In vivo biological behavior of a water-miscible fullerene: 14C labeling, absorption, distribution, excretion and acute toxicity.

Abstract

Water-soluble fullerenes have recently been shown to exhibit considerable in vitro biological activity including cytotoxicity, site-selective DNA cleavage and inhibition of HIV protease. To assess the potential of these compounds as drugs, studies on the in vivo behavior of fullerenes are needed. We therefore set out to synthesize a radiolabeled, water-soluble fullerene, in order to obtain data on the oral absorption, distribution and excretion of this class of compounds.
We synthesized a 14C-labeled water-soluble [60]fullerene using dipolar trimethylenemethane, which undergoes cycloaddition to [60]fullerene. When administered orally to rats, this compound was not efficiently absorbed and was excreted primarily in the feces. When injected intravenously, however, it was distributed rapidly to various tissues, and most of the material was retained in the body after one week. The compound was also able to penetrate the blood-brain barrier. Acute toxicity of the water-miscible fullerene was found to be quite low.
Although the water-soluble fullerenes (and possibly their simple metabolites) are not acutely toxic, they are retained in the body for long periods, raising concerns about chronic toxic effects. The fact that fullerenes distribute rapidly to many tissues suggests that they may eventually be useful to deliver highly polar drugs through membranes to a target tissue, however, and they may even have applications in the delivery of drugs to the brain. Recent advances in fullerene synthetic chemistry may also make it possible to control fullerene absorption/excretion profiles in the future.

Full text

Page 1

In viva biological
14C labeling, absorption,
behavior of a water-miscible
distribution,
and acute toxicity
fullerene:
excretion
Shigeru Yamago lt*, Hidetoshi Tokuyama’, Eiichi Nakamuralr+*, Koichi
Kikuchi**, Shinji Kananishl ‘*, Keisuke Sueki*, Hiromichi
Shuichi Enomoto3 and Fumitoshi Ambe
Nakahara*,
1 Department of Chemistry, Tokyo Institute of Technology, Meguro, Tokyo 152, Japan, 2Department of Chemistry,
Tokyo Metropolitan University, Hachioji, Tokyo 192-03, Japan and 3The Institute of Physical and
Chemical Research (RIKEN), Wako, Saitama 351-01, Japan
Background:
been shown
activity including
age and inhibition
tial of these compounds
behavior of fullerenes
synthesize
order to obtain
and excretion
Results: We synthesized
[60]fuIIerene
undergoes cycloaddition
tered orally
absorbed and was excreted primarily
injected intravenously,
Water-soluble
to exhibit
cytotoxicity,
of HIV protease. To assess the poten-
as drugs, studies on the in oiuo
are needed. We therefore
a radiolabeled, water-soluble
data on the oral absorption,
of this class of compounds.
a 14C-labeled
using dipolar trimethylenemethane,
to [60]fXIerene.When
to rats, this compound
fullerenes have recently
in vitro biological
site-selective
considerable
DNA cleav-
set out to
fuIIerene,
distribution
in
water-soluble
which
adrninis-
was not efficiently
in the feces. When
however, it was distributed rapidly
to various tissues, and most of the material was retained in
the body after one week.The
penetrate the blood-brain barrier. Acute toxicity
water-miscible lllerene was found to be quite low.
Conclusions: Although the water-soluble
possibly their simple metabolites)
they are retained in the body for long periods, raising con-
terns about chronic toxic effects.The
distribute rapidly to many tissues suggests that they may
eventually be useful to deliver highly polar drugs through
membranes to a target tissue, however, and they may even
have applications in the delivery
Recent advances in fnllerene synthetic chemistry may also
make it possible to control fnllerene
profiles in the future.
compound was also able to
of the
fuIIerenes (and
are not acutely toxic,
fact that fullerenes
of drugs to the brain.
absorption/excretion
Chemistry & Biology June 1995, 2:385-389
lipophilicity, Keywords: cycloaddition, [60lfullerene, metabolism, trimethylenemethane
Introduction
Despite
since its discovery,
realistic
demonstration
example,
able in vitro biological
DNA cleavage
recently showed
used to cleave single-
cific sites [4], further
fullerenes
studies. Current
fullerenes
mation for
biology, and eventually
systems for diagnosis
first data, to our knowledge,
tribution and
These studies
compound la
intense interest
fullerene
of scient:ific
that
in the fullerene
biology
research
water-soluble
1 and 2 in Fig. 1) show
activity, including
and anti-viral
a C&,-DNA
and double-stranded
demonstrating
be useful in biochemical
studies on the
are expected to provide
the further
to bring
and therapy.
molecule ever
has only
since
fullerenes
become
the
a
target recent
(for
compounds consider-
cytotoxicity,
[2,3].
could
DNA
tailor-made
and biological
[l]
that
activity
conjugate
We
be
at spe-
that
could
in viva behavior
fundamental
development
forth new drug
Here,
on the oral absorption,
of a water-miscible
the synthesis
(see Fig. I).
of
infor-
of fullerene
delivery
we report the
dis-
excretion
required
fullerene.
of the radioactive
The
administered
was absorbed,
When
labeled
then distributed
that, despite
not become
as the amine
and ornithine).
90 % was still retained
the chronic
an important
fullerene la was not
(p.0.).
effectively
Nonetheless,
and excreted
(i.v.),
rapidly
other
reactivity
bound to reactive
of proteins
Excretion was slow, however,
in the body
toxicity of water-miscible
focus of future
absorbed
a small
into
most
to the liver
tissues. Thus
[5], the fullerene
substances,
(for example,
when
amount
the urine.
of the radio-
and was
it seems
orally
metabolized
intravenously
compound
injected
moved
to various
its chemical
quickly
residues
does
such
lysine
and over
after one week.Thus,
fullerenes will be
work.
Results and discussion
We first investigated
soluble fullerene
mice (ddY female,
(i.p.) injection
Tween 80LSaline
Although the
the acute
2. The
18-20
(200-500
containing
mice showed
toxicity
were
of the water-
performed
intraperitoneal
doses, in
dimethylsulfoxide).
symptoms of discomfort
studies
g) by a single
mg kg-l
on
0.2 %
*Corresponding
Tokyo
Kyoto 606-01,
authors.
*Present
Japan.
+Present address:
address:
Department
Department
of Chemistry,
of Synthetic
School of Science,
and Biological
The University
Chemistry,
of Tokyo, Hongo,
Kyoto University, 113, Japan. Chemistry
0 Current Biology Ltd ISSN 1074-5521
385

