Download Disposition Characteristics of Macromolecules in

(CANCER RESEARCH 52, 4396-4401, August 15, 1992]
Disposition Characteristics of Macromolecules in the Perfused Tissue-isolated
Tumor Preparation1
Hirofumi limito, Yumi Sakamura, Kazuhiro Ohkouchi, Ryo Atsumi, Yoshinobu Takakura, Hitoshi Sezaki,
and Mitsuru Hashida2
Department of Basic Pharmaceutics, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan
ABSTRACT
Disposition characteristics of model macromolecules with different
physicochemical characteristics and macromolecular prodrugs of mitomycin ( . namely mitomycin C-dextran conjugates, were studied in tis
sue-isolated tumor preparations of Walker 256 carcinoma with the use
of a single-pass vascular perfusion technique. In constant infusion ex
periments, all radiolabeled macromolecules accumulated in the tumor
tissue, but the degree and pattern of distribution greatly varied, depend
ing on their electric charges. Positively charged macromolecules were
markedly accumulated compared with those that were neutral or nega
tively charged. In addition, their concentrations were significantly
higher in viable than in necrotic regions, while neutral and negative
compounds were distributed in necrotic rather than in viable regions.
Pharmacokinetic analysis of tissue concentration-time courses of posi
tively charged diethylaminoethyl and neutral dextrans showed that their
movement occurred by convective fluid flow, and that high tissue accu
mulation of positively charged macromolecules could be explained by
strong binding due to electrostatic interaction. For neutral and anionic
macromolecules with negligible affinity to the tissue, it was suggested
that the final concentration gradient between the viable and necrotic
regions was decided by their tissue fluid content. Thus, the present study
revealed the basic disposition characteristics of macromolecules in tu
mor tissue relative to their physicochemical properties.
INTRODUCTION
In cancer chemotherapy, it is important to deliver antitumor
drugs selectively to the tumor with minimum exposure of other
normal tissues, and various drug carrier systems have been de
veloped for achieving this. Conjugation of antitumor drugs to
macromolecules seems to be one of the most promising ap
proaches, since diverse physicochemical properties and func
tions of macromolecules can be utilized for control of drug
disposition (1-3).
In a series of investigations, we developed macromolecular
prodrugs of MMC,3 which are MMC-dextran conjugates hav
ing cationic and anionic charges as well as a variety of molec
ular weights, and examined their physicochemical, pharmacokinetic, and pharmacological
characteristics
(4-11). The
disposition and subsequent pharmacological effects can be con
trolled by selecting specific physicochemical properties. Tumor
targeting in vivo was achieved by systemic injection of
MMCDan in mice (10). These results were explained by the
Received 12/10/91; accepted 6/8/92.
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 accord
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by a Grant-in-Aid for Scientific Research from the Ministry
of Education, Science and Culture, Japan.
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: MMC, mitomycin C; MMCD, mitomycin C-dex
tran conjugate; MMCDo,,, cationic mitomycin C-dextran conjugate; MMCD„,
anionic mitomycin C-dextran conjugate; DEAED, diethylaminoethyl dextran;
CMD, carboxymethyl dextran; BSA, bovine serum albumin; cBSA, cationized bo
vine serum albumin; EB/BSA, bovine serum albumin labeled with Evan's blue;
VRS, vascular reference substance; T-10. T-70, dextrans with molecular weights of
10,000 and 70,000.
general relationships between the physicochemical characteris
tics and in vivo behavior, including tumor localization, which
we established for various model macromolecules (12). We fur
ther developed a monoclonal antibody-MMC conjugate by us
ing MMCDan as an intermediate. Immunotargeting
was
achieved, based upon these findings (13).
