Download Prolongation of Drug Exposure in Cerebrospinal

[CANCER RESEARCH 5.1. IÕW6-I598. April I. 1993]
Prolongation
DepoFoam1
of Drug Exposure
in Cerebrospinal
Fluid by Encapsulation
into
Sinil Kim,2 Shiri»Khatibi, Stephen B. Howell, Cindy McCully, Frank \I. Balis, and David G. Poplack
Division oj Hematologv/Oncoloxv, Department of Medicine, UCSD Cancer Center. University of California, San Diego. IM Joua, California 92093 ¡Si.K., Sh. K., S. B. H.j, and
the Pediatrie Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda. Maryland 20892 (C. M., F. M. B., D. G. P.¡
MATERIALS
ABSTRACT
Prolonged maintenance of a therapeutic drug concentration
Materials.
in the cere-
AND METHODS
|5-'H]Cytosine-ß-i>-arabinoside for rudioimmunoassay
was
brospinal fluid is required for optimal treatment of leptomeningeal leu
kemia or carcinomatosis with cell cycle-specific antimetabolites. The pharmacokinetics of l-ß-i>-arabinofuranosylcytosine (ara-C) encapsulated into
DepoFoam (Depo/Ara-C) was studied in six rhesus monkeys after intrathecal injection into the lumbar sac. Following a single 2-mg dose, the
Depo/Ara-C concentration decreased biexponentially with initial and ter
minal half-lives of 14.6 and 156 h. respectively. The free drug concentra
purchased from Amersham (Arlington. IL), dextran-coated charcoal suspen
sion was obtained from Wien Laboratories (Succasunna, NJ), and ara-C was
purchased from Quad Pharmaceutical (Indianapolis. IN). Anti-ara-C polyclonal antibody raised in sheep was purchased from Guildhay Antisera (Guild-
tion remained above the reported minimal cytotoxic level of 0.1 Mg/ml (0.4
UM) for more than 672 h (28 days). In contrast, the half-life of ara-C
ical Co. (St. Louis. MO); dioleoyllecithin. dipalmitoylphosphatidylglycerol,
and cholesterol were obtained from Avanti Polar-Lipids. Inc. (Birmingham,
following an intralumbar bolus dose of unencapsulated drug in a single
animal was 0.74 h. A single intrathecal injection of Depo/Ara-C can main
AL); nanograde chloroform was obtained from Mallinckrodt (Paris. KY). All
reagents were used without further purification. The vortex mixer was obtained
from American Scientific Products (McGaw Park, IL).
Synthesis of Depo/Ara-C. For each batch of Depo/Ara-C (DepoTech
Corp.) prepared, 1 ml of a 20-mg/ml ara-C solution in water (with pH adjusted
to 1.1 with I N hydrochloric acid) was added to a I-dram vial containing 9.3
tain a therapeutic drug concentration
prolonged period.
in the cerebrospinal
ford. United Kingdom), gelatin and potassium phosphate were from Fisher
Scientific (Fair Lawn. NJ). and tetrahydrouridine was from Calbiochem (La
Jolla. CA). Triolein and L-lysine. free base, were purchased from Sigma Chem
fluid for a very
INTRODUCTION
umol of dioleoyllecithin, 2.1 |jmol of dipalmitoylphosphatidylglycerol,
15
pmol of cholesterol, 1.8 umol of triolein, and 1 ml of chloroform. The vial was
attached to the head of the vortex mixer and shaken at the maximum speed for
6 min. Each half of the resulting "water-in-oil" emulsion was individually
The two drugs most frequently used for intrathecal chemotherapy,
ara-C1 and methotrexate. are both cell cycle phase-specific antime
tabolites (1, 2). Since these drugs act only during S phase, they must
be present in the environment of the cancer cells for a sufficient time
so that all or most of the cancer cells would have attempted to divide
at least once. Prolonged drug exposure is even more important in the
subarachnoid space because of the longer doubling time for leukemic
blasts in the CSF (3). Unfortunately, the half-lives of methotrexate and
ara-C after bolus intrathecal administration in humans are short (4, 5),
and frequent or continuous intrathecal administration is impractical by
the intralumbar route (4, 6). Therefore, a slow-release depot prepara
tion of methotrexate or ara-C that will maintain a therapeutic concen
tration in the cerebrospinal fluid is needed for optimal therapy.
DepoFoam, or multivesicular liposomes, is composed of micro
scopic panicles consisting of bilayer lipid membranes enclosing mul
tiple nonconcentric aqueous chambers (7). The lipids in the bilayer
membranes are identical to those found in natural cell membranes.
