Note: Descriptions are shown in the official language in which they were submitted.
CA 02354743 2001-08-07
MITOCHONDRIALLY TARGETED ANTIOXIDANTS
TECHNICAL FIELD
The invention relates to antioxidants having a lipophilic cationic group and
to uses of
these antioxidants, for example, as pharmaceuticals.
BACKGROUND OF THE INVENTION
Oxidative stress contributes to a number of human degenerative diseases
associated
with ageing, such as Parkinson's disease, and Alzheimer's disease, as well as
to
Huntington's Chorea, diabetes and Friedreich's Ataxia, and to non-specific
damage that
accumulates with aging. It also contributes to inflammation and ischaemic-
reperfusion
tissue injury in stroke and heart attack, and also during organ
transplantation and ),
surgery. To prevent the damage caused by oxidative stress a number of
antioxidant
therapies have been developed. However, most of these are not targeted within
cells
and are therefore less than optimally effective.
Mitochondria are intracellular organelles responsible for energy metabolism.
Consequently, mitochondria) defects are damaging, particularly to neural and
xrluscle
tissues which have high energy demands. They are also the major source of the
free
radicals and reactive oxygen species that cause oxidative stress inside most
cells.
Therefore, the applicants believe delivering antioxidants selectively to
mitochondria will
be more effective than using non-targeted antioxidants. Accordingly, it is
towards the
provision of antioxidants which may be targeted to mitochondria that the
present
invention is directed.
Lipophilic cations may be accumulated in the mitochondria) matrix because of
their
positive charge (Rottenberg, ( 1979) Methods Enzymol, 55, 547-560; Chen, (
1988) Annu
Rev Cell Biol 4, 155-181). Such ions are accumulated provided they are
sufficiently
lipophilic to screen the positive charge or delocalise it over a large surface
area, also
provided that there is no active efflux pathway and the canon is not
metabolised or
immediately toxic to a cell.
-1-
CA 02354743 2001-08-07
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Thiobutyl triphenylphosphonium bromide [TBTP] is disclosed in R J Burn and M P
Murphy, (1997), Archives ofBiochemi8stry and Biophysics, Vol. 339, No. 1,
March 1, pp
33-39 as a probe selectively accumulated by mitochondria in living cells that
binds
selectively to mitochondria) proteins. They also disclose that TBTP does have
some
antioxidant activity provided endogenous glutathione is depleted. RA) Smith et
al, Eur.
J. Biochem, 263, 709-716 ( 1999) "Selective targeting of an antioxidant to
mitochondria"
discloses the content of our Canadian Patent Specification No. 2311318.
US Patent 3532&67discloses triphenyl phosphonium halide compositions for use
as an
antioxidant in polyamides. (J Chem. Soc. Perkin Trans I 1984) pp 709-21
discloses a
certain triphenyl phosphoniurn bromide compound useful for the purpose of
synthesis
of an antioxidant. Tetrahedron Letters No. 23, pp 2145 - 2148 1979 discloses
certain
triphenylphosphonium bromides used in the synthesis of benzofurannes.
The focus of the invention is therefore on an approach by which it is possible
to use the
ability of mitochondria to concentrate compounds comprising triphenphosphonium
cations carbon linked to certain quinol antioxidant moieties which target to
the major
source of free radicals and reactive oxygen species causing oxidative stress
and which
compounds have a capability it is believed to anchor within mitochondria.
CA 02354743 2001-08-07
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SUMMARY OF THE INVENTION
In a first aspect the present invention is a mitochondrially-targeted
antioxidant
compound comprising or including a triphenlphosphonium cation linked by a C1
to C3o
carbon chain (optionally including none, one or more of each of or either
double or
triple bonds, and optionally including one or more subsdtuents) to an
antioxidant
moiety being
(1')m
or
OH
where m is an integer from 0 to 3, and Y is independently selected from
alkoxy,
thioalkyl, alkyl, haloalkyl, halo, amino, nitro and optionally substituted
aryl.
Preferably the compound also includes an anion (eg; that of Br).
Preferably the C1 to C3o carbon chain is an alkyl chain of the formula -(CH2)n-
where n is an integer of from 1 to 25 and preferably 2 to 25.
Preferably n is 5 or more.
Most preferably Y is independently selected from alkoxy and alkyl.
Preferably m is 2 or 3.
Preferably the compound is
OH
CH3~
Z
(CH2)n I'
CH30
OH
wherein Z is a suitable anionic species and n is ~an integer from 1 to 25.
CA 02354743 2001-08-07
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Most preferably n is 10 and the compound is
OH
CH3
CH O
3
In another aspect the invention is a method of therapy of prophylaxis of a
mammalian patient who would benefit from reduced oxidative stress which
comprises
or includes the step of administering to the patient a mitochondrially-
targeted
antioxidant of the present invention.
In still a further aspect the invention is a method of reducing oxidative
stress
in a cell which comprises or includes the step of administering to the cell a
mitochondrially targeted antioxidant of the present invention.
In yet another aspect the invention is a pharmaceutical antioxidant dosage
unit
or composition which includes a compound of the present invention.
DESCRIPTION OF DRAWINGS
In particular, a better understanding of the invention will be gained with
reference to
the accompanying drawings, in which:
Figure 1 is a graph which shows the uptake by isolated mitochondria of
compound 1,
a Vitamin E derivative (see Example 1) coupled by a carbon chain to a
triphenylphosphonium moiety, a mitochondrially-targeted antioxidant according
to our
Canadian Patent Specification No. 2311318 (Canadian NPE of PCT/NZ98/00173).
Figure 2 is a graph which shows the accumulation of compound 1 by isolated
mitochondria;
Figure 3 is a graph which shows a comparison of a compound 1 uptake with that
of the
triphenylphosphonium cation (TPMP);
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Figure 4 is a graph which shows that compound 1 protects mitochondria against
oxidative damage;
Figure 5 is a graph which compares compound 1 with vitamin E and the effect of
uncoupler and other lipophilic cations;
Figure 6 is a graph which shows that compound 1 protects mitochondria)
function from
oxidative damage;
Figure ? is a graph which shows the effect of compound 1 on mitochondria)
function;
Figure 8 is a graph which shows the uptake of compound 1 by cells;
Figure 9 is a graph which shows the energisation-sensitive uptake of compound
1 by
cells;
Figure 10 is a graph which shows the effect of compound 1 on cell viability;
Figure 11 shows the UV-absorption spectrum of [10-(6'-
ubiquinonyl)decyltriphenyl-
phosphonium bromide] (herein referred to as "mitoquinone") and of the reduced
form
of the compound (10-(6'-ubiquinolyl)decyltriphenylphosphoniurn bromide]
(herein
referred to as "mitoquinol");
Figures 12A to 12D show reactions of [ 10-(6'-
ubiquinonyl)decyltriphenylphosphonium
bromide) ("mitoquinone") and the reduced form of the compound ("mitoquinol")
with
mitochondria) membranes, both mitoquinone and mitoquinol being separately and
together a compound of the present invention;
Figure 13 shows reactions of mitoquinol and mitoquinone with pentane-extracted
mitochondria) membranes;
Figure 14 shows reduction of mitoquinone by intact mitochondria;
Figure 15 shows uptake of radiolabelled mitoquinol by energised rat liver
mitochondria
and its release on addition of the uncoupler FCCP;
Figure 16 shows the effect of mitoquinol on isolated rat liver mitochondria;
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Figure 1? shows TPMP accumulation from oral administration to mice;
Figure 18 shows compound 1 accumulation from oral administration to mice; and
Figure 19 shows uptake of mitoquinol by human osteosarcoma 143(3 cells.