On the other hand, we established an experimental system for
the evaluation of the drug disposition in the tumor at the organ
level by using a tissue-isolated tumor preparation and moment
analysis (14). This system was applied to the pharmacokinetic
analysis of the disposition of antitumor drugs, including MMC
and its lipophilic prodrugs. The present study aimed to clarify
the disposition characteristics of model macromolecules and
MMCD in tumor tissue with the use of this system and to
construct a strategy of tumor targeting with a macromolecular
drug carrier system.
MATERIALS
AND METHODS
Chemicals. MMC was kindly supplied by Kyowa Hakko Kogyo Co.,
Tokyo, Japan. Dextrans were purchased from Pharmacia Fine Chem
icals Co., Uppsala, Sweden, and had average molecular weights of about
10,000 (T-10) and 70,000 (T-70). BSA (Fraction V) was obtained from
Armour Pharmaceutical Co., United Kingdom. The sources of radiochemicals were as follows: 7-amino[t/-14C]butyric acid (74 MBq/mg);
[i/-'4C]sucrose (0.11 MBq/mmol); and [methoxy-^Cfinulm
(0.35
MBq/g), New England Nuclear, Boston, MA; potassium [l4C]cyanide
(29.5 MBq), Amersham Japan, Tokyo; indium chloride ('" In Cla),
Nihon Mediphysics, Takarazuka, Japan. All other chemicals were of
reagent grade, obtained commercially.
Preparation of Model Macromolecules. DEAED(T-70) and CMD(T-70) were synthesized by reacting (diethylamino)ethyl chloride hydrochloride and monochloroacetic acid with dextran(T-70) in alkaline
solutions, respectively (12). Radiolabeled DEAED(T-70) and CMD(T-70) were synthesized by using [carooArv-l4C]dextran(T-70). Both
[carooxy-l4C]dextran(T-10) and [carftojcy-14C]dextran(T-70) were pre
pared by reacting potassium [l4C]cyanide with dextrans according to
the method of Isbell et al. (15), as reported previously (12).
cBSA was synthesized by coupling hexamethylenediamine to BSA
with a carbodiimide-catalyzed reaction according to the published
method (12). BSA and cBSA were labeled with ' ' ' In C13, using diethylenetriaminepentaacetic acid anhydride (Dojindo Labs, Kumamoto,
Japan), according to the method of Hnatowich et al. (16).
Preparation of MMCD. MMCDcal and MMCDan were synthesized
as reported previously (4, 9). MMCD was radiolabeled by coupling
7-['4C]aminobutyric acid to dextran activated by cyanogen bromide,
together with t-aminocaproic acid (['4C]MMCDcat) or to spacer-intro
duced dextran together with MMC by a carbodiimide-catalyzed reac
tion <[14C]MMCDan) (8). The radiolabel in MMCDs was stable and had
behavior consistent with that of a dextran backbone.
Animals and Tumors. Female SLC Wistar rats weighing 100 to
120 g were obtained from Shizuoka Agricultural Association for Lab
oratory Animals, Shizuoka, Japan. The Walker 256 carcinoma was
supplied by Shionogi & Co., Osaka, Japan, and was maintained by s.c.
inoculation of rats every 2 weeks.
Preparation of Tissue-isolated Tumors. According to the method of
Cullino and Grantham (17, 18), rat ovarian tissue-isolated tumor was
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DISPOSITION
OF MACROMOLECULES
prepared as reported previously (14). In brief, three small blocks of
Walker 256 carcinoma were inoculated into the adipose tissue around
the ovary, enclosed in a Sealon film envelope (Fuji Film, Tokyo, Japan),
then placed in the s.c. pouch of the abdomen. The tumor was grown in
a "tissue-isolated" form and the film was changed after 1 week. About
2 weeks after tumor inoculation, tumor weighing 4-8 g was used for
perfusion.
