U.S. patent application number 12/985205 was filed with the patent office on 2011-11-24 for mitoquinone derivatives used as mitochondrially targeted antioxidants.
Invention is credited to Robin A.J. Smith, Kenneth Martin Taylor.
Application Number | 20110288055 12/985205 |
Document ID | / |
Family ID | 37852364 |
Filed Date | 2011-11-24 |
United States Patent
Application |
20110288055 |
Kind Code |
A1 |
Taylor; Kenneth Martin ; et
al. |
November 24, 2011 |
MITOQUINONE DERIVATIVES USED AS MITOCHONDRIALLY TARGETED
ANTIOXIDANTS
Abstract
This invention relates to pharmaceutically acceptable
amphiphilic antioxidant compounds, compositions and dosage forms
comprising said compounds, and methods and uses reliant on said
compounds. The exemplified compounds are all mitoquinone
derivatives, being methoxyphenyl alkyl triphenylphosphonium or
methoxy dioxocyclohexadiene alkyl triphenylphosphonium derivatives.
The compounds, compositions, dosage forms, uses and methods are
useful in, for example, the treatment of diseases or conditions
associated with oxidative stress.
Inventors: |
Taylor; Kenneth Martin;
(Auckland, NZ) ; Smith; Robin A.J.; (Dunedin,
NZ) |
Family ID: |
37852364 |
Appl. No.: |
12/985205 |
Filed: |
January 5, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10568655 |
Aug 31, 2006 |
7888335 |
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PCT/NZ2004/000196 |
Aug 23, 2004 |
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12985205 |
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Current U.S.
Class: |
514/125 ;
435/375; 536/103; 562/30 |
Current CPC
Class: |
A61P 25/14 20180101;
A61P 25/28 20180101; C07C 303/30 20130101; A61P 25/16 20180101;
A61K 31/724 20130101; A61K 31/66 20130101; A61P 25/00 20180101;
A61P 39/06 20180101; A61P 43/00 20180101; C07C 303/28 20130101;
C07F 9/5442 20130101; C07C 2601/16 20170501; C07C 303/28 20130101;
C07C 309/66 20130101; C07C 303/30 20130101; C07C 309/66
20130101 |
Class at
Publication: |
514/125 ; 562/30;
435/375; 536/103 |
International
Class: |
A61K 31/662 20060101
A61K031/662; A61P 39/06 20060101 A61P039/06; C08B 37/16 20060101
C08B037/16; C07F 9/54 20060101 C07F009/54; C12N 5/071 20100101
C12N005/071 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 22, 2003 |
NZ |
527800 |
Oct 23, 2003 |
NZ |
529153 |
Jun 14, 2004 |
NZ |
533556 |
Claims
1.-119. (canceled)
120. A chemically stable antioxidant compound, comprising: a
lipophilic cationic moiety linked by a linking moiety to an
antioxidant moiety; and an anionic complement for said cationic
moiety, wherein the cationic moiety is capable of mitochondrially
targeting the antioxidant moiety and is selected from the group
consisting of a substituted or unsubstituted triphenylphosphonium
cation, tribenzylammonium cation and phosphonium cation, wherein
the linking moiety has a length equivalent to a linear saturated
carbon chain of from 1 to 30 carbon atoms and comprises a straight
alkylene chain, a substituted carbon chain or a branched carbon
chain, wherein the antioxidant moiety is selected from the group
consisting of (i) a quinone or a quinol, (ii) vitamin E or a
vitamin E derivative, (iii) a chain breaking antioxidant, (iv) a
derivatized fullerene, (v) a spin trap, (vi) butylated
hydroxyanisole, (vii) butylated hydroxytoluene, (viii)
5,5-dimethylpyrroline-N-oxide, (ix) tert-butylnitrosobenzene, (x)
tert-nitrosobenzene and (xi) .alpha.-phenyl-tert-butylnitrone,
wherein the anionic complement is a pharmaceutically acceptable
anion that is not a halogen ion or a nitrate anion and is selected
from the group consisting of an alkyl sulfonate, an aryl sulfonate,
tetrafluoroborate, trifluoromethanesulfonate, hexafluoroantimonate,
hexafluoroarsenate, hexafluorophosphate, tetraphenylborate, and
tetra(perfluorophenyl)borate, and wherein said anionic complement
does not exhibit reactivity against the antioxidant moiety, the
cationic moiety or the linking moiety.
121. The compound according to claim 120 wherein the lipophilic
cationic moiety is a substituted or an unsubstituted
triphenylphosphonium cation.
122. The compound of claim 120 having the general formula I:
##STR00032## or its quinol form, wherein R.sub.1, R.sub.2, and
R.sub.3 are the same or different and are selected from C.sub.1 to
C.sub.5 alkyl, substituted C.sub.1 to C.sub.5 alkyl and H, wherein
R.sub.4 and R.sub.5 are independently selected from the group
consisting of H, hydroxyl, carboxyl, amide, unsubstituted or
substituted alkyl, unsubstituted or substituted alkenyl and
unsubstituted or substituted alkynyl, and wherein n is an integer
from 2 to 20, and wherein Z is the anionic complement.
123. The compound of claim 120 wherein the pharmaceutically
acceptable anion is not nucleophilic.
124. The compound of claim 120 wherein the pharmaceutically
acceptable anion is an alkyl sulfonate.
125. The compound of claim 120 wherein the pharmaceutically
acceptable anion is selected from the group consisting of
methanesulfonate, p-toluenesulfonate, ethanesulfonate,
benzenesulfonate and 2-naphthalenesulfonate.
126. The compound of claim 120 wherein the pharmaceutically
acceptable anion is methanesulfonate.
127. The compound according to claim 120 wherein the antioxidant
moiety is a quinone or a quinol.
128. The compound of claim 122 wherein Z is selected from the group
consisting of an alkyl sulfonate and an aryl sulfonate.
129. The compound of claim 122 wherein C of (C).sub.n is
saturated.
130. The compound of claim 122 having the formula: ##STR00033## or
its quinol form, wherein Z is the anionic complement.
131. The compound of claim 122 having the formula: ##STR00034## or
its quinol form.
132. A pharmaceutical composition, comprising: the chemically
stable antioxidant compound of claim 120; and a carrier or
excipient.
133. The pharmaceutical composition of claim 132 wherein at least
one of: (a) the lipophilic cationic moiety is a substituted or an
unsubstituted triphenylphosphonium cation; (b) the pharmaceutically
acceptable anion is selected from the group consisting of (i) an
alkyl sulfonate, (ii) a pharmaceutically acceptable anion that is
not a halogen ion, and (iii) a pharmaceutically acceptable anion
that is not nucleophilic; (c) the pharmaceutically acceptable anion
is selected from the group consisting of methanesulfonate,
p-toluenesulfonate, ethanesulfonate, benzenesulfonate and
2-naphthalenesulfonate; (d) the pharmaceutically acceptable anion
is methanesulfonate; (e) the antioxidant moiety is selected from
the group consisting of (i) a quinone or a quinol, (ii) vitamin E
or a vitamin E derivative, (iii) a chain breaking antioxidant, (iv)
a derivatized fullerene, and (v) a spin trap; (f) the compound has
the formula: ##STR00035## or its quinol form, wherein Z is the
anionic complement; and (g) the compound has the formula:
##STR00036##
134. The pharmaceutical composition according to claim 132 which
comprises cyclodextrin.
135. The pharmaceutical composition of claim 134 wherein the
compound and cyclodextrin are present at a compound-to-cyclodextrin
molar ratio that is selected from the group consisting of (i) from
about 10:1 to about 1:10, (ii) from about 5:1 to about 1:5, (iii)
from about 4:1 to about 1:4, (iv) from about 2:1 to about 1:2, (v)
about 1:1 and (vi) about 1:2.
136. The pharmaceutical composition according to claim 134 wherein
the cyclodextrin is .beta.-cyclodextrin.
137. The pharmaceutical composition according to claim 132 that is
selected from the group consisting of a pharmaceutical composition
that is formulated for oral administration and a pharmaceutical
composition that is formulated for parenteral administration.
138. The pharmaceutical composition according to claim 133(g) which
comprises cyclodextrin, and that is selected from the group
consisting of a pharmaceutical composition that is formulated for
oral administration and a pharmaceutical composition that is
formulated for parenteral administration.
139. A method of reducing oxidative stress in a cell, comprising:
contacting a cell that comprises mitochondria with the chemically
stable antioxidant compound of claim 120 under conditions and for a
time sufficient for accumulation of the antioxidant compound in the
mitochondria, and thereby reducing oxidative stress in the
cell.
140. The method of claim 139 wherein the chemically stable
antioxidant compound has the general formula I: ##STR00037## or its
quinol form, wherein R.sub.1, R.sub.2, and R.sub.3 are the same or
different and are selected from C.sub.1 to C.sub.5 alkyl,
substituted C.sub.1 to C.sub.5 alkyl and H, wherein R.sub.4 and
R.sub.5 are independently selected from the group consisting of H,
hydroxyl, carboxyl, amide, unsubstituted or substituted alkyl,
unsubstituted or substituted alkenyl and unsubstituted or
substituted alkynyl, and wherein n is an integer from 2 to 20, and
wherein Z is the anionic complement.
141. A method of therapy or prophylaxis of a patient who would
benefit from reduced oxidative stress, comprising administering to
said patient a therapeutically efficacious dose of the
pharmaceutical composition of claim 132.
142. A method of preparing an antioxidant compound that is capable
of reducing oxidative stress in a cell, comprising admixing
cyclodextrin or a cyclodextrin derivative that is selected from
.beta.-cyclodextrin, sulfobutylcyclodextrin, maltosylcyclodextrin,
and hydroxypropylcyclodextrin, with the compound of claim 122.
143. The method of claim 142 wherein the compound has the formula:
##STR00038## or its quinol form.
Description
FIELD OF THE INVENTION
[0001] The invention relates to amphiphilic antioxidant compounds
having a lipophilic cationic group, and the synthesis, formulation,
and physicochemical properties of such compounds that favour their
use as, for example, pharmaceuticals.
BACKGROUND
[0002] 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
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. Moreover,
many such antioxidants have unfavourable physicochemical properties
that limit for example, their bioavailability, and their ability to
penetrate to the target organ to exert a therapeutic effect.
[0003] Mitochondria are intracellular organelles responsible for
energy metabolism. Consequently, mitochondrial defects are
damaging, particularly to neural and muscle 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.
[0004] Lipophilic cations may be accumulated in the mitochondrial
matrix because of their positive charge (Rottenberg, 1979 Methods
Enzymol 55, 547. Chen, 1988 Ann Rev Cell Biol 4, 155). 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 cation
is not metabolised or immediately toxic to a cell.
[0005] The focus of the invention is therefore on an approach by
which it is possible to use the ability of mitochondria to
concentrate specific lipophilic cations to take up linked
antioxidants so as to target the antioxidant to the major source of
free radicals and reactive oxygen species causing the oxidative
stress.
[0006] Examples of antioxidant compounds that show good antioxidant
activity in vivo yet exhibit poor antioxidant functionality with
respect to the target compartment in vivo include Coenzyme Q (CoQ)
and Idebenone. Both of these compounds have low bioavailability and
must be administered at very high dose rates to be efficacious, and
therefore have low therapeutic efficacy when referenced to the dose
rate administered.
[0007] We believe without wishing to be bound by any theory that
for an antioxidant compound, activity in vitro or ex vivo (such as,
for example, antioxidant activity or mitochondrial accumulation) is
by no means the sole determinant of antioxidant functionality
and/or efficacy in vivo (such as, for example, therapeutic
efficacy). Whilst it is true that to be useful as a mitochondrially
targeted antioxidant compound of the present invention an
antioxidant compound must exhibit a suitable antioxidant activity
in vitro or ex vivo, to be efficacious in vivo the mitochondrially
targeted antioxidant compound must exhibit other desirable
physicochemical properties, such as, for example, suitable
bioavailability, suitable localization or distribution within the
target mitochondria, and/or suitable stability.
[0008] We believe without wishing to be bound by any theory that
the mitochondrially targeted antioxidant compounds of the present
invention exhibit advantageous antioxidant functionality, including
bioavailability, and/or mitochondrial targeting and accumulation in
vivo at least in part by virtue of their physicochemical
properties, such as, for example, their amphiphilicity, their
physical structure and/or dimensions, and/or low to moderate
hydrophobicity and/or partition coefficient. Such compounds are
thereby therapeutically efficacious at low dose rates in comparison
to other antioxidant compounds.
[0009] In U.S. Pat. No. 6,331,532 by reference to exemplifications
of compounds mitoquinol and mitoquinone (referred to collectively
herein as mitoquinone/mitoquinol) there is disclosed the prospect
of mitochondrial targeting of an antioxidant moiety reliant upon a
lipophilic cation covalently coupled to the antioxidant moiety. The
exemplified compound therein (despite generalisation of the bridge
length), is the compound mitoquinone of the formula
##STR00001##
with a carbon bridge length of 10 (i.e. C.sub.10 bridged). Its
reduced form, mitoquinol, is also C.sub.10 bridged.
[0010] Mitoquinone/mitoquinol, despite excellence in antioxidant
activity and targeting and accumulation in mitochondria in vitro
and in vivo, we have found to be somewhat unstable as the bromide
salt. We have also found that the physiochemical properties of
mitoquione/mitoquinol as disclosed in U.S. Pat. No. 6,331,532 less
appropriate for pharmaceutical formulation, for example, where
administration is to be oral or parenteral and/or where there is
targeting of the compound to mitochondria in tissues of internal
organs (for example, brain, heart, liver, or other organs).
[0011] Examples of compounds of the present invention are suitable
for pharmaceutical formulation. They may be in a form other than a
crystalline and/or solid form, but are amenable to formation of a
solid form by admixture with other agents such as for example,
carriers, excipients, complexation agents, or other additives and
the like, such as, for example, cyclodextrins. Advantageously such
agents are pharmaceutically acceptable.
[0012] We have determined a desirability to offer examples of the
amphiphilic mitochondrially targeted antioxidant compounds of the
present invention with their positive charge in association with a
suitable anion thereby to provide the compound as a general
neutralised salt form, including solid or crystalline products. In
such salt forms however certain salt forming anions we have found
to be best avoided as they exhibit reactivity against the
antioxidant compound, for example, against the antioxidant moiety,
the linking moiety, or the lipophilic cationic moiety, and/or may
lead to cleavage at or of the antioxidant moiety. Other salt
forming anions are considered pharmaceutically undesirable. For
example, nitrate moieties are considered inappropriate generally by
pharmaceutical companies as being pharmaceutically or
environmentally unacceptable, whilst a hydrogen bromide frequently
used in salt forming of such compounds we find to have nucleophilic
properties that can lead to a reactivity against the antioxidant
moiety, for example, a cleavage of a methyl group from the
antioxidant moiety of the compound of general formula (II) herein,
and/or some overall decrease in stability of the overall compound.
For example, we have determined that the bromide salt of
mitoquinone is somewhat unstable.
[0013] We believe therefore that salt forms, including salt forms
as a liquid, solid or crystalline form, of mitochondrially targeted
antioxidants are best associated with an anion or like moiety that
is not nucleophilic, and/or one which does not exhibit reactivity
against any of the moieties comprising the antioxidant compound or
complex. It is also preferable that the anion is pharmaceutically
acceptable.
OBJECT OF THE INVENTION
[0014] Accordingly, it is an object of the present invention to
provide pharmaceutically acceptable amphiphilic antioxidant
compounds and compositions, dosage forms and methods reliant on
said compounds which are, for example, useful in the treatment of
diseases or conditions associated with oxidative stress, or to
provide the public with a useful choice.
SUMMARY OF THE INVENTION
[0015] In a first aspect, the present invention consists in a
compound comprising a lipophilic cationic moiety linked by a
linking moiety to an antioxidant moiety, and an anionic complement
for said cationic moiety, wherein the cationic species is capable
of mitochondrially targeting the antioxidant species, and the salt
form is chemically stable and/or the anionic complement does not
exhibit reactivity against the antioxidant moiety, the cationic
moiety or the linking moiety.
[0016] In one embodiment the antioxidant moiety is a quinone or a
quinol.
[0017] In other embodiments the antioxidant moiety is selected from
the group comprising vitamin E and vitamin E derivatives, chain
breaking antioxidants, including butylated hydroxyanisole,
butylated hydroxytoluene, general radical scavengers including
derivatised fullerenes, spin traps including derivatives of
5,5-dimethylpyrroline-N-oxide, tert-butylnitrosobenzene,
tert-nitrosobenzene, .alpha.-phenyl-tert-butylnitrone and related
compounds.
[0018] In one embodiment, the lipophilic cationic moiety is a
substituted or an unsubstituted triphenylphosphonium cation.
[0019] In one embodiment the compound has the general formula I
##STR00002##
and/or its quinol form, wherein R.sub.1, R.sub.2, and R.sub.3,
which can be the same or different, are selected from C.sub.1 to
C.sub.5 alkyl (optionally substituted) moieties or H, and wherein n
is an integer from about 2 to about 20, and wherein Z is a
non-reactive anion.
[0020] Preferably Z is selected from the group consisting of alkyl
or aryl sulfonates or nitrates.
[0021] Preferably each C of the (C)n bridge is saturated.
[0022] In a preferred embodiment, the compound has the formula
##STR00003##
and/or its quinol form, wherein Z is a non-nucleophilic anion.
[0023] More preferably, the compound has the formula
##STR00004##
[0024] In another aspect the invention provides a pharmaceutical
composition comprising or including a compound comprising a
lipophilic cationic moiety linked by a linking moiety to an
antioxidant moiety, and an anionic complement for said cationic
moiety, wherein the cationic species is capable of mitochondrially
targeting the antioxidant species, and the salt form is chemically
stable and/or the anionic complement does not exhibit reactivity
against the antioxidant moiety, the cationic moiety or the linking
moiety.
[0025] In one embodiment the antioxidant moiety is a quinone or a
quinol.
[0026] In other embodiments the antioxidant moiety is selected from
the group comprising vitamin E and vitamin E derivatives, chain
breaking antioxidants, including butylated hydroxyanisole,
butylated hydroxytoluene, general radical scavengers including
derivatised fullerenes, spin traps including derivatives of
5,5-dimethylpyrroline-N-oxide, tert-butylnitrosobenzene,
tert-nitrosobenzene, .alpha.-phenyl-tert-butylnitrone and related
compounds.
[0027] In one embodiment, the lipophilic cationic moiety is a
substituted or an unsubstituted triphenylphosphonium cation.
[0028] In one embodiment the compound has the general formula I
##STR00005##
and/or its quinol form, wherein R.sub.1, R.sub.2, and R.sub.3,
which can be the same or different, are selected from C.sub.1 to
C.sub.5 alkyl (optionally substituted) moieties or H, and wherein n
is an integer from about 2 to 20, and wherein Z is a non-reactive
anion.
[0029] Preferably Z is selected from the group consisting of alkyl
or aryl sulfonates or nitrates.
[0030] Preferably each C of the (C)n bridge is saturated.
[0031] In a further embodiment, the compound has the formula
##STR00006##
and/or its quinol form, wherein Z is a non-nucleophilic anion.
[0032] In a further embodiment the composition comprises a compound
having the formula II and/or its quinol form, wherein Z is a
non-nucleophilic anion, and wherein the composition comprises
cyclodextrin.
[0033] In various examples the molar ratio of compound to
cyclodextrin is from about 10:1 to about 1:10, from about 5:1 to
about 1:5, from about 4:1 to about 1:4, from about 2:1 to about
1:2, or about 1:1, for example the molar ratio of compound to
cyclodextrin is about 1:2.
[0034] More preferably, the composition comprises a compound having
the formula
##STR00007##
[0035] wherein the cyclodextrin is .beta.-cyclodextrin, more
preferably the molar ratio of compound to cyclodextrin is about
1:2.
[0036] In one embodiment the pharmaceutical composition is
formulated for oral administration.
[0037] In a further embodiment the pharmaceutical composition is
formulated for parenteral administration.
[0038] In a further aspect the present invention provides a dosage
unit comprising or including a compound comprising a lipophilic
cationic moiety linked by a linking moiety to an antioxidant
moiety, and an anionic complement for said cationic moiety, wherein
the cationic species is capable of mitochondrially targeting the
antioxidant species, and the salt form is chemically stable and/or
the anionic complement does not exhibit reactivity against the
antioxidant moiety, the cationic moiety or the linking moiety,
together with any pharmaceutically acceptable diluent and/or
carrier and/or excipient.
[0039] In one embodiment the antioxidant moiety is a quinone or a
quinol.
[0040] In other embodiments the antioxidant moiety is selected from
the group comprising vitamin E and vitamin E derivatives, chain
breaking antioxidants, including butylated hydroxyanisole,
butylated hydroxytoluene, general radical scavengers including
derivatised fullerenes, spin traps including derivatives of
5,5-dimethylpyrroline-N-oxide, tert-butylnitrosobenzene,
tert-nitrosobenzene, .alpha.-phenyl-tert-butylnitrone and related
compounds.
[0041] In one embodiment, the lipophilic cationic moiety is a
substituted or an unsubstituted triphenylphosphonium cation.
[0042] In one embodiment the compound has the general formula I
##STR00008##
and/or its quinol form, wherein R.sub.1, R.sub.2, and R.sub.3,
which can be the same or different, are selected from C.sub.1 to
C.sub.5 alkyl (optionally substituted) moieties or H, and wherein n
is an integer from about 2 to 20, and wherein Z is a non-reactive
anion.
[0043] Preferably Z is selected from the group consisting of alkyl
or aryl sulfonates or nitrates.
[0044] Preferably each C of the (C)n bridge is saturated.
[0045] In a further embodiment, the compound has the formula and/or
its quinol form, wherein Z is a non-nucleophilic anion.
##STR00009##
[0046] In a further embodiment the dosage unit comprises a compound
having the formula II and/or its quinol form, wherein Z is a
non-nucleophilic anion, and wherein the composition comprises
cyclodextrin.
[0047] In various examples the molar ratio of compound to
cyclodextrin is from about 10:1 to about 1:10, from about 5:1 to
about 1:5, from about 4:1 to about 1:4, from about 2:1 to about
1:2, or about 1:1, for example the molar ratio of compound to
cyclodextrin is about 1:2.
[0048] More preferably, the dosage unit comprises a compound having
the formula
##STR00010##
[0049] wherein the cyclodextrin is .beta.-cyclodrextrin, more
preferably the molar ratio of compound to cyclodextrin is about
1:2.
[0050] In one embodiment the dosage unit is suitable for oral
administration.