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386 Chemistry & Biology 1995, Vol 2 No 6
14CH2
3 4
5
6
d
1:‘T
la: 14C
0 0
Y
OH
Fig. 1.
fullerene
Materials
Synthesis
la.
and
of the 14C-labeled
used, For
methods.
conditions see
after dosing, such as writhing
trunk, and some weight loss (5-10 % weight loss after a
day at a dose of 500 mg kg-’ of compound
survived for a period of one week.As the approximate
lethal dose must thus be more than 500 mg kg-’ i.p.,
and given similar data for the parent
fullerenes may be regarded as compounds
tively weak acute toxicity
manipulated with reasonable care. A full evaluation of
the toxicity of these compounds awaits further studies.
with stretching of the
2), all mice
C,,
with rela-
that they are
[6-81,
provided
To address basic issues of the pharmacokinetics
tribution of these compounds,
route to a labeled fullerene derivative was needed. Of
the two possible strategies
fullerenes, the simplest conceptually
the C,, core carbons with 14C. This route, however, is
difficult to perform practically
several-step synthesis of 14C-enriched
requires the handling of a large amount of radioactive
soot, from which the labeled C,; is separated. The
second route, involving attachment
fragment to the fullerene, may be far less hazardous,
more isotopically economic, and may also be applicable
to fullerenes other than C,,.
and dis-
viable a synthetically
for making
is the labeling of
labeled
[7,8]. It involves a
graphite, and
of a labeled carbon
Among the reported methods for fullerene functional-
ization, the trimethylenemethane
appeared to be ideal for rendering the second labeling
strategy practicaLThe synthesis of labeled compound la
was achieved in four synthetic steps (Fig. l).The readily
(TMM) route [9,10]
available cyclopropenone,
ated and allowed to react with 14CH,I (>97 % isotopic
purity). The methylated product, compound
merized with tert-BuOK to the methylenecyclopropane
5, and then allowed to react with C,, at 80 ‘C via the
trimethylenemethane 6.The cycloadduct 7 was purified
and esterified to obtain the labeled succinate la. The
mechanism of the TMM reaction, previously studied for
a 13C-labeled compound [12], ensures that the 14C label
is placed unambiguously at the position next to the C,,
core, where the probability of the premature biological
loss of the label is smallest.
compound 3 [ll], was lithi-
4, was iso-
Absorption of the water-soluble flerene
orally to male Fischer rats was found to be poor. Only
trace radioactivity was found in the liver and other tissues
after 3 and 6 h periods, and virtually all radioactivity
(97 % of the total) was excreted in the feces within 48 h
(Fig. 2). It is notable, however, that trace amounts of
fUerene derivatives were identified in the urine, indicat-
ing that a small amount of compound la did pass through
the gastrointestinal wall. In the O-12 h period, a small
amount of compound la itself was tentatively detected
(using HPLC) in the urine, and, in the 24-30 h period,
trace amounts of la and 7a were detected after acidifica-
tion of the urine. In the 24-30 h sample, we could not
initially detect any CH$&-soluble
the urine sample @H 8), but found, upon acidification to
pH 1 with HCl, that one third of the radioactivity did
move to the CH,Cl, extract.Two radioactive zones were
separated by HPLC (Buckyprep column, CH,Cl,).
la administered
radioactive material in
The

