U.S. patent application number 10/725667 was filed with the patent office on 2004-06-10 for use of glucose uptake enhancer for reducing post-ischemic injury of the heart.
This patent application is currently assigned to SmithKline Beecham p.l.c. & SmithKline Beecham Laboratoires Pharmaceutiques. Invention is credited to Bril, Antoine Michel Alain, Buckingham, Robin Edwin, Khandoudi, Nassirah.
Application Number | 20040110800 10/725667 |
Document ID | / |
Family ID | 27269408 |
Filed Date | 2004-06-10 |
United States Patent
Application |
20040110800 |
Kind Code |
A1 |
Bril, Antoine Michel Alain ;
et al. |
June 10, 2004 |
Use of glucose uptake enhancer for reducing post-ischemic injury of
the heart
Abstract
A method for reducing post-ischaemic injury of the heart and/or
improving the functional recovery of the heart following myocardial
ischaemia which method comprises administration of an effective,
non-toxic amount of a glucose uptake enhancer to a human or
non-human mammal in need thereof.
Inventors: |
Bril, Antoine Michel Alain;
(Rennes, FR) ; Buckingham, Robin Edwin; (Welwyn
Garden City, GB) ; Khandoudi, Nassirah; (Rennes,
FR) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property - UW2220
P.O.Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
SmithKline Beecham p.l.c. &
SmithKline Beecham Laboratoires Pharmaceutiques
|
Family ID: |
27269408 |
Appl. No.: |
10/725667 |
Filed: |
December 2, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10725667 |
Dec 2, 2003 |
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10348456 |
Jan 21, 2003 |
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6613785 |
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10348456 |
Jan 21, 2003 |
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09744118 |
Jan 19, 2001 |
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09744118 |
Jan 19, 2001 |
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PCT/GB99/02358 |
Jul 21, 1999 |
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Current U.S.
Class: |
514/342 ;
514/369 |
Current CPC
Class: |
A61K 31/00 20130101;
A61K 31/427 20130101; A61K 31/426 20130101; A61K 31/4439
20130101 |
Class at
Publication: |
514/342 ;
514/369 |
International
Class: |
A61K 031/4439; A61K
031/426 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 21, 1998 |
GB |
9815871.0 |
Jul 21, 1998 |
GB |
9815872.8 |
Claims
1. A method for reducing post-ischaemic injury of the heart and/or
improving the functional recovery of the heart following myocardial
ischaemia which method comprises administration of an effective,
non-toxic amount of a glucose uptake enhancer to a human or
non-human mammal in need thereof.
2. A method according to claim 1, wherein the glucose uptake
enhancer is a thiazolidinedione.
3. A method according to claim 2, wherein the thiazolidinedione is
Compound (I), or the tautomeric form thereof, or a pharmaceutically
acceptable derivative thereof.
4. A method according to claim 3, wherein the thiazolidinedione is
selected from:
(+)-5-[[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-
-benzopyran-2-yl)methoxy]phenyl]methyl]-2,4-thiazolidinedione (or
troglitazone),
5-[4-[(1-methylcyclohexyl)methoxy]benzyl]thiazolidine-2,4-- dione
(or ciglitazone),
5-[4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl]thiazol- idine-2,4-dione
(or pioglitazone) or 5-[(2-benzyl-2,3-dihydrobenzopyran)-5-
-ylmethyl)thiazolidine-2,4-dione (or englitazone); or a
pharmaceutically acceptable derivative thereof.
5. A pharmaceutical composition comprising a glucose uptake
enhancer, and a pharmaceutically acceptable carrier, wherein such
composition is adapted for acute administration.
Description
[0001] This invention relates to a novel method for preventing or
reducing post-ischaemic injury of the heart, in particular
myocardial infarction or for improving the functional recovery of
the heart following myocardial ischaemia.
[0002] Cardiovascular disease is a leading cause of mortality in
adult diabetics of both Type 1 and Type 2 etiologies. The
underlying presence of cardiovascular disease in diabetes means not
only that the likely incidence of myocardial infarction is higher
in the diabetic population but that its occurrence carries a
substantially greater risk of mortality for diabetics than
non-diabetics. European Patent Application, Publication Number
0,306,228 relates to certain thiazolidinedione derivatives
disclosed as having antihyperglycaemic and anti-hyperlipidaemic
activity. One particular thiazolidinedione disclosed in EP 0306228
is 5-[4-[2-(N-methyl-N-(2-pyridyl)amino)ethoxy]benzyl]thiaz-
olidine-2,4-dione (hereinafter `Compound (I)`). WO94/05659
discloses certain salts of Compound (I) including the maleate salt
at example 1 thereof.