Perfusion Experiment. The tissue-isolated tumor was perfused ac
cording to the previous method (14) with slight modifications. Fig. 1
shows a diagram of the perfusion experiment. At first, all blood vessels
supplying nontumorous tissues around the tumor (left renal artery and
vein, left adrenal artery and vein, and aorta) were ligated. Immediately
after ligation of the aorta, vinyl tubing (inside diameter, 0.5 mm; out
side diameter, 0.9 mm; Durai Plastics & Engineering, Durai, Australia)
was inserted into the aorta and the perfusate was infused at a rate of
0.8 ml/min. Vinyl tubing (inside diameter, 0.8 mm; outside diameter,
1.2 mm) was inserted into the tumor vein for outflow sampling. Finally,
the tumor was transferred onto a plate in the experimental system. The
tumor mass was perfused with Tyrode's solution (a mixture of 137 HIM
NaCl, 2.68 mM KC1, 1.80 mivi CaCl2, 11.9 mivi NaHCO3, 0.362
HIMNaH2PO4, 0.492 ITIMMgCl2, and 5.55 HIMo-glucose) containing
BSA at a concentration of 4.7% (w/v). The same medium was used for
drug administration. The perfusate was gassed with 95% O2-5% CO2
and maintained at 37°C.Viability of the perfused tumor was confirmed
by measurement of lactic acid production. The rate of production dur
ing a 3-h perfusion experiment was within the range of 1.3-2.2 mmol/
h/100 g, which was comparable to that reported by Cullino and
Grantham (17). In addition, the viable region subjected to s.c. inocula
tion to normal rats after a 3-h perfusion showed almost the same
growth rate as freshly prepared tumor, suggesting its viability.
Indicator Dilution Experiment. Drug solution (0.1 ml) was intro
duced from the arterial side of the tumor by using a six-position valve
injector as a pulse function. The concentrations of the injection solu
tions were 1 mg/ml for MMC and 100 Mg/mlfor [14C]MMCDcat(T-70).
EB/BSA was used as a VRS and was applied prior to the drug injection
to obtain basic physiological information for each tumor preparation.
The drug solution was injected into the same tumor in the second run.
The outflow was collected into previously tared tubes at appropriate
time intervals (at first 5 s, subsequently 10 to 15s). The sample volume
was calculated from the gain in weight of the tube by assuming the
density of the perfusate to be 1.0. The concentration was determined by
using a spectrophotometer at 620 nm for EB/BSA and 365 nm for
MMC. To determine [14C]MMCDcal(T-70), 5 ml of scintillation me
dium, Clear-Sol I (Nacalai Tesque, Kyoto, Japan) was added to the
sample and 14C radioactivity was measured in a liquid scintillation
system (LSC 900, Aloka, Tokyo, Japan).
Pharmacokinetic Analysis of Indicator Dilution Experiment. Mo
ment analysis was used to analyze dilution curves and the statistical
reservoir
in incubator
IN ISOLATED TUMOR
moment parameters are calculated as follows (19);
-S:
-J;
AUC=
|
Cat
(A)
tCdt / AUC
(B)
o
MTT
where t is time and C is the concentration of a test substance normalized
by injection dose with a dimension of percentage of dose/ml. AUC and
MTT are the area under the concentration-time curve and the mean
transit time, respectively. The disposition parameters representing the
recovery ratio (F) and intrinsic clearance (CL¡m)
are calculated from the
moment parameters based on well-stirred conditions as follows (19);
F=AUC/AUCV,
CIinl = (2(1 - F)IF
(O
(D)
where Q and AUCvrs are mean inflow rate and AUC of VRS.
Constant Infusion Experiment. The drug-containing perfusate (10
Mg/ml) was infused at a rate of 0.8 ml/min into the tumor preparation
for up to 180 min. At the end of infusion, drug-free perfusate was
infused for 5 min to remove drugs remaining in the intravascular space.