[0051] In a further embodiment the dosage unit is suitable for
parenteral administration.
[0052] In a further aspect the present invention provides a
compound or a pharmaceutically acceptable salt thereof, a
composition, or a dosage form of the present invention for use in
the prophylaxis or treatment of oxidative stress in a mammal by
administration of the compound or the salt thereof to said
mammal.
[0053] In one embodiment, the compound is a compound of formula II
or a pharmaceutically acceptable salt thereof.
[0054] In another embodiment, said administration is on the first
day at a dose of about 1.02 about 2.0 times the daily maintenance
dose, followed by administration of the compound or the salt
thereof at the daily maintenance dose of the subsequent days.
[0055] Preferably the salt is that of the methanesulfonate, and the
compound is combined with cyclodextrin.
[0056] More preferably the compound has the formula
##STR00011##
[0057] Preferably, the cyclodextrin is .beta.-cyclodrextrin, more
preferably the molar ratio of compound to cyclodextrin is about
1:2.
[0058] In a further aspect the present invention provides a
compound or a pharmaceutically acceptable salt thereof, a
composition, or a dosage form of the present invention for use in
the prophylaxis or treatment of symptoms of aging in a mammal by
administration of the compound or the salt thereof to said
mammal.
[0059] In one embodiment, the compound is a compound of formula II
or a pharmaceutically acceptable salt thereof.
[0060] In another embodiment, said administration is on the first
day at a dose of about 1.02 about 2.0 times the daily maintenance
dose, followed by administration of the compound or the salt
thereof at the daily maintenance dose of the subsequent days.
##STR00012##
[0061] Preferably the salt is that of the methanesulfonate, and the
compound is combined with cyclodextrin.
[0062] More preferably the compound has the formula
##STR00013##
[0063] Preferably, the cyclodextrin is .beta.-cyclodrextrin more
preferably the molar ratio of compound to cyclodextrin is about
1:2.
[0064] In a further aspect, the present invention consists in a
stable compound comprising a lipophilic cationic moiety linked by a
linking moiety to an antioxidant moiety, and an anionic complement
for said cationic moiety, wherein
[0065] the cationic species is capable of mitochondrially targeting
the antioxidant species, and
[0066] the anionic complement is not a halogen ion, and
[0067] the anionic complement is non-nucleophilic, and/or
[0068] the anionic complement does not exhibit reactivity against
the cationic moiety, the linking moiety, or the antioxidant
moiety.
[0069] In one embodiment the antioxidant moiety is a quinone or a
quinol.
[0070] In other embodiments the antioxidant moiety is selected from
the group comprising vitamin E and vitamin E derivatives, chain
breaking antioxidants, including butylated hydroxyanisole,
butylated hydroxytoluene, general radical scavengers including
derivatised fullerenes, spin traps including derivatives of
5,5-dimethylpyrroline-N-oxide, tert-butylnitrosobenzene,
tert-nitrosobenzene, .alpha.-phenyl-tert-butylnitrone and related
compounds.
[0071] In one embodiment, the lipophilic cationic moiety is a
substituted or an unsubstituted triphenylphosphonium cation.
[0072] In one embodiment the compound has the general formula
##STR00014##
and/or its quinol form, wherein R.sub.1, R.sub.2, and R.sub.3,
which can be the same or different, are selected from C.sub.1 to
C.sub.5 alkyl (optionally substituted) moieties or H, and wherein n
is an integer from about 2 to about 20, and wherein Z is a
non-reactive anion.
[0073] Preferably Z is selected from the group consisting of alkyl
or aryl sulfonates or nitrates.
[0074] Preferably each C of the (C)n bridge is saturated.
[0075] In a preferred embodiment, the compound has the formula
##STR00015##
and/or its quinol form, wherein Z is a non-nucleophilic anion.
[0076] More preferably, the compound has the formula
##STR00016##
[0077] In another aspect the invention provides a pharmaceutical
composition comprising or including a stable compound comprising a
cationic species being a lipophilic cationic moiety linked by a
linking moiety to an antioxidant moiety, and an anionic complement
for said cationic moiety, wherein
[0078] the cationic species is capable of mitochondrially targeting
the antioxidant species, and
[0079] the anionic complement is not a halogen ion, and
[0080] the anionic complement is non-nucleophilic, and/or
[0081] the anionic complement does not exhibit reactivity against
the cationic moiety, the linking moiety, or the antioxidant
moiety.
[0082] In one embodiment the antioxidant moiety is a quinone or a
quinol.
[0083] In other embodiments the antioxidant moiety is selected from
the group comprising vitamin E and vitamin E derivatives, chain
breaking antioxidants, including butylated hydroxyanisole,
butylated hydroxytoluene, general radical scavengers including
derivatised fullerenes, spin traps including derivatives of
5,5-dimethylpyrroline-N-oxide, tert-butylnitrosobenzene,
tert-nitrosobenzene, .alpha.-phenyl-tert-butylnitrone and related
compounds.
[0084] In one embodiment, the lipophilic cationic moiety is a
substituted or an unsubstituted triphenylphosphonium cation.
[0085] In one embodiment the compound has the general formula I
##STR00017##
and/or its quinol form, wherein R.sub.1, R.sub.2, and R.sub.3,
which can be the same or different, are selected from C.sub.1 to
C.sub.5 alkyl (optionally substituted) moieties or H, and wherein n
is an integer from about 2 to 20, and wherein Z is a non-reactive
anion.
[0086] Preferably Z is selected from the group consisting of alkyl
or aryl sulfonates or nitrates.
[0087] Preferably each C of the (C)n bridge is saturated.
[0088] In a further embodiment, the compound has the formula
##STR00018##
and/or its quinol form, wherein Z is a non-nucleophilic anion.
[0089] In a further embodiment the composition comprises a compound
having the formula II and/or its quinol form, wherein Z is a
non-nucleophilic anion, and wherein the composition comprises
cyclodextrin.
[0090] In various examples the molar ratio of compound to
cyclodextrin is from about 10:1 to about 1:10, from about 5:1 to
about 1:5, from about 4:1 to about 1:4, from about 2:1 to about
1:2, or about 1:1, for example the molar ratio of compound to
cyclodextrin is about 1:2.
[0091] More preferably, the composition comprises a compound having
the formula
##STR00019##
[0092] wherein the cyclodextrin is .beta.-cyclodrextrin, more
preferably the molar ratio of compound to cyclodextrin is about
1:2.
[0093] In one embodiment the pharmaceutical composition is
formulated for oral administration.
[0094] In a further embodiment the pharmaceutical composition is
formulated for parenteral administration.
[0095] In a further aspect the present invention consists of a
dosage unit comprising or including a stable compound comprising a
lipophilic cationic moiety linked by a linking moiety to an
antioxidant moiety, and an anionic complement for said cationic
moiety, together with any pharmaceutically acceptable diluent
and/or carrier and/or excipient wherein
[0096] the cationic species is capable of mitochondrially targeting
the antioxidant species, and
[0097] the anionic complement is not a halogen ion, and
[0098] the anionic complement is non-nucleophilic, and/or the
anionic complement does not exhibit reactivity against the cationic
moiety, the linking moiety, or the antioxidant moiety.
[0099] In one embodiment the antioxidant moiety is a quinone or a
quinol.
[0100] In other embodiments the antioxidant moiety is selected from
the group comprising vitamin E and vitamin E derivatives, chain
breaking antioxidants, including butylated hydroxyanisole,
butylated hydroxytoluene, general radical scavengers including
derivatised fullerenes, spin traps including derivatives of
5,5-dimethylpyrroline-N-oxide, tert-butylnitrosobenzene,
tert-nitrosobenzene, .alpha.-phenyl-tert-butylnitrone and related
compounds.
[0101] In one embodiment, the lipophilic cationic moiety is a
substituted or an unsubstituted triphenylphosphonium cation.
[0102] In one embodiment the compound has the general formula I
##STR00020##
and/or its quinol form, wherein R.sub.1, R.sub.2, and R.sub.3,
which can be the same or different, are selected from C.sub.1 to
C.sub.5 alkyl (optionally substituted) moieties or H, and wherein n
is an integer from about 2 to 20, and wherein Z is a non-reactive
anion.
[0103] Preferably Z is selected from the group consisting of alkyl
or aryl sulfonates or nitrates.
[0104] Preferably each C of the (C)n bridge is saturated.
[0105] In a further embodiment, the compound has the formula
##STR00021##
and/or its quinol form, wherein Z is a non-nucleophilic anion.
[0106] In a further embodiment the dosage unit comprises a compound
having the formula II and/or its quinol form, wherein Z is a
non-nucleophilic anion, and wherein the composition comprises
cyclodextrin.
[0107] In various examples the molar ratio of compound to
cyclodextrin is from about 10:1 to about 1:10, from about 5:1 to
about 1:5, from about 4:1 to about 1:4, from about 2:1 to about
1:2, or about 1:1, for example the molar ratio of compound to
cyclodextrin is about 1:2.
[0108] More preferably, the dosage unit comprises a compound having
the formula
##STR00022##
[0109] wherein the cyclodextrin is .beta.-cyclodrextrin, more
preferably the molar ratio of compound to cyclodextrin is about
1:2.
[0110] In one embodiment the dosage unit is suitable for oral
administration.
[0111] In a further embodiment the dosage unit is suitable for
parenteral administration.
[0112] In a further aspect, the present invention consists in a
dosage unit suitable for oral administration comprising as an
active ingredient a compound in accordance with the present
invention, the compound being of or being formulated as a
crystalline form and/or non-liquid form.
[0113] In a further aspect, the present invention consists in a
dosage unit suitable for parenteral administration comprising as an
active ingredient a compound in accordance with the present
invention.
[0114] In a further aspect, the present invention provides a
pharmaceutical composition suitable for treatment of a patient who
would benefit from reduced oxidative stress or reduced symptoms of
ageing which comprises or includes an effective amount of a
compound of the present invention in combination with one or more
pharmaceutically acceptable carriers, excipients, or diluents.
[0115] In one embodiment the compound is a compound of formula
I.
[0116] In one example, the compound is complexed with
cyclodextrin.
[0117] In various examples the molar ratio of compound to
cyclodextrin is from about 10:1 to about 1:10, from about 5:1 to
about 1:5, from about 4:1 to about 1:4, from about 2:1 to about
1:2, or about 1:1, for example the molar ratio of compound to
cyclodextrin is about 1:2.
[0118] More preferably, the compound is a compound of formula (III)
and the cyclodextrin is .beta.-cyclodrextrin, more preferably and
the molar ratio of compound to cyclodextrin is about 1:2.
[0119] In a further aspect, the invention provides a method of
reducing oxidative stress in a cell which comprises the step of
contacting said cell with a compound of the present invention.
[0120] In one embodiment the compound is a compound of formula
I.
[0121] In one example, the compound is complexed with
cyclodextrin.
[0122] In various examples the molar ratio of compound to
cyclodextrin is from about 10:1 to about 1:10, from about 5:1 to
about 1:5, from about 4:1 to about 1:4, from about 2:1 to about
1:2, or about 1:1, for example the molar ratio of compound to
cyclodextrin is about 1:2.
[0123] More preferably, the compound is a compound of formula (III)
and the cyclodextrin is .beta.-cyclodrextrin, more preferably and
the molar ratio of compound to cyclodextrin is about 1:2.
[0124] In one embodiment the pharmaceutical composition is
formulated for oral administration.
[0125] In a further embodiment the pharmaceutical composition is
formulated for parenteral administration.
[0126] In a further aspect, the present invention provides a
pharmaceutical composition suitable for treatment of a patient
suffering from or predisposed to Parkinson's disease, Alzheimer's
disease, Huntington's Chorea, or Friedreich's Ataxia, which
comprises or includes an effective amount of a compound of the
present invention in combination with one or more pharmaceutically
acceptable carriers, excipients, or diluents.
[0127] Preferably said treatment is of a patient suffering from or
predisposed to Friedreich's Ataxia.
[0128] In still a further aspect, the invention provides a method
of therapy or prophylaxis of a patient who would benefit from
reduced oxidative stress which comprises or includes the step of
administering to said patient a compound of the present
invention.
[0129] In one embodiment the compound is a compound of formula
I.
[0130] In one example, the compound is complexed with
cyclodextrin.
[0131] In various examples the molar ratio of compound to
cyclodextrin is from about 10:1 to about 1:10, from about 5:1 to
about 1:5, from about 4:1 to about 1:4, from about 2:1 to about
1:2, or about 1:1, for example the molar ratio of compound to
cyclodextrin is about 1:2.
[0132] More preferably, the compound is a compound of formula (III)
and the cyclodextrin is .beta.-cyclodrextrin, more preferably and
the molar ratio of compound to cyclodextrin is about 1:2.
[0133] In one embodiment said administration is oral
administration.
[0134] In another embodiment said administration is parenteral
administration.
[0135] In another aspect the invention provides a method of therapy
or prophylaxis of a patient who would benefit from reduced
oxidative stress, or reduced symptoms of ageing, which comprises
the step of administering to the patient a compound of the present
invention.
[0136] In yet another aspect, the invention provides a method of
therapy or prophylaxis of a patient suffering from or predisposed
to Parkinson's disease, Alzheimer's disease, Huntington's Chorea,
or Friedreich's Ataxia which comprises or includes the step of
administering to said patent a compound of the present
invention.
[0137] Preferably the method of therapy or prophylaxis is of a
patient suffering from or predisposed to Friedreich's Ataxia.
[0138] In another aspect the invention provides a method of
reducing oxidative stress in a cell, which comprises the step of
administering to the cell a compound of the present invention.
[0139] In another aspect the invention provides the use of a
compound as previously described in the preparation or manufacture
of a medicament, dosage unit, or pharmaceutical composition
effective for use in for the reduction of oxidative stress in a
patient.
[0140] In another aspect the invention provides the use of a
compound as previously described in the preparation or manufacture
of a medicament, dosage unit, or pharmaceutical composition
effective for use for the reduction of symptoms of aging in a
patient.
[0141] In a further aspect the invention provides the use of a
compound of the present invention in the preparation or manufacture
of a medicament, dosage unit, or pharmaceutical composition
effective for use in the treatment or prophylaxis of a patient
suffering from or predisposed to Parkinson's disease, Alzheimer's
disease, Huntington's Chorea, or Friedreich's Ataxia which
comprises or includes the step of administering to said patent a
compound of the present invention.
[0142] Preferably the medicament, dosage unit, or pharmaceutical
composition is effective for use in the treatment or prophylaxis of
a patient suffering from or predisposed to Friedreich's Ataxia.
[0143] In another aspect the invention provides the use of a
compound as previously described in the preparation or manufacture
of a medicament, dosage unit, or pharmaceutical composition
effective for use in the reduction of oxidative stress in a
cell.
[0144] Preferably, said preparation or manufacture is with other
material or materials, more preferably pharmaceutically acceptable
diluents, excipients, and/or carriers.
[0145] In a further aspect the present invention consists in a
method of synthesis of a compound with a moiety or the moiety of
the formula I
##STR00023##
(and/or its quinone form) wherein R.sub.1, R.sub.2, and R.sub.3,
which can be the same or different, are selected from C.sub.1 to
C.sub.5 alkyl (optionally substituted) moieties, and wherein n is
an integer from 2 to 20, said method including or comprising the
admixture of cyclodextrin.
[0146] Preferably each C of the (C)n bridge is saturated.
[0147] In a further aspect the present invention consists in a
method of synthesis of a compound having the formula
##STR00024##
[0148] said method including or comprising the admixture of
cyclodextrin.
[0149] In a further aspect the present invention consists in a
method of synthesis of a compound having the formula
##STR00025##
[0150] essentially as herein described.
[0151] Any discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is solely for the purpose of providing a context for
the present invention. It is not to be taken as an admission that
any or all of these matters form part of the prior art base or were
common general knowledge in the field relevant to the present
invention as it existed before the priority date of each claim of
this application.
[0152] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0153] Throughout this specification the term "quinone", whether
used alone or prefixed with another term to describe the oxidized
form of a compound, will be understood to include within its scope
the reduced form of that compound, that is, the quinol form.
Similarly, reference to a quinone, by structural depiction for
example, also includes within its scope the quinol form.
[0154] Throughout this specification the term "quinol", whether
used alone or prefixed with another term to describe the reduced
form of a compound, will be understood to include within its scope
the oxidised form of that compound, that is, the quinone form.
Similarly, reference to a quinol, by structural depiction for
example, also includes within its scope the quinone form.
[0155] As used herein the term "and/or" includes both "and" and
"or" as options.
[0156] As used herein, the term "partition coefficient" and
"partition coefficient (octanol:water)" refer to the
octan-1-ol/phosphate buffered saline partition coefficient
determined at 25.degree. C. or 37.degree. C. (see Kelso, G. F.,
Porteous, C. M., Coulter, C. V., Hughes, G. Porteous, W. K.,
Ledgerwood, E. C., Smith, R. A. J. and Murphy, M. P. 2001 J Biol
Chem 276 4588. Smith, R. A. J., Porteous, C. M., Coulter, C. V. and
Murphy, M. P. 1999 Eu. J Biochem 263, 709. Smith, R. A. J.,
Porteous, C. M., Gane, A. M. and Murphy, M. P. 2003 Proc Nat Acad
Sci 100, 5407.), or the octanol/water partition coefficient
calculated using Advanced Chemistry Development (ACD) Software
Solaris V4.67 as described in Jauslin, M. L., Wirth, T., Meier, T.,
and Schoumacher, F., 2002, Hum Mol Genet 11, 3055.
[0157] As used herein, the phrase "acceptable for pharmaceutical
preparation" includes within its meaning not only an acceptability
with regard to pharmaceutical administration, but also in respect
of formulation for, for example, acceptable stability, shelf life,
hygroscopicity, preparation and the like.
[0158] As used herein a "non-reactive anion" is an anion which
exhibits no reactivity against the antioxidant moiety, the
lipophilic cation, or the linking moiety. For example, if one such
moiety of the compound comprises a target of nucleophilic attack,
the anion is non-nucleophilic.
[0159] Although broadly as defined above, the invention is not
limited thereto but also consists of embodiments of which the
following description provides examples.
[0160] In particular, a better understanding of the invention will
be gained with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0161] FIG. 1 depicts the uptake of amphiphilic antioxidant
compounds by mitochondria, wherein the uptake of Mitoquinone-C10
into an energised mitochondrion is shown schematically.
[0162] FIG. 2 depicts the synthetic pathways for A: Mitoquinone-C3;
B: Mitoquinone-C5; C: Mitoquinone-C15.
[0163] FIG. 3 depicts the structure of Mitoquinone antioxidant
compounds and the related compound TPMP. A phospholipid drawn to
the same scale is aligned with the Mitoquinone antioxidant
compounds to indicate potential maximum depths of penetration of
the ubiquinol side chain into one leaflet of a phospholipid
bilayer. A: TPMP. B: Mitoquinone-C3. C: Mitoquinone-C5. D:
Mitoquininone-C10. E: Mitoquinone-C15. F: phospholipid.
[0164] FIG. 4 presents graphs showing the uptake and binding of
antioxidant compounds by mitochondria measured using an ion
selective electrode. A: Mitoquinone-C3. B: Mitoquinone-C5. C:
Mitoquinone-C10. D: Mitoquinone-C15. In the left hand panels
mitochondria (1 mg protein/ml) in the presence of rotenone were
present and then the antioxidant compounds were added as five
sequential 1 .mu.M additions (black arrowheads) to calibrate the
electrode response. For the right hand panels the electrodes were
first calibrated by five sequential 1 .mu.M additions (black
arrowheads) and mitochondria (1 mg protein/ml) were then added. In
all cases succinate was added to generate a membrane potential, and
FCCP was added to dissipate it. Data are typical traces of
experiments repeated at least 2-3 times.
[0165] FIG. 5 presents graphs showing the antioxidant efficacy of
antioxidant compounds. A: Mitochondria were energised with
succinate (black bars) or by incubation with an ATP regenerating
system consisting of ATP, phosphoenol pyruvate and pyruvate kinase
(white bars). After a 30 sec preincubation with the various
Mitoquinone analogues, TPMP or carrier, oxidative stress was
induced by addition of 50 .mu.M FeCl.sub.2 and 300 .mu.M
H.sub.20.sub.2. After 15 min incubation at 37.degree. C., lipid
peroxidation was estimated by measuring TBARs. Data are
means.+-.range of two independent experiments. The slight
protective effect of Mitoquinone-C5 on lipid peroxidation in the
presence of ATP is due to some of the Mitoquinone-C5 added from the
stock solution being in the ubiquinol form. B: The mitochondrial
membrane potential induced with succinate or with the ATP
regenerating system was measured from the accumulation of
[.sup.3H]TPMP. Data are means.+-.range of duplicate determinations
of a 25 minute incubation. The membrane potentials after a 5 minute
incubation were the same (data not shown). C: The concentration
dependence of the prevention of the accumulation of TBARs by the
antioxidant compounds was measured. All incubations were carried
out in the presence of succinate as described for A. Results are
expressed as % inhibition of TBARS formation, taking the value of a
sample exposed to FeCl.sub.2/H.sub.20.sub.2 in the absence of
Mitoquinone analogues as 0% inhibition, and a control sample (no
FeCl.sub.2/H.sub.2O.sub.2 added) as 100%. The data shown are a
typical titration with each concentration determined in triplicate
.+-.SD. D: The IC.sub.50 concentrations for prevention of lipid
peroxidation. Data are means.+-.sem, estimated from three
independent titrations as shown in C. The statistical significance
relative to the IC.sub.50 for Mitoquinone-C3 was determined using
Student's two tailed t test: *p<0.05; **p<0.005.
[0166] FIG. 6 presents a graph showing the effect of
Mitoquinone-C10 and Mitoquinone-C3 on sinus coronary flow.
[0167] FIG. 7 presents a graph showing the effect of
Mitoquinone-C10 and Mitoquinone-C3 on left ventricular diastolic
pressure.
[0168] FIG. 8 present a graph which shows the effect of
Mitoquinone-C10 and Mitoquinone-C3 on heart rate.
[0169] FIG. 9 presents graphs showing the rate of left ventricular
change.
[0170] FIG. 10 depicts graphs showing the effect of Mitoquinone-C10
and Mitoquinone-C3 on post ischaemia mitochondrial respiratory
function.
[0171] FIG. 11 is a graph depicting the stability of pure
Mitoquinone-C10 (batch no. 3) in clear glass bottles at 40.degree.
C., 75% RH; 25.degree. C., 50% RH and 5.degree. C. over silica
gel.
