FIELD OF THE INVENTION
The invention relates to a succinate dehydrogenase inhibitor or 5 a prodrug and/or a
pharmaceutically acceptable salt thereof for use in the treatment or prevention of
reperfusion injury by inhibiting the accumulation of succinate, wherein the
inhibitor or prodrug and/or pharmaceutically acceptable salt thereof is a cellpermeable
and reversible inhibitor of succinate dehydrogenase.
10
BACKGROUND TO THE INVENTION
Succinate is known to build up in anaerobic conditions. For example, diving
animals, hypoxic areas at the centre of tumours, and certain parasites can be
15 observed to show elevated succinate in conditions of hypoxia. However, these
studies are typically in the context of attempting to kill cells or to block or remove
the cells from the system under study. Moreover, these systems are not always
comparable the conditions experienced by mammalian cells under hypoxia. For
example, in bacteria the two enzymatic activities involved in succinate
20 metabolism are present in two separate enzymes [rather than in the same single
enzyme succinate dehydrogenase (SDH) in mammalian cells].
Succinate has been studied in certain in vitro systems. It is possible that reactive
oxygen species (ROS) generation via succinate has been observed in certain in
25 vitro systems. However, such observations have been largely regarded as in vitro
curiosities rather than a genuine reflection of possible conditions in vivo.
Succinate has been studied for oxygen sensing applications. There have been
attempts to block the succinate in order to try to achieve an anti-inflammatory
30 effect in the prior art.
2
There is no known experimental connection in the prior art linking succinate
accumulation in vivo to ROS production by mitochondrial complex I due to
succinate oxidation upon reperfusion.
Hu et al, Journal of Huazhong University of Science and Technology, 5 Medical
Sciences, vol. 25, 2005, pages 439-441 and Hirata et al, Transplantation, vol. 71,
2001, pages 352-359 have both shown that chemical preconditioning using 3-
nitropropionate reduced ischemia-reperfusion injury in rats. The compound 3-
nitropropionate is an inhibitor of mitochondrial complex II. Wojtovich et al,
10 Basic Research in Cardiology, vol. 104, 2009, pages 121-129 have reported that
the complex II inhibitor atpennin A5 protects the heart against simulated
ischemia-reperfusion injury through a mKATP channel dependent mechanism.
However, inhibitors such as 3-nitropropionate and atpennin A5 have the
disadvantage that they are irreversible. As a consequence, any complex II that
15 binds to such irreversible inhibitors is permanently prevented from carrying out its
normal function.
Drose et al, Molecular Pharmacology, vol. 79, 2011, pages 814-822 studied
several cardioprotective complex II inhibitors including 2-thenoyltrifluoroacetone
20 (TTFA), 3-nitropropionate, atpennin A5 and malonate. Of the inhibitors tested,
malonate required the highest concentration, in millimolar levels, to achieve halfmaximal
inhibition compared to nanomolar levels required for atepennin A5. In
addition, malonate has the disadvantage that it is not cell-permeable. The other
inhibitors TTFA, 3-nitropropionate and atpennin A5 have the disadvantage that
25 they are irreversible.
Dimethylmalonate is a known compound. Malonate and its derivatives are
industrial compounds used as feed stocks for polymer formation. There is no
known teaching in the prior art for use of malonate derivative, such as
30 dimethylmalonate, as a prodrug of an inhibitor of SDH. The prodrug dimethyl
malonate will be hydrolysed in vivo to malonate which is an inhibitor of SDH.
3
The present invention seeks to overcome problem(s) associated with the prior art.
SUMMARY OF THE INVENTION
The inventors performed an extensive metabolomic survey to identify metabolites
which are accumulated during ischaemia. As part of a carefully 5 controlled study,
this metabolomics analysis was conducted in parallel in a wide range of tissue
types. A number of metabolites were observed to vary in ischaemic conditions in
different tissues. However, despite this finding the inventors persisted with their
analysis and selected only those metabolites which were similarly affected across
10 the full spectrum of tissues studied. In this way, the inventors were ingeniously
able to select metabolites which were more likely to be common to the effect of
ischaemia.
In addition, the inventors went on to analyse the effect of reperfusion on the
15 analytes. This allowed the inventors to select only three metabolites
(hypoxanthine, xanthine and succinate) which behaved similarly across a range of
tissue types in accumulation during ischaemia. Most surprisingly, the inventors
observed that of these three, only succinate could plausibly play a role in the
generation of reactive oxygen species following reperfusion. It was these
20 dramatic insights which singled out succinate as accumulating during ischaemia,
and as being rapidly metabolised and involved in the production of reactive
oxygen species following reperfusion, upon which the invention is based.
It was a surprise to the inventors that the succinate disappeared so fast upon
25 reperfusion. It was also a surprise to discover that succinate was the source of
reactive oxygen species following restoration of metabolic function under
normoxic conditions. It was a further surprise that multiple organs share the same
metabolic signature, demonstrating that the succinate metabolism is a widely
applicable phenomenon within mammalian cells’ response to ischaemic
30 conditions.
4
Thus in one aspect the invention provides a succinate dehydrogenase inhibitor or a
prodrug and/or a pharmaceutically acceptable salt thereof for use in the treatment
or prevention of reperfusion injury by inhibiting the accumulation of succinate,
wherein the inhibitor or prodrug and/or pharmaceutically acceptable salt thereof is
a cell-permeable and reversible inhibitor of succinate 5 dehydrogenase.
In a further aspect, the invention provides a method of treating or preventing a
reperfusion injury in a subject, the method comprising administering to the subject
an effective amount of a succinate dehydrogenase inhibitoror a prodrug and/or a
10 pharmaceutically acceptable salt thereof that is a cell-permeable and reversible
inhibitor of succinate dehydrogenase.
In a further aspect, the invention provides a succinate dehydrogenase inhibitor or
a prodrug and/or a pharmaceutically acceptable salt thereof for use in the
15 treatment or prevention of reperfusion injury, wherein the inhibitor or prodrug
and/or pharmaceutically acceptable salt thereof is a cell-permeable and reversible
inhibitor of succinate dehydrogenase.
In a further aspect, the invention provides a succinate dehydrogenase inhibitor or
20 a prodrug and/or a pharmaceutically acceptable salt thereof for use in the
treatment or prevention of reperfusion injury by inhibiting the accumulation of
succinate, wherein the inhibitor or prodrug and/or pharmaceutically acceptable
salt thereof is a cell-permeable and reversible inhibitor of succinate
dehydrogenase.
25
DEFINITIONS
C1-12 alkyl: refers to straight chain and branched saturated hydrocarbon groups,
generally having from 1 to 12 carbon atoms. Examples of alkyl groups include
methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pent-1-yl, pent-
30 2-yl, pent-3-yl, 3-methylbut-1-yl, 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2,2-
trimethyleth-1-yl, n-hexyl, n-octyl, n-nonane, n-decane, n-undecane, n-dodecane
and the like.
5
“Drug”, “drug substance”, “active pharmaceutical ingredient”, and the like, refer
to a compound (e.g., compounds of Formula (I) and compounds specifically
named below) that may be used for treating a subject in need of treatment.
5
“Excipient” refers to any substance that may influence the bioavailability of a
drug, but is otherwise pharmacologically inactive.
“Pharmaceutically acceptable” substances refers to those substances which are
10 within the scope of sound medical judgment suitable for use in contact with the
tissues of subjects without undue toxicity, irritation, allergic response, and the
like, commensurate with a reasonable benefit-to-risk ratio, and effective for their
intended use.
15 “Pharmaceutical composition” refers to the combination of one or more drug
substances and one or more excipients.
The term “subject” as used herein refers to a human or non-human mammal.
Examples of non-human mammals include livestock animals such as sheep,
20 horses, cows, pigs, goats, rabbits and deer; and companion animals such as cats,
dogs, rodents, and horses.
“Therapeutically effective amount” of a drug refers to the quantity of the drug or
composition that is effective in treating a subject and thus producing the desired
25 therapeutic, ameliorative, inhibitory or preventative effect. The therapeutically
effective amount may depend on the weight and age of the subject and the route
of administration, among other things.
“Treating” refers to reversing, alleviating, inhibiting the progress of, or preventing
30 a disorder, disease or condition to which such term applies, or to reversing,
alleviating, inhibiting the progress of, or preventing one or more symptoms of
such disorder, disease or condition.
6
“Treatment” refers to the act of “treating”, as defined immediately above.
“Preventing” refers to a reduction of the risk of acquiring a given disease or
disorder, that is, causing the clinical symptoms not to 5 develop. Hence,
“preventing” refers to the prophylactic treatment of a subject in need thereof. The
prophylactic treatment can be accomplished by administering an appropriate dose
of a therapeutic agent to a subject having a predisposition to a disorder, or at risk
of developing a disorder, even though symptoms of the disorder are absent or
10 minimal, thereby substantially averting onset of the disorder.
The term “prodrug” means a compound which is, after administration to a subject,
subjected to a structural change such as hydrolysis in vivo, preferably in blood, to
produce an SDHi or a salt thereof. For example, various means for producing
15 prodrugs from pharmaceutical compounds having carboxylic acid, amino group,
hydroxyl group or the like are known, and one of ordinary skill in the art can
choose appropriate means. Types of the prodrug of an SDHi or a salt thereof are
not particularly limited. For example, where an SDHi has one or more carboxylic
acids, an example includes a prodrug wherein one or more of the carboxylic acids
20 are converted into an ester. Preferred examples include ester compounds such as
methyl ester or diemethyl esters.
“Succinate dehydrogenase inhibitor” or “SDHi” refers to compound that inhibit
the action of succinate dehydrogenase.
25
As used herein the term “comprising” means “consisting at least in part of”.
When interpreting each statement in this specification that includes the term
“comprising”, features other than that or those prefaced by the term may also be
present. Related terms such as “comprise” and “comprises” are to be interpreted
30 in the same manner.
7
SUCCINATE DEHYDROGENASE INHIBITORS (SDHi’s) OR PRODRUGS
THEREOF
The SDHi or a prodrug and/or a pharmaceutically acceptable salt thereof is useful
in the treatment or prevention of reperfusion injury by inhibiting the accumulation
of succinate. Thus, administration of the SDHi or 5 a prodrug and/or a
pharmaceutically acceptable salt thereof treats or prevents reperfusion injury in a
tissue of a subject, such as an ischaemic myocardium, by decreasing the amount
of succinate observed at reperfusion in the tissue compared to the amount of
succinate observed at reperfusion without administration of the SDHi or a prodrug
10 and/or a pharmaceutically acceptable salt thereof.
The SDHi is a reversible inhibitor of SDH. The reversibility of the SDHi is
important as this enables the inhibitor, over time, to be removed from the
mitochondrial complex by competitive binding so that its effect will be transient.
15 As a result, the complex may return to performing its normal functions.
For an SDHi that acts through the succinate site, the reversibility of the SDHi may
be determined by measuring and comparing the infarct size of (1) a heart treated
with the SDHi; (2) an untreated heart; and (3) a heart treated with the SDHi +
20 dimethyl succinate. Cardioprotection is shown if the infarct size is reduced in the
heart treated with the SDHi in comparison to that of an untreated heart. The
reversibility of the SDHi is demonstrated if the cardioprotection shown by the
heart treated with the SDHi is suppressed by adding back dimethyl succinate.
This procedure is discussed in the Examples using triphenyltetrazolium chloride
25 staining to determine the infarct size of the heart (see also Figures 4b & c).
The efficacy of an SDHi may be assessed by determining the ability of the SDHi
to prevent succinate accumulation during ischaemia. The ability of the SDHi to
prevent succinate accumulation during ischaemia may be determined by
30 measuring succinate levels in tissues by liquid chromatography/mass spectrometry
(LC/MS) as detailed in the metabolomic analysis in the Examples. The
reversibility of the SDHi may be shown by comparing the succinate levels
8
following treatment with the SDHi with the succinate levels following treatment
with the SDHi plus dimethyl succinate.
The SDHi or a prodrug and/or a pharmaceutically acceptable salt thereof is
membrane permeable. This membrane permeability enables 5 the compound to
enter the cell and accumulate.
