Plasma Pharmacokinetics and Urinary Excretion of

Hexamethylene Bisacetamide Metabolites

Merrill J. Egorin, Eleanor G. Zuhowski, Adam S. Cohen, et al.
Cancer Res 1987;47:6142-6146.

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[CANCER RESEARCH 47, 6142-6146, November 15. 1987]

Plasma Pharmacokinetics and Urinary Excretion of Hexamethylene Bisacetamide

Metabolites1
Merrill J. Egorin2, Eleanor G. Zuhowski, Adam S. Cohen, Linda A. Geelhaar, Patrick S. Callery, and
David A. Van Echo
Divisions of Developmental Therapeutics and Medical Oncology, University of Maryland Cancer Center [M. J. E., E. G. Z., A. S. C., D. A. V. E.J, Division of Medical
Oncology, Department of Medicine, University of Maryland School of Medicine [M. J. E., D. A. K E.], and Department of Medicinal Chemistry/Pharmacognosy,
University of Maryland School of Pharmacy [L. A. G., P. S. C.I, Baltimore, Maryland 21201

patient plasma equal to the concentrations required for induc

tion of differentiation in vitro (29, 30). In this regard, HMBA
In order to further understand the clinical toxicities of hexamethylene
differs from differentiating agents, such as dimethyl sulfoxide
bisacetamide (HMBA) and to allow appropriate in vitro studies, we and yV-methylformamide, that have undergone previous clinical
developed a suitable gas Chromatographie assay and quantified plasma
evaluation (21-28). However, adverse effects have been noted
concentrations and urinary excretion of four metabolites which we had
when HMBA has been administered to humans (29, 30). At
previously identified in urine of patients receiving 5-day HMBA infusions
HMBA dosages greater than or equal to 33.6 g/m2/day, meta
at 4.8-43.2 g/m2/day. 6-Acetamidohexanoic acid (AcHA) was the major
plasma metabolite and reached steady state concentration (C)by 24 h. bolic acidosis and neurotoxicity occur as dose-limiting toxici
AcHA C,, increased from 0.12 0.02 (SD) mM at 4.8 g/m2/day to 0.72
ties. Platelet count suppression, although not always dose lim
mM at 43.2 g/m2/day. The CAcHA:CHMBA ratio decreased with
iting, also occurs with HMBA therapy.
increasing HMBA dosage. At dosages below 24 g/m2/day plasma Cof
We have recently utilized gas chromatography/mass
spec/V-acetyl-1,6-diaminohexane (NADAH), the initial metabolite of HMBA,
trometry to identify five metabolites of HMBA in the urine of
were below the limit of detection of our assay. With HMBA infusions of
24, 33.6, and 43.2 g/m2/day, C,, of NADAH were 0.16 0.05, 0.14 patients treated with HMBA (31). These metabolites included
the major metabolite, AcHA; the monodeacetylated product,
0.06, and 0.19 0.04 mM, respectively. CNADAH:CHMBA ratios
NADAH; the bis-deacetylated diamine, DAH; and the amino
at 24, 33.6, and 43.2 g/m2/day were 0.18 0.06, 0.08 0.02, and 0.31
acid, AmHA and its lactam, caprolactam (Fig. 1). The impor
0.05, respectively. Plasma C,, of 1,6-diaminohexane and 6-aminohexanoic acid were below the limit of detection of our assay. Each patient's
tance of quantifying the amounts of each of these metabolites
urinary excretion of NADAH, AcHA, and 1,6-diaminohexane was con
in body fluids of patients treated with HMBA led us to develop
sistent from day to day. The fraction of dose excreted in urine as AcHA
a gas Chromatographie analysis which would allow the routine
was not affected by HMBA dosage and accounted for 12.7 3.9% of the and sensitive assay of HMBA metabolites in biological samples
daily dose. The percentage of daily HMBA dose accounted for by (32). We have now used this assay to quantify the concentra
excretion of NADAH decreased with increasing HMBA dosage (10.8 tions of various HMBA metabolites in plasma and urine of
6.0% at 4.8 g/m2/day to 4.2 1.2% at 33.6 g/m2/day). Urinary excretion
patients treated in our phase I trial of HMBA administered by
of 1.6-diaminohexane always accounted for less than 3% of the daily
5-day continuous infusion (29).
ABSTRACT

dose. Our results indicate that: (a) plasma concentrations of AcHA alone
cannot explain the degree of acidosis observed with toxic doses of HMBA;
(b) NADAH is present in plasma at concentrations that we have found
to cause differentiation in vitro-, and (c) the probable rate-limiting step
in HMBA metabolism is the initial deacetylation.