[0172] FIG. 12 is a graph depicting the stability of
Mitoquinone-C10 (batch no. 4) at 25.degree. C., 50% RH.
[0173] FIG. 13 is a graph depicting the stability of
Mitoquinone-C10 .beta.-cyclodextrin complex (1:1) at 4.degree. C.
over silica, 25.degree. C., 50% RH and 40.degree. C., 75% RH.
[0174] FIG. 14 is a graph depicting the stability of
Mitoquinone-C10 .beta.-cyclodextrin complex (1:2) at 4.degree. C.
over silica, 25.degree. C., 50% RH and 40.degree. C., 75% RH.
[0175] FIG. 15 is a graph depicting the stability of
Mitoquinone-C10 .beta.-cyclodextrin complex (1:4) at 4.degree. C.
over silica, 25.degree. C., 50% RH and 40.degree. C., 75% RH.
[0176] FIG. 16 presents a graph showing the stability of
Mitoquinone-C10 mesylate in water.
[0177] FIG. 17 presents a graph showing stability of
Mitoquinone-C10 mesylate in 0.01M HCl.
[0178] FIG. 18 presents a graph showing the stability of
Mitoquinone-C10 mesylate in IPB, pH 7.4.
[0179] FIG. 19 presents a graph showing the stability of
Mitoquinone-C10 mesylate in 50% MeOH
[0180] FIG. 20 presents a graph showing the solid state stability
of Mitoquinone-C10 mesylate at 40.degree. C., 75% RH; and
25.degree. C., 50% RH and 4.degree. C. over blue silica gel.
[0181] FIG. 21 presents a graph showing the stability of
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:2) complex in
water.
[0182] FIG. 22 presents a graph showing the stability of
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:2) complex in 0.01M
HCl.
[0183] FIG. 23 presents a graph showing the stability of
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:2) complex in IPB,
pH7.4.
[0184] FIG. 24 presents a graph showing the stability of
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:2) complex in 50%
MeOH.
[0185] FIG. 25 presents a graph showing the solid state stability
of Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:2) complex at
40.degree. C., 75% RH; at 25.degree. C., 50% RH, and 4.degree. C.
over blue silica gel.
[0186] FIG. 26 presents a graph showing the stability of
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:1) complex in
water.
[0187] FIG. 27 presents a graph showing the stability of
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:1) complex in 0.01M
HCl.
[0188] FIG. 28 presents a graph showing the stability of
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:1) complex in IPB
pH 7.4.
[0189] FIG. 29 presents a graph showing the stability of
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:1) complex in 50%
methanol.
[0190] FIG. 30 presents a graph showing the solid state stability
of Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:1) complex at
40.degree. C., 75% RH; 25.degree. C., 50% RH and 4.degree. C. over
blue silica gel.
[0191] FIG. 31 presents graphs of Rat plasma concentration-time
profiles of Mitoquinone-C10 after single IV (A) (10 mg/kg) and oral
(B) (50 mg/kg) administration to rats of Mitoquinone-C10 mesylate
in Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:2) complex
(n=5). Pharmacokinetic parameters derived from these data are given
in Table 11.
DETAILED DESCRIPTION OF THE INVENTION
[0192] As stated above, the focus of this invention is on the
mitochondrial targeting of compounds, primarily for the purpose of
therapy and/or prophylaxis to reduce oxidative stress.
[0193] 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 mitochondrial matrix.
[0194] The applicants have found that by coupling lipophilic
cations (for example the lipophilic triphenylphosphonium cation) to
an antioxidant moiety the resulting amphiphilic compound can be
delivered to the mitochondrial 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.
[0195] The applicants have now further determined that the
properties of the antioxidant compound, such as for example the
nature of the antioxidant moiety, the physical and chemical
characteristics of the linking moiety, such as, for example, the
length or lipophilicity of the linking moiety, and/or the nature of
the lipophilic cation contribute to the efficacy of the antioxidant
compound in vivo and contribute to the antioxidant functionality of
the compound. For antioxidant compounds of the present invention,
efficacy in vivo may in part comprise suitable bioavailability,
suitable stability, suitable pharmacokinetics suitable antioxidant
activity, and/or suitable mitochondrial targeting and/or
accumulation.
[0196] In principle, any lipophilic cation and any antioxidant
capable of being transported to and/or through the mitochondrial
membrane and accumulated at or within the mitochondria of intact
cells, can be employed in forming the compounds of the
invention.
[0197] It is however preferred that the lipophilic cation be the
triphenylphosphonium cation herein exemplified. Other lipophilic
cations which may covalently be coupled to antioxidants in
accordance with the present invention include the tribenzyl
ammonium and phosphonium cations. In some examples of antioxidant
compounds of the present invention, the lipophilic cation is
coupled to the antioxidant moiety by a saturated linear carbon
chain having from 1 to about 30 carbon atoms, for example from 2 to
about 20, from about 2 to about 15, from about 3 to about 10, or
from about 5 to about 10 carbon atoms. In a particularly preferred
example, the linear carbon chain comprises 10 carbon atoms.
[0198] Preferably the carbon chain is an alkylene group (for
example, C.sub.1-C.sub.20, or C.sub.1-C.sub.15), yet carbon chains
which optionally include one or more 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, such as those selected from unsubstituted or
substituted alkyl, alkenyl, or alkynyl groups. Also included are
carbon chains which comprise more than about 30 carbon atoms but
whose length is equivalent to a linear saturated carbon chain
having from 1 to about 30 carbon atoms.
[0199] It will be appreciated by those skilled in the art that
moieties other than a straight alkylene may be used to covalently
couple the antioxidant moiety to the lipophilic cation, for
example, substituted or branched alkyl groups, peptide bonds, and
the like.
[0200] In some embodiments, the lipophilic cation is linked to the
antioxidant moiety by a straight chain alkylene group having 1 to
10 carbon atoms; such as, for example an ethylene, propylene,
butylene, pentylene or decylene group.
[0201] Antioxidant moieties useful in the present invention include
those which require interaction with reductants for antioxidant
activity whether for initial antioxidant activity or for the
recycling of antioxidant activity, or both. For example,
antioxidant compounds of the present invention which comprise as
the active antioxidant moiety a quinol moiety may be administered
in the quinone form. To function as an antioxidant, that is to have
antioxidant activity, the quinone must be reduced to the quinol
form by interaction with a reductant, such as, for example, a
mitochondrial reductant such as Complex II, for initial antioxidant
activity. Subsequent interaction of the oxidised quinone form with
reductants can lead to recycling of antioxidant activity.
[0202] Other examples of antioxidant moieties useful in the present
invention include those which exist already as the reduced form and
do not require interaction with reductants for initial antioxidant
activity. Notwithstanding this, subsequent interaction of the
oxidised form of such antioxidant moieties with mitochondrial
reductants can lead to the recycling of antioxidant activity. For
example, the antioxidant moiety Vitamin E can be administered in
the reduced form and so does not require interaction with
reductants for initial antioxidant activity, but may subsequently
interact with reductants, such as, for example, the endogenous
quinone pool, thereby to recycle antioxidant activity.
[0203] Further examples of antioxidant moieties useful in the
present invention include those that are not recycled by
interaction with mitochondrial reductants.
[0204] Examples of antioxidant moieties useful in the present
invention include vitamin E and vitamin E derivatives, chain
breaking antioxidants, such as butylated hydroxyanisole, butylated
hydroxytoluene, quinols and general radical scavengers such as
derivatised fullerenes. In addition, spin traps, which react with
free radicals to generate stable free radicals can also be used.
These will include derivatives of 5,5-dimethylpyrroline-N-oxide,
tert-butylnitrosobenzene, tent-nitrosobenzene,
.alpha.-phenyl-tert-butylnitrone and related compounds.
[0205] Preferred antioxidant compounds, including those of general
formulae I and II herein, can be readily prepared, for example, by
the following reaction:
##STR00026##
[0206] The general synthesis strategy is to heat a precursor
containing a suitable leaving group, preferably a alkyl sulfonyl,
bromo or iodo precursor with greater than 1 equivalents of
triphenylphosphine under argon for several days. The phosphonium
compound is then isolated as its salt. To do this the product is
triturated repeatedly with diethyl ether until an off-white solid
remains. This is then dissolved in chloroform or dichloromethane
and precipitated with diethyl ether to remove the excess
triphenylphosphine. This is repeated until the solid no longer
dissolves in chloroform. At this point the product is
recrystallised several times from a suitable solvent such as
chloroform, acetone, ethyl acetate or higher alcohols.
[0207] A preferred synthetic method which may be used to prepare a
stable form of a preferred mitochondrially targeted antioxidant
compound of formula III (also referred to herein as Mitoquinone-C10
mesylate or Mitoquinone-C10 methanesulfonate) is as set out in
Example 1 herein.
[0208] It will also be appreciated that the anion of the
antioxidant compound thus prepared can readily be exchanged with
another pharmaceutically or pharmacologically acceptable anion, if
this is desirable or necessary, using ion exchange or other
techniques known in the art.
[0209] The applicants have determined that the stability of the
salt form of the antioxidant compound is enhanced when the anion
does not exhibit reactivity towards the antioxidant moiety, the
linking moiety, or the lipophilic cationic moiety. For example, in
the case of preferred examples of antioxidant compounds of the
invention, the anion is not nucleophilic. It is also desirable that
the anion is a pharmaceutically acceptable anion. It is also
preferred that for pharmaceutical formulation the anion does not
exhibit reactivity towards any other agents comprising the
formulation.
[0210] Examples of non-nucleophilic anions include
hexafluoroantimonate, -arsenate or -phosphate, or
tetraphenylborate, tetra(perfluorophenyl)borate or other
tetrafluoroborates, trifluoromethane sulfonate, aryl and alkyl
sulfonates such as methanesulfonate and p-toluenesulfonate, and
phosphates.
[0211] Examples of pharmaceutically acceptable anions include
halogen ions such as a fluoride ion, chloride ion, bromide ion and
iodide ion; anions of inorganic acid salts such as nitrate,
perchlorate, sulfate, phosphate, and carbonate; pharmaceutically
acceptable anions of lower alkylsulfonic acid salts such as
methanesulfonic acid, and ethanesulfonic acid salts;
pharmaceutically acceptable anions of arylsulfonic acid salts such
as benzenesulfonic acid, 2-naphthalenesulfonic acid and
p-toluenesulfonic acid salts; pharmaceutically acceptable anions of
organic acid salts such as trichloroacetic acid, trifluoroacetic
acid, hydroxyacetic acid, benzoic acid, mandelic acid, butyric
acid, propionic acid, formic acid, fumaric acid, succinic acid,
citric acid, tartaric acid, oxalic acid, maleic acid, acetic acid,
malic acid, lactic acid, and ascorbic acid salts; and
pharmaceutically acceptable anions of acidic amino acid salts such
as glutamic acid and aspartatic acid salts.
[0212] In the case of preferred examples antioxidant compounds of
the invention, the halogen anion precursor is exchanged for aryl or
alkyl sulphonate anions. Examples include, but are not limited to,
benzene sulfonate, p-toluene sulfonate, 2-naphthalene sulphonate,
methanesulfonate, ethanesulfonate, propanesulfonate. A particularly
preferred anion is the methanesulfonate anion. As described above,
an example of an antioxidant compound of the invention wherein the
anion is methanesulfonate is the particularly preferred antioxidant
compound of formula III, herein referred to as Mitoquinone-C10
methanesulfonate or Mitoquinone-C10 mesylate.
[0213] 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 (or other
lipophilic cationic) moiety or moieties. These will include a
series of vitamin E derivatives, in which the length of the bridge
coupling the Vitamin-E function with the triphenylphosphonium (or
other lipohilic cationic) moiety is varied. Other antioxidants
which can be used as R include chain breaking antioxidants, such as
butylated hydroxyanisole, butylated hydroxytoluene, quinols and
general radical scavengers such as derivatised fullerenes. In
addition, spin traps, which react with free radicals to generate
stable free radicals can also be synthesized. These will include
derivatives of 5,5-dimethylpyrroline-N-oxide,
tert-butylnitrosobenzene, tert-nitrosobenzene,
.alpha.-phenyl-tert-butylnitrone and related compounds.
[0214] It will be appreciated that for an antioxidant compound of
the present invention, as for any drug, activity in vitro is by no
means the sole determinant of functionality or efficacy in vivo.
The antioxidant activity of the antioxidant compounds of the
present invention can be determined by methods such as those
described herein using, for example, isolated mitochondria and/or
isolated cells. Whilst it is true that, to be useful as a
mitochondrially targeted antioxidant compound of the present
invention an antioxidant compound must exhibit a suitably high
antioxidant activity in such assays, to be efficacious in vivo the
mitochondrially targeted antioxidant compound must exhibit other
desirable physicochemical properties, for example, suitable
bioavailability, stability, or antioxidant functionality.
[0215] Examples of antioxidant compounds that show good antioxidant
activity yet exhibit poor bioavailability with respect to the
target compartment in vivo include Coenzyme Q (CoQ) and Idebenone.
Both of these compounds must be administered at very high dose
rates (for example, 0.5-1.2 g) to obtain minimal clinical effects
in human patients.
[0216] Examples of the mitochondrially targeted antioxidant
compounds of the present invention exhibit good antioxidant
activity and bioavailability and thereby are efficacious in vivo at
low dose rates. A determination of the bioavailability of a
preferred amphiphilic mitochondrially targeted antioxidant compound
of the present invention, Mitoquinone-C10 mesylate and a
cyclodextrin complex thereof is presented herein in Example 11. We
believe the antioxidant compounds of the present invention to be
effective at mitochondrial targeting of antioxidant activity,
whilst exhibiting one or more of the additional benefits of being
available as a crystalline or solid form or being able to be
formulated as a solid form, increased stability, enhanced
bioavailability, and/or enhanced antioxidant functionality. The
physical and chemical characteristics of the antioxidant compounds
of the present invention we believe, again without wishing to be
bound by any theory, confer upon the antioxidant compounds of the
present invention preferred characteristics, thereby enabling their
use in compositions, formulations and methods amongst other
applications to which the antioxidant compounds of the prior art
may be less suitable given their chemical and physical
properties.
[0217] In some embodiments of the invention, the antioxidant
compound is a quinol derivative of the formula II defined above.
For example, a quinol derivative of the invention is the compound
Mitoquinone-C10 (of which the compound of formula III is a specific
salt form) as defined above. A further example of a compound of the
invention is a compound of formula I in which (C).sub.n is
(CH.sub.2).sub.5, and the quinol moiety is the same as that of
Mitoquinone-C10, herein referred to as Mitoquinone-C5 (see FIG.
3C). Yet a further example of a compound of the invention is a
compound of formula I in which (C).sub.n is (CH.sub.2).sub.3, and
the quinol moiety is the same as that of Mitoquinone-C10, which is
referred to herein as Mitoquinone-C3 (see FIG. 3B). Still a further
example of a compound of the invention is a compound of formula I
in which (C).sub.n is (CH.sub.2).sub.15, and the quinol moiety is
the same as that of Mitoquinone-C10, herein referred to as
Mitoquinone-C15 (see FIG. 3E).
[0218] Once prepared, the antioxidant compound of the invention in
any pharmaceutically appropriate form and optionally including
pharmaceutically-acceptable carriers, excipients, diluents,
complexation agents, or additives, will be administered to the
patient requiring therapy and/or prophylaxis. Once administered,
the compound will target antioxidant activity to the mitochondria
within the cells of the patient.
[0219] Antioxidant compounds of the present invention may be
administered to patients by oral and/or parenteral administration
routes.
[0220] The antioxidant compound must be formulated into a stable,
safe pharmaceutical composition for administration to a patient.
The composition can be prepared according to conventional methods
by dissolving or suspending an amount of the antioxidant compound
ingredient in a diluent. The amount is from between 0.1 mg to 1000
mg per ml of diluent of the antioxidant compound. An acetate,
phosphate, citrate or glutamate buffer may be added allowing a pH
of the final composition to be from 5.0 to 9.5; optionally a
carbohydrate or polyhydric alcohol tonicifier and, a preservative
selected from the group consisting of m-cresol, benzyl alcohol,
methyl, ethyl, propyl and butyl parabens and phenol may also be
added. A sufficient amount of water for injection is used to obtain
the desired concentration of solution. Additional tonicifying
agents such as sodium chloride, as well as other excipients, may
also be present, if desired. Such excipients, however, must
maintain the overall tonicity of the antioxidant compound.
[0221] The terms buffer, buffer solution and buffered solution,
when used with reference to hydrogen-ion concentration or pH, refer
to the ability of a system, particularly an aqueous solution, to
resist a change of pH on adding acid or alkali, or on dilution with
a solvent. Characteristic of buffered solutions, which undergo
small changes of pH on addition of acid or base, is the presence
either of a weak acid and a salt of the weak acid, or a weak base
and a salt of the weak base. An example of the former system is
acetic acid and sodium acetate. The change of pH is slight as long
as the amount of hydroxyl ion added does not exceed the capacity of
the buffer system to neutralize it.
[0222] The stability of the parenteral formulation of the present
invention is enhanced by maintaining the pH of the formulation in
the range of approximately 5.0 to 9.5. Other pH ranges, for
example, include, 5.5 to 9.0, or 6.0 to 8.5, or 6.5 to 8.0, or 7.0
to 7.5.
[0223] The buffer used in the practice of the present invention is
selected from any of the following, for example, an acetate buffer,
a phosphate buffer or glutamate buffer, the most preferred buffer
being a phosphate buffer.
[0224] Carriers or excipients can also be used to facilitate
administration of the compound. Examples of carriers and excipients
include calcium carbonate, calcium phosphate, various sugars such
as lactose, glucose, or sucrose, or types of starch, cellulose
derivatives, gelatin, polyethylene glycols and physiologically
compatible solvents.
[0225] A stabilizer may be included in the present formulation but,
and importantly, is not needed. If included, however, a stabilizer
useful in the practice of the present invention is a carbohydrate
or a polyhydric alcohol. The polyhydric alcohols include such
compounds as sorbitol, mannitol, glycerol, and polyethylene glycols
(PEGs). The carbohydrates include, for example, mannose, ribose,
trehalose, maltose, inositol, lactose, galactose, arabinose, or
lactose.
[0226] Suitable stabilizers include, for example, polyhydric
alcohols such as sorbitol, mannitol, inositol, glycerol, xylitol,
and polypropylene/ethylene glycol copolymer, as well as various
polyethylene glycols (PEG) of molecular weight 200, 400, 1450,
3350, 4000, 6000, and 8000).
[0227] The United States Pharmacopeia (USP) states that
anti-microbial agents in bacteriostatic or fungistatic
concentrations must be added to preparations contained in multiple
dose containers. They must be present in adequate concentration at
the time of use to prevent the multiplication of microorganisms
inadvertently introduced into the preparation while withdrawing a
portion of the contents with a hypodermic needle and syringe, or
using other invasive means for delivery, such as pen injectors.
Antimicrobial agents should be evaluated to ensure compatibility
with all other components of the formula, and their activity should
be evaluated in the total formula to ensure that a particular agent
that is effective in one formulation is not ineffective in another.
It is not uncommon to find that a particular agent will be
effective in one formulation but not effective in another
formulation.
[0228] A preservative is, in the common pharmaceutical sense, a
substance that prevents or inhibits microbial growth and may be
added to a pharmaceutical formulation for this purpose to avoid
consequent spoilage of the formulation by microorganisms. While the
amount of the preservative is not great, it may nevertheless affect
the overall stability of the antioxidant compound. Thus, even
selection of a preservative can be difficult.
[0229] While the preservative for use in the practice of the
present invention can range from 0.005 to 1.0% (w/v), the preferred
range for each preservative, alone or in combination with others,
is: benzyl alcohol (0.1-1.0%), or m-cresol (0.1-0.6%), or phenol
(0.1-0.8%) or combination of methyl (0.05-0.25%) and ethyl or
propyl or butyl (0.005%-0.03%) parabens. The parabens are lower
alkyl esters of para-hydroxybenzoic acid.
[0230] A detailed description of each preservative is set forth in
"Remington's Pharmaceutical Sciences" as well as Pharmaceutical
Dosage Forms: Parenteral Medications, Vol. 1, 1992, Avis et al. For
these purposes, the crystalline trientine dihydrochloride salt may
be administered parenterally (including subcutaneous injections,
intravenous, intramuscular, intradermal injection or infusion
techniques) or by inhalation spray in dosage unit formulations
containing conventional non-toxic pharmaceutically-acceptable
carriers, adjuvants and vehicles.
[0231] It may also be desirable to add sodium chloride or other
salt to adjust the tonicity of the pharmaceutical formulation,
depending on the tonicifier selected. However, this is optional and
depends on the particular formulation selected. Parenteral
formulations must be isotonic or substantially isotonic otherwise
significant irritation and pain would occur at the site of
administration.
[0232] The desired isotonicity may be accomplished using sodium
chloride or other pharmaceutically acceptable agents such as
dextrose, boric acid, sodium tartrate, propylene glycol, polyols
(such as mannitol and sorbitol), or other inorganic or organic
solutes. Generally, the composition is isotonic with the blood of
the subject.
[0233] If desired, the parenteral formulation may be thickened with
a thickening agent such as methyl cellulose. The formulation may be
prepared in an emulsified form, either water in oil or oil in
water. Any of a wide variety of pharmaceutically acceptable
emulsifying agents may be employed including, for example, acacia
powder, a non-ionic surfactant or an ionic surfactant.
[0234] It may also be desirable to add suitable dispersing or
suspending agents to the pharmaceutical formulation these may
include, for example, aqueous suspensions such as synthetic and
natural gums i.e. tragacanth, acacia, alginate, dextran, sodium
carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone or
gelatin.
[0235] The vehicle of greatest importance for parenteral products
is water. Water of suitable quality for parenteral administration
must be prepared either by distillation or by reverse osmosis. Only
by these means is it possible to separate adequately various
liquid, gas and solid contaminating substances from water. Water
for injection is the preferred aqueous vehicle for use in the
pharmaceutical formulation of the present invention. The water may
be purged with nitrogen gas to remove any oxygen or free radicals
of oxygen from the water.
[0236] It is possible that other ingredients may be present in the
parenteral pharmaceutical formulation of the present invention.
Such additional ingredients may include wetting agents, oils (e.g.,
a vegetable oil such as sesame, peanut or olive), analgesic agents,
emulsifiers, antioxidants, bulking agents, tonicity modifiers,
metal ions, oleaginous vehicles, proteins (e.g., human serum
albumin, gelatin or proteins) and a zwitterion (e.g., an amino acid
such as betaine, taurine, arginine, glycine, lysine and histidine).
