ICANCER RESEARCH54, 5374—5379,
October 15, 1994]
Effects of Photodynamic Therapy Using Mono-L-aspartyl Chiorin e6 on
Vessel Constriction, Vessel Leakage, and Tumor Response1
Kimberly
S. McMahon,
T. Jeffery
Wieman,
Pamela
H. Moore,
and Victor H. Fmgar@
Department of Surgery, Division of Surgical Oncology, University of Louisville, Louisville, Kentucky 40292
ABSTRACT
damage is further supported by work by Gomer and Ferrario (4) and
The effect of photodynamic therapy using mono-L-aspartylchiorin e6
(NPe6) on both direct cytotoxicity and vascular damage was examined.
Sprague-Dawley
jections
rats bearing
chondrosarcoma
tumor
were given
of 5 or 10 mg/kg NPe6 and exposed to 135-i/cm2
i.v. in
664-nm laser
light either 4 or 24 h after NPe6 injection. The percentage of viable tumor
cells was estimated either immediately after the completion of light treat
ment or 24 h after treatment using a 3-(4,5-dimethylthiazol-2-yl).2,5diphenyltetrazolium bromide assay. Measurements of arteriole constric.
lion and venule leakage in normal cremaster tissues were made during
and 1 h after the light treatment.
Tumor response
was evaluated
for the
4 different NPe6 dose and time combinations. Both direct tumor cytotox
icity
and
vascular
stasis
were
observed
during
light
treatment.
leakage did not occur. Blood flow stasis was a result ofplatelet
Vessel
aggregation
and the mechanical obstruction of flow rather than vessel constriction.
The magnitude ofdirect cytotoxicity and vascular response was dependent
on both the amount ofNPe6 delivered and the delay between Injection and
light treatment. Tumor cure was found in animals either when given high
NPe6
doses
or when
treated
early
after
NPe6
injection.
Treatment
regi
mens which maximized the effect of both vascular stasis and direct tumor
cytotoxicity
were found to produce
Ferrario
et al. (6), who found that treatment
efficacy
was best when
light treatment was delivered between 2 and 6 h after NPe6 injection.
Significant decreases in treatment efficacy occurred if light treatment
was given 12—24h after NPe6 injection, even though tumor levels of
photosensitizer
were greatest
at this time. The contribution
of direct
tumor cytotoxicity on tumor response in vivo is not known, but it is
likely to play some role because high levels of drug are bound to
tumor cells.
This study was designed to evaluate the events which lead to blood
flow stasis after NPe6 activation using an intravital microscopy model
and compare the sequence to that observed in tissues treated with
other photosensitizers. The role of direct tumor cytotoxicity in PDT
using NPe6 was evaluated using a modification of the MiT assay.
Tumor cytotoxicity after PDT using NPe6 is expected based on the
tissue distribution studies done by Gomer and Ferrario (4) and Fer
rario Ct a!. (6), however, direct cytotoxicity may be inhibited as a
result of tissue hypoxia. Observation of the timing required before
vascular stasis may provide information regarding this inhibition.
the best tumor response. Dose combi
nations which produced vascular stasis with minimal early cytotoxicity
did not result in cure. The combined mechanisms of damage after photo
dynamic therapy using NPe6 suggests that this photosensitizer may have
specific advantages for clinical use and provides a benchmark for the
development of new photosensitizers.
MATERIALS AND METhODS
Photosensitizer
NPe6 NPe6 was a generous gift from Beckloff Associ
ates, Kansas City, MO, and Nippon Petrochemicals Company, Tokyo, Japan.
NPe6 was received in a lyophilized powder and was reconstituted using 0.9%
sodium chloride injection (United States Pharmacopoeia)
INTRODUCTION
2.0 mg/nil.
NPe63 is a promising new photosensitizer for use in PDT. NPe6 has
many characteristics which make it an attractive photosensitizer. It is
a chemically pure agent, it absorbs light at long wavelengths (664 tim)
(1), and PDT using NPe6 has produced tumor cure in experimental
tumor models (2, 3). Furthermore, studies have demonstrated a re
duced duration of cutaneous photosensitivity compared to Photofrin
(4). NPe6
is currently
undergoing
phase
I clinical
trials
for use in
photodynamic therapy.