[0003] Compound (I) is an example of a class of anti-hyperglycaemic
agents known as `insulin sensitisers`. In particular Compound (I)
is a thiazolidinedione insulin sensitiser. Thiazolidinedione
insulin sensitisers include compounds comprising a
2,4-thiazolidinedione moiety.
[0004] European Patent Applications, Publication Numbers: 0008203,
0139421, 0032128, 0428312, 0489663, 0155845, 0257781, 0208420,
0177353, 0193256, 0319189, 0332331, 0332332, 0528734, 0508740;
International Patent Application, Publication Numbers 92/18501,
93/02079, 93/22445 and U.S. Pat. Nos. 4,687,777, 5,104,888 and
5,478,852, also disclose certain thiazolidinedione insulin
sensitisers.
[0005] Another series of compounds generally recognised as having
insulin sensitiser activity are those typified by the compounds
disclosed in International Patent Applications, Publication Numbers
WO93/21166 and WO94/01420. These compounds are herein referred to
as `acyclic insulin sensitisers`. Other examples of acyclic insulin
sensitisers are those disclosed in U.S. Pat. No. 5,232,945 and
International Patent Applications, Publication Numbers WO92/03425
and WO91/19702.
[0006] Examples of other insulin sensitisers are those disclosed in
European Patent Application, Publication Number 0533933, Japanese
Patent Application Publication Number 05271204 and U.S. Pat. No.
5,264,451.
[0007] The above mentioned publications are incorporated herein by
reference.
[0008] It is suggested by Shimabukuro et al (Diabetes 44[Suppl
1]:797 (Abstract) 1995) that long term treatment with the
thiazolidinedione, troglitazone, preserves cardiac function of the
diabetic heart. Also, Eckel et al (Diabetes, 46 [Suppl 1]: 575
(Abstract) 1997) have suggested that chronic exposure to
troglitazone may exert a cardioprotective effect by increasing
glucose supply to the myocytes of the diabetic heart.
[0009] It is now surprisingly indicated that the acute
administration of Compound (I) exerts a cardioprotective effect on
the diabetic heart and is therefore effective at preventing or
reducing post-ischaemic injury, such as myocardial infarction. The
acute administration of Compound (I) is also indicated to improve
the functional recovery of the diabetic heart following myocardial
ischaemia.
[0010] In addition, and perhaps more surprisingly, it is indicated
that administration, especially acute administration, of Compound
(I) exerts a particularly effective cardioprotective effect on the
non-diabetic heart.
[0011] Accordingly, the present invention provides a method for
reducing post-ischaemic injury of the heart, in particular
myocardial infarction, which method comprises administration,
especially acute administration, of an effective, non-toxic amount
of a glucose uptake enhancer to a human or non-human mammal in need
thereof.
[0012] The invention also provides a method for improving the
functional recovery of the heart following myocardial ischaemia
which method comprises administration, especially acute
administration, of an effective, non-toxic amount of a glucose
uptake enhancer to a human or non-human mammal in need thereof.
[0013] In one particular aspect the invention provides a glucose
uptake enhancer, such as Compound (I) or a tautomeric form thereof
or a pharmaceutically acceptable derivative thereof, for use in
reducing post-ischaemic injury of the heart, in particular
myocardial infarction or for use in improving the functional
recovery of the heart following myocardial ischaemia.
[0014] Certain of the human or non-human mammals may be suffering
from diabetes mellitus or a related disorder. Particularly, the
diabetes mellitus is Type 1 diabetes mellitus. Particularly, the
diabetes mellitus is Type 2 diabetes mellitus.
[0015] A suitable glucose uptake enhancer is an insulin
sensitiser.
[0016] A suitable glucose uptake enhancer is a
thiazolidinedione.
[0017] Suitable thiazolidinediones are those disclosed in the above
mentioned publications.
[0018] A preferred thiazolidinedione is Compound (I), or the
tautomeric form thereof, or a pharmaceutically acceptable
derivative thereof.