INTRODUCTION
HMBA3 (NSC 95580) induces in vitro morphological

and

functional differentiation of murine and human leukemic and

solid tumor cell lines (1-13). Among the class of agents that
have the potential for inducing the differentiation of tumor cells
and that represent an exciting and novel approach to the
chemotherapy of neoplasia (14-19), HMBA has a number of
characteristics which render it of greatest potential clinical use
(20). HMBA was selected for introduction into clinical trials
because, of a series of bisacetamides tested, it approached
maximum differentiation potency (1-5). In addition, carefully
conducted clinical and pharmacokinetic studies have docu
mented the ability to achieve concentrations of HMBA in
Received 3/5/87; revised 8/17/87; accepted 8/24/87.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported in part by American Cancer Society Institutional Research Grant
IN-174D, Maryland Cancer Program/University of Maryland, and Contract
NO1-CM47734 awarded by the National Cancer Institute. Department of Health
and Human Services.
2To whom requests for reprints should be addressed, at Division of Develop
mental Therapeutics, University of Maryland Cancer Center, 655 W. Baltimore
St., Baltimore. MD 21201.
3 The abbreviations used are: HMBA, hexamethylene bisacetamide: NADAH,
A'-acetyl- 1,6-diaminohexane; DAH, 1,6-diaminohexane; AcHA, 6-acetamidohexanoic acid; AmHA, 6-aminohexanoic acid; C,steady state concentration.

MATERIALS

AND METHODS

Patient Selection and Evaluation. All patients entered into this study
had histolgica! proof of malignant disease for which conventional
chemotherapy had proven ineffective and for which no other investigational therapy with established efficacy was available. Before entry
into this study, each patient had the investigational nature of the
treatment explained, and an informed consent, approved by the Insti
tutional Review Board, was signed. Additional details of eligibility
criteria and the patient population treated have been published previ
ously (29).
Reagents. DAH, AcHA, AmHA, acetic anhydride, trifluoroacetic
anhydride, 2,2,2-trifluoroethanol, 1,2-diphenylethylamine, and cadav
erine were obtained from the Aldrich Chemical Co., Inc. (Milwaukee,
WI). NADAH was synthesized from DAH and acetic anhydride as
described previously (31).
Drug Schedule and Administration. HMBA was supplied by the
Investigational Drug Branch of the National Cancer Institute (Bethesda, MD). Each 500-ml bottle contained 15 g of HMBA as a solution
in 0.154 M NaCl. HMBA was administered by continuous infusion
through a free-flowing peripheral or central venous catheter. The rate
of HMBA infusion was controlled by a Travenol Flo-Gard 8000 volu
metric infusion pump (Travenol Laboratories, Inc., Deerfield, IL). The
starting dose was 4.8 g/m2/day for 5 consecutive days and approximated
one-half of the canine toxic dose low (20). Doses were escalated to 43.2
g/m2/day for 5 days according to a modified Fibonacci schedule (29).
Doses were not escalated within individual patients.
Sample Acquisition. Heparinized blood samples were obtained before
and at multiple times during and after the HMBA infusion. Specifically,
samples were obtained at 0, 1, 3, 5, 8, 12, 16, 23, 47, 71, 95, and 120
h during the 5-day infusion and at 5, 15, 30, 60, 120, 240, 360, and

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PHARMACOKINETICS
AcNHCH2(CH2)4
hexomethylene

AND EXCRETION

m2/day, and the single patient treated with 43.2 g/m2/day.

