pharmacokinetics of 9-methoxy-n, n-dimethyl-5...
TRANSCRIPT
Vol. 1, 831-837, August 1995 Clinical Cancer Research 831
Pharmacokinetics of 9-Methoxy-N, N-dimethyl-5-nitropyrazolo[3,4,5-
kl]acridine-2(6H)-propanamine (PZA, PD 115934, NSC 366140) in
Mice: Guidelines for Early Clinical Trials1
Brenda J. Foster,2 Richard A. Wiegand,
Patricia M LoRusso, and Laurence H. Baker
Wayne State University School of Medicine, Department of
Internal Medicine, Division of Hematology and Oncology, Detroit,
Michigan 48202-0188
ABSTRACT
Pharmacokinetic studies that consisted of measuring
the plasma drug profile, tissue drug distribution, and elim-
ination in urine and feces were performed in female
C57BL/6 x DBA/2 (hereafter called B6D2F1) and male
B6D2F1A/2 and C57BL/6 x CH3 (hereafter called B6C3F1)
mice following treatment with a 1-h i.v. infusion of the PZA,
PD115934 (NSC 366140). This drug is the first of a new classof cytotoxic agents and was selected for clinical trials be-
cause of both its broad antitumor activity in vivo against
murine solid tumors and human xenografts, and its in vivo
toxicity profile that was predictable based on drug dose and
schedule of administration. The pharmacokinetic results ob-
tamed here in mice have been used to facilitate the doseescalations during the Phase I trial and to determine phar-
macokinetic drug exposure targets for its acute and sub-
acute toxic effects. Plasma samples from three to four mice
per time point were pooled, and then individual tissue sam-
ples from the same mice were collected at specified times
following treatment. All samples were prepared using solid-
phase extraction and assayed using high pressure liquid
chromatography. The acute dose-limiting toxicity was neu-
robogical and occurred immediately after treatment at 300
mg/rn2. The peak plasma level range at the acute maximum
tolerated dose was 1040-1283 ng/ml. Thus, peak plasma
levels < 1000 ng/ml were the acute toxicity target. Variations
in the area under the plasma drug concentration X the timecurve were observed that did not appear to be related to sex
or age. The previously defined subacute dose-limiting toxic-
ity was myebosuppression that occurred at a maximum tol-
erated dose of 600 mg/rn2 (300 mg/rn2 X 2) in B6D2F1
females. Thus, the area under the plasma drug concentra-
tion X the time curve in B6D2F1 females at this dose (1048
�ig/ml x mm) was the area under the plasma drug concen-
Received 1/23/95; revised 4/17/95; accepted 4/19/95.
I This work was supported by Grants CA 46560 and CA 46560-04S1
awarded by the National Cancer Institute, Department of Health and
Human Services.
2 To whom requests for reprints should be addressed, at Department ofInternal Medicine, Division of Hematology/Oncology, Wayne State
University School of Medicine, P. 0. Box 02188, Detroit, MI 48202-
0188.
tration X the time curve target. Drug bevels were detected at
60 mm following treatment in all tissues examined with a
plasma:tissue ratio as high as 1:500. The organs with thehighest levels were kidney, pancreas, liver, lung, and brain.
Fecal excretion was low (range, 0.04-0.20% of the dose
administered) and was not clearly different between males
and females. Urinary excretion was higher (range, 5-28% of
the dose administered) and did show evidence of sex-rebated
differences, with male urinary drug excretion being higher
than female urinary drug excretion. The drug was �95%
protein bound. Preliminary evidence for drug metabolism
was found in urine and feces and will be further explored.
INTRODUCTION
Common solid cancers such as lung, colon, and breast
carcinomas are frequently diagnosed in stages that dictate sys-
temic treatment if cure or significant palliation is possible. Such
systemic treatment most often involves cytotoxic drugs, yet the
armamentarium of useful drugs, i.e., those expected to improve
quality of life and/or survival of the cancer patient, remains
limited. Thus, the search for drugs with preferential antitumor
activity for these cancers is a worthwhile endeavor.
Multiple classes of synthetic polycyclic aromatic corn-
pounds that contain a chromophore resembling the three-ringed
anthracenedione nucleus, shown in Fig. 1, have antitumor ac-
tivity, e.g., anthracyclines (1, 2), anthraquinones (3), and an-
thrapyrazoles (4). Some are used in standard regimens for treat-
ing advanced solid tumor cancers (1, 5). A newer class of
compounds, the PZAs,3 shown in Fig. 1, also contain a nucleus
resembling the anthracenedione as well as the addition of (a) a
pyrazole ring, (b) a nitro group, (c) a nitrogen substituted into
the middle ring, and (d) a variety of different substitutions
indicated by R2. The PZAs have shown preferential activity
against a broad spectrum of solid tumors as compared to leu-
kemias (6-8). Other interesting characteristics of the PZAs,
which may prove useful in treating solid cancers, include
antitumor activity against hypoxic and noncycling cells as well
as evidence suggestive of a lack of cross-resistance with Adria-
mycin-resistant cells (9, 10). The first compound from this
new class of antitumor drugs selected for clinical development was
9-methoxy-N, N-dimethyl-5-nitropyrazolo[3,4,5-kl]acnidine-2 (oH)-
propanamine (PZA,3 PD 115934, NSC 366140).