Page 3

Pharmacokinetics of a water-miscible fullerene Yamago et al.
387
When the dose was delivered intravenously (i.v.), com-
pound la was excreted extremely slowly (Fig. 2). After
160 h, only 5.4 % was eliminated into the feces and the
remainder stayed in the body, In both p.o. and i.v. experi-
ments, virtually all of the excretion occurred via the
feces, and little radioactivity was seen in the urine. The
strong retention of the i.v.-dosed fullerenes is consistent
with the high lipophilicity of the molecule. Compound
1 is unusually lipophilic for a carboxylic acid derivative,
as evidenced by its high log P,
coefficient in octanol/water
which is in the same range as that of phenanthrene
Pow = 4.5) and 1,2-diphenylethane
L
16 12
Time (h)
value (the partition
[13]) of 4.5 (free acid form),
(log
12
30 36 42 48 72
(log P,,, = 4.8).
18 d0 18 h 46
As the i.v.-administered
in the body, we examined the time-dependent
the concentration of the radioactivity in tissues in sacri-
ficed animals (Table 1). The dosed fullerene quickly
passed from the blood to the liver, leaving 1.6 % of the
fullerene la is strongly retained
change of
Fig.
intravenous
2. Fecal excretion
(red) dosing of compound
of radioactivity
1
after oral (blue) and
la.
: :
may have been present as the carboxylate salt at pH 8, in
which case the lack of extraction may simply reflect a lack
of solubility in the organic solvent used.
retention times of these compounds were consistent with
those of compounds 7a and la, as determined by com-
parison with the retention times of authentic samples (in a
7.5:1 ratio, giving retention
respectively).The fact that compounds la and 7a could
be extracted only at pH 1 suggested that, in the urine,
they existed as acid-labile metabolites, as is often observed
for lipophilic drugs. It is also possible that compound la
times of 10 and 18 min,
Table 1. Distribution of radioactivity in rats.
% total dosed radioactivity
(concentrations of radioactivity (pg equiv/g))
tissue lh 3h 6h
16h
30 h 160h
liver 72.9 f 13.7
(57.7 f 4.8)
84.7 i 10.4
(57.3 i 8.3)
86.7 f 20.2
(62.2 f 14.6)
91.7 f 8.0
(61.9 i 6.3)
80.1 f 13.4
(54.5 zt 7.6)
1.62 zrz 0.31
(0.29 f 0.03)
spleen 0.74 f 0.63
(11.5 f 2.2)
1.4 f 0.1
(10.6 i 0.7)
1.2 f 0.3
(8.7 f 2.1)
1.6 f 0.2
(11.4 f 2.4)
2.0 f 0.4
(12.4 * 1 .O)
0.05 f 0.00
(0.12 f 0.01)
lungs 1.4 1 0.3
(5.5 * 0.7)
5.0 f 3.3
(22.9 f 13.5)
1.4 f 0.3
(5.9 i 1.2)
1 .o f 0.3
(4.2 f 0.6)
3.2 zt 2.3
(12.7 f 9.2)
0.05 f 0.02
(0.06 i 0.02)
kidney 0.50 f 0.04
(1.3 10.1)
0.58 zt 0.15
(1.5 f 0.2)
0.99 f 0.18
(2.5 f 0.4)
1 .o f 0.3
(2.7 f 0.8)
3.2 f 0.1
(1.8 i 0.2)
0.41 f 0.14
(0.35 f 0.10)
heart 0.31 zt 0.25
(1.8 :t 1.3)
0.44 f 0.65
(3.7 i 5.7)
0.24 f 0.08
(1.5 f 0.5)
1.07 i 0.16
(5.8 f 1.3)
0.26 zt 0.13
(1.5 f 0.8)
0.01 f 0.00
(0.03 f 0.00)
brain 0.20 f 0.04
(0.30 i 0.06)
0.15 f 0.24
(0.24 zt 0.37)
0.84 f 0.70
(1.2 * 1 .O)
0.57 f 0.19
(0.85 f 0.24)
0.10 f 0.11
(0.14 zt 0.16)
0.00 f 0.00
(0.00 f 0.00)
testicles 0.03 * 0.00
(0.12 i 0.01)
0.04 f 0.02
(0.14 f 0.07)
0.06 f 0.01
(0.20 * 0.03)
0.09 f 0.01
(0.26 i 0.02)
0.08 zt 0.02
(0.21 f 0.05)
0.07 f 0.01
(0.05 f 0.00)
blood 1.6 f 0.6
(1.6 f 0.3)
1 .o f 0.2
(0.87 f 0.05)
0.50 * 0.12
(0.45 f 0.11)
0.57 i 0.27
(0.45 f 0.20)
0.50 f 0.08
(0.38 f 0.04)
0.00 f 0.00
(0.00 f 0.00)

