Download Invited Review Structure and Biodistrlbution Relationships of Ross

Photochemistry and Photobiology, 1996, 64(3):
469-485
Invited Review
Structure and Biodistrlbution Relationships of
Photodynamic Sensitizers*
Ross W. Boylet' and David
Dolphin~1,2
'Department of Chemistry, University of British Columbia, Vancouver, B.C., Canada and
2QLT PhotoTherapeutics, Vancouver, B.C., Canada
Received 8 February 1996j accepted 31 May 1996
ABSTRACT
e.g. rose bengal and the hypericins, have also been omitted
to allow meaningful comparisons to be made between different compounds. As the intracellular distribution of photosensitizers to organelles and other subcellular structures
can have a large effect on PDT efficacy, a section will be
devoted to this topic.
Photodynamic therapy (PDT) has, during the last quarter
century, developed into a fully fledged biomedical field
with its own association, the International Photodynamic
Association (IPA) and regular conferences devoted solely
to this topic. Recent approval of the first PDT sensitizer,
Photofrin® (pommer sodium), by health boards in Canada, Japan, the Netherlands and United States for use
against certain types of solid tumors represents, perhaps,
the single most significant indicator of the progress of PDT
from a laboratory research concept to clinical reality.
The approval of Photofrin® will undoubtedly encourage
the accelerated development of second-generation photosensitizers, which have recently been the subject of intense
study. Many of these second-generation drugs show significant ditTerences, when compared to Photofrin®, in
terms of treatment times postinjection, light doses and
drug doses required for optimal results. These differences
can ultimately be attributed to variations in either the
quantum efficiency of the photosensitizer in situ, which is
in turn affected by aggregation state, localized concentration of endogenous quenchers and primary photophysics
of the dye, or the intratumoral and intracellular localization of the photosensitizer at the time of activation with
light. The purpose of this review is to bring together data
relating to the biodistribution and pharmacokinetics of
second-generation sensitizers and attempt to correlate this
with structural and electronic features of these molecules.
As this requires a clear knowledge of photosensitizer
structure, only chemically well-characterized compounds
are included, e.g. Photofrin® and crude sulfonated phthalocyanines have been excluded as they are known to be
complex mixtures. Nonporphyrin-based photosensitizers,
INTRACELLULAR DISTRIBUTION OF
PHOTOSENSITIZERS
Intracellular distributions in cultured cells have been determined for a range of photosensitizers with widely differing
patterns of substitution. Roberts and Berns (I) reported that
mono-aspartyl chlorin e6 (MACE)§ (1) localized in Iyso-
(1)
§Abbreviations: AlPeS •• sulfonated aluminum phthalocyanines;
Bch-a, bacteriochlorophyll a; BPD, benzoporphyrin derivative;
BPD-DA, BPD diacid; BPD-MA, BPD monoacid; BPD-MB,
BPD-MA ring B; DMSO, dimethylsulfoxide; DPPC, 1,2-dipalmitoylphosphatidylcholine; EDA, ethylene diamine; GePcOBu8'
germanium(lV)-octabutoxy phthalocyanine; ia, intraarterial; ID,
injected dose; ip, intraperitoneal; isoBosinc, Bis(diisobutyloctadecylsiloxy)silicon-2,3-naphthalocyanine; iv, intravenous; LDL,
low-density lipoprotein; MACE, mono-aspartyl chlorin e6; MF,
monomeric fraction; Nc, naphthalocyanine; OOPS, 1,2-dioleoylphosphatidylserine; PBS, phosphate-buffered saline; Pc, phthalocyanine; PDT, photodynamic therapy; PEG, polyethylene glycol;
Phe-a, pheophorbide a; pi, postinjection; POPC, I-palmitoyl-2-
*This Review is dedicated to the memory of Dr. Brian E. Johnson.
tPresent address: Department of Biological and Chemical Sciences,
University of Essex, Central Campus, Wivenhoe Park, Colchester
C04 3SQ, UK
tTo whom correspondence should be addressed at: Department of
Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, B.C. V6T IZI, Canada. Fax: 604-822-9678;
e-mail: [email protected].
© 1996 American Society for Photobiology 0031-8655196
$5.00+0.00
469
470 Ross W. Boyle and David Dolphin
somes of Chinese hamster ovary cells (CHO-KI) after ingestion by endocytosis. This was attributed to the hydrophilic nature of MACE, which presents four ionizable carboxyl groups in an asymmetric arrangement around the central chlorin ring. It was suggested that these polar characteristics prevent passive diffusion of MACE into the cytoplasm
and subsequent migration to the mitochondrion. A comparative study of meso-tetraphenylporphyrins (TPP) substituted
to different degrees at the para positions of the phenyl rings
with sulfonic acid groups (2) revealed a similar pattern of
localization in cervical carcinoma cells (NHIK 3025) to that
found above for MACE (1), although the tetrasulfonate
(TPPS 4 ) (2) was also detected, to a lesser extent, in the
R,
croscopy to determine intracellular distribution of aluminum
phthalocyanine, sulfonated to different levels, in human melanoma cells (LOX). It was found in this case that the tetraand trisulfonated phthalocyanines (AIPcS4 and AIPcS) (6,7)
localized in the Iysosomes, whereas the lower sulfonates
(AIPcS2 and AIPcSI) (8,9) exhibited a diffuse distribution
throughout the cytoplasm. The power of the confocal technique was elegantly demonstrated by its ability to resolve
(6)
(7)
R1 = R2 = R.:s= R... = S0.:sH
R1 = H, R2 = R3= R4= 50 3 H
(8)
R,= R2 = H.
(9)
(2)
(3)
(4)
(5)
R 1= R 2 = R3 = 50 3 H
R1 = R3 =H, R2 = 50 3 H
RI = R2 = H,
R3= 50 3H
R1 = R2 = R3= H
nucleus and cytoplasm. The distribution of both reglOlsomers of the disulfonate (TPPS 20 and TPPS 2a ) (3,4), bearing
sulfonic acid moieties on the opposing and adjacent phenyl
rings respectively, was found to mimic that of TPPS4 (2);
however, the monosulfonate (TPPS 1) (5) was detected diffusely through the cell but more strongly in the perinuclear
area. Interestingly, a subsequent study by this group (3)
found that exposure of cells of the same line, preincubated
with TPPS 4 (2) or TPPS 2 (3,4), to light doses that inactivated
20% of the cells resulted in relocalization of the sensitizers
from the lysosomes to the cytoplasm in general, and, more
specifically, the nucleus. This behavior was attributed to
photodynamic permeabilization of the lysosomal membrane,
thus allowing small molecules, including the photosensitizers, to leak out. The same effect was not observed for TPPS 1
(5), again suggesting that this sensitizer does not localize in
lysosomes. Peng et al. (4) used confocal laser scanning mioleoylphosphatidy\Choline; PV A, polyvinyl alcohol; SiNe, silicon
naphthalocyanine; m- THPC, meta-tetrahydroxyphenylchlorin;
3,1-Tpro, 3,I-meso-tetrakis(o-propionamidophenyl)porphyrin:
TPP, telraphenylporphyrin; TPPS n, sulfonated TPP; ZnNe, zine
Ne; ZnNcA4' tetraamino ZnNe; ZnNeM4' tetramethoxy ZnNe;
ZnNeMA4 • tetra(methylamido) ZnNe.
R3= R4= 50 3 H
R 1= R2 = R3= H, R4= 50 3 H
in three dimensions the granular pattern of lysosomal localization for AIPcS4 (6) and AlPeS) (7), a pattern that had
previously been described as diffuse when using conventional fluorescence microscopy (5). The distribution of
these anionic dyes suggests a relationship between the
number of negatively charged groups attached to the photosensitizing "core" and its mode of entry into the cell,
which, in tum, determines the intracellular distribution pattern. Neither the pKa of the acidic group nor charge distribution on the molecule seem to be critical. Rather, the number of negative charges is the determining factor. The exact
amount of negative charge that prevents diffusion across
the cell membrane and allows endocytosis to become the
dominant mode of entry seems to be around - 2; however,
the situation for AIPcS2 (8) is confused by conflicting reports of lysosomal localization (6) and diffuse distribution
throughout the cytoplasm (4). This situation is most likely
a reflection of difficulties in purifying this compound.
Woodburn et al. (7) studied intracellular localization, in
V79 Chinese hamster lung fibroblasts and C6 glioma cells,
of a series of porphyrins derived from hemato- and protoporphyrin [X with side chains chemically modified to give
hydrophobic and anionic or cationic residues at physiological pH (10-17). Compounds were selected to represent all
combinations of these characteristics and it was found that
those with a net cationic character localized in mitochondria,
while those with net anionic character localized in Iysosomes. As the anionic porphyrins all bore two negative
charges, these results are in accord with previous work suggesting that sensitizers with a net charge of - 2 or greater
accumulate in Iysosomes. A porphyrin with both two anionic
carboxylate residues and hydrophilic chains, however,
showed a diffuse distribution in the cytoplasm, indicating a
subtle involvement of the charge-to-mass ratio in the distribution of anionic sensitizers. A time-dependent redistribu-
Photochemistry and Photobiology, 1996, 64(3) 471
tion of two cationic sensitizers from the mitochondria to the
lysosomes was attributed to internalization of porphyrinloaded mitochondria by the Iysosomes.
Recently, a dynamic method for following the uptake and
intracellular distribution of photosensitizers was reported (8).