In preclinical toxicological evaluations, the dose-limiting
toxicities in rodents and dogs were neurological (lethargy,
ataxia, tachypnea, and convulsion) and myelosuppression (leu-
3 The abbreviations used are: PZA, pyrazoloacnidine; AUC, area under
the plasma drug concentration X time curve; HPLC, high-pressure
liquid chromatography; SPE, solid-phase extraction; Cl, clearance.
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Anthracenedione Ring System
Pyrazoloacridine Ring System
832 Pharmacokinetics of Pyrazoloacridine in Mice
�/CH3
�9-methoxy-N,N-dimethyl-5-nitropyrazolo-
I3,4,5-kllacridine-2(6H)propanamine
Fig. 1 Structures of the anthracenedione ring system, the PZA ring
system, and the 9-methoxy-N, N-dimethyl-5-nitropyrazolo[3,4,5-kl]acni-
dine-2(6H) propanamine.
copenia, erythropenia, and thrombocytopenia; Ref. 1 1). How-
ever, dose schedules predicted to be associated with lower peak
plasma levels than bolus treatment (1-24-h infusions, split
doses) allowed the administration of higher total drug doses (7,
11). Thus, the initial clinical schedule chosen to be studied was
a 1-h infusion. In preparation for the Phase I trial, plasma
pharmacokinetics, tissue distribution, and urine and fecal excre-
tion of PZA were studied in B6D2F1 female mice. These initial
studies were to determine peak levels, drug distribution, and
elimination, as well as the AUC at the maximum tolerated dose
in these mice. The latter has been proposed as a possible target
AUC for patients to guide dose escalation and facilitate Phase I
trials (12). The usefulness of this target would be assessed
during the Phase I trial. PZA was then studied using the same
dose and schedule in B6D2F, and B6C3F1 males after pharma-
cokinetic variations were noted at the early levels of the Phase
I trial to determine whether strain and sex variations existed.
This is a report of the pharmacokinetic studies in three mice
strains and will be used as a basis for comparison with early
clinical trial results.
MATERIALS AND METHODS
Drug. The PZA was supplied in powder form as the
monomethansulfonate salt (Mr 463.5) by the Developmental
Therapeutics Program of the National Cancer Institute (Be-
thesda, MD). The appropriate amount of drug was weighed and
then solubilized with sterile 0.9% NaCl or 5% dextrose in water.
Drug solution was made just prior to treatment for each exper-
iment.
Chemicals and Solvents. Al! chemicals and solvents
were either analytical reagent grade or HPLC grade. Methanol,
acetonitrile, and acetic acid were obtained from J. T. Baker, Inc.
(Phil!ipsburg, NJ). HC1 was obtained from Fisher Scientific
Company (Fair Lawn, NJ). Ammonium acetate was obtained
from Aldrich Chemical Company (Milwaukee, WI).
Animals. Female and male C57BL/6 X DBA/2 (hereaf-
ter called B6D2F1) and male C57BL/6 X C3H (hereafter called
B6C3F1) mice were obtained from the Frederick Cancer Re-
search and Development center of the National Cancer Institute
and were 85, 98, 112-116 days old, respectively, at the time of
treatment with PZA. The first generation (F,) hybrid mice were
used because they are cheaper and hardier than inbred mice and
because they were the hosts for the antiturnor studies (7). The
weight range ofthe mice at the time of treatment was 15-23g for
the females and 24-33g for males. Animals were kept in stan-
dard cages and allowed to consume a standard pellet diet and
water ad libitum until time of treatment with PZA.
Treatment and Sample Collection. Each mouse re-
ceived, based on its weight, either 150 or 300 mg/m2 (50 or 100
mg/kg) PZA by 1-h infusion into a tail vein using the technique
previously described (7). Upon completion of the infusion, the
mice were placed in cages pre!abe!ed with the time of blood
collection (three to four mice per time point), except for mice
designated for the 8-, 12-, or 24-h time points. These latter mice
were placed in metabolic cages (Lab Products, Inc., Maywood,
NJ) for collection of urine and feces until the time of blood
collection. Urine and feces were each separately pooled over the
24-h time period following treatment. Blood collection time
points were 5, 10, 15, 30, 60, 120, 240, 480, or 720 and 1440
mm following completion of the infusion. Blood was collected
by open chest intracardiac puncture from each mouse then
immediately transferred to a heparinized microtest tube. Test
tubes containing heparinized blood were centrifuged for 10 mm
in a fixed angle microcentrifuge. Plasma samples were pooled
based on the blood sample collection time point.