Such additional ingredients, of course, should not adversely affect
the overall stability of the pharmaceutical formulation of the
present invention.
[0237] Containers are also an integral part of the formulation of
an injection and may be considered a component, for there is no
container that is totally insoluble or does not in some way affect
the liquid it contains, particularly if the liquid is aqueous.
Therefore, the selection of a container for a particular injection
must be based on a consideration of the composition of the
container, as well as of the solution, and the treatment to which
it will be subjected.
[0238] In order to permit introduction of a needle from a
hypodermic syringe into a multiple-dose vial and provide for
resealing as soon as the needle is withdrawn, each vial is sealed
with a rubber closure held in place by an aluminum band.
[0239] Stoppers for glass vials, such as, West 4416/50, 4416/50
(Teflon faced) and 4406/40, Abbott 5139 or any equivalent stopper
can be used as the closure for the dose vial. These stoppers pass
the stopper integrity test when tested using patient use patterns,
e.g., the stopper can withstand at least about 100 injections.
[0240] Each of the components of the pharmaceutical formulation
described above is known in the art and is described in
Pharmaceutical Dosage Forms: Parenteral Medications, Vol. 1, 2nd
ed., Avis et al. Ed., Mercel Dekker, New York, N.Y. 1992, which is
incorporated by reference in its entirety herein.
[0241] The manufacturing process for the above formulation involves
compounding, sterile filtration and filling steps. The compounding
procedure, may for example, involve the dissolution of ingredients
in a specific order, such as the preservative first followed by the
stabilizer/tonicity agents, buffers and then the antioxidant
compound or dissolving all of the ingredients forming the
parenteral formulation at the same time. An example of one method
of preparing a parenteral formulation for administration is the
dissolution of the antioxidant compound form, for example,
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:2), in water and
diluting the resultant mixture in a phosphate buffered saline.
[0242] Alternatively, parenteral formulations of the present
invention are prepared by mixing the ingredients following
generally accepted procedures. For example, the selected components
may be mixed in a blender or other standard device to produce a
concentrated mixture which may then be adjusted to the final
concentration and viscosity by the addition of water, a thickening
agent, a buffer, 5% human serum albumin or an additional solute to
control tonicity.
[0243] Alternatively, the antioxidant compound can be packaged as a
dry solid and/or powder to be reconstituted with a solvent to yield
a parenteral formulation in accordance with the present invention
for use at the time of reconstitution.
[0244] In addition the manufacturing process may include any
suitable sterilization process when developing the parenteral
formulation of the present invention. Typical sterilization
processes include filtration, steam (moist heat), dry heat, gases
(e.g., ethylene oxide, formaldehyde, chlorine dioxide, propylene
oxide, beta-propiolacctone, ozone, chloropicrin, peracetic acid
methyl bromide and the like), radiant exposure and aseptic
handling.
[0245] Suitable routes of parenteral administration include
intravenous, intramuscular, subcutaneous, intradermal, sub dermal,
intraarticular, intrathecal, intraperitoneal, and the like. The
intravenous route of administration is preferred. Mucosal delivery
is also permissible. The dose and dosage regimen will depend upon
the weight and health of the subject.
[0246] Pharmaceutically acceptable carriers, excipients, diluents,
complexation agents, or additives may be chosen so as to, for
example, enhance the stability of the antioxidant compound,
facilitate synthesis or formulation of a pharmaceutical
formulation, and/or to enhance the bioavailability of the
antioxidant compound.
[0247] For example, carrier molecules such as cyclodextrin and
derivatives thereof are well known in the art for their potential
as complexation agents capable of altering the physicochemical
attributes of drug molecules. For example, cyclodextrins may
stabilize (both thermally and oxidatively), reduce the volatility
of, and alter the solubility of, active agents with which they are
complexed. Cyclodextrins are cyclic molecules composed of
glucopyranose ring units which form toroidal structures. The
interior of the cyclodextrin molecule is hydrophobic and the
exterior is hydrophilic, making the cyclodextrin molecule water
soluble. The degree of solubility can be altered through
substitution of the hydroxyl groups on the exterior of the
cyclodextrin. Similarly, the hydrophobicity of the interior can be
altered through substitution, though generally the hydrophobic
nature of the interior allows accommodation of relatively
hydrophobic guests within the cavity. Accommodation of one molecule
within another is known as complexation and the resulting product
is referred to as an inclusion complex. Examples of cyclodextrin
derivatives include sulfobutylcyclodextrin, maltosylcyclodextrin,
hydroxypropylcyclodextrin, and salts thereof.
[0248] Methods of forming pharmaceutically acceptable composition
comprising an inclusion complex of a mitochondrially targeted
antioxidant compound, in this case Mitoquinone-C10 in complex with
.beta.-cyclodextrin, are disclosed herein in Example 1 and Example
7. Methods of forming pharmaceutically acceptable compositions
comprising an inclusion complex of a preferred mitochondrially
targeted antioxidant compound Mitoquinone-C10 mesylate in complex
with .beta.-cyclodextrin are disclosed herein in Example 9 and
Example 10.
[0249] The physicochemical properties, including for example the
pharmaceutical properties, of the antioxidant compound-cyclodextrin
complex can be varied by, for example, variation of the molar ratio
of antioxidant compound to cyclodextrin, or variation of the
cyclodextrin itself. For example, for the preferred antioxidant
compounds of general formula I, the molar ratio of antioxidant
compound to cyclodextrin (antioxidant compound:cyclodextrin) may be
from about 10:1 to about 1:10, from about 5:1 to about 1:5, from
about 4:1 to about 1:4, from about 2:1 to about 1:2, or about 1:1.
In a further example, the preferred molar ratio of exemplary
antioxidant compound Mitoquinone-C10 to cyclodextrin is 1:2 and the
cyclodextrin is .beta.-cyclodextrin.
[0250] Alternatively, the pharmaceutically appropriate form of
antioxidant compound may be formulated so as to enhance the
stability and bioavailability of the antioxidant compound. For
example, enteric coatings may be applied to tablets to prevent the
release of the antioxidant compound in the stomach either to reduce
the risk of unpleasant side effects or to maintain the stability of
the antioxidant compound which might otherwise be subject to
degradation by exposure to the gastric environment. Most polymers
that are used for this purpose are polyacids that function by
virtue of the fact that their solubility in aqueous medium is
pH-dependent, and they require conditions with a pH higher than
normally encountered in the stomach.
[0251] One preferable type of oral controlled release structure is
enteric coating of a solid dosage form. Enteric coatings promote
the compounds remaining physically incorporated in the dosage form
for a specified period when exposed to gastric juice, yet the
enteric coatings are designed to disintegrate in intestinal fluid
for ready absorption. Delay of absorption is dependent on the rate
of transfer through the gastrointestinal tract, and so the rate of
gastric emptying is an important factor. For some administrations,
a multiple-unit type dosage form, such as granules, may be superior
to a single-unit type. Therefore, in one embodiment, antioxidant
compounds of the invention may be contained in an enterically
coated multiple-unit dosage form. In a more preferable embodiment,
the antioxidant compound dosage form is prepared by producing
particles having an antioxidant compound-enteric coating agent
solid on an inert core material. These granules can result in
prolonged absorption of the antioxidant compound with good
bioavailability.
[0252] Typical enteric coating agents include, but are not limited
to, hydroxypropylmethylcellulose phthalate, methacrylic
acid-methacrylic acid ester copolymer, polyvinyl acetate-phthalate
and cellulose acetate phthalate.
[0253] Examples of preferred antioxidant compounds of the present
invention and/or formulations and/or complexes thereof exhibit
advantageous pharmaceutical properties. For example, they are
readily formulatable, are chemically and physically stable, are
readily water soluble, have low hygroscopicity and exhibit good
shelf life.
[0254] The invention will now be described in more detail with
reference to the following non-limiting experimental section.
Example 1
Synthesis of Mitoquinone-C10
[0255] The following describes a preferred method of synthesis of a
preferred stable salt form of the exemplary mitochondrially
targeted antioxidant compound Mitoquinone-C10, Mitoquinone-C10
mesylate, and a cyclodextrin complex thereof.
Stage 1
##STR00027##
[0256] Step:
[0257] 1. Idebenone (A1, 0.25 kg, 0.74 mol) is dissolved in 2.5 L
of reaction grade DCM, and the mixture is then cooled to
10.+-.3.degree. C. under an inert atmosphere.
[0258] 2. Triethylamine (0.152 kg, 1.5 mol) is added in one portion
at ambient temperature and the mixture allowed to re-equilibrate to
10.+-.3.degree. C.
[0259] 3. A solution of methanesulfonyl chloride (0.094 kg, 0.82
mol) in 0.5 L of DCM is then added gradually at such a rate as to
maintain an internal temperature of approx. 10-15.degree. C. (On
this scale the addition was complete after 75 minutes).
[0260] 4. The reaction mixture is agitated for a further 15-30
minutes,
[0261] 5. IPC checked for completion by TLC (R.sub.f 0.65 5%
Ethanol/Dichloromethane).
[0262] 6. The mixture is then washed with water (0.85 L) and
saturated aqueous sodium bicarbonate solution (0.85 L).
[0263] 7. The organic layer is evaporated to a red liquid under
reduced pressure at 40-45.degree. C. After drying for an additional
2-4 hours under high vacuum at ambient temperature, the crude A2 so
obtained is used directly in the next step. Yield unknown as
solvent was trapped in the liquid.
Stage 2
##STR00028##
[0264] Step:
[0265] 1. Idebenone mesylate (A2, assume 100% yield from last step,
0.31 kg, 0.74 mol) is dissolved in 2 L of methanol and the mixture
then cooled to 0-5.degree. C. under an inert atmosphere.
[0266] 2. Sodium borohydride (0.03 kg, 0.79 mol) is added
portionwise at such a rate as to ensure that the internal
temperature doe not exceed 15.degree. C. Completion of reaction
will be accompanied by a colour change: red yellow (On this scale
the addition was complete after 20 minutes),
[0267] 3. The reaction mixture is agitated for a further 10-30
minutes,
[0268] 4. IPC checked for completion by TLC (A3 R.sub.f 0.60 5%
Ethanol/Dichloromethane, A2 R.sub.f 0.65).
[0269] 5. The mixture is then quenched with 2 L of 2M hydrochloric
acid solution and extracted three times with 1.2 L of
dichloromethane.
[0270] 6. The combined organic phases are then washed once with 1.2
L of water and dried over anhydrous magnesium sulfate (0.24
kg).
[0271] 7. The organic phase is then evaporated to a yellow/brown
syrup under reduced pressure at 40-45.degree. C. After drying for
an additional 2-8 hours under high vacuum at ambient temperature,
the crude product A3, 0.304 kg 98% yield, so obtained is used
directly in the next step.
Stage 3
##STR00029##
[0272] Step:
[0273] 1. Triphenylphosphine chunks (0.383 kg 1.46 mol) are added
to Idebenol mesylate (A3, 0.304 kg, 0.73 mol) in a suitably sized
round-bottomed flask.
[0274] 2. The flask is then attached to a rotary evaporator and the
contents heated under vacuum to a bath temperature of 80-85.degree.
C.
[0275] 3. The mixture should form a homogeneous melt at this
temperature. Once a melt has formed and degassing is no longer
evident, the vacuum is displaced with an inert atmosphere and the
mixture is spun gently in a bath set to 80-85.degree. C. for
approx. 3 days.
[0276] 4. IPC check for completion by .sup.1H and .sup.31P NMR. A
minimum of 95% conversion is required before workup can take
place.
[0277] 5. The mixture is then cooled to near room temperature and
dissolved in 0.8 L of dichloromethane.
[0278] 6. 3.2 L of ethyl acetate is then added in portions with
gentle warming to precipitate the desired product away from excess
triphenylphosphine.
[0279] 7. A small volume of solvent is removed by evaporation under
reduced pressure (to remove DCM) and the remaining mixture is then
cooled to near ambient temperature and decanted.
[0280] 8. The remaining syrupy residue is then subjected to the
same washing procedure twice more and then finally dried under high
vacuum to constant weight to afford a tan foam 0.441 kg 89% yield
(NOTE: product still contained some solvent, see nmr). A4 so
obtained is used directly in the next step.
Stage 4
##STR00030##
[0281] Step:
[0282] 1. The crude mitoQuinol mesylate salt (0.44 kg, assume 0.65
mol) is dissolved in 6 L of anhydrous DCM and the flask is purged
with oxygen.
[0283] 2. The flask contents are vigorously stirred under an oxygen
atmosphere for 30 minutes to ensure saturation of the solvent with
the gas.
[0284] 3. A 0.1 L solution of 0.65M NO.sub.2 in dry DCM (2 mol %
NO.sub.2) is added rapidly in one portion and the mixture is
vigorously stirred under an oxygen atmosphere for 4-8 hours at
ambient temperature.
[0285] 4. An IPC check for completion (by .sup.1H NMR and
optionally .sup.31P NMR) is then carried out.
[0286] 5. If the oxidation is incomplete a further 2 mol % of
NO.sub.2 as a solution in DCM is added. This should drive the
reaction to completion. IPC check as above. On this scale 8 mol %
of NO.sub.2 as a solution in DCM was required for the reaction to
reach completion.
[0287] 6. The solvent is then removed by evaporation under reduced
pressure to afford a red syrupy residue. This residue is dissolved
in 2 L of dichloromethane at 40-45.degree. C.
[0288] 7. 3.2 L of ethyl acetate is then added in portions with
gentle warming to precipitate the desired product. A small volume
of solvent is removed by evaporation under reduced pressure (to
remove DCM) and the remaining mixture is then cooled to near
ambient temperature and decanted.
[0289] 8. The oily residue is then finally dried under high vacuum
to constant weight to afford a red glass (419 g, 94% yield). A5 so
obtained is used directly in the next step.
Stage 5
##STR00031##
[0290] Step:
[0291] 1. The crude mitoQuinone mesylate salt (A5 0.419 kg) is
dissolved in 6 L of water with gentle heating at 40-43.degree.
C.
[0292] 2. The beta-cyclodextrin, 1.24 kg, is separately dissolved
in 20 L of water, with heating at 60.degree. C.
[0293] 3. These two solutions are cooled to approximately room
temperature and combined to form a homogeneous mixture. This
solution should be stored at <5.degree. C.
[0294] 4. This orange solution is then frozen at -20.degree. C. and
lyophilised in batches to constant weight (at least 48 hours).
[0295] 5. The resultant solid is then gently crushed to form a
uniform free flowing yellow/orange powder (1.433 kg).
[0296] An alternative synthetic method has been performed wherein
the oxidation step 3 of stage 4 of the synthetic method described
above was achieved by bubbling oxygen through the solution,
indicating the oxidation reaction may be driven substantially to
completion by oxidative means other than oxidation with
NO.sub.2.
Example 2
Synthesis of Mitochondrially Targeted Antioxidant Compounds
[0297] The chemical syntheses of Mitoquinone-C3, Mitoquinone-C5 and
Mitoquinone-C15 are outlined in FIG. 2 and are described below.
Nuclear magnetic resonance spectra were acquired using a Varian 300
MHz instrument. For .sup.1H-NMR tetramethylsilane was the internal
standard in CDCl.sub.3. For .sup.31P NMR 85% phosphoric acid was
the external standard. Chemical shifts (.delta.) are in ppm
relative to the standard. Elemental analyses were done by the
Campbell Microanalytical Laboratory, University of Otago.
Electrospray mass spectrometry was done using a Shimadzu
LCMS-QP800X liquid chromatography mass spectrometer. Stock
solutions were prepared in absolute ethanol and stored at
-20.degree. C. in the dark.
[0298] Mitoquinone-C3 (6). The synthetic route to Mitoquinone-C3 is
shown in FIG. 2A. The starting material, 2, 3, 4,
5-tetramethoxytoluene (1) (Lipshutz, B. H., Kim, S.-k., Mollard; P.
and Stevens, K. L. (1998) Tetrahedron 54, 1241-1253) was prepared
by reducing 2,3-dimethoxy-5-methyl-1,4-benzoquinone (CoQ.sub.0) to
the hydroquinol (Carpino, L. A., Triolo, S. A. and Berglund, R. A.
(1989) J. Org. Chem. 54, 3303-3310) followed by methylation to give
1 (Lipshutz, B. H., Kim, S.-k., Mollard, P. and Stevens, K. L.
(1998) Tetrahedron 54, 1241-1253). A solution of 1 (6.35 g, 29.9
mmol) in dry hexane (80 mL) and
N,N,N',N'-tetramethylethylenediamine (8.6 mL) was placed with a
flame-dried stirrer bar in a flame-dried Schlenk tube under
nitrogen. A hexane solution of n-butyl lithium (1.6 M, 26.2 mL) was
slowly added at room temperature and the mixture was cooled and
stirred at 0.degree. C. for 1 hr. After being cooled to -78.degree.
C., dry tetrahydrofuran (THF; 250 mL) was added, and a small
aliquot of the reaction mixture was removed, quenched with D.sub.2O
and examined by .sup.1H NMR to assure complete metallation. The
yellow suspension was then transferred to a second flame-dried
Schlenk tube containing CuCN (0.54 g, 6.03 mmol) under nitrogen at
-78.degree. C. The mixture was warmed to 0.degree. C. for 10 mins,
then cooled to -78.degree. C. and allyl bromide (3.62 mL) was added
and the reaction was stirred overnight (19 his) and allowed to warm
to room temperature. The reaction was quenched with 10% aqueous
NH.sub.4Cl (75 mL), and extracted with ether (2.times.200 mL). The
combined ethereal extracts were washed with H.sub.2O (2.times.150
mL), 10% aqueous NH.sub.4OH (200 mL) and saturated aqueous NaCl
(200 mL). The organic solvents were dried over MgSO.sub.4, filtered
and the solvent removed by rotary evaporation in vacuo to give a
crude product (7.25 g). Column chromatography on silica gel and
elution with 20% ether/hexane gave pure
1,2,3,4-tetramethoxy-5-methyl-6-(2-propenyl)benzene (2) (Yoshioka,
T., Nishi, T., Kanai, T., Aizawa, Y., Wada, K., Fujita, T. and
Horikoshi, H. (1993), Eur. Pat. Appl. EP 549366 A1) (6.05 g,
83.5%). .sup.1H NMR .delta. 5.84-5.98 (1H, m, --CH.dbd.C),
4.88-5.03 (2H, m, .dbd.CH.sub.2), 3.78, 3.80, 3.90, 3.92 (12H, s,
OMe), 3.38 (2H, d, J=7.0 Hz, Ar--CH.sub.2), 2.14 (3H, s, Ar-Me)
ppm.
[0299] A solution of 2 (8.0 g, 33.05 mmol) in dry THF (45 mL) was
added dropwise over 20 mins under argon to a stirred suspension of
9-borabicyclo[3,3,1]nonane in THF (79 mL, 39.67 mmol, 0.5 M) at
25.degree. C. The resulting solution was stirred overnight at room
temperature and for a further 2 hrs at 65.degree. C. under argon.
The mixture was then cooled to 0.degree. C. and 3 M NaOH (53 mL)
was then added dropwise followed by 30% aqueous H.sub.2O.sub.2 (53
mL). After 30 mins stirring at room temperature, the water phase
was saturated with NaCl and extracted 3 times with THF. The
combined organic fractions were washed with saturated aqueous NaCl,
dried (Na.sub.2SO.sub.4), filtered and evaporated to give an oily
residue (11.5 g) which was purified by column chromatography on
silica gel (200 g, packed with ether/hexane 1:9). Elution with
ether/hexane 1:4 gave pure
3-(2,3,4,5-tetramethoxy-6-methyl-phenyl)-propan-1-ol (3) as a
viscous, colourless oil (6.85 g, 80%). .sup.1H NMR .delta. 3.91,
3.90, 3.84, 3.79 (12H, s, OMe), 3.56 (2H, t, J=7.0 Hz,
--CH.sub.2--OH), 2.72 (2H, t, J=7.0 Hz, Ar--CH.sub.2), 2.17 (3H, s,
Ar-Me), 1.74 (2H, quintet, J=7.0 Hz, --CH.sub.2--) ppm. Anal.
calcd. for C.sub.14H.sub.22O.sub.5: C, 62.2; H, 8.2. Found: C,
62.2; H, 8.4%.
[0300] A solution of 3 (3.88 g, 15 mmol) and triethylamine (3.0 g,
30 mmol, 4.2 mL) in CH.sub.2Cl.sub.2 (50 mL) was stirred at room
temperature for 10 mins. Methanesulfonyl chloride (1.8 g, 1.20 mL,
15.75 mmol) in CH.sub.2Cl.sub.2 (50 mL) was added dropwise over 20
mins and the reaction mixture stirred at room temperature for 1 hr.
The mixture was then diluted with CH.sub.2Cl.sub.2 (50 mL) and the
organic layer was washed with H.sub.2O (5.times.100 mL), 10%
aqueous NaHCO.sub.3 (100 mL); dried (MgSO.sub.4), filtered and the
solvent removed in vacuo by rotary evaporation to afford
1-(3-methanesulfonyloxypropyl)-2-methyl-3,4,5,6-tetramethoxybenzene
(4) as a liquid (4.8 g, 95%). .sup.1H NMR .delta. 4.27 (2H, t,
J=7.0 Hz, --CH.sub.2--O--SO.sub.2-Me), 3.91, 3.89, 3.82, 3.78 (12H,
s, OMe), 3.03 (3H, s, O--SO.sub.2-Me), 2.70 (2H, t, J=7.0 Hz,
Ar--CH.sub.2--), 2.17 (3H, s, Ar-Me), 1.9 (2H, m, --CH.sub.2--)
ppm.
[0301] The crude methanesulfonate 4 (3.30 g, 9.8 mmol) was used
directly in the following reaction by mixing with a freshly ground
mixture of triphenylphosphine (4.08 g, 15.6 mmol) and NaI (7.78 g,
51.9 mmol) in a Kimax tube and sealed under argon. The mixture was
then held at 70-74.degree. C. with magnetic stirring for 3 hrs
during which time the mixture changed from a molten thick liquid
into a glassy solid. The tube was cooled to room temperature and
the residue stirred with CH.sub.2Cl.sub.2 (30 mL). The suspension
was then filtered and the filtrate evaporated in vacuo. The residue
was dissolved in a minimum amount of CH.sub.2Cl.sub.2 and
triturated with excess ether (250 mL) to precipitate the white
solid. The solid was filtered and washed with ether, dried in vacuo
to give pure
[3-(2,3,4,5-tetramethoxy-6-methyl-phenyl)-propyl]triphenylphosphonium
iodide (5) (5.69 g, 90%). .sup.1H NMR .delta. 7.82-7.65 (15H, m,
Ar--H), 3.88, 3.86, 3.74, 3.73 (12H, s, OMe), 3.76-3.88 (2H, m,
CH.sub.2--P.sup.+), 2.98 (2H, t, J=7.0 Hz, CH.sub.2--Ar), 2.13 (3H,
s, Ar-Me), 1.92-1.78 (2H, m, --CH.sub.2--) ppm. .sup.31P NMR (121.4
MHz) .delta. 25.32 ppm. Anal. calcd. for C.sub.32H.sub.36IO.sub.5P:
C, 59.8; H, 5.7; P, 4.8; Found: C, 59.8; H, 5.8; P, 4.5%.