The authors followed the uptake of two cationic meso-tetra-
Rl
R2
Rl
(10) HC(CH..,)OCH..,
OCH..,
NH(CH 2 )2 N(CH "')2
(12) HC(CH.., )O(CH 2)2 OCH 2 CH.., OH
(13) HC(CH.., )O(CH 2 )2 OCH 2 CH.., NH(CH 2 ) 2 N(CH..,h
(14) HC(CH..,)SCH 2 CONHNH(CH 2 )2 N(CH..,)2
-(CH2 )2 N(CH"')2
(11) CH=CH 2
(15) HC(CH..,)S(CH 2hNO
OH
(16) HC(CH l )S(CH 2 hNO
(17) HC(CH l )O(CH 2 h N(CH l)2
NH(CH 2 ) 2 N(CH..,h
OH
pyridyl porphyrins, quartemized with methyl (18) and hexyl
(19) residues, in human dermal fibroblasts (0532). Important
IN VIVO PHARMACOKINETICS AND
BIODISTRIBUTION OF PHOTOSENSITIZERS
In reviewing the in vivo pharmacokinetic and biodistribution
behavior of photodynamic therapy (PDT) sensitizers, it soon
becomes apparent that direct correlation between photosensitizer structure and these biological parameters is complicated for many compounds; this is especially true for the
"hydrophobic" sensitizers, which, due to lack of water solubility, must be injected in a suitable transport vehicle, e.g.
liposomes, oil in water emulsions and nanoparticles. As it
has been shown that the nature of the delivery vehicle affects
the relative transfer of the drug to different serum proteins
(10-12), which can in tum alter the in vivo pharmacokinetics
and biodistribution, wherever possible, comparisons will be
made among similar modes of delivery. The use of different
tumor types and animals presents yet another problem of
heterogeneity, so these parameters will be quoted in every
case. Finally, certain physical characteristics of the photosensitizer have been used to classify them into three major
groups: (l) Hydrophobic sensitizers are defined here as those
bearing no charged peripheral substituents and which have
negligible solubility in water or alcohol. (2) Hydrophilic sensitizers have three or more charged substituents and are freely soluble in water at physiological pH. (3) Amphiphilic sensitizers have two or less charged substituents and are soluble
in alcohol or water at physiological pH.
The above classifications are, of course, only intended to
make comparison between in vivo results more clear, and
some photosensitizers may be on the borderline between
groups. Amphiphilic character is especially difficult to define, as it involves not only the number of hydrophilic substituents, but also their spacial distribution, e.g. both isomers
of TPPS 2 are amphiphilic by the above definition and only
one atropisomer of m-tetrahydroxyphenylchlorin could truly
be described as amphiphilic, yet it is assigned to this subsection. These ambiguities will. however, be addressed individually in the text and where appropriate cross reference
will be made between groups.
HYDROPHOBIC SENSITIZERS
(18) R = CH J
(19) R = n-C S H1J
differences were found. with the methyl derivative being
taken up faster and localizing in the nucleus, while the hexyl
analogue entered the cells much more slowly and exhibited
a particulate distribution in the cytoplasm. These results
again illustrate the importance of charge/mass ratio in determining intracellular distribution. A more comprehensive
review of the role of lysosomal localization has been published by Geze et al. (9).
The most prevalent family of hydrophobic PDT sensitizers
are the phthalocyanines (Pc) and closely related naphthalocyanines (Nc). these compounds have a basic photosensitizing core that is formally a tetrabenztetraazaporphyrin or tetranaphthotetraazaporphyrin (13). The structure of the molecules allows substitution around the periphery of the macrocycle on the four benzenoid or naphthalenoid rings. Further
substitution is also possible by chelation of metals bearing
axial ligands within the central cavity of the Pc or Nc. The
Pc and Nc used for PDT are, without exception, metallated.
This is in contrast with the porphyrin-based sensitizers,
which are most often unmetallated.
One of the simplest of these compounds, zinc phthalocyanine (ZnPc) (20), has no peripheral substituents and a centrally chelated zinc ion and has been the subject of several
in vivo studies. Reddi et al. performed a series of studies
using ZnPc encapsulated in small unilamellar liposomes of
dipalmitoylphosphatidylcholine (OPPC). Female BALB/C
mice bearing MS-2 fibrosarcoma were used in all these experiments; however. the mode of administration was intra-
472 Ross W. Boyle and David Dolphin
peritoneal (ip) in the first case (14) and this was altered to
intravenous (iv) subsequently. For ip injection of 0.5 mg
kg-I body weight a maximal tumor concentration of 0.6 V-g
g-I tissue was reported at 24 h postinjection (pi) representing
a tumor/muscle ratio of -7.5. Levels of ZnPc (20) in skin
(20)
M =Zn, R1 = R2= RJ= R4= H
(25)
M = 67GaCI,
R1= R2 =R3 =R4 = O(n-C ta H37)
(26)
M = Ge(OSiPh 2 (Cholesterol»2
never exceeded 0.1 j.Lg g-I tissue between I and 168 h pi.
Interestingly, a comparison can be made between these results and others obtained by the same group using iv injection (15), although a lower dose of 0.12 mg kg-I was used.
Under these conditions maximal tumor concentrations of
-0.3 V-g g-I tissue were reached 3 h pi and this level was
maintained up to 45 h pi. This is in contrast with ip injection
where tumor concentrations were less than half the maximal
by 45 h pi. Tumor/muscle ratio at 24 h pi was 3.71; however.
the peak value for this parameter was 3.84 attained 168 h
pi. Skin levels of ZnPc (20) again never exceeded 0.1 V-g
g-I tissue between 1 and 168 h pi. The authors also investigated the possibility of manipulating ZnPc (20) biodistribution by: (a) complexation with human low-density lipoprotein (LDL) (15) and (b) addition of 15% mole cholesterol
to the DPPC liposomes (16).
The results show an increased maximal tumor concentration (0.55 V-g g-I tissue at 24 h pi) using LDL and also a
higher tumor/muscle ratio (5.71); however, the maxima were
more transient and by 45 h pi the tumor concentration had
fallen to -0.43 j.Lg g<1 tissue. Levels of ZnPc (20) in liver
and muscle were reported to be similar whether delivered by
DPPC liposomes or LDL. Incorporation of 15% mole cholesterol in the Iiposomes resulted in a maximal uptake of
0,42 V-g g-I tissue at 24-48 h pi with a corresponding tumor/muscle ratio of 5.
Other research groups have studied ZnPc (20) distribution
in different animals, bearing a variety of tumors. Shopova et
al. (17) used male golden hamsters with 7,12-dimethylbenz(a)-anthracene-induced tumors and compared these with
a similar population bearing transplanted tumors derived from
the original induced neoplasms. Injection (iv) of 0.2 mg kg-I
ZnPc (20) in DPPC Iiposomes gave a maximal concentration
of ~ US V-g g-I tissue for the induced tumor 24 h pi, while
the transplanted tumor accumulated -0.7 V-g g-I tissue but at
the later time of 72 h pi. Clearance of ZnPc (20) from the
induced tumor was essentially linear from the maximum and
had dropped to 0.9 V-g g-I tissue by 72 h pi. Tumor/muscle
ratios for the induced and transplanted tumors were 1.05 and
0.8, respectively, at 24 h pi, while by 72 h the corresponding
values were 1.29 and 0.97. Intratumoral distribution in vivo
of ZnPc (20) in DPPC liposomes has been reported (18) and,
although no absolute values are given. relative fluorescence
from tumor, vessels and normal tissue are supplied for female
WAGIRIJ rats with isogeneic mammary carcinoma. Twentyfour hours after iv injection of 0.14 mg kg-I body weight the
fluorescence intensity from tumor and vessels was almost
identical but is double that of normal tissue.
Perhaps the most comprehensive study of pharmacokinetics
and biodistribution of ZnPc (20) was published recently by
lsele et at. (19). In this report ZnPc (20) was delivered in a
liposomal formulation consisting of a mixture of I-palmitoyl2-0leoylphosphatidylcholine (POPC) and 1.2-dioleoylphosphatidylserine (OOPS) (9: I). this is the same formulation used
for the only clinical trials on humans conducted to date with
ZnPc (20). By altering the formulation procedure, different
degrees of aggregation of the ZnPc (20) within the liposomes
was induced and this was correlated with pharmacokinetic and
biodistribution parameters. Aggregation state was expressed
as percentage monomeric fraction (% MF) of ZnPe (20) and
four states were obtained: 28, 78, 88 and 100% MF. Female
BALB/e mice bearing Meth-A sarcoma were injected iv with
0.25 mg kg-I body weight of each aggregate fraction. Both
tumor uptake and plasma elimination curves had similar kinetic profiles; however. maximal tumor uptake increased linearly with increasing % MF (670 ng g-I tissue for 100% MF
vs 200 ng g-I for 28% MF, both at 48 h pi) and elimination
of ZnPc (20) from the liver was similarly affected by aggregation state with 28% MF hardly eliminated after 7 days,
while the 100% MF eliminated with a half life of 4-12 days.
