Download 1993 Syntex A ward Lecture Photomedicine and photodynamic therapy!

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1993 Syntex A ward Lecture
Photomedicine and photodynamic therapy!
DAVID DOLPHIN
2
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver; BC V6T 1Z1, Canada
Received June 4, 1993
DAVID DOLPHIN. Can. J. Chern. 72,1005 (1994).
Photodynamic therapy (PDT) involves the treatment of diseased tissue and cells using a photosensitizer and visible light. Such
photomedical treatments have been known since the time of the ancient Egyptians but it was only just this year that this therapeutic modality was made available to modern medicine with the approval, in Canada, of Photofrin® for the treatment of bladder
cancer. This paper reviews PDT with an emphasis on drug development, particulary for the second generation drugs. especially
BPDMA (benzoporphyrin derivative-mono acid). which is now in human clinical trials.
DAVID DOLPHIN. Can. J. Chern. 72,1005 (1994).
La therapie photodynamique (TPD) implique Ie traitement des tissus et des cellules malades en utilisant un photosensibilisateur et la lumiere visible. De tels traitements photomedicaux sont connus depuis Ie temps des anciens Egyptiens, mais la medecine modeme n'a eu acces a cette modalite therapeutique que cette annee seulement avec I'approbation, au Canada, de la
Photofine® pour Ie traitement du cancer de la vessie. Cet article revoit la TPD en mettant l' emphase sur la mise au point des medicaments, particulierement pour la deuxieme generation de medicaments et specialement la BPDMA (Ie derive mono-acide de la
benzophorphyrine) qui est a I'essai c1inique chez les humains.
[Traduit par la Redaction]
Photo medicine has been practiced since at least the time of
the ancient Egyptians (1). Psoralins from orally ingested plants
accumulated in the skin and when activated by sunlight brought
about repigmentation of the skin. This 4000-year-old treatment
for vitilago is still used today with psoralins and UV exposure
and represents the best known treatment even though it has only
limited success. Indeed, a number of skin diseases, including
acne, eczema, herpes simplex, and psoriasis, have been treated
in a similar manner.
Niels Finsen won the Nobel Prize in Medicine in 1903 for his
treatment of cutaneous tuberculosis by UV radiation. However,
the most promising areas of photomedicine are those that use a
photosensitizer. Indeed, it was believed in the 19th century that
all efficacious drugs were colored and while this belief led to the
discovery of the sulfa drugs (Prontosil is an azo dye) it was not
until the beginning of the 20th century that the medicinal properties of photosensitizers were explored. Raab (2) in 1900
showed that acridine dyes and light effectively killed Paramecia. In 1925 Policard (3) examined the ability of porphyrins,
including hematoporphyrin (1), to produce a phototoxic effect.
IThis Syntex Award Lecture was delivered by Professor Dolphin at
the Annual Conference of the Canadian Society for Chemistry, Sherbrooke, Quebec, June 2, 1993.
2David Dolphin is the NSERC Industrial Research Chairholder in
Photodynamic Technologies at the University of British Columbia and
Vice President, Technology Development, at Quadra Logic Technologies Inc., Vancouver.
Mayer-Betz injected himself with 200 mg of hematoporphyrin
(4) and suffered no ill effects until he exposed himself to sunlight, whereupon he suffered extreme swelling and remained
photosensitive for several months. Similar accumulation ofporphyrins in the skin of porphyric patients may cause severe skin
necrosis upon exposure to strong light (5). Auler and Banzer in
1946 (6) showed that hematoporphyrin accumulated in cancerous tissues and, since it exhibits strong fluorescence, its localization into neoplastic and rapidly dividing tissue could be
quantitatively assessed (7).