[0302] A solution of the iodide form of 5 (4.963 g, 7.8 mmol) in
CH.sub.2Cl.sub.2 (80 mL) was shaken with 10% aqueous NaNO.sub.3 (50
mL) in a separatory funnel for 5 mins. The organic layer was
separated, dried (Na.sub.2SO.sub.4), filtered and evaporated in
vacuo to give the nitrate salt of 5 (4.5 g, 7.8 mmol, 100%), which
was dissolved in a mixture of CH.sub.3CN and H.sub.2O (7:3, 38 mL)
and stirred at 0.degree. C. in an ice bath.
Pyridine-2,6-dicarboxylic acid (6.4 g, 39 mmol) was then added
followed by dropwise addition of a solution of ceric ammonium
nitrate (21.0 g, 39 mmol) in CH.sub.3CN/H.sub.2O (1:1, 77 mL) over
5 mins. The reaction mixture was stirred at 0.degree. C. for 20
mins and then at room temperature for a further 10 mins. The
reaction mixture was then poured into H.sub.2O (200 mL) and
extracted with CH.sub.2Cl.sub.2 (200 mL), dried (Na.sub.2SO.sub.4),
filtered and evaporated in vacuo to give a crude
[3-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)propyl]-
triphenylphosphonium (6) nitrate. The total product was dissolved
in CH.sub.2Cl.sub.2 (100 mL) and shaken for 10 mins with 20%
aqueous KBr (50 mL). The organic layer was separated, dried and
evaporated in vacuo to give the bromide salt of 6 (4.1 g, 93.6%).
.sup.1H NMR .delta. 7.90-7.65 (15H, m, Ar--H), 4.15-4.05 (2H, m,
CH.sub.2--P), 3.96, 3.95, (6H, s, OMe), 2.93 (2H, t, J=7.0 Hz,
CH.sub.2--Ar), 2.15 (3H, s, Ar-Me), 1.85-1.70 (2H, m, --CH.sub.2--)
ppm. .sup.31P NMR .delta. 25.29 ppm.
[0303] A solution of 6 bromide (3.65 g, 6.5 mmol) in
CH.sub.2Cl.sub.2 (75 mL) was shaken with a 10% w/v aqueous solution
of sodium methanesulfonate (100 mL) in a separatory funnel for 5
mins. The CH.sub.2Cl.sub.2 layer was separated, dried
(Na.sub.2SO.sub.4), filtered and evaporated in vacuo to give
[3-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)propy-
l]triphenylphosphonium methanesulfonate salt (6) (3.7 g, 98%).
.sup.1H NMR .delta. 7.88-7.60 (15H, m, Ar--H), 3.93, 3.92, (6H, s,
OMe), 3.90-3.78 (2H, m, CH.sub.2--P.sup.+), 2.85 (2H, t, J=7.0 Hz,
CH.sub.2--Ar), 2.70 (3H, s, OSO.sub.2CH.sub.3), 2.09 (3H, s,
Ar-Me), 1.82-1.68 (2H, m, --CH.sub.2--) ppm. .sup.31P NMR (121.4
MHz) .delta. 25.26 ppm. Anal. calcd. for C.sub.31H.sub.33O.sub.7PS:
C, 64.1; H, 5.7; P, 5.3; S, 5.5. Found: C, 63.8; H, 5.9; S, 5.3; P,
5.2%.
[0304] Mitoquinone-C5 (14). The synthetic route to Mitoquinone-C5
is shown in FIG. 2B. Dihydropyran (46.83 g, 0.55 mol) was added to
2,3-dimethoxy-5-methyl-1,4-benzohydroquinone (CoQ.sub.0) (50 g,
0.275 mol) dissolved in acetic acid (500 mL) and stirred at room
temperature for 10 minutes. To this solution was added
BF.sub.3.Et.sub.2O (38.57 g, 0.271 mol). The resulting solution was
stirred for 18 hours at room temperature. After this time the crude
reaction mixture was poured into iced water (500 mL) and extracted
with chloroform (1000 mL). The organic extract was washed with
brine (500 mL) and dried (MgSO.sub.4). The solvent was removed in
vacuo to give the crude
2,3-dimethoxy-5-methyl-6-(tetrahydro-pyran-2-yl)-4-(tetrahydro-pyran-2-yl-
oxy)-phenol(7) as a red oil (115 g) which was used without further
purification. A solution of crude 7 (110 g) in a mixture of acetic
acid/perchloric acid (97.5:2.5, 500 mL) was hydrogenated over 5%
palladium/charcoal (5.42 g) at atmospheric pressure and room
temperature until hydrogen uptake ceased (three days). The reaction
mixture was then filtered through a pad of Celite, and the solid
residue washed with ethanol (500 mL). The combined filtrate was
divided into three equal portions and each portion added to
distilled water (1000 mL) and extracted with CH.sub.2Cl.sub.2
(2.times.200 mL). The combined organic extracts were washed with
brine (500 mL), saturated sodium bicarbonate (500 mL), brine (300
mL) and then dried (MgSO.sub.4). The mixture was then filtered and
solvents were removed in vacuo to give crude
4-acetoxy-3-(5-acetoxy-pentyl)-5,6-dimethoxy-2-methyl-phenyl
acetate (8) as a red oil (110 g) which was used in the subsequent
step without further purification. .sup.1H NMR .delta. 4.0-4.15
(2H, m, --CH.sub.2--O), 3.86 (6H, s, 2.times.OMe), 2.58 (2H, t,
J=7.0 Hz, --CH.sub.2--Ar), 2.12 (3H, s, Ar-Me), 2.06 (6H, s,
2.times.CH.sub.3--C.dbd.O), 2.02 (3H, s, CH.sub.3--C.dbd.O),
1.35-1.70 (6H, m, --CH.sub.2CH.sub.2CH.sub.2--) ppm.
[0305] Lithium aluminium hydride (8.0 g, 0.21 mol) was added to dry
THF (500 mL) in a 1 L round bottomed flask equipped with a magnetic
stirrer, reflux condenser and surrounded by a room temperature
water bath. A solution of crude 8 (74 g) in dry freshly distilled
THF (100 mL) and was added dropwise to the THF/LiALH.sub.4 mixture
over a period of 25-30 minutes. Additional dry THF (200 mL) was
added, to facilitate stirring, and the reaction was left stirring
for 3 hours at room temperature. The reaction was then quenched by
the dropwise addition of 3 M HCl (20 mL) followed by the slow
addition of distilled water (70 mL). The reaction mixture was then
filtered and the filtrate was washed with brine (2.times.300 mL),
dried (MgSO.sub.4), filtered and the solvent removed in vacuo. The
green residue remaining in the filter funnel was dissolved in 15%
HCl (500 mL) and extracted with CH.sub.2Cl.sub.2 (1.times.300 ml,
2.times.200 ml). The organic fractions were combined and washed
with brine (400 ml), dried (MgSO.sub.4), filtered and evaporated in
vacuo. This extract was combined with the material from the
filtrate workup to give crude
245-hydroxypentyl)-5,6-dimethoxy-3-methyl-benzene-1,4-diol (9)(68.3
g) as a red oil. This product 9 was purified using column
chromatography on silica gel, (600 g, packed in 10%
ether/CH.sub.2Cl.sub.2). Elution with 10% ether/CH.sub.2Cl.sub.2
gave some unreacted 8 and
2,3-dimethoxy-5-methyl-1,4-benzohydroquinone starting material.
Elution with 20% ether/CH.sub.2Cl.sub.2, gave a mixture of 9 and
the quinone 10 (14.14 g, 19% from
2,3-dimethoxy-5-methyl-1,4-benzoquinol). Compound 9 was slowly
converted to the quinone 10 on standing in air and satisfactory
elemental analysis could not be obtained. .sup.1H NMR .delta. 5.41
(1H, s, Ar--OH), 5.38 (1H, s, Ar--OH), 4.88 (6H, s,
2.times.Ar--OMe), 3.65 (2H, t, J=6.3 Hz, CH.sub.2--OH), 2.61 (2H,
t, J=6.4 Hz, Ar--CH.sub.2), 2.14 (3H, s, Ar-Me), 1.42-1.68 (6H, m,
3.times.--CH.sub.2--) ppm.
[0306] A solution of the quinol 9 (7.5 g, 27.7 mmol) in
CH.sub.2Cl.sub.2 (150 mL) was saturated with oxygen gas at
atmospheric pressure and a solution of NO.sub.2 in CH.sub.2Cl.sub.2
(1 ml, 1.32 M) was added. The reaction was stirred at room
temperature under an oxygen atmosphere for 18 hours by which time
TLC (40% ether/CH.sub.2Cl.sub.2) showed the formation of the
quinone
2-(5-hydroxypentyl)-5,6-dimethoxy-3-methyl-[1,4]benzoquinone (10)
was complete. The solvent was then removed in vacuo to afford the
product 10 (Yu, C. A. and Yu, L. (1982) Biochemistry 21,
4096-4101)(7.40 g) as a red oil. .sup.1H NMR .delta. 3.99 (6H, s,
2.times.Ar--OMe), 3.65 (2H, t, J=6.3 Hz, CH.sub.2--OH), 2.47 (2H,
t, J=6.3 Hz, Ar--CH.sub.2), 2.01 (3H, s, Ar-Me), 1.52-1.60 (2H, m,
--CH.sub.2--), 1.37-1.43 (4H, m, --CH.sub.2CH.sub.2--) ppm.
[0307] A solution of 10 (7.40 g, 27.3 mmol) in CH.sub.2Cl.sub.2
(150 mL) and triethylamine (5.46 g, 5.46 mmol) was prepared and a
solution of methanesulfonyl chloride (2.48 g, 30 mmol) in
CH.sub.2Cl.sub.2 (50 mL) was added over 30 minutes with stirring.
After stirring for an additional 1.5 hours at room temperature the
reaction mixture was washed with distilled water (5.times.100 mL),
saturated sodium bicarbonate (150 mL) and dried (MgSO.sub.4). The
mixture was filtered and solvent removed in vacuo to give the crude
methanesulfonate (9.03 g) as a red oil. .sup.1H NMR .delta. 4.19
(2H, t, J=7.5 Hz, --CH.sub.2--OMs), 3.95 (6H, s, 2.times.Ar--OMe),
2.98 (3H, s, OSO.sub.2CH.sub.3), 2.44 (2H, t, J=7.5 Hz,
Ar--CH.sub.2--), 1.98 (3H, s, Ar-Me), 1.75 (2H, quintet, J=7.5 Hz,
--CH.sub.2--), 1.38-1.48 (4H, m, --CH.sub.2--CH.sub.2--) ppm. The
methanesulfonate was dissolved in 10% (w/v) NaI in acetone (100 mL)
and stirred for 44 hours at room temperature. The mixture was then
concentrated in vacuo and H.sub.2O (100 mL) was added to the
residue. The mixture was extracted with CH.sub.2Cl.sub.2
(3.times.70 mL) and the combined organic extracts were washed with
brine, dried (MgSO.sub.4), filtered and the solvent was removed in
vacuo to give crude
2-(5-iodopentyl)-5,6-dimethoxy-3-methyl-[1,4]benzoquinone (11).
This product was purified by column chromatography on silica gel
(150 g). Elution with CH.sub.2Cl.sub.2 and 10%
ether/CH.sub.2Cl.sub.2 gave pure 11 (7.05 g, 69%) as a red oil.
.sup.1H NMR .delta. 3.99 (6H, s, 2.times.Ar--OMe), 3.18 (2H, t,
J=6.9 Hz, CH.sub.2--I), 2.47 (2H, t, J=7.2 Hz, Ar--CH.sub.2), 2.02
(3H, s, Ar-Me), 1.85 (2H, quintet, J=7.5 Hz, --CH.sub.2--),
1.38-1.48 (4H, m, --CH.sub.2--CH.sub.2--) ppm. Anal. calcd. For
C.sub.14H.sub.19IO.sub.4: C, 44.5; H, 5.1; I, 33.6. Found: C, 44.6;
H, 5.1; I, 33.4%.
[0308] A solution of 11 (1.14 g, 2.87 mmol) in methanol (20 ml) was
treated with NaBH.sub.4 (0.16 g, 4.3 mmol) and the mixture turned
colourless within 1 minute. After 5 minutes at room temperature 5%
aqueous HCl (100 mL) was added and the solution was extracted with
CH.sub.2Cl.sub.2 (2.times.50 mL). The organic fractions were
combined, dried (MgSO.sub.4), filtered and the solvent removed in
vacuo to give 12 (1.15 g, 100%) as an oxygen sensitive yellow oil
which was used without delay. .sup.1H NMR .delta. 5.36, 5.31 (2H,
s, Ar--OH), 3.89 (6H, s, 2.times.Ar--OMe), 3.20 (2H, t, J=7.5 Hz,
--CH.sub.2--I), 2.62 (2H, t, J=7.5 Hz, --CH.sub.2--Ar), 2.15 (3H,
s, Me), 1.82-1.92 (2H, m, --CH.sub.2--), 1.45-1.55 (4H, m,
--CH.sub.2--CH.sub.2--) ppm. A mixture of 12 (1.15 g, 2.87 mmol)
and triphenylphosphine (1.2 g, 4.31 mmol) was placed in a Kimax
tube with a stirrer bar. The tube was flushed with argon, firmly
sealed and heated and stirred for 14 hrs at 70.degree. C. A dark
solid was formed which dissolved in CH.sub.2Cl.sub.2 (10 mL) and
triturated in ether (200 mL) and the white precipitate formed was
filtered rapidly. The precipitate, which became sticky on exposure
to air, was re-dissolved in CH.sub.2Cl.sub.2 and evaporated in
vacuo to give the crude product
[5-(2,5-dihydroxy-3,4-dimethoxy-6-methyl-phenyl)-pentyl]triphenylphosphon-
ium iodide (13) (2.07 g, 115%) as a brown oil. The material was not
stable on storage for extended periods and was used as soon as
practicable for subsequent reactions. .sup.1H NMR .delta. 7.84-7.68
(15H, m, Ar--H), 5.45 (1H, s, Ar--OH), 5.35 (1H, s, Ar--OH), 3.89
(3H, s, Ar--OMe), 3.87 (3H, s, Ar--OMe), 3.65 (2H, m,
--CH.sub.2.sup.-+PPh.sub.3), 2.54 (2H, t, J=7.0 Hz, Ar--CH.sub.2),
2.08 (3H, s, Ar-Me), 1.65-1.75 (2H, m, --CH.sub.2--), 1.45-1.55
(4H, m, --CH.sub.2CH.sub.2--) ppm. .sup.31P NMR .delta. 25.43
ppm.
[0309] A solution of 13 (2.07 g) in CH.sub.2Cl.sub.2 (50 mL) was
saturated with oxygen gas and a solution of NO.sub.2 in
CH.sub.2Cl.sub.2 (0.5 mL, 1.32 M) was added. The reaction was then
stirred at room temperature under an oxygen atmosphere for 18 hrs.
The solvent was removed in vacuo to afford the crude product
[5-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)pentyl]triphe-
nylphosphonium iodide (14) as a red oil. This residue was
re-dissolved in CH.sub.2Cl.sub.2 (10 mL) and triturated in ether
(200 mL) to give an initial yellow precipitate which congealed into
a red oil in a few minutes. The solvents were decanted and the
precipitate dissolved in CH.sub.2Cl.sub.2 and the solvent removed
in vacuo to give the product (14) (1.866 g) as a red oil. An
aliquot (0.880 g) of 14 was purified by column chromatography on
silica gel (20 g). Elution with CH.sub.2Cl.sub.2 gave some
unidentified purple coloured material. Elution with 5%
ethanol/CH.sub.2Cl.sub.2 gave the pure iodide product 14 (0.606 g)
as a red oil. .sup.1H NMR .delta. 7.84-7.68 (15H, m, Ar--H) 3.98
(6H, s, 2.times.Ar--OMe), 3.65 (2H, m, CH.sub.2--P.sup.+), 2.40
(2H, t, J=7.5 Hz, Ar--CH.sub.2), 2.00 (3H, s, Ar-Me), 1.71 (4H, m,
--CH.sub.2--), 1.43 (2H, m, --CH.sub.2--) ppm. .sup.31P NMR (121.4
MHz) .delta. 25.47 ppm. Anal. calcd. for C.sub.32H.sub.36IO.sub.4P:
C, 59.8; H, 5.7; I, 19.8; P, 4.8; Found: C, 60.0; H, 5.3; I, 19.7;
P, 4.7%.
[0310] Mitoquinone-C15 (16). The synthetic route to Mitoquinone-C15
is shown in FIG. 2C. A solution of K.sub.2S.sub.2O.sub.8 (0.450 g,
1.66 mmol) in H.sub.2O (25 mL) was added dropwise over 2.5 hours to
a stirred suspension of AgNO.sub.3 (0.262 g, 1.54 mmol),
16-hydroxyhexadecanoic acid (0.408 g, 1.50 mmol), and
2,3-dimethoxy-5-methyl-1,4-benzoquinone (0.271 g, 1.49 mmol) in
H.sub.2O:CH.sub.3CN (1:1, 36 mL) held at 75.degree. C. After
stirring for 30 minutes the mixture was cooled and extracted with
ether (4.times.30 mL). The combined organic phase was washed with
H.sub.2O (2.times.100 mL), NaHCO.sub.3 (1 M, 2.times.50 mL) and
saturated NaCl (2.times.50 mL). The organic phase was dried
(Na.sub.2SO.sub.4), filtered and concentrated in vacuo to give a
red oil (0.444 g). Column chromatography of the crude oil (silica
gel, 15 g) and elution with mixtures of CH.sub.2Cl.sub.2 and ether
(0%, 5% 20%) gave
2-(15-hydroxypentadecyl)-5,6-dimethoxy-3-methyl-[1,4]benzoquinone
(15) (0.192 g, 33%) as a red oil. .sup.1H NMR .delta. 3.99, 3.98
(6H, s, OMe), 3.64 (2H, t, J=6.5 Hz, --CH.sub.2OH), 2.45 (2H, t,
J=7.5 Hz, --CH.sub.2-ring), 1.4-1.2 (26H, m,
--(CH.sub.2).sub.13--). Anal. Calcd. for C.sub.24H.sub.40O.sub.5:
C, 70.6; H, 9.9. Found: C, 70.5; H, 9.8%.
[0311] A mixture of triphenylphosphine (0.066 g, 0.25 mmol),
Ph.sub.3PHBr (0.086 g, 0.25 mmol) and 15 (0.101 g, 0.25 mmol) was
stirred under argon in a sealed Kimax tube at 70.degree. C. for 24
hours, by which time it had turned into a viscous red oil. The
residue was dissolved in minimum CH.sub.2Cl.sub.2 (0.5 mL) and
poured into ether (10 mL) to produce a red oily precipitate. The
solvents were then decanted the residue was dissolved in CH.sub.3OH
(0.5 mL) and diluted with H.sub.2O (10 mL) containing 48% HBr (1
drop). A red precipitate formed and after the precipitate had
settled the supernatant was poured off and the residue was washed
with H.sub.2O (5 mL). The residue was then dissolved in ethanol (5
mL) and the solvent removed in vacuo. The residue was redissolved
in CH.sub.2Cl.sub.2 (0.5 mL), diluted with ether (5 mL) and the
solvent was decanted and the residue placed in a vacuum system (0.1
mbar) for 24 hours to give
[15-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)pentadecyl]t-
riphenylphosphonium bromide (16) (0.111 g, 61%) as a yellow foam
which turned to a red oil on contact with air. .sup.1H NMR (299
MHz) .delta. 7.6-8.0 (15H, m, Ar--H), 3.89 (6H, s, OMe), 3.9 (2H,
m, --CH.sub.2--P), 2.6 (2H, m, --CH.sub.2-ring), 1.7-1.1 (26H, m,
--(CH.sub.2).sub.13--) ppm. .sup.31P NMR (121.4 MHz) .delta. 25.71
ppm. Electrospray mass spectrometry found (M.sup.+) 653, calculated
for C.sub.42H.sub.54O.sub.4P.sup.+653. Combustion analytical
results were unsatisfactory due to inconsistent levels of solvent
inclusion.
Example 3
Properties of Exemplary Mitochondrially Targeted Antioxidant
Compounds
[0312] The present invention recognises that, in order to be
suitable in a wide variety of applications, for example the
formulation of dosage forms such as tablets, there is advantage in
being able to form a crystalline or solid form of the
mitochondrially targeted antioxidant compound. Similarly, it is
believed, without wishing to be bound by any theory, that the
antioxidant functionality of the compounds of the present invention
are at least in part determined by their physicochemical
properties.
[0313] The partition coefficients for a variety of antioxidant
compounds are shown in Table 1. Octan-1-ol/PBS partition
coefficients were determined by adding 400 nmol of the compound to
2 ml PBS-saturated octan-1-ol and mixing for 30 min at 37.degree.
C. with 2 ml octan-1-ol saturated PBS. The concentrations of the
compound in the two phases were measured by UV absorption at 268 nm
and quantitated from standard curves of the compound in octan-1-ol
saturated PBS, or PBS-saturated octan-1-ol (Kelso, G. F., Porteous,
C. M., Coulter, C. V., Hughes, G., Porteus, W. K., Ledgerwood, E.
C., Smith, R. A. J., and Murphy, M. P., 2001, J Biol Chem 276,
4588; Smith, R. A. J., Porteous, C. M., Coulter, C. V., and Murphy,
M. P. 1999 Eur J Biochem 263, 709). Stock solutions of compounds
were prepared in absolute ethanol and stored at -20.degree. C. in
the dark. [.sup.3H]TPMP was from American Radiolabelled Chemicals
Inc, (MO, USA).
