U.S. patent application number 09/873599 was filed with the patent office on 2001-10-25 for photothermal vascular targeting with bioreductive agents.
Invention is credited to Durville, Frederic M., McMillan, Kathleen.
Application Number | 20010034319 09/873599 |
Document ID | / |
Family ID | 23048771 |
Filed Date | 2001-10-25 |
United States Patent
Application |
20010034319 |
Kind Code |
A1 |
McMillan, Kathleen ; et
al. |
October 25, 2001 |
Photothermal vascular targeting with bioreductive agents
Abstract
A method of selectively enhancing the effect of photothermal
vascular targeting on tumor regression by administration of a
bioreductive agent is disclosed.
Inventors: |
McMillan, Kathleen;
(Concord, MA) ; Durville, Frederic M.;
(Chelmsford, MA) |
Correspondence
Address: |
John P. Iwanicki
BANNER & WITCOFF, LTD.
28th Floor
28 State Street
Boston
MA
02109
US
|
Family ID: |
23048771 |
Appl. No.: |
09/873599 |
Filed: |
June 4, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09873599 |
Jun 4, 2001 |
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09274576 |
Mar 23, 1999 |
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6240925 |
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Current U.S.
Class: |
514/1 ;
514/410 |
Current CPC
Class: |
A61N 2005/0645 20130101;
A61B 2018/00458 20130101; A61B 2018/00452 20130101; A61B 18/203
20130101; A61N 5/062 20130101 |
Class at
Publication: |
514/1 ;
514/410 |
International
Class: |
A61K 031/00; A61K
031/409 |
Claims
What is claimed is:
1. A method of treating a tumor characterized by nutrient-providing
blood vessels comprising irradiating the tumor or nutrient
providing blood vessels in the presence of a bioreductive agent,
providing an environment of hypoxia for the bioreductive agent, and
allowing the bioreductive agent to act as a cytotoxic agent in a
manner to reduce tumor size.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] Embodiments of the present invention relate in general to
the combined use of laser therapy and cytotoxic agents in the
treatment of vascular lesions including tumors. More particularly,
embodiments of the present invention relate to methods of reducing
or eliminating growth of tumors using laser light and bioreductive
agents without significant harm to surrounding normal tissue.
[0003] 2. Description of Related Art
[0004] Vascular targeting is an anticancer strategy in which the
endothelial wall of the blood vessels supplying the tumor is the
treatment target, and tumor cell hypoxia is a therapeutic goal.
Because of the interdependence of tumor cells and tumor
vasculature, prolonged ischemia secondary to irreversible loss of
tumor blood vessel function will result in tumor cell necrosis.
Vascular targeting has as its goal the destruction of existing
tumor vasculature and so differs from anti-angiogenic therapies
aimed at preventing the growth of new vessels within a tumor.
[0005] The function of the tumor vascular supply has significance
both in the natural progression of a malignancy and in the outcome
of treatment. The chaotic vascular architecture and abnormalities
of blood flow within tumor microvessels are associated with regions
of tumor hypoxia that limit the efficacy of both chemotherapy and
radiotherapy. Similarly, the effectiveness of photodynamic therapy
depends on an adequate oxygen level within the tumor for generation
of cytotoxic species.
[0006] The application of phototherapy to tumor treatment has been
actively pursued because of both the incidence and health
significance of tumors that arise in the skin or mucosal surfaces,
including the gastrointestinal tract, aerodigestive tract, urinary
bladder, and cervix, and therefore are readily accessible to
radiation. The phototherapeutic approach typically involves
administering a photosensitizer, allowing a suitable period of time
for uptake of this photosensitizer by the tumor tissue, and
irradiation at a wavelength absorbed by the photosensitizer to
produce cytotoxic species. Although preferential accumulation of
photosensitizer in tumor versus normal tissue has been demonstrated
for certain photosensitizers, prevention of damage to normal
vasculature typically requires spatial confinement of the
irradiated zone to the target tumor tissue.