Though preliminary data yield promising results, the mechanism of
tumor destruction in vivo using NPe6 has yet to be completely
resolved. Damage to tissue after PDT using NPe6 appears to be
secondary to vascular damage and hemostasis. Nelson et a!. (3, 5)
found RBC extravasation and hemorrhagic necrosis after treatment
that was correlated with damage to the subendothelial zone of capil
lanes. This damage was shown to result from collagen disruption and
fragmentation rather than the collagen coalescence observed after
treatment with PDT using Photofrmn. A vascular mechanism of
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
investigation
was
supported
by
USPHS
Grant
CA51771
awarded
by
the
Na
tional Cancer Institute, Department of Health and Human Services, by the Department of
Surgery, and by the James Graham Brown Cancer Center.
2 To
whom
requests
for
reprints
should
be
addressed,
at
University
of
Louisville,
Department of Surgery, James Graham Brown Cancer Center, 529 South Jackson Street,
Louisville, KY 40292.
3 The
abbreviations
used
are:
NPe6,
mono-L-aspartyl
chiorin
e6; PDT,
intervals
to a concentration of
and stored in the dark at
4°C.NPe6 was injected into the tail vein of rats at doses of 5.0 or 10.0 mg/kg,
4 or 24 h before the light treatment.
Tumor
Model.
A chondrosarcoma
tumor line maintained
was used in this study (7). Approximately
in our laboratory
500 mg of nonnecrotic tumor from
donor animals were minced in 2 ml of Hanks' balanced salt solution containing
2000 units streptomycin and 2 mg penicillin (Sigma Chemical Co., St. Louis,
MO). The suspension was passed through a tissue sieve (Cellector tissue sieve,
30-mesh screen; Sigma) and then through graded needles. For tumor implan
tation, 0.3 ml (3 X i0@cells) tumor suspension was injected s.c. into the center
of the
on the right hind limb of Sprague-Dawley rats (100—130g; Harlan
Laboratories, Indianapolis, IN) with a 21-gauge needle. Tumors were used for
experimentation when they had reached a surface diameter of 6—10
mm and a
thickness
of 2—3mm. Tumors
were
free of evident
necrosis
at the time of
treatment.
Animals were given injections of the photosensitizer
before treatment. Prior
to treatment, rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.).
The right hind limbs of tumor-bearing or control rats were shaved and depi
lated. Tumors and a surrounding area of normal skin were illuminated as
described below.
Light
Received 12/14/93; accepted 8/16/94.
The costs of publication of this article were defrayed in part by the payment of page
NPe6 was prepared at weekly
Treatment
Protocol.
For photosensitizer
activation,
an argon
dye
laser (models 165 and 375B; Spectra Physics, Mountain View, CA) with a
fiber optic light delivery system was used to illuminate a 2.0-cm-diameter field
centered on the cremaster or the tumored right hind limb. The wavelength was
adjusted to 664 am. The wavelength was verified by a scanning monochro
mator (model DMC1—02;
Optometrics, Ayer, MA). The power density of light
was adjusted to 75 mW/cm2 as measured
by a thermopile
(model 210;
Coherent, Auburn, CA). This power density was not sufficient to induce a
significant increase in tissue temperature (8). A microlens (gradient index lens;
General Fiber Optics, Inc., Cedar Grove, NJ) was attached to the end of the
fiber to ensure uniform light distribution. Treatment areas were illuminated
photodynamic
therapy; FITC, fluorescein isothiocyanate; MT1', 3-(4,5-dimethylthiazol-2-yl)-2,5-diphen
yltetrazolium bromide; RBCC, RBC column.
with 135-i/cm2 light (30-min treatment).
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EFFECI@SOF PDT USING NPe6
animal. This data established tumor cell kill due to hypoxia and nutrient
Cremaster Muscle Preparation for Microvascular Studies. Sprague
Dawley rats (100—150 g) were anesthetized with sodium pentobarbital (50
deprivation.
MU
mg/kg, i.p.) and placed on their backs on a temperature-controlled heating pad.
Rectal temperature was maintained at 37°Cand back temperature was moni
tored with a thermocouple to avoid local overheating of the skin. The right
cremaster muscle was prepared for microvascular observations in the manner
reported previously (8). The muscle was slit on the ventral midline and spread
with sutures over a cover glass which was positioned
in the bottom of a 60-ml
(Sigma)
was
prepared
by adding
sodium
bicarbonate
buffer
(pH
adjusted to 7.4) with a final concentration of 5.0 mg/ml. The solution was
filtered through a 0.2-p.m syringe filter (sterile Millex-GS; Millipore, Bedford,
MA), and stored
until used.