[0314] Of particular note is the low partition coefficient of
compounds with small numbers of carbon atoms bridging the
antioxidant moiety and the phosphonium. For example, a compound
within the present invention, herein referred to as Mitoquinone-C3,
which has a 3 carbon bridge has a partition coefficient
approximately 50-fold lower than that observed for the related
compound, Mitoquinone-C10 (Table 1).
TABLE-US-00001 TABLE I Partition coefficients of antioxidants and
related compounds Compound Partition coefficient
Methyltriphenylphosphonium (TPMP) .sup.a0.35 .+-. 0.02 MitoVit E
.sup.b7.4 .+-. 1.6 4-Bromobutyltriphenylphosphonium .sup.b3.83 .+-.
0.22 4-Iodobutyltriphenylphosphonium .sup.c4.0 .+-. 0.4
Mitoquinone-C15 Mitoquinone-C10 .sup.a160 .+-. 9 Mitoquinone-C5
13.9 .+-. 1.9 Mitoquinone-C3 .sup.c2.8 .+-. 0.3 .alpha.-Tocopherol
.sup.b27.4 .+-. 1.9 Bromodecylubiquinone .sup.d310 .+-. 60
Idebenone .sup.d3.1 .times. 10.sup.3 Decylubiquinone .sup.d3.1
.times. 10.sup.5 Coenzyme Q.sub.0 .sup.d1.33 Coenzyme Q.sub.1
.sup.d409 Coenzyme Q.sub.2 .sup.d4.44 .times. 10.sup.4 Ubiquinone
(Coenzyme Q.sub.10) .sup.d1.82 .times. 10.sup.20 Ubiquinol
.sup.d4.53 .times. 10.sup.20 Decylubiquinol .sup.d7.91 .times.
10.sup.5 Idebenol .sup.d7.82 .times. 10.sup.3 Data.sup.a-c are
octan-1-ol/phosphate buffered saline partition coefficients
determined at 25.degree. C. or 37.degree. C. as described above, or
octanol/water partition coefficients.sup.d calculated using
Advanced Chemistry Development (ACD) Software Solaris V4.67 as
described in Jauslin, M. L., Wirth, T., Meier, T., and Schoumacher,
F., 2002, Hum Mol Genet 11, 3055. .sup.aKelso, G. F., Porteous, C.
M., Coulter, C. V., Hughes, G., Porteus, W. K., Ledgerwood, E. C.,
Smith, R. A. J., and Murphy, M. P., 2001, J Biol Chem 276, 4588.
.sup.bSmith, R. A. J., Porteous, C. M., Coulter, C. V., and Murphy,
M. P. 1999 Eur J Biochem 263, 709. .sup.cSmith, R. A. J., Porteous,
C. M., Gane, A. M., and Murphy, M. P. 2003 Proc Nat Acad Sci 100,
9, 5407.
[0315] From their octan-1-ol/PBS partition coefficients it is clear
that Mitoquinone-C3, Mitoquinone-C5, Mitoquinone-C10 and
Mitoquinone-C15 span a wide range of hydrophobicities. That of
Mitoquinone-C3 is similar to the simple, relatively water soluble
TPMP cation, while that of Mitoquinone-C15 indicates that it has
very low water solubility. Alkyltriphenylphosphonium cations such
as Mitoquinone are reported to adsorb onto phospholipid bilayers
with the cation at the level of the carboxylic acid groups while
the hydrophobic alkyl group penetrates into the hydrophobic core of
the membrane. It is believed that the longer the methylene bridge
the deeper the antioxidant ubiquinol will penetrate into the
hydrophobic core of membrane. The maximum extent to which
penetration into one leaflet of the membrane we believe will occur
for these compounds is illustrated in FIG. 3, which shows the
Mitoquinone variants aligned with a typical phospholipid. This
modelling indicates that the ubiquinol moiety of Mitoquinone-C3
only penetrates close to the membrane surface while those of
Mitoquinone-C10 and Mitoquinone-C15 penetrate close to the core of
the phospholipid bilayer.
[0316] We have synthesised a series of antioxidant compounds with a
range of hydrophobicities and depths of penetration into the
phospholipid bilayer.
Example 4
Mitochondrial Uptake of Mitochondrially Targeted Compounds
[0317] To demonstrate that mitochondrial targeting is effective,
the uptake by mitochondria in response to the membrane potential of
exemplary antioxidant compounds Mitoquinone-C3, Mitoquinone-C5,
Mitoquinone-C10, and Mitoquinone-C15 was determined.
[0318] To measure the uptake of antioxidant compounds by energised
mitochondria, an ion-selective electrode was constructed (Smith, R.
A., Kelso, G. F., James, A. M. and Murphy, M. P. (2004) Meth.
Enzymol. 382, 45-67; Davey, G. P., Tipton, K. F. and Murphy, M. P.
(1992) Biochem. J. 288, 439-443; Kamo, N., Muratsugu, M., Hongoh,
R. and Kobatake, Y. (1979) J. Membr. Biol. 49, 105-121). The
electrode and a Ag/AgCI reference electrode were inserted through
the air-tight Perspex lid of a stirred and thermostatted 3 ml
incubation chamber at 30.degree. C., provided with an injection
port for the addition of substrates. To measure antioxidant
compound uptake, rat liver mitochondria (1 mg protein/ml) were
incubated at 30.degree. C. in KCl medium (120 mM KCl, 10 mM HEPES,
pH 7.2, 1 mM EGTA) and nigericin (1 .mu.g/ml) and rotenone (8
.mu.g/ml). Succinate (10 mM) and FCCP (500 nM) were added where
indicated. The output from the ion-selective electrode was passed
to a PowerLab Data acquisition system via a front-end pH amplifier
and analyzed using Chart software, all from ADInstruments.
[0319] Rat liver mitochondria were prepared by homogenisation
followed by differential centrifugation in ice-cold buffer
containing 250 mM sucrose, 5 mM Tris-HCl, 1 mM EGTA, pH 7.4
(Chappell, J. B. and Hansford, R. G. (1972) in: Subcellular
components: preparation and fractionation, pp. 77-91 (Birnie, G.
D., Ed.) Butterworths, London). The protein concentration was
determined by the biuret assay using BSA as a standard (Gornall, A.
G., Bardawill, C. J. and David, M. M. (1949) J. Biol. Chem. 177,
751-766). Mitochondrial membrane potential was measured by adding
500 nM TPMP supplemented with 50 nCi [.sup.3H]TPMP to mitochondria
suspended in KCl medium (120 mM KCl, 10 mM HEPES, pH 7.2, 1 mM
EGTA) at 25.degree. C. (Brand, M. D. (1995) in: Bioenergetics--a
practical approach, pp. 39-62 (Brown, G. C. and Cooper, C. E.,
Eds.) IRL, Oxford). After incubation, the mitochondria were
pelleted by centrifugation and the amounts of [.sup.3H]TPMP in the
supernatant and pellets were quantitated by scintillation counting
and the membrane potential calculated assuming a mitochondrial
volume of 0.5 .mu.l/mg mitochondrial protein and a TPMP binding
correction of 0.4 (Brown, G. C. and Brand, M. D. (1985) Biochem. J.
225, 399-405).
[0320] We constructed ion-selective electrodes to measure their
steady-state concentrations (Smith, R. A., Kelso, G. F., James, A.
M. and Murphy, M. P. (2004) Meth. Enzymol. 382, 45-67; Davey, G.
P., Tipton, K. F. and Murphy, M. P. (1992) Biochem. J. 288,
439-443; Kamo, N., Muratsugu, M., Hongoh, R. and Kobatake, Y.
(1979) J. Membr. Biol. 49, 105-121). The response of these
electrodes to simple triphenylphosphonium cations such as TPMP is
Nernstian, with a linear response of electrode voltage to
log.sub.10[cation concentration] and a slope of .about.60 mV at
30.degree. C. (Davey, G. P., Tipton, K. F. and Murphy, M. P. (1992)
Biochem. J. 288, 439-443; Kamo, N., Muratsugu, M., Hongoh, R. and
Kobatake, Y. (1979) J. Membr. Biol. 49, 105-121). The most
hydrophilic compound, Mitoquinone-C3, also gave a Nernstian
electrode response with a slope close to 60 mV at concentrations
above 10 W. This is illustrated in FIG. 4A, right hand side, by the
logarithmic electrode response to sequential additions of 1 pM
Mitoquinone-C3 in the absence of mitochondria. For Mitoquinone-C5,
Mitoquinone-C10 and Mitoquinone-C15 the electrode also responded
rapidly and stably to sequential additions in the absence of
mitochondria (FIGS. 4B, 4C, and 4D, respectively, right hand side
panels). However in these cases the electrode responses were not
Nernstian, we believe due to the greater hydrophobicity of these
compounds. Even so, for all four antioxidant compounds the
ion-selective electrode enabled the measurement of the free
concentrations of the compounds and thus their uptake by
mitochondria in real time.
[0321] To measure antioxidant compound uptake, mitochondria were
added to the electrode chamber in the presence of rotenone to
prevent formation of a membrane potential (left side of FIG. 4). We
then made five sequential 1 .mu.M additions of antioxidant compound
to calibrate the electrode response, followed by the respiratory
substrate succinate to generate a membrane potential. Mitochondrial
energization led to the rapid uptake of all the antioxidant
compound variants by the mitochondria, and subsequent addition of
the uncoupler FCCP abolished the membrane potential and led to
their rapid release from the mitochondria (FIG. 4A-D, left side).
These experiments clearly show mitochondrial membrane
potential-dependent uptake of Mitoquinone-C3, Mitoquinone-C5, and
Mitoquinone-C10. While Mitoquinone-C15 was also taken up by
mitochondria on induction of a membrane potential, the electrode
response to Mitoquinone-C15 in the presence of mitochondria was
weaker, noisier and more prone to drift. This contrasts with the
electrode response to Mitoquinone-C15 in the absence of
mitochondria (cf. right hand panels), and is due to its low free
concentrations in the presence of mitochondria.
[0322] The extent of antioxidant compound binding to deenergised
mitochondria was then determined (FIG. 4, right hand side). For
these experiments the antioxidant compound variants were first
added to the electrode chamber and then mitochondria were added in
the presence of rotenone to prevent formation of a membrane
potential. The decrease in antioxidant compound concentration on
adding mitochondria is due to binding of antioxidant compound to
the deenergised mitochondria. The subsequent addition of succinate
to generate a membrane potential indicates the membrane potential
dependent uptake of the compounds, which is then reversed by
addition of FCCP to abolish the membrane potential.
[0323] The free concentration of Mitoquinone-C3 was unaffected by
addition of mitochondria, indicating that negligible amounts of
Mitoquinone-C3 bound to deenergised mitochondria (FIG. 4A, right
hand side). The FCCP-sensitive uptake of mitoquinone-C3 on
energization with succinate was about 3.7 nmol mitoquinone-C3/mg
protein, corresponding to an accumulation ratio of
2.times.10.sup.3. This is consistent with that expected from the
Nernst equation and a mitochondrial membrane potential of about 180
mV, allowing for corrections for intramitochondrial binding.
[0324] For Mitoquinone-C5 there was some binding of the compound to
the deenergised mitochondria (.about.0.6 nmol/mg protein), however
this was negligible compared to its subsequent uptake on
energization with succinate, of about 2.8 nmol Mitoquinone-C5/mg
protein, corresponding to an accumulation ratio of about
1.4.times.10.sup.3 (FIG. 4B, right hand side).
[0325] For Mitoquinone-C10 there was significant binding to
deenergised mitochondria of about 2.6 nmol Mitoquinone-C10, and
this was followed by further uptake of about 1 nmol/mg protein on
addition of succinate (FIG. 4C, right hand side).
[0326] Nearly all of the free Mitoquinone-C15 was bound to the
deenergised mitochondria, but there was some further uptake on
energization with succinate. The membrane potential-dependent
uptake of Mitoquinone-C15 was clear on the left hand panel of FIG.
4D, where the electrode response was highly sensitive to enable
measurement of the small amount of free Mitoquinone-C15 when the
electrode was calibrated in the presence of mitochondria. In
contrast, the uptake of Mitoquinone-C15 is difficult to see on the
right hand side of FIG. 4D, where the electrode response was far
less sensitive to enable measurement of Mitoquinone-C15 in the
absence of mitochondria.
[0327] These experiments show that the length of the methylene
bridges of the antioxidant compounds at least in part determines
their extents of adsorption to mitochondrial membranes (right hand
side of FIG. 4). The adsorption ranges from negligible for
Mitoquinone-C3, to almost complete binding for Mitoquinone-C15. On
addition of Mitoquinone-C15 to deenergised mitochondria essentially
all the compound binds, distributed across both surfaces of the
inner and outer membranes. When a membrane potential is induced we
believe there will be significant redistribution of the compound to
the matrix-facing surface of the inner membrane from the outer
surface of the inner membrane and from the outer membrane. In
summary, all the antioxidant compound variants are taken up into
mitochondria driven by the membrane potential, and the longer the
methylene bridge the greater their adsorption to phospholipid
bilayers.
Example 5
Antioxidant Efficacy of Exemplary Mitochondrially Targeted
Compounds
[0328] The compounds of the invention are also highly effective
against oxidative stress. To measure antioxidant efficacy, the
ability of the antioxidant compounds to prevent lipid peroxidation
in mitochondria, measured from the accumulation of TBARS in
mitochondria exposed to ferrous iron and hydrogen peroxide (FIG.
5).
[0329] To quantitate lipid peroxidation, the TBARS assay was used.
Rat liver mitochondria (2 mg protein/ml) were incubated in 0.8 ml
medium containing 100 mM KCl, 10 mM Tris-HCl, pH 7.6 at 37.degree.
C., supplemented with either 10 mM succinate and 8 mg/ml rotenone,
or an ATP regenerating system of 2.5 mM ATP, 1 mM
phosphoenolpyruvate and 4 U/ml pyruvate kinase. The mitochondria
were then exposed to oxidative stress by addition of 50 mM
FeCl.sub.2/300 mM H.sub.2O.sub.2 for 15 min at 37.degree. C. After
the incubation, 64 ml 2% (w/v) butylated hydroxytoluene in ethanol
was added, followed by 200 ml 35% (v/v) HClO.sub.4 and 200 ml 1%
(w/v) thiobarbituric acid. Samples were then incubated for 15 min
at 100.degree. C., centrifuged (5 min at 12,000.times.g) and the
supernatant transferred to a glass tube. After addition of 3 ml
water and 3 ml butan-1-ol, samples were vortexed, and the two
phases allowed to separate. 200 ml aliquots of the organic layer
were then analyzed in a fluorometric plate reader
(.lamda..sub.Ex=515 nm; .lamda..sub.Em=553 nm) for thiobarbituric
acid reactive species (TBARS) and compared with a malondialdehyde
(MDA) standard curve prepared from 0.01-5 mM
1,1,3,3-tetraethoxypropane (Kelso, G. F., Porteous, C. M., Coulter,
C. V., Hughes, G., Porteous, W. K., Ledgerwood, E. C., Smith, R. A.
J. and Murphy, M. P. (2001) J. Biol. Chem. 276, 4588-4596).
[0330] For mitochondria energised with succinate, the background
level of TBARS was negligible, but it increased to about 3.75 nmol
MDA/mg protein on exposure to oxidative stress (FIG. 5A; filled
bars). High concentrations (5 .mu.M) of any of the antioxidant
compounds largely prevented the accumulation of TBARS, while the
simple cation TPMP did not. This confirms that it was the ubiquinol
side chain of the Mitoquinone antioxidant compounds that was
responsible for the antioxidant action, and not any non-specific
interactions of the cation with mitochondria.
[0331] In these experiments, succinate will both maintain a
membrane potential to drive the uptake of the cations into
mitochondria, and also recycle the ubiquinone form of the
Mitoquinone antioxidant compounds to the active antioxidant
ubiquinol form (Kelso, G. F., Porteous, C. M., Coulter, C. V.,
Hughes, G., Porteous, W. K., Ledgerwood, E. C., Smith, R. A. J. and
Murphy, M. P. (2001) J. Biol. Chem. 276, 4588-4596). To see if
reduction by the respiratory chain was required for the antioxidant
efficacy of the Mitoquinone antioxidant compounds, we incubated
mitochondria in the presence of ATP and an ATP regenerating system.
ATP hydrolysis and reversal of the mitochondrial ATP synthase led
to extensive proton pumping which built up a membrane potential
similar to that generated by succinate (FIG. 5B). This will lead to
the same Mitoquinone antioxidant compound uptake as for
mitochondria energised by succinate, but now the Mitoquinone
antioxidant compounds will no longer be recycled to their active
ubiquinol forms by the respiratory chain. The Mitoquinone
antioxidant compounds were ineffective at preventing lipid
peroxidation in mitochondria energised by ATP hydrolysis (FIG. 5a,
white bars), compared with the dramatic protection seen in
mitochondria energised by succinate (FIG. 5b, black bars).
Therefore reduction of Mitoquinone antioxidant compounds by the
respiratory chain, as well as accumulation by the mitochondrial
membrane potential are required for the antioxidant efficacy of the
Mitoquinone antioxidant compounds.
[0332] Lower levels of lipid peroxidation were observed in the
control samples of mitochondria energised with succinate, compared
to those energised with ATP (FIG. 5A). This is due to the
protective antioxidant effect of the endogenous mitochondrial
Coenzyme Q pool which is held reduced by in the presence of
succinate but oxidised in the presence of ATP (James, A. M., Smith,
R. A. and Murphy, M. P. (2004) Arch. Biochem. Biophys. 423, 47-56;
Ernster, L., Forsmark, P. and Nordenbrand, K. (1992) Biofactors 3,
241-8). In summary, all the Mitoquinone antioxidant compounds
require activation by the respiratory chain to be effective
antioxidants.
[0333] For FIG. 5A a single concentration of 5 pM was used for all
the Mitoquinone antioxidant compounds. To compare their relative
antioxidant efficacies we titrated the compounds in the presence of
succinate: a typical titration is shown in FIG. 5C. This experiment
suggests that the antioxidant efficacy of these compounds
correlates with the length of the methylene bridge. To quantitate
this we calculated the IC50 values for the prevention of lipid
peroxidation by the four exemplary Mitoquinone antioxidant
compounds (FIG. 4D). These measurements confirmed that the order of
antioxidant efficacy was:
Mitoquinone-C15>Mitoquinone-C10>Mitoquinone-C5>Mitoquinone-C3.
[0334] All the Mitoquinone antioxidant compounds were accumulated
into mitochondria driven by the mitochondrial membrane potential.
For the most hydrophobic compound, Mitoquinone-C15, this effect was
largely masked by extensive binding to phospholipid bilayers. All
of the compounds were effective antioxidants and for persistent
antioxidant activity over 15 minutes all required the action of the
respiratory chain to recycle the Mitoquinone antioxidant compound
to its active antioxidant form after having detoxified lipid
peroxidation intermediates.
Example 6
Effect of Mitochondrially Targeted Antioxidant Compounds on Cardiac
Haemodynamics and Mitochondrial Function
[0335] The effect of administration of mitochondrially targeted
antioxidant compounds, in particular Mitoquinone-C10 and
Mitoquinone-C3, on cardiac function was assessed using the
Langendorf isolated heart perfusion model. Rats were assigned to
the following four administration groups: Control (placebo), TPMP
(methyltriphenyl phosphonium), Mitoquinone-C10, and Mitoquinone-C3.
Following the treatment period, rats were humanely sacrificed and
the isolated hearts were connected to the Langendorf isolated
perfusion system. This system uses retro-perfusion through the
aorta to maintain the heart while cardiac function is measured.
Left ventricular pressure was measured with a left ventricular
balloon. Coronary flow was also measured.
[0336] FIG. 6 depicts the coronary flow at 10 mmHg left ventricular
pressure for each of the treatment groups. Coronary flow was
measured pre-ischaemia and again at zero minutes, 20 minutes, 40
minutes and 60 minutes following induction of ischaemia. A one way
ANOVA with bonferroni post hoc test was performed. Significance
versus pre-ischaemic control: * P<0.05; ** P<0.01; ***
P<0.001. Significance versus respective time control: .dagger.
P<0.05; .dagger..dagger. P<0.01; .dagger..dagger..dagger.
P<0.001.
[0337] The results show that treatment with Mitoquinone-C10
significantly reduces the ischaemia-induced reduction in coronary
flow. Mitoquinone-C3 has a lesser but still significant effect at
the later time points. The absence of any effect with
administration of TPMP indicate that it is the antioxidant moiety
of Mitoquinone-C10 and Mitoquinone-C3, and not the
triphenylphosphonium cation, that is responsible for the effects
observed with the mitochondrially targeted antioxidant
compounds.
[0338] FIG. 7 depicts the effects of treatment on left ventricular
diastolic pressure at 10 mmHg. Left ventricular diastolic pressure
was measured prior to induction of ischaemia and again at zero
minutes, 20 minutes, 40 minutes and 60 minutes following the
induction of ischaemia. Statistical analysis was an ANOVA on ranks
with Dunns post hoc test. Significance verses pre-ischaemic
control: * P<0.05. .dagger. represents P<0.05 versus 60 min
post ischaemic control. The results show that treatment with
Mitoquinone-C10 results in a statistically significant increase in
left ventricular diastolic pressure verses untreated rats, reducing
the ischaemia-induced reduction in left ventricular diastolic
pressure. The absence of any effect with administration of TPMP
indicate that it is the antioxidant moiety of Mitoquinone-C10, and
not the triphenylphosphonium cation, that is responsible for the
effects observed with the mitochondrially targeted antioxidant
compounds.
[0339] The effect of administration of Mitoquinone-C10 and
Mitoquinone-C3 on heart rate was then determined. FIG. 8 depicts
the heart rate for each of the treatment groups pre-ischaemia, and
zero minutes, 20 minutes, 40 minutes and 60 minutes following the
induction of ischaemia. Results shown are one way ANOVA followed by
bonferroni post hoc test. *** represents P<0.001 versus
pre-ischaemic control. .dagger..dagger. represents P<0.05 versus
respective post ischaemic control. The results show that treatment
with Mitoquinone-C10 significantly reduces the ischaemia induced
reduction in heart rate compared to control rats. The absence of
any effect with administration of TPMP indicate that it is the
antioxidant moiety of Mitoquinone-C10, and not the
triphenylphosphonium cation, that is responsible for the effects
observed with the mitochondrially targeted antioxidant
compounds.
[0340] Cardiac function was further assessed by determining the
effect of administration of mitochondrially targeted antioxidant
compounds on the rate of contraction and relaxation of the heart.