[0007] When the photosensitizer is administered intravenously, it
has been observed that vascular damage follows irradiation. This
vascular damage has been exploited in ophthalmology for treatment
of pathologic ocular neovascularization and in dermatology for
treatment of benign vascular lesions. Photodynamic therapy
specifically designed to target tumor vasculature has been
described. However, the mechanism of photodynamic therapy leads to
vascular damage in any tissue that is exposed to radiation, in the
presence of sufficient oxygen. Tumor blood vessels and the vessels
supplying normal tissue surrounding the tumor are both susceptible
to damage by the cytotoxic species produced by the intravenous
photosensitizer during illumination.
[0008] Selective photothermolysis is a method of causing selective
and irreversible photothermal damage to tissue structures
containing a chromophore that can be used to distinguish that
target structure from surrounding tissue. For a light source,
typically a laser, to be useful for selective photothermolysis, it
must emit with sufficient intensity at a wavelength preferentially
absorbed by the target chromophore. The pulse duration or exposure
time of the source must be less than the thermal relaxation time of
the target, to minimize temperature increases in tissue surrounding
the target. Techniques based on this concept using well known laser
systems are well established for treatment of benign cutaneous
vascular lesions such as portwine stain (PWS), birthmarks,
telangiectasias, hemangiomas, warts, psoriasis, arthritis in which
hemoglobin in the abnormal, ectatic lesional vasculature serves as
the chromophore and the target is the vessel wall, as well as,
atherosclerotic plaque and other desired applications. See U.S.
Pat. No. 5,312,395; U.S. Pat. No. 5,749,868; U.S. Pat. No.
5,257,970; U.S. Pat. No. 5,066,293, U.S. Pat. No. 5,346,488,
"Selective Photothermolysis: Precise Microsurgery by Selective
Absorption of Pulsed Radiation", Anderson et al., Science,
220:524-527 (1983); Spears et al. J. Clin. Invest, 71, 39-399
(1983) each of which are hereby incorporated by reference in their
entireties for all purposes. The deepest blood vessels contributing
to the color of PWS lesions are approximately 1 mm below the skin
surface, and are accessible to selective photothermal targeting
using available lasers such as the 585 nm pulsed dye laser. The
theoretical advantages of selective photothermolysis have been
borne out in clinical studies showing that PDL treatment of benign
cutaneous vascular lesions is associated with very low risk of
scarring. However, photothermolysis techniques would be more
effective if the results of damage to surrounding tumor vasculature
and other blood vessels primarily responsible for maintaining
growth of the tumor could be advantageously used to promote the
efficacy of cytotoxic agents which are activated by hypoxic
conditions produced as a result of tumor vascular damage.
[0009] Accordingly, there is a need in the art to provide methods
of treating tumors combining photothermolysis and cytotoxic species
that are activated under hypoxic conditions. There is also a
further need in the art to selectively localize the effects of
photothermolysis to target tumors and their associated
microvasculature without significantly harming surrounding normal
tissue.
BRIEF SUMMARY OF THE INVENTION
[0010] Embodiments of the present invention are directed to methods
in mammals including a human which are useful in selectively
treating tumors or other vascular lesions surrounded by normal or
otherwise healthy tissue without significantly harming or otherwise
adversely affecting the surrounding normal tissue. A tumor is
treated according to the invention in a manner to reduce or regress
existing tumor size, to inhibit the growth of an existing tumor or
to prevent establishment of a vascularized tumor mass. According to
the methods of the present invention, a tumor or other area
selected for treatment is irradiated with laser light having a
wavelength, duration, fluence and spot size selected to
preferentially heat blood vessels that supply the tumor with
sufficient blood and oxygen to support its growth in the presence
of a bioreductive agent or hypoxic cytotoxin. According to one
embodiment, the blood vessels are heated to the point of
denaturation and to effectively prevent blood and oxygen flow
through the blood vessels to the tumor, thereby depriving the tumor
of nourishment and accordingly leading to tumor necrosis, reduction
or regression of tumor size, inhibit of the growth of an existing
tumor or prevention of establishment of a vascularized tumor mass.
In general, according to the present invention, the blood vessels
are heated as a result of irradiation in a manner to create a
region of hypoxia, i.e. reduced oxygen content.