Immediately following or 24 h after treatment, tumor cells were excised,
weighed, and carefully minced under subdued lighting. Tumor was transferred
capacity Plexiglas bath. This bath was filled with a modified Krebs solution.
to a 15-ml polypropylene centrifuge tube containing a 0.1-mi MiT solution
The bath was constantly mixed by aeration with CO2 and N2 gases. The pH of
the Krebs solution was maintained at 7.40 ±0.02 by adjusting the flow of CO2
and 1.0 ml culture medium (RPM! 1640; Sigma). Cells were incubated in the
dark for 4 h at 37.5°C.
After incubation, cells were centrifuged at 2500 rpm for
into the bath. An indwelling heater maintained the bath temperature at 35°C. 10 min. The supernatant was poured off and 5.0 ml dimethyl sulfoxide were
The right carotid artery was cannulated for the measurement of mean arterial
blood pressure and heart rate, and for the infusion of fluorescein isothiocya
nate-labeled albumin. Blood pressure and heart rate were monitored using a
blood pressure analyzer (MicroMed Instruments, Louisville, KY). Arteriole
added. The cells were allowed to stand for 10 mm with intermittent vortexing,
followed by sonication for 1 mm (Sonic Dismembrator model 150; Fisher
Scientific, Pittsburgh, PA). Samples were centrifuged at 10,000 X g for 15 min
following the addition of 15 ml distilled water. Sample absorbance at 570 nm
and venule pairs were chosen for the study based on their diameter (20—30 was measured in the supernatant using a spectrophotometer (DU-62 Spectro
g.tm) and branching order (in general, fourth-order vessels were chosen al
though occasionally third-order vessels were studied). Arterioles could be
photometer; Beckman Instruments, Inc., Palo Alto, CA). Cells with intact,
viable mitochondria turn the solution from yellow to purple. Sample absor
easily discerned from other vessels by the presence of a smooth muscle wall,
rapid blood flow, and blood flow in the direction from large vessels to small
vessels. Venules had no muscle wall, exhibited slower blood flow, and had
bance was compared to a standard curve in order to calculate percent cell
flow in the direction from small vessels to larger vessels. RBCC diameter and
arteriole wall diameter were measured every 5 min for the 30 min of light
treatment, and every 10 mm for a 1-h observation period following light
therapy. The RBCC diameter is a measurement of the inner lumen of the
vessel. Reductions in RBCC diameter without a change in wall diameter
indicate that aggregates have formed at the inner vessel wall. Nine to 11
animals were used in each different experimental group for measurement of
vessel diameters.
Light and fluorescent Microscopy and Image Analysis. In order to
visualize the cremaster muscle under the intravital miCroscope, transmitted
light and fluorescence microscopy were used (8, 9). For the fluorescence study,
0.5 lad/kg FITC-labeled albumin (10) was injected into a cannula leading into
the right carotid artery (equivalent to 26 pg/kg fluorescein and 10 mg/kg
albumin). Epiillumination using blue light (450—490am) was used to stimu
late the FITC for brief periods (<5 s) every 5 mm during light therapy, and
every 10 nm for a 1-h observation period after light treatment. These short
durations of illumination did not induce any vascular changes (11). In the
viability. The standard curve was prepared by measuring
MTT conversion
from different weights of viable tumor and verified with tumor standards
containing known percentages of viable and dead cells (different amounts of
tumor killed by heat sterilization were mixed into minced suspensions of viable
cells to produce standards containing viable/nonviable ratios of 20:80, 50:50,
and 80:20).
The reproducibility
and sensitivity
of the assay was increased
by
dividing the viability data from the treated tumor by its untreated internal
control.
Statistical
Analysis.
Data from experiments were grouped and the means
calculated. Data are presented as the mean ±2 X SEM. The experimental data
set was compared using one-way analysis of variance. Dunnett's test was used
to compare the results of experiments to the controls and a multiple compar
isons test was done to evaluate data groupings (14, 15). Type II error was held
to 0.10 for the multiple comparisons testing. Differences between groups were
considered statistically significant if P < 0.05.
RESULTS
V using a 10-ng/ml fluorescein diacetate standard. A 1-h equilibration period
preceded each experiment.
Examination of macromolecular leakage was done using digital image
Alterations in Vessel Diameter and Vessel Lumen Diameter.
Profound changes were observed in normal cremasteric vessels
treated with NPe6 and light. For each of the experimental treatment
groups, significant reductions in the diameter of the lumen of arte
rioles (RBCC diameter) were observed 1 h after treatment compared
to the initial value before treatment or to the controls (P < 0.0001).