After blood collection, liver and kidneys were removed at
30, 60, 240, and 1440 mm following treatment, and lungs, heart,
pancreas, spleen, thigh muscle and brain were removed at 60
and 1440 mm after treatment. Samples of control plasma, urine,
feces, and organs were obtained from mice of the same strain,
sex, and age that had not received treatment with PZA. All
samples from controls and treated mice were frozen immedi-
ately after collection and stored (-20#{176}C) until time of sample
preparation and analysis. Samples were stored for up to 6
months, and PZA has demonstrated stability under these storage
conditions for > 1 year. This stability is based on the analysis of
control plasma, urine, and feces samples immediately after the
addition of a known quantity of PZA and after storage under
similar conditions at 4-month intervals up to 24 months of
storage.
Sample Preparation. Plasma samples were slowly
thawed in an ice-water bath, and then a 0.5-mb aliquot was
deproteinized using chilled methanol (1:2, v/v). Urine and feces
samples were similarly thawed and diluted with deionized water
(1:9; v/v, w/v, respectively) prior to the addition of methanol.
Organs were allowed to thaw, then weighed, and tissue homo-
genates (w/v) were made using buffer (Tris-HC1, pH 7.4, or
phosphate buffered 0.9% NaCl, pH 7.4). The tissue homoge-
nates were deproteinized in a manner similar to plasma. The
methanolic mixtures of plasma, urine, feces, and tissue homo-
genate were chilled on ice for 10 mm, centrifuged at 1000 X g
for 10 mm, and the methanolic supernatant of the plasma, feces,
and tissue homogenate mixture was removed. Deionized water
(5 ml) was added to the methanolic supernatants and urine/
methanol mixture. The drug was then removed from each sam-
ple by SPE using a 1.0-ml cyano column (J. T. Baker, Inc.). The
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Clinical Cancer Research 833
SPE column was solvated using 2 ml methanol followed by 5 ml
methanol/water (2:5, v/v). Five ml of the water/methanolic
sample mixture were passed through the solvated column, and
the liquid was discarded. One ml of HCI (10.2 N)/methanol
mixture (1:19, v/v) was used to elute the drug. The eluate was
dried at 37#{176}Cunder a gentle stream of N2 and then reconstituted
in mobile phase (250 p.1) for injection (100 p.1).
in Vitro Plasma Protein Binding. The PZA was dis-
solved in freshly prepared control mouse plasma, human plasma
from four healthy volunteers, and buffer (0.25 M ammoniurn
acetate, pH 3.5) at concentrations of0.25, 0.5, 0.75, 1.0, 5.0, and
10 p.g/ml. The concentrations in buffer solution were used both
as nonfiltered controls and to determine the degree of nonspe-
cific binding of drug to the filter of the separation system. A
I .0-mb aliquot of each sample was incubated at 37#{176}Cfor 1 h,
then 0.5 ml of each was placed onto an Arnicon Centrifree
micropartition system (Amicon Division, W. R. Grace and Co.,
Beverly, MA) and centrifuged for 30 mm at 3000 X g using a
fixed angle rotor. Aliquots (100 pA) of the ultrafiltrate and
unfiltered buffer concentrations were assayed directly (without
sample preparation as described above) using the HPLC assay
described below. The percentage of PZA protein binding was
determined by comparing the peak area of the plasma ultrafib-
trate to the peak area of the unfiltered buffer control using the
equation:
% protein binding = 100 X 1
Iconcentration in filtered sample�
- [concentration in unfiltered sample]
after correction for nonspecific drug binding to the filter using
the results obtained from the drug dissolved in buffer following
ultrafiltration.
HPLC Apparatus and Assay. The HPLC system con-
sisted of a Waters Maxima Workstation and chromatography
program (Waters, Milford, MA), a WISP 710B autosampler,
two Model SlOB solvent pumps, and a Model 773 detector
(Kratos Inc., Westwood, NJ) set at 460 nm. The analytical
column consisted of a 0.46- x 15-cm ultrasphere cyano column
(Beckman Instruments, Inc., San Ramon, CA) fitted with a
resolve cyano precolumn cartridge in a Guard-Pak (Millipore
Corporation, Milford, MA). The mobile phase was 90% of 0.25
M ammonium acetate (pH 3.5)/i0% acetonitrile with a flow rate
of 1.0 ml/min and a run time of 20 mm in ambient temperature.