FIG. 9A depicts the rate of contraction in each of the four
treatment groups pre-ischaemia, and zero minutes, 20 minutes, 40
minutes and 60 minutes following induction of ischaemia. FIG. 9B
depicts the rate of relaxation in each of the four treatment groups
pre-ischaemia, and zero minutes, 20 minutes, 40 minutes and 60
minutes following induction of ischaemia. In each case ANOVA was
performed on ranks with Dunns post hoc test performed. * represents
significance with P<0.05 verses pre-ischaemia control. .dagger.
represents significance with P<0.05 versus respective post
ischaemic time controls. .dagger..dagger. represents significance
with P<0.01 versus respective post ischaemic time control.
[0341] The results show that administration of Mitoquinone-C10 has
a statistically significant effect, reducing the ischaemia-induced
reduction in the rate of contraction and relaxation of the left
ventricle when compared to control rats.
[0342] The above data clearly show the beneficial effect of
administration of mitochondrially targeted antioxidant compounds on
cardiac function. In order to determine whether the observed
effects on cardiac function were due to the effect of the
mitochondrial targeted antioxidant compound on mitochondrial
function, mitochondrial activity pre-ischaemia and post-ischaemia
was assessed for each of the treatment groups. FIG. 10A depicts
NAD.sup.+ linked respiratory function of mitochondria pre and
post-ischaemia for each of the four treatment groups. FIG. 10B
presents FAD linked respiratory function pre and post-ischaemia for
each of the four treatment groups. *** represents significance with
P<0.001 versus pre-ischaemic control. .dagger..dagger..dagger.
represents significance with P<0.001 versus post ischaemic
control.
[0343] These data show that Mitoquinone-C10 has a statistically
significant beneficial effect on mitochondrially respiratory
function following ischaemia compared to control rats. These
results support the conclusion that the effects of administration
of mitochondrially targeted antioxidant compounds on cardiac
function is due to a protective effect on mitochondrial
function.
Example 7
Stability of Mitoquinone-C10 Complexes with .beta.-Cyclodextrin
[0344] In preformulation studies Mitoquinone-C10 as the bromide
salt was found to degrade over time in the solid state when stored
at 25.degree. C., 50% RH and 40.degree. C., 75% RH. The objective
of the present study was to establish whether the solid state
stability of Mitoquinone-C10 could be improved by complexing with
.beta.-cyclodextrin.
[0345] Mitoquinone-C10 batch no. 6 and idebenone were supplied by
Industrial Research Limited (New Zealand). .beta.-cyclodextrin (lot
no. 70P225) was purchased from ISP technologies Inc. NaCl, NaH PU
and methanol (HPLC) were purchased from BDH.
[0346] Solid State Stability Study of Pure Mitoquinone-C10
[0347] Samples of Mitoquinone-C10 (approximately 5 mg) were
accurately weighed into clear bottles and exposed to 25.degree. C.,
50% RH, 40.degree. C., 75% RH and 4.degree. C. over silica. The
bottles were removed after 1, 2, 4, 8, 16, 32 and 64 days and
analyzed for Mitoquinone-C10 by a validated HPLC method using
Mitoquinone-C10 stored at -20.degree. C. over silica as
control.
[0348] Preparation of Mitoquinone-C10: .beta.-Cyclodextrin
Complexes
[0349] Three complexes with different molar ratios (Mitoquinone-C10
bromide: .beta.-cyclodextrin 1:1, 1:2 and 1:4) were prepared using
Mitoquinone-C10 batch no. 6.
[0350] Preparation of .beta.-Cyclodextrin Solution in Water
[0351] .beta.-cyclodextrin (1.1397 g, equal to 1.0361 g after
correction for moisture content) was accurately weighed and
dissolved in double distilled water by sonicating for 10 min. The
volume was made up to 100 ml with water.
[0352] Preparation of Mitoquinone-C10: .beta.-cyclodextrin (1:1
Molar Ratio) Complex
[0353] An ethanolic solution of Mitoquinone-C10 bromide (90 mg
equal to 59.95 mg of Mitoquinone-C10) was evaporated under nitrogen
on a hot plate maintained at 40-50.degree. C. for 8 min.
.beta.-cyclodextrin solution (10 ml) and double distilled water (30
ml) were added to the beaker which was then sonicated for 40
min.
[0354] Preparation of Mitoquinone-C10: .beta.-Cyclodextrin (1:2
Molar Ratio) Complex
[0355] An ethanolic solution of Mitoquinone-C10 bromide (89.8 mg
equal to 59.82 mg of Mitoquinone-C10) was evaporated under nitrogen
on a hot plate maintained at 37-45.degree. C. for 10 min followed
by 3 min at 50.degree. C. .beta.-cyclodextrin solution (20 ml) and
double distilled water (20 ml) were added to the beaker which was
then sonicated for 30 min.
[0356] Preparation of Mitoquinone-C10: .beta.-Cyclodextrin (1:4
Molar Ratio) Complex
[0357] An ethanolic solution of Mitoquinone-C10 bromide (90 mg
equal to 59.95 mg of Mitoquinone-C10) was evaporated under nitrogen
on a hot plate maintained at 37-50.degree. C. for 12 min.
.beta.-cyclodextrin solution (40 ml) was added to the beaker which
was then sonicated for 20 min.
[0358] All the above solutions were frozen by storing at
-18.degree. C. overnight. The frozen solutions were freeze-dried
for 2 days using the LABCONO freeze drier. The lyophilized
compounds were stored at -20.degree. C.
[0359] Differential Scanning Calorimetry of the Freeze-Dried
Mitoquinone-C10: .beta.-Cyclodextrin complexes
[0360] Differential scanning calorimetry (DSC) of the three
freeze-dried complexes was carried out using a Perkin Elmer
Differential Scanning calorimeter PYRIS-1. A Mitoquinone-C10 sample
was prepared by evaporating an ethanolic solution under nitrogen
gas at 35-50.degree. C. for 10 min.
[0361] Aluminium pans (No. 0219-0041, supplied by Perkin-Elmer)
were used. The analysis was carried out under nitrogen purge. Empty
pans were used to set the baseline.
[0362] Scanning temperature range was 50-160.degree. C. with an
initial hold at 50.degree. C. for 1 min followed by an increase of
10.degree. C./min up to 160.degree. C.
[0363] HPLC Assay
[0364] An HPLC method for Mitoquinone-C10 was developed using
methanol and 0.01M sodium dihydrogen phosphate (85:15) as mobile
phase at a flow rate of 1 ml/min and using UV-VIS detection at 265
nm. The internal standard was idebenone. The column was Prodigy
ODS3 100A (Phenomenex) with particle size 5.mu.. Later this method
was modified after the arrival of a new column. The mobile phase
used in the modified method was methanol and 0.01M sodium
dihydrogen phosphate (80:20). This method was validated.
[0365] Interference by .beta.-cyclodextrin in the HPLC method was
checked before analyzing the Mitoquinone-C10: .beta.-cyclodextrin
complexes. It was shown that .beta.-cyclodextrin does not interfere
in the Mitoquinone-C10 HPLC assay.
[0366] Stability Study of Mitoquinone-C10: .beta.-Cyclodextrin
Complexes
[0367] As there were three complexes of Mitoquinone-C10 with
.beta.-cyclodextrin, the amount of Mitoquinone-C10 in 5 mg samples
from the different complexes was different. In order to expose
equal amounts of Mitoquinone-C10 in all three complexes, different
weights of complexes were taken: 4 mg of 1:1 complex containing
1.473 mg of Mitoquinone-C10; 6.5 mg of 1:2 complex containing 1.469
mg of Mitoquinone-C10; and 11.5 mg of 1:4 complex containing 1.467
mg of Mitoquinone-C10 were taken and used in the stability study as
per the Standard Operating Procedure.
[0368] Aliquots of HPLC water (1.5 ml) were added to each sample
bottle to completely dissolve the Mitoquinone-C10:
.beta.-cyclodextrin complexes. Aliquots (50 .mu.l) of these
solutions were diluted to 1 ml with water. Aliquots (100 .mu.l) of
these diluted solutions of Mitoquinone-C10: .beta.-cyclodextrin
complexes were vortexed with 200 .mu.l of a 10 .mu.g/ml solution of
internal standard in methanol. The samples were centrifuged for 10
min at 10000 rpm and 50 .mu.l of the supernatants injected into the
HPLC system. A standard curve was prepared using solutions of
Mitoquinone-C10 in the concentration range 2.5 to 120 .mu.g/ml
containing 5 mg/ml solutions of .beta.-cyclodextrin.
[0369] All the compounds were slighty orange-yellow in colour and
very fluffy in appearance. The colour was not uniform and was more
concentrated towards the bottom of the freeze drying flasks.
The results of DSC are given as follows:
[0370] Mitoquinone-C10: When a pure sample of Mitoquinone-C10 was
analyzed, peaks were observed above 120.degree. C. With one sample
of Mitoquinone-C10, two prominent peaks were observed between
130.degree. C. and 140.degree. C. When another sample was analyzed,
no such prominent peaks were observed but small peaks were observed
above 120.degree. C. After analysis, the pans were cut and the
samples examined. The samples were dark green to black in colour in
both cases.
[0371] .beta.-Cyclodextrin: There was a broad peak between
70.degree. C. and 85.degree. C.
[0372] Mitoquinone-C10: .beta.-cyclodextrin (1:1) complex: No
significant peaks were observed. After analysis the pan was cut and
examined. The sample colour had undergone a slight change to light
brown (not a significant change).
[0373] Mitoquinone-C10: .beta.-cyclodextrin (1:2) complex: No
significant peaks were observed. After analysis, no colour change
in the sample was observed.
[0374] Mitoquinone-C10: .beta.-cyclodextrin (1:4) complex: No
significant peaks were observed but a very small exothermic peak
was observed at 120.degree. C. After analysis, no colour change in
the sample was observed.
[0375] The appearance of peaks in the Mitoquinone-C10 pure sample
indicates that changes in the compound are taking place with
temperature. However, as there were many peaks and also colour
changes in the sample, these could have arisen due to degradation.
When a second sample of Mitoquinone-C10 was analyzed, it gave a
different thermogram to the first sample. In the case of the
complexes, there were no significant peaks or any colour
changes.
[0376] The results of the solid state stability study of pure
Mitoquinone-C10 (batch no. 3) are given in Table 2 and FIG. 11.
TABLE-US-00002 TABLE 2 Solid state stability of Mitoquinone-C10
(batch no. 3). Clear Glass Day Day Day Day Day Day Bottles Day 1 2
4 8 16 32 64 40.degree. C., 75% 98.90 101.9 102.8 94.07 83.22 76.70
67.25 RH 25.degree. C., 50% 95.11 97.46 95.06 97.52 102.8 40.76
18.37 RH 5.degree. C., silica 97.04 102.8 92.97 95.67 98.37 67.36
63.70 gel Solid state stability of Mitoquinone-C10 (batch no. 3) in
the absence of light at 40.degree. C., 75% RH; 25.degree. C., 50%
RH and 5.degree. C. over blue silica gel. Data are means of two
values expressed as percentage of original content.
[0377] Due to the significant instability at 25.degree. C., 50% RH
compared to 40.degree. C., 75% RH, the stability study was repeated
at 25.degree. C., 50% RH with Mitoquinone-C10 batch no. 4. The
second stability study was conducted both in clear and amber
bottles and the results are given in Table 3 and FIG. 12.
TABLE-US-00003 TABLE 3 Solid state stability of Mitoquinone-C10
(batch no. 4) Time (days) 1 2 4 8 16 32 64 Clear Glass Bottles
88.21 93.19 92.65 93.10 94.47 62.05 57.94 Dark Amber Glass 94.84
94.52 100.28 97.65 98.03 61.48 58.66 Bottles Solid state stability
of Mitoquinone-C10 (batch no. 4) was measured in the absence of
light at 25.degree. C., 50% RH. Data are means of three values
expressed as percentage of the initial content.
[0378] Both batches (batches no. 3 and 4) of Mitoquinone-C10
supplied by the Chemistry Department showed a sudden drop in
content after 16 days. However, for batch no. 4 the degradation was
not as great after 32 to 64 days compared to batch no. 3. Also it
was observed whether the bottles were clear or amber had no effect
on Mitoquinone-C10 stability.
[0379] The Mitoquinone-C10 supplied from IRL was used for the
preparation of the Mitoquinone-C10: .beta.-cyclodextrin complexes.
The Mitoquinone-C10 supplied from IRL was a reddish-yellow syrup in
ethyl alcohol. The stability of the Mitoquinone-C10:
.beta.-cyclodextrin complexes is given in Table 4 and in FIGS. 13,
14 and 15. Because of the small amounts of Mitoquinone-C10:
.beta.-cyclodextrin complexes available for study, there are no
results for day 1 and day 4.
TABLE-US-00004 TABLE 4 Solid state stability of Mitoquinone-C10:
.beta.-cyclodextrin complexes Time (days) 2 8 16 32 64 1:1 complex
4.degree. C., silica 106.38 110.97 101.71 101.71 102.68 25.degree.
C., 50% RH 95.65 93.00 101.15 101.15 108.89 40.degree. C., 75% RH
129.22 108.77 113.48 113.49 89.25 1:2 complex 4.degree. C., silica
105.48 101.23 105.08 111.21 101.16 25.degree. C., 50% RH 108.16
95.46 105.41 108.55 99.78 40.degree. C., 75% RH 115.99 110.22
114.03 101.50 99.44 1:4 complex 4.degree. C., silica 105.10 115.86
100.25 107.63 107.63 25.degree. C., 50% RH 111.46 116.03 96.61
92.40 92.40 40.degree. C., 75% RH 108.85 100.01 87.34 71.13 71.13
Solid state stability of Mitoquinone-C10: .beta.-cyclodextrin
complexes in the absence of light at 40.degree. C., 75% RH;
25.degree. C., 50% RH and 5.degree. C. over blue silica gel. Data
are means of two values expressed as percentage.
[0380] The results show that Mitoquinone-C10 can effectively form
complexes with .beta.-cyclodextrin, and can be stabilized by
complexing with .beta.-cyclodextrin. The results show that
Mitoquinone-C10 in the 1:1 and 1:2 .beta.-cyclodextrin complexes
was stable under various conditions. The results also show that the
stability of Mitoquinone-C10 in the 1:4 complex was decreased
relative to the stability of Mitoquinone-C10 in the 1:1 and 1:2
.beta.-cyclodextrin complexes.
Example 8
Stability Studies of Mitoquinone-C10 Mesylate
Solution Stability of Mitoquinone-C10 Mesylate
[0381] The solution stability of Mitoquinone-C10 mesylate was
determined in five solvents; water, 0.01M HCl, 0.01M NaOH, IPB (pH
7.4) and 50% MeOH at two temperatures 25.degree. C. and 40.degree.
C., under two atmospheric conditions, air and nitrogen, for 125
days as per the Applicant's standard operating procedure.
[0382] Mitoquinone-C10 mesylate solutions (100 .mu.g/ml) in the
five solvents were prepared by dilution of a stock solution of 1
mg/ml Mitoquinone-C10 mesylate in water. Solutions (5 ml) were
placed in glass vials, flushed with air or nitrogen, sealed and
placed on storage. Aliquots (0.25 ml) were collected at 0, 1, 2, 4,
8, 16, 32, 64 and 125 days and the concentration of Mitoquinone-C10
determined by HPLC.
[0383] The results are given in Table 5. The stability of
Mitoquinone-C10 mesylate in 0.01M NaOH is not included because
Mitoquinone-C10 mesylate decomposed in this solvent within 15
minutes. The results show that (a) solution stability is
independent of the atmosphere above the solution and (b)
temperature has a significant effect on the stability of
Mitoquinone-C10 in all solvents except HCl.
TABLE-US-00005 TABLE 5 Solution stability of Mitoquinone-C10
mesylate in 4 different solvents under different conditions. Time
(Days) Conditions 1 2 4 8 16 32 64 125 Water, Air, 99.6 98.3 98.1
91.2 93.8 98.0 94.7 91.7 25.degree. C. Water, Air, 98.1 95.0 94.6
92.1 92.1 91.5 58.3 21.8 40.degree. C. 0.01M HCl, 103.7 107.6 102.8
98.3 98.8 98.6 98.6 92.5 Air, 25.degree. C. 0.01M HCl, 98.4 98.8
99.2 96.5 100.6 104.9 94.0 82.6 Air, 40.degree. C. IPB, Air, 95.6
95.8 98.6 94.5 93.7 90.4 89.6 100.3 25.degree. C. IPB, Air, 95.7
95.5 94.1 92.3 91.2 89.5 68.7 40.0 40.degree. C. 50% MeOH, 97.7
97.1 106.9 103.6 104.5 98.6 102.8 98.0 Air, 25.degree. C. 50% MeOH,
99.2 98.8 99.4 98.5 100.5 101.5 81.9 60.5 Air, 40.degree. C. Water,
106.7 114.9 96.8 96.7 99.7 97.6 100.8 92.7 N.sub.2, 25.degree. C.
Water, 97.0 97.5 98.3 93.0 90.4 87.3 59.8 22.0 N.sub.2, 40.degree.
C. 0.01M HCl, 102.7 112.0 103.6 98.8 101.7 98.9 99.6 93.1 N.sub.2,
25.degree. C. 0.01M HCl, 99.3 99.6 100.2 98.3 100.4 102.9 94.1 88.8
N.sub.2, 40.degree. C. IPB, N.sub.2, 99.6 96.5 96.9 95.3 96.6 91.9
90.9 97.5 25.degree. C. IPB, N.sub.2, 92.8 90.9 92.7 91.2 93.3 88.1
68.0 40.3 40.degree. C. 50% MeOH, 101.2 97.3 105.1 104.2 102.0
105.0 101.0 100.2 N.sub.2, 25.degree. C. 50% MeOH, 99.8 100.5 99.3
98.9 101.5 103.6 83.8 63.6 N.sub.2, 40.degree. C. Data are the mean
of two values expressed as percentage of the time zero value. The
solution stability of Mitoquinone-C10 mesylate in four solvents is
also shown in FIGS. 16, 17, 18, and 19.
Solid State Stability of Mitoquinone-C10 Mesylate
[0384] Solid state stability of Mitoquinone-C10 mesylate was
studied in the absence of light under three different conditions;
40.degree. C., 75% RH; 25.degree. C., 50% RH; and 4.degree. C. over
blue silica gel as per the Applicant's standard operating
procedure.
[0385] A known weight of Mitoquinone-C10 mesylate was put in clear
glass bottles and stored under different conditions. Duplicate
samples were withdrawn at 1, 2, 4, 8, 16, 32, 64 and 125 days and
the concentration of Mitoquinone-C10 mesylate determined by HPLC
after dissolving the samples in water. The results are given in
Table 6 and in FIG. 20.
[0386] Mitoquinone-C10 mesylate was stable (<10% decomposition)
at 4.degree. C. over silica gel for 125 days and at 25.degree.
C./50% RH for 60 days.
TABLE-US-00006 TABLE 6 Solid state stability of Mitoquinone-C10
mesylate at 40.degree. C., 75% RH; 25.degree. C., 50% RH; and
4.degree. C. over blue silica gel. Time (Days) 1 2 4 8 16 32 64 125
4.degree. C. over 101.6 103.4 102.4 108.2 113.5 96.6 98.2 96.0
silica 25.degree. C./50% 109.2 110.7 110.2 108.1 107.6 95. 91.6
73.7 RH 40.degree. C./75% 98.0 101.7 101.3 98.5 93.1 86.1 82.1 59.9
RH The data are the mean of two values expressed as percentage of
the time zero value.
Example 9
Stability Studies of Mitoquinone-C10 Mesylate-.beta.-Cyclodextrin
(1:2) Complex
Solution Stability of Mitoquinone-C10 Mesylate-(3-Cyclodextrin
(1:2) Complex
[0387] The solution stability of Mitoquinone-C10
mesylate-.beta.-cyclodextrin (1:2) complex was determined in five
solvents; water, 0.01M HCl, 0.01M NaOH, IPB (pH7.4) and 50% MeOH at
two temperatures 25.degree. C. and 40.degree. C., under two
atmospheric conditions, air and nitrogen, for 64 days as per the
Applicant's standard operating procedure.
[0388] Mitoquinone-C10 mesylate-3-cyclodextrin (1:2) complex
solutions (100 .mu.g/ml as Mitoquinone-C10 mesylate) in the five
solvents were prepared by dilution of a stock solution of
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:2) complex (1 mg/ml
as Mitoquinone-C10 mesylate) in water. Solutions (5 ml) were placed
in glass vials, flushed with air or nitrogen, sealed and placed on
storage. Aliquots (0.25 ml) were collected at 0, 1, 2, 4, 8, 16,
32, 64 and 125 days and the concentration determined by HPLC.
[0389] The results are given in Table 7 and in FIGS. 21, 22, 23,
and 24. The stability of Mitoquinone-C10
mesylate-.beta.-cyclodextrin (1:2) complex in 0.01M NaOH is not
included because Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:2)
complex decomposed in this solvent within 15 minutes. The results
show that (a) solution stability is independent of the atmosphere
above the solution and (b) temperature has a significant effect on
the stability of Mitoquinone-C10 mesylate in the 1:2 complex with
.beta.-cyclodextrin in all solvents except HCl.
TABLE-US-00007 TABLE 7 Solution stability of Mitoquinone-C10
mesylate-.beta.-cyclodextrin (1:2) complex in 4 different solvents
under different conditions. Time (Days) Conditions 1 2 4 8 16 32 64
125 Water, Air, 25.degree. C. 106.5 103.3 96.7 98.1 97.9 97.5 100.0
105.8 Water, Air, 40.degree. C. 102.0 98.7 101.3 101.4 92.8 80.5
55.9 17.2 0.01M HCl, Air, 25.degree. C. 96.4 100.5 99.9 98.6 101.5
97.6 104.5 101.6 0.01M HCl, Air, 40.degree. C. 96.6 98.4 107.1
104.0 100.4 96.4 101.0 96.2 IPB, Air, 25.degree. C. 97.4 94.3 98.3
96.1 107.5 97.3 97.3 99.5 IPB, Air, 40.degree. C. 95.2 93.2 97.8
98.8 91.7 87.5 74.2 55.9 50% MeOH, Air, 25.degree. C. 97.8 96.2
95.8 97.5 102.8 98.3 99.7 104.5 50% MeOH, Air, 40.degree. C. 99.5
98.1 106.5 106.5 102.9 92.6 84.6 64.6 Water, N.sub.2, 25.degree. C.
