Phase I and Pharmacokinetics Study of Crotoxin (Cytotoxic PLA
2
,
NSC-624244) in Patients with Advanced Cancer
1
Jorge E. Cura, Daniel P. Blanzaco,
Cecilia Brisson, Marco A. Cura, Rosa Cabrol,
Luis Larrateguy, Carlos Mendez,
Jose Carlos Sechi, Jorge Solana Silveira,
Elvira Theiller, Adolfo R. de Roodt, and
Juan Carlos Vidal
2
Department of Medical Oncology Hospital San Martin, Parana ´, Entre
Rios, Argentina 3100 [J. E. C., D. P. B., C. B., M. A. C., R. C., L. L.,
C. M., J. C. S., J. S. S., E. T., J. C. V.] and Instituto “Dr. Carlos G.
Malbran” Administracion Nacional de Laboratorios e Institutos de
Salud, Buenos Aires, Argentina 1281 [A. R. d R.]
ABSTRACT
A Phase I clinical trial was performed on patients with
solid tumors refractory to conventional therapy. Crotoxin
was administered i.m. for 30 consecutive days at doses rang-
ing from 0.03 to 0.22 mg/m
2
. Patients entered the study after
providing a written informed consent. Although 26 patients
were entered only 23 were evaluated. Reversible, nonlimit-
ing neuromuscular toxicity evidenced as diplopia because of
pareses of the external ocular muscles was present in 13
patients. It started at doses of 0.18 mg/m
2
and lasted from 2
to 6 h. These episodes did not require dose adjustment and
disappeared in 1–3 weeks of treatment. Three patients ex-
perienced palpebral ptosis, nystagmus (grade 2), and anxiety
(grade 2–3) at the dose-limiting toxicity of 0.22 mg/m
2
. Also
at dose-limiting toxicity, 1 patient showed nystagmus (grade
2) and anxiety (grade 3) without evidence of palpebral pto-
sis. Transient increases (grades 1–3) in the levels of creati-
nine kinase, aspartate aminotransferase, and alanine trans-
aminase attributed to crotoxin myotoxicity were observed
but returned to normal by the last week of treatment. At
0.21 mg/m
2
there was a case of grade-3 anaphylactic reac-
tion on day 31, which required treatment. Hypersensitivity
was regarded as an adverse drug-related reaction, and the
patient was removed from the protocol. Two patients at
different doses (0.12 mg/m
2
and 0.22 mg/m
2
) had sialorrhea.
Four patients had asymptomatic transient increase in blood
pressure (up to 20 mm Hg) 12 h after the first injection,
which lasted 24 h. No treatment was required and toxicity
did not reappear. Six patients experienced slight eosino-
philia during the first 2 weeks. The maximum tolerated dose
was set at 0.21 mg/m
2
. Objective measurable partial re-
sponses (>50% reduction of tumor mass) were noted in 2
patients treated at 0.21 mg/m
2
and 1 at 0.12 mg/m
2
. One
patient (at 0.21 mg/m
2
) presented a complete response on
day 110. Crotoxin pharmacokinetics showed rapid absorp-
tion from the injection site to blood (t
1/2 A
5.2 0.6 min).
Plasma concentration reached a peak (C
max
0.79 0.1
ng/ml) at
max
19 3 min. The half-life of the distribution
() phase is 22 2 min. Starting at 1.5 h after injection, the
decrease in plasma concentration becomes slower, reaching
14 3 pg/ml 24 h after injection. The profile is dominated
by the elimination () phase with a half-life of 5.2 0.6 h.
Consequently, 24 h after the injection (5 half-life) 97% of
the product was eliminated. The area under plasma concen-
tration versus time curve was 0.19 0.05 g/min/ml. As-
suming availability (F) 1, the clearance is C
L
26.3 7
ml/min, and the apparent volume of distribution is V
d
12 3 liter/kg. The recommended dose for a Phase II study
is 0.18 mg/m
2
.
INTRODUCTION
Crotoxin is a cytotoxic PLA
2
3
compound isolated from a
South American snake, Crotalus durissus terrificus, venom. It is a
noncovalent complex formed by two nonidentical subunits, one
acidic (subunit A 9.5 kDa) and one basic (subunit B 14.5 kDa).
Subunit B is a PLA
2
formed by a single chain of 122 amino acid
residues cross-linked by seven disulfide bonds (1– 6). Subunit A is
formed by three polypeptide chains cross-linked by seven disulfide
bonds (7), is devoid of catalytic activity, has no affinity for mem-
branes, and is nontoxic (LD
50
i.v. mice 20 mg/kg; Ref. 6). The
two subunits form spontaneously a tight 1:1 complex (6, 8). Com-
plex formation inhibits the PLA
2
activity but increases toxicity by
at least one order of magnitude (6, 9 –11). Two mechanisms, acting
synergistically, may explain this phenomenon: (a) subunit A acts as
a “chaperone” preventing the nonspecific binding of subunit B to
membranes (2, 6, 12); and (b) the specific binding of
125
I-crotoxin
to target membranes (13, 14) suggests that subunit A may be
involved in target recognition. Thus, crotoxin circulates nondisso-
ciated (i.e., as a complex) until it recognizes specific “acceptor
sites” on the target membranes. Some of these sites have been
identified (15, 16). On binding, crotoxin dissociates into their
subunits. Subunit B remains bound, whereas the subunit A is
released to the medium (2, 17). Thus, subunit A transforms
the PLA
2
from an unspecific cytotoxin into a self-target toxin
compound.
Received 3/26/01; revised 9/1/01; accepted 1/9/02.
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.
1
This work was supported by the Argentine National Council for
Science and Technology (CONICET).
2
To whom requests for reprints should be addressed, at Av. Libertador
4980, Floor 2 A, C1426 BWX Buenos Aires, Argentina. Phone: 5411-
4-409-6748; Fax: 5411-4777-0781; E-mail: Etche@tiac.net.
3
The abbreviations used are: PLA
2
, phospholipase A
2
; MTD, maximum
tolerated dose; CK, creatinine kinase; AST, aspargine transaminase;
ALT, alanine transaminase; CAT, computer-assisted tomography;
ECOG, Eastern Cooperative Oncology Group; DLT, dose-limiting tox-
icity; LD
50
, lethal dose 50%; CI, confidence interval; FVC, forced vital
capacity; AUC, area under curve.
1033 Vol. 8, 1033–1041, April 2002 Clinical Cancer Research
Research.
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