[CANCER RESEARCH 33, 906-914,
April 1973]
Diffusion in Vitro and in Vivo of
l-(2-ChloroethyI)-3-(ira«s-4-methylcyclohexyl)-l-nitrosourea
from Silicone Rubber Capsules, a Potentially New Mode of
Chemotherapy Administration
Mark L. Rosenblum, Donald L. Bowie, and Michael D. Walker
Section of Neurosurgery, Baltimore Cancer Research Center, National Cancer Institute, NIH, Baltimore, Maryland 21211
resulting in dose-limiting systemic toxicity. The purpose of
this study is to present an alternative mode of administration
Diffusion of drugs from silicone rubber capsules is a that will permit continuous infusion of chemotherapeutic
potentially new mode of chemotherapy administration. Dry drugs over long periods of time and avoid all these difficulties.
crystalline
l-(2-chloroethyl)-3-(/rara-4-methylcyclohexyl)-lAgents that are either insoluble or unstable in aqueous
nitrosourea, a very hydrophobic and unstable nitrosourea, was solutions may also be given in this manner. Moreover, the
encapsulated and permitted to diffuse into various solutions at possibility of drug infusions directly at tumor sites may result
37°for 30 days. Significant and relatively constant diffusion
in greater effectiveness and less systemic toxicity than with the
was reproducible and predictable. The diffusion rate was present modes of therapy.
The diffusion of drugs from silicone rubber (dimethylpolyindependent of the amount of drug packed inside capsules,
directly proportional to the available surface area, and affected siloxane) capsules may permit continuous administration of
many agents after systemic capsule implantation.1 The tissue
by membrane thickness and temperature. l-(2-Chloroethyl)-3(fra/is-4-methylcyclohexyl-l -nitrosourea
remained dry and compatibility
of the polymer makes such implantations
reasonably stable inside capsules for 30 days at physiological feasible (7, 9). The physical characteristics of the silicone
temperatures. Capsule implantation s.c. in rats resulted in 50 polymer have been shown to permit significant passage of
to 60% greater cumulative diffusion than observed in vitro into many agents from membrane enclosures (1—3,5, 6,9. 11, 18,
27, 28, 29), and it has been implied that more
0.9% NaCl solution. The techniques presented are applicable 21-23,
to other chemotherapeutic
agents, especially those that are lipid-soluble substances diffuse more readily (8, 10, 12, 13, 24,
non-ionized and lipid soluble. Administration by diffusion 31). This investigation determines the in vitro and in vivo
diffusion of methyl-CCNU,2
a highly lipid-soluble and
from capsule implants may have several advantages. Continu
ous long-term infusion of drugs is possible and avoids the unstable chemotherapeutic
agent, through silicone capsules
difficulties inherent with p.o. and i.v. therapy. Substances that and explores the influence of factors affecting its dynamics.
are insoluble or unstable in aqueous media may be readily
administered. Furthermore, capsule implantation directly at MATERIALS AND METHODS
tumor sites may result in greater effectiveness and less
systemic toxicity.
General. Methyl-CCNU (obtained from Cancer Chemo
therapy National Service Center, who also provided the infor
mation on its characteristics) is very insoluble and unstable
INTRODUCTION
in aqueous solutions and shows a 50% decomposition within
Continous long-term therapy with certain cancer chemo
53 min when dissolved in an ethanol:pH 7.4 buffer (1:19)
solvent at 37°.Dry crystalline methyl-CCNU is stable, with
therapeutic agents may be advantageous (30, 32). However,
resulting from storage at room
the currently available modes of administration present many only 4% decomposition
temperature for 30 days. The breakdown products of the drug
difficulties. Therapy p.o. introduces such variables as absorp
tion irregularities and possible agent decomposition from have not yet been fully elucidated.
Methyl-CCNU 2-chloroethyl-14C3 was prepared with noninteraction with gastrointestinal enzymes and bacterial flora.
Frequent administration by this route also requires strict labeled drug to provide a specific activity ranging from 10 to
patient cooperation and assumes that emesis and gastroin
1V. Schmidt, W. Zapol, W. Prensky, T. Wonders, I. Wodinsky, and R.
testinal distress, common side effects from many chemother
apeutic agents, are not complicating factors. The hazards of Kitz. Continuous Cancer Chemotherapy: Nitrosourea Diffusion through
long-term i.v. administration,
with its local and systemic
Implanted Silicone Rubber Capsules. Abstract presented at Meeting of
infections and required patient immobilization, are well American Society for Artificial Internal Organs, Seattle, Wash., April
16,1972.
known. In addition, several drugs cannot be given i.v. because
"The abbreviation used is: methyl-CCNU, l-(2-chloroethyl)3-(f/-flHS-4-methylcyclohexyI)-l-nitrosourea (NSC 95441).
of their hydrophobic characteristics and aqueous instability.