C
bisocetomide

There were adequate urine samples but inadequate plasma

samples for one additional patient treated at 33.6 m/m2/day.
In accordance with the results observed in our development of
assay methodology for HMBA metabolites (32), there were no
endogenous materials in any patient's plasma or urine that

AcNHCH2(CH2)4CH2NH2
/V - ocelyl -1,6 - diominohexone

interfered with analysis of NADAH, AcHA, DAH, or AmHA.

At all dosages, AcHA was the major plasma metabolite of
HMBA. Plasma concentrations of AcHA were at steady state
(Css) by 24 h after the start of the HMBA infusion, and, within
any patient, there was little day-to-day variation in the plasma
CMof AcHA (Fig. 2). For individual patients, the daily variation
about the mean CS5of HMBA in plasma obtained at 24,47, 71,
95, and 120 h was usually less than 25% (Fig. 2). As observed
with HMBA (29), there was variation among the AcHA CM
achieved in the individual patients treated at each dosage (Fig.
2). Plasma AcHA Css increased with increased rate of delivery
of HMBA. AcHA CK of 0.125 0.02 (SD) mM were obtained
with an HMBA dosage of 4.8 g/m2/day and increased to a Css
of 0.72 mM in the one patient treated with 43.2 g/m2/day of

[AcNHCH2(CH2)4CHO]
6- ocetomidohexonol

H2NCH2

(CH2)4CH2NH2

1,6 - diominohexone

OF HMBA METABOLITES

AcNHCHj(CH2)4COOH

6-ocetomtdohexonoic acid
(major metabolite)

H2NCH2 ( CH2>4 COOH

6"ominohexonoic ocid

Fig. 1. Proposed metabolic pathways leading to the metabolites of HMBA in

humans, a. deacetylase: b, monoamine oxidase; <. aldehyde dehydrogenase or
aldehyde oxidase: il. diamine oxidase and aldehyde oxidase. Ac. acetyl.

480 min after cessation of the infusion. Blood samples were immedi
ately centrifuged at 1000 x g for 10 min, and the resulting plasma
supernatants were immediately removed and stored at -20C until
analyzed. Urine specimens were collected at 4-h intervals for the first
24 h and as pooled 24-h collections for the ensuing 4 days. Urine was
stored at 4Cuntil the 4- or 24-h collection was complete. At the end
of each collection, the volume of urine was measured immediately, and
a portion of the urine was frozen and stored at
20*C until analysis.
Extraction and Analysis of HMBA and Metabolites. HMBA was
quantified with a gas Chromatographie analysis based on the method
originally described by Kelley et al. (33). The specifics of our modifi
cations to the method and the quantitative characteristics of the assay
in our laboratory have been published previously (29).
Individual HMBA metabolites were quantified in plasma and urine
by gas chromatography (32). Briefly, 100-jil samples of plasma or urine
were mixed with 50 n\ of either 6 mM 1,2-diphenylethylamine or 3 mM
cadaverine internal standard. Ethanol (900 u\) was added to denature
proteins and the mixture was vortexed and centrifuged at 10,000 x g
for 10 min at room temperature. A 700-j/l portion of the resulting
supernatant was evaporated to dryness under nitrogen and derivatized
with trifluoroacetic anhydride and 2,2,2-trifluoroethanol. This final
reaction mixture was redissolved in 800 //I of ethyl acetate and 1 n\ of
the resulting solution was injected into a Hewlett-Packard 5840A gas
Chromatograph (Hewlett-Packard, Palo Alto, CA), fitted with a glass
column ( 1.8 m x 2 mm) containing 3% SP-2250-DB on 100-120 mesh
Supelcoport (Supelco, Bellfonte, PA). For analysis of NADAH and
AcHA, the oven was maintained at 180'C. For analysis of DAH and
AmHA, the oven was maintained at 140"( '. In all analyses, the injection
port was maintained at 255"C and nitrogen, at a flow rate of 30 ml/