Membrane permeability may be assayed in cell experiments by measuring oxygen
consumption rate (OCR) and by driving this with dimethyl succinate and then
10 inhibiting this by adding an SDHi or a prodrug and/or pharmaceutically
acceptable salt thereof (such as dimethyl malonate) so the efficacy and timescale
of uptake and hydrolysis of the SDHi or a prodrug and/or pharmaceutically
acceptable salt thereof can be assessed. In addition, as shown in Figure 5a the cell
permeability of the compound may be determined by comparing the level of the
15 compound in an untreated cell with that of a treated cell. Typically, the level of
the compound may be determined using LC/MS as detailed in the metabolomic
analysis in the Examples.
More suitably, the SDHi or a prodrug and/or a pharmaceutically acceptable salt
20 thereof is a prodrug of an SDHi or a pharmaceutically acceptable salt thereof.
Most suitably, the SDHi or a prodrug and/or a pharmaceutically acceptable salt
thereof is a prodrug.
25 Suitably the SDHi binds to SDH.
Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof
has the general formula (I):
30 (I)
9
wherein:
n is 0 or 1;
R1 is selected from C1-12 alkyl;
R2 is selected from H and OH;
R3 is selected from CO2R4, and 5 C(O)-CO2R4; and
R4 is selected from C1-12 alkyl.
Suitably the succinate dehydrogenase inhibitor or a prodrug and/or
pharmaceutically acceptable salt thereof is selected from R1O2C-CH2-CO2R4,
R1O2C-CH(OH)-CH2-CO2R4, R1O2C-CH2-C(O)-CO2R410 .
More suitably the succinate dehydrogenase inhibitor or a prodrug and/or
pharmaceutically acceptable salt thereof is selected from R1O2C-CH2-CO2R4,
wherein R1 and R4 are independently selected from methyl, ethyl, n-propyl, iso15
propyl, n-butyl, s-butyl, t-butyl.
Most suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt
thereof is a malonate compound.
20 Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof
may comprise a cell permeable malonate.
Suitably the malonate SDHi or a prodrug and/or pharmaceutically acceptable salt
thereof of the invention is an ethyl ester malonate.
25
Most suitably the succinate dehydrogenase inhibitor or a prodrug and/or
pharmaceutically acceptable salt thereof is dimethyl malonate.
Dimethylmalonate is advantageous as it has extremely low toxicity. For example,
30 the LD50 of dimethylmalonate is approximately 5gms per kilogram when
administered orally.
10
Suitably binding of the SDHi to SDH is at the succinate binding site. Suitably
this is competitive binding with succinate.
Suitably the SDHi that binds SDH at the succinate binding site or a prodrug
and/or pharmaceutically acceptable salt thereof is selected 5 R1O2C-CH2-CO2R4,
R1O2C-CH(OH)-CH2-CO2R4 and R1O2C-CH2-C(O)-CO2R4.
Suitably the SDHi binds SDH at the ubiquinone binding site. Suitably this
binding is competitive binding with quinone.
10
Most suitably SDHis useful in the invention inhibit re-oxidation of succinate
under normoxic conditions.
Suitably the SDHi of the invention is a “complex II” inhibitor.
15
The SDHi of the invention is a reversible inhibitor.
Suitably the SDHi of the invention is a reversible SDHi.
R1 20
Suitably R1 is selected from methyl, ethyl, propyl, butyl, pentyl and hexyl.
More suitably R1 is selected from methyl and ethyl.
25
R2
Suitably R2 is H.
R3
Suitably R3 is CO2R430 .
R4
11
Suitably R4 is selected from methyl, ethyl, propyl, butyl, pentyl and hexyl.
More suitably R4 is selected from methyl and ethyl.
ADMINISTRATION 5 AND TIMING
Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof is
administered orally, topically, subcutaneous, parenterally, intramuscular, intraarterial
and/or intravenously.
10
More suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt
thereof is administered intravenously.
The SDHi or a prodrug and/or pharmaceutically acceptable salt thereof may be
15 administered by direct injection into the coronary artery.
Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of
the invention is administered before ischaemia. In such circumstances the SDHi
or a prodrug and/or pharmaceutically acceptable salt thereof is administered to a
20 subject at risk of ischaemia.
Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of
the invention is administered following ischaemia.
25 Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of
the invention is administered before reperfusion.
Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of
the invention is administered as soon as possible after the initiation of reperfusion.
30
Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of
the invention is administered within five minutes of reperfusion commencing.
12
Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of
the invention is administered within four minutes of reperfusion commencing;
within three minutes of reperfusion commencing; within two minutes of
reperfusion commencing; or within one minute of reperfusion commencing.
5
Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of
the invention is administered before surgery.
Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of
10 the invention is administered during surgery.
Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of
the invention is administered to organs intended to be preserved before ischaemia.
15 Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of
the invention is administered to organs to be preserved as soon as possible after
ischaemia.
Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of
20 the invention is administered to organs to be preserved as soon as possible after
reperfusion.
Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of
the invention is administered to organs to be preserved within five minutes of
25 reperfusion. Suitably the SDHi or a prodrug and/or pharmaceutically acceptable
salt thereof of the invention is administered within four minutes of reperfusion
commencing; within three minutes of reperfusion commencing; within two
minutes of reperfusion commencing; or within one minute of reperfusion
commencing.
30
Suitably the SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of
the invention finds application in organ transplantation.
13
It is an advantage of the invention that a protective treatment is provided. There
are no licensed protective compounds on the market at present.
Suitably the SDHi or a prodrug and/or pharmaceutically acceptable 5 salt thereof of
the invention is quickly metabolised in mammals.
It is an advantage of malonate SDHi’s or a prodrug and/or pharmaceutically
acceptable salt thereof that they are metabolised in mammalian cells. Cell
10 permeable malonate derivatives are suitably metabolised in order to release
malonate once inside the cell. Malonate itself is advantageously metabolised by
natural metabolic pathways within the cell. More specifically, malonate is
metabolised into part of the fat decomposition pathway naturally present in
mammalian cells. This offers the advantage that there are no side effects or by
15 products of metabolites of the SDHi or a prodrug and/or pharmaceutically
acceptable salt thereof of the invention when the SDHi or a prodrug and/or
pharmaceutically acceptable salt thereof is a malonate compound.
Suitably the succinate dehydrogenase inhibitor or a prodrug and/or
20 pharmaceutically acceptable salt thereof may be administered at a dose of 0.1-50
mg/kg of subject/min for 1 to 30 minutes. More suitably, the succinate
dehydrogenase inhibitor or a prodrug and/or pharmaceutically acceptable salt
thereof may be administered at a dose of 0.5-20 mg/kg of subject/min for 1 to 30
minutes. More suitably, the succinate dehydrogenase inhibitor or a prodrug
25 and/or pharmaceutically acceptable salt thereof may be administered at a dose of
1-10 mg/kg of subject/min for 1 to 30 minutes.
DETAILED DESCRIPTION OF THE INVENTION
30 From the candidate metabolites analysed, the inventors excluded those which
varied differently in different tissues. For example, some metabolites were
elevated in one tissue yet decreased in another following ischaemic conditions.
14
Some metabolites were affected to different levels. Other metabolites did not
appear to be affected at all. Further metabolites did not appear affected in all of
the tissue types studied. The inventors systematically examined the data and
removed each of the metabolites possessing these diverse criteria so as to select
only those metabolites exhibiting common properties across ischaemia 5 of a range
of different tissues studied.
A key finding upon which the invention is based is the rapid return of succinate to
normal levels following reperfusion. This is illustrated for example in Figure 1E
10 where it can be seen that succinate levels descend sharply to normal within only
five minutes of reperfusion. The rapidity of this response is remarkable. This
contributes to the evidence accumulated by the inventors that succinate is forming
the reservoir of electrons which is created during ischaemic conditions.
Moreover, the timing is extraordinary since it overlaps precisely with the time
15 course of damage inflicted on tissue from a reperfusion. It is this same five
minute window observed by the inventors during which most damage is caused,
which also follows exactly the time course of succinate metabolism following
reperfusion.
20 APPLICATIONS
Suitably, the invention relates to a succinate dehydrogenase inhibitor or a prodrug
and/or a pharmaceutically acceptable salt thereof for use in the treatment or
prevention of reperfusion injury by inhibiting the accumulation of succinate.
25
Suitably, the invention relates to a succinate dehydrogenase inhibitor or a prodrug
and/or a pharmaceutically acceptable salt thereof for use in the treatment or
prevention of reperfusion injury by inhibiting succinate transport.
30 Suitably, the invention relates to a succinate dehydrogenase inhibitor or a prodrug
and/or a pharmaceutically acceptable salt thereof for use in the treatment or
prevention of reperfusion injury by inhibiting succinate oxidation.
15
The invention finds application in surgery.
The invention finds application in organ preservation.
5
The invention finds application in the reduction or inhibition of ROS production.
The invention finds application in the inhibition or prevention of succinate
accumulation.
10
The invention finds application in slowing the release of ROS following
reperfusion.
The invention finds application in all forms of the elective surgery. In these
15 applications of the invention, the treatment may be preventative, i.e. preventative
against production of ROS following reperfusion.
REPERFUSION INJURY
20 Suitably, the reperfusion injury is an ischaemia-reperfusion injury.
Suitably, the reperfusion injury is a result of a disorder selected from an
abdominal aortic aneurysm, atherosclerosis, artery disease, burns, cancer, cardiac
arrest, cerebrovascular disease, cerebral edema secondary to stroke, cerebral
25 damage, chronic obstructive pulmonary disease, congestive heart disease,
constriction after percutaneous transluminal coronary angioplasty, coronary
disease, diabetes, hypertension, hypoxia during/after childbirth, mechanical
trauma resulting from crush injury or surgery, migraine, myocardial infarction,
(non-fatal) drowning, peripheral vascular disease, pulmonary vascular disease,
30 regenerative medicine, reperfusion after cardiac surgery, reperfusion after stroke,
retinal vascular disease, stroke and surgical tissue reperfusion injury.
16
Suitably the surgical tissue reperfusion injury may be the result of liver, kidney or
bowel surgery.
Surgical tissue reperfusion injury may be the result of elective surgical operations
where an organ is exposed to a period of ischaemia include liver 5 surgery (when
the blood supply to the liver is temporarily clamped to enable dissection/partial
resection) and kidney surgery (e.g., partial resection of a tumour).
A common cause of cerebral damage is systemic hypotension during surgery,
10 resulting in temporary hypoperfusion of the brain.
More suitably, the reperfusion injury is a result of a disorder selected from an
abdominal aortic aneurysm, atherosclerosis, artery disease, cardiac arrest,
cerebrovascular disease, cerebral edema secondary to stroke, congestive heart
15 disease, constriction after percutaneous transluminal coronary angioplasty,
coronary disease, mechanical trauma resulting from crush injury or surgery,
myocardial infarction, peripheral vascular disease, pulmonary vascular disease,
reperfusion after cardiac surgery, reperfusion after stroke, retinal vascular disease,
stroke and surgical tissue reperfusion injury.
20
Suitably the surgery is vascular surgery, heart bypass surgery or transplant
surgery.
More suitably, the reperfusion injury is a result of a disorder selected from
25 reperfusion after stroke, stroke and myocardial infarction.
Suitably the reperfusion injury is a reperfusion injury in an organ to be preserved.
Suitably organs to be preserved may be selected from heart, intestine, kidney,
liver, lung, pancreas and skin.
30
Organs may be preserved for the purpose of transplantation, or as a source of cells
and tissues for use in regenerative medicine (for example, isolation of stem cells).
17
More suitably, the reperfusion injury is a reperfusion injury in regenerative
medicine. The future of regenerative medicine is based on the principle of (cryo-)
preserving stem cells and stem cell-derived cells and tissues under likely anoxic
condition, with subsequent adoptive transfer and/or transplantation 5 into an
oxygenated recipient.
COMBINATIONS
10 The SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of the
invention may be administered in combination with a treatment used or intended
to remove a blockage in blood flow.
The SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of the
15 invention may be administered in combination with one or more of stents,
MitoSNO, blood thinning agents, lysis agents, blood preservation solutions, organ
preservation solutions or any other treatment used or intended to remove a
blockage in blood flow.
20 Suitably blood or organ preservation solutions may include crystalloid and noncrystalloid
solutions including but not limited to saline, human albumin solution,
Hartmann’s solution and gelofusin.
MitoSNO may be prepared as described in WO2008039085.
25
Suitably the blood thinning agents may be selected from CoumadinTM (warfarin);
PradaxaTM (dabigatran); XareltoTM (rivaroxaban) and EliquisTM (apixaban),
Fondaparinux, unfractionated heparin, low molecular weight heparins including
but not limited to enoxaparin and deltaparin, thrombolytic agents including but
30 not limited to Streptokinase (SK), Urokinase, Lanoteplase, Reteplase,
Staphylokinase and Tenecteplase.