[0019] Other suitable thiazolidinediones include
(+)-5-[[4-[(3,4-dihydro-6-
-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]phenyl]methyl]-2-
,4-thiazolidinedione (or troglitazone),
5-[4-[(1-methylcyclohexyl)methoxy]- benzyl]thiazolidine-2,4-dione
(or ciglitazone), 5-[4-[2-(5-ethylpyridin-2--
yl)ethoxy]benzyl]thiazolidine-2,4-dione (or pioglitazone) or
5-[(2-benzyl-2,3-dihydrobenzopyran)-5-ylmethyl)thiazolidine-2,4-dione
(or englitazone); or a pharmaceutically acceptable derivative
thereof.
[0020] A suitable pharmaceutically acceptable derivative is a
pharmaceutically acceptable salt or a pharmaceutically acceptable
solvate, including a pharmaceutically acceptable solvate of a
pharmaceutically acceptable salt.
[0021] Suitable pharmaceutically acceptable derivatives, including
pharmaceutically acceptable salts and pharmaceutically acceptable
solvates, of the glucose uptake enhancer, for example the
thiazolidinediones, are as described in the above mentioned
publications and standard reference texts such as the British and
US Pharmacopoeias, Remington's Pharmaceutical Sciences (Mack
Publishing Co.), Martindale The Extra Pharmacopoeia (London, The
Pharmaceutical Press).
[0022] Suitable pharmaceutically acceptable salts of Compound (I)
include those described in EP 0306228 and WO94/05659. A preferred
pharmaceutically acceptable salt is a maleate.
[0023] Suitable pharmaceutically acceptable solvated forms of
Compound (I) include those described in EP 0306228 and WO94/05659,
in particular hydrates.
[0024] Certain of the glucose uptake enhancers, such as the
thiazolidinediones, for example Compound (I), may exist in one of
several tautomeric forms, all of which are encompassed by the
method of the invention, either as individual tautomeric forms or
as mixtures thereof.
[0025] Certain of the glucose uptake enhancers, such as the
thiazolidinediones, for example Compound (I), may also contain
chiral carbon atoms, and hence can exist in several stereoisomeric
forms, all of which are encompassed by the method of the invention
whether as individual isomers or as mixtures of isomers.
[0026] The glucose uptake enhancers, such as the
thiazolidinediones, including the pharmaceutically acceptable
derivatives thereof, are prepared using conventional methods; for
example the thiazolidinediones are conveniently prepared according
to the methods disclosed in the above mentioned publications: Thus
Compound (I), or the tautomeric form thereof, or a pharmaceutically
acceptable derivative thereof, such as a salt thereof or a
pharmaceutically acceptable solvate thereof, may be prepared using
the processes described in EP 0306228 and WO94/05659.
[0027] The above mentioned stereoisomeric forms, such as those of
the thiazolidinediones, may be prepared and separated as required,
according to known methods such as those disclosed in the above
mentioned publications.
[0028] The above-mentioned feature of the acute administration of
glucose uptake enhancer, especially of the thiazolidinediones such
as Compound (I), is considered to comprise in its own right a
further part of the present invention. Accordingly, the invention
further provides a glucose uptake enhancer, such as a
thiazolidinedione for example Compound (I) or a tautomeric form
thereof or a pharmaceutically acceptable derivative thereof, for
use as an acutely administerable therapeutic substance.
[0029] The present invention also provides a glucose uptake
enhancer, such as a thiazolidinedione for example Compound (I) or a
tautomeric form thereof or a pharmaceutically acceptable derivative
thereof, for use as an acutely administrable cardioprotective
agent, especially for preventing or reducing post-ischaemic injury
of the heart, in particular myocardial infarction,
[0030] The present invention also provides a glucose uptake
enhancer, such as a thiazolidinedione for example Compound (I) or a
tautomeric form thereof or a pharmaceutically acceptable derivative
thereof, for acute administration for improving the functional
recovery of the heart following myocardial ischaemia.
[0031] In all of the above-mentioned treatments, the glucose uptake
enhancer such as Compound (I) or a tautomeric form thereof or a
pharmaceutically acceptable derivative thereof, may be administered
per se or, preferably, as a pharmaceutical composition also
comprising a pharmaceutically acceptable carrier.
[0032] Accordingly, the present invention also provides a
pharmaceutical composition comprising a glucose uptake enhancer,
such as Compound (I) or a tautomeric form thereof or a
pharmaceutically acceptable derivative thereof, and a
pharmaceutically acceptable carrier wherein such composition is
adapted for acute administration.
