using a novel benzophenothiazine photosensitizer ...

Photodynamic therapy of naturally occurring tumors in animals using a novel benzophenothiazine photosensitizer. A E Frimberger, A S Moore, L Cincotta, et al. Clin Cancer Res 1998;4:2207-2218. Published online September 1, 1998.

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Downloaded from clincancerres.aacrjournals.org on July 14, 2011 Copyright © 1998 American Association for Cancer Research

Vol. 4, 2207-2218,

Septenther

Photodynamic Using a Novel

Angela

Louis James

E. Friniberger,2

Cincotta,

Susan

1998

Clinical

Therapy of Naturally Benzophenothiazine

Antony

M. Cotter,

S. Moore,

Occurring Photosensitizer’ has

activity

objective

Research

2207

in Animals

naturally

response

occurring

rate

tumors,

(partial

response

+

CR) of 61.5%.

W. Foley

The Harrington Oncology Program, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536 [A. E. F., A. S. M., S. M. C.]; The University of Massachusetts Cancer Center, Worcester, Massachusetts 01605 [A. E. F.]; and The Rowland Institute for Science, Cambridge, Massachusetts 02142 EL. C., J. W. F.]

INTRODUCTION PDT3 is a local antineoplastic interaction photodynamic greatly

expanded

ments,

chiefly

and

fiberoptics

z-rthylamino--diethyIaminobenzo[a]phenothiazinium (EtNBS) is a novel photodynamic therapy (PDT) photosensitizer with efficacy against experimental murine tumors. In this preliminary study, dogs and cats with naturally occurring tumors were treated with EtNBS-PDT to determine safety and efficacy. Fifteen treatments were performed on 13 animals (9 treatments in 8 cats and 6 treatments in 5 dogs), generally using 400 J of 652 urn light. Two feline sublingual squamous cell carcinomas (SCCs) responded briefly (minor response). Six feline facial SCCs were treated, resulting in two partial responses and four long-term complete responses (CR). Two canine intraoral SCCs were treated; one responded minimally for 2 weeks (minor response), and one achieved long-term CR. One canine cutaneous mast cell tumor achieved CR, and one canine ocular mast cell tumor responded briefly. One canine ocular melanoma did not respond to treatment. Systemic

apy,

PDT

reactions

because

chloride

nausea

associated

with

photosensitizer

in two cats and two dogs, elevated body temperatures during treatment in two dogs, elevated body temperature 2 days after PDT in one cat, and inappetance for 2 weeks in one cat. A peripheral neuropathy of undetermined cause occurred in one cat 2 weeks after PDT and resolved without treatment. Local reaction was well tolerated in 13 of 15 treatments. All animals were exposed to normal daylight after less than 5 days (mean, 3.5 days) without residual photosensitization. EtNBS-PDT is safe for dogs and cats and injection

in recent in the area (1-4).

that is based on the

photosensitizers

in tumor

cells.

years

because

of technical

develop-

delivery

systems

as lasers

safer

with

for the

ideally

It also

radiation

surrounding

fewer

ther-

normal

are preferentially

requires

should be wavelength.

such

ionizing

tissues

accumulated

treatments,

but because of the sparing of the normal repeated if needed. The photosensitizer icalby and appropriate

light. The concept of its utility has been

of light Compared

is generally

because

often

tissues, is usually

only

one,

it can be safely given system-

inert unless activated by light of Upon activation, the photosensitizer

the

can undergo type I reaction in which the excited photosensitizer reacts directly with substrates to produce free radicals. Alternatively,

the

oxygen

to generate

singlet radiation,

by,

genation is critical for limits efficacy because light

are

the

peutic excited

red

The

to infrared

window”);

absorption

oped,

band

in the

oxybulk and

mm,

even

at the

best

penetrate

tissue

best

600-900

nm

further PDT) is repaired

of second

limited

benzoporphyrins,

“thera-

would Photofrin

window,

be and

by a relatively

therapeutic

ineffiand

more

photosensitization (1-3). of these photosensitizers

to the tumor vasculature, which (and thus resistance to ionizing

and risk through

(2-3). generation

(the

photosensitizer

cutaneous of action

more on damage both hypoxia

photocytotoxicity including

are

by prolonged the mechanism

to rely increase

radiation and the vasculature number

tissue

that

an ideal

limiting

effect (8). Tumor to tissue hypoxia, 5-10

wavelengths,

derivative,

ionizing

is not only required

by a wavelength in this range. The best characterized photosensitizers,

importantly, Furthermore,

direct

primarily

reaction,

of light

therefore,

hematoporphyrin cient

oxygen

is only

molecular

(5). As with

photodynamic

wavelengths

with species,

(7) are important

a photodynamic it contributes

penetration

wavelengths.