18
The SDHi or a prodrug and/or pharmaceutically acceptable salt thereof of the
invention may be administered as a pharmaceutical composition comprising an
SDHi and a pharmaceutically acceptable excipient, carrier or diluent.
Suitable excipients, carriers and diluents can be found in standard 5 pharmaceutical
texts. See, for example, Handbook for Pharmaceutical Additives, 3rd Edition (eds.
M. Ash and I. Ash), 2007 (Synapse Information Resources, Inc., Endicott, New
York, USA) and Remington: The Science and Practice of Pharmacy, 21st Edition
(ed. D. B. Troy) 2006 (Lippincott, Williams and Wilkins, Philadelphia, USA)
10 which are incorporated herein by reference.
Excipients for use in the compositions of the invention include, but are not limited
to microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium
phosphate and glycine may be employed along with various disintegrants such as
15 starch (and preferably corn, potato or tapioca starch), alginic acid and certain
complex silicates, together with granulation binders like polyvinylpyrrolidone,
sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium
stearate, sodium lauryl sulfate and talc are often very useful for tabletting
purposes. Solid compositions of a similar type may also be employed as fillers in
20 gelatin capsules; preferred materials in this connection also include lactose or
milk sugar as well as high molecular weight polyethylene glycols. When aqueous
suspensions and/or elixirs are desired for oral administration, the active ingredient
may be combined with various sweetening or flavouring agents, colouring matter
or dyes, and, if so desired, emulsifying and/or suspending agents as well, together
25 with such diluents as water, ethanol, propylene glycol, glycerin and various like
combinations thereof.
Pharmaceutical carriers include solid diluents or fillers, sterile aqueous media and
various non-toxic organic solvents, and the like.
30
Pharmaceutically acceptable carriers include gums, starches, sugars, cellulosic
materials, and mixtures thereof. The compound can be administered to a subject
19
by, for example, subcutaneous implantation of a pellet. The preparation can also
be administered by intravenous, intra-arterial, or intramuscular injection of a
liquid preparation oral administration of a liquid or solid preparation, or by topical
application. Administration can also be accomplished by use of a rectal
suppository or a urethral 5 suppository.
Further, as used herein “pharmaceutically acceptable carriers” are well known to
those skilled in the art and include, but are not limited to, 0.01-0.1M and
preferably 0.05M phosphate buffer or 0.9% saline. Additionally, such
10 pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are propylene
glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered media.
15
Pharmaceutically acceptable parenteral vehicles include sodium chloride solution,
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
Intravenous vehicles include fluid and nutrient replenishers, electrolyte
replenishers such as those based on Ringer's dextrose, and the like. Preservatives
20 and other additives may also be present, such as, for example, antimicrobials,
antioxidants, collating agents, inert gases and the like.
Pharmaceutically acceptable carriers for controlled or sustained release
compositions administerable according to the invention include formulation in
25 lipophilic depots (e.g. fatty acids, waxes, oils). Also comprehended by the
invention are particulate compositions coated with polymers (e.g. poloxamers or
poloxamines) and the compound coupled to antibodies directed against tissuespecific
receptors, ligands or antigens or coupled to ligands of tissue-specific
receptors.
30
Pharmaceutically acceptable carriers include compounds modified by the covalent
attachment of water-soluble polymers such as polyethylene glycol, copolymers of
20
polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran,
polyvinyl alcohol, polyvinylpyrrolidone or polyproline are known to exhibit
substantially longer half-lives in blood following intravenous injection than do the
corresponding unmodified compounds (Abuchowski and Davis, Soluble Polymer-
Enzyme Adducts, Enzymes as Drugs, Hocenberg and Roberts, 5 eds., Wiley-
Interscience, New York, N.Y., (1981), pp 367-383; and [65]). Such modifications
may also increase the compound's solubility in aqueous solution, eliminate
aggregation, enhance the physical and chemical stability of the compound, and
greatly reduce the immunogenicity and reactivity of the compound. As a result,
10 the desired in vivo biological activity may be achieved by the administration of
such polymer-compound abducts less frequently or in lower doses than with the
unmodified compound.
Further particular and preferred aspects are set out in the accompanying
15 independent and dependent claims. Features of the dependent claims may be
combined with features of the independent claims as appropriate, and in
combinations other than those explicitly set out in the claims.
Isomers, Salts and Solvates
20
Certain compounds may exist in one or more particular geometric, optical,
enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric,
conformational, or anomeric forms, including but not limited to, cis- and transforms;
E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and
25 meso-forms; D- and L-forms; d- and l- forms; (+) and (-) forms; keto-, enol-, and
enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; alpha- and
beta-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and
halfchair-forms; and combinations thereof, hereinafter collectively referred to as
“isomers” (or “isomeric forms”).
30
Note that, except as discussed below for tautomeric forms, specifically excluded
from the term “isomers”, as used herein, are structural (or constitutional) isomers
21
(i.e. isomers which differ in the connections between atoms rather than merely by
the position of atoms in space). For example, a reference to a methoxy group, -
OCH3, is not to be construed as a reference to its structural isomer, a
hydroxymethyl group, -CH2OH.
5
A reference to a class of structures may well include structurally isomeric forms
falling within that class (e.g. C1-67 alkyl includes n-propyl and iso-propyl; butyl
includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and
para-methoxyphenyl).
10
The above exclusion does not apply to tautomeric forms, for example, keto-, enol-
, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol,
imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime,
thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
15
Note that specifically included in the term “isomer” are compounds with one or
more isotopic substitutions. For example, H may be in any isotopic form,
including 1H, 2H (D), and 3H (T); C may be in any isotopic form, including 12C,
13C, and 14C; O may be in any isotopic form, including 16O and 18O; and the like.
20
Unless otherwise specified, a reference to a particular compound includes all such
isomeric forms, including (wholly or partially) racemic and other mixtures
thereof.
25 Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g.
fractional crystallisation and chromatographic means) of such isomeric forms are
either known in the art or are readily obtained by adapting the methods taught
herein, or known methods, in a known manner.
30 Unless otherwise specified, a reference to a particular compound also includes
ionic, tautomeric, salt, solvate, protected forms, and combinations thereof, for
example, as discussed below.
22
An example of a combination thereof is where a reference to an SDHi includes a
salt of a tautomer. For example, a reference to the compound diethyl malate
includes the sodium salt of the enolate form as shown below:
5
.
SDHi’s, which include compounds specifically named above, may form
pharmaceutically acceptable complexes, salts, solvates and hydrates. These salts
10 include nontoxic acid addition salts (including di-acids) and base salts.
If the compound is cationic, or has a functional group which may be cationic (e.g.
-NH2 may be -NH3
+), then an acid addition salt may be formed with a suitable
anion. Examples of suitable inorganic anions include, but are not limited to, those
15 derived from the following inorganic acids hydrochloric acid, nitric acid, nitrous
acid, phosphoric acid, sulfuric acid, sulphurous acid, hydrobromic acid,
hydroiodic acid, hydrofluoric acid, phosphoric acid and phosphorous acids.
Examples of suitable organic anions include, but are not limited to, those derived
from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic,
20 benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic,
ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic,
hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic,
lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic,
pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,
25 succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable
polymeric organic anions include, but are not limited to, those derived from the
following polymeric acids: tannic acid, carboxymethyl cellulose. Such salts
include acetate, adipate, aspartate, benzoate, besylate, bicarbonate, carbonate,
bisulfate, sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate,
23
fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate,
hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate,
lactate, malate, maleate, malonate, mesylate, methylsulfonate, naphthylate, 2-
napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate,
hydrogen phosphate, dihydrogen phosphate, pyroglutamate, saccharate, 5 stearate,
succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts.
For example, if the compound is anionic, or has a functional group which may be
anionic (e.g. -COOH may be –COO-), then a base salt may be formed with a
10 suitable cation. Examples of suitable inorganic cations include, but are not
limited to, metal cations, such as an alkali or alkaline earth metal cation,
ammonium and substituted ammonium cations, as well as amines. Examples of
suitable metal cations include sodium (Na+) potassium (K+), magnesium (Mg2+),
calcium (Ca2+), zinc (Zn2+), and aluminum (Al3+). Examples of suitable organic
cations include, but are not limited to, ammonium ion (i.e. NH4+15 ) and substituted
ammonium ions (e.g. NH3R+, NH2R2
+, NHR3
+, NR4
+). Examples of some suitable
substituted ammonium ions are those derived from: ethylamine, diethylamine,
dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine,
diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline,
20 meglumine, and tromethamine, as well as amino acids, such as lysine and
arginine. An example of a common quaternary ammonium ion is N(CH3)4
+.
Examples of suitable amines include arginine, N,N'-dibenzylethylenediamine,
chloroprocaine, choline, diethylamine, diethanolamine, dicyclohexylamine,
ethylenediamine, glycine, lysine, N-methylglucamine, olamine, 2-amino-2-
25 hydroxymethyl-propane-1,3-diol, and procaine. For a discussion of useful acid
addition and base salts, see S. M. Berge et al., J. Pharm. Sci. (1977) 66:1-19; see
also Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties,
Selection, and Use (2011)
30 Pharmaceutically acceptable salts may be prepared using various methods. For
example, one may react SDHi with an appropriate acid or base to give the desired
salt. One may also react a precursor of the compound of SDHi with an acid or
24
base to remove an acid- or base-labile protecting group or to open a lactone or
lactam group of the precursor. Additionally, one may convert a salt of the
compound of SDHi to another salt through treatment with an appropriate acid or
base or through contact with an ion exchange resin. Following reaction, one may
then isolate the salt by filtration if it precipitates from solution, 5 or by evaporation
to recover the salt. The degree of ionization of the salt may vary from completely
ionized to almost non-ionized.
It may be convenient or desirable to prepare, purify, and/or handle a
10 corresponding solvate of the active compound. The term “solvate” describes a
molecular complex comprising the compound and one or more pharmaceutically
acceptable solvent molecules (e.g., EtOH). The term “hydrate” is a solvate in
which the solvent is water. Pharmaceutically acceptable solvates include those in
which the solvent may be isotopically substituted (e.g., D2O, acetone-d6, DMSO15
d6).
A currently accepted classification system for solvates and hydrates of organic
compounds is one that distinguishes between isolated site, channel, and metal-ion
coordinated solvates and hydrates. See, e.g., K. R. Morris (H. G. Brittain ed.)
20 Polymorphism in Pharmaceutical Solids (1995). Isolated site solvates and
hydrates are ones in which the solvent (e.g., water) molecules are isolated from
direct contact with each other by intervening molecules of the organic compound.
In channel solvates, the solvent molecules lie in lattice channels where they are
next to other solvent molecules. In metal-ion coordinated solvates, the solvent
25 molecules are bonded to the metal ion.
When the solvent or water is tightly bound, the complex will have a well-defined
stoichiometry independent of humidity. When, however, the solvent or water is
weakly bound, as in channel solvates and in hygroscopic compounds, the water or
30 solvent content will depend on humidity and drying conditions. In such cases,
non-stoichiometry will typically be observed.
25
Further particular and preferred aspects are set out in the accompanying
independent and dependent claims. Features of the dependent claims may be
combined with features of the independent claims as appropriate, and in
combinations other than those explicitly set out in the claims.
5
A wide variety of compounds of SDHi’s are commercially available. Derivatives
of such commercially available compounds may be prepared by carrying out
functional group interconversions or making substitutions and carrying out
common reactions as are known in the art. Common techniques and reactions,
10 including oxidations, reductions, and so on, separation techniques (extraction,
evaporation, precipitation, chromatography, filtration, trituration, crystallization,
and the like), and analytical procedures, are known to persons of ordinary skill in
the art of organic chemistry. The details of such reactions and techniques can be
found in a number of treatises, including Richard Larock, Comprehensive Organic
15 Transformations, A Guide to Functional Group Preparations, 2nd Ed (2010), and
the multi-volume series edited by Michael B. Smith and others, Compendium of
Organic Synthetic Methods (1974 et seq.). Starting materials and reagents may be
obtained from commercial sources or may be prepared using literature methods.
In addition, in some instances, reaction intermediates may be used in subsequent
20 steps without isolation or purification (i.e., in situ).