[0033] More particularly, the present invention provides a
pharmaceutical composition for use as an acutely administerable
cardioprotective agent, especially for preventing or reducing
post-ischaemic injury of the heart, in particular myocardial
infarction, which composition comprises a glucose uptake enhancer,
such as Compound (I) or a tautomeric form thereof or a
pharmaceutically acceptable derivative thereof, and a
pharmaceutically acceptable carrier.
[0034] The invention further provides a pharmaceutical composition
for acute administration for improving the functional recovery of
the heart following myocardial ischaemia, which composition
comprises a glucose uptake enhancer, such as Compound (I) or a
tautomeric form thereof or a pharmaceutically acceptable derivative
thereof, and a pharmaceutically acceptable carrier.
[0035] It is also envisaged that the acute cardioprotective effect
of a glucose uptake enhancer would be useful for enhancing
post-surgical recovery. Accordingly, the invention further provides
a method for enhancing recovery after surgery, especially major
surgery, for example cardiac surgery, which method comprises
administration, generally acute administration, of an effective,
non-toxic amount of a glucose uptake enhancer such as Compound (I),
or a tautomeric form thereof or a pharmaceutically acceptable
derivative thereof. Said administration of the glucose uptake
enhancer may be before or after surgery. Particular patient groups
include the elderly such as post-60 year age groups.
[0036] As used herein the term "pharmaceutically acceptable"
embraces compounds, compositions and ingredients for both human and
veterinary use: for example the term `pharmaceutically acceptable
salt` embraces a veterinarily acceptable salt.
[0037] As used herein "post-ischaemic injury of the heart" includes
myocardial infarction and certain arrhythmias, especially due to
myocardial infarction.
[0038] As used herein "improving the functional recovery of the
heart" includes improving or restoring cardiac output and/or
enhancing the recovery, especially the rate of recovery, of cardiac
output.
[0039] As used herein "acute administration" or phrases or terms
used to convey an equivalent meaning to acute administration refer
to a single administration of the medicament or the short term use.
Short term use of a thiazolidinedione insulin sensitiser means a
period of time less than that associated with an antihyperglycaemic
effect. A suitable short term use period is 3-4 weeks.
[0040] As used herein "glucose uptake enhancer" means an agent
which increases basal (insulin independent) or insulin-stimulated
uptake of glucose into a cell.
[0041] In the method of the invention, the active medicaments are
preferably administered in pharmaceutical composition form.
[0042] Usually the compositions are adapted for oral
administration. However, they may be adapted for other modes of
administration, for example parenteral administration, sublingual
or transdermal administration.
[0043] The compositions may be in the form of tablets, capsules,
powders, granules, lozenges, suppositories, reconstitutable
powders, or liquid preparations, such as oral or sterile parenteral
solutions or suspensions.
[0044] In order to obtain consistency of administration it is
preferred that a composition of the invention is in the form of a
unit dose.
[0045] Unit dose presentation forms for oral administration may be
tablets and capsules and may contain conventional excipients such
as binding agents, for example syrup, acacia, gelatin, sorbitol,
tragacanth, or polyvinylpyrrolidone; fillers, for example lactose,
sugar, maize-starch, calcium phosphate, sorbitol or glycine;
tabletting lubricants, for example magnesium stearate;
disintegrants, for example starch, polyvinylpyrrolidone, sodium
starch glycollate or microcrystalline cellulose; or
pharmaceutically acceptable wetting agents such as sodium lauryl
sulphate.
[0046] The compositions are preferably in a unit dosage form in an
amount appropriate for the relevant daily dosage.
[0047] Suitable dosage regimens, including details of unit dosages,
for the thiazolidinediones include those described in the above
mentioned publications or in reference texts such as the British
and US Pharmacopoeias, Remington's Pharmaceutical Sciences (Mack
Publishing Co.), Martindale The Extra Pharmacopoeia (London, The
Pharmaceutical Press).
[0048] The compositions are preferably in a unit dosage form in an
amount appropriate for the relevant daily dosages. For example, for
compound (I), unit doses suitably contain up to 12 mg of Compound
(I).