During these procedures, the venous outflow was collected into tared
counting vials at appropriate time intervals. The sum of the outflow
perfusate and exúdate from the tumor was almost the same as
the inflow rate and no significant weight gain of the tumor during the
perfusion was detected. After washing the intravascular space, the tissue
was excised and carefully divided into viable and necrotic regions based
upon macroscopic observation. The viable region is the whitish periph
eral part of the tumor while the reddish and flabby necrotic region was
usually found in the center. Histológica! observation confirmed that
both samples essentially represented viable and necrotic tissues. These
samples were minced into small pieces. To measure the level of 14Clabeled macromolecules, tissue sample (approximately 0.1 g) was
placed into a counting vial and 0.7 ml of Soluene-350 (Packard, Down
ers Grove, IL) was added. The mixture was heated overnight at 50°C,
cooled to room temperature, and 0.18 ml of 2 N hydrogen chloride was
added. The radioactivity in the tissue sample and outflow were deter
mined in a liquid scintillation counter after adding 5 ml of scintillation
fluid. The radioactivity of the "'In-BSA was directly determined by a
Nal well-type scintillator (ARC-500, Aloka, Tokyo, Japan).
Pharmacokinetic Analysis of Constant Infusion Experiment. Time
courses of accumulation of [l4C]dextran(T-70) and [I4C]DEAED(T-70)
in the tumor tissue were analyzed based upon a parallel flow model
(Fig. 2). Here, a test compound, which is initially found in the vascular
space at a fixed concentration (Co), permeates across the vascular wall,
moves through the interstitial space, and finally oozes from the tumor
tissue along with the convectional fluid flow. In this model, the macromolecule is directly distributed to the viable and necrotic regions by
an independent convection flow with rates of Jv and Jn (ml/min/g). The
osmotic reflection coefficient for the macromolecule is assumed to be
zero throughout all processes. The macromolecule passes through the
interstitial water space with a volume of V and interacts with tissue
components. The degree of the interaction is expressed in terms of the
distribution volume (K). Based upon the model, the following equations
were derived to describe the concentration change in the viable (v) and
necrotic (n) regions.
dCv
dt
dCv'
~dT
= Jv(Co - Cv')
(E)
(F)
Fig. 1. Perfusion system of the tissue-isolated tumor preparation.
where Cvand Cn are concentrations in the viable and necrotic regions
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DISPOSITION
OF MACROMOLECULES
Constant Infusion
than 10% of the MMC was extracted by the tumor, whereas no
significant difference was observed in the uptake amounts of
[14C]MMCDca,(T-70) and VRS during a single passage after a
Vv
bolus arterial injection.
Constant Infusion Experiment. Fig. 4 shows a typical out
flow pattern of [14C]DEAED(T-70) during constant infusion
Cv1
Viable
Region
Kv
Co
IN ISOLATED TUMOR
Cv'
for 180 min. The outflow concentration reached a plateau of
almost 100% of the inflow concentration in three minutes, re
mained at the same level during infusion, then declined imme
diately after vascular washing. Essentially similar results were
obtained for other macromolecules (data not shown).
Fig. 5 illustrates the concentrations of all tested macromol
ecules in the tumor tissue after a 180-inin infusion. Marked
accumulation was observed in positively charged macromole
cules such as [14C]MMCDcat(T-10), [I4C]MMCDca,(T-70),
[14C]DEAED(T-70), and ['"InJcBSA.
Furthermore,
they
Necrotic
Region
Kn Cn'
Fig. 2. Pharmacokinetic models for analysis of the disposition of macromolecules in tissue-isolated tumor preparations after constant infusion.
normalized by inflow concentration; Co is the inflow concentration
(100%); J is the rate of convection flow (ml/min/g); C is the concen
tration in the interstitial space (%/ml); and (V+ K) then corresponds to
the apparent total distribution volume of the macromolecule per unit
weight (ml/g). Integration of Equations E and F give.
Jv
= Co(Vv + Kv)\ 1 -exp
Cn = Co(Vn + Kn)\ 1 -exp
Vv + Kv
-
Jn
Vn + Kn
(G)
showed higher concentrations in the viable than in necrotic
regions. In contrast, the concentrations of neutral ([14C]dextran(T-lO), [14C]dextran(T-70), and [l4C]inulin) and negatively
charged ([14C]MMCDan(T-70), [14C]CMD(T-70), and ['" In]BSA) compounds in the necrotic were relatively higher than
those in the viable region.