Complete blood flow stasis was observed in 20—30-sm arterioles in
animals given 5 mg/kg NPe6, 4 h before the light treatment. Stasis
analysis
occurred
absence of F!TC-albumin in the interstitium, the images of the blood vessels
appear bright white on a dark background. The images were recorded on
videotape with a closed-circuit television system. A Cohu silicon intensifying
target television camera (Cohu Electronics, San Diego, CA) was used to work
with very low fluorescent
as described
light intensities.
previously
The camera voltage
(8). Changes
in FITC-albumin
was set at 0.10
leakage
from
vessels were quantitated by selecting areas of interest adjacent to vessels and
by measuring the alterations in the gray level of pixels in this area. Each set of
experimental data was corrected for any fluctuations in the intensity of the
fluorescence light source or in the camera sensitivity that may have occurred.
Eight to 10 animals were used in each different experimental group.
Assessment
of Tumor Response.
Rats were examined for tumor regrowth
daily for the first 14 days after treatment, and weekly thereafter for a total of
42 days. Treatment areas lacking a macroscopically
visible and palpable tumor
were considered flat. Animals free of tumor after this time were considered
cured. Twenty to 35 animals were used in each treatment group.
MTT Assay. Tumor cell viability was assessed after treatment using a
variation of a colorimetric MiT assay (12, 13). Animals were prepared with
chondrosarcoma
tumor in both the right and left hind limbs for these studies.
The tumor in the right limb was given treatment as necessary and the tumor in
the left limb served as an additional control. Animals were given injections of
NPe6 at either 5 or 10 mg/kg, and light treatment was administered at either 4
or 24 h after NPe6 injection. Groups of 9 animals were used. Additional
control groups consisted of animals given NPe6 alone, animals given light
alone, or animals given neither light nor drug. In addition, a group of 5 animals
were given no photosensitizer and sacrificed without light treatment. Tumor
tissue was prepared for the assay immediately or 24 h after sacrificing the
by the end of the light treatment
and blood
flow
did not
resume during the observation period (Fig. la). Blood flow stasis was
a result of aggregation and coagulation of platelets and other blood
constituents. Vessel constriction was not observed in animals given
NPe6 and light. Animals given either NPe6 alone (5.0 mg/kg i.v., 4 h),
or light treatment (135 J/cm2) alone showed no apparent change in
vessel diameter or change in the RBCC diameter within arterioles. In
animals given 10 mg/kg, i.v., 4 h before the light treatment, the vessel
response was more dramatic. Blood flow ceased in arterioles within
the first one-third of the light treatment (45 J/cm2) (Fig. ib). The
mechanism of stasis was similar to animals treated with 5 mg/kg
NPe6, i.e., stasis resulted from platelet aggregation and coagulation of
blood elements rather than by vessel constriction or a combination of
these effects. Platelet aggregation and thrombi formation were con
firmed
by electron
microscopic
study
of treated
tissues
(data
not
shown).
The vascular response to PDT using NPe6 was diminished when
rats were treated 24 h after injection of 5 mg/kg NPe6 (Fig. lc).
Reductions in the diameter of the RBC column were observed in
vessels, but stasis was a rare event. By the completion of the light
5375
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@
I@
EFFECt'S OF POT USING NPC6
b.@
@
t@
lot
10
0
0.-S
0
0__@ &
.@
Fig. 1. Change in the RBCC diameter within
&8 4C
20—[email protected]
arterioles during and after PDT. Mi
mals were given injections of 5 or 10 mg/kg NPe6
.@
at either 4 or 24 h before light treatment. Light
treatment was 135 J/cm2, delivered during the first
a
a
30 mm of the experiment. Vessel measurements
were continued for an additional 1 h after the corn
pletion of the light treatment. a, animals given 5
mg/kg NPe6, 4 h before light treatment; b, animals
given 10 mg/kg NPe6, 4 h before light treatment; c,
@
2
@,
0
animals given 5 mg/kg NPe6, 24 h before light
treatment; d, animals given 10 mg/kg NPe6, 24 h
before light treatment. Points, means of data from
experiments in 9—1
1 animals, calculated as a per
centage of the initial RBCC diameter. Bars,
i'o8o
10 2030405060
; r@o
102030
II.