3Methyl-CCNU-2-chloroethyl-14C was supplied by the Drug Devel
Furthermore,
most agents lack tumor specificity thereby
SUMMARY
Received July 5,1972;accepted
906
January 3, 1973.
opment Branch, National Cancer Institute, under contract with
Monsanto Research Corporation, Dayton, Ohio (Contract NIH 723715).
CANCER
RESEARCH
VOL. 33
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Methyl-CCNU Silicone Diffusion
12 X IO4 dpm/mg.
Silicone capsules were prepared
from
silicone tubing (Silastic; Dow Corning Co., Midland, Mich.)
with dimensions of 3.2 mm outside diameter x 2.0 mm inside
diameter. One end was sealed with Silastic type A Medical
Adhesive (Dow Corning), a product that polymerizes to
silicone rubber and binds intimately with the capsule wall. The
adhesive was permitted to cure for 2 to 4 days at room
temperature. Because of the marked instability of methylCCNU in aqueous solutions, dry crystalline powder was used
as the packing material. After gravimetric weighing of the
empty capsules, they were packed firmly and evenly to various
lengths and reweighed. Capsules are denoted by the length of
packed drug. The open end was then sealed with the same
adhesive and allowed to cure for an additional 2 to 4 days.
Just prior to the start of each experiment, the adhesive ends of
each capsule were arbitrarily cut to 6-mm lengths. Each
capsule was then rinsed briefly in absolute methanol to remove
any methyl-CCNU that may have diffused to the external
capsule surface and was subsequently washed extensively with
water. The capsule was placed into a stainless steel wire mesh
basket, which was suspended by means of silk suture to the
cap of a 20-ml vial. The vial was then filled with 20 ml of
various prewarmed fluids. With the cap secured, this apparatus
provided an airtight unit which, when agitated in a water bath,
allowed gentle mixing of the internal solution. Unless
otherwise stated, all experiments were performed at 37°.The
sampling procedure was performed every 24 hr for 30 days as
follows. The basket was removed from the solution of the
previous day, throughly washed in running water, and then
replaced into fresh, prewarmed, solvent-filled vials. Duplicate
0.2-ml samples of the desorbing solution were placed in
scintillation vials to which were added 18 ml of scintillation
fluid [4.2% Liquifluor (New England Nuclear. Boston,
Mass.):30% absolute methanol:65.8% toluene]. All samples
were counted in a Packard Tri-Carb Model 3375 liquid
scintillation spectrometer, with internal and external stan
dardization. Results were corrected for quench and dilution
and expressed as mg of methyl-CCNU. The data were plotted
as the cumulative diffusion versus time, and the slope of the
resultant linear regression analysis was defined as the diffusion
rate in mg/day.
Determination of Sampling Frequency. To determine a
sampling frequency that would permit the maximum rate of
diffusion (quasi-steady state), capsules of 3 different lengths
(2.5, 5.0, and 7.5 mm) were prepared and allowed to diffuse
into 0.9% NaCl solution at 37°.Two groups of 3 capsules, 1 of
each size, were investigated over a 48-hr period. One group was
sampled every 3 hr, and the desorbing solution was changed to
fresh, prewarmed 0.9% NaCl solution at each sampling
interval. The other group was sampled every 3 hr, but the
desorbing solution was never changed. Comparison of the
curves, resulting from the plot of the cumulative mg of
methyl-CCNU diffused against time, determined the optimum
sampling frequency for quasi-steady-state diffusion.
Diffusion into 0.9% NaCl Solution. Experiments were
performed to determine the diffusion rate of methyl-CCNU
into 0.9% NaCl solution, to evaluate the reproducibility of
these results, and to see whether the quantity of drug present
within a capsule affects its diffusion rate. Five capsules of each
of 3 different lengths (2.5, 5.0, and 7.5 mm) were prepared in
the usual manner by firm packing of the crystalline drug.
Several additional capsules of each length were packed to
different degrees of firmness. All of the above capsules were
permitted to diffuse into 0.9% NaCl solution at 37°during 3
separate experiments, and the results were tabulated and
compared.
An experiment was performed to determine the dependence
of diffusion on surface area. Capsules of 1.25 to 15.0-mm
lengths were prepared in the usual manner except that glass
rods of 2.0-mm diameter were used to seal the capsule ends.
Silastic Medical Adhesive, which adheres firmly to glass, was
used to connect the rods to the capsule wall. It was assumed
that methyl-CCNU does not diffuse significantly through glass.
The resultant diffusion rates were plotted against surface area
and were also compared to the rates observed from similar
length capsules made in the usual manner.