DESCRIPTION OF THE INVENTION
As stated above, the focus of this invention is on the mitochondria) targeting
of
compounds, primarily for the purpose of therapy and/or prophylaxis to reduce
oxidative stress.
Mitochondria have a substantial membrane potential of up to 180 mV across
their
inner membrane (negative inside). Because of this potential, membrane
permeant,
lipophilic cations accumulate several-hundred fold within the mitochondria)
matrix.
The applicants have found that by covalently coupling lipophilic cations
(preferably the
lipophilic triphenylphosphonium cation) to an antioxidant the compound can be
delivered to the mitochondria) matrix within intact cells. The antioxidant is
then
targeted to a primary production site of free radicals and reactive oxygen
species within
the cell, rather than being randomly dispersed. See our Canadian Patent
Application
No. 2311318 (Canadian NPE of PCT/NZ98/00173).
While it is generally preferred that the carbon chain is an alkyl chain (eg -
(CI-i2)n-)
(preferably C1-C2o, more preferably C1-C1;), carbon chains which optionally
include
none, one or more of each of or both double or triple bonds are also within
the scope
of the invention. Also included are carbon chains which include one or more
substituents (such as hydroxyl, carboxylic acid or amide groups), and/or
include one
or more side chains or branches (for example, selected from unsubstituted or
substituted alkyl, alkenyl or alkynyl groups).
In some particularly preferred embodiments, the linking group is an ethylene,
propylene, butylene, pentylene or decylene group.
Preferred antioxidant compounds of the invention, can be readily prepared, for
example,
by the following reaction:
CA 02354743 2001-08-07
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I \
//~ B r / -
+P ~ ~ ~ Br
\ P \
The general synthesis strategy is to heat a halogenated precursor, preferably
a
brominated or iodinated precursor (RBr or RI) in an appropriate solvent with 2-
3
equivalents of triphenylphosphine under argon for several days. The
phosphonium
compound is then isolated as its bromide or iodide salt. To do this the
solvent is
removed, the product is then triturated repeatedly with diethyl ether until an
off white
solid remains. This is then dissolved in chloroform and precipitated with
diethyl ether
to remove the excess triphenylphasphine. This is repeated until the solid no
longer
dissolves in chloroform. At this point the product is recrystallised several
times from
methylene chloride/diethyl ether.
It will also be appreciated that the anion of the antioxidant compound thus
prepared,
which will be a halogen when this synthetic procedure is used, can readily be
exchanged with another pharmaceutically or pharmacologically acceptable anion,
if this
is desirable or necessary, using ion exchange chromatography or other
techniques
known in the art.
The same general procedure can be used to make a wide range of mitochondrially
targeted compounds with different antioxidant moieties R attached to the
triphenylphosphonium cation.
In some preferred embodiments of the invention, the antioxidant compound is a
quinol
derivative of the formula II defined above. A particularly preferred quinol
derivative of
the invention is the compound mitoquinol as defined above. Another preferred
compound of the invention is a compound of formula II in which (C)n is (CHZ)5,
and the
quinol moiety is the same as that of mitoquinol.
CA 02354743 2001-08-07
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Once prepared, the antioxidant compound of the invention, in any
pharmaceutically
appropriate form and optionally including pharmaceutically-acceptable carriers
or
additives, will be administered to the patient requiring therapy and/or
prophylaxis.
Once administered (eg; orally, parenterally or otherwise - preferably orally)
the
compound will target the mitochondria within the cell.
The invention will now be described in more detail with reference to the
following non-
limiting examples.
EXAMPLES
Example 1
Experimental
1. Synthesis of a mitochondrially-targeted vitamin-E derivative (Compound I)
The synthesis strategy for a mitochondrially-targeted vitamin-E derivative
(compound
1) is as follows. The brominated precursor (compound 2) 2-(2-bromoethyl)-3,4-
dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran was synthesized by
bromination of the corresponding alcohol as described by Grisar et al, ( 1995)
(J Nled
Chem 38, 2880-2886). The alcohol was synthesized by reduction of the
corresponding
carboxylic acid as described by Cohen et al., (1979) (J. AmerChem Soc 101,
6710-6716).
The carboxylic acid derivative was synthesized as described by Cohen et al., (
1982) (Syn
Commun 12, 57-65) from 2,6-dihydroxy-2,5,7,8-tetramethylchroman, synthesized
as
described by Scott et al., (1974) (J. Amer. Oil Chem. Soc. 101,6710-6716).
0
Br
25
Compound 1
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CH3
HC
CH3
Br
CH3
Compound 2
For the synthesis of compound 1, 1g of compound 2 was added to 8 ml butanone
containing 2.5 molar equivalents of triphenylphosphine and heated at
100°C in a sealed
Kimax tube under argon for 7-8 days. The solvent was removed under vacuum at
room temperature, the yellow oil triturated with diethyl ether until an off
white solid
remained. This was then dissolved in chloroform and precipitated with diethyl
ether.
This was repeated until the solid was insoluble in chloroform and it was then
recrystallised several times from methylene chloride/diethyl ether and dried
under
vacuum to give a white hygroscopic powder.
2. Mitochoadrial uptake of compound 1
To demonstrate that this targeting is effective for compounds our Canadian
Patent
Specification No. 2311318 and of the present invention, the exemplary vitamin
E
compound 1 was tested in relation to both isolated mitochondria and isolated
cells. To
do this a [3H]-version of compound 1 was synthesized using (3H]-
triphenylphosphine
and the mitochondria) accumulation of compound 1 quantitated by scintillation
counting (Fig. 1) (Burns et al., 1995, Arch Biochem Biophys 332,60-68; Burns
and
Murphy, 1997, Arch Biochem Biophys 339, 33-39). To do this rat liver
mitochondria
were incubated under conditions known to generate a mitochondria) membrane
potential of about 180 mV (Burns et al., 1995; Burns and Murphy, 1997). Under
these
conditions compound 1 was rapidly (< 10 s) taken up into mitochondria with an
accumulation ratio of about 6,000. This accumulation of compound 1 into
mitochondria was blocked by addition of the uncoupler FCCP (carbonyl cyanide-p-
trifluoromethoxyphenylhydrazone) which prevents mitochondria establishing a
membrane potential (Figs. 1 and 2) (Burns et al., 1995). Therefore compound 1
is
CA 02354743 2001-08-07
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rapidly and selectively accumulated into mitochondria driven by the
mitochondria)
membrane potential and this accumulation results in a concentration of the
compound
within mitochondria several thousand fold higher than in the external medium.