Page 4

388
Chemistry & Biology 1995, Vol 2 No 6
total
after 6 h. In the liver, 73 % of the radioactivity
after 1 h, and the concentration
6-16 h. About 80 %
retained in the liver
was mostly eliminated
radioactivity in the blood after 1 h and only 0.5 %
was found
was maximal
radioactivity
after
was of the total
30 h, but the radiolabel
(1.6 %) after 160 h.
even after
The
kidney
of the
radioactivity
lipophilicity
fullerene
this large molecule
blood-brain
in the various
manner
organs listed
excretion
skeletal muscle
concentrations
were highest
total
of compound
after 30 h and accounted
radioactivity dosed
was also found
and the rather
la must be responsible
(molecular
barrier. From
organs decreased
and, after 160 h, the radioactivity
in Table 1 mostly
from the body,
and hair (data not shown).
la in the spleen and
for 2-3 %
Notably,
brain. The
structure
for the transportation
weight 995)
30 to 160 h, the radioactivity
in a time-dependent
initially.
the
the
high
of the
in
compact
of
through the
found in the
without disappeared
became and distributed to
Significance
Fullerenes
since their first synthesis ten years ago, partly for
the intrinsic interest of their unusual structure,
also because of increasing
be biologically active themselves,
as cages or carriers for other
molecules. To determine
ever be useful as drugs or drug delivery devices, it
is necessary to understand
and toxicity of these compounds.
have attracted considerable interest
but
evidence that they can
and can be used
biologically
whether fullerenes
active
will
the pharmacokinetics
Although
possibly their simple metabolites)
toxic, they are not free from
such as pain and weight loss. Our observation
the administered fullerene
for long periods may also raise new concerns
about chronic toxicity. From a more positive per-
spective, however, our data suggest that it may in
the future be possible to use fullerenes
the delivery of highly polar drugs attached
fullerene (such as a &,-oligo
capable of site-specific
through membranes to a target tissue. In addition,
these studies suggest that water-soluble
may enable the delivery
in the form of endohedral
offering new possibilities
Given the recent advances
chemistry [15-171, it should also soon be possible
to alter fullerene absorption/excretion
we have found that the fullerenes (and
are not acutely
undesirable effects
that
is retained in the body
to mediate
to the
DNA
cleavage)
conjugate
DNA [1,4]
derivatives
of metals into the brain
metallofullerenes
for imaging
in fullerene
[14],
agents.
synthetic
profiles.
Materials
Synthesis
The labeled
dure previously
and methods
of 74C-labeled
fullerene
reported
fullerene
synthesized
for an unlabeled
la
was according to the proce-
[l], as compound
summarized
using
acid la under
(silica
column,
(u) compound
methylphosphoric
-78 ‘C,
-78 ‘C
Et20/dimethylsulfoxide,
1,2-dichlorobenzene,
(1.0 mmol),
r.t., overnight.
to inadvertent
transformations
matography.
natural
411 % of a faster-moving
HPLC (Buckyprep
with 30 % i-PrOH
in Fig. 1. Product
samples
different
identification
stage
was
alcohol
achieved
‘*C-authentic at the
chromatographic
toluene)
and
mmol),
triamide
14CH,I
temperature
r.t.,
80 “C,
4-dimethylaminopiridine