Certain compounds can be prepared using protecting groups, which prevent
undesirable chemical reaction at otherwise reactive sites. Protecting groups may
also be used to enhance solubility or otherwise modify physical properties of a
25 compound. For a discussion of protecting group strategies, a description of
materials and methods for installing and removing protecting groups, and a
compilation of useful protecting groups for common functional groups, including
amines, carboxylic acids, alcohols, ketones, aldehydes, and so on, see T. W.
Greene and P. G. Wuts, Protecting Groups in Organic Chemistry, 4th Edition,
30 (2006) and P. Kocienski, Protective Groups, 3rd Edition (2005).
26
Generally, chemical transformations may be carried out using substantially
stoichiometric amounts of reactants, though certain reactions may benefit from
using an excess of one or more of the reactants. Additionally, many of the
suitable reactions may be carried out at about room temperature (RT) and ambient
pressure, but depending on reaction kinetics, yields, and so 5 on, some reactions
may be run at elevated pressures or employ higher temperatures (e.g., reflux
conditions) or lower temperatures (e.g., -78°C. to 0°C.). Any reference in the
disclosure to a stoichiometric range, a temperature range, a pH range, etc.,
whether or not expressly using the word “range,” also includes the indicated
10 endpoints.
BRIEF DESCRIPTION OF THE DRAWINGS
Embodiments of the present invention will now be described further, with
15 reference to the accompanying drawings, in which:
Figure 1: Comparative metabolomics identifies succinate as a potential
mitochondrial metabolite that drives reperfusion ROS production. (a) Outline of
comparative metabolomics strategy for identification of metabolites that
20 accumulate in vivo under ischaemic conditions.
Figure 1: (b) HIVE plot comparative analysis of metabolites significantly
accumulated across all ischaemic tissues in vivo. Metabolites accumulated
commonly across all tissues are highlighted.
Figure 1: (c) Prevalence of accumulation of metabolites in murine tissues during
25 ischaemia.
Figure 1: (d) Profile of mitochondrial citric acid cycle (CAC) metabolite levels
following ischaemia across five ischaemic tissue conditions. n = 3-4 mice per
tissue, per group.
Figure 1: (e) Time course of CAC metabolite levels during myocardial ischaemia
30 and reperfusion for 5 min in the ex vivo heart. n = 4 mouse hearts per group.
27
Figure 1: (f) CAC metabolite levels during in vivo myocardial IR in at risk and
peripheral heart tissue following ischaemia and after 5 min reperfusion. n = 3-5
mice per group.
Figure 1: (g) CAC metabolite levels during in vivo brain IR immediately
following ischaemia and following 5 min reperfusion. n = 5 3 rats per group.
Figure 1: (h) CAC metabolite levels during in vivo kidney IR immediately
following ischaemia and after 5 min reperfusion. n = 3 mice per group.
Figure 1: (i) Effect of succinate on ROS production assessed by dihydroethidium
(DHE) oxidation in adult primary cardiomyocytes during IR. n = 3 independent
10 cardiomyocyte preparations per group. * p < 0.05, ** p < 0.01, *** p < 0.001.
Data are shown as the mean ± s.e.m of at least three replicates.
Figure 2: Reverse SDH activity drives ischaemic succinate accumulation by the
reduction of fumarate produced by the malate-aspartate shuttle and the purine
nucleotide cycle. (a) Potential inputs to succinate-directed ischaemic flux.
Figure 2: (b) 1315 C-glucose metabolic labelling strategy.
Figure 2: (c) 13C isotopic labelling profile of succinate in the normoxic and
ischaemic myocardium. n = 4 hearts per group.
Figure 2: (d) Effect of inhibition of ı-aminobutyric acid (GABA) shunt with
vigabatrin on GABA and succinate levels in the ischaemic myocardium. n = 3
20 hearts per group.
Figure 2: (e) Summary of in silico metabolic modelling of potential drivers of
ischaemic succinate accumulation driven by reverse SDH operation; and 13Caspartate
metabolic labelling strategy to determine the contribution of malate- and
adenylosuccinate-linked pathways on ischaemic succinate accumulation.
25 Figure 2: (f) Effect of SDH inhibition by dimethyl malonate on succinate
accumulation and CAC metabolite abundance in the ischaemic myocardium in
vivo. n = 3 mice per group.
Figure 2: (g) Relative ischaemic flux of 13C-aspartate to the indicated CAC
metabolites in the normoxic and ischaemic myocardium. n = 4 hearts per group.
30 Figure 2: (h) Effect on CAC metabolite abundance in the ischaemic myocardium
in vivo of blocking NADH-dependent aspartate entry into the CAC, through
aminooxyacetate (AOA)-mediated inhibition of aspartate aminotransferase, or
28
blocking PNC by inhibition of adenylosuccinate lyase with AICAR. n = 3 mice
per group. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are shown as the mean ±
s.e.m of at least three replicates.
Figure 3: Ischaemic succinate levels control ROS production, mitochondrial
membrane polarisation, and NAD(P)H reduction state at reperfusion 5 in adult
primary cardiomyocytes. (a) Effect of manipulation of ischaemic succinate levels
on ROS production measured as DHE oxidation during late ischaemia and early
reperfusion. n = 3 independent cardiomyocyte preparations per group.
Figure 3: (b) Determination of mitochondrial complex I RET contribution to
10 succinate-driven ROS production at reperfusion through selective inhibition with
MitoSNO. n = 3 independent cardiomyocyte preparations per group.
Figure 3: (c) Effect of manipulation of ischaemic succinate levels on NAD(P)H
oxidation during late ischaemia and early reperfusion. n = 3 independent
cardiomyocyte preparations per group.
15 Figure 3: (d) Effects of manipulation of ischaemic succinate levels on
mitochondrial membrane potential following late ischaemia (left panels) and early
reperfusion (right panels) using TMRM fluorescence in de-quench mode (high
fluorescence = low membrane potential).
Figure 3: (e) Effects of manipulation of ischaemic succinate levels on the initial
20 rate of inner membrane re-polarisation following 40 min ischaemia as determined
by the rate of TMRM quenching at reperfusion. n = 3 independent cardiomyocyte
preparations per group.
Figure 3: (f) Effect of dimethyl succinate and oligomycin on mitochondrial ROS
in the aerobic C2C12 myoblasts. Mitochondria within myoblasts under normoxic
25 incubation were hyperpolarised with oligomycin and dimethyl succinate and the
rate of MitoSOX oxidation assessed to measure mitochondrial ROS production. n
= 4 independent experiments of 10-12 myoblasts per trial. * p < 0.05, ** p < 0.01.
Data are shown as the mean ± s.e.m of at least three replicates.
Figure 4: NADH and AMP sensing pathways drive ischaemic succinate
30 accumulation to control reperfusion pathologies in vivo through mitochondrial
ROS production. (a) Model of succinate accumulation during ischaemia and
superoxide formation by reverse electron transport (RET) during reperfusion.
29
Figure 4: (b) Representative images of cross-sections from mouse hearts
following myocardial infarction ± inhibition of ischaemic succinate accumulation
by i.v. injection of dimethyl malonate, or reintroduction of ischaemic succinate by
i.v. injection of dimethyl malonate and dimethyl succinate. Infarcted tissue is
white, the rest of the area at risk is red, and non-risk tissue 5 is dark blue.
Figure 4: (c) Quantification of myocardial infarct size carried out as described in
b. Each open circle represents data from a single mouse, and filled circles
represent the mean values of all mice for a particular condition. n = 6 mice per
group.
10 Figure 4: (d) Protection by dimethyl malonate against brain IR injury in vivo.
Representative images of cross-sections from rat brains after undergoing tMCAO
in vivo with or without treatment with dimethyl malonate. Brains were treated
with hematoxylin and eosin to delineate infarcted tissue. Quantification of brain
infarct volume
15 Figure 4: (e) and rostro-caudal infarct distribution
Figure 4: (f) ± dimethyl malonate following brain IR injury by tMCAO in vivo. n
= 3-6 rats per group.
Figure 4: (g) Neurological scores for rats following tMCAO ± dimethyl
malonate. n = 3-6 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are
20 shown as the mean ± s.e.m, except for (g) for which data are median ± C.I.
Figure 5: Dimethyl malonate and dimethyl succinate treatment of cells and in
vivo results in intracellular accumulation of malonate and succinate. a,
Intravenous infusion of dimethyl malonate in vivo results in accumulation of
malonate in the ischaemic myocardium. b, C2C12 cells were incubated with: no
25 additions, glucose, 5 mM dimethyl succinate, 5 mM dimethyl malonate, or 5 mM
dimethyl malonate and 5 mM dimethyl succinate. Cellular oxygen consumption
rate due to ATP synthesis and c, maximal rates were determined using a Seahorse
XF96 analyser. n = 3-4, *, p < 0.05; ***, p < 0.001.
Figure 6: Summary of the three potential metabolic inputs for succinate-directed
30 ischaemic flux. To understand the metabolic pathways that could contribute to
succinate production under ischaemia, an updated version of the iAS253 model of
cardiac metabolism was employed to simulate ischaemia using flux balance
30
analysis. The model showed three possible mechanisms for producing succinate:
from alpha-ketoglutarate produced by the CAC, derived from glycolysis and
glutaminolysis (grey box), from succinic semialdehyde produced from the GABA
shunt (blue box), and from fumarate produced from the malate-aspartate shuttle
and purine nucleotide cycle (red box) via the 5 reversal of SDH.
Figure 7: Metabolic labelling of CAC and proximal metabolites by 13C glucose
in the ischaemic and normoxic myocardium. Proportional isotopic labelling
profile of CAC and proximal metabolites during normoxic and ischaemic
myocardial respiration. Mouse hearts were perfused with 11 mM 13C glucose for
10 10 min followed by either 30 min no flow ischaemia or 30 min normoxic
respiration followed by snap freezing and LC-MS metabolite analysis. n = 4, * p <
0.05, ** p < 0.01, *** p < 0.001.
Figure 8: Metabolic labelling of CAC and proximal metabolites by 13C
glutamine in the ischaemic and normoxic myocardium. Isotopic flux from
15 glutamine to CAC and proximal metabolites during normoxic and ischaemic
myocardial respiration. Mouse hearts were perfused with 4 mM 13C glutamine
(+5 labelled) for 10 min followed by either 30 min no flow ischaemia or 30 min
normoxic respiration followed by snap freezing and metabolomics analysis. (a)
The isotopic profile for each metabolite is expressed as a proportion of the total
20 pool.
Figure 8: (b,) Additionally, flux to ıKG was determined relative to the
proportion of the +5 glutamine pool in the heart. n = 4, * p < 0.05.
Figure 9: Effect of GABA transaminase inhibition by vigabatrin on GABA shunt
and CAC metabolites in the ischaemic myocardium. Perfused mouse hearts were
25 subjected to 30 min no flow ischaemia ± continuous infusion of vigabatrin (Vig;
100, 300, and 700 ıM) 10 min prior to ischaemia. Heart tissue was snap frozen
and a, GABA and b, succinate abundance quantified relative to normoxic levels
by LC-MS. n = 3, * p < 0.05.
Figure 10: Unabridged metabolic model identifying pathways that can become
30 activated by tissue ischaemia to drive succinate accumulation. To identify the
metabolic pathways that could contribute to succinate production under
ischaemia, we simulated these conditions using flux balance analysis in
31
conjunction with an expanded version of the iAS253 mitochondrial model of
central cardiac metabolism. The major pathways contributing to succinate
accumulation (bold red lines) were via fumarate feeding into the reverse activity
of SDH. This was produced by the purine nucleotide cycle (PNC) and the malateaspartate
shuttle (MAS), which consumed glucose and aspartate, 5 and also led to
significant production of lactate and alanine. Lesser sources of succinate (thin red
lines) included glycolysis and glutaminolysis but this was relatively minor as this
route was constrained by the overproduction of NADH. In addition a small
amount of fumarate was generated by pyruvate carboxylase activity. The GABA
10 shunt did not contribute (black dashed line).
Figure 11: Comparison of 13C ischaemic metabolite fluxes to succinate relative
to isotopic pools. Mouse hearts were perfused with 13C-glucose (+6 labelled) ,
13C-glutamine (+5 labelled), or 13C-aspartate (+1 labelled) for 10 min followed
by 30 min no flow ischaemia or 30 min normoxic respiration, and snap freezing
15 and metabolomics analysis. To compare the relative magnitude of metabolite flux
from each carbon source, ischaemic 13C incorporation to succinate compared to
the unlabelled succainate pool was determined relative to normoxia, and
expressed relative to the total labelled pool of 13C-infused precursor. Data are
plotted on a linear (left) and logarithmic (right) scale.