This
accumulation is rapidly (< 10 s) reversed by addition of the uncoupler FCCP to
dissipate
the mitochondria) membrane potential after accumulation of compound 1 within
the
mitochondria. Therefore the mitochondria) specific accumulation is solely due
to the
mitochondria) membrane potential and is not due to specific binding or
covalent
interaction.
The mitochondria) specific accumulation of compound 1 also occurs in intact
cells.
This was measured as described by Burns and Murphy, 1997 and the accumulation
was prevented by dissipating both the mitochondria) and plasma membrane
potentials.
In addition, compound 1 was not accumulated by cells containing defective
mitochondria, which consequently do not have a mitochondria) membrane
potential.
Therefore the accumulation of compound 1 into cells is driven by the
mitochondria)
membrane potential.
The accumulation ratio was similar across a range of concentrations of
compound 1
and the amount of compound 1 taken inside the mitochondria corresponds to an
intramitochondrial concentration of 4-8 mM (Fig 2). This uptake was entirely
due to the
membrane potential and paralleled that of the simple triphenylphosphonium
cation
TPMP over a range of membrane potentials (Fig 3). From comparison of the
uptake of
TPMP and compound 1 at the same membrane potential we infer that within
mitochondria about 84% of compound 1 is membrane-bound (cf. About 60% for the
less
hydrophobic compound TPMP).
Further details of the experimental procedures and results are given below.
Figure 1 shows the uptake of 10 ~M [3H] compound 1 by energised rat liver
mitochondria (continuous line and filled symbols). The dotted line and open
symbols
show the effect of addition of 333 nM FCCP at 3 min. Incubation with FCCP from
the
start of the incubation led to the same uptake as for adding FCCP at 3 min
(data not
shown). Liver mitochondria were prepared from female Wistar rats by
homogenisation
followed by differential centrifugation in medium containing 250 mM sucrose,
10 mM
Tris-HCL (pH 7.4) and 1 mM EGTA and the protein concentration determined by
the
biuret assay using BSA as a standard. To measure [3H] compound 1 uptake
mitochondria (2 mg protein/ml) were suspended at 25°C in 0.5 - 1 ml 110
mM KCl, 40
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mM Hepes-KOH, pH 7.2, 0.1 mM EDTA supplemented with nigericin (1 ~g/ml), 10 mM
succinate, rotenone 1.33 ~g/ml and 60 nCi/ml [3H] compound 1 and 10 ~M
compound
1. After the incubation mitochondria were pelleted by centrifugation and the
[3H)
compound 1 in the supernatant and pellet quantitated by scintilation counting.
Figure 2 shows the mitochondrial accumulation ratios [(compound 1 /mg
protein)/(compound 1/~1)] obtained following 3 min incubation of energised rat
liver
mitochondria with different concentrations of compound 1 (filled bars) and the
effect
of 333 nM FCCP on these (open bars). The dotted line and open circles show
compound
1 uptake by mitochondria, corrected for FCCP-insensitive binding. To measure
[3H)
compound 1 accumulation ratio mitochondria (2 mg protein/ml) were suspended at
25°C in 0.5 - 1 ml 110 mM KCI, 40 mM Hepes-KOH, pH 7.2, 0.1 mM EDTA
supplemented with nigericin (1 ~g/ml), lOmM succinate, rotenone 1.33 ~g/ml and
6 -
60 nCi/ml [3H] compound 1 and 1-50 ~.M compound 1. After the incubation
mitochondria were pelleted by centrifugation and the [3H] compound 1 in the
supernatant and pellet quantitated by scintillation counting.
Figure 3 shows a comparison of compound 1 uptake with that of TPMP at a range
of
mitochondria) membrane potentials. Energised rat liver mitochondria were
incubated
for 3 min with 10 ~uM compound 1 and 1 ~M TPMP and different membrane
potentials
established with 0-8 mM malonate or 333 nM FCCP. The accumulation ratios of
parallel incubations with either 60 nCi/ml [3H] compound 1 or 50 nCi/ml [3H)
TPMP
were determined, and the accumulation ratio for compound 1 is plotted relative
to that
of TPMP at the same membrane potential (slope = 2.472, y intercept = 319, r =
0.97).
Mitochondria (2 mg protein/ml) were suspended at 25°C in 0.5-1 ml 110
mM KCl, 40
mM Hepes-KOH, pH 7.2, 0.1 mM EDTA supplemented with nigericin (lug/ml), 10 mM
succinate, rotenone 1.33 ~g/ml.
3. Anti-oxidant efficacy of compound 1
The compounds of the present invention are highly effective against oxidative
stress.
To demonstrate this, compound 1 was further tested using rat brain
homogenates. The
rat brain homogenates were incubated with or without various concentrations of
the
test compounds (compound l; native Vitamin E (a-tocopherol), bromobutyl
triphenylphosphonium bromide, Trolox (a water soluble form of Vitamin E) and
compound 2, ie2-(2-bromoethyl)-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-
6-
ol, the precursor of compound 1 ("Brom Vit E")) and the oxidative damage
occurring
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over the incubation was quantitated using the TBARS assay (Stocks et al.,
1974, Clin
Sci Mol Med 47,215-222). From this the concentration of compound required to
inhibit
oxidative damage by 50% was determined. In this system 210 nM compound 1
inhibited oxidative stress by 50% while the corresponding value for native
Vitamin E
was 36 nM. The value for bromobutyltriphenylphosphonium bromide, which
contains
the triphenylphosphonium moiety but not the antioxidant Vitamin E moiety was
47 ~ M.
These data show that compound 1 is an extremely effective antioxidant, within
an
order of magnitude as effective as Vitamin E. Comparison with
bramobutyltriphenylphosphonium bromide shows that the antioxidant capability
is
due to the Vitamin E function and not to the phosphonium salt. Further details
of the
experimental procedures and results are set out below.