Under these conditions, the PZA peak eluted near 15 mm. The
lower limit of quantitation was 50 ng/mb, and the assay was
linear over the range tested (up to 20,000 ng/ml). PZA concen-
tration was quantitated using a standard curve (five to six
concentrations) constructed from the results of the monorneth-
ansubfonate salt of PZA dissolved in control matrix (plasma,
urine, feces, tissue-specific organ) and extracted in a manner
similar to the samples from treated mice. The standard curve
linear regression range was 0.9952-0.9999. Assay validations
were accomplished using run standards (drug dissolved in mo-
bile phase) analyzed after every four samples. The intraassay
coefficient of variation range was 0.11-1.93%, and the interas-
say coefficient of variation was 5.25%. Drug recovery from the
SPE column was determined by using drug dissolved in the
mobile phase as control and was >90% over the range of
50-10,000 ng/ml and >80% from 10,000 to 20,000 ng/ml.
Representative HPLC tracings are shown in Fig. 2, a (blank) and
b (50 ng/mb).
Pharmacokinetic Data Analysis. The pharmacokinetic
data from plasma were analyzed by a computer-generated non-
linear least-squares regression analysis with a weighting of l/(y
+v)2 (13, 14). The computer program CRVF!T (kindly pro-
vided by Dr. L. Hart, Institute of Cancer Research, Sutton,
Surrey, United Kingdom) was used to facilitate these calcuba-
tions. The data points were fitted to a rnonoexponential model to
facilitate comparisons using the equation:
C = Ae’#{176}’
where C is the plasma concentration of the PZA at time t, A is
the concentration constant, and a is the first order rate constant
(15). The AUC was determined using the trapezoidal rule. The
plasma Cl was calculated using the equation:
Cl = Dose/AUC.
The volume of distribution at steady state (V��) was calculated
using the equation:
Vss = Dose/A.
The half-life (t,,,) was calculated from the first-order rate con-
stant using the equation:
�I/2 0.693/first-order rate constant.
The computer program, PK2 (kindly provided by Dr. D. Newell,
Newcastle University, Newcastle-Upon-Tyne, United King-
darn) was used to facilitate calculations of Cl, � and t112.
RESULTS
Plasma Pharmacokinetics. A representative graph of
plasma concentrations (B6D2F, males) and the corresponding
monoexponential line are shown in Fig. 3. The plasma PZA
pharmacokinetic summary in mice is shown in Table 1 . Al-
though the peak plasma bevels from each strain were similar
following treatment with 300 mg/rn2, the AUC variation was
more than 2-fold. The corresponding Cl and t,,2 also varied. The
ti!2, AUC, and Cl in B6D2F, female mice indicated dose bin-
earity at the two doses of 150 and 300 mg/rn2.
Tissue Distribution. Summaries of simultaneous plasma
and tissue levels of PZA are given in Tables 2 and 3. Tissue
bevels, except at the 1440-mm time point for heart and muscle,
were consistently higher than those detected at the simultaneous
time point in plasma. Kidney, liver, pancreas, and lung had the
highest levels. The levels at 60 mm increased over the 30-mm
levels in liver and kidney, then declined thereafter. Brain levels
were easily demonstrated, which indicate that PZA does cross
the blood-brain barrier.
Urinary and Fecal Excretion. The percentage of the
PZA dose recovered in the urine and feces of mice during the
first 24 h after treatment is shown in Table 4. Urinary excretion
consistently exceeded fecal excretion, and fecal excretion was
highest in B6C3F, males. Urinary excretion in males was higher
than in females.
Research. on June 12, 2018. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
a
Cl)
0
> 2.54-so
x
2.53-
Fig. 2 High-pressure liquid chro-matograms obtained followingsample preparation using the PZAassay. a, blank plasma and b, 50 2.55ng/mI PZA control.
Co
0> 2.54-
‘01�
x
2.53-
10000
E
� ::�-�-�
5 10 15
Minutes
CO�
C’)
N\ ‘I�‘m” “ �“#{176}‘ � ‘ � “-j �“ ‘ ‘ ‘ �‘.�‘V’#{149}I.’�’ � � ‘5�’,�’ ‘ ‘“ � .‘-‘ .,4. � , s.,..,_,.,_, � � . 5, 4,
Minutes
834 Pharmacokinetics of Pynazoboacridine in Mice
�vo � 500 1000 1500 2000
Time (minutes)
Fig. 3 Graph of plasma levels obtained from B6D2F, males followingiv. treatment with 300 mg/rn2 PZA and the corresponding monoexpo-nential line.
Protein Binding. The percentage of in vitro binding of
PZA to plasma proteins from mice and humans is shown in
Table 5. The percentage bound was 100% at the two lower
concentrations. There was only a very slight difference at the
other levels between mice and humans.