B'B 4C
.@
treatment, vessel lumen diameters had reduced to roughly 60% of
controls. The reduction in the vascular lumen was a result of adher
ence of platelet thrombi. Platelet aggregation and adherence to vessels
continued after the completion of the light treatment until complete
stasis was observed. This occurred within the 1-h observation period
after the completion of PDT. A similar reduction in the vascular
response to PDT and timing required before stasis did not occur in
animals given 10 mg/kg NPe6 24 h before light treatment (Fig. id).
The vascular response to PDT in these animals was nearly identical to
the reaction observed in animals given 10 mg/kg only 4 h before the
light treatment.
Macromolecular Leakage from Venules. Leakage of fluorescent
labeled albumin from normal cremasteric venules into the extravas
cular space was measured in animals given PDT using NPe6 and in
30C
25
h
I_/.
80 4i
lot
20
@,0
Time (minutes)
ci.
70
o_.. 8C
.2
C.)
@
60
C)
m
cannot be seen fall within the size of the points.
50
d.!
a
0
0
0@..
mean ±2 SEM. Bars for later time points which
40
Time(minutes)
C, 10 20 30 40 50 60 10 8090
Time(minutes)
controls. Leakage of the albumin macromolecule was not observed in
animals given either NPe6 alone (5.0 mg/kg i.v., 4 h) or light alone
(135 J/cm2). Furthermore, leakage did not occur in animals given 5 or
10 mg/kg NPe6 4 h before light treatment of 135 J/cm2 (Fig. 2, a and
b). Minimal values of leakage were observed in animals treated with
either 5 or 10 mg/kg NPe6 when given 24 h before the light treatment
(Fig. 2, c and d).
Cytotoxicity. Cell kill was estimated immediately after light treat
ment and 24 h after light treatment using a modification of the MTF
assay. Table 1 lists the results of the cytotoxicity assay. Analysis using
a multiple comparisons test revealed 4 distinct groupings of the data.
No decrease in viability was observed in control tumors or in tumor
from animals given drug or light treatment alone. In all cases, tumor
given PDT had significantly less tumor cell viability compared to
3°
251
@20
@
15
154
L@@°
0
C.
10
20
30
40 50 60
Time (minutes)
70
80
CI
90
ib
@o @o40
Fig. 2. Alterations in venule permeability to flu
orescein-labeledalbumin.Animalswere given injec
tions of5 or 10 mg/kg NPe6 at either4or 24 h before
light treatment. Light treatment was 135 J/cm2, de
livered during the first 30 rain of the experiment.
@0 @0 @0 @0 @0 Venule leakage was measuredfor an additional1 h
after the completion of the light treatment. a, animals
given 5 mg/kg NPe6, 4 h before light treatment; b,
d.
30
animals given 10 mg/kg NPe6, 4 h before light
treatment; c, animals given 5 mg/kg NPe6, 24 h
before light treatment; d, animals given 10 mgJkg
251
@
@
NPe6, 24 h before light treatment. Points, means of
experiments in 8-10 animals; bars, mean ±2 SEM.
Bars for time points which cannot be seen fall within
1St
151
the size of the points.
i@10
@@++*H+IH+H
r@
0
10
20
30
40 50
Time@)
60
70
80
@0
0
10
20
30
40
50
60
70
80
90
5376
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@
7@
EFFECTS OF PDT USING NPe6
cell viability of 72.5% immediately after treatment and 18.3% when
measured 24 h later.
In a second experiment, a group of animals was sacrificed by CO2
asphyxiation and the tumors were excised either immediately or 24 h
Table 1 Tumor cell viability after photodynamic therapy
Animals were injected iv. with 5 or 10 mg/kg NPe6 at either 4 or 24 h before light
treatment of 135 J/cm2. Light treatment was 75 mW/cm2 664 nm to a 2-cm diameter spot
centered on the animal's right hind limb bearing chondrosarcoma tumor (6—10mm
diameter) for a total of 30 main.Data represent the mean of 9 animals; the range represents
the 95% confidence interval of the data. Comparison of animals given both NPe6 and light
to controls by analysis of variance found a significant reduction in survival in the treated
animals(P < 0.001; ? = 0.926). Comparisons ofindividual data sets ofanimals given both
after sacrifice.