[0011] According to one embodiment of the present invention, a
chromophore, such as a dye, or other photoactive agent is
administered to the site of irradiation. The chromophore acts to
selectively absorb the chosen wavelength of laser light thereby
enhancing the effectiveness of the irradiation. Other chromophores
or photoactive compounds can be used which themselves act as
therapeutic or cytotoxic agents upon irradiation.
[0012] Methods of the present invention also include the
administration of a cytotoxic agent or hypoxic cytotoxin which is
preferentially activated under conditions of hypoxia to effectively
prevent or eliminate tumor growth or reduce tumor size or otherwise
lead to tumor necrosis, or otherwise prevent establishment of a
vascularized tumor mass. Such cytotoxic agents or hypoxic
cytotoxins are commonly known as bioreductive agents. According to
the present invention, a method of treating a tumor characterized
by nutrient-providing blood vessels is provided including
irradiating the tumor or nutrient providing blood vessels in the
presence of a bioreductive agent, providing an environment of
hypoxia for the bioreductive agent, and allowing the bioreductive
agent to act as a cytotoxic agent in a manner to effectively reduce
tumor size, eliminate tumor growth or otherwise lead to tumor
necrosis, or otherwise prevent establishment of a vascularized
tumor mass.
[0013] Other features and advantages of certain embodiments of the
present invention will become more fully apparent from the
following description taken in conjunction with the accompanying
figures and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] In the course of the detailed description of certain
preferred embodiments to follow, reference will be made to the
attached figures, in which,
[0015] FIG. 1 is a graph of mean tumor volumes of experimental
mouse groups as a function of time after administration of
different dosages of the bioreductive agent porfiromycin (POR).
Error bars indicate +1 standard deviation.
[0016] FIG. 2 is a graph of mean tumor volumes of four experimental
mouse groups as a function of time after first treatment of the
tumor only and excluding the tumor margin, one group including POR
only, one group including vascular targeting only, one group
including POR with vascular targeting and one control group. Error
bars indicate +1 standard deviation.
[0017] FIG. 3 is a graph of mean tumor volumes of four experimental
mouse groups as a function of time after first treatment of the
tumor and tumor margin, one group including POR only, one group
including vascular targeting only, one group including POR with
vascular targeting and one control group. Error bars indicate +1
standard deviation.
[0018] FIG. 4 is a graph of mean tumor volumes of four experimental
mouse groups as a function of time after first treatment of the
tumor and tumor margin and including only the largest third of
tumors measured on day 0, one group including POR only, one group
including vascular targeting only, one group including POR with
vascular targeting and one control group. Error bars indicate +1
standard deviation.
[0019] FIG. 5 is a graph of mean tumor volume of four experimental
hamster groups as a function of time after first treatment of the
tumor and tumor margin, one group including POR only, one group
including vascular targeting only, one group including POR with
vascular targeting and one control group.
DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS
[0020] The principles of the present invention may be applied with
particular advantage to treat tumors or other lesions which have
associated blood vessels which provide nourishment to the tumor or
other lesion. According to the teachings of the present invention,
a tumor is irradiated with a laser light source having a
wavelength, duration, fluence and spot size sufficient to render
blood vessels associated with the tumor incapable of providing
continued and sufficient blood and/or oxygen for sustained or
continued growth.
[0021] In one embodiment, the laser light is produced by a tunable
pulsed dye laser system and is characterized in having a wavelength
corresponding to that which is absorbed by hemoglobin found in the
blood vessels. The wavelength produced by the pulsed dye laser is
well absorbed by oxyhemoglobin in blood, and its pulse duration
(300 to 500 .mu.s) is short enough that heat is largely confined to
ectatic microvessels during the laser pulse. Consequently,
irreversible vascular damage may be achieved with little or no
damage to surrounding tissue structures.
[0022] In general, suitable pulsed dye laser systems useful in the
present invention include a power source, a flashlamp capable of
emitting multiple pulses of light, a dye reservoir containing a dye
suitable for stimulated emission of light, and an optical resonator
having an output coupler. The power source, flashlamp, dye
reservoir and optical resonator are operatively connected so as to
generate multiple pulses of laser light having a defined wavelength
and pulse duration. An optical fiber is operatively coupled to the
optical resonator in a manner to allow the multiple pulses of laser
light to travel from the optical resonator through the optical
fiber to the tumor or tissue margin area to be irradiated with a
defined pulse fluence. A handpiece delivery system incorporating
the terminal end of the optical fiber is used to effectively direct
the laser light source to the target area in the mammal.