20 Figure 12: Predicted changes in pathways of succinate and OXPHOS metabolism
during ichaemia and following reperfusion. To determine possible changes in
succinate metabolism during ischaemia, reperfusion and normoxia, cardiac
metabolism was simulated in these conditions using an expanded version of the
iAS253 model with flux balance analysis. The simulations predicted that: (a)
25 under ischaemia, complex II ran in reverse by using ubiquinol produced by
complex I to reduce fumarate to succinate, thereby acting as a terminal electron
acceptor instead of oxygen. Fumarate was produced from the purine nucleotide
cycle (PNC) and reversal of the citric acid cycle (CAC). Flux through the rest of
the respiratory chain was diminished and AMP was produced from ADP due to
30 insufficient ATP production.
Figure 12: (b) With oxygen restored complex II metabolised the excess succinate.
A delay in regenerating AMP to ADP, as typified in the first minute of
32
reperfusion, limited the flux through ATP-synthase. This in turn prevented
complex III consuming all the ubiquinol generated by complex II, as the
membrane became hyper-polarised. The excess flux of ubiquinol and protons
forced complex I to run in reverse, which would generate ROS.
Figure 12: (c) Once the flux of succinate was reduced to normal 5 levels, as in the
transition from late reperfusion to normoxia, the fluxes through the respiratory
chain and citric acid cycle returned to normal.
Figure 13: Tracking cardiomyocytes ROS levels in primary cardiomyocytes
during in situ IR. Primary rat cardiomyocytes were subjected to 40 min ischaemia
10 and reoxygenation with ROS levels tracked throughout the experiment by
ratiometric measurement of dihydroethidium (DHE) oxidation. Ischaemic buffer
contained either no additions, 4 mM dimethyl malonate, or 4 mM dimethyl
succinate. Representative traces of each condition are shown. The highlighted
window indicates the period of the experiment expanded in detail in Fig. 3.a.
15 Figure 14: Tracking NADH reduction state in primary cardiomyocytes during in
situ IR. Primary rat cardiomyocytes were subjected to 40 min ischaemia and
reoxygenation and NADH reduction state was tracked throughout the experiment
by measurement of NAD(P)H autofluorescence. Ischaemic buffer contained either
no additions, 4 mM dimethyl malonate, or 4 mM dimethyl succinate.
20 Representative traces of each condition are shown. The highlighted window
indicates the period of the experiment expanded in detail in Fig. 3c.
Figure 15: Tracking mitochondrial membrane potential in primary
cardiomyocytes during in situ IR. Primary rat cardiomyocytes were subjected to
40 min ischaemia and reoxygenation and mitochondrial membrane potential was
25 tracked throughout the experiment by measurement of tetramethylrhoadmine
(TMRM) fluorescence. Ischaemic buffer contained either no additions or 4 mM
dimethyl malonate. Average traces from at least three replicate experiments are
shown. (a) TMRM signal throughout the entire experiment.
Figure 15: (b) TMRM signal during the transition from ischaemia to
30 reoxygenation. n = 3-4 experiments from independantly isolated primary rat
cardiomyocytes per group.
33
DESCRIPTION OF THE EMBODIMENTS
Mitochondrial ROS production is a critical early driver of ischaemia-reperfusion
(IR) injury, but has been considered a non-specific consequence of the interaction
of a dysfunctional respiratory chain with oxygen during reperfusion5 1-4. The
inventors investigated an alternative hypothesis: that mitochondrial ROS during
IR are generated by a specific metabolic process. To do this the inventors
developed a comparative metabolomics approach to identify conserved metabolic
signatures in tissues during IR that might indicate the source of mitochondrial
10 ROS (Fig. 1a). Liquid chromatography-mass spectrometry (LC-MS)-based
metabolomic analysis of murine brain, kidney, liver and heart subjected to
ischaemia in vivo (Fig. 1a) revealed changes in several metabolites. However,
only three were elevated across all tissues (Fig. 1b, c and Table 1).
15 Various murine tissues exposed to sufficient periods of ischaemia to prime for
reperfusion ROS production were subjected to metabolomic analysis and
comparison of metabolites that accumulated significantly when compared to
normoxic levels. Following this, metabolites were scored according to the
prevalence of their accumulation across five ischaemic pro-ROS tissue conditions
20 (see Table 1).
34
Table 1 Full comparative analysis of metabolites significantly accumulated in
ischaemic pro-ROS conditions. B = brain, H = whole heart ischaemia ex vivo,
HL = LAD ischaemia in vivo, K = 5 kidney, L = liver
35
Two metabolites were well-characterised by-products of ischaemic purine
nucleotide breakdown, xanthine and hypoxanthine6, corroborating the validity of
our approach. Xanthine and hypoxanthine are metabolised by cytosolic xanthine
oxidoreductase and do not contribute to mitochondrial metabolism7. The third
metabolite, the mitochondrial citric acid cycle (CAC) intermediate 5 succinate,
increased 3- to 19-fold across the tested tissues (Fig. 1d) and was the sole
mitochondrial metabolic feature of ischaemia that occurred universally in a range
of metabolically diverse tissues. Therefore the inventors focused on the potential
role of succinate in mitochondrial ROS production during IR.
10
Since mitochondrial ROS production occurs early in reperfusion1-4,8,9, it follows
that metabolites fuelling ROS should be oxidised quickly. Strikingly, the
succinate accumulated during ischaemia was restored to normoxic levels by 5
minutes reperfusion ex vivo in the heart (Fig. 1e), and this was also observed in
15 vivo in the heart, brain and kidney (Fig. 1f-h). Furthermore, these succinate
changes were localised to areas of the tissues where IR injury occurred in vivo,
and took place in the absence of the accumulation of other CAC metabolites (Fig.
1d, f). To further assess the role of succinate in driving ROS production, the
inventors administered a cell permeable derivative of succinate, dimethyl
succinate (Fig. 5), to ischaemic primary cardiomyocytes8 20 and found that this
significantly increased mitochondrial ROS production during reperfusion (Fig.
1i). These data suggest that the succinate accumulated during ischaemia then
fuels ROS production upon reperfusion.
25 To determine the mechanisms responsible for succinate accumulation during
ischaemia and explore its role in IR injury the inventors focused on the heart,
because of the many experimental and theoretical resources available. In
mammalian tissues succinate can be generated through α-ketoglutarate-dependent
CAC flux from pyruvate or glutamate, or the ı-aminobutyric acid (GABA) shunt,
(Fig. 2a and 6)10,1130 . Ischaemic CAC flux to succinate via α-ketoglutarate was
investigated by measuring its isotopologue distribution following infusion with
13C-labelled glucose or glutamine, which enter the CAC from pyruvate and
36
glutamate, respectively. The 13C-isotopologue distribution of succinate upon
perfusion with U-13C-glucose was significantly reduced in ischaemic hearts
indicating that pyruvate and α-ketoglutarate-linked CAC flux to succinate
decreases during ischaemia (Fig. 2b, c, and 7). Glutamine was not a major carbon
source for CAC metabolites in normoxia or ischaemia (Fig. 8a). 5 Furthermore, the
minimal 13C-glutamine incorporation to α-ketoglutarate observed was decreased
in ischaemia (Fig. 8b), and inhibition of the GABA shunt with vigabatrin10 did not
decrease ischaemic succinate accumulation (Fig. 2d and 9). Therefore the build
up of succinate during ischaemia is not caused by conventional operation of
10 cardiac metabolism.
To explore other mechanisms that could lead to succinate accumulation during
ischaemia, the inventors considered whether during anaerobic metabolism SDH
might act in reverse to reduce fumarate to succinate12-14. While SDH reversal has
15 not been demonstrated in ischaemic tissues, in silico flux analysis determined
succinate production by SDH reversal during ischaemia as an optimal solution
(Fig. 2e and 10). This approach also suggested that fumarate supply to SDH
could come from two converging pathways: the malate/aspartate shuttle (MAS),
where the high NADH/NAD ratio drives malate formation that is converted to
fumarate14-1620 ; and AMP-dependent activation of the purine nucleotide cycle
(PNC) that drives fumarate production17,18 (Fig. 2e and 10). To test this
prediction experimentally, the inventors infused mice with dimethyl malonate, a
membrane-permeable precursor of the SDH competitive inhibitor malonate (Fig.
5)19,20. Surprisingly, dimethyl malonate infusion strikingly decreased succinate
25 accumulation in the ischaemic myocardium (Fig. 2f). This result indicates that
SDH operates in reverse in the ischaemic heart, as inhibition of SDH operating in
its conventional direction would have further increased succinate (Fig. 2a and 10).
Therefore succinate accumulates during ischaemia from fumarate reduction by the
reversal of SDH.
30
Since aspartate is a common carbon source for fumarate in both the PNC and the
MAS pathways (Fig. 2e), the inventors used 13C-labelled aspartate to evaluate the
37
contribution of these pathways to succinate production during ischaemia (Fig. 6
and 10). 13C-aspartate infusion significantly increased 13C-succinate content of
the ischaemic myocardium compared to normoxia (Fig. 2g). In fact, 13C-asparate
was the only 13C-carbon donor that incorporated significantly into succinate
during ischaemia (Fig. 11). To characterise the relative contributions 5 of the MAS
and PNC to ischaemic succinate accumulation the inventors used
aminooxyacetate (AOA), which inhibits aspartate aminotransferase in the MAS21
(Fig. 2e) and 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR),
which inhibits adenylosuccinate lyase in the PNC18,22 (Fig. 2e). Both inhibitors
10 decreased ischaemic succinate levels (Fig. 2h). Therefore, these results indicate
that during ischaemia both the MAS and PNC pathways increase fumarate
production, which is then converted to succinate by SDH reversal.
To investigate the potential mechanisms underlying succinate-driven
15 mitochondrial ROS production the inventors modelled how ischaemic cardiac
metabolism alters upon reperfusion (Fig. 12). This analysis suggested that SDH
would oxidise the accumulated succinate and thereby drive reverse electron
transport (RET) through mitochondrial complex I23-26. Intriguingly, succinate
drives extensive superoxide formation from complex I by RET in vitro, making it
a compelling potential source of mitochondrial ROS during IR2620 . However, the
role of complex I RET in IR injury has never been investigated. To test whether
the succinate accumulated during ischaemia could drive complex I RET upon
reperfusion, the inventors tracked mitochondrial ROS with the fluorescent probe
dihydroethidine (DHE) and mitochondrial membrane potential from the potential25
sensitive fluorescence of tetramethylrhodamine (TMRM), in a cardiomyocyte
model of IR injury27. The rate of oxidation of DHE remained stable during
ischaemia but rose rapidly upon reperfusion, consistent with increased superoxide
production (Fig. 3a and 13)27. Inhibition of SDH-mediated ischaemic succinate
accumulation with dimethyl malonate reduced DHE oxidation upon reperfusion
30 (Fig. 3a), whereas increasing succinate further during ischaemia with dimethyl
succinate amplified reperfusion DHE oxidation, indicating that succinate levels
controlled the extent of reperfusion ROS (Fig. 3b). Importantly, selective
38
blocking of complex I RET with MitoSNO8 abolished dimethyl succinate-driven
DHE oxidation upon reperfusion (Fig. 3b), indicating that ischaemic succinate
levels drove superoxide production through complex I RET. Succinate-dependent
RET was further supported by the observation that NAD(P)H oxidation at
reperfusion was suppressed by increasing succinate levels with 5 dimethyl succinate
(Fig. 3c and 14). Tracking the mitochondrial membrane potential revealed that
inhibition of ischaemic succinate accumulation diminished the rate of
mitochondrial polarisation upon reperfusion (Fig. 3d,e and 15), consistent with
succinate-dependent enhanced complex I RET. Finally, elevating succinate in
10 C2C12 mouse myoblast cells with dimethyl succinate while hyperpolarising
mitochondria with oligomycin increased MitoSOX oxidation independently of IR
(Fig. 3f), suggesting that combining high succinate levels with a large
protonmotive force is sufficient to drive complex I ROS production by RET.