[0049] In the acute treatment of the invention, the glucose uptake
enhancer, such as Compound (I), or the tautomeric form thereof, or
a pharmaceutically acceptable derivative thereof, is generally
administered as a single dose. However, if required, additional
doses may be administered to provide suitable short-term,
non-chronic treatments, for example to prevent or reduce
post-ischaemic injury, such as myocardial infarction, due to a
subsequent ischaemic event and/or to prevent or reduce the severity
of such an event and/or its re-occurrence.
[0050] In the above mentioned acute administration of glucose
uptake enhancers, for example thiazolidinediones, dosages are
envisaged to include higher doses than those associated with an
anti-hyperglycaemic effect.
[0051] In a further aspect the treatment comprises the sequential
administration or the co-administration of a thrombolytic agent,
such as streptokinase, with the glucose uptake enhancer, such as
Compound (I) or the tautomeric form thereof, or a pharmaceutically
acceptable derivative thereof.
[0052] Accordingly, the invention also comprises a pharmaceutical
composition comprising a glucose uptake enhancer, such as Compound
(I) or the tautomeric form thereof, or a pharmaceutically
acceptable derivative thereof, and a thrombolytic agent, such as
streptokinase, and a pharmaceutically acceptable carrier.
[0053] The particular thrombolytic agent and its required dosage
include those described in reference texts such as the British and
US Pharmacopoeias, Remington's Pharmaceutical Sciences (Mack
Publishing Co.), Martindale The Extra Pharmacopoeia (London, The
Pharmaceutical Press). The solid oral compositions may be prepared
by conventional methods of blending, filling or tabletting.
Repeated blending operations may be used to distribute the active
agent throughout those compositions employing large quantities of
fillers. Such operations are of course conventional in the art. The
tablets may be coated according to methods well known in normal
pharmaceutical practice, in particular with an enteric coating.
[0054] Oral liquid preparations may be in the form of, for example,
emulsions, syrups, or elixirs, or may be presented as a dry product
for reconstitution with water or other suitable vehicle before use.
Such liquid preparations may contain conventional additives such as
suspending agents, for example sorbitol, syrup, methyl cellulose,
gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium
stearate gel, hydrogenated edible fats; emulsifying agents, for
example lecithin, sorbitan monooleate, or acacia; non-aqueous
vehicles (which may include edible oils), for example almond oil,
fractionated coconut oil, oily esters such as esters of glycerine,
propylene glycol, or ethyl alcohol; preservatives, for example
methyl or propyl p-hydroxybenzoate or sorbic acid; and if desired
conventional flavouring or colouring agents.
[0055] For parenteral administration, fluid unit dosage forms are
prepared utilizing the compound and a sterile vehicle, and,
depending on the concentration used, can be either suspended or
dissolved in the vehicle. In preparing solutions the compound can
be dissolved in water for injection and filter sterilized before
filling into a suitable vial or ampoule and sealing.
Advantageously, adjuvants such as a local anaesthetic, a
preservative and buffering agent can be dissolved in the vehicle.
To enhance the stability, the composition can be frozen after
filling into the vial and the water removed under vacuum.
Parenteral suspensions are prepared in substantially the same
manner, except that the active compound may be suspended in the
vehicle instead of being dissolved, and sterilization cannot be
accomplished by filtration. The compound can be sterilized by
exposure to ethylene oxide before suspending in the sterile
vehicle. Advantageously, a surfactant or wetting agent is included
in the composition to facilitate uniform distribution of the
compound.
[0056] Compositions may contain from 0.1% to 99% by weight,
preferably from 10-60% by weight, of the active material, depending
upon the method of administration.
[0057] Compositions may, if desired, be in the form of a pack
accompanied by written or printed instructions for use.
[0058] Finally, the cardioprotective effects, especially the acute
cardioprotective effects, of a glucose uptake enhancer, such as
Compound (I) or the tautomeric form thereof, or a pharmaceutically
acceptable derivative thereof, are also considered to provide
potential for use as a cardioplegic agent. Accordingly, the present
invention also provides a glucose uptake enhancer, such as Compound
(I) or the tautomeric form thereof, or a pharmaceutically
acceptable derivative thereof for use as a cardioplegic agent,
especially in cardioplegic solutions, to preserve cardiac function
during surgery.
[0059] Particular uses of a cardioplegic agent include use in
cardiac by-pass surgery. Particular uses of a cardioplegic agent
include use in cardiac transplant surgery for maintaining cardiac
viability.