[0023] According to one embodiment, useful wavelengths are between
about 560 nm to about 610 nm, preferably between about 570 nm to
about 600 nm and more preferably about 585 nm or about 590 nm. The
laser light has a pulse duration less than the thermal relaxation
time of the volume of tissue being irradiated. Specific pulse
durations include between 0.1 msec to about 60 msec, preferably
between 0.1 to about 10 msec and more preferably about 0.3 msec.
The delivered fluence of the pulsed laser light is between about 1
J/cm.sup.3 to about 30 J/cm.sup.3, preferably between about 5
J/cm.sup.3 to about 15 J/cm.sup.3 and more preferably about 10
J/cm.sup.3. The irradiated spot size is sufficient to include the
tumor as a whole or portions thereof According to an additional
embodiment, the spot size is sufficient to include not only the
tumor but an area of normal tissue adjacent to or surrounding the
tumor within which is included blood vessels which nourish the
tumor with blood and oxygen sufficient for its continued growth.
The area of normal tissue adjacent to or surrounding the tumor
within which is included blood vessels which nourish the tumor is
referred to herein as the "margin" or "margin of tissue".
Alternatively, the laser light has a spot size sufficient to
irradiate only the margin or portions thereof, or part of the
margin and part of the tumor. Spot sizes in accordance with the
present invention include those between about 1 mm to about 20 mm,
preferably about 5 mm to about 15 mm and more preferably about 10
mm.
[0024] It is to be understood that other lasers, such as yellow,
green and blue wavelength lasers with or without suitable exogenous
chromophores, are useful within the scope of the present invention
and include Argon ion lasers, Copper-vapor lasers, alexandrite
lasers, ruby lasers, semiconductor diode lasers, frequency-doubled
Nd:YAG lasers, and other dye lasers pumped by a Nitrogen laser or
Argon-ion laser and the like. The lasers within the scope of the
present invention are pulsed but may also operate in a
continuous-wave (cw) mode with a scanner to automatically scan the
treatment area and provide temporal modulation of the laser
intensity on the treatment site.
[0025] In addition, one or more exogenous agents and/or dyes are
also administered when necessary to enhance the absorption of the
laser light at the site of irradiation and to also aid in the
selective absorption of laser light. Administration of exogenous
chromophores are used to take advantage of the deeper tissue
penetration of longer visible or near-infrared wavelengths. Such
exogenous chromophores include the lipophilic dye indocyanine green
(ICG) and the like. One or more photoactivated compounds can also
be administered as necessary to therapeutically treat the tumor.
The amount, duration and mode of administration of the chromophore
or other photoactive agent will depend on its properties and the
makeup of the individual on which the treatment is to be carried
out. Typically, between about a 50 mg and about a 2000 mg,
preferably 500 mg dose of ICG for a patient of 100 kg of body
weight is injected intravenously to the patient immediately prior
to laser irradiation.
[0026] According to the present invention, the area of the
individual to be treated should be irradiated at least once with
laser light having the above parameters, with the appropriate
number of pulses necessary to treat the entire area. When deeper
penetration of the laser light is desired, the area may be
irradiated several times, and the complete treatment may be
repeated up to five times with at least one week between each
treatment.
[0027] The potential advantage of selective photothermal vascular
targeting over conventional anticancer strategies is the
preservation of normal tissue surrounding the tumor. The basis of
the ability of photothermal techniques to discriminate between
lesion and normal skin may be due to differences in size between
pathologic and normal cutaneous vasculature; it is known that for
effective and selective photothermal injury to the vessel wall the
pulse duration of the laser should be equal to or slightly less
than the thermal relaxation time of the targeted vessel. According
to the present invention, a small margin of skin at the tumor
periphery is irradiated. While not wishing to be bound to any
scientific theory, this peripheral region or "margin" is believed
to be the location of relatively large caliber vessels that supply
and drain the tumor microvasculature. Successful tumor treatment
may be the result of selective damage to the arterioles and venules
at the periphery, rather than direct damage to the much smaller
capillaries, i.e. microvasculature, within the tumor itself. The
existence of large caliber feeding vessels as an integral part of
the tumor microvascular architecture has been documented in
corrosion cast studies of human colorectal carcinomas, cutaneous
basal cell tumors, and xenotransplanted human tumors grown in mice.