The ICSO values for inhibition of lipid peroxidation were determined in rat
brain
homogenates, and are means + , SEM or range of determinations on 2-3 brain
preparations. Octan-1-ol/PBS partition coefficients are means ~ SEM for three
independent determinations. N.D. not determined. Partition coefficients were
determined by mixing 200 ~M of the compound in 2 ml water-saturated octanol-1-
0l
with 2 ml octanol-saturated-PBS at room temperature for 1 h, then the two
layers were
separated by brief centrifugation and their concentrations determined
spectrophotometrically from standard curves prepared in PBS or octanol. To
measure
antioxidant efficacy four rat brains were hornogenised in I5 ml 40 mM
potassium
phosphate (pH 7.4), 140 mM NaCl at 4°C, particulate matter was pelleted
(1,000 x g at
4°C for 15 min) and washed once and the combined supernatants stored
frozen.
Aliquots were rapidly thawed and 5 mg protein suspended in 800 ~l PBS
containing
antioxidant or ethanol carrier and incubated at 37°C for 30 min.
Thiobarbituric acid
reactive species (TBARS) were quantitated at 532 nm by adding 200 ~1 conc.
HC104 and
200 ~l 1% thiobarbituric acid to the incubation, heating at 100°C for
15 min and then
cooling and clarification by centrifugation (10,000 x g for 2 min). The
results are shown
in Table 1 below.
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Table 1. Partition coefficients and antioxidant efficacy of compound 1 and
related compounds
Compound ICso for inhibition Octanol:PBS partition
of lipid coefficient
peroxidation (nM)
Compound 1 210 + 58 7.37 + 1.56
Bromo Vit E 45 + 26 33.1 + 4.4
a-Tocopherol 36 22 27.4 1.0
Trolox 18500 + 5900 N.D.
BrBTP 47000 + 13000 3.83 + 0.22
When mitochondria were exposed to oxidative stress corripound 1 protected them
against oxidative damage, measured by lipid peroxidation and protein carbonyl
formation (Fig 4). This antioxidant protection was prevented by incubating
mitochondria with the uncoupler FCCP to prevent uptake of compound l, and
lipophilic cations alone did not protect mitochondria (Fig 5). Most
importantly, the
uptake of compound 1 protected mitochondria) function, measured by the ability
to
generate a membrane potential, far more effectively than Vitamin E itself (Fig
6). This
shows that the accumulation of compound 1 into mitochondria selectively
protects their
function from oxidative damage. In addition, we showed that compound 1 is not
damaging to mitochondria at the concentrations that afford protection (Fig 7).
The next step was to determine whether compound 1 was accumulated by intact
cells.
Compound 1 was rapidly accumulated by intact 143B cells, and the amount
accumulated was greater than that by p° cells derived from 143B cells.
This is
important because the p° cells lack mitochondria) DNA and consequently
have far lower
mitochondria) membrane potential than the 143B cells, but are identical in
every other
way, including plasma membrane potential, cell volume and protein content (Fig
8); this
suggests that most of the compound 1 within cells is mitochondria). A
proportion of
this uptake of compound 1 into cells was inhibited by blocking the plasma and
mitochondria) membrane potentials (Fig. 9). This energisation-sensitive uptake
corresponds to an intra mitochondria) concentration of compound 1 of about 2-
4 mM,
which is sufficient to protect mitochondria from oxidative damage. These
concentrations of compound 1 are not toxic to cells (Fig. 10).
Further details of the experimental procedures and results are discussed
below.
CA 02354743 2001-08-07
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Figure 4 shows the protection of mitochondria against oxidative damage by
compound
1. Mitochondria were exposed to oxidative stress by incubation with
iron/ascorbate
and the effect of compound 1 on oxidative damage assessed by measuring TBARS
(filled
bars) and protein carbonyls (open bars). Rat liver mitochondria (10 mg
protein) were
incubated at 25°C in a shaking water bath in 2 ml medium containing 100
mM KCl, 10
mM Tris, pH 7.7, supplemented with rotenone (1.33 ~ug/ml), 10 mM succinate,
500 ~M
ascorbate and other additions. After preincubation for 5 min, 100 ~uM FeS04
was
added and 45-55 min later duplicate samples were removed and assayed for TBARS
or
protein carbonyls.
Figure 5 shows a comparison of compound 1 with vitamin E and the effect of
uncoupler
and other lipophilic cations. Energised rat liver mitochondria were exposed to
tert-
butylhydroperoxide and the effect of compound 1 (filled bars), a-tocopherol
(open bars),
compound 1 + 333 nM FCCP (stippled bars) or the simple lipophilic cation
bromobutyl
triphenylphosphonium (cross hatched bars) on TBARS formation determined. Rat
liver
mitochondria (4 mg protein) were incubated in 2 rnl medium containing 120 mM
KCl,
10 mM Hepes-HCl pH 7.2, 1 mM EGTA at 37°C in a shaking water bath for 5
min with
various additions, then tert butyl hydroperoxide (5 mM) was added, and the
mitochondria incubated for a further 45 min and then TSARS determined.
Figure 6 shows how compound 1 protects mitochondrial function from oxidative
damage. Energised rat liver mitochondria were incubated with iron/ascorbate
with no
additions (stippled bars), 5 ~M compound 1 (filled bars), 5 ~M a-tocopherol
(open bars)
or 5 ~M TPMP (cross hatched bars), and then isolated and the membrane
potential
generated by respiratory substrates measured relative to control incubations
in the
absence of iron/ascorbate. Rat liver mitochondria were incubated at
25°C in a shaking
water bath in 2 ml medium containing 100 mM KCl, 10 mM Tris, pH 7.7,
supplemented
with rotenone (1.33 ug/ml), 10 mM succinate, 500 ~M ascorbate and other
additions.
After preincubation for 5 min, 100 ~M FeS04 was added and after 30 min the
incubation was diluted with 6 ml ice-cold STE 250 mM sucrose, 10 mM Tris-HCL
(pH
7.4) and 1 mM EGTA, pelleted by centrifugation (5 min at 5,000 x g) and the
pellet
resuspended in 200 u1 STE and 20 ~l (= 1 mg protein) suspended in 1 ml 110 mM
KCl,
mM HEPES, 0.1 M EDTA pH 7.2 containing 1 ~M TPMP and 50 nCi/ml [3H] TPMP
35 either 10 mM glutamate and malate, 10 mM succinate and rotenone, or 5 mM
ascorbate/ 100 ~uM TMPD with rotenone and myxothiazol (2 ~g/ml), incubated at
25°C
for 3 min then pelleted and the membrane potential determined as above and
compared
with an incubation that had not been exposed to oxidative stress.