Hydrophobic Pes with peripheral substituents have also
been studied. Cuomo et al. (20) injected female BALB/c
mice bearing MS-2 fibrosarcoma iv with 0.5 mg kg-I body
weight germanium(IV)-octabutoxy phthalocyanine (GePcOBus) (21) in DPPC liposomes. Maximal tumor uptake of
(21) M
Ge(OSi(C 2Hs h h
(22) M
Ge(OSi(CH 3 h (CH 2 h OOCCH 3 h
(23) M
Ge(OSi(n-C6Hl~J h
(24) M
Ge(OSi(n-C,oH21 )J h
0.42 V-g g-1 tissue was attained 12 and 48 h pi, this represented tumor/muscle ratios of 42 and 3.2. Rapid clearance
from the serum, liver and spleen was also reported. This was
attributed to the eight aJkoxy residues imparting some polar
Photochemistry and Photobiology, 1996, 64(3) 473
character on the hydrophobic Pc. In an extension of this work
a series of GePcOBug derivatives were synthesized in which
the triethylsiloxy axial ligands were exchanged for dimethyl(3-acetoxypropyl)-. trihexyl- and tridecyl-siloxy ligands
(22.23,24) (21). Using the same animals, tumor cell line and
liposomal formulation as for the original study 0.35 IJ.mol
kg-' body weight was injected iv. Maximal tumor sensitizer
levels were achieved 24 h pi, with the trihexyl and tridecyl
analogues giving the highest uptake (0.15 nmol g' tissue),
whereas the triethyl and dimethyl(3-acetoxypropyl) derivatives achieved 0.11 nmol g'" tissue and 0.09 nmol g~' tissue,
respectively. Converting previous results for ZnPc (20) in
DPPC liposomes to molarity allows comparisons to be made:
0.21 IJ.mol kg~' ZnPc (20) injected dose equated to a maximal
tumor uptake of 0.52 nmol g-I tissue at 24 h pi (15). Tumor/muscle ratios at 24 h pi were lower for the dimethyl(3acetoxypropyl) and triethyl GePc (22.21) (2.61 and 2.2) but
higher for trihexyl and tridecyl GePc (23,24) (5A and 6.26)
than ZnPc (20) (3.71). The four GePc (21-24) derivatives
were also delivered in Cremophor EL emulsions and this resulted in an increased tumor uptake by all sensitizers of between 3.3 (tridecyl) and 5.6 (trimethYH3-acetoxypropyl])
times. Tumor/muscle ratios were also increased between 1.57
(trihexyl) and 3.35 (triethyl) times.
Rousseau et ai. (22) used radiolabeled [67Galchlorogallium
tetraoctadecyloxyphthalocyanine (25) to determine the biodistribution of this compound in male C3H mice bearing RIF
tumors at an ip injected does of 8 mg kg-I. The maximal tumor
concentration, attained at 24 h pi, was 1.05% of the injected
dose
tissue. and this corresponded to a tumor/muscle ratio
of 0.73. Sulfonation of this compound and delivery iv in saline
doubled the tumor maximum to 2.16% and increased the tumor/muscle ratio by almost an order of magnitude to 7.2.
Germanium phthalocyanine. with two axially ligated cholesterol moieties (26), was encapsulated in liposomes composed of a 9: I mixture of POPC and OOPS (the same formulation used for ZnPc (20) clinical trials and in the study
of ZnPc (20) aggregation by Isele et al. (19» and studied
by Segal\a et al. (23). Two doses, 0.76 and 1.52 mg kg-I
body weight, were injected iv in female BALB/c mice bearing MS-2 fibrosarcoma and biodistribution was measured.
For the lower dose maximal tumor concentrations were attained at 3 h pi (~0.9 IJ.g g~1 tissue); however, levels were
similar at 24 h pi (0.74 IJ.g g-' tissue), for the higher dose
maximal tumor drug levels were measured at 24 h pi (1.87
IJ.g g~' tissue). Although doubling the drug dose resulted in
a 2.5 times increase in tumor concentration at 24 h the corresponding tumor/muscle ratios (4.35 for the lower dose and
5.67 for the higher) increased by a factor of only 1.3. The
authors also report low concentrations of sensitizer in the
brain and skin and rapid clearance from the liver and spleen.
Two reports have been published on the potential use of
zinc hexadecafluorophthalocyanine (ZnPcF I6 ) (27) delivered
to male BALB/c bearing EMT -6 mammary tumors. The
compound was formulated in Cremophor (24) and polyethyleneglycol (PEG)-coated poly(iactic acid) nanoparticles (25).
In the first study ZnPcF'6 (27) and ZnPc (20) were labeled
with radioactive MZn and comparative biodistribution data
were obtained. The maximal tumor concentration obtained
for [65ZnjZnF,6 (27), at 48 h pi, represented 11% of the
injected dose gl tissue (% ID g~I). compared to 8% ID g-'
for [65ZnlZnPc (20) at 24 h pi. The rates of clearance from
(27)
blood were high, resulting in a tumorlblood ratio of 14 at
72 h pi, tumor/muscle and tumorlskin ratios at this time poim
were 12 and 5, respectively, suggesting that this sensitizer
has better characteristics for activation at longer intervals
after injection than ZnPc (20). The second study with this
compound. using PEG-coated nanopanicles as the delivery
vehicle, indicated a similar distribution to the original Cremophor study, with maximal tumor uptake at 48 h (20% ID
g-I tissue) and favorable tumor/muscle ratio at 72 h pi
(11.03); however, direct comparison between the two studies
was difficult due to the use of extraction/fluorescence measurements in the nanoparticle work.
Naphthalocyanines (Nc) have been proposed as PDT sensitizers due, mainly, to their intense absorption in the 750800 nm region of the spectrum. The Nc are similar to Pc;
however, the four additional benzenoid rings make these
compounds even more hydrophobic. Biodistribution of
bis(diisobutyloctadecylsiloxy)silicon-2,3-naphthalocyanine
(isoBosinc) (28) in both female BALB/c mice with MS-2
R
(28)
(29)
(30)
(31)
(32)
M
M
M
M
M
(33) M
R
Si(OSi(i-C 4 Hgh n-C,aHJ7h, R
In, R - H
H
In, R- NHCOCHJ
In, R= NH2
In, R = OCH J
Si(OSi(CH J )2 n-C s Huh, R=H
fibrosarcoma (26) and male Fischer 344 CrlBR rats with
AY-27 FANFf-induced urothelial tumors (27) have been reported. The mouse study used iv-injected doses of 2 and 0.5
474 Ross W. Boyle and David Dolphin
mg kg-I body weight in DPPC liposomes and maximal tumor concentrations of 1.2 and 0.34 f.1g g-I tissue were reported at 24 h pi. The corresponding tumor/muscle ratios
were 6.32 and 17, respectively. Serum clearance was rapid
with 90% eliminated at 24 h pi and skin accumulation with
the lower dose never exceeded 0.03 f.1g g-I tissue, equating
to a tumor/skin ratio at 24 h pi of 17. In the rat model
accumulation in the tumor at 24 h pi was ~0.61 f.1g g-I tissue
for a dose of 0.25 mg kg-I body weight injected iv in 10%
Tween. This was the maximum achieved and represented a
tumor/muscle ratio of 8.7. Serum clearance was again rapid
(85% at 24 h) but skin levels were high at 24 h pi (0.42 f.1g
g-I tissue) and the tumor/skin ratio (1.45) was correspondingly low, compared with liposomally formulated material.
The isoBosinc was also the subject of a study involving the
highly pigmented B 16 melanoma neoplasm grown on female
C57BLl6 mice (28). Two doses (0.5 and 1.0 mg kg-I) were
injected iv in DPPC liposomes and maximal tumor levels of
0.35 f.1g g-I tissue at 24 h pi for the 0.5 mg kg-I dose and
-0.57 f.1g g-I tissue at 24-48 h pi for the 1.0 mg kg-I dose
were found. Corresponding tumor/skin ratios, which, in this
case, represented the tumor/peri tumoral tissue ratio more
commonly expressed by tumor/muscle, were -1.1 for both
injected doses at the time of maximal tumor accumulation.
Serum clearance was rapid, with isoBosinc levels falling by
95% (1.0 mg kg-lID) and 90% (0.5 mg kg-lID) between
I and 24 h pi. Liver uptake was high throughout the experiment, -10-50 times tumor levels.
WhorIe et al. (29) and Shopova et al. (30) determined
biodistributionlpharmacokinetic parameters for zinc naphthalocyanine (ZnNc) (29) and three tetrasubstituted derivatives (30-32) in DPPC liposomes. In golden hamsters bearing DMBA-induced rhabdomyosarcoma 0.7 f.1g g-I tissue
accumulated in the tumor 24 h after injection ip of 0.15 mg
kg-I body weight ZnNe (29), and corresponding tumor/muscle and tumor/liver ratios were 1.4 and I. I, respectively. No serum or skin data was reported. In the second
study, biodistribution of ZnNc (29) was compared with tetra(methylamido)(ZnNcMA4 ) (30), tetraamino (ZnNcA4) (31)
and tetramethoxy (ZnNcM 4 ) (32) derivatives in male
C57/BL mice with implanted Lewis lung carcinomas. Intraperitoneal injection of 0.25 mg kg-I body weight of each of
the sensitizers gave the following tumor maximal concentrations: ZnNc (29): -1.5 f.1g g-I tissue at 16 h pi, ZnNcMA4
(30): -1.15 f.1g g-I tissue at 16 h pi, ZnNcA4 (31): -0.9 f.1g
g-I tissue at II h pi and ZnNcM4 (32): -1.2 f.1g g-I at 16
h pi. Tumor/liver and tumor/skin data were provided for 20
h pi and were similar for all the sensitizers tested with tumor/liver ratios varying from -1.2 (ZnNcMA 4) (30) to -1.6
(ZnNcA 4 ) (31) and tumor/skin ratios from -3.0 (ZnNc) (29)
to 4.0 (ZnMA4) (30).
In a wide-ranging study of the effects of varying the axial
ligands on silicon naphthalocyanine (SiNc) Brasseur et al.
(31) studied PDT effects of several compounds; however,
only the bis(dimethylhexylsiloxy) analogue (33) was examined with respect to biodistribution. Injection of I mg kg-I
drug in Cremophor emulsion iv in male BALB/c mice bearing EMT-6 mammary tumors gave maximal tumor concentrations of 0.9 f.1g g-I tissue at 12 h pi and a tumor/muscle
ratio of 10 at this time. Tumor/skin ratios were relatively
low, 1.6 compared with 17 for isoBosinc (28), at this point,
and the tumor/plasma ratio at the same time (0.16) was also
poor.