NPe6 and light to the controls showed reductions in survival significant at the P < 0.01
level.
controls)Immediately
Tumor viability (percentage of
afterTreatment
treatmentNo
regimentreatment24
drug, no light
6.5610
No
drug, 135 i/cm2 light101.5
mg/kg NPe6 24h, no light
5 mg/kg NPe6 4h, 135 J/cm2 light
5 mg/kg NPe6 24h, 135 J/cm2 light
10 mg/kg NPe6 4h, 135 J/cm2 light
10 mg/kg NPe6 24h, 135 J/cm2 light100.5
h after
±9.28
100.6 ±
±8.49
±5.28
76.3 ±5.51
81.8 ±8
53.3 ±5.76
21.0 ±6.65
13.6 ±5.91
72.5 ±5.4224.1
18.3 ±3.74
tumor in the control animals (P < 0.05). Tumor viability measured
immediately after light treatment was not statistically different be
tween groups of animals given 5 or 10 mg/kg NPe6 24 h or those
given 5 mg/kg 4 h. Animals given 10 mg/kg NPe6 4 h has signifi
cantly reduced survival (P < 0.05) compared to the other 3 experi
mental groups assayed immediately after the light treatment. Signuf
icant reductions in tumor viability occurred in all groups over 24 h
after the completion of treatment when compared to viability mean
ured 4 h after treatment (P < 0.05). There was no significant differ
ence between each of the experimental groups when measured 24 h
after light treatment. Animals given 5 mg/kg NPe6, 4 h before light
treatment of 135 J/cm2, left 76% cells viable in the tumor (compared
to controls) when assayed immediately after the light treatment, and
only 24.1% of cells viable when tumors were excised and assayed 24
h after PDT. A treatment of 5 mg/kg NPe6, 24 h before light treat
ment, left 81.8% cells viable when assayed immediately after the
completion of PDT and only 21% of cells viable when assayed 24 h
later. Cell viability immediately after treatment with 10 mg/kg NPe6,
i.v., 4 h was 53.3% and fell to 13.6% when tumors were assayed 24
h later. Injection of 10 mg/kg NPe6 24 h before light treatment gave
No tumor
cytotoxicity
was observed
when the tumors
were assayed immediately after sacrificing the animals; however,
tumor cell viability fell to 16.8 ±6.92% (n = 5) when the tumors
were assayed 24 h after animal death. The tumor cell survival meas
ured 24 h after PDT using NPe6 is not statistically different from that
of these tumors made hypoxic 24 h earlier.
Tumor Response. The efficacy of treatment using NPe6 and light
was assessed in the chondrosarcoma tumor model. Animals treated 4
h after injection of 5 mg/kg NPe6 showed complete tumor destruction
in all cases within 48 h after PDT (Fig. 3a). Tumor regrowth was
evident in selected animals by 13 days after treatment. Tumor cure
was observed in 33% of these animals. Animals treated 24 h after
injection of 5 mg/kg NPe6 showed initial flattening of tumor within
48 h of treatment; however, all of these animals showed tumor
regrowth in the treatment area within 14—21days of PDT (Fig. 3c).
The magnitude of the tumor response to PDT was greater when 10
mg/kg NPe6 was administered at both 4 and 24 h before light
treatment. Animals treated 4 h after injection of 10 mg/kg NPe6
showed complete tumor destruction as seen at the lower NPe6 dose,
however, tumor regrowth was not detected until 21—28days had
passed after treatment (Fig. 3b). Tumor cure was observed in 83% of
animals treated at this dose regimen. Reductions in the tumor response
in animals given injections of 10 mg/kg NPe6 24 h before light
treatment were noted when compared to the 4-h data. Although initial
tumor destruction was observed in all animals, regrowth began at 1
week after PDT and only 12% of animals remained tumor free at the
conclusion of the experiment (Fig. 3d).
DISCUSSION
The goal of this study was to evaluate the effects of PDT using
NPe6 on vascular damage and direct tumor cytotoxicity in vivo.
Evaluation of these events was found to provide important clues
concerning the mechanisms of damage after PDT using NPe6.
a.
[email protected]
.@
@
74
z@
z
.@54
a
@
44
11.34
Fig. 3. Tumor response and cure following PDT.
@
Animals were given injections of 5 or 10 mg/kg
NPe6 at either 4 or 24 h before light treatment.
E
Light treatment was 135 i/cm2 664 am to a 2-cm
diameter spot centered over the tumor. Animals
were considered cured of tumor if no regrowth was
observed before 35 days after treatment. a, animals
given 5 mg/kg NPe6, 4 h before light treatment
I-
(n
@
24); b, animals given 10 mg/kg NPe6, 4 h
before light treatment (n = 12); c, animals given 5
mg/kg NPe6, 24 h before light treatment (n = 16);
2(
,2 1(
0
7
14
21
28
35
42
@Di41@i8@542
Time (days)
C.