CA 02354743 2001-08-07
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Figure ~ shows the effect of compound 1 on the membrane potential (filled
bars) and
respiration rate of coupled (open bars), phosphorylating (stippled bars) and
uncoupled
mitochondria {cross hatched bars), as a percentage of values in the absence of
compound 1. The effect of various concentrations of compound 1 on the membrane
potential of isolated mitochondria was determined from the distribution of
[3H] TPMP
by incubating rat liver mitochondria (2 mg protein/ml) in 0.5 ml medium as
above
containing 1 ~M TPMP and 50 nCi/ml [3H] TPMP at 25°C for 3 min. After
the
incubation mitochondria were pelleted by centrifugation and the [3H] TPMP in
the
supernatant and pellet quantitated by scintilation counting and the membrane
potential calculated assuming a volume of 0.5 ~rl/mg proteins and that 60% of
intramitochondrial TPMP is membrane bound. To measure the effect of compound 1
on
coupled, phosphorylating and uncoupled respiration rates, mitochondria (2 mg
protein/ml) were suspended in 120 mM KCI, 10 mM Hepes-HCl pH 7.2, 1 mM EGTA,
10 rnM K Pi in a 3 ml Clark oxygen electrode then respiratory substrate, ADP
(200~M)
and FCCP (333 nM) were added sequentially to the electrode and respiration
rates
measured.
Figure 8 shows the uptake of compound 1 by cells. Here 106 143B cells (closed
symbols) or p° cells (open symbols) were incubated with 1 ~M [3H]
compound l and the
compound 1 accumulation ratio determined. Human osteosarcoma 143B cells and a
derived p° cell line lacking rnitochondrial DNA were cultured in DMEM/
10 % FCS (foetal
calf serum) supplemented with uridine and pyruvate under an atmosphere of 5%
COZ/95% air at 37°C, grown to confluence and harvested for experiments
by treatment
with trypsin. To measure [3H] compound 1 accumulation cells (106) were
incubated in
1 ml HEPES-buffered DMEM. At the end of the incubation, cells were pelleted by
centrifugation, the cell pellet and the supernatant prepared for scintillation
counting
and the accumulation ratio [compound 1 / mg protein) / (compound 1 / ~ul)]
calculated.
Figure 9 shows the amount of compound 1 taken up by 106 143B cells over 1 h
incubation, corrected for inhibitor-insensitive binding. Human osteosarcoma
143B
cells were incubated in 1 ml HEPES-buffered DMEM with 1-50 ~M compound 1
supplemented with 6-60 nCi/ml [3H] compound 1. To determine the energistration-
dependent uptake, parallel incubations with 12.5 uM oligomycin, 20 pM FCCP, 10
~M
myxathiazol, 100 nM valinomycin and 1mM ouabain were carried out. At the end
of the
incubation, cells were pelleted by centrifugation and prepared for
scintillation counting
and the energisation-sensitive uptake determined.
CA 02354743 2001-08-07
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Figure 10 shows the effect of compound 1 on cell viability. Here, confluent
143B cells
in 24 well tissue culture dishes were incubated with various concentrations of
compound 1 for 24 h and cell viability measured by lactate dehydrogenase
release.
Figure 11 shows the UV-absorption spectrum of [10-(6'-
ubiquinonyl)decyltriphenyl-
phosphonium bromide] (herein referred to as "mitoquinone") and of the reduced
form
of the compound [10-(6'-ubiquinolyl)decyltriphenylphosphonium bromide] (herein
referred to as "mitoquinol"), both separately or together being a compound of
the
present invention;
Figures 12A to 12D show reactions of [10-(6'-
ubiquinonyl)decyltriphenylphosphonium
bromide] ("mitoquinone") and the reduced form of the compound ("mitoquinol")
with
mitochondria) membranes. Beef heart mitochondria) membranes (20 ~g/ml) were
suspended in 50 mM sodium phosphate, pH 7.2 at 20°C. In panel A
rotenone and
antimycin were present and for the t = 0 scan, then succinate (5 mM) was added
and
scans repeated at 5 minute intervals as indicated. In panel B A27swas
monitored in the
presence of rotenone and antimycin and then mitoquinone (50 ~uM) was added,
followed
by succinate (5 mM) and malonate (20 mM) where indicated. In Panel C rotenone,
ferricytochrome c (50 ~M) and malonate (20 mM) were present, A2~swas monitored
and
mitoquinol (50 ~M) and myxathiazol (10 ~M) were added where indicated. In
panel D
Assowas monitored and the experiment in Panel C was repeated in the presence
of KCN.
Addition of myxathiazol inhibited this rate by about 60 - 70%. There was no
reaction
between mitoquinone and succinate or NADH in the absence of mitochondria)
membranes, however mixing 50 ~uM mitoquinone, but not mitoquinol, with 50 ~M
ferricytochrome c led to some reduction of Asso;
Figure 13 shows reactions of mitoquinol and mitoquinone with pentane-extracted
mitochondria) membranes. Pentane extracted beef heart mitochondria ( 100~g
protein/ml) were suspended in 50 mM sodium phosphate, pH 7.2 at 20°C.
In Panel A
NADH (125 ~M) was added and A34o was monitored and ubiquinone-1 (UQ-1; 50 ~M)
added where indicated. This was repeated in Panel b, except that
mitoubiquinone (50
~M) was added. In Panel C pentane extracted mitochondria were incubated with
mitoquinone (50 ~uM), A2,swas monitored and succinate (5 mM) and malonate (20
mM)
added where indicated. In Panel D pentane-extracted mitochondria were
incubated
with NADH (125 ~M), ferricytochrome c (50 ~M) and Asso was monitored and
mitoquinone (50 ~M) was added where indicated. Addition of myxathiazol
inhibited the
CA 02354743 2001-08-07
-17-
rate of reduction by about 60 - 70 %;
Figure 14 shows reduction of mitoquinone by intact mitochondria. Rat liver
mitochondria (100 ~g/ml) were incubated in 120 mM KCl, 10 mM HEPES, 1 mM EGTA,
pH 7.2 at 20°C and A275 monitored. In panel A rotenone and succinate (5
mM) were
present and mitoquinone (50 ~M) was added where indicated. This experiment was
repeated in the presence of malonate (20 mM) or FCCP (333 nM). In panel B
glutamate
and malate (5 mM of each) were present from the start and and mitoquinone (50
~uM)
was added where indicated. This experiment was repeated in the presence of
FCCP or
with rotenone and FCCP. Addition of TPMP (50 ~M) instead of mitoquinone did
not lead
to changes in A27s;
Figure 15 shows uptake of radiolabelled mitoquinol by energised rat liver
mitochondria
and its release on addition of the uncoupler FCCP;
Figure 16 shows the effect of mitoquinol on isolated rat liver mitochondria.
In A rat
liver mitochondria energised with succinate were incubated with various
concentrations
of mitoquinol and the membrane potential determined as a percentage of control
incubations. In B the respiration rate of succinate energised mitochondria
under state
4 (black), state 3 (white) and uncoupled (stippled) conditions, as a
percentage of control
incubations.
Distribution of TPMP ovithia mice:
Pairs of mice were supplied with drinking water supplemented with 500~M TPMP
spiked with tritiated TPMP for various times. The mice were then killed and
the amount
of TPMP in each organ quantitated by homogenisation followed by scintillation
counting. This showed that substantial amounts of TPMP did accumulate in the
major
organs, in particular there was substantial uptake by the brain and heart.