HMBA (Fig. 2). Although AcHA CK increased with increased

HMBA dosage, this rate of increase was less than that of the
increase in C^ HMBA. As a result, the plasma C
AcHA:plasma Css HMBA ratio decreased with increasing
HMBA dosage (Fig. 3).
Plasma concentrations of NADAH were less than plasma
concentrations of either HMBA or AcHA. At HMBA dosages
of 4.8,9.6, and 16 g/m2/day, plasma concentrations of NADAH
were less than the limit of detection of our assay. In the three
patients treated with 24 g/m2/day, plasma NADAH C5Swere
0.05 0.02,0.19 0.08, and 0.23 0.01 mM. In the 2 patients
treated with 33.6 g/m2/day and 1 patient treated with 43.2 g/
m2/day NADAH Css were 0.08 0.01, 0.21 0.04, and 0.19
0.04 mM, respectively. These concentrations represented
between 33 and 80% of concomitant plasma concentrations of
AcHA and between 6 and 38% of concomitant plasma concen
trations of HMBA.
In all patients, plasma concentrations of DAH and AmHA
were below the limits of detection of our assay.
Upon completion of the 120-h infusion of HMBA, plasma
concentrations of HMBA and NADAH declined monoexponentially with half-lives of approximately 2-4 h (29,30). Plasma
09 0807 -

; 06-

min, was used as a carrier gas. Detection was accomplished with a

nitrogen-phosphorus detector that was maintained at 275'C with an

' OS-

air flow rate of 90 ml/min, a hydrogen flow rate of 3.5 ml/min, and a
bead voltage of 16-18 V. Peaks were recorded and integrated with a
Hewlett-Packard 5840A GC terminal. Concentrations of each metab
olite were calculated by comparison of the area of the metabolite peak
with that of the internal standard peak in each sample, and referenced
to concomitantly performed standards.

[ 04

I
0.3-

RESULTS

02O.I

10

Adequate plasma and urine samples were available to allow

analysis of samples from 15 of the 20 patients treated in our
phase I trial (29). These patients included 3 each treated with
4.8, 9.6, 16, and 24 g/m2/day, 2 patients treated with 33.6 g/

20

3O

40

50

HMBA DOSAGE (g/mz/d)

Fig. 2. Relationship of steady state concentrations of 6-acetamido-hexanoic
acid in plasma to HMBA dosage. Points, means for individual patients; bars. SD.
Concentrations of 6-acetamidohexanoic acid in plasma samples obtained at 24,
47, 71, 95, and 120 h in the 120-h infusion of HMBA were used to calculate
means and SD.

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PHARMACOKINETICS

AND EXCRETION

09-08-07-CD

OF HMBA METABOLITES
Table 2 Urinary concentrations of HMBA and metabolites

Dosage4.89.6162433.643.2Patient12312312.11231231HMBA(mm)3.3
0.8"7.9

0.65.5

0.61.6

0.310.8

0.83.6

1.92.8

3.622.0

1.620.7

0.715.9

06-J05-^I

04-UJ03-

' I
3I

02-0.1-

o*I1

10

20
30
HMBA DOSAGE (g/mz/d)

40

so

Fig. 3. Relationship of steady state concentrations of 6-acetamidohexanoic

acid to those of HMBA at different HMBA dosages. Points, means for individual
patients; bars, SD. Ratios of plasma concentrations of 6-acetamidohexanoic acid
to those of HMBA in plasma samples obtained at 24, 47, 71, 95. and 120 h in
the 120-h infusion of HMBA were used to calculate means SD.

Table 1 Urinary excretion of HMBA and metabolites

Dosage
(g/m2/day)4.8
9.616

asHMBA36.9

11.65.8

2.78.8

0.911.8

10.815.2

6.25.5
0.510.2

8.58.5
4.46.9

0.712.7

0.60.7
0.70.6

0.21.0

8.538.0
+
9.365.5

36.474.4

1.628.8

3.516.2

4.316.8

0.47.4
1.414.5

3.812.0

0.900.4

48.620.0

0.990.6

22.916.9

1.111.5

5.626.8
11.515.3

0.48.7
3.627.8

11.06.6

13.262.5

11.157.5

16.812.1

4.521.5
2.711.2

1.921.6

1.27.2
7.524.1*NADAH(min)3.3
2.914.4*DAH(mM)0001.5

0.000.5

0.61.0

0.20000

1.6AcHA(HIM)3.8
" Mean SD of concentration of HMBA and metabolites in 24-h urine
collections obtained from individual patients on days 2, 3, 4, and 5 of their 5-day
continuous infusions of HMBA.
4 Urinary concentrations of HMBA and metabolites in the one patient treated
with 43.2 g/m2/day. Values represent urinary concentrations on day 1 of treat
ment only because insufficient material remained from urine voided on days 2, 3,
4. and 5.