Hematoporphyrin derivative HpD was first prepared by
Schwartz and co-workers and its potential as a radiosensitizer
was examined (8), but in 1964 Lipson et al. showed that HpD
preferentially accumulated in cancerous tissue rather than in the
surrounding healthy tissue (9-11). Interest in photomedicine
and particulary photodynamic therapy (PDT) was rekindled
when Dougherty (12) showed that HpD could be purified by gel
exclusion chromatography, which removed monomeric porphyrins. The remaining oligomeric material is known as Photofrin®
(2) and Quadra Logic Technologies, Vancouver (QLT), in partnership with Lederle Laboratories have just received a notice of
Compliance from the Canadian Health Protection Branch for the
use of Photofrin® in the treatment of superficial bladder cancer.
Photodynamic therapy (PDT) requires that the photoactive
agent first absorb a photon of a specific wavelength and in general the action spectra correlate well with the absorption spectra.
As can be seen from the modified Yablonski diagram in Fig. 1,
the first excited singlet state (SI) can fluoresce or this excited
state can participate in an electron transfer process with a biological substrate, resulting in the photobleaching of the photosensitizer and modification (destruction/inactivation) of the
substrate. This is known as a Type I photoprocess (13). PUVA
(psoralin, 8-methoxypsoralin, plus UV radiation using UVA)
relies on this photochemistry where the psoralin is believed to
undergo photo addition to thymidine bases of DNA (14). In
addition to being the most widely used treatment of psoriasis,
PUVA also gives rise to cutaneous cancers (15). Photosynthesis
using reduced porphyrins (chlorophylls, bacteriochlorophylls)
makes use of a Type I process but the initially oxidized "porphyrin", at the reaction center, is reduced by another porphyrin
1006
CAN. 1. CHEM. VOL. 72,1994
R
=
HO-CH-
or
-
I
CH=CH2
and n = 0-7
CH3
PHOTOFRIN® (Profimer Sodium) (2)
Sn _ __
S4 _ __
S3--S2 _ __
(1+) S, S,
>-
T, (tt)
'-'
0:::
W
Z
W
Singlet
1
2
4
Triplet
So
, 2 3 -
Absorption - Depends upon exti nction coefficient and wavelength
Fluorescence - Lifetime depends on molecular interactions
Intersystem crossing
4
5 -
Phosphorescence
,
Conversion of triplet oxygen 3~ to singlet oxygen 02
FIG. 1. Modified Yablonski diagram.
(cytochrome) such that no net destruction of the photoactive
species occurs.
A second, and far more interesting, photochemical process
known as a Type II photoprocess results in the conversion of
stable triplet oxygen
2 ) to the short-lived but highly reactive
(toxic) singlet oxygen (102 ), This reaction occurs, as shown in
Fig.!, when the S 1 state of the photosensitizer undergoes intersystem crossing to its first excited triplet state (T 1) followed by a
triplet-triplet reaction with 302 to regenerate the photosensitizer in its ground state and singlet oxygen.
Singlet oxygen has a lifetime of ~6 /Ls in water and a little
longer in lipid and cell membranes, which means that it cannot
diffuse more than a single cell length. Singlet oxygen is a powerful, fairly indiscriminant, oxidant that reacts with a variety of
e0
biological molecules and assemblies. As shown in Fig. 2, oxygen atom transfer can result in the oxidation of both carbon and
sulfur and in the formation of hydroperoxides from a variety of
substrates including cholesterol and phosopholipids. The site of
the greatest accumulation of the phototoxin may not define the
site of cell death since, while a single oxidation of an amino acid
in a protein may results in the shutdown of critical biological
process (16--18), greater destruction ofless critical sites may be
required for cell death. Be that as it may, Oleinick and colleagues (19) have shown that, in vitro, cells die by apoptosis 3
3 Apoptosis is programmed cell death involving endonucleosis and
chromatin condensation.
DOLPHIN
~H
~
02 H
NHl
CH2-CH-COO-
~
lllstidine endoperoxides
NH3+
NH3+
~-CH-COO-
~-CH-COO-
~_OH
H
H
Tryptophan
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Tryptophan
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o
NH3+
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o
o
Methionine sulfoxide
NHl
HOS-CH2-CH-COO-
Cysteine
sulfenate
NH3+
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II
Methionine sulfone
NHt
H~S-CH2-CH-cOOCysteine
sulfinate
NHt
HO;sS-CH2-CH-COO-
Cysteine
sulfonate
FIG. 2. Typical examples of the reactions of singlet oxygen with
biomolecules.
after PDT. A dramatic example of cell membrane damage is
shown in Fig. 3. This damage was observed (20) using
BPDMA, which is discussed below.