This
corresponds to about 5-10 ~M in the brain and about 15-20 ~M in the heart. See
Figure 17.
Distribution of Mit Vit E within mice:
Pairs of mice were supplied with drinking water supplemented with 500 ~uM Mit
Vit E
spiked with tritiated Mit Vit E for various times. The mice were then killed
and the
amount of Mit Vit E in each organ quantitated by homogenisation followed by
scintillation counting. Uptake is expressed as nmol Mit Vie E/g wet weight of
the
CA 02354743 2001-08-07
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organ, except for blood where the uptake is in nmol/ml. This led to
substantial uptake
into their major organs. The uptake was less than for TPMP but was still
substantial
in the brain and heart. This corresponds to about 1 ~M in the brain and about
5-10
~M in the heart, which are sufficient to offer protection from oxidative
stress.
Example 2
Synthesis of [10-(6'-ubiquinolyl)decyltriphenplphosphonium bromide] (herein
referred to as "mitoquinol")
Synthesis of precursors
To synthesise 11-bromoundecanoic peroxide 11-bromoundecanoic acid (4.00 g,
15.1
mmol) and SOCl2 (1.6 mL, 21.5 mmol) were heated, with stirring, at 90°C
for 15 min.
ExcessSOCl2 was removed by distillation under reduced pressure (15 mm Hg,
90°C)
and the residue (IR; 1799 crri') was dissolved in diethyl ether (20 mL) and
the solution
cooled to 0°C. Hydrogen peroxide (30%, 1.8 mL) was added, followed by
dropwise
addition of pyridine (1.4 mL) over 45 min. Diethyl ether (10 mL) was added and
the
mixture was stirred for 1 h at room temperature then diluted with diethyl
ether ( 150
mL) and washed with H20 (2 x 70 mL), 1.2 M HCI (2 x 70 mL), H20 (70 mL), 0.5 M
NaHC03 (2 x 70 mL) and H20 (70 mL). The organic phase was dried over MgS04 and
the solvent removed at room temperature under reduced pressure, giving a white
solid
(3.51 g). IR (nujol mull) 1810, 1782.
6-( 10-bromodecyl)ubiquinone was synthesised by mixing crude material above
(3.51
g, 12.5 mmol max), (ubiquinoneo, 1.31 g, 7.19 mmol, Aldrich) and acetic acid
(60 mL)
and stirring the mixture for 20 h at 100°C. The mixture was diluted
with diethyl ether
(600 mL) and washed with H20 (2 x 400 mL), 1 M HCI (2 x 450 mL), 0.50 M NaHC03
(2
x 450 mL) and H2O (2 x 400 mL). The organic phase was dried over MgS04. The
solvent was removed under reduced pressure, giving a reddish solid (4.31 g).
Column
chromatography of the crude solid on silica gel (packed in CHZC12) and elution
with
CH2Cl2 gave the product as a red oil (809 mg, 28%) and unreacted ubiquinone as
a red
solid (300 mg, 1.6 mmol, 13%). TLC: Rf (CH2CI2, diethyl ether 20:1) 0.46; IR
(neat)
2928, 2854, 1650, 161 l, 1456, 1288; ~m~ (ethanol): 278 nm; 1H NMR (299.9 MHz)
3.99
(s, 6H, 2 x-OCH3), 3.41 (t, J= 6.8 Hz, 3H, -CH2-Br), 2.45 (t, J= 7.7 Hz, 2H,
ubquinone-
CHZ-), 2.02, (s, 3H, -CH3). 1.89 (quin, J= 7.4 Hz, 3H, -CHZ -CHZ -Br), 1.42-
1.28 (m,
20H, -(CHZ)7-); 13C NMR (125.7 MHz) 184.7 (carbonyl), 184.2 (carbonyl), 144.3
(2C, ring),
143.1 (ring), 138.7 (ring), 61.2 (2 x-OCH3), 34.0 (-CH2-); 32.8 (-CHZ-), 29.8
(-CH2-), 29.4
(2 x -CH2-), 29.3 (-CH2-), 28.7 (2 x -CH2-), 28.2 (-CH2-), 26.4 (-CH2-), 11.9
(-CH3). Anal.
CA 02354743 2001-08-07
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Calcd. For C19H2902Br:C, 56.86; H, 7.28; Found: C, 56.49, H, 7.34; LREI mass
spectrum: calcd. For C19H2902Br 400/402; Found 400/402.
To form the quinol, 6-(10-bromodecyl)-ubiquinol, Na.BH4 (295 mg, 7.80 mmol)
was
added to a solution of the quinone (649 mg, 1.62 mmol) in methanol (6 mL) and
stirred
under argon for 10 min. Excess NaBH4 was quenched with 5% HCI (2 mL) and the
mixture diluted with diethyl ether (40 mL). The organic phase was washed with
1.2 M
HCl (40 mL) and saturated NaCI (2 x 40 mL), and dried over MgS04. The solvent
was
removed under reduced pressure, giving a yellow oily solid (541 mg, 83%). 1H
NMR
(299.9 MHz) 5.31 (s, 1H, -OH), 5.26 (s, 1H, -OH), 3.89 (s, 6H, 2 x-OCH3), 3.41
(t, J=
6.8 Hz, 2H, -CH2 -Br), 2.59 (t, J = 7.7 Hz, 2H ubquinol-CH2-), 2.15 (s, 3H,
CH3) 1.85
(quin, J =7.4 Hz, 2H, -CHZ -CH2 -Br), 1.44-1.21 (m, 19H, -CH2)~-).
Synthesis of 10-(6'-ubiquinolyl)decyltriphenylphosphonium bromide
('mitoquinol')
To synthesise 10-(6~-ubiquinolyl)decyltriphenylphosphonium bromide. To a 15 mL
Kimax tube were added 6-( 10-bromodecyl)ubiquinol (541 mg, 1.34 mmol), PPH3
(387
mg, 1.48 mmol), ethanol (95%, 2.5 mL) and a stirnng bar. The tube was purged
with
argon, sealed and the mixture stirred in the dark for 88 h at 85°C. The
solvent was
removed under reduced pressure, giving an oily orange residue. The residue was
dissolved in CH2C12 (2 mL) followed by addition of pentane (20 mL). The
resultant
suspension was refluxed for 5 min at 50°C and the supernatant decanted.
The residue
was dissolved in CH2C12 (2 mL) followed by addition of diethyl either (20 mL).
The
resultant suspension was refluxed for 5 min at 40°C and the supernatant
decanted.