Unoptimized
material loss
took place
The optimized
abundance.The
of both 7 and
(TLC two conditions
HPLC
were
(0.15
tetrahydrofuran,
0.21
(b) t-BuOK
(c) C,,
(d) succinic
(1.0 mmol),
0.4 %.The
at stage
(a) and
cleanly, as monitored
yield was 13 % for
radiolabeled
radioactive impurity
Nakalai tesque,
in CH,Cl,).
gel, 10 % EtOAc
CH,Cl,)).
in and (Buckyprep
as follows:
mmol),
Reagents
(0.15
conditions
n-BuLi
(0.30
(0.10
(r.t.);
1 h;
overnight;
3 hexa-
mmol),
mmol, 0.5 h, then
+ room
GBq/mmol),
(0.05
(0.10
anhydride
CH,Cl,,
yield
the remaining
by
a sample
sample
as analyzed
Japan,
mmol),
mmol),
yield low is due
chro-
of
isotopic contained
by
column, elution
Fecal excretion
compound
The excretion
(Fischer
la, dissolved
0.18 MBq
(9.6 kBq).
tillation
with 1N
excreted
the radioactivity
Excretion
tillation
(O-12 h period)
(taken
-0.2 % of the dosed
la and 7a were
24-30 h period,
tion was
against authentic samples (Buckyprep
of radioactivity after po. and i.v. dosing of
la
of compound
la was
case.The
studied for three male rats
rats; 85-95 g) in each 14C-labeled compound
(1:1:23,
or
a liquid
were
p.o. dosed
48 h, only
96 h in the
also measured using
p.o. dosing experiment,
excretion of compound
by extraction), which
radioactivity.Trace amounts
found in the acidified
as described in the text.
by comparison of
column, CH,Cl,).
in EtOH/PEG400/5
ml-‘),
The radioactivity
counter for
NaOH at 60 ‘C
97 % of the total
was
into the urine
counter.
urinary
up in CH,Cl,
% aqueous albumin
(18 l&q)
using
which
the
was administrated
was
samples
for
radioactivity
excreted
was
the
p.o. i.v.
measured
of the
2 days. While
scin-
feces, treated
rats
after 5.4 % of
after i.v. dosed
a liquid
rats.
scin-
initial
found
In an
la was
accounted
of compounds
sample
identifica-
retention
for
also urine of the
Compound
HPLC achieved times
Distribution
The distribution
with male rats (Fischer rats; 85-95
kidney, heart, brain,
fullerene
la (14 kBq)
ene glycol (50 pL) and 4 % albumin
by i.v. injection. The distribution
after 1,3,6,16,30
of compound la was administered).The
the blood was removed
tissues was homogenized
radioactivity was measured
of radioactivity
of the labeled
in rats
firllerene
g) for the liver, spleen, lungs,
and blood. A
of ethanol (50 p,L), polyethyl-
(115 pL) was administered
in each tissue was analyzed
and 160 h periods (In the 160 h run, 18 kBq
rats were dissected after
with a hypodermic
in 2N NaOH at 60 ‘C
using a liquid scintillation
la was investigated
testicles
in a mixture
solution of the
syringe. Each
for
counter.
of the
24 h.The
Acknowledgments:
Education,
thank Prof.
Dr. R. Ando
ments. Provision
AG is gratefully
This work was supported
(to S.Y., E.N. and K.K.). We
treatment of radioactivity,
discussions on the
of a [60]fullerene sample
acknowledged.
by the Ministry of
Science and Culture
M. Katada
for helpful
for the and
toxicity experi-
to E.N. by Hoescht
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Pharmacokinetics of a water-miscible fullerene Yamago et a/. 389
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revisions
April
received:
28th 1995; revisions
1995.
requested:
Accepted:
May 18th
May 30th
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1995. May 30th