Several other hydrophobic PDT sensitizers have been the
subject of biodistributionlpharmacokinetic studies. Tetra-npropylporphycene (34), a structural isomer of tetra-n-pro-
(34)
pylporphyrin, has been incorporated in DPPC liposomes and
injected into female BALB/c mice with MS-2 fibrosarcoma
at a dose of 2 mg kg-I (32). Resulting maximal tumor levels
of 1.5 f.1g g-I tissue were found at 24 h pi. This corresponded
to tumor/muscle, tumor/skin and tumor/liver ratios of 16.67,
3.41 and 0.14, respectively.
Tetraphenylporphyrins conjugated to a carotenoid moiety
(35) and the corresponding TPP without the carotenoid (36)
R,
R,
(35)
(36) R,= OCH 3 • R2 =
o
NA.
H
were injected iv as Cremophor emulsions in female BALB/c
mice with MS-2 fibrosarcoma at a dose of 4.2 f.1mol kg-I
body weight (33). Twenty hours pi tumor accumulation of
the TPP (36) was -10 f.1mol kg-I vs -9 f.1mol kg-I for the
carotenoid-TPP (35). These were the maximum values attained and corresponded to tumor/muscle ratios of 94 and
27, while tumor/skin ratios were 29 and 7, respectively.
Clearance of the TPP-carotenoid (35) from serum was reported to be rapid, a feature common to most hydrophobic
sensitizers and associated with sequestration by components
Photochemistry and Photobiology, 1996, 64(3) 475
of the reticuloendothelial system; however, no comparison
was made with the nonconjugated TPP analogue (36).
An unusual hydrophobic sensitizer comprising one atropisomer of a "picket fence" porphyrin, 3,I-meso-tetrakis(opropionamidophenyl)porphyrin (3,1-Tpro) (37) has recently
(37)
been radiolabeled with 14C and studied in female Fischer rats
bearing R3230AC mammary adenocarcinoma (34). A dose
of 33.75 mg kg-I body weight was administered ip in Cremophor emulsion. Tumor levels of sensitizer were low at 4,
24 and 48 h pi, 2.7, 1.4 and 0.5 fLg g-I tissue, respectively,
compared to most other normal tissue. The liver and spleen
initially took up large amounts of drug (23 and 52 fLg g-I
tissue) and the skin. in contrast to all other tissue. continued
to accumulate drug throughout the course of the experiment,
rising from 3 fLg g 1 tissue at 4 h pi to 12 fLg g-I tissue at
48 h pi. Tumor/muscle and tumor/skin ratios never exceeded
I. The authors believed, however, that some in vivo metabolism of the radiolabeled sensitizer may have taken place.
Analysis of biodistribution data for hydrophobic PDT sensitizers (Table I) suggests that the maximal concentration in
the tumor is affected by the nature of the delivery vehicle,
which expresses its effect by altering the relative transfer to
different serum components. Not surprisingly, a concentration dependency can also be seen. however, in cases where
both delivery vehicle and concentration are constant, but
sensitizer structure is altered, a clear difference can be seen
in tumor concentrations. It is probable that this is a reflection
of the aggregation state of the drug within the lipid environment of the delivery vehicle.
HYDROPHILIC SENSITIZERS
Hydrophilic sensitizers are typified by ionic substituents.
which lend the compounds their "water-loving" character.
In the case of PDT drugs these ionic groups are most often
anionic in nature, and the most frequently encountered of
these are sulfonic and carboxylic acids. The Pcs once again
provide a major part of the available data. due to the ready
availability of sulfonated analogues. long known in the dye
and pigment industry. The most widely studied of these are
the sulfonated aluminum phthalocyanines (AIPcS n) and the
tetra- and trisulfonates (AIPcS4 and AIPcS,) (6,7). which fall
under our classification of hydrophilic sensitizers. Aluminum. being trivalent, bears one axial ligand when chelated
by Pc. which. depending upon the method of preparation, is
usually a chloride of hydroxide moiety.
Intravenous injection of 9.1 fLmol kg-I body weight
AIPcS 4 (6) and AIPcS, (7) into BALB/c mice bearing Colo
26 tumors (35) gave maximal tumor concentrations of II
nmol g-I tissue at 24 h pi for AIPcS4 (6) and 7 nmol g-I
tissue at 3-50 h pi for AlPcS, (7). These values were much
higher than for the mono- and disulfonated AIPc (AIPcS I
and AIPcS2) (9,8), which gave concentrations of < I and 5
nmol g-I tissue, respectively. Tumor/muscle and tumor/skin
ratios were 28 and 2 for AIPcS4 (6) at 24 h pi and 14 and
2.8 for AIPcS, (7) at the same time point. Comparing these
values with the corresponding ratios for AIPcS I (9) and
AIPcS2 (8) the monosulfonate levels were so low that they
could not be accurately measured. and for the disulfonate
the tumor/muscle ratio was 10 while the tumor/skin ratio was
-5. The liver and spleen concentrated much lesser amounts
of the drug with the hydrophilic AIPcS4 and S, (6,7) (I-IO
nmol g-I tissue) vs the amphiphilic AlPeS 1 and S2 (9,8)
(- 100-200 nmol g-I tissue). Initial plasma levels, however,
reversed this trend with AIPcS4 (6) and S, (7), much higher
than AlPcSI (9) and S2 (8). By 48 h pi serum levels for all
the compounds had fallen to below I nmol mL -I.
Peng et al. (36) studied biodistribution of AlPcS 4 (6) in
female BALB/c nu/nu mice bearing the LOX human melanoma. Twenty mg kg-I body weight of AIPcS 4 (6) was injected ip and a maximal tumor concentration of 21 fLg g- 1
tissue was found -40 min after injection. This concentration
was, however. transient and by 24 h it had fallen to 3.2 fLg
g -1 tissue. Tumor/muscle ratio rose from < 1 at 30 min pi to
a maximum of 10 at 15 h pi. Tumor/skin ratios showed less
variability. increasing from 0.8 at 30 min pi to 1.1 at 4 h.
Both tumor/muscle and tumor/skin ratios decreased rapidly
after attaining the maxima and fell to 3 at 120 h pi for the
former and 0.3 for the latter at 75 h pi. Surprisingly, the tumor/skin ratio rose again to 0.8 at 120 h. To allow comparisons with the previous results for AIPcS4 (6) to be made. the
injected dose here equated to 20 fLmOI kg-I body weight and
the tumor concentration at 24 h pi was 3.3 nmol g-I tissue.
Intratumoral distributions of AIPcS n have been the subject
of several studies; however, little quantitative data exist with
regard to this topic. Peng et al. (37-39) used female BALB/c
nu/nu athyrnic nude mice implanted with LOX human malignant melanoma and laser scanning fluorescence microscopy to
study AlPcS n distribution. In the first two of these reports
(37,38) a relatively simple distribution amongst the compartments of the tumor was found, in which AIPcS4 and S, (6.7)
localized extracellularly and were. presumably. weakly bound
as the fluorescence decreased from 4 to 48 h pi. This was in
contrast with AIPcSI (9) and S2 (8), which localized intracellularly and gave increasing fluorescence over this time period.
At 120 h pi significant amounts of fluorescence were detected
with AlPcSI (9) and S2 (8) while no fluorescence was detected
for AIPcS 4 (6) and S, (7). The same authors (39) extended this
work for AIPcS4 (6) and found strong fluorescence in specific
extracellular compartments. i.e. the subcutaneous fatty tissue
surrounding the tumor, stroma and blood vessels. Once again
no fluorescence was detected 72-120 h pi.
Sulfonated aluminum and ZnPc were compared with respect to localization in tumor, tumor neovasculature and subcutis in female W AGIRIJ rats bearing transplanted isogeneic
mammary carCinomas (40). Although no absolute concentrations were reported. relative measures of fluorescence were
provided. The compound AIPcS 4 (6) achieved a tu-
476 Ross W. Boyle and David Dolphin
Table I.
