compound providing for
more accurate sampling for detection of gold levels in serum. Results indicated
A two-compartment IV bolus model with
first-order kinetics best described the eliminathat variability in the concentration of the gold
tion properties of MU-Gold for the dogs in
compound did not affect the serum gold conthis study. The mean elimination half-life (t1⁄2)
centrations achieved.
was 40 hours and the mean volume of distriThe relatively low volume of distribution
and long plasma t1⁄2 for MU-Gold in these three
bution was 0.6 L/kg. Serum gold concentranormal dogs may be beneficial in terms of its
tions ranged from 10 to 50 µg/ml, and these
ability to act as a cytotoxic agent for milevels were sustained for 2 or 3 days (Figure 3).
crometastatic disease. Unfortunately, there is
■ DISCUSSION
no significant evidence that chemotherapeutic
The purpose of this study was to conduct a
agents are effective at controlling macropreliminary evaluation of the tolerance and
metastatic disease.22 Metastatic cells present in
pharmacokinetics following a single IV dose of
the vascular system may be exposed to the cySerum Creatinine (mg/dl)
Serum Urea Nitrogen (mg/dl)
35
80
M. L. Higginbotham, C. J. Henry, K. V. Katti, S. W. Casteel, P. M. Dowling, and N. Pillarsetty
in preparation for large-scale toxicity and organ distribution studies.
Dog 1
In addition, this study was de50
Dog 2
signed to evaluate tolerance and
Dog 3
40
not delayed toxicity. Delayed toxi30
cities are possible with this compound and further in vivo studies
20
are required to evaluate for poten10
tial long-term toxicity associated
with MU-Gold. Data were insuffi0 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78 81 84 87 90
cient for statistical analysis of pharmacokinetic properties or laboratoTime after Treatment (min)
A
ry tests. Future studies with larger
numbers of dogs will be designed
to allow for statistical analysis of the
20
Dog 1
data of interest.
18
Dog 2
16
Further pharmacokinetic evalua14
Dog 3
tion of MU-Gold elimination and
12
organ distribution following re10
peated dosing is warranted. Addi8
tional studies to determine dose6
limiting toxicities as well as studies
4
to determine the optimal dosage of
2
MU-Gold are planned. In addition,
0
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72
in vitro studies to reveal the mechTime after Treatment (hr)
B
anism of action of MU-Gold are
needed before this compound can
Figure 3. Serum gold levels (A) 0 (immediately after material was injected)
to 90 minutes and (B) 2 to 72 hours after IV administration of different be considered for practical use in
concentrations of MU-Gold to healthy dogs. Dog 1 received 10 mg/ml con- the clinic setting. If the hypothesis
of G1-phase arrest proves to be corcentration. Dogs 2 and 3 received 100 mg/ml concentration.
rect, there is potential for synergism
totoxic agent over a more prolonged period of
between MU-Gold and either nonphase-speciftime as compared with drugs with a short t1⁄2.
ic or G1-phase–specific cytotoxic drugs, which
Gold levels that correspond to previously dewill cause the arrest of cycling cells in the G1
termined cytotoxic concentrations (in vitro)14
phase and expose them to a compound that is
were sustained for 2 to 3 days in these dogs.
cytotoxic within this phase. If such synergism is
The authors acknowledge the small number
found to exist, it would potentially increase the
of dogs and brief posttreatment evaluation
overall efficacy of cytotoxic therapy to cells that
period are shortcomings to this study. However,
are sensitive to MU-Gold. Further in vitro
because MU-Gold had not yet been evaluated
evaluation for cytotoxicity of MU-Gold against
in this species, the minimum number of subhuman non-Hodgkin’s lymphoma is ongoing
jects necessary was used to determine prelimias well as a Phase I clinical trial in dogs
nary pharmacokinetic properties and tolerance
with lymphoma.
Gold Level (µ/ml)
Gold Level (µ/ml)
60
81
Veterinary Therapeutics • Vol. 4, No. 1, Spring 2003
■ CONCLUSION
In this preliminary pilot study, MU-Gold
administered at 10 mg/kg by IV injection to
three normal laboratory dogs was well tolerated
and did not produce signs of toxicity over a
30-day period. Administration of MU-Gold
possesses pharmacokinetic properties consistent
with a two-compartment IV bolus model with
first-order kinetics. Based on in vitro results in
earlier studies and preliminary pharmacokinetic
data collected in the present study, Phase I
clinical trials should be conducted to define
the optimal dosage, dose-limiting toxicities,
and other characteristics of MU-Gold that will
be used to design Phase II clinical trials.
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