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.

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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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