.) v� � � � � � . �.V&’� .� � ,�e- � �& ‘l� . � �
0 � � lb 15
DISCUSSION
The effort to select drugs for clinical development that are
preferentially active against solid tumors is of major interest as
a result of the paucity of currently available drugs that have
antitumor activity which results in improved quality of life
and/or overall survival in patients with advanced common solid
tumors. The ‘ ‘soft-agar-cobony formation disc-diffusion’ ‘ assay
developed by Corbett and coworkers (16, 17) involves simulta-
neous use of munine leukemia and solid tumor cells. This was
cost effective when compared to other discovery methods (18)
and formed a basis by which a large number of compounds
(synthetics and natural products) could be evaluated relatively
quickly, with the intent to select agents that showed preferential
activity against the solid tumor cells for further evaluation and
possible clinical development.
The PZAs are a new class of polycyclic compounds that
were first described by Sebolt et a!. (6) as having solid tumor-
selective antitumor activity. This has been confirmed by others
in different tumor cells of murine (7) and human (8) origin.
However, the cytotoxicity spectrum of the PZAs is not limited
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B6D2F, female
B6D2F1 male
B6C3F, male
300
300
300
Dose
(mg/m2)
Time
(mm)
Plasma”
(ng/ml)
Liven
(p.gjg5)
Kidney(p�gfgb)
150 30
60
240
1440
30
60
240
1440
30
60
240
1440
30
60
240
1440
625
578
306
40
802
796
480
66
1090
930
819
310
986
630
585
227
108
120
96
3.6
168
160
156
48
164
196
120
24
76
156
128
52
144
168
60
5.4
198
186
120
1.8
488
492
200
80
378
468
372
132
4’ Plasma results are from pooled plasma (three to four mice per time point), and tissue results are the mean values of tissues from three to four
mice per time point.
1� Tissue results are expressed as p.g/g wet weight.
Clinical Cancer Research 835
Ta ble I Plas ma PZ A pharmaco kinetics summar y in mice foll owing treatment with a 1 -h iv. infusion
Dose
mg/rn2Total
(mg”)
Peak
(ngjml)
t,12
(h)
AUC(p.g/ml X mm)
Cl(mb/mm)
V�
(liters/kg)
B6D2F1 female 150
300
1.0
2.0
842
1140
5.7
7.8
254
524
3.1
3.1
75
103
B6D2F1 male 300 2.0 1283 12.3 1133 1.7 89
B6C3FJ male 300 2.0 1040 11.3 631 2.3 110
“ Total dose based on a 20-g mouse. Each mouse was weighed and the total dose was adjusted based on the actual weight of each mouse.
Table 2 Plasma versus tissue levels of PZA in mice following treatment with a 1-h iv. infusion
Table 3 Plasma versus tis sue levels of PZ A in mice folbowi ng treatment wit h a 1-h iv. infusion
Dose (mg/m2)
Time
(mm)
Plasmaa
(ng/ml)
Pancreas
(p.g/gh)
Brain(pg/g”)
Lung(p.gjgb)
Heart(pg/gb)
Muscle(p.g/gb)
Spleen(p.g/gh)
B6D2F1 female
150 60
1440
578
40
111
1
12
1
88
10
4
2
1
1
1
3
300 60
1440
796
66
231
13
16
8
99
4
11
BLQC
2
BLQ4
2B6D2F1 male
300 60
1440
930
310
276
42
40
3
253
44
55
1
4
1
88
22
B6C3F, male300 60
1440
630
227
286
77
36
4
110
2
11
1
5
2
40
60
a Plasma results are from pooled plasma (three to four mice per time point), and tissue results are the mean values of tissues from three to four
mice per time point.b Tissue results are expressed as p.g/g wet weight.
( BLQ, below the limit of quantification, 50 ng/ml.
to cells from common solid tumors. As possible mechanisms of
cytotoxicity, the PZAs have been shown to intercalate into DNA
(19) and cause protein-associated DNA strain breaks character-
istic of topoisomerase I! inhibitors (8). Those PZAs that showed
the most potent L1210 murine leukemia cytotoxicity showed the
highest number of DNA strand breaks (8), whereas those less
potent against L1210 showed solid tumor activity (6), albeit at
higher concentrations. These results led to the conclusion that
there might be more than one mechanism of action for the PZAs.
Horwitz et al. (20) analyzed 44 PZA structures and their growth
inhibitory ability against HC’T-8 cells (human colon tumor)
s’ersus L1210. The results indicated that steric and electrostatic
fields alone, in comparative molecular field analysis, did not
elucidate what conveyed solid tumor selectivity for these corn-
pounds. Therefore, even though the mechanism of action ap-
pears to be related to reactivity with DNA, directly or indirectly,
the means of solid tumor versus leukemia selectivity is poorly
understood. Nevertheless, the PZA compound chosen for initial
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Table 6 PZA AUC values in mice, monkeys, and dogs followingtreatment with a 1-h iv. infusion
Table 5 PZA protein binding in fresh plasma of mice and humans
a Human values arehealthy volunteers.
a AUC value units are �sg/ml X mm.
h no results available.