The CH2C12/diethyl ether reflux was repeated twice more. Residual solvent was
removed under reduced pressure, giving crude product as a cream solid (507
mg). 1H
NMR (299.9 MHz) 7.9-7.6 (m, 20H, -P+ Ph3), 3.89 (s, 6H, 2 x-OCH3), 3.91-3.77
(m, 2H,
-CH2-P+Ph3), 2.57 (t, J= 7.8 Hz, 2H ubquinol-CH2-), 2.14 (s, 3H, CH3), 1.6-1.2
(m, 23H,
-(CH2)$-). 31P NMR (121.4 MHz) 25.1.
The crude product (200 mg) was oxidized to 10-(6'-
ubiquinonyl)decyltriphenylphosphonium bromide (the oxidised form) by stirring
in
CDC13 under an oxygen atmosphere for 13 days. The oxidation was monitored by'H
NMR and was complete after 13 days. The solvent was removed under reduced
pressure and the resultant residue dissolved in CH2CI2 (5 mL). Excess diethyl
ether (15
mL) was added and the resultant suspension stirred for 5 min. The supernatant
was
decanted and the CHzCI2/diethyl ether precipitation repeated twice more.
Residual
CA 02354743 2001-08-07
-20-
solvent was removed under reduced pressure, giving crude product as a brown
sticky
solid (173 mg).
The quinone was reduced to the quinol by taking a mixture of crude quinor~e
and
quinol (73 mg, ca. 3:1 by 1 H NMR) in methanol ( 1 mL) was added NaBH4 (21 mg,
0.55 mmol). The mixture was stirred slowly under an argon atmosphere for 10
min.
Excess NaBH4 was quenched with 5% HBr (0.2 mL) and the mixture extracted with
CHZCI2. The organic extract was washed with H20 (3 x 5 mL). Solvent was
removed
under reduced pressure, giving a mixture of quinone and quinol (ca 1:5 by'H
NMR)
as a pale yellow solid (55 mg).For routine preparation of the quinol form the
ethanolic solution, dissolve in 5 vols of water, (= 1 ml) add a pinch of NaBH4
leave
on ice in the dark for 5 min, then extract 3 x 0.5 ml dichloromethane, Wash
with
water/HCl etc blow off in nitrogen; dissolve in same vol of etoh and take
spectrum
and store at -80 under argon. Yield about 70 - 80%. Oxidises rapidly in air so
should be prepared fresh. vortex with 1 ml 2M NaCl. Collect the upper organic
phase and evaporate to dryness under a stream of N2 and dissolve in 1 ml
ethanol
acidified to pH 2.
Synthesis of [ 3HJ-10-(6'-ubiquinonyl)decyltriphenylphosphonium bromide
To a Kimax tube was added 6-(10-bromodecyl)ubiquinol ( 6.3 mg; 15.6 ~mol)
triphenylphosphine (4.09 mg; ' 15.6 ~mol) and 100 ~1 ethanol containing [3H]
triphenylphosphine (74 ~Ci custom synthesis by Moravek Biochemicals, Brea, CA,
USA, Spec Ac 1 Ci/mmol) and 150 ~ul ethanol added. The mixture was stirred in
the
dark under argon for 55h at 80°C. Then it was cooled and precipitated
by adition of 5
ml diethyl ether. The orange solid was dissolved in few drops of
dichloromethane and
then precipitated with diethyl ether and the solid was washed (x4) with ~ 2 ml
diethyl
ether. Then dissolved in ethanol to give a stock solution of 404 ~M which was
stored
at -20°C. The UV absorption spectrum and TLC were identical to those
of the
unlabelled 10-(6'-ubiquinonyl)decyltriphenylphosphonium bromide and the
specific
activity of the stock solution was 2.6 mCi/mmol.
Distributioa of Mitoquinol within Mice:
Mice supplied with mitoquinol orally were subject to dosage related
distribution of
mitoquinol into the organs similarly to TPMF and Mit Vit E (compound 1).
CA 02354743 2001-08-07
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With the reduced water solubility of mitoquinol with respect to compound 1, an
oil
suspension delivery into the mice was adopted in preferenceto a supplemented
drinking
water delivery. Nevertheless uptake into the organs and flushing therefrom was
demonstrated as was a ability with ongoing dosage of maintaining organ levels
of the
mitoquinol.
Extinction coe~cients
Stock solutions of the quinone in ethanol were stored at -80°C in the
dark and their
concentrations confirmed by 31P nmr. The compound was converted to the fully
oxidised form by incubation in basic 95% ethanol over an hour on ice or by
incubation
with beef heart mitochondria) membrane at room temperature, either procedure
leading
to the same extinction coefficient of 10,400 M-1 cm 1 at the local maximum of
275 nrn,
with shoulders at 263 and 268 nm corresponding to the absorption maxima of the
triphenylphosphonium moiety (Smith et al, Eur. J. Biochem., 263, 709-716,
1999;
Burns et al; Archives of Biochemistry and Biophysics, 322, 60-68, 1995) and a
broad
shoulder at 290 nm due to the quinol (Crane et al, Meth. Enzymol., 18C, 137-
165,
1971). Reduction by addition of NaBH4 gave the spectrum of the quinol which
had the
expected peak at 290 nm with an extinction coefficient of 1800 M-1 cm-1 and
the
extinction coefficient for at 268nm was 3,000 M-' cm-1 the same as that for
the
phosphonium moiety alone (Burns, 1995 above). The extinction coefficient of
10,400
M-1 cm-1 at 275nm was lower than that for other quinones which have values of
14,600 M-1 cm-i in ethanol (Crane, 1971 above) and 12,250 M-1 cm-1 in aqueous
buffer
(Cabririi et al, Arch. Biochem Biaphys, 208, 11-19, 1981). While the
absorbance of the
quninone was about 10% lower in buffer than in ethanol, the discrepancy was
not due
to an interaction between the phosphonium and the quinone as the absorbance of
the
precursor quinone before linking to the phosphonium and that of the simple
phosphonium methyltriphenylphosphonium were additive when 50 uM of each were
mixed together in either ethanol or aqueous buffer. The DEoX _ rea was 7,000 M-
lcrri 1
The spectrum of fully oxidised mitoquinone (50 ~M) in 50 mM sodium phosphate,
pH
7.2 is shown in Figure 11. Addition of NaBH4 gave the fully reduced compound,
mitoquinol. The UV absorption spectrum of the reduced (quinol) and oxidised
(quinone)
mitoquinone/ol are shown in Figure 11. To determine whether the mitochondria)
respiratory chain could also oxidise or reduce the compound mitoquinone was
incubated with beef heart mitochondria) membranes (Figure 12). In panel A the
CA 02354743 2001-08-07
-22-
spectrum of fully oxidised mitoquinone in the presence of antimycin inhibited
membranes is shown (t = 0; Fig 12A). Addition of succinate led to the gradual
reduction
of the mitoquinol as measured by repeating the measurement every five minutes
and
showing that the peak at 275 nm gradually disappeared, the presence of
antimycin
prevented the oxidation of the quinol by mitochondria) complex III. Succinate
did not
lead to the complete reduction of mitoquinone to mitoquinol, as can be seen by
comparing the complete reduction brought about by borohydride (Fig 11),
instead it
reduced about 23 % of the added ubiquinone (Fig 12A). This is presumably due
to
equilibration of the quinol/quinone couple with the succinate/fumarate couple
(Em Q=
40 mV at pH 7, Em Suc = 30 mV), hence this proportion corresponds to an Eh of
about
+8 mV.