For determination of the effect of membrane thickness,
silicone adhesive was molded around tubing to obtain a
capsule with a wall thickness of 6.0 mm. Diffusion was
permitted into 0.9% NaCl solution at 37°,and the results were
compared to the diffusion rates of capsules with 0.6-mm side
walls.
For determination of the effect of temperature, 5.0-mmlong capsules were prepared in the usual manner and permitted
to diffuse into 0.9% NaCl solution at 21.5°and compared with
the diffusion results for similar capsules at 37°.
Diffusion into Plasma and Lipid. In order to determine
whether different desorbing solutions affect the diffusion
process, we made capsules in the usual manner and permitted
them to diffuse into human plasma and a fat emulsion at 37°.
Capsules 2.5 and 5.0 mm long diffused into citrated human
blood plasma obtained from healthy blood donors. The plasma
had a pH of 7.66, an osmolality of 300 mosm, and a sodium
and chloride content of 170 and 70 mg/100 ml, respectively.
It contained 3.6 g of albumin, 6.1 g of total protein, and 190
mg of total lipid per 100 ml. All experiments were performed
aseptically. In addition, capsules 5.0 mm long were diffused
into Intralipid (Cutter Laboratories, Berkeley, Calif.), a fat
emulsion containing 10 g soybean oil, 1.2 g phospholipids, and
2.3 g glycerol per 100 ml of aqueous solution. The diffusion
results were compared between both these solutions and 0.9%
NaCl solution at 37°.
Water Diffusion and Stability Studies. It has been reported
previously (26) that water diffused rapidly through silicone
membranes. That finding and the marked aqueous instability
of methyl-CCNU have prompted experiments to determine the
actual water diffusion under the conditions of this study and
the stability of methyl-CCNU inside capsules. Capsules 5.0 and
7.5 mm long were prepared with nonradiolabeled methylCCNU. Five capsules of each of these lengths were suspended
into vials containing 20 ml of 0.9% NaCl solution that had
been prepared with tritiated water to a specific activity of 1.16
X 10s dpm/ml. Both vials were gently agitated at 37°.One
capsule of each length was removed after 5 min (control) and
weekly thereafter for 4 weeks. After extensive washing in
running water, the capsules were cut open, and the internal
contents and capsule walls were analyzed separately. The
internal contents, primarily methyl-CCNU crystal, were placed
directly into scintillation vials. The capsule walls were
completely dissolved in 1.0 ml of NCS solubilizer (Nuclear
Chicago, Chicago, 111.)by agitation at 50°for 48 hr. After the
addition of 18 ml of scintillation fluid, samples were analyzed
APRIL 1973
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907
M. L. Rosenblum, D. L. Bowie, and M. D. Walker
separately for tritiated water content with a standard tritium
quench curve.
The content of two 7.5-mm-long capsules containing
radiolabeled drug were analyzed after 30 days of diffusion into
0.9% NaCl solution at 37° to determine the amount of
the 1st day and decreased to a relatively constant rate by
about the 5th day. Linear regression analysis of all points
yielded curves with an average correlation coefficient of 0.988.
The diffusion rate (D) of each experiment is determined by
the resulting slope in mg/day. Due to the initially high
decomposition of the remaining methyl-CCNU. Ether:water
diffusion rate, when extrapolated to zero time, all curves had y
partitioning combined with thin-layer chromatography has intercepts significantly greater than zero (D0). This value is
been used to determine the decomposition of other related proportional to the initial rate of diffusion. The results of
nitrosoureas by Oliverio et al. (25), and a modification of their analysis for 7 different capsules in each of 3 different lengths
technique was used in this study. The capsules were cut open, are shown in Table 1. Also included are the weights of packed
and the contents were dissolved in 10 ml of ether to which 10 drug and the 21-day cumulative diffusions. In each group there
ml of distilled water were added. The solutions were also was little variability in diffusion rates. No significant differ
added in the reverse order. Partitioning was permitted for 30 ences in the diffusion rate were observed over the range of
min with frequent shaking. After separation of the 2 phases. drug weights tested. With the exception of the very loosely
0.2 ml of each solvent was analyzed in the usual way for packed capsules, the values for D0 were relatively constant
radioactivity and corrected to total volume. The percentage of over the weights tested, and the cumulative 21-day diffusions
drug in the ether phase was calculated by the formula: [dpm are about the same.