II reaction

molecular the

react

oxygen

(6) and hypoxia

Because

consumed

can

reactive

in the type

bulk

for PDT. but

photosensitizer cytotoxic

(‘02),

tumor

factors for,

excited

oxygen

appears would Received 5/5/98; revised 6/23/98; accepted 6/24/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I Funding for this research was provided by The Rowland Foundation and The Rowland Institute for Science. Partial salary support for A. E. F. was provided by the University of Massachusetts Cancer Center, NIH Grants P01 HL56920-0l and DK 94-05, NASA Grant NRA94-OLMSA-02, and the Our Danny Cancer Fund. 2 To whom requests for reprints should be addressed, at University of Massachusetts Cancer Center, Two Biotech, Suite 202, 373 Plantation Street, Worcester, MA 01605. Phone: (508) 856-1316; Fax: (508) 8561310; E-mail: [email protected].

therapy

of a photosensitizing drug with therapy is not new; however,

ABSTRACT

included

selected

against

with an overall

and

Tumors

Cancer

To

of tumor recurrence angioneogenesis, address

photosensitizers chlonns,

these have purpurins,

when than on

problems, been

a

devebnaphtha-

The abbreviations used are: PDT, photodynamic therapy; EtNBS, 5-ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride; CR, complete response; PR, partial response; MR. minor response; SCC, squamous cell carcinoma; MCT, mast cell tumor; ALT, alanine amino3

transferase.


2208 Novel

Photodynamic

Therapy:

Natural

Animal

Tumors

at Tufts

University

The

tumors

and

re-evaluation

Each

tumor

tobogical

School

were

and

was

the

locyanines,

and

derived have

The molecular

I

phthalocyanines

from dissimilar different

complement

structure

and

another

photosensitizers

(9). Despite

families

efficacy one

that

clinical

the fact

that

drugs

the vast

developed

majority

are porphyrin

other photosensitizers. noxazine dyes were chemotherapeutic possess long-term erty oftumor photoactive

Photodynamicalby originally investigated

computed

tomography

scan,

depth

determined

by ocular

agents, and although they were not found to antitumor efficacy, they did exhibit the prop-

celllocalization red-absorbing

(10-12). derivatives

Because ofthis property, of the benzophenoxazines

have been synthesized in recent potential as PDT photosensitizers empirical ment.

findings, Those

tissue

EtNBS

selected

studies

in mice,

dosimetry

years and (13-18).

was

preliminary

distribution

light

inactive benzophein the l940s as

studied On the

for

further

demonstrated

as well

for the treatment

its

smear,

was

as its efficacy

of RIF

and

cell

and

by an

assigned

criteria

a

(31)

situation.

and hospitalized of the Foster

from

and

other

The

animals

under Hospital

Drug

in dog

by

cytology.

using calipers determined by

where

5,

treatment.

EtNBS

was

and

as described

magnetic

Thereafter, purified

by col-

to be homogeneous

resonance

Institute for Science. Treatment. Each

below

in the clinical wards at Tufts University

synthesized,

shown

spectroscopy

animal

with the ani-

by the individual

treated

Medicine until discharged. cared for by their owners.

Source.

tumor

healthy, determined by blood cell count, serum

as indicated

were

the Animals

In addition,

standard conditions for Small Animals

chromatography, nuclear

aspiration

ultrasonography.

tests

profile,

clinical

and

optimal

was

WHO

marrow

chemistry

develop-

tumor

excluded

School of Veterinary they were housed and

and

diagnosed

Each

were taken directly tumor thickness was

otherwise complete

umn

and

EMT-6

were

bone

mals were required to be general clinical examination,

for their basis of safety

his-

histopathology,

and/or

of

to

was

rate.

tumors,

lymph node palpation and fine needle where indicated, thoracic radiographs, and mast cell tumors, abdominal radiography,

tumor measurements in cat 3, where the

benzophenoxunrelated

metastatic

physical examination, aspiration cytology in the case of the

metastases (Fig. 1) is a novel, nonporphyrin, photosensitizer that is chemically

treated.

of treatment

cell

and

which

to established

coat

(9). EtNBS azine-derived

according

Primary except

based

expected the mast

on its appearance.

buffy

would be expected profiles, and therefore

effectively,

been

melanoma,

based

stage

for

were

for ease

by biopsy

to to

of dyes

toxicity

more

have

of EtNBS.

a low

(and

assigned)

intraocular

ophthalmologist Fig.