‘ Mean results of four male monkeys at 300 mg/m2 and two males
at 600 mg/rn2.
d Mean results of two male and two female dogs in each treatment
group.
836 Pharmacokinetics of Pyrazoloacnidine in Mice
5 Unpublished observations.
Table 4 PZA excretion in urine and feces from mice in the first 24
h following treatment with a 1-h iv. infusion”
% of Dose administered
recovered fromDose
(mg/rn2) Urine Feces
0.06
0.05
0.04
0.20
B6D2F1 female 150 6.2300 5.0
B6D2F1 male 300 28
B6C3F1 male 300 25
a Urine and feces were collected from 4 to 10 mice placed together
in metabolic cages following treatment.
PZAconcentration
(p.g/ml)
% Bound to plasma proteins from
Mice Men” Women”
0.25 100 100 100
0.50 100 100 1001.0 97 98 99
5.0 96 99 9910.0 95 98 98
the mean of duplicate samples from two
clinical studies was one that demonstrated a relatively high
degree of activity in solid tumor models (6, 7).
Previously published toxicity studies in B6D2F1 females
indicated that acute dose-limiting toxicity was neurological (7),
but when given by infusion of at least 60-mm duration, similar
B6D2F1 mice tolerated two doses of 300 mg/m2 (100 mg/kg)
without treatment-related deaths (0/4), and myelosuppression
was dose limiting. B6D2F1 males treated with 60-mm infusions
of either (a) two (slightly higher) doses of 375 mg/rn2 (125
mg/kg) or (b) two (slightly lower) doses of 252 mg/m2 (82
mg/kg) showed 7 of 7 and 5 of 8 treatment-related deaths,
respectively, that occurred on days 18-21, indicating something
other than acute neurotoxicity as the cause of death (7). Given
that myelosuppression was a dose-limiting toxic effect (7, 1 1),
these mice likely died as a direct result of subacute myelosup-
pression. These differences in the presence and absence of
subacute treatment-related deaths fo!!owing the infusions could
be related to a difference in tissue sensitivity, pharmacokinetics,
or both. The pharmacokinetic results reported herein showed a
2-fold difference in AUC at 300 mg/rn2 in B6D2F1 mice (fe-
males, 524 p.g/ml; males, 1133 p.g/ml X mm), which clearly
implicates drug exposure differences as a major factor in drug
tolerance for subacute deaths. During the Phase I trial, AUC was
related to drug dose, and patients treated with the same dose
who had a higher AUC also had a higher degree of myelo-
suppression.4 Since neurotoxicity was similar in B6D2F1
male and female mice, it was not surprising that the peak
Dose (mg/m2)
Reference150 300 600
Mice
B6D2F1 254” 524 h Table 1
female
B6D2F, - 1 133 - Table 1
male
B6C3F1 - 631 - Table 1
male
Monkeys” - 354 616 22Dogs” - 95 124 II
plasma levels were also similar (females, 1140 ng/m!; males,
1283 ng/ml X mm).Pharmacokinetic results at the second (60 mg/rn2) and
subsequent dose levels of the Phase I trial at our institution
showed variation that did not appear to be sex related.4 The
AUC level from B6C3F1 males was different from that observed
in the B6D2F1 mice but more closely resembled that obtained
from the B6D2F1 females. Again indicating that the variation in
AUC was not likely sex related. The intraspecies differences in
AUC and drug tolerance in mice confirmed our early Phase I
results. Berg et al. (21) reported pharmacokinetic studies in
male rhesus monkeys and also demonstrated intraspecies van-
ations in AUC that were not sex related. In addition to intraspe-
cies variations in AUC, a comparison of the mean results from
monkeys (21), dogs (10), and mice (Table 1) also demonstrates
interspecies variations as shown in Table 6. Intraspecies and
interspecies variations in drug handling could be explained by
metabolism; as suggested by Berg et al. (21), as yet no metab-
olite(s) has been reported from plasma studies. However, we
observed additional peaks on the HPLC tracings from mice
urine and feces that may be PZA rnetabolites.5 Isolation and
characterization of these are ongoing. Accordingly, if similar
peaks are observed from the urine of patients, these will be
likewise characterized.