Since most photosensitizers currently in clinical trials for
PDT (21) have quantum yields for singlet oxygen production of
~0.5 or higher, considerable amounts of singlet oxygen can be
produced when the photosensitizer is illuminated with light of
the appropriate wavelength. The penetration of light through
tissue is attenuated by a number of factors but absorption and
scattering are the most important in limiting the penetration
depth for effective treatment. Heme proteins account for most
of the absorption of light in the visible region. Since this drops
off rapidly beyond 550 nm, the effective depth of penetration
doubles in going from 550 to 630 nm (where Photofrin® is activated) and doubles again in going to 700 nm. However, beyond
700 nm only an additional 10% increase in penetration is
achieved by moving into the infrared (Fig. 4). The clinical activation of Photofrin® at 630 nm was chosen since it has an
absorption band there (albeit weak) that is stronger than the
absorption of oxyhemoglobin at that wavelength (Fig. 5).
Porphyrins (3) (Fig. 6) are 1871"-electron aromatic macrocycles that exhibit characteristics optical spectra with a very
strong 71"-71"* transition around 400 nm (known as the Soret
band) and four Q-bands in the visible region. As can be seen in
Fig. 6, two of the peripheral double bonds, in opposite pyrrolic
rings, are crossed conjugated and are not required to maintain
1007
aromaticity. Thus reduction of one or both of these cross-conjugated double bonds (to give chlorins (4) and bacteriochlorins
(5) maintains aromaticity but the change in symmetry results in
bathochemically shifted Q-bands with high extinction coefficients (Fig. 6). Nature uses these optical properties of the
reduced porphyrins to harvest solar energy via photosynthesis
with chlorophylls and bacteriochlorophylls as both antennae
and reaction center pigments. The long-wavelength absorptions
of these chromophores naturally led to explorations of their use
as phototoxins in PDT where they have exhibited some interesting properties (22, 23). Nevertheless, the complexity of these
natural pigments and the considerable problems in their isolation, separation, and purification add to the difficulties in their
development for clinical use. Moreover, chlorins and bacteriochlorins, by virtue of their di- and tetrahydro reduction states,
can be readily oxidized back to the parent porphyrin accompanied by the loss of their long-wavelength absorption bands.
This potential lack of stability has led us to examine other ways
of producing stable chlorin-like chromophores.
Protoporphyrin IX as its iron complex (heme) is the prosthetic group of heme proteins and is thus readily available from
a number of animal sources. As a result of the cross-conjugated
nature of the peripheral double bonds it seemed likely that one
of these double bonds and a conjugated vinyl group of protoporphyrin IX might act as a diene in a Diels-Alder reaction
(24).
When protoporphyrin IX dimethyl ester (6) is treated with a
strong dienophile such as tetracyanoethylene, both 2 + 2 and 2 +
4 cycloadditions take place on either or both rings A and B
(Scheme 1) (24). Acetylene dicarboxylic ester, on the other
hand, brings about a Diels-Alder reaction at either ring A or B
but we have not seen reaction at both rings (25). The initially
formed cycloadducts (7,8) are 1,4-cyclohexadienes having
strong absorptions at 666 nm consistent with their chlorin-like
chromophores (Fig. 7). Treatment of the 1,4-diene with triethylamine tautomerizes it to the 1,3-diene (9). This conjugated
chlorin absorbs at 686 nm and turns out to be a kinetically controlled product having a cis arrangement of the angular methyl
and methoxycarbonyl groups. Treatment of this cis-l ,3-diene or
the original l,4-dienes with 1,5-diazobicyclo[5.4.0lundec-5ene (DBU) generates the thermodynamically more stable trans
product (11). X-ray crystallography confirms the structure and
stereochemical assignments made from previous NMR studies
(Fig 8) (26). The cis and trans isomers both exhibit strong
absorption around ~690 nm (Fig. 7). Prolonged treatment of
the 1,3-dienes with TEA eliminates the angular methyl group
and generates the benzoporphyrin (10), which has a characteristic porphyrin spectrum (Fig. 7).