[0060] The amount of active agent required for cardioplegic use
will be provided by standard tests methods such as those described
herein, for example cardioplegic solutions of Compound (I) are
envisaged to contain between 0.01 .mu.M and 10 .mu.M of Compound
(I).
[0061] In a further aspect, the invention also provides a
pharmaceutical composition adapted for use as a cardioplegic agent,
which comprises a glucose uptake enhancer, such as Compound (I) or
a tautomeric form thereof, or a pharmaceutically acceptable
derivative thereof and a pharmaceutically acceptable carrier.
[0062] The compositions are prepared and formulated according to
conventional methods, such as those disclosed in standard reference
texts, for example the British and US Pharmacopoeias, Remington's
Pharmaceutical Sciences (Mack Publishing Co.), Martindale The Extra
Pharmacopoeia (London, The Pharmaceutical Press and Harry's
Cosmeticology (Leonard Hill Books) or the above mentioned
publications.
[0063] The cardioprotective effects of the invention may be
identified by using test methods such as those provided hereinafter
or those known in the art such as those disclosed in Khandoudi N,
Bernard M, Cozzone P, Feuvray D (Intracellular pH and role of
Na+/H+ exchange during ischaemia and reperfusion of normal and
diabetic rat hearts. Cardiovasc Res 24: 873-878, 1990) or in
Khandoudi N, Laville M P, Bril A (Protective effect of the
Sodium/Hydrogen exchange inhibitors during global low
flow-ischemia. J Cardiovasc Pharmacol 28: 540-546, 1996).
[0064] No adverse toxicological effects have been established for
the compositions or methods of the invention in the above mentioned
dosage ranges.
[0065] In the Tables and Figures shown below:
[0066] Table 1: shows baseline ventricular function of isolated
working hearts from male Wistar rats: with vehicle or Compound (I)
added to the perfusate pre-ischaemia;
[0067] Table 2: shows baseline ventricular function of isolated
working hearts from STZ-diabetic rats: with vehicle or Compound (I)
added to the perfusate pre-iscaemia;
[0068] FIG. 1: shows the effect of Compound (I) on post-ischaemic
functional impairment of normal Male Wistar rat working hearts;
and
[0069] FIG. 2: shows the effect of Compound (I) on post-ischaemic
functional impairment of STZ-diabetic male Wistar rat working
hearts.
[0070] The following example illustrates the invention but does not
limit it in any way
Materials & Methods
[0071] Materials: Stock solutions of Compound (I) were prepared
freshly in dimethylsulphoxide (DMSO) and further dilutions were
made in the perfusion buffer. The maximum vehicle DMSO
concentration was 0.001% which was without effect on any parameters
when added by itself in control experiments.
[0072] A solution of streptozotocin (STZ commercially available)
was prepared in citrate buffer (40 mg/ml).
[0073] Test Systems: Male Wistar rats (Charles River; St Aubin ls
Elbeuf, France), with a body weight ranging from 260 to 280 g were
housed on a 12 h/12 h light-dark cycle with access to water and
standard rat chow ad libitum. An acclimatisation period of at least
one week was allowed prior to experiment.
[0074] Induction of experimental diabetes: Male Wistar rats
weighing between 300 and 320 g were fasted overnight and made
diabetic by a single intravenous injection of STZ, 40 mg/kg body
weight. The development of diabetes and its persistence were
monitored by serial quantitative measurements of glucose in the
urine with reagent strips. On the day of the experiment, the
severity of diabetes was assessed by measuring glucose
concentrations from blood samples collected at the time of heart
excision. Only rats with plasma glucose levels exceeding 20 mM were
considered diabetic and included in these experiments.
[0075] Experimental Procedures
[0076] Perfusion of isolated hearts: Rats were anaesthetized using
thiopental sodium (50 mg/kg body weight intraperitoneally). Hearts
from normoglycaemic and one-month STZ-induced diabetic rats, were
quickly removed and immersed in ice-cold buffer to produce an
immediate cessation of contractility. The aorta was dissected free
and then mounted onto a cannula attached to a perfusion apparatus.