There was marked tissue uptake of PZA in all mice strains,
indicating a large volume of distribution, i.e., the mean ± SD
apparent volume of distribution at steady state was 94 ± 16
liters/kg. A review of the volume of distribution for other drugs
(22) showed that most were much lower, i.e., cisplatin, 0.28 ±
0.07; phenytoin, 0.64 ± 0.04; nifedipine, 0.78 ± 0.22; 1-�3-o-
arabinofuranosylcytosine, 3.0 ± 1.9, and doxorubicin = 25
liters/kg. Kidney, liven, pancreas, and lung had the highest PZA
levels and these were up to 500 times greater than simultaneous
levels in plasma. Although liver drug levels were fairly constant
across strains, other tissue levels generally ranked in the same
4 P. LoRusso, B. J. Foster, E. Poplin, M. Kraut, L. Flaherty, L. K.Heilbrun, M. Valdivieso, and L. Baker. Phase I clinical trial of pyra-
zoloacnidine NSC 366140 (PD 115934), submitted for publication.
Research. on June 12, 2018. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Clinical Cancer Research 837
order as AUC values, i.e., B6D2F, males > B6C3F1 males >
B6D2F, females. Brain PZA levels were clearly demonstrated
in each strain with a range of 20-60 for the brain tissue:plasma
ratios at 60 mm following treatment with 300 mg/rn2. Urinary
excretion was �5-fold higher in male mice as compared to
females, while the fecal excretion and percentage of protein
bound in vitro showed no consistent sexual differences.
The results of these preclinical PZA studies in mice mdi-
cate a linear relationship between dose and drug exposure as
measured by AUC following treatment with 150 and 300 rng/
m2. Thus, dose escalations during the Phase I trial up to 300
mg/m2 should not produce nonlinear effects, and none were
observed over this dose range during the Phase ! trial.4 Since
this dose could be administered twice in B6D2F1 females and
produce tolerable toxicity without treatment-related deaths,
twice their AUC at 300 mg/rn2 (1048 p.g/ml X mm) was
selected as the target AUC for the Phase I trial and was expected
to be associated with myelosuppression. Intraspecies and inter-
species variations in plasma AUC values may be related to
metabolic differences as indicated by evidence of metabolites in
the urine and feces. The intraspecies variations of plasma AUC
values correlate inversely with subacute drug tolerance at the
same dose, i.e. , higher plasma AUC values were associated with
lower drug tolerance. Acute drug effects of neurotoxicity were
related to peak plasma levels which are related to dose and the
speed of injection/infusion and showed minimal intraspecies
variation. Based on the results from mice, when given as a 1-h
infusion, peak plasma PZA levels of > 1000 ng/ml were pre-
dicted to be associated with acute neurological symptoms in
patients and neuromotor, neurosensory, and mood affects were
observed during our Phase I trial.4 High-affinity tissue uptake of
PZA in mice may be an important component of its preferential
solid tumor activity and will require monitoring of patients for
subacute or chronic organ toxicity.
ACKNOWLEDGMENTS
We thank Susan Pugh for her valuable expertise and technical
assistance.
REFERENCES
1. Young, R. C., Ozols, R. F., and Myers, C. E. The anthracycline
antineoplastic drugs. N. Engl. J. Med., 305: 139-153, 1981.
2. Weiss, R. B., Sarosy, G., Clagett-Carr, K., Russo, M., and Leyland-
Jones, B. Anthracycline analogs: the past, present and future. Cancer
Chemothen. Pharmacol., 18: 185-197, 1986.
3. Shenkenbeng, T. D., and Von Hoff, D. D. Mitoxantrone: a new
anticancer drug with significant clinical activity. Ann. Intern. Med.,
105: 67-81, 1986.
4. Talbot, D. C., Smith, I. E., Mansi, J. L., Judson, I., Calvert, A. H., and
Ashley, S. E., Anthrapyrazole CI-941: a highly active new agent in the
treatment of advanced breast cancer. J. Clin. Oncol., 9: 2141-2147,
1991.
5. Bennett, J. M., Byrne, P., Desai, A., White, C., De Conti, R., Vogel,
C., Drementz, E., Muggia, F., Doroshow, V., Plotkin, D., Golomb, H.,
Muss, H., Brodovski, H., Gams, R., Horgan, L. R., Bryant, S., Weiss,
A., Cartwnight, K., and Dukart, G. A randomized multicenter trial of
cyclophosphamide, novantrone and 5-fluorounacil (CNF) versus cyclo-
phosphamide, adniamycin and 5-fluonouracil (CAF) in patients with
metastatic breast cancer. Invest. New Drugs, 3: 179-185, 1985.
6. Sebolt, J. S., Scavone, S. V., Pinter, C. D., Hamelehle. K. L., Von
Hoff, D. D., and Jackson, R. C. Pyrazoboacnidines, a new class of
anticancer agents with selectivity against solid tumors in vitro. Cancer
Res., 47: 4299-4304, 1987.
