+
+
Systematic Study on the Chemical Stability of the Prodrug Antitumor Agent
Carzelesin (U-80,244)
X
†
†
J. D. JONKMAN
-DE
V
RIES* , W. G. DOPPENBERG†, R. E. C. HENRAR‡, A. BULT
,
AND J. H. BEIJNEN
*
Received January 8, 1996, from the *Department of Pharmacy, Slotervaart Hospital/Netherlands Cancer Institute, Louwesweg 6, 1066 EC
Amsterdam, The Netherlands, †Department of Pharmaceutical Analysis, Faculty of Pharmacy, Utrecht University, Sorbonnelaan 16, 3584
CA Utrecht, The Netherlands, and ‡European Organization for the Research and Treatment of Cancer
s
New Drug Development Office,
Free University Hospital, Gebouw Zuid, Amstelveenseweg 601, 1081 BT Amsterdam, The Netherlands.
Final revised manuscript
received July 9, 1996 .
Accepted for publication July 10, 1996X.
of the drug in this formulation was dependent upon the
interbatch variability of the excipients used in the vehicle.
Accelerated stability studies (at 40 °C) of carzelesin in the
PET formulation showed that the variable stability charac-
teristics were caused mainly by the pH differences between
batches of PEG 400.7 This stimulated us to investigate
systematically the chemical stability of carzelesin in solutions,
which can aid to further optimize the pharmaceutical formu-
lation, and to get more insight into the degradation mecha-
nism.
Abstract
in aqueous buffer/acetonitrile (1:1, v/v) mixtures has been investigated
utilizing stability-indicating reversed-phase high-performance liquid
chromatographic assay. The degradation kinetics of carzelesin has been
studied as a function of pH, buffer composition, ionic strength, and
temperature. Degradation of carzelesin follows (pseudo-) first-order
0 The chemical stability of the novel anticancer agent carzelesin
a
kinetics. A pH
buffer concentration, was constructed demonstrating that carzelesin is
most stable in the pH region 1 4. The degradation rate of carzelesin
−rate profile, using rate constants extrapolated to zero
−
was not significantly affected by buffer components and by the ionic
strength. In addition to the formation of the degradation products U-76,-
073, U-76,074, and aniline in alkaline medium and in acetate buffer
solution, another degradation product was formed in acetate buffer
solution. In perchloric acid buffer solution (pH* < 3), U-76,073 and U-76,-
074 could not be detected as degradation products.
Experimental Section
Ma ter ia lssChemicalssCarzelesin, U-76,073, and U-76,074 were
synthesized by The Upjohn Company (Kalamazoo, MI)8,9 and provided
through the New Drug Development Office (NDDO) of the EORTC
(Amsterdam, The Netherlands). Aniline was obtained from Aldrich
Chemical Co. Ltd. (Axel, The Netherlands). All other chemicals used
were of analytical grade, and deionized water was used through-
out.
Buffer SolutionssDue to the poor aqueous solubility of carzelesin,
the kinetic studies were performed in water/acetonitrile mixtures (1:
1, v/v). The aqueous components were 0.02 or 0.03 M buffer solutions,
except in the experiments where the effects of buffer concentration
were studied. The following buffers were used: 0 e pH < 3, perchloric
acid; 3 e pH < 6, acetate; 6 e pH < 9, phosphate; and pH g 9,
carbonate. The pH values of the aqueous buffer solutions were
measured using an Ingold Semimicro combination pH electrode and
a Model 654 pH meter (Metrohm AG, Herisau, Switzerland), which
had been calibrated on fully aqueous standard solutions. The pH
values of the buffer solutions were adjusted with either 0.1 M
perchloric acid solution or 0.1 M sodium hydroxide solution to the
appropriate pH values. Then, acetonitrile was added to the buffer
solutions and the apparent pH values (called pH*) of these mixtures
(except for pH* 0, which was calculated), at the study temperature,
were measured using the same electrode system.
Introduction
Carzelesin (U-80,244; NSC 619029; Figure 1) is a syntheti-
cally derived cyclopropylpyrroloindole (CPI) analog of the
highly potent, alkylating, antitumor agent antibiotic CC-
1065.1 The CPI analogs are DNA minor-groove binders
containing a cyclopropyl group, which mediates N3-adenine
covalent adduct formation in a sequence-selective fashion
with no intercalation.1 It has been speculated that these
CPI derivatives selectively inhibit the binding of regulatory
proteins to DNA, thereby modifying the transcription of
specific genes that are important for neoplastic cell growth.2
Carzelesin showed activity against a broad panel of human
tumor xenografts in preclinical in vivo studies in mice1
and in vitro in a number of gynecologic cancer cell lines.3
Carzelesin was designed to be an inactive prodrug, requir-
ing chemical or enzymatic activation to the DNA-reactive
form.1 Activation of carzelesin requires two steps, i.e., (a)
hydrolysis of the carbanilate substituent to form U-76,073
followed by (b) ring closure of the chloromethyl group to
form the cyclopropyl-containing DNA-reactive U-76,074 (Fig-
ure 1).
A constant ionic strength (µ) of 0.3 was maintained for each solution
by addition of the appropriate amount of sodium chloride, except in
experiments where the effect of the ionic strength on the degradation
of carzelesin was investigated and where the OH- and H+ concentra-
tions were higher than 0.3 M.
Degr a d a tion Kin eticssKinetic MeasurementssThe kinetic stud-
ies were carried out over at least 3 half-lives in the dark (in order
to prevent possible photolytic degradation), and the temperature
of the study was 25 °C in the pH* range 6-10, 60 °C in the pH*
range 1.5-5.5, and 70 °C at pH* 0. The reaction was initiated by
addition of 100 µL of a solution of carzelesin in absolute ethanol (1
mg/mL) to 5.0 mL of preheated buffer/acetonitrile solution (1:1, v/v)
to give an initial concentration of approximately 20 µg/mL (2.7 × 10-5
M). The reaction solutions were kept in screw-capped brown glass
vials in a thermostatically controlled water bath. At appropriate time
intervals 200-µL samples were withdrawn and directly analyzed for
undegraded carzelesin and the degradation products U-76,073 and
U-76,074, by a stability-indicating high-performance liquid chroma-
tography (HPLC) assay. All kinetic studies were performed in
duplicate.
Recently, a study4 has been conducted dealing with the
characterization and pharmaceutical formulation of carzelesin
according to the “Guidelines for the formulation of investiga-
tional cytotoxic drugs”, which were drawn up by the J oint
Formulation Working Party of the EORTC/CRC/NCI.5
A
stable parenteral formulation of carzelesin in a polyethylene
glycol 400 (PEG 400)/absolute ethanol/Tween 80 (6:3:1, v/v/v;
PET formulation) solution was developed,4 which is now being
tested in phase I clinical trials.6 During the manufacturing
process of clinical batches of carzelesin in the PET formu-
lation, observations were made indicating that the stability
Influence of TemperaturesThe effect of temperature on the
degradation of carzelesin was studied at pH* 1.5 (0.1 M perchloric
X Abstract published in Advance ACS Abstracts, October 1, 1996.
© 1996, American Chemical Society and
American Pharmaceutical Association
S0022-3549(96)00005-6 CCC: $12.00
Journal of Pharmaceutical Sciences / 1227
Vol. 85, No. 11, November 1996