Other factors may be at work, for example the relative fragility of
tumor microvasculature and the closer dependence of proliferating
tumor cells on their vascular supply.
[0028] According to the invention, the blood and oxygen supplying
vessels of a tumor are irradiated to the extent to cause
irreversible ischemic damage to tumor cells but also in a manner to
spare normal surrounding tissue. This method is implemented,
depending on the size and depth of the tumor and its vascular
architecture, by means of the pulsed dye laser or any other source
of radiation preferentially absorbed by the endogenous chromophores
of blood, or by use of other, more deeply penetrating radiation
sources in combination with an exogenous chromophore such as
ICG.
[0029] According to an addition embodiment of the present
invention, one or more bioreductive agents are administered to
enhance the effect of photothermal vascular targeting on tumor
regression. Bioreductive agents according to the present invention
include porfiromycin (POR), mitomycin C, tirapazamine,
indoloquinone and the like. POR is a prodrug that is activated by
bioreductive enzymes in the absence of oxygen to form cytotoxic
species. Mitomycin C, which is structurally related to POR, is also
a useful bioreductive agent within the scope of the present
invention. The preferential toxicity for hypoxic cells is greater
for POR than mitomycin C.
[0030] The following examples are set forth as representative of
the present invention. These examples are not to be construed as
limiting the scope of the invention as these and other equivalent
embodiments will become apparent in view of the present disclosure,
figures and accompanying claims.
EXAMPLE I
Cell Culture
[0031] KB cells (human pharyngeal SCC, American Type Culture
Collection, Rockville, Md.) were maintained in culture medium
consisting of 90% Minimal Essential medium (Gibco BRL, Grand
Island, N.Y.) and 10% fetal bovine serum (Hyclone, Logan, Utah)
with the addition of 10 nM nonessential amino acid (Gibco BRL) and
antibiotics (penicillin, streptomycin: Gibco BRL). The cells were
grown as monolayers in Falcon T150 flasks (Becton Dickinson,
Franklin Lakes, N.J.) in a 37.degree. C. humidified incubator with
5% CO.sub.2.
[0032] Prior to inoculation, the cells were rinsed with PBS lacking
calcium and magnesium (Gibco BRL), trypsinized (Gibco BRL),
counted, centrifuged at 1000 rpm for 6 min., and resuspended in
normal saline for a final concentration of 1,000,000 cells/ml.
EXAMPLE II
Tumor Model
[0033] Six- to seven-week-old nude mice (Charles River Labs,
Wilmington, Mass.) were sterilely housed and fed ad libitum.
Intradermal inoculation of 0.05 ml of cell suspension (50,000
cells) was performed under general anesthesia (ether) using a 30
gauge hypodermic needle. After 5 days, the majority of the tumors
were visually apparent and palpable.
EXAMPLE III
Statistical Analysis
[0034] The effect of treatment on tumor volumes was determined
using analysis of variance. Multiple comparisons were performed
using the Student-Newman-Keuls (SNK) test. P values less than 0.05
are considered significant.
EXAMPLE IV
Effect of POR Alone
[0035] Fifteen mice, each with 4 tumor inoculations, were divided
into 4 treatment groups and 1 control group. Five days after tumor
inoculation, the 4 treatment groups received 2.50, 5.0, 7.5, and
10.0 mg/kg intraperitoneal (ip) injections of POR (Vion
Pharmaceuticals, Inc., New Haven, Conn.) dissolved in sterile
water. (The LD.sub.50 of POR in mice is 50 mg/kg.) The control
group received ip saline injections. The 3 orthogonal diameters of
the tumors were measured at days 0, 2, 4, 6, 8, 10, and 13
following POR injection. All measurements included the thickness of
the overlying skin. All mice were anesthetized during ip injections
and tumor measurements. The tumor volume was estimated as the
product of the three orthogonal diameters multiplied by .pi./6. The
mice were weighed at the time of each tumor measurement to monitor
for any systemic toxicity of POR.