Selected biodistribution data for hydrophobic photosensitizers
Sensitizer
(animal)
ZnPc
ZnPc
ZnPc
ZnPc
(20)(mouse)
(20)(mouse)
(20)(mouse)
(20)(mouse)
ZnPc (20)(hamster)
ZnPc (20)(100% MF)(mouse)
ZnPc (20)(28% MF)(mouse)
GePc(OBu)s-Et (21)(mouse)
Delivery vehicle
(route)
DPPC Iiposomes (ip)
DPPC 1iposomes (iv)
LDL (iv)
DPPC liposomes + 15%
mole cholesterol (iv)
DPPC liposomes (iv)
POPC/OOPS liposomes
(iv)
POPC/OOPS liposomes
(iv)
DPPC lipsomes (iv)
Injected
dose
(mg kg-I)
0.25
1.05
0.97
N/A
19
0.25
0.2 (24)
N/A
19
0.5
0.35
0.35
0.35
0.35
0.35
0.35
0.35
0.35
0.76
1.52
0.87
0.87
0.42 (24)
0.42 (48)
0.14(24)
0.18 (24)
0.26 (24)
0.31 (24)
0.76 (24)
0.8 (24)
1.19(24)
1.05 (24)
0.9 (3)
1.87 (24)
2.2 (48)
4.01 (48)
42
3.2
2.61
2.2
5.4
6.26
5.68
7.37
8.48
11.81
9.0
5.7
13.0
11.0
20
20
21
21
21
21
21
21
21
21
23
0.34 (24)
1.2 (24)
0.61 (24)
0.35 (24)
0.57 (24--48)
0.7 (24)
1.5 (16)
1.15(16)
0.9 (II)
1.2 (\6)
0.9 (12)
17.0
6.32
8.7
1.I
l.l
1.4
N/A
N/A
N/A
N/A
10.0
26
29
30
30
30
30
31
1.5 (24)
9.0 (24)
2.7 (4)
\6.7
27
0.6
32
33
34
Tween 80 (iv)
DPPC Iiposomes (iv)
211Nc (29)(hamster)
ZnNe (29)(mouse)
ZnNe-M~ (30)(mouse)
ZnNc_A4 (31)(mouse)
ZnNc-M4 (32)(mouse)
SiNe
Bis(dimethylhexylsiloxy) (33)(mouse)
Tetra-n-propylporphyeene (34 )(mouse)
TPP-carotenoid (35)(mouse)
3,1-TPro (37)(rat)
DPPC Iiposomes (ip)
DPPC Iiposomes (ip)
DPPC liposomes (ip)
DPPC Iiposomes (ip)
DPPC Iiposomes (ip)
Cremophor EL (iv)
DPPC Iiposomes (iv)
Cremophor EL (i v)
Cremophor EL (ip)
2.0
5.2
33.75
M = Gael, R1 = R2 = H. R,3= R4 = So,3 H
M= 55Fe, R1= R2 = R,3= R4 = SO,3H
17
LIS (24) Induced tumor
0.7 (72) Transplanted tumor
0.67 (24)
IsoBosine (28)(rat)
IsoBosine (28)(mouse)
M=Zn. R1= R2= R3= R4= S03 H
M=GaCI. R 1= H, R2= R,3= R4= S03 H
14
15
15
16
0.2
0.5
2.0
0.25
0.5
1.0
0.15
0.25
0.25
0.25
0.25
1.0
(38)
(39)
(40)
(41)
7.5
3.71
5.71
5.0
0.6
0.3
0.55
0.42
IsoBosinc (28)(mouse)
ZnPcF I6 (27)(mouse)
ZnPcF I6 (27)(mouse)
(24)
(24)
(24)
(24)
Reference
0.5
0.12
0.12
0.12
DPPC liposomes (iv)
DPPC liposomes (iv)
DPPC Iiposomes (iv)
DPPC liposomes (iv)
Cremophor EL (iv)
Cremophor EL (iv)
Cremophor EL (iv)
Cremophor EL (iv)
POPC/OOPS
Liposomes (iv)
Cremophor EL (iv)
PEG-Coated polylactic
acid nanoparticles (iv)
DPPC liposomes (iv)
GePc(OBu)s-OAc (22)(mouse)
GePc(OBu)g-Et (21)(mouse)
GePc(OBukhex (23)(mouse)
GePc(OBu)g-dec (24)(mouse)
GePc(OBu)g-OAc (22)(mouse)
GePc(OBu)g-Et (21)(mouse)
GePc(OBu)g-hex (23)(mouse)
GePc(OBu)g-dec (24)(mouse)
GePc-cho1esterol (26)(mouse)
Maximal tumor uptake
(J.Lg g-I tissue)
(time attained pi in hours)
Tumor/
peritumoral
tissue
ratio
24
25
27
28
mor/normal tissue ratio of >3 by 15 min pi and this was
maintained to 6 h pi. The compound ZnPcS4 (38) exhibited
a similar pattern with the tumor/normal tissue ratio exceeding 2 from 5 min to 7 h pi.
Purified GaPeS) (39) radiolabeled with 14C was compared
with GaPCS2 (40) in C3H mice inoculated with RIF tumors
(41). Nine mg kg-I body weight of each compound in phosphate-buffered saline (PBS) was administered by iv injection
and resulted in maximal tumor concentrations of 11.2% ID
at 48 h pi for the trisulfonate and 7.64% ID for the disulfonate with corresponding tumor/muscle ratios of 15.3 and
6.9, and tumor/skin ratios of 4.7 and 2.8.
Eckhauser et al. (42) used radiolabeled 55Fe(H) phthalocyanine tetrasulfonate (55FePcS4) (41) to study biodistribution in male AJ mice with implanted Ta,Ha mouse mammary carcinoma. Two doses, 5 mg kg~ 1 and 10 mg kg I.
were injected ip and resulted in maximal tumor uptake be-
Photochemistry and Photobiology, 1996, 64(3) 477
tween 24 and 48 h pi in both cases. Tumor/muscle ratios of
5.1 and 6 were found for the two doses 24 h pi, while tumor/skin ratios peaked later at 5 days pi with values of 7.6
and 4.
Biodistribution of TPP sulfonated to differing degrees has
been studied. Kessel et al. (43) reported concentrations between 30 and 50 fLg g-l tissue 48 h pi, regardless of sulfonation level, in Panc 02 pancreatic tumors implanted in female C57BU6 mice injected with 10 mg kg-I body weight
porphyrin. Fluorescence microscopic investigation of the tumors revealed a similar intratumoral distribution to that
found with the sulfonated phthalocyanines, with the tetraand trisulfonated TPP (2,42) located in the stroma, while the
mono- and disulfonates (5,3.4) were within the neoplastic
cells. These results were later confirmed by Peng et al. (37)
who detected TPPS 1 (5) and TPPS 2• (4) in LOX malignant
melonoma cells of female BALB/c nu/nu athymic mice,
while TPPS 20 (3), TPPS 3 (42) and TPPS 4 (2) were visualized
declined by 40% from the maximal value. Gomer and Ferario (45) circumvented the need for artificially high injected
doses of MACE (1) by radiolabeling the compound with 14C.
Intravenous injection of 5 mg kg- 1 in female C3H1HeJ mice
bearing BA mammary carcinoma gave peak concentrations
in the tumor of 3.84 ILg g-I tissue at 2 min pi; however,
levels of - 3 ILg g-I tissue were maintained to 8 h pi resulting
in tumor/muscle and tumor/skin ratios of 10.6 and 2.2, respectively. Plasma levels fell from 42.74 fLg g-I tissue at 2
min pi to 0.64 ILg g-l at 8 h pi. A comparative study of iv
vs ip administration indicated that, while at 4 h pi iv injection resulted in 25% higher tumor concentrations. by 24 h
pi no difference could be detected. In a subsequent report
Ferrario et ai. (46) studied MACE (1) with 14C incorporated
into the pyrrolic rings of the sensitizer vs 14C in the aspartyl
residue and found similar biodistribution and pharmacokinetics, indicating that MACE (1) was not significantly metabolized during the course of the experiment.
Chlorin e6 (43) and pheophorbide a (53) chelated with
M
2H,
= OH
Rl = H, R 2 = OH
(45) M
2H,
R,= CH 3 ,
(43) M =48 V, R, = H, R 2
(44)
(42)
only in the extracellular space. The more hydrophilic sensitizers also cleared more quickly from the tumor and could
no longer be detected by 120 h pi.
Mono-L-aspartyl chlorin e6 (1) is a derivative of chlorin
e6 in which an L-aspartic acid residue has been attached via
an amide bond to the propylcarboxy group on the o-pyrrolic
ring of the chlorin. The effect of the modification, in terms
of hydrophilicity, is to increase this parameter by adding an
additional ionizable carboxyl residue. Being water-soluble,
MACE (1) falls under the definition of a hydrophilic sensitizer for the purposes of this review; however, it should be
appreciated that MACE (1) also has considerable amphiphilic character by virtue of the "clustering" of the four
carboxyl groups on one side of the otherwise hydrophobic
chlorin molecule.
Preliminary work on biodistribution of MACE (1) was
conducted by Aizawa et al. (44) using female BALB/c mice
bearing m-KSA mouse kidney sarcoma. As this study was
conducted using fluorescence, and consequently a high iv
injected dose of 200 mg kg I was used. only relative values
were obtained. Some selected values were, at 3 h pi: tumorlliver. 1.7; tumor/skin, 1.0; after 12 h pi fluorescence
values from skin and muscle were 40% and 30% relative to
the sarcoma. By 72 h pi little fluorescence was detected in
normal tissue; however, fluorescence in the tumor had only
= NH(CH 2 ) 2NH2
R2
positron-emitting [48V]vanadyl chloride have been injected
iv in male C3HIHe mice implanted with FM3A mammary
carcinoma (47). Tumor/muscle and tumor/skin ratios were
higher, at 24 h pi. for the chlorin (l2.{)6 and 6.68% ID g-l
tissue) compared with the pheophorbide (8.89 and 5.66% ID
g-l tissue); however, maximal tumor concentrations of
chlorin were attained 6 h pi (4.87% ID) with corresponding
tumor/muscle and tumor/skin ratios of 9.19 and 5.66. No
time-dependent data were reported for the pheophorbide.
A comparison of chlorin e 6 (44) with an ethylene diamine
(EDA) (45) derivative in which the EDA moiety was attached by an amide linkage to the carboxyl group on the
pyrroltc C ring of the chiorin has been made by Gurinivich
et al. (48). Ten mg kg-I body weight of each chlodn was
injected ip in PBS into Af mice bearing transplanted methyIcholanthrene-induced sarcomas. For the chlorin e6 the
maximal tumor uptake (\.5 ILg g-I tissue) occurred at 6 h
pi, compared with the EDA derivative, which attained a
maximal tumor concentration of 40 ILg g-I tissue but at the
later time of 18 h pi. This disparity in accumulated dose and
kinetics of uptake translated into a large difference in tumor/muscle ratios at the time of maximal tumor uptake with
this parameter for chlorin e6 being - 3 compared to 20 for
chlorin e6 EDA. A comprehensive study of biodistribution
478 Ross W. Boyle and David Dolphin
and photodynamic effects of chlorin e6 has been published
recently (49) in which Wi star rats bearing sarcoma M-I and
sarcoma 45 tumors were injected ip with 10 mg kg~ I body
weight of dye. Maximal tumor concentrations were attained
at different times pi for the different tumors with sarcoma
M-I accumulating 4.4 /-Lg g-I tissue at 18 h pi, while the
sarcoma 45 gave a peak value of 3.6 /-Lg g-I tissue at 12 h
pi. Tumor/muscle ratios were 8.8 for the M-I and 6 for the
sarcoma 45 at these times. Concentrations of dye in the
blood exceeded those in all internal organs, muscular tissues
and neoplastic tissues at all time points to 72 h pi.