Time (days)
10
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d, animalsgiven 10 mg/kg NPe6, 24 h before light
(n
17).
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42
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14
21
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EFFECFS OF PDT USING NPe6
Blood flow stasis occurred rapidly during light treatment in animals
exposed to NPe6 and light. Stasis was observed in the cremaster
arterioles of all animals given injections of 10 mg/kg NPe6 either 4 or
24 h before light treatment or injected with 5 mg/kg NPe6, 4 h before
light treatment. Animals given 5.0 mg/kg i.v. 24 h showed a less
severe arteriole response and timing before blood flow stasis was
delayed. This response confirms the work by Gomer and Ferrario (4)
and Nelson et a!. (5), who concluded that vascular damage was
responsible for tumor response. Blood flow stasis was the first mor
phological change that we observed during light treatment. This
response
is an early step in the cascade
of events
which
Minimal macromolecular leakage from cremaster venules was ob
served during light treatment or the 1-h observation period following
in animals
may lead to
the RBC extravasation and hemorrhagic necrosis found by Nelson et
a!. (3) in their studies using NPe6. The steps which lead to blood flow
stasis were unusual compared to our studies using other photosensi
tizers.
Blood
flow
stasis
did not result
from
vessel
constriction
microvasculature
as
observed in Photofrmn-treated animals (7); rather, blood flow stasis
was a consequence of the aggregation of blood components, namely,
platelets. No vessel constriction was observed. This leads us to believe
that the events incurred using NPe6 differ from those causing vessel
stasis in Photofrin-treated animals. With Photofrin, large quantities of
thromboxane and leukotrienes are released and are believed to cause
vessel constriction. The release of these vasoactive compounds is
thought to result from the early aggregation and adhesion of platelets
to a damaged endothelial lining. Since no constriction is observed
after PDT using NPe6, it suggests that endothelial cells are not
damaged during treatment by the same mechanism observed with
Photofrin (16) or that the cascade of events leading to vessel constric
tion is somehow blocked. In earlier studies, Nelson et a!. (3) found
damage to the subendothelial zone of capillaries, edema, and frag
mentation of collagen and fiber elements (3). Endothelial cells along
capillaries were elongated compared to controls but were otherwise
normal. The platelet aggregation leading to blood flow stasis may
result from damage to the platelet itself rather than the endothelial
cell. Damage to platelet membranes has been shown by Zieve et a!.
(17) and Soloman et a!. (18) for hematoporphyrin derivative and
Henderson et a!. (19) for Photofrmn. We believe that a similar series of
events may occur during PDT using NPe6. A complete
lack of
endothelial cell damage during PDT is unlikely, however, since plate
let aggregation was found near the vessel wall as well as within the
luminal space. It is more likely that damage occurs to both platelets
and endothelial cells, but that the platelet response to therapy is
greater. Although measurements of the release of arachidonic acid
metabolites were not done in this study, it is unlikely that these
metabolites play a significant role in the response. Although throm
boxane will cause vessel constriction and platelet aggregation when
released into the blood, these effects can be blocked or reduced by a
concomitant release of prostacyclin, a product of endothelial cells (20,
21). The release of both thromboxane and prostacyclin has been
observed during PDT using tin etiopurpurin, and the net result is no
vessel constriction and no platelet aggregation (22). Since we observe
platelet aggregation but no vessel constriction during PDT using
NPe6, it suggests that neither prostacyclin nor thromboxane is re
leased. The vascular nature of the observed tissue response to PDT
using NPe6 is confirmed by the data from the MTI' assay. When
tumors were assayed for cell survival 24 h after PDT treatment using
5 or 10 mg/kg NPe6, the resulting cell survival was the same as that
from tumors made hypoxic 24 h earlier and not given PDT treatment.
Since there are significant differences in the number of tumor cures
between animals given 5 and 10 mg/kg NPe6 4 h before light treat
ment, it suggests that the early tumor response may result from
vascular stasis and the consequences of tissue anoxia and that the later
response includes contributions from other factors including direct
cytotoxicity and possibly immune effects.
given NPe6. This is in contrast
to observations
of macro
molecular leakage from venules after PDT using Photofrmn (8). Al
though leakage from venules is not a major observation in NPe6treated animals, it does not imply that damage has not occurred. It is
possible that even if venule damage occurred, vessel stasis and platelet
aggregation precluded the escape of macromolecules such as albumin
into the extravascular space.