15 The inventors’ findings may be explained by the following model (Fig. 4a):
during ischaemia fumarate production increases, through activation of the MAS
and PNC, and is then reduced to succinate by SDH reversal. Upon reperfusion,
the accumulated succinate is rapidly oxidised, thereby sustaining a large
protonmotive force and driving RET at complex I to produce the mitochondrial
ROS that initiate IR injury2620 . This model provides a unifying framework for
many hitherto unconnected aspects of IR injury, such as the requirement for
priming during ischaemia to induce ROS upon reperfusion, protection against IR
injury by the inhibition of complexes I8 and II28, and by mild uncoupling29.
25 Intriguingly, the inventors’ model also generates an unexpected, but testable,
prediction. Manipulation of the pathways that increase succinate during
ischaemia and oxidise it upon reperfusion should determine the extent of IR
injury. Since the reversible inhibition of SDH blocks both succinate accumulation
during ischaemia (Fig. 2b) and its oxidation upon reperfusion, it should protect
30 against IR injury in vivo. Intravenous (i.v.) infusion of dimethyl malonate, a
precursor of the SDH inhibitor malonate, during an in vivo model of cardiac IR
injury was protective (Figs. 4b,c). Importantly, this cardioprotection was
39
suppressed by adding back dimethyl succinate (Figs. 4b,c), indicating that
protection resulted solely from blunting succinate accumulation. Finally, i.v.
infusion of dimethyl malonate during rat brain transient middle cerebral artery
occlusion (tMCAO), an in vivo model of brain IR injury during stroke, was
protective, reducing the pyknotic nuclear morphology and 5 vacuolation of the
neuropil (Fig. 4d), decreasing the volume of infarcted brain tissue caused by IR
injury (Figs 4e,f) and preventing the decline in neurological function associated
with stroke (Fig 4g and Table 2).
MCAO Control Baseline Day 1 Day 2 Day 3
Median Neurological Score 31 16 19 20
Average Neurological Score 30.67 16.33 19.00 20.33
Alpha 0.05 0.05 0.05 0.05
n 6.00 6.00 6.00 6.00
Standard Deviation 0.82 2.25 2.37 2.34
Confidence Interval 0.29 1.10 1.06 1.02
St Error 0.33 0.92 0.97 0.95
10
MCAO + Dimethyl malonate Baseline Day 1 Day 2 Day 3
Median Neurological Score 32.00 22.00 24.00 25.00
Average Neurological Score 31.67 22.67 24.00 24.33
Alpha 0.05 0.05 0.05 0.05
n 3.00 3.00 3.00 3.00
Standard Deviation 0.58 2.08 2.00 1.15
Confidence Interval 0.20 0.87 0.80 0.45
St Error 0.33 1.20 1.15 0.67
Table 2: Extended summary of neurological scores of rats subjected to
tMCAO IR in vivo ± dimethyl malonate infusion. Extended data summary of
15 median neurological scores in the three days following tMCAO IR ± dimethyl
malonate described in Fig. 4g.
40
These findings validate the inventors’ model of succinate-driven IR injury,
demonstrating that succinate accumulation underlies IR injury in the heart and
brain and validate the therapeutic approach of decreasing succinate accumulation
and oxidation to 5 treat IR injury.
The inventors have demonstrated that accumulation of succinate, via fumarate
production and reversal of SDH, is a universal metabolic signature of ischaemia in
vivo. In turn, succinate is a primary driver of the mitochondrial ROS production
10 upon reperfusion that underlies IR injury in a range of tissues. Ischaemic
accumulation of succinate may be of further relevance via its role in inflammatory
and hypoxic signalling10. Thus succinate could contribute to both the acute
pathogenesis of IR injury by mitochondrial ROS, and then upon secretion also
trigger inflammation and neovascularisation30. Besides elucidating the metabolic
15 responses that underlie IR injury, the inventors’ have demonstrated that
preventing succinate accumulation during ischaemia is protective against IR
injury in vivo, thus, providing novel therapeutic targets for IR injury in
pathologies such as heart attack and stroke.
20 METHODS SUMMARY
The following murine models of warm ischaemia and/or IR injury were used:
global ischaemia of ex vivo perfused mouse heart; in vivo mouse left descending
coronary artery (LAD) ligation; in vivo rat brain transient middle cerebral artery
25 occlusion (tMCAO); in vivo mouse kidney ischaemia by monolateral occlusion of
the renal hilum; in vivo global ischaemia of the liver following cervical
dislocation. The modelling of metabolic pathways in the ischaemic myocardium
was done as described previously11. The cell models of IR injury used were adult
isolated primary rat cardiomyocytes and the mouse C2C12 myoblast cell line.
30 Fluorescence within cardiomyocytes was assessed by laser scanning confocal
microscopy. DHE fluorescence was used to measure ROS production, TMRM
fluorescence in de-quench mode was used for mitochondrial membrane potential
41
and NAD(P)H autofluorescence was used to assess the reduction potential of the
NAD(P)H pools8. To assess metabolic pathways in the ex vivo perfused mouse
heart 13C-labelled glucose, glutamine or aspartate were infused into the isolated
heart and the 13C isotopologues of CAC metabolites were then quantified by LCMS.
The effects of metabolic inhibitors on ischaemic metabolite 5 accumulation
and the distribution of 13C-labels were assessed by i.v. infusion of metabolic
inhibitors followed by LC-MS analysis of tissue metabolites. The redox state of
the cardiac CoQ pool was assessed by LC. The amelioration of in vivo IR injury
by metabolic inhibitors was assessed by i.v. administration of the inhibitors
10 followed by measurement of the infarct size in the heart by triphenyltetrazolium
chloride staining and in the brain by determining neurological function and infarct
size by histology.
EXAMPLES
15
METHODS
In vivo mouse myocardial experiments. The mice used were C57BL/6J.
20 In vivo mouse myocardial experiments. For the in vivo heart IR model an openchest,
in situ heart model was used31,32. Male mice (8–10 weeks; Charles River
Laboratories, UK) were anaesthetised with sodium pentobarbital (70 mg/kg
intraperitoneally (i.p.)), intubated endotracheally and ventilated with 3 cm H2O
positive-end expiratory pressure. Adequacy of anaesthesia was monitored using
25 corneal and withdrawal reflexes. Ventilation frequency was kept at 110 breaths
per minute with tidal volume between 125 - 150 μL. A thoracotomy was
performed and the heart exposed by stripping the pericardium. A prominent
branch of the left anterior descending coronary artery (LAD) was surrounded by a
7-0 Prolene suture that was then passed through a small plastic tube. Ischaemia
30 was induced by tightening the tubing against the heart surface. To assess
metabolites during IR in vivo, mice were divided into three groups: 30 min
ischaemia, 30 min ischaemia plus 5 min reperfusion and 30 min sham-operation
42
in which the suture was placed but the LAD was not occluded. At the end of each
protocol tissue was removed from the at risk and peripheral areas of the heart,
selected visually by comparing white versus red tissue, and snap-frozen in liquid
nitrogen. Sham-operated tissue was removed from the presumed risk zone.
5
Infarct size was assessed after 30 min of ischaemia followed by 120 min
reperfusion using 2% triphenyltetrazolium chloride staining and is expressed as a
percentage of the risk zone33. Metabolic inhibitors (all from Sigma) in sterile
saline were infused i.v. via a tail vein 10 min prior to and throughout ischaemia at
10 the following doses: dimethyl malonate (4 mg/kg/min), AOA (50 μg/kg/min;
Fluorochem) and AICAR 10 mg/kg/min). Dimethyl succinate (8 mg/kg/min) was
infused in combination with dimethyl malonate. Control mice were infused with
sterile saline. The total volume administered never exceeded 200 μL/mouse.
15 Ex vivo Langendorff heart experiments for metabolomic analysis. Mice were
heparinised (200 U i.p.) and anaesthetised with sodium pentobarbital (100 mg/kg
i.p.). The chest was then opened and the heart rapidly excised and arrested in cold
Krebs-Henseleit (KH) buffer (0.5 mM EDTA, 118 mM NaCl, 4.7 mM KCl, 25
mM NaHCO3, 11 mM glucose, 1.2 mM MgSO4, 1.2 mM KH2PO4 and 2 mM
20 CaCl2) at pH 7.4. The aorta was then cannulated with a 22 G blunt needle and
transferred to a perfusion apparatus. The heart was perfused with 37°C KH buffer
(95% O2/5% CO2) at a constant pressure of 80 mm Hg. After 20 min
equilibration hearts were separated into four groups: 60 min normoxic perfusion;
30 min global ischaemia; 30 min global ischaemia plus 5 min reperfusion; and 30
25 min global ischaemia plus 30 mins reperfusion. Metabolic inhibitors were infused
for 10 min prior to ischaemia through a side port above the aortic cannula at 1%
of coronary flow. At the end of the experiments the hearts were snap-frozen in
liquid nitrogen and stored at -80°C.
1330 C metabolite labelling in ex vivo Langendorff heart experiments. Mice were
anaesthetised with sodium pentobarbital (~140 mg/kg). Hearts were rapidly
excised, cannulated and perfused in the isovolumic Langendorff mode at 80 mm
43
Hg perfusion pressure, at 37ºC with KH buffer continuously gassed with 95%
O2/5% CO2 (pH 7.4)34. Cardiac function was assessed using a fluid-filled balloon
inserted into the left ventricle (LV), and connected to a pressure transducer and a
PowerLabTM system (ADInstruments, UK). Balloon volume was adjusted to an
initial LV diastolic pressure of 4 - 9 mm Hg34 and all hearts 5 were paced at 550
bpm. Left ventricular developed pressure (LVDP) was calculated from the
difference between systolic (SP) and diastolic pressures (DP). Functional
parameters (SP, end diastolic pressure, heart rate, LVDP, coronary flow, perfusion
pressure) were recorded continuously using LabChartTM software v.7
10 (ADInstruments, UK).
After 20 min equilibration with standard KH buffer, hearts were divided into the
following groups: perfused with KH buffer containing 11 mM U-13C Glucose
followed by 30 min normoxic respiration (n=4/group); perfused for 10 min with
KH buffer containing 11 mM U-1315 C glucose and then subjected to 30 min global
normothermic ischaemia (n=4/group); perfusion of KH buffer containing 1 mM 5-
13C L-glutamine for 10 min followed by standard normoxic perfusion for 30 min
with unlabeled KH buffer (n=4);10 min perfusion with 1 mM 13C5 L-glutamine,
followed by 30 min global ischaemia (n=4); 10 min perfusion of 1mM 1-13C L20
aspartic acid, followed by normoxic perfusion for 30 min with unlabeled KH
buffer; 10 min perfusion with 1mM 1-13C L-aspartic acid, followed by 30 min
global ischaemia. At the end hearts were snap frozen in liquid nitrogen and stored
at -80°C.
25 In vivo rat brain ischaemia and reperfusion. Male spontaneously hypertensive
stroke prone (SHRSP) rats from the colony maintained at the University of
Glasgow (270–310 g) were anaesthetised with 5% isoflurane in oxygen and were
intubated and ventilated throughout surgery (~2.5% isoflurane/oxygen). Body
temperature was maintained at 37 ± 0.5°C. Animals underwent pre-stroke
burrhole surgery35 30 before transient middle cerebral artery occlusion (tMCAO, 45
min). Briefly, a silicone-coated monofilament was advanced (Doccol
Corporation, USA) was advanced through the common carotid artery to block the
44
origin of the MCA36. Animals were maintained under anaesthesia during
ischaemia. Immediately following removal of the filament, or after 5 mins of
reperfusion, the brain was removed following cervical dislocation and infarct
tissue separated from surrounding tissue on the ipsilateral side and snap frozen in
liquid nitrogen for metabolomic analysis. Corresponding regions 5 were taken from
the contralateral side. A separate group was infused with dimethyl malonate (6
mg/kg/min) by i.v. infusion 10 min prior to and during tMCAO) or carrier,
allowed to recover for 3 days, over which time they were scored for neurological
function37 as modified38. These rats were then sacrificed by transcardiac perfusion
10 fixation and the infarct area was assessed across 7 coronal levels following
hematoxylin and eosin staining39.
In vivo mouse renal ischaemia and reperfusion. Under isofluorane general
anaesthesia, mice underwent laparotomy and exposure of the renal hilum
15 bilaterally. Vascular clips (8 mm, InterFocus Fine Science Tools, Cambridge,
UK) were placed over one renal hila to induce unilateral renal ischaemia. At the
end of 45 min ischaemia the clip was removed and reperfusion of the kidney
noted as return of blush colour and visualisation of flow from the renal vein.
Kidneys were taken at the end of ischaemia, or following 5 min reperfusion and
20 snap-frozen in liquid nitrogen for metabolomic analysis.