of daily dose excreted

manyfold greater than the plasma concentrations

pound measured in the same patient.

18.4'
2.4
6.0
0.3
27.0
18.3 7.9
18.7 10.7 14.2 6.9 1.4 0.6 68.4 21.7
30.7 9.7
14.5 6.2
9.7 4.1 0.4 0.6 84.2 21.2
41. 6 20.7
8.3 4.9 0.3 0.3 84.8 17.2
9.6 3.8
44.3 18.7 10.4 5.1
4.2 1.2
79.9 19.8
0
46.7'AcHA10.1 12.7NADAH10.6
5.5DAH0.2 0Total74.4 64.9

24
33.6
43.2%
" Mean SD of mean daily excretion by 3 patients at each dosage except 43.2
g/m2/day. Mean daily excretion for each patient was based on the 24-h excretion
of HMBA and each metabolite on days 2. 3. 4. and 5 of the 5-day continuous
infusion.
* Daily excretion by the one patient treated with 43.2 g/m2/day. Values
represent the excretion from day I of treatment only because insufficient material
remained from urine voided on days 2. 3. 4, and 5.

concentrations of NADAH declined in parallel with the decline

in plasma concentrations of HMBA. In contrast, plasma con
centrations of AcHA declined little, if at all, during the 6 to 8
h after the infusion from which samples were available.
Within each patient, the daily urine excretion of AcHA,
NADAH, and DAH was consistent from day to day. As in
plasma, AcHA was the major HMBA metabolite present in
urine. Urinary excretion of AcHA accounted for 12.7 3.9%
of the daily dose of HMBA and the percentage of the dose
excreted as AcHA did not change with HMBA dosage (Table
1). In contrast, the percentage of the daily HMBA dose that
was excreted as NADAH decreased with increased HMBA
dosage, from 10.8 6.0% at 4.8 g/m2/day to 4.2 1.2% at
33.6 g/m2/day (Table 1). Urinary excretion of DAH was not

of each com

DISCUSSION
HMBA has been introduced into phase I clinical trials with
the hope of developing an antineoplastic agent the mechanism
of action of which differs from the traditional cytotoxic effects
of most standard and investigational antitumor drugs (14-20).
Although numerous in vitro studies have documented the ability
of HMBA to induce differentiation of tumor cell lines (1-13),
there is much less evidence of in vivo activity (20). In addition,
phase I trials have documented metabolic acidosis and neurotoxicity as dose-limiting toxicities associated with HMBA dos
ages greater than or equal to 33.6 g/m2/day (29, 30). Our

dose related, never accounted for more than 3% of the daily

dose of HMBA, and in most cases accounted for less than 1%
of the daily HMBA dose (Table 1). Consideration of each
patient's aggregate urinary excretion of metabolites and parent
compound allowed the majority of each day's HMBA dosage

previous demonstration of multiple acidic and amine metabo

lites of HMBA (31) and the development of analytical meth
odology suitable for quantifying them in biological fluids (32)
has allowed us to develop data that may facilitate investigation
of: (a) the discrepancy between in vitro and in vivo activity of
HMBA; (b) the toxicities attendant upon HMBA use; and (c)
the characteristics of the interconversion of HMBA and its
catabolites.
Our studies document AcHA as the major plasma metabolite
of HMBA. This quantification of plasma concentrations of
AcHA has direct bearing on attempts to understand the doselimiting anin gap. metabolic acidosis associated with dosages
that produced plasma HMBA concentrations equivalent to
those required in vitro for differentiation (29, 30). The plasma
concentrations of AcHA measured in patients treated with 33.6
and 43.2 g/m2/day are insufficient by themselves to account for