Pharmacokinetic Analysis of Disposition of Macromolecules.
Tissue concentration-time
profiles of neutral [14C]dextran(T-70) and cationic [14C]DEAED(T-70) in viable and necrotic
regions were determined during constant infusion for up to
180 min (Fig. 6). Both compounds accumulated in the tumor
(H)
=-
The concentration profiles were then fitted to Equations G and H by
using the nonlinear regression program MULTI (20), to estimate the
pharmacokinetic parameters.
Vascular and interstitial water space volumes of the tissue-isolated
tumor preparation were determined by using ¡l4C]dextran(T-70)
and [14C]sucrose as intravascular and extracellular markers, respec
tively. The tumor preparation was infused with perfusate contain
ing [l4C]dextran for 5 min or [l4C]sucrose for 60 min, and the radio
activity in each region of the tumor and in the outflow samples
was measured without washing with drug-free perfusate. Vascular
and interstitial water space volumes were calculated by using the
following equations
0>
(0
o
-o
C
o
co
1-
—¿
o
U
14C]Dextran in out flow (dpm/ml)
o
O
O
[I4C]Sucrose in tissue (dpm/g)
Extracellular space (ml/g) =
100 •¿
I+—
[l4C)Dextran in tissue (dpm/g)
Vascular space imi gì
=
1000
60
[l4C)Sucrose in outflow (dpm/ml)
120
180
240
300
Time (sec)
Fig. 3. Typical dilution curves of [I4C]MMCDC,,(T-70) together with VRS
(EB/BSA). •¿.
EB/BSA; O. |14ClMMCDc.,(T-70). Each compound was injected
separately into the same tumor.
Interstitial space (ml/g) = Extracellular space —¿
vascular space.
RESULTS
Table I Moment and disposition parameters for MMC. ['<C¡MMCDM (T-70),
unii I'RS (EB/BSA) from the indicator dilution experiment
Indicator Dilution Experiment. Fig. 3 shows a typical dilu
tion curve of [14C]MMCDcal(T-70) together with that of VRS
in a separate run of the same tumor preparation. The outflow
pattern of [14C]MMCDt.„,(T-70)was almost identical to that of
VRS. On the other hand, the concentration profile of MMC in
the outflow was lower in the initial phase and higher in the later
phase than that of VRS (data not shown). Table 1 summarizes
the moment and disposition parameters for MMC, [I4C]MMCDcal(T-70), and VRS. The analysis revealed that more
Values arc calculated from the dilution curves obtained after a bolus arterial
injection. Values are expressed as means of at least three experiments.
doses/ml)7774.26850.37528.77538.4MTT(s)44.370.135.332.4F10
of
DrugsEB/BSAMMCEB/BSA[I4C]MMCDC.,
(T-70)ADC(%
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DISPOSITION
OF MACROMOLECULES
IN ISOLATED TUMOR
In Fig. 6, simulation curves generated by using the parame
ters in Table 2 are also shown and relatively good agreement
with experimental data was obtained.
DISCUSSION
o
U
120
60
Time
180
(min)
Fig. 4. Typical outflow pattern of (14C]DEAED(T-70) in constant infusion
experiment. Perfusate containing [I4C]DEAED(T-70) at a concentration of 10
¿ig/mlwere infused into tissue-isolated tumors for 180 min and the tumor was
subsequently washed with drug-free perfusate for 5 min. The solid lines drawn
through the points were fit by eye, postulating that the equilibrium was at a
concentration of 100%.