Retrograde perfusion of the heart was started for 10 min by the
Langendorff method and then switched to perfusion using the
working-heart technique [16]. The perfusion fluid was
Krebs-Henseleit buffer (pH 7.4) of the following composition (mM):
NaCl 118, NaHCO.sub.3 23, KCl 4.7, KH.sub.2PO.sub.4 1.2, MgCl.sub.2
1.2, CaCl.sub.2 1.25, glucose 11, pyruvate 2. The buffer was
continuously gassed with a 95% O.sub.2/5% CO.sub.2 mixture and the
entire system was thermoregulated at 37.degree. C. The perfusate
was not recirculated. Preload was held at a pressure of 15 cm
H.sub.2O and afterload, as well as coronary perfusion pressure,
were kept constant at 80 cm H.sub.2O.
[0077] Measurement of cardiac function: Both heart rate (beat/min)
and peak systolic pressure (mm Hg) were monitored continuously via
the fluid-filled side-arm on the aortic cannula connected to a
pressure transducer (Statham P23 Db) and recorded on a Gould
pen-recorder (model 8188.602). Aortic and coronary flows (ml/min)
were measured by timed collection. Cardiac output (m/min) was
derived from the sum of the aortic and coronary flows. Stroke
volume (ml/beat) was derived by dividing cardiac output by heart
rate.
[0078] Induction of global ischaemia and re-perfusion: Total
ischaemia was initiated by clamping the left atrium and the aortic
perfusion tubes and reducing coronary flow to zero for 30 minutes.
The hearts were then re-perfused at 37.degree. C. in working heart
mode and recovery of ventricular function was followed for 30
minutes.
[0079] To investigate the action of Compound (I), this agent was
added to the perfusate 15-min prior to the induction of ischaemia
and then maintained throughout the re-perfusion phase.
[0080] Data Handling & Analysis: The data are presented as the
mean .+-.SEM. Statistical significance of differences was
determined using Student's t-test. Differences with p.ltoreq.0.05
were considered to be statistically significant.
[0081] Results: Baseline functional parameters for perfused normal
male Wistar rat hearts ex vivo are shown in Table 1. In the
perfusion system used here, at a constant outflow resistance,
aortic flow reflects ventricular contractility [16].
[0082] The effects on cardiac function of inclusion of Compound (I)
(1 uM) in the perfusate 15 min prior to zero-flow ischaemia (30
min) and subsequent re-perfusion, are shown in FIG. 1. The data
demonstrate that recovery of post-ischaemic control hearts is
relatively slow and not all functional parameters (e.g. cardiac
output) return to pre-ischaemic levels, even after 30 min
re-perfusion. Inclusion of Compound (I) in the perfusate prior to
ischaemia significantly enhanced the rate of recovery of each of
the functional indices. For example, inclusion of Compound (I) (1
.mu.M) in the perfusate for 15 min prior to, during ischaemia and
during the subsequent re-perfusion phase, enhanced the recovery in
cardiac output and heart rate.
CONCLUSIONS
[0083] The results of this study suggest that Compound (I)
possesses protective properties of rapid onset in both normal and
diabetic rat hearts subjected to zero-flow ischaemia in vitro.
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Tables and Figures
[0102]
1 TABLE 1 Male Wistar Rats: Male Wistar Rats: Control Compound (I)
(1 .mu.M) Aortic Flow 36.5 .+-. 1.6 34.0 .+-. 0.7 (ml/min) Coronary
Flow 15.0 .+-. 0.6 16.6 .+-. 0.6 (ml/min) Cardiac Output 52 .+-. 2
51 .+-. 1 (ml/min) Peak Systolic Pressure 79 .+-. 1 73 .+-. 1
(mmHg) Stroke Volume 0.16 .+-. 0.01 0.16 .+-. 0.01 (ml/beat) Heart
Rate (beats/min) 314 .+-. 10 317 .+-. 15
[0103]
2 TABLE 2 Male Wistar STZ-Diabetic Male Wistar STZ-Diabetic Rats:
Control Rats: Compound (I) (1 .mu.M) (n = 6) (n = 6) Aortic Flow
42.5 .+-. 1.7 42.3 .+-. 1.5 (ml/min) Coronary Flow 13.6 .+-. 0.5
12.1 .+-. 0.4 (ml/min) Cardiac Output 56.2 .+-. 2.0 54.4 .+-. 1.7
(ml/min) Peak Systolic 77 .+-. 2 76 .+-. 2 Pressure (mm Hg) Stroke
Volume 0.24 .+-. 0.02 0.25 .+-. 0.01 (ml/beat) Heart Rate 242 .+-.
19 225 .+-. 14 (beats/min)
* * * * *