[0036] FIG. 1 shows the mean tumor volumes of each experimental
group as a function of time after the administration of POR.
Analysis of variance indicates no significant effect of treatment
at any point in time. There is also no significant effect of
treatment on body weight, or any other indication of acute toxicity
related to POR administration. Tumors ranged in volume at time of
treatment on day 0 (5 days after tumor cell inoculation) from 0.5
to 20.9 mm.sup.3, with a mean volume of 8.2 mm.sup.3 and standard
deviation (SD) of 5.3 mm.sup.3.
EXAMPLE V
POR with PDL Limited to Tumor Area
[0037] Sixteen mice, each with 4 tumor inoculations, were divided
into 3 treatment groups and 1 control group. On day 6 following
tumor inoculation, the treatment groups received (1) ip POR only
(10 mg/kg), (2) ip saline and PDL limited to tumor area (585 nm, 10
J/cm.sup.2, 300-500 .mu.s; Model SPTL-1a, Candela Corporation,
Wayland, Mass.), or (3) ip POR (10 mg/kg) and PDL limited to the
tumor area (585 nm, 10 J/cm.sup.2, 300-500 .mu.s). The control
group received ip saline injections only. PDL irradiation was
performed 15 min. after administration of POR or vehicle, using a
laser handpiece that produced a 5 mm diameter irradiated spot with
a uniform energy distribution on the tumor surface. An aluminum
plate with holes of various diameters was used to shield areas
beyond the tumor border. For those tumors larger than 5 mm,
multiple partially overlapping exposures were necessary to
irradiate the entire tumor area. Treatments were repeated in all
groups two days later. The orthogonal diameters of the tumors were
measured and the mice were weighed on days 0, 2, 4, 6, 8, 10, and
13 following the first treatment. One mouse from the control group
died during anesthesia before day 10 and was therefore excluded
from analysis.
[0038] Following the final measurement, tumors in control and
treatment groups were fixed with formalin, embedded in paraffin,
sectioned, and stained with hematoxylin and eosin (H&E) for
histologic evaluation.
[0039] In Example IV, the difference in growth of tumors in mice
receiving POR alone at any dosage within the range tested and the
growth of tumors in control mice was not statistically significant,
therefore in this Example V any difference between groups receiving
PDL treatment with and without POR in this dosage range was
expected to indicate supra-additivity. The highest POR dosage
tested in Example IV (10 mg/kg) was used to increase the likelihood
of detecting a difference between the two laser treatment groups.
FIG. 2 shows the mean tumor volumes for each experimental group
after the first treatment session At the start of treatment (day 0,
6 days after tumor cell inoculation) the mean volume of all tumors
was 14.8 mm.sup.3 (SD 12.5 mm.sup.3). Analysis of variance
indicates no significant effect of treatment at any point in time.
There was no gross evidence of skin sloughing or any histological
evidence of epithelial necrosis in the laser treatment groups.
[0040] Dermal skin flaps containing the tumor masses were examined
in 2 animals from control and PDL groups following the final tumor
measurement on day 13. While a small portion of the blood supply
appeared to originate in the subcutaneous tissue, a notable aspect
of the tumors was the presence of radially organized large caliber
microvessels in the dermis. It is believed that these supplying
vessels had been shielded by the template during PDL
irradiation.
EXAMPLE VI
POR with PDL Extending Beyond Tumor Area
[0041] Twelve mice with 6 tumor inoculations were divided into 3
treatment groups and 1 control group. On day 5 following
inoculation, the 4 groups were treated as in Example V except that
the field of irradiation was extended to include a 2 to 3 mm
periphery beyond the tumor edge. In addition, on the second
treatment day, the fluence was raised from 10 to 15 J/cm.sup.2.