to have

were

Medicine

to be superficial

diagnosed

grades

except

of Veterinary

required

was

by TLC

at the

examined

Rowland

physically

and

lines, both in vitro and as solid tumors in mice (17, 18). No disadvantages relative to other photosensitizers were identified. The therapeutic margin is wide because of a tumor:normal cell

tested as described above. Animals were admitted either the night before or the morning of the procedure to the Foster Hospital. Animals were not fed the morning of the procedure, as routine preparation for general anesthesia. Prior to treatment, a

distribution

by

blood

increased deactivation of the drug in normal skin by enzymes (17). The absorption spectrum peaks at 652 nm

Heinz

relatively NADH

ratio

of approximately

[extinction coefficient (#{128}), 68,600 reaction spares the vasculature, good

enough

enough up

to prevent

by

mice),

tumor

L/cm

the

excellent

easy

tissues

rapidly

M], the photocytotoxic

lipophilicity

tissue

aqueous

.

preclinical

naturally tumors suitable

of the drug

distribution

solution,

and

(3 h after

is

but not strong the drug

s.c.

administration

trial

of

EtNBS-PDT

in dogs

and

in

cats

tumors

as animal

models

accepted

in

for

recent

human years

disease (19-22).

PDT trials of other photosensitizers

have been

spontaneous

(23-30).

were

animal

tumor

to determine

the tolerance

PDT and to determine occurring

models

the efficacy

of dogs

has

with

become

Specifically, carried out using

The

goals

and cats

of this treatment

of this

to EtNBSin naturally

EtNBS 25-30 of

is taken

tumors

that were

The dose weight,

of EtNBS

except

dim

lighting

cats

consisted

diazepam

and

positioned

METHODS

Thirteen presented

pet

animals

with

to the Harrington

naturally Oncology

occurring Program

the

was either first

with

administered

i.v.

i.v. propofol,

and

(cat 7) also

received

anesthesia

consisted

phanol,

with

administered which

were

i.v. via

with

and pentothal,

and

endotracheal was Corp.,

for

with

Another i.m.

combination

except

Systems,

Santa Barbara,

cat

For dogs, of butor-

administered

i.m.,

with

halothane

dogs

2 and

for

Plainfield,

using NJ)

a diode

CA).


Because

laser

producing

length of 652 ± 3 rim wavelength unless otherwise “Results”) and a 4O0-pm fiberoptic fitted with

(PDT

isoflurane

1 cat (cat 6), was induced

5,

isoflurane.

performed

South

under and

for i.m.,

maintenance

tube,

with

which

of ketamine

isoflurane.

glycopyrrolate

in of

Anesthesia

buprenorphine

of a preanesthetic

maintained

Optronics

maintained

1),

anesthetized

maintenance

tube, except butorphanol

preanesthetic

acepromazine,

induction

were

(cat

a combination

and

administered via endotracheal which received preanesthetic with

and placed.

or 2.0 mg/kg

2.5

animal

for photoirradiation.

of induction

Photoirradiation

AND

used

for

received 5.0 mg/kg (see “Results”). Three h after the infusion, animals

plied Animals.

of methemoglobin i.v. catheter was

was administered i.v. in a solution of 1.25 mg/ml in water as a constant rate infusion for a period

mm.

body

tumors.

SUBJECTS

sample was drawn for evaluation body bevels, and an indwelling

dextrose

5%

occurring tumors. Dogs and cats develop spontaneous that are similar to those in humans, with characteristics for PDT. The use of pet animals with naturally occur-

increasingly

trial

augmented

and cleared from all tissues of mice within 24 h (17, 18). The encouraging results of those studies bed to this prelim-

mary,

ring

to allow

1, perhaps

3.5:

(Ap-

a wave-

noted (see a microlens

of availability

of

Clinical

lasers,

the first

animal

(cat

nm

wavelength,

and

light

of 652

nm wavelength.

activation rate

of EtNBS,

but double

animals.

a fluence light

and “Results”).

beam

directed

ments

were

mined

extent

not taken

in murine

responsible

of 200

mW/cm2

i/cm2, to the

at the midpoint of the beam of tumor.

during

systems

of the

temperature because

thermal

of the 1 cm

data

effects

tested

by abdominal

not

cytology

(18), and we

light

until

evidence had

was

been

seen

in the urine

excreted,

Thereafter,

the

released weekly

or

for

animals

were

from the hospital. for the first month

month,

every

months

thereafter.

exposed

for

the

Complete

occurred normal

6 months,

cell counts

1 and

panels Toxicity

and Response.

At each

re-examination,

ubar

attention

was

owner’s

description

to the

2 months

mal’s appetite, energy, and subjective physical examination was performed,

after

serum

istry

paid

and

and every

and

6

chem-

well-being. and tumors

before

detected

anesthesia

tissue murine i/cm2

panicani-

of cardiac

Toxicity.