101.1 98.3 102.4 103.5 109.3 96.8 100.3 91.3 Water, N.sub.2,
40.degree. C. 100.6 100.3 104.8 101.6 94.1 78.1 52.8 14.9 0.01M
HCl, N.sub.2, 25.degree. C. 100.1 100.7 97.9 101.0 103.0 100.6
104.3 100.0 0.01M HCl, N.sub.2, 40.degree. C. 98.6 96.8 103.7 104.2
100.6 97.8 100.3 95.14 IPB, N.sub.2, 25.degree. C. 102.0 97.9 99.6
95.6 104.6 93.9 96.9 98.7 IPB, N.sub.2, 40.degree. C. 92.1 93.7
95.2 93.1 90.3 86.6 73.9 54.8 50% MeOH, N.sub.2, 25.degree. C.
105.0 96.0 94.1 96.1 106.4 97.0 100.0 105.7 50% MeOH, N.sub.2,
40.degree. C. 98.3 98.9 104.2 105.3 99.4 94.9 88.8 64.4 Data are
the mean of two values expressed as percentage of the time zero
value.
Solid State Stability of Mitoquinone-C10
Mesylate-.beta.-Cyclodextrin (1:2) Complex
[0390] Solid state stability of Mitoquinone-C10
mesylate-.beta.-cyclodextrin (1:2) complex was studied in the
absence of light under three different conditions; 40.degree. C.,
75% RH; 25.degree. C., 50% RH; and 4.degree. C. over blue silica
gel as per the Applicant's standard operating procedure.
[0391] A known weight of Mitoquinone-C10 mesylate-3-cyclodextrin
(1:2) complex was put in clear glass bottles and stored under
different conditions. Duplicate samples were withdrawn at 1, 2, 4,
8, 16, 32, 64 and 125 days and the concentration of Mitoquinone-C10
mesylate determined by HPLC after dissolving the samples in water.
The results are given in Table 8 and in FIG. 25. The results show
that Mitoquinone-C10 mesylate was stable in Mitoquinone-C10
mesylate-.beta.-cyclodextrin (1:2) complex at 4.degree. C. over
blue silica gel and at 25.degree. C., 50% RH. At 40.degree. C., 75%
RH, 37% of the Mitoquinone-C10 mesylate was degraded in
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:2) complex on
storage for 64 days.
TABLE-US-00008 TABLE 8 Solid state stability of Mitoquinone-C10
mesylate-.beta.-cyclodextrin (1:2) complex at 40.degree. C., 75%
RH; 25.degree. C., 50% RH and 4.degree. C. over blue silica gel.
Time (Days) 1 2 4 8 16 32 64 125 4.degree. C. over 97.6 107.6 111.8
106.3 106.8 97.7 96.8 99.9 silica 25.degree. C./50% 96.0 99.7 101.0
104.1 102.9 98.1 98.7 99.6 RH 40.degree. C./75% 105.5 109.7 110.6
114.3 110.5 92.0 65.5 51.5* RH The data are the mean of two values
expressed as percentage of the time zero value. *Mean of two very
different values (71.9 and 31.1%).
Example 10
Stability Studies of Mitoquinone-C10 Mesylate (3-cyclodextrin (1:1)
Complex
Solution Stability
[0392] The solution stability of Mitoquinone-C10
mesylate-.beta.-cyclodextrin (1:1) complex was determined in five
solvents; water, 0.01M HCl, 0.01M NaOH, IPB (pH7.4) and 50% MeOH at
two temperature 25.degree. C. and 40.degree. C., under two
atmospheric conditions, air and nitrogen, for 64 days as per the
Applicant's standard operating procedure.
[0393] Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:1) complex
solutions (100 .mu.g/ml in Mitoquinone-C10 mesylate) in the five
solvents were prepared by dilution of a stock solution of
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:1) complex (1 mg/ml
as Mitoquinone-C10 mesylate) in water. Solutions (5 ml) were placed
in glass vials flushed with air or nitrogen, sealed and placed on
storage. Aliquots (0.25 ml) were collected at 0, 1, 2, 4, 8, 16,
32, 64 and 125 days and the concentration determined by HPLC.
[0394] The results are given in Table 9 and in FIGS. 26, 27, 28,
and 29. The stability of Mitoquinone-C10
mesylate-.beta.-cyclodextrin (1:1) in 0.01M NaOH is not included
because Mitoquinone-C10 mesylate decomposed in this solvent within
15 minutes. The results show that (a) solution stability is
independent of the atmosphere above the solution and (b)
temperature has a significant effect on the stability of
Mitoquinone-C10 mesylate in the 1:1 complex with
.beta.-cyclodextrin in water and IPB but not in HCl or 50%
MeOH.
TABLE-US-00009 TABLE 9 Solution stability of Mitoquinone-C10
mesylate-.beta.-cyclodextrin (1:1) in 4 different solvents under
different conditions. Time (Days) Conditions 1 2 4 8 16 32 64
Water, Air, 25.degree. C. 101.3 98.2 99.5 99.0 94.0 92.7 89.8
Water, Air, 40.degree. C. 89.4 87.2 90.4 88.1 89.8 83.9 55.2 0.01M
HCl, Air, 25.degree. C. 103.0 104.3 109.4 104.6 99.1 102.2 100.0
0.01M HCl, Air, 40.degree. C. 94.9 88.22 91.17 99.76 99.74 108.5
102.6 IPB, Air, 25.degree. C. 97.9 95.7 96.1 97.1 96.1 97.1 95.5
IPB, Air, 40.degree. C. 93.5 94.1 99.4 105.4 93.6 86.9 75.3 50%
MeOH, Air, 25.degree. C. 104.8 103.7 108.7 106.4 97.6 96.9 98.1 50%
MeOH, Air, 40.degree. C. 89.3 85.68 91.01 93.0 93.1 92.9 85.9
Water, N.sub.2, 25.degree. C. 101.7 101.3 106.4 102.7 92.0 92.4
89.6 Water, N.sub.2, 40.degree. C. 96.2 91.7 95.9 101.6 88.0 84.5
56.5 0.01M HCl, N.sub.2, 25.degree. C. 103.7 106.5 108.7 108.2
102.7 97.2 100.3 0.01M HCl, N.sub.2, 40.degree. C. 96.2 90.9 97.5
98.5 98.5 106.7 104.8 IPB, N.sub.2, 25.degree. C. 100.1 99.2 100.4
97.1 96.4 98.2 95.5 IPB, N.sub.2, 40.degree. C. 98.4 95.3 102.7
101.0 91.4 87.9 75.7 50% MeOH, N.sub.2, 25.degree. C. 101.2 101.4
104.5 102.4 97.6 96.4 99.0 50% MeOH, N.sub.2, 40.degree. C. 94.7
86.4 90.0 86.4 92.1 97.4 87.7 Data are the mean of two values
expressed as percentage of the time zero value.
Solid State Stability
[0395] Solid state stability of Mitoquinone-C10
mesylate-.beta.-cyclodextrin (1:1) complex was studied in the
absence of light under three different conditions; 40.degree. C.,
75% RH; 25.degree. C., 50% RH; and 4.degree. C. over blue silica
gel as per the Applicant's standard operating procedure.
[0396] A known weight of Mitoquinone-C10
mesylate-.beta.-cyclodextrin (1:1) complex was put in clear glass
bottles and stored under different conditions. Duplicate samples
were withdrawn at 1, 2, 4, 8, 16, 32, 64 and 125 days and the
concentration of Mitoquinone-C10 mesylate determined by HPLC after
dissolving the samples in water. The results are given in Table 10
and in FIG. 30. The results show that Mitoquinone-C10 mesylate was
stable at 4.degree. C. over silica gel and at 25.degree. C., 50% RH
but 37% of the Mitoquinone-C10 mesylate was degraded in
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:1) on storage for
125 days at 40.degree. C., 75% RH.
TABLE-US-00010 TABLE 10 Solid state stability of Mitoquinone-C10
mesylate-.beta.-cyclodextrin (1:1) complex at 40.degree. C., 75%
RH; 25.degree. C., 50% RH and 4.degree. C. over blue silica gel.
Time (Days) 1 2 4 8 16 32 64 125 4.degree. C. over 102.1 97.7 100.0
98.5 103.4 101.4 100.6 102.3 silica 25.degree. C./50% 99.7 101.6
104.2 101.8 102.4 100.7 95.2 101.9 RH 40.degree. C./75% 98.2 101.6
98.3 97.8 98.8 96.0 87.2 66.7 RH The data are the mean of two
values expressed as percentage of the time zero value.
Example 11
Pharmacokinetic Study of a Single IV and Oral Dose of
Mitoquinone-C10 Mesylate-.beta.-Cyclodextrin (1:2) Complex in Rat
(P2 & P3)
[0397] Based on the results of a previous pharmacokinetic study of
Mitoquinone-C10 bromide and an acute oral toxicity study of
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:2) complex, doses
of Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:2) complex for
the pharmacokinetic study were 50 mg/kg Mitoquinone-C10 mesylate
for the oral dose and 10 mg/kg Mitoquinone-C10 mesylate for the IV
dose.
[0398] Ten female Wistar rats (mean weight approximately 236 g)
were cannulated with Silastic tubing in the right jugular vein
under halothane anesthesia 48 h before an experiment. An aqueous
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:2) complex solution
(10 mg/ml as Mitoquinone-C10 mesylate) was freshly prepared and
administered by either the oral (n=5) or IV route (n=5). Blood
samples (0.2 ml) were collected at 0, 5, 10, 20, 30, 45, 60, 90,
120, 180, 240, 360, 720, and 1440 (24 h) min after the IV dose and
at 0, 15, 30, 60, 90, 120, 150, 180, 240, 300, 420, 540, 720, and
1440 (24 h) min after the oral dose. Blood samples were centrifuged
and plasma samples (0.1 ml) were stored in the -20.degree. C.
freezer. Samples of 24 h urine and feces were also collected.
[0399] The Mitoquinone-C10 mesylate concentration in plasma was
determined by ESR using LC/MS (Table 12).
Pharmacokinetic Analysis
[0400] The pharmacokinetics of Mitoquinone-C10 were analyzed by
iterative unweighted non-linear least-squares regression analysis
using MINIM. The IV data were fitted using one-, two- and
three-compartment models. The model giving the best fit was the one
with the minimum value according to Akaike's information criterion
(A.I.C). The plasma drug concentration-time curves after drug
administration were found to be best and adequately fitted by a
three-compartment open model described by the following
equation
C=Ae.sup.-.alpha.t+Be.sup.-.beta.t+Ee.sup.-.gamma.t
where C is the plasma drug concentration, A, B and E are
mathematical coefficients, .alpha. is the rate constant for the
distribution phase, .beta. is the rate constant for an intermediate
phase (distribution or elimination) and .gamma. is the rate
constant for a terminal, slower elimination phase. Drug elimination
half-life (t.sub.1/2) in the terminal phase was calculated as
t.sub.1/2=0.693/.gamma.. Oral data (post 4 h) were fitted with a
one-compartment model. The peak concentration (C.sub.max) and time
to reach C.sub.max (t.sub.max) were obtained directly from the
concentration-time profile. The area under the plasma
concentration-time curve (AUC) was estimated using the linear
trapezoidal rule, with extrapolation from the last measured
concentration to infinity determined by use of the terminal
elimination rate constant (.gamma.). The total plasma clearances
after intravenous (CL) and oral (CL/F) administration were
estimated as CL=dose/AUC. Volumes of distribution were calculated
as V.sub..beta.=dose/(AUC.beta.) and V.gamma.=dose/(AUC.gamma.).
Absolute bioavailability (F) was calculated as:
F=AUC.sub.po.times.Dose.sub.iv/AUC.sub.iv.times.Dose.sub.po. Mean
residence time (MRT) was calculated as AUMC/AUC. Apparent volume of
distribution at steady state (Vss) was calculated as
dose.sub.iv.times.AUMC/(AUC).sup.2.
Results and Discussion
[0401] The mean plasma concentration-time profiles of
Mitoquinone-C10 mesylate after IV and oral administration of
Mitoquinone-C10 mesylate-.beta.-cyclodextrin (1:2) complex are
shown in FIG. 31 and the mean pharmacokinetic parameters are listed
in Table 11. Original data of plasma levels of Mitoquinone-C10
mesylate are attached (Table 12).
TABLE-US-00011 TABLE 11 Pharmacokinetic parameters of
Mitoquinone-C10 mesylate administered as Mitoquinone-C10
mesylate-.beta.-cyclodextrin (1:2) complex in rat after single IV
(10 mg/kg) and oral (50 mg/kg) doses IV Mitoquinone-C10 Oral
Mitoquinone-C10 (n = 5) (n = 5) Body Weight (g) 236.8 .+-. 21.0
236.8 .+-. 22.9 C.sub.max (ng/ml) -- 35.1 t.sub.max (min) -- 30
t.sub.1/2.alpha. (min) 1.6 .+-. 0.3 -- t.sub.1/2.beta. (min) 10.4
.+-. 3.2 -- t.sub.1/2.gamma. (h) 1.83 .+-. 0.44 -- t.sub.1/2 (h)
14.3* 13.9** AUC (.mu.g min/ml) 47.3 .+-. 11.1 29.3 .+-. 2.7 AUMC
(.mu.g min.sup.2/ml) 5292 .+-. 831 7477 .+-. 365 F (%) 100 12.4 CL
(l/min/kg) 0.22 .+-. 0.04 -- CL/F (l/min/kg) -- 13.7 .+-. 1.3
V.sub..beta. (l/kg) 3.33 .+-. 1.46 -- V.sub..gamma. (l/kg) 24.04
.+-. 18.3 -- MRT (h) 4.2 .+-. 0.5 9.5 .+-. 2.2 Vss (l/kg) 25.2 .+-.
6.5 -- *t.sub.1/2 value obtained from mean concentrations at times
>4 h **t.sub.1/2 value obtained from mean concentrations at
times >4 h
TABLE-US-00012 TABLE 12 Mitoquinone-C10 concentration in Rat Plasma
for the P2-IV and P3-PO study 01 IV 2 IV 3 IV 4 IV 5 IV Time (min)
(ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml) Time (Hr) Mean 0 0.495
0.348 30.4 1.39 1.39 0 5 1010 867 1550 2300 1640 0.1 1473.4 10 306
391 572 641 476 0.2 477.2 20 199 186 221 251 192 0.3 209.8 30 132
111 117 158 111 0.5 125.8 45 90.5 87.5 70 113 72.3 0.8 86.7 60 62.1
59.4 56.4 72.5 48.7 1.0 59.8 90 36.4 37.9 38.9 57.4 29.4 1.5 40.0
120 23.2 25.3 24.2 54.8 18.8 2.0 29.3 180 17.8 21.3 20.7 30.2 22
3.0 22.4 240 13.1 12.9 16.4 27.4 9.63 4.0 15.9 360 7.01 8.89 11.5
16.7 7.46 6.0 10.3 720 2.8 3.44 5.66 4.07 2.19 12.0 3.6 1440 1.49
1.63 1.47 1.37 1.96 24.0 1.6 Urine 19.3 52.6 48.5 10.1 12.9 06 PO
07 PO 08 PO 09 PO 10 PO Time (min) (ng/ml) (ng/ml) (ng/ml) (ng/ml)
(ng/ml) Time (Hr) Mean 0 0.878 2.15 1.35 1.39 0.279 0 1.2 15 18.3
21.2 17.5 18.3 44.8 0.3 24.0 30 29.9 36.9 26.7 28 54 0.5 35.1 60 16
25.4 19.5 20.3 40.8 1.0 24.4 90 10.7 25.3 20.2 21.1 25.3 1.5 20.5
120 24.3 23.2 24.5 25.6 13.5 2.0 22.2 150 20.1 23.8 25.7 26.9 10.4
2.5 21.4 180 21 22.4 19.2 20.1 8.72 3.0 18.3 240 33.9 20.9 12 22
7.67 4.0 19.3 300 22.4 17.4 13 13.5 17.3 5.0 16.7 420 9.19 21.8
5.03 5.24 21.4 7.0 12.5 540 6.27 20.1 14.5 15.1 15.2 9.0 14.2 720
8.25 7.31 4.38 4.57 6.08 12.0 6.1 1440 2.12 0.418 2.93 3.05 1.71
24.0 2.0 Urine 1.54 35.4 4.42 4.61 20.7
[0402] Following IV administration, a very rapid distribution phase
is followed by a slower distribution or initial elimination phrase
followed at approx 4 h by a prolonged elimination phase. The
concentration-time profile of Mitoquinone-C10 was fitted to a
three-compartment model with a terminal half-life of 1.8 h,
although the half-life based on data called post 4 h dose is 14.3 h
(Table 13).
[0403] Following oral administration, the absorption of
Mitoquinone-C10 from the rat GI tract was fast. The peak plasma
concentration of Mitoquinone-C10 occurred within 1 h of oral
administration and then declined slowly over time with an
elimination half-life based on post 4 h data of about 14 h.
[0404] The estimated F value is 12.4%.
TABLE-US-00013 TABLE 13 Pharmacokinetics of IV (P2) and Oral (P3)
Mitoquinone-C10 mesylate-.beta.-cyclodextrin 1:2) complex K1 B Rat
Code final final K2 C K3 final value A SD value SD value SD final
value SD final value SD final value SD r2 A.I.C 3 compartment model
P2 01 IV 22766.1 12412.7 0.7080 0.1143 378.5 23.2 0.0456 0.0047
49.6 13.2 0.0057 0.0016 0.9999 69.21 P2 02 IV 2868.7 1502.0 0.3945
0.1855 591.0 325.4 0.0902 0.0317 105.0 26.2 0.0100 0.0024 0.9996
85.45 P2 03 IV 6736.1 1535.2 0.4168 0.0738 1029.7 248.0 0.0945
0.0128 72.2 11.4 0.0067 0.0013 0.9999 77.63 P2 04 IV 13002.7 880.7
0.3973 0.0198 591.4 96.3 0.0639 0.0092 88.5 13.7 0.0050 0.0010
1.0000 82.32 P2 05 IV 9353.2 943.4 0.4073 0.0257 510.0 80.0 0.0638
0.0094 50.4 14.0 0.0060 0.0020 0.9999 77.42 Mean 10945.3 3454.8
0.4648 0.0838 620.1 154.6 0.0716 0.0136 73.1 15.7 0.0067 0.0017
0.9999 78.4 SD 7573.8 5016.9 0.1362 0.0686 244.9 126.6 0.0204
0.0105 24.1 6.0 0.0020 0.0005 0.0001 6.1 2 compartment model P2 01
IV 8722.0 2340.9 0.4962 0.0583 320.2 28.9 0.0267 0.0027 0.9990
96.46 P2 02 IV 2131.2 135.1 0.2295 0.0158 219.5 27.7 0.0199 0.0029
0.9986 97.56 P2 03 IV 4787.7 303.8 0.2642 0.0173 313.8 61.8 0.0289
0.0056 0.9990 108.04 P2 04 IV 10519.2 755.6 0.3306 0.0165 316.4
46.5 0.0203 0.0038 0.9992 117.86 P2 05 IV 7711.2 581.3 0.3466
0.0187 324.2 46.0 0.0317 0.0044 0.9995 100.53 Mean 6774.2 823.3
0.3334 0.0253 298.8 42.2 0.0255 0.0039 0.9991 104.1 SD 3324.2 881.7
0.1028 0.0185 44.53 14.17 0.0052 0.0012 0.0003 8.9
[0405] All patents, publications, scientific articles, and other
documents and materials referenced or mentioned herein are
indicative of the levels of skill of those skilled in the art to
which the invention pertains, and each such referenced document and
material is hereby incorporated by reference to the same extent as
if it had been incorporated by reference in its entirety
individually or set forth herein in its entirety. Applicants
reserve the right to physically incorporate into this specification
any and all materials and information from any such patents,
publications, scientific articles, web sites, electronically
available information, and other referenced materials or
documents.
[0406] The specific methods and compositions described herein are
representative of various embodiments or preferred embodiments and
are exemplary only and not intended as limitations on the scope of
the invention. Other objects, aspects, examples and embodiments
will occur to those skilled in the art upon consideration of this
specification, and are encompassed within the spirit of the
invention as defined by the scope of the claims. It will be readily
apparent to one skilled in the art that varying substitutions and
modifications may be made to the invention disclosed herein without
departing from the scope and spirit of the invention. The invention
illustratively described herein suitably may be practiced in the
absence of any element or elements, or limitation or limitations,
which is not specifically disclosed herein as essential. Thus, for
example, in each instance herein, in embodiments or examples of the
present invention, any of the terms "comprising", "consisting
essentially of" and "consisting of may be replaced with either of
the other two terms in the specification. Also, the terms
"comprising", "including", containing", etc. are to be read
expansively and without limitation. The methods and processes
illustratively described herein suitably may be practiced in
differing orders of steps, and that they are not necessarily
restricted to the orders of steps indicated herein or in the
claims. It is also that as used herein and in the appended claims,
the singular forms "a," "an," and "the" include plural reference
unless the context clearly dictates otherwise. Thus, for example, a
reference to "a host cell" includes a plurality (for example, a
culture or population) of such host cells, and so forth. Under no
circumstances may the patent be interpreted to be limited to the
specific examples or embodiments or methods specifically disclosed
herein. Under no circumstances may the patent be interpreted to be
limited by any statement made by any Examiner or any other official
or employee of the Patent and Trademark Office unless such
statement is specifically and without qualification or reservation
expressly adopted in a responsive writing by Applicants.
[0407] The terms and expressions that have been employed are used
as terms of description and not of limitation, and there is no
intent in the use of such terms and expressions to exclude any
equivalent of the features shown and described or portions thereof,
but it is recognized that various modifications are possible within
the scope of the invention as claimed. Thus, it will be understood
that although the present invention has been specifically disclosed
by preferred embodiments and optional features, modification and
variation of the concepts herein disclosed may be resorted to by
those skilled in the art, and that such modifications and
variations are considered to be within the scope of this invention
as defined by the appended claims.
[0408] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0409] Other embodiments are within the following claims. In
addition, where features or aspects of the invention are described
in terms of Markush groups, those skilled in the art will recognize
that the invention is also thereby described in terms of any
individual member or subgroup of members of the Markush group.
[0410] The compounds of the invention have application in selective
antioxidant therapies for human patients to prevent mitochondrial
damage. This can be to prevent the elevated mitochondrial oxidative
stress associated with particular diseases, such as Parkinson's
disease or diseases associated with mitochondrial DNA mutations.
They could also be used in conjunction with cell transplant
therapies for neurodegenerative diseases, to increase the survival
rate of implanted cells.
[0411] 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.
[0412] 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.
* * * * *