Generally, tumor tissues are characterized by enhanced vas
cular permeability, high interstitial diffusion coefficients of
macromolecules, and lack of a functional lymphatic system
(21, 22). These characteristics seem to act as favorable factors
for passive transport of blood-oriented large molecules to the
tumor, and several investigators have reported the accumula
tion of macromolecules in tumors after i.v. injection (12, 2325). Thus, tumor targeting using macromolecular drug carriers
is rationalized by the physiological and anatomical character
istics of tumor tissues. On the other hand, it has also been
suggested that elevated interstitial pressure in tumor tissues
restricts the extravasation and interstitial transport of macromolecules and leads to their heterogeneous distribution (2628). For monoclonal antibodies, furthermore, antigen-antibody
interaction was shown to result in retarded penetration and
nonuniform distribution in the tumor (29-32). However, de
tailed information on the actual disposition of macromolecules
in the tumor tissue remains to be elucidated. We performed
perfusion experiments by using tissue-isolated tumor prepara
tions to assess the effect of physicochemical properties of mac
romolecules on their disposition in the tumor.
2 i oo
e
1 50
rflrfl
MMCDcal
(T-10)
MMCDcat MMCDan
(T-70)
(T-70)
Dexlran
IT-10)
- H
r-n
Dexlran
(T-70)
DEAED CMD
{T-70) (T-70)
Fig. 5. Concentrations of radiolabeled macromolecules in viable (D) and necrotic i! il regions of tissue-isolated tumors after constant infusion for 180 min
and subsequent washing for 5 min. Data are expressed as mean ±SD of the
percentage of radioactivity in the tissue to that in the inflow perfusate, assuming
the specific gravity of the tissue to be 1.0. *,statistically significant difference (P
< 0.05, t test) between concentrations in viable and necrotic regions.
tissue with time, and cationic [14C]DEAED(T-70) was accumu
lated at higher levels.
Prior to the analysis, the vascular volumes and the interstitial
water space volumes of viable and necrotic regions were deter
mined. The vascular volume in the viable region [0.0372 ±
0.0200 (SD) (N = 3)] was slightly larger than that in the necrotic
region [0.0282 ±0.0178 (N = 3)] (no significant difference),
whereas the extracellular space volume determined as equili
brated sucrose space at 60 min in the necrotic region [0.281 ±
0.048 (N = 3)], was significantly larger (P < 0.05) than that in
the viable region [ 0.194 ±0.019 (N = 3)]. Interstitial water
space volumes calculated as the difference between these vol
umes are shown in Table2 and are used for the pharmacokinetic
analysis.
Table 2 summarizes the parameters estimated by data fitting
the tumor concentration profiles based on the pharmacokinetic
model. The total distribution volumes of [14C]dextran(T-70) for
viable and necrotic regions were 71.7 and 57.7% of the inter
stitial water space volumes, respectively. On the other hand,
those of [I4C]DEAED(T-70) were 9.8- and 7.0-fold larger than
the interstitial water space volumes, respectively. The convec
tion flow rates were estimated to be 0.011 and 0.005 ml/min/g
in the viable and necrotic regions, respectively.
120
240
Time
360
(min)
Fig. 6. Concentration-time profiles and simulation curves of neutral [I4C]dextran(T-70) and cationic (I4C]DEAED(T-70) in the tissue-isolated tumor prep
aration after constant infusion. •¿,
[14C]dextran(T-70) (viable region); O,
|14C]dextran(T-70) (necrotic region); •¿.
[I4C]DEAED(T-70) (viable region);
D, [14C]DEAED(T-70) (necrotic region). Each value represents the mean ±SD of
the percentage of radioactivity in the tissue compared with that in the inflow
perfusate after infusion for each time period and subsequent washing. The specific
gravity of the tissue was assumed to be 1.0. '.statistically significant difference
(P < 0.05, f test) between concentrations in viable and necrotic regions. The
simulation curves were generated by using the pharmacokinetic parameters
from Table 2.