Though tumor vessels are believed to be inherently more fragile
and react differently to stimuli than vessels in muscle, previous studies
confirm that similar microvascular damage occurs in tumor vessels
(for tumors implanted in the cremaster) compared to normal cremaster
(23). One exception
to this appears
to involve
leu
kocyte adhesion which is found in normal microvasculature but not
within tumor microvasculature (24). Any differences between the
response
expected
in normal
or neoplastic
microvasculature
are likely
to be minimized during PDT using NPe6 since the therapy appears to
target elements in the blood rather than on the blood vessels. Vessel
stasis results from platelet aggregation and thrombus formation; a
response mediated by platelets and other blood elements.
Although vessel damage is a dominant factor in the mechanisms of
tumor destruction following PDT using NPe6, other components
including direct cytotoxicity were found to play a role in the observed
response to therapy. Measurable estimates of direct tumor cytotoxicity
were found after PDT using NPe6. Although the MU assay used in
this study does not measure tumor cell viability directly, Carmichael
et a!. (13) showed a good correlation between clonogenic and MU
assays when measured 24 h after treatment. Measurements of viability
using MU within the first few hours are less predictive since we
cannot demonstrate in our model that a given amount of enzyme
inhibition corresponds directly with a corresponding level of cytotox
icity. It is possible that PDT causes a slight reduction in mitochondrial
enzyme activity in many cells, and that certain of these cells survive
the initial depression of enzyme activity. We expect that inhibition of
succinate
dehydrogenase
should significantly
alter cellular
respiration
in affected cells and that this event is likely to precede cell death.
Inhibition
of succinate
dehydrogenase
has been shown
by Hilf et a!.
(25) during PDT using Photofrmn,and this is believed to represent an
early step in cytotoxicity.
Estimates
of direct
cell kill were greatest
when
the animals
were
given NPe6 4 h before light treatment (47% kill at 4 h compared to
27% kill at 24 h for 10 mg/kg NPe6). The increased
early cytotoxicity
as a result of PDT may be one of the reasons why tumor cure is higher
among animals treated at 4 h compared to 24 h after drug injection.
This conclusion is supported by data from the microvascular studies.
Vessel stasis was complete during the 1-h observation period for PDT
using a NPe6 dose of 10 mg/kg when delivered either 4 or 24 h before
treatment. Despite this, greater tumor cure was observed in the group
given drug 4 h before the light treatment. The increased efficacy of
treatment may be related to the greater direct tumor cytotoxicity
observed at the 4-h time point.
PDT using NPe6 may have specific advantages compared to other
photosensitizers
since
tumor
regression
can result
both from
direct
cytotoxicity and from the consequences of blood flow stasis. It is not
known whether the direct cytotoxicity observed at the 10-mg/kg dose
of NPe6 could be increased by delaying the induction of blood flow
stasis and resulting depletion of oxygen in the tissue. If direct cyto
toxicity is limited at this NPe6 dose by the availability of oxygen in
the tissue, it may be possible to increase the magnitude of direct
cytotoxicity by delaying the vascular response (26). Certain eico
sanoid inhibitors and inhibitors of platelet aggregation are currently
5378
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EFFECTS OF PDT USING NPe6
6. Ferrario, A., Kessel, D., and Gomer, C. J. Metabolic properties and photosensitizing
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The vascular and direct cytotoxic response to PDT using NPe6
followed the same time dependence as tumor response previously
described by Gomer and Ferrario (4) and Ferrario et a!. (6). Both
vascular damage and tumor response were greater when treated 4 h
after administration of 5 mg/kg NPe6 compared to the response
observed 24 h after injection. Differences in tumor response alone
were found in animals given doses of 10 mg/kg NPe6 at 4 and 24 h
before light treatment. The lack of difference in the vascular response
between these time points after injection of 10 mg/kg NPe6 is likely
due to the maintenance of sufficient levels of NPe6 in plasma to evoke
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ACKNOWLEDGMENTS
The authorswish to thankDr. PatriciaCerritofor herexpertassistancewith
the statistical analysis of the data. We also acknowledge the efforts of Nippon
Petrochemicals
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and Beckloff Associates for providing the NPe6 used in this
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Effects of Photodynamic Therapy Using Mono-l-aspartyl Chlorin
e6 on Vessel Constriction, Vessel Leakage, and Tumor
Response
Kimberly S. McMahon, T. Jeffery Wieman, Pamela H. Moore, et al.
Cancer Res 1994;54:5374-5379.
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