(ether)]/[total
dpm (ether + water) X 100]. Thin-layer
For analysis of the effect of surface area on diffusion,
chromatography was performed on both the ether and water capsules were made with glass rather than silicone adhesive
extracts and compared to known methyl-CCNU. Samples of ends. The diffusion results are shown in Table 2. The diffusion
50 ¿u!were spotted on Silica Gel F-254 plates (Brinkman rate is directly proportional to the surface area of the
Instruments, Inc., Westbury, N. Y.) and developed in absolute membrane. A plot of the surface area against diffusion rate
methanol. The RF values of these chromatograms were resulted in a linear regression with a slope of 0.26 and a y
determined both by the fluorometric method as well as intercept of 0.02. When one notes the significantly higher
radiochromatogram
analysis with a Packard Model 7201 diffusion rates for capsules of similar length with silicone
adhesive ends, as shown in Table 1, it becomes obvious that
radiochromatogram scanner.
In Vivo Studies. Silicone capsules 2.5, 5.0, and 7.5 mm long the adhesive ends contribute significantly to methyl-CCNU
were prepared in the usual manner. Capsules were implanted diffusion.
Actual physical changes inside the capsules were noted over
s.c. into the flanks of 110 male Fischer CDF rats, weighing
the
30-day period of each study; examples are shown in Fig. 1.
150 to 250 g (purchased from Charles River Breeding
From about the 4th day, a "clear space" began to appear
Laboratories, Wilmington, Mass.). After various periods of in
vivo diffusion, the capsules were removed and dissolved in 1.0 between a plug, formed of the packed drug, and the internal
ml of NCS solubilizer by agitation at 50°for 48 hr. Analysis capsule wall. Over time this plug decreased in volume and a
for the amount of drug remaining was performed in the usual larger space progressively developed between the plug and the
manner. The difference between the amount of methyl-CCNU entire internal capsule surface, including the silicone adhesive
ends (Fig. 1, A to D). The formation of this "clear space" was
present before and after implantation was considered the
amount of drug diffused. Five capsules of each length were
studied for periods of up to 7 days. Morphological study of
the implantation sites was not performed since all animals
were followed at least 60 days for evidence of drug toxicity.
O 16
ut
RESULTS
Determination
of Sampling Frequency. The cumulative
diffusion was plotted against time for capsules suspended in
0.9% NaCl solution at 37°.No significant difference (Student's
t test, p < 0.05) was found between the slopes (diffusion
rates) for capsules of similar length whenever the daily
cumulative diffusion was less than 1.3 mg. Maximal diffusion
was prevented when greater than that amount accumulated in
the desorbing solution over 24 hr. Therefore, all subsequent
experiments were performed with daily sampling, and any
capsule that permitted greater than 1.3 mg of drug to diffuse
into 0.9% NaCl solution over 24 hr was excluded from
analysis.
Diffusion into 0.9% NaCl Solution. An example of
methyl-CCNU diffusion is shown in Chart 1 in which the
cumulative mg of drug diffused is plotted against the time in
days. In all experiments, the diffusion rate was greatest during
908
a 4
5
s
o
IO
20
30
TIME (days)
40
Chart 1. An example of the diffusion results obtained in this study.
Methyl-CCNU was permitted to diffuse from a capsule 5.0 mm long
into 0.9% NaCl solution at 37°for 30 days. The cumulative amount of
drug diffused is plotted against the time in days; a linear regression
analysis is superimposed on the daily points. The diffusion rate is the
slope of this curve in mg/day.
CANCER RESEARCH VOL. 33
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Methyl-CCNU Silicone Diffusion
Table 1
A nalysis of methyl-CCNU diffusion into 0.9% NaCl solution at 37°
Internal contents
Capsule
length
(mm)2.5D°
, days0
(mg/day)0.150.130.150.140.150.170.16D0°(mg)0.51.21.21.51.51.51.5Q,
(mg)3.44.04.44.44.65.24.9Wt
(mg)4.06.87.88.08.28.310.9Firmness
packingVery
of
looseFirmFirmFirmFirmFirmVery
firm
0.15±0.01&
5.0
0.19
0.20
0.23
0.21
0.20
0.23
0.23
1.1
1.9
2.1
2.0
1.8
1.8
2.0
5.0
6.1
7.0
6.6
6.1
6.7
6.9
6.6
11.7
12.6
13.1
13.3
13.3
17.6
Very loose
Firm
Firm
Firm
Firm
Firm
Very firm
1.0
1.8
2.5
2.6
2.4
2.3
2.9
6.9
9.0
9.8
8.5
9.4
8.6
9.4
9.0
18.1
18.5
18.9
19.2
20.9
25.5
Very loose
Firm
Firm
Firm
Firm
Firm
Very firm
0.21 ±0.01b
7.5
0.28
0.34
0.34
0.27
0.32
0.29
0.30
0.31 ±0.01b
" Calculated from straight-line analysis of cumulative amount diffused versus time, Q( =
DT + D0, where Q is the cumulative mg diffused over time T in days, D is the diffusion rate,
and D0 is the extrapolated^ intercept.
b Mean ±S.E.
different for glass-end capsules, where the space formed only
between the drug and the side walls of the capsule (Fig. \F).