Hydrolysis of the dimethyl ester in concentrated HClleaves
the ester groups on the exocyclic ring intact owing to steric hindrance experienced by the group close to the angular methyl
and the conjugation of the other. The propionic esters do hydrolyze to give a mixture of the diacid (14) and the two regioisomeric monacid monoesters (12, 13). Cytotoxicity studies using
the diacid showed that it was 10-70 times more cytotoxic than
hematoporphyrin against a variety of normal and malignant cell
lines (27). Surprisingly, a comparison of the photo toxicity of
the monoacids showed that they were five times more cytotoxic
than the corresponding diacids (28) for both the ring A and ring
B analogs (Fig. 9). We choose to proceed towards clinical trials
using the two regioisomeric monoacids of the ring A analog (12
and 13) that we call BPDMA (benzoporphyrin derivative ring
o
o
00
n
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z
~
n
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tTl
s:
o<
r-
-.J
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-0
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FIG. 3. Feline T cells were treated with 2 j.Lg/mL of BPDMA and light. A: uninfected cells and light show no damage; B. infected (with FeIV) cells after early treatment with light show small
holes in the membrane, as light treatment continues, the holes increase in size (C) until the membrane eventually appears to be completely destroyed (D). Reprinted by permission form Blood
Cells (20).
1009
DOLPHIN
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700
900
1100
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Q)
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FIG. 5. Optical spectra of oxyhemoglobin (--) and Photofrin®
+'
(----).
(1j
Q)
0:
550
630
800
700
nm
FIG. 4. Relative depth of penetration, in muscle, of light of various
wavelengths.
A mono acid), whose optical spectrum (Fig. 10) shows a maximum absorption at the wavelength (~690 nm) where blood has
a minimum absorption.
While BPDMA does not show specific affinity for tumors, it
exhibits significantly higher concentrations in tumors than in
the surrounding healthy tissues (29). BPDMA localized in
tumors better than in other tissues except liver, kidney, and
300
400
500
600
WAVEl..£NGTH (nm)
700
400
spleen. BPDMA is a chemically stable powerful phototoxin (its
quantum yield for singlet oxygen is 0.46 (30)); however, before
proceeding with the extremely expensive and time- consuming
preclinical studies needed before a health board will issue an
IND (investigation of a new drug), which allows the undertaking of human clinical trials, one is well advised to examine the
extent of prolonged skin photosensitivity.
Photofrin® causes skin photosensitivity in patients for 4-6
weeks after treatment. This is a minor side effect compared to
those experienced with chemotherapy or radiation therapy;
nevertheless, a successful second generation phototoxin for
PDT must exhibit dramatically reduced skin photosensitivity
compared to Photofrin®. As can be seen from Fig. 11, compounds 12, 13, and 14 all showed rapidly decreasing skin photo-
500
600 700
WAIJEl.ENGTH (nm)
500
600
700
WAVElENGTH (nm)
Porphyrin
Chlorin
B acteriochlorin
3
4
5
BOO
FIG. 6. Structures of the porphyrin (3), chlorin (4), and bacteriochlorin (5) macrocycles. The cross-conjugated double bonds in 3 can be
reduced, leaving the dihydro (4) and tetrahydro (5) analogs aromatic. Increasing reductions result in an increasing bathochromic shift of the lowest
energy absorption band.
1010
CAN. J. CHEM. VOL. 72,1994
NC
CN
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NC
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8
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DBU
SCHEME 1
BPDMA is virtually insoluble in water and it has been formulated in unilamellar liposomes to make it water soluble.