A series of porphyrin dimers linked by aliphatic carbon
chains of varying lengths (46) were studied by Kessel et al.
OH
o
ip and iv to CBA mice implanted with C 6 glioma intracerebrally. Uptake and release of drug from the neoplasm was
quite rapid for the lower dose, with peak concentrations of
17.5 /-Lg g-I tissue at 24 h pi for ip injection and 40 /-Lg g - I
tissue at the same pi for iv injection. The higher dose gave
similar uptake kinetics but slower release of drug from the
tumor. Peak concentrations in this case were 90 /-Lg g-I tissue
and -100 /-Lg g~1 tissue for ip and iv routes, respectively, at
24 h pi. High tumor/normal brain ratios were obtained with
this compound, the maximum being 402 at 48 h after iv
injection of 100 mg kg-I body weight; however, at 24 h pi,
when the tumor concentrations were maximal, these values
were: 157 (ip; 10 mg kg-I), 136 (iv; 10 mg kg-I), 203 (ip;
100 mg kg-I) and 238 (iv; 100 mg kg-I).
A series of three hematoporphyrin analogues with side
chain modifications such that the porphyrin bore four carboxyl, four amino or two carboxyl and two amino groups
(48-50) were synthesized and investigated (52). The highest
R,
(46)
(50). Four /-Lmol kg~1 body weight of each of the dimers was
injected iv, in isotonic aqueous saline solution, into C3H1HeJ
mice bearing RIF tumors. At 4 hand 24 h pi no significant
differences were detected in tumor concentrations for dimers
with 3, 4 and 5 carbon chains; however, both 6 and 13 carbon chain dimers gave significantly higher tumor concentrations at both time points. Plasma levels at 4 h pi, however,
showed a steady increase in going from 3 carbons to 13
carbons. This trend was present, but less accentuated, for the
same parameter measured at 24 h pi.
In an innovative approach to combining photodynamic
and neutron capture therapies Hill et ai. (51) investigated the
biodistribution of a tetrakis carboranecarboxylate ester of
2,4-his(a,j3-dihydroxyethyl)deuteroporphyrin IX (47). Doses
R
O={
o
OH
(47)
OH
R=C2 B,aH, 1
of 10 and 100 mg kg I body weight were administered both
(48) R, = CH(CH 3 )SCH 2 COOH,
= OH
R, = CH(CH 3 )S(CH2 h N
R2
(49)
~
0
'--I'
R2 = OH
'
= CH(CH )S(CH h Nb
= NH(CH 2 )2 N(CH 3 )2
(50) R,
R2
3
2
'--I' '
concentrations found, in POVD small cell lung carcinoma
grown on male athymic Swiss mice injected iv with 20 mg
kg-I body weight of each porphyrin, were 5 /-Lg g~1 tissue
at 6 h pi for the tetracarboxylate and the same value, but at
24 h pi, for the amphoteric compound. The tumor concentration for these two drugs exceeded that for the tetraamine
by a factor of > 3 at all time points. Tumor/muscle ratios for
these compounds were 5.3 at 6 h pi for the tetracarboxylate;
however, this rose to 16.7 at 24 h pi, 8.3 at 24 h pi for the
amphoteric porphyrin and for the tetraamine a maximal value of 1.1 was attained at 6 h pi. Tumorlblood ratios increased
throughout the course of the experiment and by 24 h pi these
values were 12.5 for the tetracarboxylate and 0.94 for the
tetraamine, while for the amphoteric drug sensitizer levels
in the blood were below measurable levels. These results are
in contrast with a previous study on these compounds by
Woodburn et al. (53) who found that the tetraamine had the
most favorable localization characteristics (8.23 IJ.g g~ I tissue at 24 h pi; tumor/peritumoral tissue ratio: 41.2) in female
CBA mice bearing C 6 glioma.
The cationic sensitizer meso-tetra(4N-methylpyridyl)
Photochemistry and Photobiology, 1996, 64(3} 479
porphyrin (18) were studied by Villanueva and Jori (54). A
dose of 2.1 mg kg-I body weight was administered iv to
female BALB/c mice bearing MS-2 fibrosarcoma. A peak
tumor concentration of 1.19 /kg g~ I tissue was attained at 24
h pi, and at this time no drug could be detected in the blood
or skin. The liver and spleen were. however, major reservoirs for this sensitizer with levels of 2.64 and 1.29 /kg g-I
tissue at 24 h pi.
In an example of a water-soluble photosensitizer without
ionic groups. tritiated benzoporphyrin derivative (BPD) was
conjugated with polyvinyl alcohol (PVA) (51) at a ratio of
o
cation of the chlorin e6 structure by esterification of the
meso-carboxyl group and amidation of the D ring propionic
acid group with ethylene diamine increased the maximal tumor concentration by a factor of 27. Another amphoteric
sensitizer also gave the higher tumor/peritumoral tissue ratio
when compared to fully cationic or anionic sensitizers derived from the same porphyrin. Finally, of the two tetracationic drugs studied, the formally quarternized compound.
i.e. one bearing four positive charges independent of pH,
concentrated in tumor tissue at a much higher concentration
compared with a porphyrin analogue bearing four tertiary
amino residues, which were free to establish a pH-dependent
equilibrium between charged and neutral species.
AMPHIPHILIC PHOTOSENSITIZERS
(51) R
Polyvinyl alcohol
(56) R
H
25 (BPD) : 1 (PV A) and injected iv in PBS into male DBAI2
mice bearing P815 mastocytoma (55). Administration of a
dose equivalent to 0.5 mg kg I body weight BPD resulted
in a tumor/muscle ratio of 4 at 3 h pi and this had risen
slightly to 4.5 by 24 h pi. Corresponding results for nonconjugated, and therefore water-insoluble. BPD injected in Iiposomes were 2.75 and 3.0. The most striking differences
were seen in the tumorlbrain ratios, which were 3.75 and
3.0 at 3 and 24 h pi for liposomal BPD, while for the PVAconjugated BPD the corresponding values were 7.25 and 9.5.
These large differences were attributed to altered bloodlbrain
barrier kinetics.
Comparison of biodistribution data for the hydrophilic
photosensitizers is. generally, more easy than for their hydrophobic counterparts as one of the variables, delivery vehicle, has been eliminated. However, as in all cases differences in tumor morphology, vascularization and host tissue
must be taken into account, although a rigorous treatment of
this subject could constitute a review in itself, it is outside
the scope of this work. Differences in tumor type notwithstanding, some general structure/tumor accumulation trends
can be seen. Amongst the tetraanionic sensitizers the highest
% ID in the tumor was found for AlPcS4 (6), while a tetracarboxyl porphyrin gave a maximal tumor concentration approximately half that of AIPcS4 (6), but at twice the injected
dose. The compound MACE (1), however, which is also a
tetracarboxylated compound. gave tumor concentrations
only slightly lower than AIPcS4 (6), albeit at 2 min ip, thus
highlighting the importance of regioisomerization in biodistribution. Trianionic compounds again revealed a sulfonated
AIPc, AIPcS) (7) to be the most efficient, with tumor concentrations approximately four times that for the tricarboxyl
chlorin eo (44) at similar injected doses. However, modifi-
Amphiphilic photosensitizers are generally recognized to be
compounds that have present in their structure both a hydrophilic and a hydrophobic region. In the case of the porphyrin-based PDT agents this often results from an asymmetric
distribution of charge groups around the periphery of the
macrocycle. The region of the porphyrin most distant from
the charged groups then acts as the hydrophobic region. This
class of sensitizer is, arguably. the most important of the
three drug types described in this review, as it includes the
most potent of PDT agents, with respect to "direct" activity
on neoplastic cells, and also two of the compounds on which
most clinical data have been amassed, meta-tetrahydroxyphenylchlorin (m-THPC) (52) and benzoporphyrin derivative
(52)
monoacid ring A (verteporfin. BPD-MA) (56).
Once again the sulfonated Pc were the focus of much
of the original work relating to amphiphilic sensitizers;
however, as most of these data have been covered in the
context of comparisons with their hydrophilic higher sulfonated analogues, only a brief summary will be included
here. The lower sulfonated Pc (PcS I and PCS2) generally
give lower maximal tumor concentrations, when compared to PcS} and PCS4 (see Tables 2 and 3); however,
intratumoral distribution studies suggest that the more hydrophilic sulfonates localize in the extracellular space and
associate with connective tissue in the tumor, while the
amphiphilic dyes penetrate and accumulate within the
neoplastic cells themselves. Localization within the various compartments of the tumor is thought to be influenced
by the relative association of differently sulfonated Pc
with serum components and the amount of free sensitizer
480 Ross W. Boyle and David Dolphin
Table 2.