This is a visual display of significant diffusion through the
silicone-adhesive ends. When capsules were cut open, the
"clear space" was found to be devoid of obvious solid or liquid
medium.
Analysis of the effect of membrane thickness is difficult
when dealing with cylindrical capsules, for as the membrane
thickness is increased so is the surface area. For elimination of
the surface area as a factor, the diffusion rate is divided by the
external surface area to give a specific rate of diffusion
expressed as mg/sq cm/day. For determination of the effect of
membrane thickness, a capsule was prepared with silicone
adhesive molded to provide a wall thickness of 6.0 mm. The
specific diffusion through the previously described glass-end
capsules with a membrane thickness of 0.6 mm was 0.277
mg/sq cm/day. If a true inverse relationship exists between
diffusion rate and membrane thickness, the specific diffusion
rate of the thicker membrane should be one-tenth of this latter
value. However, the actual specific diffusion rate observed for
the 6.0-mm membrane capsule was 0.063 mg/sq cm/day, or
about one-fifth of the thinner membrane.
The dependence of the methyl-CCNU diffusion on tempera
ture is illustrated in Table 3. The diffusion rate for capsules
5.0 mm long into 0.9% NaCl solution at 21.5° was 0.08
mg/day as compared to 0.21 mg/day for diffusion at 37°.As
expected, these rates are significantly different (Student's t
test, p < 0.05).
In order to be able to anticipate the cumulative diffusion of
methyl-CCNU for any length of capsule over any period of
time, graphs were made of both the mean diffusion rate (D)
and the mean diffusion extrapolated to zero time (D0) versus
capsule length. Both resultant functions were linear regressions
with correlation coefficients of 0.99 and 0.96, respectively,
and fit the following formulas:
D = 0.033 L + 0.066
(A)
were D is expressed in mg/day and L is the capsule length in
mm; and
= 0.247 L + 0.564
(B)
where D0 is expressed in mg.
When these values for D and D0 are substituted in the linear
regression formula for methyl-CCNU diffusion:
(C)
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909
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M. L. Rosenblum, D. L. Bowie, and M. D. Walker
Table 2
Relationship of methyl-CCNU diffusion to surface area for
glass-end capsules diffusing 0.9% NaCl solution at 37°
we obtain:
ß= [0.033 (¿)+ 0.066] T + [0.247 (L) + 0.564]
(D)
area
area
rate
Capsule
(mg/sq
cm-day)0.290.360.320.280.270.270.31
where Q is the cumulative
(D) (mg/day)0.-040.090.160.210.270.41£)/surface
length
(mm)1.252.55.07.510.015.0Surface
(sqcm)0.130.250.500.751.001.50Diffusion
±0.02°
" Mean ±S.E.
diffusion (mg) from a capsule of
any length over any period of time, T (days). The average
percentage of difference between the calculated and actual
values for capsules of 2.5-, 5.0-, and 7.5-mm lengths over the
entire study period was only ±4.0%,which is well within
experimental variation.
Diffusion into Plasma and Lipid. The rates of methyl-CCNU
diffusion into human blood plasma and a fat emulsion at 37°
are listed in Table 3. No significant difference is observed
between the diffusion rates into plasma and 0.9% NaCl
solution for capsules 2.5 or 5.0 mm long (Student's t test,
p > 0.05). However, diffusion from 5.0-mm long capsules into
fat emulsion was 0.31 mg/day as compared to 0.21 mg/day for
diffusion into 0.9% NaCl solution. This difference is statis
tically significant (Student's t test, p < 0.05).
Water Diffusion and Stability Studies. The results of the
water diffusion experiment are listed in Table 4. The diffusion
rate of water into capsules containing crystalline methylCCNU was only 0.1 ¿d/dayafter the 14th day. Water diffused
into the silicone walls and inside the capsules at approximately
the same rate after the 1st week. The cumulative water
diffusion for each of the above components is plotted against
time (Chart 2).
Essentially no water diffuses inside capsules for 2 weeks,
and after 30 days only 1.2 and 2.1 n\ were found inside
capsules of 5.0- and 7.5-mm lengths, respectively. Water
droplets became visible inside a capsule only after all the
methyl-CCNU had diffused out as shown in Fig. \E.
The 2 capsules containing radiolabeled methyl-CCNU which
were analyzed for stability of the remaining drug by
ethenwater
partitioning
showed 95.5 to 97.4% of all
radioactivity in the ether phase. Thin-layer chromatography of
this extract disclosed an RF value by both fluorescence and
radiochromatogram
scanning that was identical to control
methyl-CCNU. It was concluded that less than 5% of the
methyl-CCNU remaining inside the capsules was a decomposi
tion product after 30 days of in vitro diffusion at 37°.