There is evidence to suggest that tumor selectivity with hydrophobic photosensitizers may be mediated by plasma proteins,
especially low-density lipoproteins (LDL) (31-33). BPDMA
associates predominantly with LDL (34) and, when preassociated with LDL improved delivery to tumors in a mouse tumor
model was seen (35). The unilamellar liposomes rapidly
deliver the BPDMA to the lipoprotein fraction when placed in
serum, and PDT in a mouse tumor model showed that the liposomally formulated material accumulated more rapidly when
delivered in this fashion than when injected in DMSOIPBS
(36).
300
400
500
600
WAVELENGTH (nm)
700
FIG. 7. Optical spectra of the 1,4-diene (7,··· .), the 1,3-diene (11,
--), and the benzoporphyrin (10, - - - -).
sensitivity compared to Photofrin®, and after 72 skin photosensitivity had almost returned to normal with the monoacids. At
this stage we were anxious to see if this minimal skin photosensitivity would also be seen with humans.
As long as a phototoxin preferentially accumulates in diseased tissue, or in a pathogen, then differential damage occurs
when the system is illuminated. Under such conditions an appropriate drug dose - light energy combination can be found
whereby the diseased tissue or pathogen can be destroyed without causing irreversible damage to healthy tissues. Photobleaching (37), a type I photoprocess that occurs more readily
in vivo than in pure organic or aqueous solvents, may assist in
minimizing damage to healthy tissue if photobleaching of low
concentrations of the phototoxin in healthy tissue is complete
1011
DOLPHIN
FIG. 8. X-ray structure of a BPD derivative drawn by Biosym's InsightII.
100
.....
-0
IS
(])
0
80
...I
~
(fJ
:::2l
60
(])
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U
~
10
40
I
I
5
~
,\
tV
'..1I "
,
J
:x2
20
10
100
1000
Concentration (ng/mL)
FIG. 9. In vitro cytotoxicity BPDMA (12 and 13, .), the corresponding monoacid monoester derivatives of the ring B analog ( ), the
diacid (14, e), and the analogous ring B diacid (D) examined under
the same conditions (28).
before irreversible damage can take place. Photofrin® and
BPDMA photobleach at similar rates. 4
While we are just beginning to understand the mechanisms
by which phototoxins biodistribute, few structure-activity relationships are known. Nevertheless, BPDMA has a great propensity to accumulate in diseased cells and in the neovasculature of
hypoproliferic tissues. Measurements of biodistribution are
aided by the strong fluorescence at 690 nm shown by BPDMA
(Fig. 12). Fluorescence-activated cell-sorting (FACS) analysis
following excitation in the UV at 420 nm showed dramatic differences between several leukemic cell lines, leukemic clinical
isolates, and normal bone marrow cells incubated with BPDMA
(38). Indeed BPDMA shows great potential for the ex-vivo
purging of residual tumor cells in autologous bone marrow
grafts. A four-log elimination of tumor cells occurred after a l-h
incubation with 75 ng/mL BPDMA followed by exposure to
white light; at the same time pluripotent stems, the progenitors
for all hemopoietic cells, survived the purging regime (39).
Human clinical trial using BPDMA to purge leukemic autolysis
grafts wiII begin this year under the direction of QLT.
Numerous viruses, pathogenic to humans, contain an outer
hydrophobic membrane (envelope). Included amongst these
viruses are cytomegatovirus (CMV), herpes simplex virus
4W.R. Potter. Personal communication.
Wavelength (nm)
FlO. 10. Optical spectra of oxyhemoglobin (--) and BPDMA
(-
-).
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';;;:
:;::
'iii
c:
5
~
4
Q.
3
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..c:
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D Photofrin®
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o
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72
24
3
Time post injection (h)
FlO. 11. Skin photosensitivity in mice following Lv. injection of the
photosensitizers (4 mglkg) and exposure to white light (95 J/cm\
Severe damage resulting in eschar formation was scored as 5 (47).
Compounds are described in Fig. 9.