to be accounted for (Table 1). The urinary excretory data can

be viewed in another way (Table 2). Despite the fact that urinary
concentrations of HMBA and metabolites reflect both the ab
solute amount of material excreted and the volume of urine
produced, the data define the range of concentrations of HMBA
and each metabolite achieved in urine when various dosages of
HMBA are administered by continuous infusion (Table 2). For
HMBA, AcHA, and NADAH, these urinary concentrations are

the aningap encountered in those patients (29). Even doubling

the plasma concentration of AcHA, to account for the obligate
mole of acetate liberated in the production of each mole of
AcHA, does not produce a milliequivalent per liter acid load
equal to the aningap noted in our patients. These observations
reduce, somewhat, the attractiveness of the potentially straight
forward strategy of blocking AcHA production with monoamine oxidase inhibitors (34). However, catabolism of HMBA

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PHARMACOKINETICS

AND EXCRETION

beyond AcHA could yield a number of acid species such as

AmHA, 7-aminobutyric acid, adipic acid, acetate, and glycine.
Our failure to document AmHA in plasma does not preclude
such catabolism of AcHA but might reflect rapid catabolism of
AmHA to 7-aminobutyric acid, adipic acid, acetate, and glycine.
Since these latter compounds are endogenous materials with
regulated pool sizes, measurement of changes in their plasma
concentrations need not necessarily reflect the degree of acid
load generated from HMBA. On the other hand, the ability to
account for most of a day's dose of HMBA by urinary excretion
of HMBA, AcHA, NADAH, and DAH makes it less likely that
substantial amounts of other metabolites are produced. It may
be that HMBA-associated acidosis is produced at least in part
by other mechanisms such as precipitation of lactic acidosis,
generation of ketoacidosis, or alteration of renal function.
Our quantification of plasma AcHA concentrations and char
acterization of their relationship to those of HMBA have other
practical implications for the clinical use of HMBA. The results
of our initial phase I trial of HMBA (29) led us to propose the
concept of a maximum tolerated concentration of HMBA
rather than a traditional maximum tolerated dose. This concept
could be logically extended to monitoring Css AcHA if in fact
AcHA was responsible for HMBA-associated acidosis or if its
Css bore a consistent relationship to the degree of systemic
acidosis present. Unfortunately neither of these possibilities
was observed.
The concentrations of AcHA and NADAH measured in
plasma and urine of our patients are of interest in terms of in
vitro studies of HMBA. They have allowed studies examining
the potential differentiating activity of each metabolite at con
centrations which mimic those generated in vivo (35) and have
allowed appropriate combinations and concentrations of these
agents to be studied in vitro for their effect on HMBA-induced
differentiation (35, 36). In addition, the urinary concentrations
of HMBA and metabolites measured in our current studies
might argue for a trial of HMBA in patients with superficial
carcinoma of the bladder.
When considered as a group, our studies of the relationships
of plasma concentrations and urinary excretion of various
HMBA metabolites may provide some insight into the interconversion of these species. We have previously demonstrated
HMBA clearance to have two components, one being renal and
proportional to creatinine clearance and the other being satu
rable and having Michaelis-Menten characteristics (37). An
obvious candidate for this saturable component would be an
enzyme system, and, based on the relationships observed be
tween plasma concentrations of HMBA, AcHA, and NADAH,
the most likely rate-limiting step in HMBA metabolism is its
initial deacetylation. At present, characterization of the distri
bution and capacity of the enzymes responsible for the various
metabolic interconversions of HMBA and its metabolites is an
ongoing effort in our laboratory.

10.
11.
12.
13.
14.
15.

16.
17.
18.
19.
20.

21.
22.
23.
24.
25.
26.

ACKNOWLEDGMENTS
We thank B. Knickman for excellent secretarial assistance in prepa
ration of this manuscript and M. Y. Whitacre for assistance is procuring
plasma and urine samples. We also thank Drs. Alan Forrest and Jerry
Collins for stimulating discussions during preparation of this manu
script.

27.
28.

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