Fig. 1. Changes observed in capsules after various periods of
diffusion into 0.9% NaCl solution at 37°.The dark area within a
capsule is dry crystalline methyl-CCNU. Capsules are shown prior to
initiation of study (A); after 1 week (B); after 2 weeks (O; and after 3
weeks (D) of diffusion. The plug, formed of crystalline drug, decreases
in volume as a "clear space" between the plug and the entire internal
capsule wall progressively increases in size. Only after all drug has
diffused out [after 60 days (£')]are water droplets visible within a
capsule. After 21 days of diffusion from a capsule made with glass ends
(F), a "clear space" develops only between the drug and the exposed
side walls.
910
In Vivo Studies. The results of in vivo methyl-CCNU
diffusion is shown in Chart 3, in which the cumulative
diffusion is plotted against time for 3 capsules of different
lengths. The observed diffusion rates (slopes) were 0.40, 0.59,
and 0.68 mg/day for capsules 2.5, 5.0, and 7.5 mm long,
respectively. A comparison of the in vivo and in vitro results is
shown in Table 5. The in vivo cumulative diffusion was about
50 to 60% greater than in vitro diffusion into 0.9% NaCl
solution. However, when a comparison is made to the in vitro
diffusion into a fat emulsion, a difference of only 21% is
observed.
Preliminary investigation of drug toxicity was based upon
animal survival. Eighteen of 110 rats died subsequent to drug
implantation with a median day of death of 40 days after
capsule implantation and a range of 33 to 57 days. No deaths
were noted if a cumulative dose of less than 3.5 mg of
methyl-CCNU was administered over any period of time. The
percentage of deaths observed for groups receiving larger
amounts of drug appeared dose related.
CANCER RESEARCH VOL. 33
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Methyl-CCNU Silicone Diffusion
Table 3
Methyl-CCNU diffusion into various desorbing solutions
ofsamples15060150606060Diffusion
rate
ature373737373721.5No.
(mg/day)0.15
Capsule
solutionNaCl
length(mm)2.55.0Desorbing
solutionPlasmaNaCl
±0.01°0.1
±0.010.21
4b
solutionPlasmaFat
±0.010.25b
0.020.31e
±
0.020.08e
±
±0.01
emulsionNaCl
solutionTemper
0 Mean ±S.E.
b Not significantly different than the diffusion rate into 0.9% NaCl solution at 37°
(Student's t test, p > 0.05).
c Significantly different than the diffusion rate into 0.9% NaCl solution at 37°(Student's t
test, p < 0.05).
Table 4
Analysis of the diffusion of water into silicone capsules containing methyl-CCNU
of
Capsule
rate"
methyl-CCNU(mg)15.4
length
(ml/day)0.07
(mm)5.07.5Wt
20.1Diffusion
diffusion
(0)b(ml)1.25
(ml/cu
mm)0.08
0.12Cumulative 2.120/volume
0.090/wt
(ml/mg)0.08
0.11
0 Rate determined for period after 14 days.
b Over a 30-day period.
1
is
_.# 7.5 mm
-•5.0 mm
2 5mm
Z 2
UJ
16
20
TIME (days)
24
28
32
Chart 2. Curve showing the cumulative amount of water diffused
into capsule walls (A) and inside capsules (B) for 2 capsule lengths. For
details see "Materials and Methods."
DISCUSSION
The difficulties inherent with the presently available modes
of chemotherapy administration, as indicated earlier, has led
to this study of capsular diffusion. Methyl-CCNU, a recently
developed nitrosourea, has been found effective in the
treatment of several animal tumor systems (16, 17, 19) and is
presently being investigated in Phase II and III human studies.
Furthermore, its relatives, l,3-bis(2-chloroethyl)-l-nitrosourea
and l-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea,
have shown
effectiveness in the treatment of several human tumors (14,
15, 20, 33, 34). Methyl-CCNU has been chosen for study for 2
reasons: (a) the diffusion of a substance that is insoluble and
unstable in aqueous media could be investigated; (b), because
of the ability of the drug to cross the blood-brain barrier
I
g
6
2345
TIME
(days)
Chart 3. The cumulative diffusion of methyl-CCNU in vivo from
capsules of 3 different lengths is plotted against time; linear regression
analyses are superimposed for each size capsule. Each point represents
the mean of 5 samples. For details see "Materials and Methods."
(A. F. Reynolds, M. L. Rosenblum, D. L. Bowie, and M. D.
Walker, unpublished results), both systemic and intracranial
cancers may be treated by s.c. capsule implantation.