(HSV), several of the hepatitis viruses, and retroviruses such as
the human T cell leukemic virus (HTLV) and human immunodeficiency virus (HIV). These, and other viruses, may be transmitted in transfused blood but it has been shown, using
1012
CAN. 1. CHEM. VOL. 72,1994
100
t..J
,
t)
,1,
I
Z
t..J
,I'I
t)
(/)
t..J
c::::
50
=>
I
I'
I
I
I
I
I
'
1
I
I
I
I
•
,
I
1
I
I
0
....J
I..&...
I
II
I
.
•
I
\
I
"
... _-.....
O~TTTlnn,.TTrrMnIlTTrrM
300
400 500 600 700
WAVELENGTH (nm)
800
FIG. 12. Fluorescence (- - - -) excitation (--) spectrum of
BPDMA
-:::J 1,000
E 500
"
0>
C
'-'
Z
<0
200
100
::::el(/)<
50
o..Z
20
~g:
l.LI
()
z
0
()
10
5
a
6
12
18
24
TIME (h)
FIG. 13. Plasma concentrations of BPDMA in patients following
an i. v. infusion of the drug at 2.5 mg/kg.
vesicular stomatitis virus (VSV) and feline leukemia virus
(FeLV) as models, that BPDMA and light were effective in
eliminating both cell-free virus and virally infected cells while
at the same time leaving the red cells apparently undamaged
(20, 40). Further studies have shown that BPDMA is effective
for the elimination of both free and virally infected leukocytes
in whole blood from virally infected humans under conditions
that appeared to spare both the red cells and uninfected leukocytes (41). On the basis of these results and other preclinical
studies, QLT plan to initiate clinical trials for the extracorporeal
treatment of HIV -infected patients. The benefits of reducing the
viral load must await the results of the clinical trials. In addition
to reducing the viremia, other immunologic effects may be
found as were observed after PDT with Photofrin®. Immunosuppression, as indicated by sensitivity to dinitrofluorobenzene
(42), was observed in mice after PDT and an increase in chronic
inflammatory cells was observed in patients after treatment for
bladder cancer using Photofrin®. Moreover, the release of
cytokines such as interleukin-l band -2 and the release of tumor
necrosis factor by macrophages (43,44) and of thromoxane (45)
have all been observed shortly after PDT.
While several second generation drugs are in clinical trials
(21), the most advanced and most promising is BPDMA,
which is being used on a number of cutaneous cancers including basal cell carcinoma, metastatic breast and gastrointestinal
carcinomas, and metastatic amelanotic melanoma as well as
for the treatment of psoriasis. At the present time the drug is
infused in doses ranging from 1.5 to 5.0 mg/kg and light doses,
administered 3 h later, have ranged from 25 to 150 J/cm 2 • More
than 70 cutaneous tumors have been treated and followed for
several months after treatment. Complete responses were
observed for the higher drug and light doses, and overall tumor
responses of greater than 60% has been recorded (46). These
exciting and promising results are being paralleled in the treatment of psoriasis where only a few patients have so far been
treated.
We noted above that any successful second generation photosensitizer must exhibit minimally prolonged skin photosensitivity if it is to be successful in the clinic. The pharmacokinetic
profile for BPDMA in human serum concentrations is shown in
Fig. 13 where rapid clearance from the system is clearly
observed. This decrease in serum levels is paralleled by a lowering of skin photosensitivity with time. A major purpose of the
first phase of the clinical trial was a measurement of skin photosensitivity, and extensive studies using UVA and visible light
from a solar simulator (using a filtered xenon arc lamp) have
been carried out (46). Marked skin photosensitivity was
observed shortly after treatment but photosensitivity shows a
logarithmic decrease with time and it is expected that skin photosensitivity will return to normal (pre-BPDMA infusion)
within 1-2 days at the expected clinical dose of BPDMA.
BPDMA thus seems to be meeting all of the necessary and
hoped-for requirements of a successful second generation photosensitizing drug and, as the only non-mutagenic, non-carcinogenic, non-(dark) toxic anti-cancer drug known, we anticipate
its use in a variety of clinical situations.
Acknowledgements
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and Quadra
Logic Technologies Inc.
L
2.
3.
4.
5.
6.
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