Selected biodistribution data for hydrophilic photosensitizers
Delivery
vehicle
(route)
Sensitizer
(animal)
AIPcS4 (6)(mouse)
AIPcS3 (7)(mouse)
AIPcS 4 (6)(mouse)
55Fe(II)PcS4 (41)(mouse)
PBS
PBS
PBS
PBS
(iv)
(iv)
(ip)
(ip)
MACE (l)(mouse)
Chlorin e6 (44)(mouse)
Chlorin e6-EDA (4S)(mouse)
Chlorin e6 (44)(rat)
PBS
PBS
PBS
PBS
(iv)
(ip)
(ip)
(ip)
Tetrakiscarborane
Deuteroporphyrin (47)(mouse)
PBS (iv)
PBS (ip)
Hp(COOH)4 (48)(mouse)
Hp(COOH)iaminoh (49)(mouse)
Hp(amino)4 (SO)(mouse)
Meso-tetra(4N-methylpyridyl) porphine
(18)(mouse)
PBS
PBS
PBS
PBS
(iv)
(iv)
(iv)
(iv)
Injected
dose
(mg kg-I)
Maximal tumor uptake
(fLg g-I tissue)
(time attained pi in hours)
9.0
8.0
20
5
10
5
10
10
10
10.8 (24)
6.2 (24)
(0.5)
21
0.4 (24)
1.0 (24)
3.84 (2 min)
1.5 (6)
(18)
40
4.4 (18) M 1 sarcoma
3.6 (12) sarcoma 45
(24)
40
(24)
100
17.5 (24)
(24)
90
(6)
5
(24)
5
(2)
-1
1.2 (24)
10
100
10
100
20
20
20
2.1
dissociated from these components within the extracellular space (56).
Pheophorbide a (phe-a) (53) and chemically modified derivatives have been the subject of several biodistribution
studies. Nishiwaki et al. (57) compared methods of delivery
for this compound by injecting in either PBS or a lipoidal
solution. Male New Zealand white rabbits inoculated with
YX-2 liver tumors were injected iv or directly into the hepatic artery (ia) with 1.0 mg kg-I body weight Phe-a. After
24 h ia delivery of the lipoidal solution gave a tumor concentration of -85 I-lg g-I tissue and the same delivery route
with PBS solution gave -129 I-lg g-I tissue. Intravenous
OH
(53) R, = CH=CH2. R2 = COOH
(54) R,
R2
= CH(CH3)O(n-C6 H'3).
=H
injection, however, resulted in only -5 I-lg g-I tissue. Tumor/liver ratios were 19 (lipoidal; ia), 20 (PBS; ia) and 0.3
(PBS; iv), while levels in skin and blood were below detection limits for ia lipoidal administration, 0.75 and 0.41 I-lg
g-I tissue for ia PBS and 1.89 I-lg g-I tissue for skin with iv
PBS solution, but blood levels were below detection limits.
Iwai et al. (58) chelated Phe-a (53) with positron emitting
48y and investigated its biodistribution in C3H/He mice im-
Tumor/peritumoral
tissue ratio
Reference
28
14
< 1.0
5.1
6.0
2.6
3.0
20
8.8
6.0
136
238
157
203
5.3
8.3
-1.0
Peri tumoral below
detection limit
35
35
36
42
45
48
48
49
51
52
52
52
54
planted with FM3A mammary carcinoma or MH 134 heptoma. Male ddY mice with transplanted S 180 sarcoma were
also used in the study. Injected doses were based on radioactivity, rather than directly on the sensitizer concentration.
so all data were reported as % ID g-I tissue. However,
controls with inorganic 48YOCI 2 indicated no demetallation
took place in vivo. Interestingly all three tumor types exhibited very similar % ID g-I tissue values at 24 h pi and
these were all peak levels, suggesting common pharmacokinetics. Tumor/muscle and tumor/skin ratios for the FM3A
carcinoma were 8.9 and 5.7 at 24 h pi. A parallel study
with 14C-Iabeled Phe-a (53) gave similar biodistribution results, confirming that tumor localization was controlled by
the nature of the macrocyclic ligand, rather than the metal.
Pheophorbide a (53) has also been studied in Lewis rats
with azaserine-induced acinar pancreatic tumors (59). Injection of 3 mg kg-I body weight drug in ethanollPBS (1:
I vol/vol) iv led to a maximal tumor concentration of 0.98
I-lg g-I tissue I h after injection; however. the maximal
tumor/pancreas ratio of 7.8 was attained at 48 h pi and
tumor concentration at this time was 0.39 I-lg g-I tissue.
Skin levels of drug were very low 48 h pi (0.04 I-lg g-I),
while blood and muscle showed somewhat higher levels of
0.26 and 0.47 I-lg g-I tissue. The chlorin 2-[I-hexyloxyethYll-2-devinyl pyropheophorbide a (54), a chemically
modified derivative of Phe-a (53) has been labeled with 14C
and studied in female C3H mice bearing RIFI tumors and
female DBAl2 mice bearing SMT-F tumors (60). One mg
kg-I body weight drug formulated in 0.1 % Tweenl2% ethanol/5% dextrose in water was injected iv and resulted in
a maximum concentration in the tumor of 2.5 I-lg g-I tissue
7 h pi and a corresponding tumor/skin ratio of 5.6. Drug
uptake in SMT-F tumors was measured by fluorescence;
however. it was only reported that relative fluorescence in
this tissue increased steadily to 24 h pi.
Bacteriochlorin a (Bch-a) (55), a derivative of the photo-
Photochemistry and Photobiology, 1996, 64(3) 481
Table 3.
Selected biodistribution data for amphiphilic photosensitizers
Sensitizer
(animal)
AIPeS2 (8)(mouse)
AIPeS , (9)(mouse)
Pheophorbide a (53)(rabbit)
Pheophorbide a (53)(rabbit)
Pheophorbide a (53)(rabbit)
Pheophorbide a (53)(rat)
2-[ I-Hexyloxyethyl]-2-devinyl
pyropheophorbide a (54)(mouse)
Ketochlorin (57)
Ketoehlorin (57)
m-THPC «52)(mouse)
BPD-MA (56)(mouse)
BPD-MA (56)(mouse)
BPD-MA (56)(mouse)
Delivery
vehicle
(route)
Injected
dose
(mg kg I)
PBS (iv)
40% EtOHIPBS (iv)
PBS (ia)
PBS (iv)
Lipoidol (ia)
EtOHIPBS (I:I)(iv)
0.1 % Tween 80/2%
EtOW5% dextrose in
water (iv)
Tween 80IPBS (iv)
Cremophor EL (iv)
EtOHIPEG 400/water
(2:3:5)(ip)
10% DMSOIPBS (iv)
DMPC/egg PG liposomes
(iv)
6% DMSOIPBS (iv)
6.8
6.0
1.0
1.0
1.0
3.0
1.0
5.0
5.0
0.3
Maximal tumor uptake
(f.l.g g-I tissue)
(time attained pi in hours)
2.6
-0.5
129
-5
-85
0.98
2.5
(24)
(24)
(24)
(24)
(24)
(I)
-17
synthetic pigment bacteriochlorophyll a, has been labeled
with radioactive 99mtechnetium and studied in male Syrian
golden hamsters implanted with Greene melanoma (6\). Intravenous injection of 20 mg kg- ' body weight in dimethylsulfoxide (DMSO)IPBS (25:75) resulted in a peak tumor
concentration of 0.8% of radioactivity g-I wet tissue at 2 h
pi, tumor/skin and tumorlblood ratios at this time point were
1.6 and 0.3, respectively. However, maximal values for these
parameters were found at later time points, 1.67 for tumor/skin at 3 h pi and 0.92 for tumorlblood at 24 h. No data
could be recorded after 24 h pi due to the short half life of
99mTc (tl/2 = 6 h). Relative intratumoral distribution of Bch-a
in isogeneic RMA mammary tumors grown on female
W AGIRJJ rats has been reported by van Leengoed et al.
(62). A dose of 20 mg kg-I body weight was injected ip as
a Cremophor EL aqueous emulsion and time-dependent distribution was followed by fluorescence. Tumor/normal tissue
ratios rose from 1.9 at 5 min pi to a maximum of 2.5 at 1530 min pi and then decreased steadily to a value of 0.5 at
24 h pi. The tumorlblood vessel ratios were maximal 5 min
pi (2.5) and also declined throughout the course of the experiment, reaching a value of 0.8 at 24 h pi.
Woodburn et al. (63) synthesized a ketochlorin with propionic acid groups on rings C and D and octyl chains on
rings A and B (57). Injection of female C3H mice bearing
N/A
4.2
4.0
1.3
63
63
67
-2.8
1.6
72
76
3.8
76
20
0.3
18
1.72
(7)
3.5 (24)
6.7 (24)
57.5 (36)
4.0
4.0
2.94 (3)
2.0 (0.25)
4.0
1.5 (3)
Referenee
35
35
57
57
57
59
60
N/A
OH
(55)
Tumorl
peri tumoral
tissue ratio
OH
(57)
RIF tumors iv with 5 mg kg-I body weight drug in either
Tween 80 or Cremophor EL gave maximal tumor concentrations of 6.69 j.1g g-I tissue for Cremophor and 3.5 j.1g g-I
for Tween, both at 24 h pi. The corresponding tumor/muscle
and tumor/skin ratios were 4.0 and 3.0 for Cremophor and
4.2 and 2.4 for Tween. Plasma concentrations were greater
than those in the tumor at both 3 and 24 h pi.
The last two photosensitizers covered in this section are,
perhaps, the most important drugs currently being studied.
Both are in advanced clinical trials on human patients and,
therefore, one of these compounds will most likely be the
first second-generation photosensitizer to be brought before
a regulatory board for approval. The first of these drugs, mTHPC (52) is a totally synthetic analogue of meso-tetraphenylporphyrin, one of the simplest "model" porphyrins.