In vivo mouse liver warm ischaemia. Mice were killed by cervical dislocation
to ensure cessation of blood flow. Liver tissue was maintained in situ in the body
cavity for 45 min at 37°C through use of a thermostated heat pad followed by
25 removal and snap-freezing on liquid nitrogen for subsequent metabolomic
analysis.
Metabolomic analyses. Equal amounts wet weight murine tissue were lysed in
250 μL extraction solution (ES; 30% acetonitrile, 50% methanol and 20% water)
30 per 10 mg tissue in Precellysis 24 vials, following the manufacturer’s instructions.
The suspension was immediately centrifuged (16,000 g, 15 min at 0°C) and the
supernatant used for LC-MS analysis. For the LC separation, column A was
45
Sequant Zic-Hilic (150 mm × 4.6 mm, internal diameter 3.5 μm) with a guard
column (20 mm × 2.1 mm 3.5 μm) from HiChrom, Reading, UK. Mobile phases.
A: 0.1% formic acid (v/v) in water. B: 0.1% formic acid (v/v) in acetonitrile.
Flow rate 300 μL/min. Gradient: 0-3 min 80 % B, 25 min 20% B, 26 min 80 % B,
36 min 80% B. Column B was sequant Zic-pHilic (150 mm × 2.1 5 mm i.d. 3.5 μm)
with guard column (20 mm × 2.1 mm i.d. 3.5 μm) from HiChrom, Reading, UK.
Mobile phases. C: 20 mM ammonium carbonate plus 0.1% ammonium hydroxide
in water. D: acetonitrile. Flow rate 100 μL/min. Gradient: 0 min 80% D, 28 min
20% D, 29 min 80% D, 45 min 80% D. The mass spectrometer (Thermo
10 QExactive Orbitrap) was operated in full MS and polarity switching mode.
Samples were randomised in order to avoid machine drifts. Spectra were
analysed using both targeted and untargeted approaches. For the targeted
approach spectra were analysed using XCalibur Qual Browser and XCalibur Quan
Browser softwares (Thermo Scientific) by referencing to an internal library of
compounds. For the untargeted approach spectra were processed with SieveTM 15
2.0 software (Thermo Scientific) and spectral peaks were extracted. The arrays of
spectra were then statistically analysed using the functions explore.data and
univariate of the R package muma40.
20 In situ ischaemia and reperfusion of adult rat primary cardiomyocytes. Male
Sprague-Dawley rats (300-370 g) were terminally anaesthetised via IP injections
of 200 mg/kg sodium pentobarbitone and 330 U/kg heparin. Hearts were excised
and retrograde perfused on a Langendorff-perfusion system with 13 mL/min
oxygenated KH buffer at 37°C. Cells were isolated by collagenase digestion
using standard methods4125 . Briefly, hearts were perfused for 5 min with KH
buffer, then 5 min with Ca2+-free KH buffer containing 100 μM EGTA, followed
by 8 min with KH buffer containing 100 μM CaCl2 and 0.5 mg/ml collagenase II
(Worthington). The heart was removed from the cannula and ventricles quickly
chopped and bathed in 20 mL of the same collagenase buffer for 15 min.
30 Digested tissue was passed through a 100 μm cell filter, and cells were collected
by gravity. The supernatant was removed and cells were washed with KH buffers
containing first 0.5 mM CaCl2, then 1 mM CaCl2. Typical yields were 2 x 106
46
cells/heart with 90% viable, rod-shaped cells. The cells were resuspended in
Medium 199 (supplemented with 5 mM creatine, 2 mM carnitine, 5 mM taurine,
and 100 μg/mL penicillin/streptomycin) and plated onto coverslips coated with
laminin (Sigma). After 1 h incubation at 37°C/5% CO2, unattached cells were
washed off, and fresh Medium 199 was added to each well for 5 at least 4 h at
37°C/5% CO2.
Cells were imaged within 36 hours of plating. Images using a ZeissTM LSM 510
META confocal microscope with a Fluar 20x/0.75NA UV objective, or a
microscope equipped with an OrcaTM 10 ER cooled CCD camera (Hamamatsu), a
monochrometer (Cairn Research) and emission filter wheel (Prior) with a Fluar
20x/0.75NA objective. Cells attached to coverslips, which formed the base of
custom built imaging chambers, were placed on a heated stage at 37°C on the
microscope with normoxic recording buffer (156 mM NaCl, 3 mM KCl, 2 mM
15 MgSO4, 1.25 mM K2HPO4, 2 mM CaCl2, 10 mM Hepes, 10 mM D-Glucose; pH
7.4). Simulated ischaemia was achieved by replacing the buffer with a pregassed,
hypoxic recording buffer simulating ischaemia (as above but lacking
glucose and containing 10 mM sodium lactate, 14.8 mM KCl; pH 6.4) and by
covering the heated stage with a transparent, gas-impermeant lid, forming a small
20 chamber into which argon was forced to maintain hypoxia. pO2 was routinely
measured as < 2.0 mm Hg during simulated ischaemia. To simulate reperfusion,
the lid was removed from the chamber, and the buffer replaced with normoxic
recording buffer.
25 Mitochondrial membrane potential was measured using tetramethylrhodamine,
methyl ester (TMRM, Life Technologies) in dequench mode. In this mode,
mitochondrial depolarisation causes redistribution of a high concentration of
quenched TMRM from mitochondria to cytosol, where the lower concentration
results in dequenching and an increase in fluorescence27. Cells were loaded at
30 room temperature with normoxic recording buffer containing 3 μM TMRM for 30
min. Prior to imaging, loading buffer was removed and replaced with normoxic
47
recording buffer. TMRM fluorescence was excited at 543 nm and emission was
collected using a LP 560 filter.
ROS production was estimated by oxidation of DHE. For this cells were loaded
with 5 μM dihydroethidium (DHE, Invitrogen), which 5 remained present
throughout normoxic and ischaemic conditions. DHE was excited at 351 nm and
the emitted signal was acquired with a BP 435-485 IR filter. Oxidised DHE was
excited at 543 nm and emission was collected with a LP 560 filter. NADH
autofluorescence was excited at 351 nm and the emitted signal was collected
10 using a BP 435-485IR filter. All measured cell parameters were analysed with
Fiji image processing software.
Assessment of succinate-dependent mitochondrial superoxide production in
myoblasts. C2C12 myoblasts were seeded in 35 mm glass bottom culture dishes
15 (MatTek) and incubated for 24 h in low glucose (1 g/L) DMEM. 2 h prior to
imaging DMEM was removed, and replaced with imaging buffer (132 mM NaCl;
10 mM HEPES; 4.2 mM KCl; 1 mM MgCl2 1mM CaCl2 adjusted to pH 7.4 with
Tris base and supplemented with 2-deoxyglucose (25 μM), and sodium pyruvate
(10 mg/L or 4 μM oligomycin as indicated). Myoblasts were pre-incubated with
20 2 μM MitoSOX for 15 min prior to imaging. MitoSOX fluorescence was
monitored using a Nikon Eclipse Ti confocal microscope at 37°C on a temperature
controlled stage for 30 min. MitoSOX was excited at 510 nm and the emitted
signal collected with a LP 560 filter following the indicated additions.
25 In silico analysis of metabolic flux during ischaemia and reperfusion.
Simulations were performed using an expanded version of the myocardial
mitochondrial metabolic model iAS25311. The model was expanded to include
additional mitochondrial reactions by using the latest version of MitoMiner, a
mitochondrial proteomics database42. MitoMiner was used to identify new
30 mitochondrial reactions for inclusion by cross-referencing these data with
information from BRENDA43, HumanCyc44 and relevant literature to confirm that
the new reactions are present in human, expressed in heart tissue and localised to
48
the mitochondrial matrix. In addition, cytosolic reactions were included that
could contribute to energy production, such as amino acid degradation and
conversion reactions as well as the purine nucleotide cycle. Protonation states of
metabolites in the model were calculated by using the Marvin suite of
computational chemistry software (ChemAxon Ltd, Budapest, 5 Hungary).
Reactions were then charge-balanced according to the protonation state of the
major microspecies found at pH 8.05 for the mitochondrial matrix45 and pH 7.30
for the cytosol. In addition directionality constraints were imposed based upon
general rules of irreversibility, thermodynamics and information from public
10 resources such as BRENDA and HumanCyc and capacity constraints were taken
from the literature11. The final model contained 227 mitochondrial matrix
reactions, 76 cytosolic reactions, 91 transport steps between the two
compartments and 84 boundary conditions representing inputs and outputs into
the system. The expanded model is a manually curated and highly refined model
15 of the mitochondrion, and as with iAS253, no metabolite dead ends were present
and all reactions were capable of having flux.
To represent ischaemia, the maximum uptake of oxygen was reduced to 5% of its
level under normal conditions (0.99 vs. 19.8 μmol/min/gram dry weight). To
20 represent reperfusion, the oxygen level was restored to its normal level and the
availability of succinate, lactate, pyruvate and NADH was increased to various
levels so to reflect the ischemic accumulation of these metabolites. The flux
capacity of ATP synthase was reduced by up to 50% to represent the delay in
generating ADP from AMP required for ATP synthase to function and also to
25 model hyper-polarisation of the mitochondrial membrane, in effect by
constraining the efficiency of the other proton pumping complexes of the electron
transport chain.
Metabolism of the mitochondrial network was simulated using flux balance
analysis, a technique that has been described in detail elsewhere4630 . The objective
function used to optimise the reaction fluxes was maximum ATP production. All
the FBA simulations were carried out using MATLABTM R2012b (Math Works,
49
Inc, Natick, MA) with the COBRA Toolbox47, and the linear programming solver
GLPK (http://www.gnu.org/software/glpk).
50
REFERENCES
1. Murphy, E. & Steenbergen, C. Mechanisms underlying acute protection
from cardiac ischemia-reperfusion injury. Physiol. Rev. 88, 581-609,
5 (2007).
2. Yellon, D. M. & Hausenloy, D. J. Myocardial reperfusion injury. N. Engl.
J. Med. 357, 1121-1135, (2007).
3. Burwell, L. S., Nadtochiy, S. M. & Brookes, P. S. Cardioprotection by
metabolic shut-down and gradual wake-up. J. Mol. Cell. Cardiol. 46, 804-
10 810, (2009).
4. Eltzschig, H. K. & Eckle, T. Ischemia and reperfusion--from mechanism
to translation. Nat. Med. 17, 1391-1401, (2011).
5. Timmers, L. et al. The innate immune response in reperfused myocardium.
Cardiovasc. Res. 94, 276-283, (2012).
15 6. Harmsen, E., de Jong, J. W. & Serruys, P. W. Hypoxanthine production by
ischemic heart demonstrated by high pressure liquid chromatography of
blood purine nucleosides and oxypurines. Clin. Chim. Acta 115, 73-84
(1981).
7. Pacher, P., Nivorozhkin, A. & Szabo, C. Therapeutic effects of xanthine
20 oxidase inhibitors: renaissance half a century after the discovery of
allopurinol. Pharmacol. Revs. 58, 87-114, (2006).
8. Chouchani, E. T. et al. Cardioprotection by S-nitrosation of a cysteine
switch on mitochondrial complex I. Nat. Med. 19, 753-759, (2013).
9. Zweier, J. L., Flaherty, J. T. & Weisfeldt, M. L. Direct measurement of
25 free radical generation following reperfusion of ischemic myocardium.
Proc. Natl. Acad. Sci. USA 84, 1404-1407 (1987).
10. Tannahill, G. M. et al. Succinate is an inflammatory signal that induces
IL-1beta through HIF-1alpha. Nature 496, 238-242, (2013).
11. Smith, A. C. & Robinson, A. J. A metabolic model of the mitochondrion
30 and its use in modelling diseases of the tricarboxylic acid cycle. BMC Syst.
Biol. 5, 102, (2011).
12. Pisarenko, O. I. et al. On the mechanism of enhanced ATP formation in
hypoxic myocardium caused by glutamic acid. Basic Res. Cardiol. 80,
126-134 (1985).
35 13. Taegtmeyer, H. Metabolic responses to cardiac hypoxia. Increased
production of succinate by rabbit papillary muscles. Circ. Res. 43, 808-815
(1978).
14. Hochachka, P. W. & Storey, K. B. Metabolic consequences of diving in
animals and man. Science 187, 613-621 (1975).