The reduction of mitoquinone can be followed continuously at A2,5 nm (Fig
12B). On
addition to rotenone inhibited mitochondria) membranes the small amount of
mitoquinol remaining was oxidised leading to a slight increase in A275, but on
addition
of the Complex II substrate succinate mitoquinone was rapidly reduced and this
reduction was blocked by malonate, an inhibitor of Complex II (Fig 12B). The
rate of
reduction of mitoquinone was 51 ~ 9.9 nmol /min/mg protein, which compares
with
the rate of reduction of cytochrome c by succinate in the presence of KCN of
359
nmol/min/mg. Allowing for the 2 electrons required for mitoquinone reduction
compared with 1 for cytochrome c the rate of electron flux into the
mitoquinone pool
is of similar order to the electron flux through the respiratory chain.
To determine whether mitoquinol was oxidised by Complex III of the respiratory
chain,
mitoquinol was added to beef heart membranes which had been inhibited with
rotenone
and malonate (Fig 12C). The mitoquinol was oxidised rapidly by membranes at an
initial rate of about 89 ~ 9 nmol mitoquinol/min/mg protein (mean of 2 +/-
range) and
this oxidation was blocked by myxathiazol an inhibitor of complex III (Fig
12C). To
confirm that these electrons were being passed on to cytochrome c, mitoquinol
was
then added to membrane supplemented with ferricytochrome c and the rate of
reduction of cytochrome c monitored (Fig 12D). Addition of mitoquinol led to
reduction
of cytochrome c at an initial rate of about 93 +/- 13 nmol/min/mg (mean +/-
range).
This rate was largely blocked by myxathiazol, although a small amount of
cytochrome
c reduction (about 30 - 40%) was not blocked by myxathiazol.
Mitoquinone/ol may be picking up and donating electrons directly from the
active sites
of the respiratory complexes, or it could be equilibrating with the endogenous
CA 02354743 2001-08-07
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mitochondrial ubiquinone pool. To address this question the endogenous
ubiquinone
pool was removed from beef heart mitochondria by pentane extraction. In the
absence
of endogenous ubiquinone as an electron acceptor the pentane extracted beef
heart
mitochondria could not oxidise added NADH, but addition of ubiquinone-l, a
ubiquinone analogue that can pick up electrons from the active site of complex
I, the
oxidation of NADH is partially restored (Fig 13A). Similarly, addition of
mitoquinone
also restored NADH oxidation indicating that mitoquinone can pick up electrons
from
the complex I active site (Fig 13B). Succinate could also donate electrons to
mitoquinone in pentane extracted beef heart mitochondrial in a malonate
sensitive
manner, suggesting that mitoquinone could also pick up electrons from the
active site
of Complex II (Fig 13C). Finally, the effect of the quinone on the flux of
electrons to
cytochrome c was detemined and it was shown that there was no NADH-
ferricytochrome c activity until mitoquinone was added (Fig 13D), and this was
partially
inhibited by myxathizol (60 - 70 %).
The next step was to see if mitoquinone also accepted electrons within intact
mitochondria (Fig 14). When mitoquinone was added to intact energised
mitochondria
it was rapidly reduced (Fig 14A). In the presence of the uncoupler FCCP to
dissipate the
membrane potential the rate was decreased about 2-3 fold, presumably due to
the
prevention of the uptake of the compound in to the mitochondria (Fig 14A). The
complex II inhibitor malonate also decreased the rate of reduction of
mitoquinone (Fig
14A). Use of the NADH-linked substrates glutamate/malate also led to the rapid
reduction of mitoquinone by intact mitochondria which again was decreased by
addition of the uncoupler FCCP (Fig 14B). The Complex I inhibitor rotenone
also
decreased the rate of reduction of mitoquinone (Fig 14B).
The next step was to see if mitoquinol was accumulated by energised
mitochondria.
To do this a tritiated version of the compound was made, incubated with
energised
mitochondria and the amount taken up into the mitochondria determined. It can
be
seen that the compound is accumulated rapidly and that this accumulation is
reversed
by addition of the uncoupler FCCP (Fig 15).
The next assays were to determine the toxicity of these compounds to
mitochondria and
cells. To determine the toxicity to isolated mitochondria the effect on
membrane
potential and respiration rate were measured (Fig 16). It can be seen from
Figure 16
that 10 ~M mitoquinol had little effect on mitochondrial function and at 25 ~M
and
above there was some uncoupling and inhibition of respiration.
CA 02354743 2001-08-07
-24-
F"agure 19 shows the uptake of mitoquinol by Human osteosarcoma 143B cells (5
x 106)
were incubated with 5 ~M (3H]-mitoquinol (filled squares) or with 4 ~M FCCP
(open
triangle) or with a range of inhibitors (open circles).
INDUSTRIAL APPLICATION
The compounds of the invention have application in selective antioxidant
therapies for
human patients to prevent mitochondria) damage. This can be to prevent the
elevated
mitochondria) oxidative stress associated with particular diseases, such as
Parkinson's
disease, diabetes or diseases associated with mitochondria) DNA mutations.
They
could also be used in conjunction with cell transplant therapies for
neurodegenerative
diseases, to increase the survival rate of implanted cells.
In addition, these compounds could be used as prophylactics to protect organs
during
transplantation, or ameliorate the ischaemia-reperfusion injury that occurs
during
surgery. The compounds of the invention could also be used to reduce cell
damage
following stroke and heart attack or be given prophylactically to premature
babies,
which are susceptible to brain ischemia. The methods of the invention have a
major
advantage over current antioxidant therapies - they will enable antioxidants
to
accumulate selectively in mitochondria, the part of the cell under greatest
oxidative
stress. This will greatly increase the efficacy of antioxidant therapies.
Related lipophilic
canons are being trialed as potential anticancer drugs and are known to be
relatively
non-toxic to whole animals, therefore these mitochondrially-targeted
antioxidants are
unlikely to have harmful side effects.
Any appropriate delivery technique can be utilised ranging from orally,
parenterally,
etc. in the mammalian patient to in vitro into tissues or cells to be
transplanted into the
mammalian patient.
Those persons skilled in the art will appreciate that the above description is
provided
by way of example only, and that different lipophilic cation/antioxidant
combinations
can be employed without departing from the scope of the invention.