7. LoRusso, P., Wozniak, A. J., Polin, L., Capps, D., Leopold, W. R.,
Werbel, L. M., Biennat, L., Dan, M. E., and Conbett, T. H. Antitumor
efficacy of PD115934 (NSC 366140) against solid tumors of mice.
Cancer Res., 50: 4900-4905, 1990.
8. Jackson, R. C., Sebolt, J. S., Shillis, J. L., and Leopold, W. R. The
pyrazoloacnidines: approaches to the development of a carcinoma se-
lective cytotoxic agent. Clin. Invest., 8: 39-47, 1990.
9. Sebolt, J., Havlick, M., Hamelehle, K., Nelson, J., Leopold, W., and
Jackson, R. Activity of the pyrazoloacnidines against multidnug-nesistant
tumor cells. Cancer Chemother. Pharmacob., 24: 219-224, 1989.
10. Politi, P. M., Berg, S. L., Balis, F. M., Poplack, D. G., Allegra, C. J.,
and Grem, J. L. Cytotoxicity and cell associated levels of pyrazoloacni-
dine in human multidnug resistant tumor cell lines. Proc. Am. Assoc.
Cancer Res., 33: 524, 1992.
11. Clinical Brochure for Pyrazoloacnidine (NSC 366140), IND 36325.
Division of Cancer Treatment, National Cancer Institute March, 1993.
12. Collins, J. M., Zaharko, D. S., Dednick, R. L., and Chabner, B. A.
Potential, roles for preclinical pharmacology in phase I clinical trials.
Cancer Treat. Rep., 70: 73-80, 1986.
13. Jennnich, R. I., and Sampson, P. F. Application of stepwise negres-
sion to non-linear least squares estimation. Techometnics, 10: 63-72,
1968.
14. Ottaway, J. H. Normalization in the fitting of data by iterative
methods. Biochem. J., 134: 729-736, 1973.
15. Wagner, J. G. Fundamentals of Clinical Pharmacokinetics, pp.38-
58. Chicago: Drug Intelligence Publications, 1975.
16. Corbett, T. H. A selective two-tumor soft assay for drug discovery.
Proc. Am. Assoc. Cancer Res., 25: 325, 1984.
17. Corbett, T. H., Valeniote, F. A., Polin, L., Panchapon, C., Pugh, S.,White, K., Lowichik, N., Knight, J., Bisseny, M. C., Wozniak, A.,
LoRusso, P., Biernat, L., Polin, D., Knight, L., Piggar, S., Looney, D.,
Demchick, L., Jones, J., Blair, S., Palmer, K., Essenmacher, S., Lisow,
L., Mattes, K. C., Cavanaugh, P. F., Rake, J. B., and Baker, L. Discoveryof solid tumor active agents using a soft-agar-colony formation-disk-
diffusion-assay. In: F. A. Valeniote, T. H. Conbett, and L. H. Baker
(eds.), Cytotoxic Anticancer Drugs: Models and Concepts for Drug
Discovery and Development, pp. 50-58. Boston: Kiuwer Academic
Publishers, 1990.
18. Conbett, T. H. A selective two-tumor soft assay for drug discovery.
Proc. Am. Assoc. Cancer Res., 26: 332, 1985.
19. Sebolt-Leopold, J. S., and Scavone, S. Biochemistry of the intenac-
tions between DNA and the pynazoloacnidines, a series of biologically
novel anticancer agents. Proc. Am. Assoc. Cancer Res. 32: 334, 1991.
20. Horwitz, J. P., Massova, I., Wiese, T. E., Wozniak, A. J., Conbett,
T. H., Sebolt-Leopold, J. S., Capps, D. B., and Leopold, W. R. Com-
parative molecular field analysis of in vitro growth inhibition of L1210and HCT-8 cells by some pyrazoloacnidines. J. Med. Chem., 36: 3511-
3516, 1993.
21. Berg, S. L., Balis, F. M., McCully, C. L., Godwin, K. S., and
Poplack, D. G. Pharmacokinetics of pynazoloacnidine in the rhesus
monkey. Cancer Res., 51: 5467-5470, 1991.
22. Pharmacokinetic Data, Table A-Il-i. In: A. G. Gilman, T. W. Rail,
A. S. Nies, and P. Taylor (eds.), Goodman and Gilman’s: The Pharma-
cological Basis of Therapeutics, Ed. 8, pp. 1655-1715. New York:
Pergamon Press, 1990.
Research. on June 12, 2018. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
1995;1:831-837. Clin Cancer Res B J Foster, R A Wiegand, P M LoRusso, et al. 366140) in mice: guidelines for early clinical trials1.[3,4, 5-kl]acridine-2(6H)-propanamine (PZA, PD 115934, NSC Pharmacokinetics of 9-methoxy-N,N-dimethyl-5-nitropyrazolo
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