The m- THPC (52) is a single substance but due to rotation
around the porphyrin/phenyl bond four atropisomers are possible. However, at in vivo temperatures (-37°C), rotation is
fast enough to prevent different spatial orientations of the
four hydroxy groups from being recognized by biological
components. This freedom of movement of peripheral hydroxy groups has been demonstrated to have important implications with respect to in vivo activity for tetrahydroxyphthalocyanines (64). Preliminary results of relative values
482 Ross W. Boyle and David Dolphin
for tumor, skin and muscle uptake in human patients were
reported by Braichotte et al. (65). Patients were injected iv
with 0.3 mg kg-I body weight, three patients had malignant
mesothelioma, while a fourth had a malignant fibrous histiocytoma. Comparing patients with the same cancer, fluorescence in the tumor increased steadily from 24 h pi to 72 h
pi, as did the tumor/muscle ratios (1.5 at 24 h pi and 6 at
72 h pi); however, the tumor/skin ratio of the patient from
whom the 72 h biopsy was extracted was low (-0.9) compared with biopsies taken at 24 (1.5) and 61 (3.25) h pi. The
patient with the histiocytoma showed a maximal tumor uptake of more than twice that of the other three patients at 72
h pi and also very high tumor/muscle and tumor/skin ratios
of 14.3 and 10.8 respectively. It should be stressed that these
results were obtained from individual patients for each time
point and therefore no statistical analysis of the data was
possible. Another study of pharmacokinetics in human patients was conducted a year later (66). Two patients, one
with invasive epidermoid carcinoma on the tongue and the
other with a microinvasive carcinoma on the palatoglossal
arch were injected iv with a dose of 0.15 mg kg- I body
weight and fluorescence of m-THPC (52) was measured,
noninvasively, in tumor, skin and normal tissue as a function
of time. Maximal tumor fluorescence was obtained between
25 and 50 h pi for the patient with the epidermoid carcinoma, while skin and normal tissue maxima occurred at 4090 h pi and 50 h pi respectively. Tumor/normal tissue ratios
increased rapidly over the first 3 h, reaching values of 14,
then fell to values of 5 and 3 at 24 and 48 h pi. Results for
the second patient showed similar kinetics, with maximal
tumor fluorescence at 30 h pi and normal tissue at 45 h pi.
Maximal tumor/normal tissue ratios were lower at 6 (1-6 h
pi) and this had fallen to 2.5 by 45 h pi. More conventional
biodistribution studies have also been performed in mice
(67) and rats (68). In the first of these reports male nude
BALB/c mice bearing human malignant mesothelioma xenographs were injected ip with 0.3 mg kg I body weight m·
THPC (52) in ethanollPEG 400/water (2:3:5). At 36 h pi a
maximal concentration in the tumor of 57.7 ng g-I tissue
was found. Uptake kinetics were similar for both tumor and
normal tissue; however, the drug persisted for longer in the
normal tissue such that, while maximal tumor/normal tissue
ratio (1.3) coincided with the highest concentration in the
tumor, by 72 h pi this had fallen below I. The authors be·
lieve this effect may be due to drug dilution on tumor-cell
doubling. Laser-induced fluorescence was used to study
biodistribution in male WistarlFurth rats bearing colon adenocarcinoma. A dose of 1.3 mg kg-I body weight m-THPC
(52) was injected iv in the same formulation medium as used
above for mice. At 24 hand 48 pi relative measurements of
drug concentrations in various tissues were made. Maximal
tumor/muscle ratio was 9 at 24 h pi and this had fallen to 7
by 48 h pi; however. while ratios were still high at 48 h the
tumor fluorescence had fallen to -37% and -50% of the 24
h values for tumor surface and tumor interior, respectively.
Fluorescence signals from the liver, lung and spleen dropped
more rapidly, compared with the tumor, such that only 7~
14% of the 24 h levels were detected at 48 h pi. The drop
in liver values, which had been relatively high (-63% of
tumor surface and 130% of tumor interior) at 24 h pi, was
attributed to metabolism of the drug in this organ. No skin
values were reported in this study. Recently Morlet el aJ.
(69) injected male Swiss nude/nude mice bearing human co10rectal adenocarcinoma ip with increasing doses (0.05-1.6
mg kg- I body weight) of m-THPC (52) in ethanollPEG
400/water (2:3:5) and biodistribution was studied by fluorescence. Maximal tumor fluorescence was obtained 72 h
after injection of 1.6 mg kg-I; however, tumor/muscle and
tumor/skin ratios decreased between 24 and 72 h from 15 to
2.1 for the former and 15 to 1.1 for the latter, indicating that
tumor selectivity decreased with time after injection. In contrast with these results Whelpton et al. (70) reported that
tumor/muscle ratio increased from 24 h pi (3.5) to 72 h pi
(5.0) when female BALB/c mice bearing Colo 26 colorectal
carcinomas were injected iv with 0.51 mg kg- I m-THPC.
The authors report high tumor/intestine and tumorlliver ratios of 8.6 and 6.9, respectively, at 96 h pi. while a tumor/muscle ratio of 4.6 was maintained at this time. Absolute tumor concentrations remained between 1 and 2 JLg g-I
tissue from 20 to 96 h pi. Discrepancies between these two
sets of results may be due to the use of chromatographic vs
spectrof!uorometric detection methods.
The final photosensitizer to be covered in this review.
BPD-MA (verteporfin) (56). is a chlorin with extended conjugation formed by an electrocyclic Diets Alder reaction on
protoporphyrin IX dimethyl ester. followed by base-induced
isomerization of an isolated double bond to bring it into
conjugation and hydrolysis of one methoxycarbonyl group
(71). The BPD-MA (56) is currently in phase II clinical trials.
Richter et at. (72) synthesized tritiated BPD·MA (56) for
the purpose of preliminary biodistribution studies on female
DBAl2J mice bearing P815 mastocytoma. Intravenous injection of 4 mg kg-I body weight in DMSOIPBS (I: 10) gave
a maximal tumor concentration of 2.94 f,Lg g-I tissue at 3 h
pi; however, while tumor concentrations fell over the following 46 h to l.0 f,Lg g-I tissue, tumor/muscle ratios remained
essentially the same over this period (-2.8). Tumor/skin ratios fell from 3 h pi (2.57) to 24 h pi 0.64), but then rose
again at 48 h pi (1.95). Blood levels of BPD·MA (56) fell
steadily although the maximal tumorlblood ratio (l,45) was
attained at 24 h pi. Comparing these results with I4C-Iabeled
BPD-MA (56) (5 mg kg-I) administered in the same delivery
vehicle to the same strain of mice, but bearing M I rhabdomyosarcoma (73), peak tumor concentration was again
found 3 h pi (2.7% ID g-I tissue); however, tumor/muscle
ratios at this time (l.08) were quite different but by 8 h pi
(2.71) were similar to those found with tritiated BPD-MA
(56). Tumor/skin ratios also rose from 3 h pi (1.68) to 8 h
pi (3.17). Biodistribution results for two BPD-MA analogues, BPD·MA ring B (BPD·MB) and BPD diacid (BPDDA), have been reported and compared with BPD-MA in
male DBAl2 mice bearing MI rhabdomyosarcoma (74). All
BPD analogues were injected iv in 10% DMSO in PBS at a
dose of 3.5 mg kg- l body weight and no significant differences were found between them at 24 h pi. These data are
interesting in view of the fact that BPD-MA is consistently
a much more effective PDT agent, compared with BPD-MB
and BPD-DA, and once again highlights the complexity of
the in vivo situation. In an even mote in·depth study Richter
et al. (75) examined biodistribution and photosensitizing efficiency of two regioisomers of BPD·MA (56), one with the
Photochemistry and Photobiology, 1996, 64(3) 483
free propionic acid residue located on the pyrrolic C ring
(A I), and the other in which this group is attached on the D
ring (A2). Similar mice, tumor type and injection protocols
were used as for the previous study; however, detection of
A I and A2 regioisomers was achieved by chromatographic
analysis. Tumor concentrations of both regioisomers remained essentially equivalent up to 3 h pi; however, a selective clearance from the blood was detected with ~3.6
times more A I present at 3 h pi. Initially the authors thought
the effect was mediated by specific esterases, but subsequent
plasma studies indicated that this was unlikely. Finally a
comparative study of BPD-MA (56) delivered in unilamellar
liposomes (dimyristoyl phosphatidyl choline/egg phosphatidyl glycerol; <200 nm average particle size) or 6%
DMSOIPBS was conducted (76). Male BALB/c mice bearing MI rhabdomyosarcoma were injected iv with 4 mg kg-I
body weight of 14C-labeled BPD-MA (56) in each delivery
vehicle. Peak concentrations in the tumor were obtained at
15 min pi (2 J..Lg g- I tissue) for Iiposomal BPD-MA (56) but
at 3 h pi (1.5 J..Lg g-I tissue) for DMSOIPBS administered
material. Tumor/tissue ratios showed both formulations attained maximal tumor/muscle ratios at 24 h pi, 19.2 for Iiposomal and 12.6 for DMSOIPBS, while tumor/skin ratios
peaked at 3 h pi, 4.3 for liposomal and 5.9 for DMSOIPBS.
Significant accumulations of BPD-MA (56) in the liver and
gall bladder were also reported, but no absolute data were
given.
Comparisons between biodistribution and pharmacokinetic data for different amphiphilic photosensitizers are, once
again, complicated by differences in delivery vehicles and,
as can be seen from Table 3, this problem is worse for amphiphilic than for hydrophobic compounds. In fact, in this
section no two data sets utilize the same delivery protocol
in the same animal! This disparity between delivery vehicles,
animals and tumor types is a consistent limitation in analysis
of results with PDT drugs. It is, therefore, of considerable
importance to establish common screening protocols, both
in vitro and in vivo. for new PDT agents, as has been established by the National Cancer Institute for screening conventional cancer chemotherapeutics (77). Only in this way
will it be possible to extrapolate results to predict ideal structures for future photodynamic sensitizers.
Acknowledgement-This work was supported by the Natural Sciences and Engineering Research Council of Canada.
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