Table 2 Interstitial space volumes and pharmacokinetic parameters for
["CJdextran (T-70) and ["CJDEAED (T-70) in the tissue-isolated tumor
Numbers in parentheses are percentages to the respective interstitial space
volume. The abbreviations are: J, convection flow rate; y + K, total distribution
volume.
K(ml/g)
J (ml/min/g)
y+ tf(ml/g)
[l4C]dextran (T-70)
¡14C]DEAED(T-70)Viable
region0.157
region0.253
0.01120.113(71.7)
0.00500.146(57.7)
1.530(975)Necrotic
1.73 (701)
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DISPOSITION
OF MACROMOLECULES
We at first performed indicator dilution experiments, with
which we previously evaluated the disposition of low molecular
weight antitumor drugs (14), whose extraction by the tumor
occurred depending on their lipophilicity as shown for MMC
(Table 1). However, the macromolecules tested in this study
had similar dilution curves to that of VRS (Fig. 2), indicating
similar behavior to VRS at the extraction step. In addition, only
a slight decrease in the perfusate concentration of cationic mac
romolecules was observed during single passage under the
steady-state condition in the constant infusion (Fig. 4). These
results are contrasted to our previous findings in the liver,
where cationic macromolecules were extensively taken up
through discontinuous endothelium, which allows free contact
of macromolecules with the surface of the parenchymal cells
(33). The present facts indicated that architecture of vascular
wall is essentially tight in the tumor, unlike the liver, although
tumors should have permeable vasculature. The binding of mac
romolecules to capillary endothelium was also suggested to be
negligible.
In the tumor tissue, convection is considered to be the dom
inant mechanism of macromolecular transport (34), although it
is a relatively slow process. Therefore, we measured the accu
mulation of macromolecules in the constant infusion system to
elucidate the effects of their physicochemical properties (Figs. 5
and 6). Positively charged macromolecules were markedly ac
cumulated in the tumor, in particular in the viable region, which
was a well-perfused part. In contrast, those negatively charged
or neutral macromolecules were accumulated at lower levels,
and were characterized by being at a relatively higher level in
the necrotic region. The molecular weight had little effect in the
tested range (A/r 5,000-70,000).
From the data in Fig. 5, the apparent tissue uptake clearance
can be calculated by dividing the tissue concentration by the
AUC of the compound in the perfusate (11). The values for
cationic [14C]DEAED(T-70), which was markedly accumulated
in the tumor, were 6.2 and 3.8 ^l/min/g for the viable and
necrotic regions, respectively. Then, recovery ratio (F) of a macromolecule in the outflowed perfusate during the constant in
fusion is calculated as follows
Inflow rate - tissue uptake clearance
F =
Inflow rate
Assuming the tumor weight to be 6 g and weight ratio of viable
and necrotic regions to be 5:1, F is calculated to be 0.957 for
[14C]DEAED(T-70). At first glance, this value appears to be
inconsistent with the results described in Fig. 4, where the
outflow concentration was almost equal to inflow concentra
tion. Furthermore, indicator dilution experiments showed no
significant difference in uptake of [14C]MMCDca,(T-70) and
VRS. These discrepancies can be explained by the hypothesis
that the macromolecule is conveyed into the interstitial space by
convective flow. The concentration in the perfusate would not
change before and after passage through the tumor and the
macromolecule would have the same dilution curves as that of
VRS.
The tissue uptake clearance is an apparent index offering
only phenomenalistic information equivalent to that in Fig. 5
and no information about the accumulation mechanism. There
fore, we analyzed the accumulation process of macromolecules
with a pharmacokinetic model (Fig. 2), assuming convective
transport and tissue binding.