Tumor volume measurements were performed on days 0, 2, 4, 6, 8, 10,
and 12 following the first treatment. Mice were weighed on day 0
and 6 following the first treatment. There were two anesthesia
related deaths: one mouse from the control group and one mouse from
the group receiving both PDL and POR died before day 10 and were
therefore excluded from analysis.
[0042] Following the final tumor measurement, control tumors,
residual treated tumors, and areas previously exhibiting tumors
were processed as before for histologic evaluation.
[0043] When PDL treatment was extended to a small margin of tissue
beyond the tumor edge encompassing the supply vessels, tumor growth
was significantly affected as shown in FIG. 3. At the time of
treatment, the mean volume of all tumors was 15.2 mm.sup.3 (SD 8.3
m.sup.3). An effect of treatment was seen at all days beginning
with day 2 (day 2, p<0.05; day 4, p<0.01, days 6 through 13,
p<0.0001). The SNK test indicated that all 3 treatments,
including POR alone, differed from the controls from day 6 onward.
There was no significance between the effects of PDL irradiation
with and without POR at any time, however.
[0044] FIG. 4 shows the results of the experiment of Example VI
when only the largest 33% of tumors on day 0 are included in the
analysis. This subset of tumors has a mean volume of 23.9 mM.sup.3
and SD of 8.2 mm.sup.3 on day 0. In contrast to the all-inclusive
analysis, the SNK tests here shows no difference between the
control and PDL groups at any point in time, and does demonstrate a
significant difference between PDL with and without POR at all days
after day 6.
[0045] Both the area of irradiation and fluence were increased in
Example VI relative to Example V experiments. In the majority of
animals this treatment resulted in a darkening of skin tissue
overlying the tumor, beginning on day 3, that was suggestive of
epithelial injury. There was no tissue sloughing. By day 10, these
darkened areas exhibited near normal pigmentation with minimal
scarring.
EXAMPLE VII
POR with PDL Treatment of Normal Skin Tissue
[0046] The effect on normal skin tissue of PDL irradiation with or
without POR was determined using 6 adult Syrian golden hamsters.
The hamsters were divided into two groups, and hair of each animal
was shaved from an area of the flank. One group was irradiated with
the PDL 15 min. after ip injection of POR, and the other group was
irradiated without POR administration. Biopsies of treated sites
were taken 10 min. or 2 days after irradiation. Specimens were
fixed with formalin, embedded in paraffin, sectioned, and stained
(H&E) for histologic evaluation.
[0047] The acute and delayed results of PDL irradiation of hamster
skin after administration of POR were observed as a result of
histologic studies. Damage appeared limited to the larger
microvessels visible at the magnification. Clinically, there was
production of slight purpura beginning a few minutes after PDL
irradiation that had largely resolved by day 2. Regardless of
whether POR had been administered, there was no microscopic or
clinical evidence of epithelial necrosis or other damage to tissue
structures beyond the blood vessels resulting from single PDL
irradiation sufficient to damage microvessels in this animal
model.
[0048] It is significant that normal skin was unaffected by
administration of POR before PDL irradiation, at laser fluences
sufficient to cause microvascular damage. Hypoxia-induced
cytotoxicity would be an adverse effect if occurring in normal
tissue. Most blood vessels in the papillary dermis of normal human
skin have outer diameters of 17 to 22 .mu.m and only a small
fraction of blood vessels 20 .mu.m or smaller can be damaged by the
pulsed dye laser. Consequently, the degree of hypoxia induced in
normal skin by irradiation with the pulsed dye laser is
advantageously slight providing for a high degree of selectivity of
photothermal vascular targeting for tumor tissue using bioreductive
agent administration.
EXAMPLE VIII
PDL and POR on Human Squamous Cancer Cells
[0049] In this example, tumors were produced in the cheek pouches
of hamsters by transplantation of human squamous cancer cells
according to methods well known in the art. Hamsters were divided
into four treatment groups: (1) control, (2) bioreductive agent
porfiromycin only (POR), (3) 588 nm pulsed dye laser only (PDL),
and (4) combined PDL and POR. The results are presented in FIG. 5
which demonstrates that tumor regression and remission following
laser vascular targeting is enhanced by administration of a
bioreductive cytotoxin.
* * * * *