Local

for activation

toxicity

in painless

as a

murmurs

that

was

dependent

on

the

and the of local

1. As noted above, the duration of for this cat compared with subse-

tissues

exposure

This

dosage laser,

treatment

surrounding

of maxillary

alveolar

bone

and,

10 days

assessed

in terms

2.5 mg/kg, and the dosage of bight was reduced to 400 i/cm2 with a fluence rate of 200 mW/cm2 for 33 mm, using a wavelength of 652 run. After six treatments had been performed at

the diameter criteria for

tumor

than

reduction

namic

response one that could tumor

was

tumor

bed),

treatment

(the product

perpendicular tumor response

detectable 50%

of area

after

were

healing

MR

to accurately

or a response field;

that

no response,

than

response,

measure

did not last beyond no change

in the

2, skin or mucous

pared

in serum

or pro-

tissue toxicity alopecia only; membrane

edema; and grade 3, full-thickness skin or ulceration or corneal erosion or ulcer. Any chemistry

with

pretreatment

the animal’s

condition

or hematobogical

levels were

that dosage,

and any other

parameters adverse

and

com-

changes

in

noted.

for seven untoward

of the remaining effects had been

exposure

while

effects

to

acceptable effects

the

treatments were performed were performed in five dogs

treated

for

facial

treated

for sublingual

SCC

(one 5CC.

SCC (one treated twice),

treated Two

dogs

in eight cats, and six (Table 1). Six cats were

twice), were

and treated

two

cats

were

for intraoral

one dog for grade H cutaneous

MCT

of

and

of EtNBS

eight seen,

efficacy.

generally

and 400

normal except

consisted

reduced

tongue.

reduced

to

were

dem-

to 2.0 mg/kg

treatments, although no further in an attempt to minimize drug

of EtNBS

cases

was

toxicity

further

2.5 mg/kg surrounding

of the

of EtNBS

tolerable

was

maintaining

in all

i/cm2

tissues

1 (dog

of edema

were

1). On

of light, found

skin,

and erythema

the

to

the

be

local

(grade

the first week, then localized alopecia with regrowth 1-6 months. When oral mucous membranes were

1) for

of hair over treated, the

surrounding normal tissues in the field became ulcerated (grade 3), with rapid complete healing in less than 4 weeks, even for the largest

ulceration.

Halitosis

only lasted approximately sboughed. In one dog (dog

this

animals. The eyelids

did were

using

a photo-opaque

any

visual

problems MCT

not

and

in some the (cat

to

cause

shield.

the treatment.

experienced

cases,

any

a deep


pain

gingiva bone. to

the

2) and one cat (cat

Neither The

but

necrotic tissue 1), gingiva was

and in both cases the exposing the alveolar

in one dog (dog comeal

after

severe

appear

treated

6),

cutaneous

was

1 week, until 1) and one cat

in the treatment field, necrotic and sboughed,

Surprisingly,

Nine treatments

efficacy

the dose

present became RESULTS

good

onstrated,

At 2.0 and

of the

tumor;

or edema;

changes

photody-

healing

grade

grade

of

greater

or was postoperative

local normal or localized

or corneal membrane

absence

reduction of 50% (the initial

The criteria for grade 0, normal

1, erythema

The

PR,

gressive disease. were as follows: erosion mucous

and

volume.

field;

or brief

were

diameter

CR,

of treatment

in tumor;

they

of the largest

to it, in cm2) rather were as follows:

that resulted in tumor not clearly be determined

too small

plaque-like,

the dosage

third

resulting

many

this experience,

rostra!

severe

bed,

Because

tumors

of the

produced

the tumor

uated at each visit (except for one intraocular tumor, which was re-evaluated by ultrasonography only at 8 weeks after PDT).

Following

sboughing

of 800 i/cm2, 670 nm, is not

after

of the treated

treatment,

to rule

dosage used, extrapolated from the was 5 mg/kg of EtNBS and 800 light with a fluence rate of 200

of EtNBS.

of the normal

aspira-

and light, the size of the patient, field. See Fig. 2 for examples

mW/cm2 for 66 mm in cat photoirradiation was doubled

optimal

its

examination.

reactions. The initial preclimcal studies, of 670-nm wavelength

burns

A complete were re-eval-

marrow

echocardiograms

because

on physical

that

one dog

examination

underwent

quent animals to provide a total light because the wavelength of the available

the treatment. of the

precaution

and 4 cats

and ocular

showed

bone

smear

a

in one cat

Furthermore,

and

coat

were

above,

mm,

5) tumor wide.

Of

three

lesion