IN ISOLATED TUMOR
We determined the vascular and interstitial water volumes of
viable and necrotic regions for comparison with the apparent
distribution volumes of macromolecules. The reported values of
the vascular space in Walker 256 carcinoma varied to some
extent, depending on the marker substance (35). Song and Lev
itt (23) reported that the average vascular volume of s.c. im
planted Walker 256 carcinoma (0.01-4 g tumor) was 0.8%,
using 51Cr-labeled erythrocytes. Cullino and Grantham (36)
determined the vascular volume of tissue-isolated Walker 256
carcinoma (3.5-13 g tumor) with dextran 500 (Mr 37,500) tobe
10.8%. In a previous study, we reported the vascular volume of
tissue-isolated Walker 256 carcinoma (1-20 g tumor; mean,
8.71 g) to be 7.0% using EB/BSA (17). The vascular volumes
determined in the present study with dextran(T-70) (3.7 and
2.8% for viable and necrotic regions, respectively) were within
these values. However, leakage of dextran, especially in the
necrotic region, or contamination between these regions, might
be included in these values.
On the other hand, the interstitial water spaces determined in
the present study (15.7 and 25.3% for viable and necrotic re
gions, respectively) were smaller than that reported by Cullino
et al. (37) (38.7%) obtained by direct measurement of 24Na in
tissue fluid of Walker 256 carcinoma (4.7-10.4 g). Although
some differences were observed between these studies, we used
our values since those for viable and necrotic regions were sep
arately available.
Analysis of the accumulation pattern of cationic [I4C]DEAED(T-70) revealed that the apparent distribution volumes
in the viable and necrotic regions were 7 to 10 times higher than
those of the interstitial water volumes (Table 2), indicating
strong binding to the tissue. On the other hand, the apparent
distribution volumes for neutral [14C]dextran(T-70) were
smaller than the interstitial volumes.
Our previous studies in vitro demonstrated that MMCDcal
and other cationic macromolecules bound with leukemia cells
(8) and solid tumor cell layers grown on microporous mem
branes (38) due to electrostatic interactions. The same mecha
nism can account for the interaction of positively charged mac
romolecules with the tumor cell surface and components in the
interstitium. In contrast, negatively charged or neutral macromolecules would only be distributed in the interstitial fluid,
without any tissue interaction.
The sum of the convective flow (0.061 ml/min) calculated in
the viable and necrotic regions with a weight ratio of 5:1 ap
proximately corresponds to 7.7% of perfusion flow rate for 6 g
of tumor. In another study, we determined the rate of fluid loss
in isolated tumors with a mean weight of 6.36 g to be 16.6%
from the outflow recovery. Butler et al. (39) reported that extravascular fluid loss was 10.2% of the perfused plasma volume
in Walker 256 carcinoma (mean, 3.8 g tumors). These values
essentially correspond to our estimated convective flows, sug
gesting the reliability of considering convective flow as a driving
force of macromolecular movement.
The heterogeneous distribution in the tumor shown for cationic macromolecules in this study may be an obstacle to tumor
targeting with macromolecular carrier systems including mon
oclonal antibodies (26). A possible approach to overcome this
problem may be the use of the prodrug concept; i.e., regenera
tion of the parent drug in the interstitial space. The regenerated
free drug can readily distribute over the whole tumor mass by
diffusion, even if the movement of the prodrug (drug-macromolecule conjugate) itself is prevented by strong binding or a
4400
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DISPOSITION
OF MACROMOLECULES
high interstitial pressure. MMCD was shown to liberate free
MMC by simple chemical hydrolysis (5), and it is implied that
local constant infusion of MMCDcal might be effective for
intensive exposure of the tumor tissue to MMC irrespective of
its limited movement. However, further studies are required to
confirm whether regenerated MMC actually reaches into the
tumor cell itself.
Thus, the present study demonstrates the basic disposition
characteristics of macromolecules in a tissue-isolated tumor
preparation and the importance of their electric charge. These
findings will be useful for the development of macromolecular
prodrugs aiming at targeted cancer chemotherapy.
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Disposition Characteristics of Macromolecules in the Perfused
Tissue-isolated Tumor Preparation
Hirofumi Imoto, Yumi Sakamura, Kazuhiro Ohkouchi, et al.
Cancer Res 1992;52:4396-4401.
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