DepoFoam (7) is distinct from other drug delivery systems (8-13) in
that each DepoFoam particle encloses multiple nonconcentric internal
chambers, the capture efficiency is high (14), and the average size of
DepoFoam particles and their internal chambers can be reproducibly
adjusted (7). DepoFoam is especially suitable as a slow-release mi
crocapsule for hydrophilic molecules such as ara-C, because of its
slow release rate and high stability in storage ( 15). We report here the
results of pharmacokinetic studies of intrathecally administered ara-C
encapsulated into DepoFoam in a nonhuman primate model.
Received 11/9/92; accepted 1/26/93.
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.
' Supported in part by Grants CH-368 and CH-484 from American Cancer Society and
Grants CA 23100 and 35309 from the NIH. This work was conducted in part by the
Clayton Foundation for Research-California Division. S. B. H.' is a Clayton Foundation
investigator.
2 To whom requests for reprints should be addressed.
' The abbreviations used are: ara-C, 1-ß-D-arabinofuranosylcytosine; CSF, cerebrospi
nal fluid; Depo/Ara-C. ara-C encapsulated into DepoFoam; AUC. area under the curve.
squirted rapidly through a narrow-tip Pasteur pipet into 1-dram vials, each
containing 2.5 ml of water, glucose (3.2 g/100 ml), and free-base lysine (40
m.M), and then shaken on the vortex mixer for 3 s at setting 5 to form
chloroform spherules. The chloroform spherule suspensions in the two vials
were transferred into the bottom of a 250-ml Erlenmeyer flask containing 5 ml
of water, glucose (3.2 g/100 ml), and free-base lysine (40 HIM).A stream of
nitrogen gas at 7 liters/min was flushed through the flask to evaporate chlo
roform for 10 to 15 min at 37°C.The Depo/Ara-C particles were then isolated
by centrifugation at 600 X g for 5 min and washed with 0.97r NaCl solution
3 times. Sterile procedures were used for Depo/Ara-C preparation. The average
volume-adjusted size of the DepoFoam particles was 19 urn. the percentage of
capture was 597r. and capture volume was 36 |al/mg of total lipids used (14).
Animals. Seven adult male rhesus monkeys (Macaca mulatta) weighing 9
to 12 kg were obtained from the Hazelton Labs Primate Products. Animals
were singly housed and were fed NIH Open Formula Extruded Nonhuman
Primate Diet twice daily in accordance with the "Guide for the Care and Use
of Laboratory Animals" (16). Animals were given general anesthesia with
ketamine and Rompun. After sterile preparation of the lumbosacral region, a
22-gauge spinal needle was inserted into the subarachnoid space at the L5-L(,
intervertebral space. A zero time point CSF sample was obtained. In 6 mon
keys, 2 mg of Depo/Ara-C was injected intrathecally followed by a 0.4-ml
flush of Elliott's B solution. One animal received a 2-mg intralumbar (intra
thecal) dose of unencapsulated
ara-C as a control. CSF samples were obtained
from the lumbar thecal sac under general anesthesia at 0.33, 24, 48. 96, 120,
192, 336. 504, and 672 h in the animals given injections of Depo/Ara-C and at
0.25, 0.5, 1. 2, 3, 4, 6, and 8 h in the single animal given an injection of
unencapsulated ara-C. A drop of undiluted CSF was mounted on a hemocytometer and DepoFoam particles and WBC were counted manually. The De
poFoam particles in CSF were separated by centrifugation in an Eppendorf
microfuge for 5 min. The supernatant was separated from the pellet and both
were kept frozen until assayed. A simultaneous blood sample was obtained in
a heparini/ed tube. To prevent degradation of ara-C. all blood and CSF samples
were collected into tubes containing 40 msi (final concentration) tetrahydrou
ridine.
The concentration of ara-C in monkey CSF was determined in triplicate by
radioimmunoassay according to a previously published method (17, 18). The
buffered diluent used throughout the assay was 0.05 M phosphate buffer.
1596
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[NTRATHECAL
DEPO/ARA-C
PHARMACOKINETICS
100000
containing 0.6% (w/v) NaCI and 0.1% (w/v) gelatin, pH 7.6. The limit of
detection was 20 ng/ml and the coefficient of variation was generally less than
10%.
The pharmacokinetic curves were fitted to the biexponential function
E 10000
C(t) = Ae~"' + Be~e'
where Ctt) is the concentration at time /. A and B are coefficients, and a and
ßare the initial and terminal rate constant. The RSTRIP computer program
(MicroMath Scientific Software, Salt Lake City, UT) was used for curve fitting
by iterative nonlinear regression. The AUC was determined by linear trape
zoidal rule up to the last measured concentration and extrapolated to infinity.
The CSF clearance of the drug was calculated by dividing the dose of ara-C by
the AUC.
1000i
200
400
600
800
HOURS
Fig. 2. Number of DepoFoam particles in CSF over time. Data from 6 monkeys. Bars
SEM.