Diffusion of a drug through a polymer capsule has been
generally accepted to occur in 3 steps: absorption of the drug
into the wall at its internal surface, diffusion across the wall,
and desorption at the outer surface (12, 13). The rates of
absorption and desorption are proportional to the solubility of
the drug in the wall and the desorbing solution, respectively
(12, 13, 18, 31). However, the diffusion of methyl-CCNU
through silicone capsules results in the progressive formation
of a "clear space," devoid of obvious liquid or solid media,
between the enclosed drug and the internal capsule wall. This
APRIL 1973
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911
M. L. Rosenblum, D. L. Bowie, and M. D. Walker
Table 5
Comparison between the cumulative diffusion (Q) of methyl-CCNU
in vivo and in vitro over 7 days
similarly fabricated capsules are reproducible. The diffusion
rate has been shown to be directly proportional to the surface
area of the membrane. In other studies with silicone capsules,
the assumption has generally been made that diffusion through
In vitro
capsule ends is insignificant when compared to the contribu
vivo
Capsule
tion of the much thinner capsule walls (18,21,31).
However,
différence052612160
solution"SSFSQ (mg)b2.53.64.84.7%
length
(mm)2.55.07.5In
(Q)(mg)3.85.87.5Desorbing
with methyl-CCNU we have shown that significant diffusion
does occur through capsule ends that are 10 times thicker than
the side walls. In addition we have found that, although
diffusion decreases with increased membrane thickness, a
true inverse relationship does not exist, as has been shown for
" S, 0.9% NaCl solution; F, fat emulsion, both at 37°.
other more polar substances (12, 18, 31). Both these findings
h Mean cumulative diffusion for 5 capsules.
illustrate that methyl-CCNU readily diffuses across a silicone
c Calculated from the formula (Qin vivo - Qin „¿/(„,)/((?,„
vitro~>
membrane.
x loo.
The marked difference in the diffusion rates observed for
experiments that were performed at 21.5°and 37°,points out
phenomenon has not been described previously for capsule
diffusion, although a similar observation was noted with a the necessity for strict temperature regulation in studies of this
drug-silicone matrix (27). This possibly results from a rapid nature.
rate of methyl-CCNU absorption. The mechanism of this
When drugs in solution are placed inside a membrane
diffusion out of silicone capsules must therefore include enclosure, the resulting diffusion rate has been found to
additional factors: the release of molecules from the drug depend on its concentration
(2, 12, 13, 18). This is
surface and its diffusion across the observed "clear space." If understandable when one recalls the dependence of diffusion
this space is indeed rate limiting, its abolition should result upon concentration
gradients. However, when only dry
subsequently in a higher diffusion rate. This was found, in crystalline drug is packed within a capsule, the usual concept
fact, when the contents of a capsule that had been diffusing of concentration is not valid, and the diffusion rate may be
into 0.9% NaCl solution at a rate of 0.2 mg/day was dispersed independent of the quantity of drug present. This was found,
by severe capsule agitation on the 18th day of diffusion. The in fact, when the diffusion rate over 30 days was shown to be
amount of methyl-CCNU diffused during the subsequent 24 hr independent of the amount of methyl-CCNU placed inside
was 1.2 mg, the same amount observed on the 1st day of the capsules. However, a lower initial rate, reflected in a smaller
experiment. Thus, a possible explanation for the observed value for D0, was observed occasionally for very loosely
decreasing rates of diffusion is that the "clear space" becomes
packed capsules and a slightly smaller cumulative diffusion was
progressively larger during the course of an experiment. Since noted. Perhaps this resulted from incomplete initial utilization
the drug must diffuse across an increasing distance, a of the available internal surface area.
progressive decrease in diffusion rate is expected. This
Analysis of diffusion rates from different size capsules has
decreasing rate, most obvious during days 1 through 5 is permitted the formulation of an equation that accurately
observed also during the period of relatively constant diffusion predicts methyl-CCNU diffusion into 0.9% NaCl solution. The
and is valuable because it permits the diffusion of methyl- equation can be solved for any of its 3 variables: cumulative
CCNU over long periods of time. A mathematical model is diffusion, time, and capsule length. By this means, diffusion
currently being formulated to better explain this phenomenon can be calculated for a capsule of any length over any period
and will be presented in a subsequent publication.
of time. Alternatively, capsules of appropriate lengths can be
A true steady-state measurement of drug diffusion requires fabricated to permit the diffusion of a specified amount of
the desorbing solution to act as an "infinite sink" for the
drug over a desired interval. Also, in the same way, the time
diffusing substance. Since the diffusion of an agent depends can be anticipated for a desired amount to diffuse from a
upon the concentration gradient (2, 12, 13, 18), to allow capsule of a specific size.