40 15. Easlon, E., Tsang, F., Skinner, C., Wang, C. & Lin, S. J. The malateaspartate
NADH shuttle components are novel metabolic longevity
regulators required for calorie restriction-mediated life span extension in
yeast. Genes Dev. 22, 931-944, (2008).
16. Barron, J. T., Gu, L. & Parrillo, J. E. Malate-aspartate shuttle, cytoplasmic
45 NADH redox potential, and energetics in vascular smooth muscle. J. Mol.
Cell. Cardiol. 30, 1571-1579, (1998).
51
17. Van den Berghe, G., Vincent, M. F. & Jaeken, J. Inborn errors of the
purine nucleotide cycle: adenylosuccinase deficiency. J. Inherit. Metab.
Dis. 20, 193-202 (1997).
18. Sridharan, V. et al. O2-sensing signal cascade: clamping of O2 respiration,
reduced ATP utilization, and inducible fumarate respiration. 5 Amer. J.
Physiol. 295, C29-37, (2008).
19. Dervartanian, D. V. & Veeger, C. Studies on succinate dehydrogenase. I.
spectral properties of the purified enzyme and formation of enzymecompetitive
inhibitor complexes. Biochim. Biophys. Acta 92, 233-247
10 (1964).
20. Gutman, M. Modulation of mitochondrial succinate dehydrogenase
activity, mechanism and function. Mol. Cell. Biochem. 20, 41-60 (1978).
21. Bunger, R., Glanert, S., Sommer, O. & Gerlach, E. Inhibition by
(aminooxy)acetate of the malate-aspartate cycle in the isolated working
15 guinea pig heart. Hoppe Seylers Z. Physiol. Chem. 361, 907-914 (1980).
22. Swain, J. L., Hines, J. J., Sabina, R. L., Harbury, O. L. & Holmes, E. W.
Disruption of the purine nucleotide cycle by inhibition of adenylosuccinate
lyase produces skeletal muscle dysfunction. J. Clin. Invest. 74, 1422-1427,
(1984).
20 23. Hirst, J., King, M. S. & Pryde, K. R. The production of reactive oxygen
species by complex I. Biochem. Soc. Trans. 36, 976-980 (2008).
24. Kussmaul, L. & Hirst, J. The mechanism of superoxide production by
NADH:ubiquinone oxidoreductase (complex I) from bovine heart
mitochondria. Proc. Natl. Acad. Sci. USA 103, 7607-7612 (2006).
25 25. Pryde, K. R. & Hirst, J. Superoxide is produced by the reduced flavin in
mitochondrial complex I: a single, unified mechanism that applies during
both forward and reverse electron transfer. J. Biol. Chem. 286, 18056-
18065, (2011).
26. Murphy, M. P. How mitochondria produce reactive oxygen species.
30 Biochem. J. 417, 1-13, (2009).
27. Davidson, S. M., Yellon, D. & Duchen, M. R. Assessing mitochondrial
potential, calcium, and redox state in isolated mammalian cells using
confocal microscopy. Methods Mol. Biol. 372, 421-430, (2007).
28. Wojtovich, A. P., Smith, C. O., Haynes, C. M., Nehrke, K. W. & Brookes,
35 P. S. Physiological consequences of complex II inhibition for aging,
disease, and the mKATP channel. Biochim. Biophys. Acta 1827, 598-611,
(2013).
29. Brennan, J. P. et al. Mitochondrial uncoupling, with low concentration
FCCP, induces ROS-dependent cardioprotection independent of KATP
40 channel activation. Cardiovas. Res. 72, 313-321, (2006).
30. Hamel, D. et al. G-Protein-coupled receptor 91 and succinate are key
contributors in neonatal postcerebral hypoxia-ischemia recovery.
Arterioscler. Thromb. Vasc. Biol. 34, 285-293, (2014).
31. Schmidt, K. et al. Cardioprotective effects of mineralocorticoid receptor
45 antagonists at reperfusion. Eur. Heart J. 31, 1655-1662, (2010).
32. Methner, C. et al. Protection through postconditioning or a mitochondriatargeted
S-nitrosothiol is unaffected by cardiomyocyte-selective ablation
of protein kinase G. Basic Res. Cardiol. 108, 337, (2013).
52
33. Nadtochiy, S. M., Redman, E., Rahman, I. & Brookes, P. S. Lysine
deacetylation in ischaemic preconditioning: the role of SIRT1.
Cardiovasc. Res. 89, 643-649, (2011).
34. Ashrafian, H. et al. Fumarate is cardioprotective via activation of the Nrf2
antioxidant pathway. Cell Metab. 15, 5 361-371, (2012).
35. Ord, E.N.J., Shirley, R., van Kralingen, J.C., Graves, A., McClure, J.D.,
Wilkinson, M., McCabe, C., Macrae, I.M. & Work, L.M. Positive impact
of pre-stroke surgery on survival following transient focal ischemia in
hypertensive rats. J. Neurosci. Methods 211, 305-308, (2012).
10 36. Koizumi, J., Yoshida, Y., Nakazawa, T. & Ooneda, G. Experimental
studies of ischemic brain edema: a new experimental model of cerebral
embolism in rats in which recirculation can be introduced in the ischemic
area. Jpn. J. Stroke 8, 1-8 (1986).
37. Hunter, A. J., et al., Functional assessments in mice and rats after focal
15 stroke. Neuropharmacology. 39, 806-816, (2000).
38. Ord, E. N. J. et al., Combined antiapoptotic and antioxidant approach to
acute neuroprotection for stroke in hypertensive rats. J. Cereb. Blood Flow
Metab. 33, 1215-1224, (2013).
39. Osborne, K. A. et al. Quantitative assessment of early brain damage in a
20 rat model of focal cerebral ischaemia. J. Neurol. Neurosurg. Psychiatry
50, 402-410 (1987).
40. Gaude, E. et al. muma, An R Package for metabolomics univariate and
multivariate statistical analysis. Current Metabol. 1, 180-189 (2013).
41. Davidson, S. M. & Duchen, M. R. Effects of NO on mitochondrial
25 function in cardiomyocytes: Pathophysiological relevance. Cardiovasc.
Res. 71, 10-21, (2006).
42. Smith, A. C., Blackshaw, J. A. & Robinson, A. J. MitoMiner: a data
warehouse for mitochondrial proteomics data. Nucleic Acids Res. 40,
(2012).
30 43. Chang, A., Scheer, M., Grote, A., Schomburg, I. & Schomburg, D.
BRENDA, AMENDA and FRENDA the enzyme information system: new
content and tools in 2009. Nucleic Acids Res. 37, D588-592, (2009).
44. Romero, P. et al. Computational prediction of human metabolic pathways
from the complete human genome. Genome Biol. 6, R2, (2005).
35 45. Abad, M. F., Di Benedetto, G., Magalhaes, P. J., Filippin, L. & Pozzan, T.
Mitochondrial pH monitored by a new engineered green fluorescent
protein mutant. J. Biol. Chem. 279, 11521-11529, (2004).
46. Orth, J. D., Thiele, I. & Palsson, B. O. What is flux balance analysis? Nat.
Biotechnol. 28, 245-248, (2010).
40 47. Becker, S. A. et al. Quantitative prediction of cellular metabolism with
constraint-based models: the COBRA Toolbox. Nat. Prot. 2, 727-738,
(2007).
All publications mentioned in the above specification are herein incorporated by
45 reference. Although illustrative embodiments of the invention have been
disclosed in detail herein, with reference to the accompanying drawings, it is
understood that the invention is not limited to the precise embodiment and that
53
various changes and modifications can be effected therein by one skilled in the art
without departing from the scope of the invention as defined by the appended
claims and their equivalents.

CLAIMS
1. A succinate dehydrogenase inhibitor or a prodrug and/or a
pharmaceutically acceptable salt thereof for use in the treatment or prevention of
reperfusion injury by inhibiting the accumulation of succinate, 5 wherein the
inhibitor or prodrug and/or pharmaceutically acceptable salt thereof is a cellpermeable
and reversible inhibitor of succinate dehydrogenase.
2. A cell-permeable and reversible succinate dehydrogenase inhibitor or
10 prodrug and/or a pharmaceutically acceptable salt thereof according to claim 1
that has the general formula (I):
(I)
wherein:
15 n is 0 or 1;
R1 is selected from C1-12 alkyl;
R2 is selected from H and OH;
R3 is selected from CO2R4 and C(O)-CO2R4; and
R4 is selected from C1-12 alkyl.
20
3. A cell-permeable and reversible succinate dehydrogenase inhibitor or a
prodrug and/or a pharmaceutically acceptable salt thereof according to claim 2
that is selected from R1O2C-CH2-CO2R4, R1O2C-CH(OH)-CH2-CO2R4, R1O2CCH2-
C(O)-CO2R4.
25
4. A cell-permeable and reversible succinate dehydrogenase inhibitor or a
prodrug and/or a pharmaceutically acceptable salt thereof according to any of the
preceding claims that is selected from R1O2C-CH2-CO2R4, wherein R1 and R4 are
independently selected from methyl, ethyl, propyl and butyl.
30
55
5. A cell-permeable and reversible succinate dehydrogenase inhibitor or a
prodrug and/or a pharmaceutically acceptable salt thereof according to any of the
preceding claims that is dimethyl malonate.
6. A cell-permeable and reversible succinate dehydrogenase 5 inhibitor or a
prodrug and/or a pharmaceutically acceptable salt thereof according to any of the
previous claims, wherein the reperfusion injury is an ischaemia-reperfusion
injury.
10 7. A cell-permeable and reversible succinate dehydrogenase inhibitor or a
prodrug and/or a pharmaceutically acceptable salt thereof according to any one of
claims 1 to 5, wherein the reperfusion injury is a result of disorder selected from
an abdominal aortic aneurysm, atherosclerosis, artery disease, burns, cancer,
cardiac arrest, cerebrovascular disease, cerebral edema secondary to stroke,
15 chronic obstructive pulmonary disease, congestive heart disease, constriction after
percutaneous transluminal coronary angioplasty, coronary disease, diabetes,
hypertension, mechanical trauma resulting from crush injury or surgery, migraine,
myocardial infarction, peripheral vascular disease, pulmonary vascular disease,
reperfusion after cardiac surgery, reperfusion after stroke, retinal vascular disease,
20 stroke and surgical tissue reperfusion injury.
8. A cell-permeable and reversible succinate dehydrogenase inhibitor or a
prodrug and/or a pharmaceutically acceptable salt thereof according to any one of
claims 1 to 5, for use in the treatment or prevention of reperfusion injury in
25 elective surgery by inhibiting the accumulation of succinate.
9. A cell-permeable and reversible succinate dehydrogenase inhibitor or a
prodrug and/or a pharmaceutically acceptable salt thereof according to any one of
claims 1 to 5, for use in the treatment or prevention of reperfusion injury in an
30 organ to be preserved.
56
10. A cell-permeable and reversible succinate dehydrogenase inhibitor or a
prodrug and/or a pharmaceutically acceptable salt thereof according to any one of
claims 1 to 5, for use in the treatment or prevention of reperfusion injury in organ
transplantation.
5
11. A cell-permeable and reversible succinate dehydrogenase inhibitor or a
prodrug and/or a pharmaceutically acceptable salt thereof according to any one of
claims 1 to 5, wherein the succinate dehydrogenase inhibitor is administered in
combination with a treatment used or intended to remove a blockage in blood
10 flow.
12. A cell-permeable and reversible succinate dehydrogenase inhibitor or a
prodrug and/or a pharmaceutically acceptable salt thereof according to claim 11,
wherein the treatment used or intended to remove a blockage in blood flow is
15 selected from blood thinning agents, lysis agents, MitoSNO and stents.
13. Use of a succinate dehydrogenase inhibitor or a prodrug and/or a
pharmaceutically acceptable salt thereof for the manufacture of a medicament for
treating or preventing reperfusion injury by inhibiting the accumulation of
20 succinate, wherein the inhibitor or prodrug and/or pharmaceutically acceptable
salt thereof is a cell-permeable and reversible inhibitor of succinate
dehydrogenase.
25
30
57
14. A method of treating or preventing a reperfusion injury in a subject, the
method comprising administering to the subject an effective amount of succinate
dehydrogenase inhibitor or a prodrug and/or a pharmaceutically acceptable salt
thereof, wherein the inhibitor or prodrug and/or pharmaceutically acceptable salt
thereof is a cell-permeable and reversible inhibitor of succinate 5 dehydrogenase.