RESULTS
Fig. 1 shows the CSF concentration curves of both free and DepoFoam-entrapped ara-C following intrathecal injection of a single 2-mg
dose of Depo/Ara-C (n = 6) and the curve of free ara-C following
injection of unencapsulated ara-C (n = 1). The entrapped ara-C con
centration in CSF decreased in a biexponential fashion with an initial
half-life of 14.6 h and a terminal half-life of 156 h. The free drug
200
400
600
800
HOURS
1000
—¿. 100i
E
at
~~~
o
o
101
1;
concentration remained above the reported minimal cytotoxic level of
0.1 ug/ml or 0.4 UM(19) for greater than 672 h. In contrast, the free
ara-C CSF concentration in the control animal (given an injection of
unencapsulated ara-C) decreased in a biexponential fashion with an
initial half-life of 0.13 h (8 min) and a terminal half-life of 0.74 h (44
min). The ara-C concentration remained above the minimum cyto
toxic level for 11 h in the control animal. The AUC of free ara-C for
the Depo/Ara-C group was 334-ug-h/ml and that for the control ani
mal was 337 ug-h/ml. The free-drug clearance for the Depo/Ara-C
group was 0.100 ml/min versus 0.099 ml/min for the control animal.
The CSF bulk flow rate for the model is 0.040 ml/min. No ara-C was
detectable in blood (limit of detection, 20 ng/ml).
The DepoFoam particle count over time is plotted in Fig. 2. The
DepoFoam count decreased with an initial half-life of 20 h and a
terminal half-life of 277 h, similar to the half-life of encapsulated
ara-C depicted in Fig. 1.
Although it was difficult to differentiate Depofoam particles from
WBC, the animals appeared to develop a moderate pleocytosis, peak
ing with a median of 400 cells/mm3 at 2 to 3 days. No animals
demonstrated evidence of neurotoxicity or systemic toxicity attribut
able to intrathecal Depo/Ara-C.
.11
DISCUSSION
.01
200
400
600
800
HOURS
Fig. 1. CSF pharmacokinetics after a single 2 mg dose of Depo/Ara-C {lop) and
unencapsulated ara-C (bottom). •¿Depo/Ara-C concentration; O. free ara-C concentra
tion. Data from 6 monkeys for Depo/Ara-C group and one animal for unencapsulated
ara-C. Ban. SEM.
Lumbar punctures for intrathecal chemotherapy are uncomfortable
for patients and time consuming for physicians. Even with an Ommaya reservoir, frequent or continuous administration is impractical
and may carry an increased risk of infection. These studies, performed
in a well-established nonhuman primate model that has proved to be
predictive of CSF pharmacokinetics in humans, demonstrated that a
single intrathecal injection of DepoFoam encapsulated ara-C results in
the maintenance of cytotoxic drug concentrations in the CSF for a
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INTRATHECAL
DEPO/ARA-C
prolonged period. There was a 200-fold increase in the terminal halflife of the drug from 0.74 to 156 h compared with unencapsulated
ara-C. This is comparable to the results in rats where the intrathecal
half-life increased from 2.7 to 148 h (15).
Although an efficacy study has not been done in an animal leptomeningeal tumor model, a single dose of ¡.p.Depo/Ara-C resulted in
100% cures in mice inoculated i.p. with 1 million L1210 cells 1 day
previously. In contrast, no cures were observed with a single dose of
unencapsulated ara-C (20).
We have not performed histopathological examinations in these
primates. However, a previously reported histopathological study in
rats revealed no abnormalities with the exception of a single rat that
developed a small focus of infiltration by neutrophils in a spinal nerve
root, presumably due to inadvertent introduction of bacteria at the
time of intrathecal injection (15).
It has been reponed that intrathecal injection into the lumbar sac
may result in poor distribution into rostral CSF compartments (21).
One contributing factor for this limited distribution is rapidity of drug
efflux from the CSF relative to the slower rate of CSF circulation into
the rostral compartments. We speculate that entrapment of drug in
DepoFoam could allow for a more uniform distribution of the drug
throughout the entire neuraxis because of the slower encapsulated
drug efflux from the CSF. Furthermore, since the drug-containing
DepoFoam particles are heavier than CSF, there is a possibility that
this approach would permit a gravitational effect on CSF drug distri
bution, in which rostral movement of drug could be facilitated after
injection in the lumbar sac by simply positioning the patient in a
Trendelenburg position.
The use of a slow-releasing depot permits maintenance of pro
longed therapeutic drug concentrations in the CSF. This may both
optimize drug exposure to tumor and avoid the need for frequent
lumbar punctures. Further studies are being planned to explore the
distribution of DepoFoam-encapsulated drug in the CSF. The efficacy
and toxicity of intrathecal Depo/Ara-C in humans will be addressed in
a Phase I clinical trial.
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Prolongation of Drug Exposure in Cerebrospinal Fluid by
Encapsulation into DepoFoam
Sinil Kim, Shirin Khatibi, Stephen B. Howell, et al.
Cancer Res 1993;53:1596-1598.
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