Stability of the encapsulated drug is required for long-term
maximum rates of diffusion the drug concentration in the
desorbing solution should approximate zero at all times. treatment. Despite the previously reported rapid diffusion rate
Furthermore, if one is to record true unindirectional permea of water through silicone membranes, very little permeated
the
tion, the drug should not be permitted to diffuse back into the into capsules containing methyl-CCNU. Furthermore,
capsule. This may be prevented by total ionization of the drug amount of decomposition
of the drug remaining inside
in the desorbing solution, since only nonionized molecules capsules after 1 month of in vitro diffusion was identical to
diffuse (12, 13, 24). Under present conditions, with a the amount observed for dry crystalline drug stored at room
nonpolar agent such as methyl-CCNU, neither circumstance is temperature.
Therefore, it appears that methyl-CCNU is
possible. However, a situation may be obtained where these reasonably stable inside silicone capsules at physiological
factors are kept to insignificant levels. Such was the case in temperatures for long periods of time.
this study, where sampling every 24 hr permitted quasi-steadyMethyl-CCNU has been found to diffuse into both plasma
and 0.9% NaCl solution at the same rate. However, the rate of
state diffusion.
This study has shown that methyl-CCNU readily diffuses diffusion into a fat emulsion was significantly greater. This
through silicone rubber and that the amounts diffusing from may be explained by an increased rate of drug desorption due
912
CANCER RESEARCH VOL. 33
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Methyl-CCNU Silicone Diffusion
to the greater solubility of methyl-CCNU in a lipid medium.
However, since lipid-soluble substances diffuse readily into
silicone, there is also the possibility that the lipid diffused into
the wall and changed the diffusion properties of the capsules
themselves. The fact that lipids accumulate in silicone after
long-term implantantion in humans (4) makes this possibility
feasible. This may also explain the observed more rapid
diffusion of methyl-CCNU in vivo.
We have shown that a lipid-soluble, unstable chemotherapeutic agent is readily and predictably released by capsular
diffusion over long periods of time. Studies are in progress to
evaluate the effectiveness of this mode of administration for
methyl-CCNU in the treatment of animal tumor models. The
techniques used in this study may be applied to other
chemotherapeutic
agents and therefore is potentially very
rewarding.
12. Garrett, E. R., and Chemburkar, P. B. Evaluation, Control and
Prediction of Drug Diffusion through Polymeric Membranes II. J.
Pharm. Sci., 57: 944-959, 1968.
13. Garrett, E. R., and Chemburkar, P. B. Evaluation, Control and
Prediction of Drug Diffusion through Polymeric Membranes III. J.
Pharm. Sci., 57: 1401-1409, 1968.
14. Hansen, H. H., Selawry, O. S., Muggia, F. M., and Walker, M. D.
Clinical Studies with l-(2-Chloroethyl)-3-cyclohexyl-l-nitrosourea
(NSC 79037). Cancer Res., 31: 223-227, 1971.
15. Iriarte, P. V., Hananian, J., and Cortner, J. A. Central Nervous
System Leukemia and Solid Tumors of Childhood: Treatment with
l,3-Bis(2-chloroethyl)-l-nitrosourea.
Cancer, 19: 1187-1194,
1966.
16. Johnston, T. P., McCaleb, G. S., Opliger, P. S., Aster, W. R., Jr.,
and Montgomery, J. A. Synthesis of Potential Anticancer Agents
38. ./V-Nitrosoureas 4. Further Synthesis and Evaluation of
Haloethyl Derivatives. J. Med. Chem., 14: 600-614, 1971.
17. Johnston, T. P., McCaleb, G. S., Opliger, P. S., and Montgomery, J.
A. Synthesis of Potential Anticancer Agents XXXVI JV-Nitrosoureas II Haloalkyl Derivatives. J. Med. Chem., 9: 892-911,
ACKNOWLEDGMENTS
1966.
18.
Kind,
F. A., Benagiano, G., and Angee, I. Sustained Release
We are indebted to Dr. Carl C. Levy, Chief, Laboratory of
Hormonal Preparations 1. Diffusion of Various Steroids through
Pharmacology, Baltimore Cancer Research Center, for his assistance in
Polymer Membranes. Steroids, //. 673-680, 1968.
the preparation of this manuscript.
19. Laster, W. R., Jr., Mago, J. G., Andrews, C. M., and Schabel, F. M.,
Jr. Lewis Lung Carcinoma as an Experimental Model for Study
with Anticancer Drugs as Surgical Adjuvants. Proc. Am. Assoc.
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913
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CANCER RESEARCH
VOL. 33
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Diffusion in Vitro and in Vivo of 1-(2-Chloroethyl)-3-(trans
-4-methylcyclohexyl)-1-nitrosourea from Silicone Rubber
Capsules, a Potentially New Mode of Chemotherapy
Administration
Mark L. Rosenblum, Donald L. Bowie and Michael D. Walker
Cancer Res 1973;33:906-914.
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