Note: Descriptions are shown in the official language in which they were submitted.
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w o 97/38685 PCT~EPg7/0186
USE OF AN OSMOLYTE IN THE PREPARATION OF A MEDICAMENT FOR TREATING
COMPLICATIONS RESULTING FROM ISCHEMIA
- Field of invention
s
The present invention relates to the use of organic osmolytes in the manufacture of
a therapeutic agent capable of treating or preventing complications resulting from
ischemia, hypoxia or oxidative stress.
10 Background of the invention
In recent studies it has been revealed that, immune competent cells with macrophage
activity such as the Kupffer cells have a remarkably sensitive and potent osmoregulation,
see e.g. Biochem J. 1995, Vol. 312, pag. 135-142, F Zhang et al. The studies suggest that
15 cell volume homeostasis is a critical factor for the cellular function of Kupffer cells. This
type of organic osmolytes need to be non-perturbing solutes that do not interfere with
protein function even when occurring at high intracellular concentrations. Such a
prerequisite may explain why only a few classes of organic compounds, viz. polyols (e.g.
inositol and sorbitol), methylamines (betaine, a-glycerophosphorylcholine) and certain
20 amino acids such as taurine have evolved as osmolytes in living cells.
In m~mm~, osmolytes have been identified in astrocytes, renal medulla cells and lens
epithelia. The need for osmolytes in renal medulla cells is obvious, because ambient
medullary osmolarity can increase up to 3800 mosmol/l during antidiuresis and decrease to
170 mosmolA during diuresis. In the antidiuretic state (high extracellular osmolarity),
25 intracellular osmolarity increases in renal medullary cells as the result of the intracellular
accumulation of inositol and betaine which are taken up via sodium ion dependenttransporters. These sodium ion dependent transporters are induced upon hyperosmotic
exposure in renal cells and astrocytes. Recent studies with Madine-Darby canine kidney
(MDCK) cells have identified a hypertonic stress-responsive element in the 5'-flanking
30 region of the mammalian BGT- I gene (betaine transporter).
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In a study disclosed in FEBS Letters, 1995, Vol. 377, pages 47-50, U Warskulat et
al., betaine is identified as osmolyte in mouse macrophages. The betaine uptake in mouse
macrophages was significantly stimulated when the cells were exposed to a hyperosmotic
(405 mosm/l) medium. From the results of this study it was concluded that betaine
5 availability could be a potential site for the regulation of macrophage cell function.
Certain organic osmolytes have previously been suggested in the
International Patent Application WO 91/14435 as supplements to protect cells in a
dehydrated environment from volume changes. Also in Biochem. Journal, 1992, Vol. 282,
pages 69-73, it is demonstrated that SV-3T3 cells (fibroblasts) subjected to hyperosmotic
10 conditions may retain normal function in terms of rate of cell proliferation and protein
synthesis in the presence of an osmolyte. Even if these publications may consider a
therapeutic utility of certain osmolytes, there are no disclosures of how osmolytes can
affect cells which mediates pathological events resulting from ischemia, hypoxia or
oxidative stress, both during hyperosmolar conditions and in conditions with normal
l 5 osmolarity.
Organ transplantation has become an established therapy for end stage liver and
heart disease, although primary graft non-function or dysfunction is serious clinical
problem. Cold ischemic storage and the following reperfusion of the donated organ are
identified as major contributors to failing primary graft function and is shown to have a
20 detrimental impact on endothelial and immune competent cells, injuries to the endothelial
cells precipitates a malfunction vascular system and consequently, an inadequate oxygen
and substrate delivery, as well as an impaired waste product clearance. Furthermore, the
challenged endothelium enhances the expression of adhesive molecules facilitating the
binding and infiltration of immune competent cells in the tissue area at risk. Immune
25 competent cells respond to ischemia and reperfusion by producing a number of
biologically toxic mediators, again leading to the dysfunction of surrounding cells,
including the vascular endothelium and in certain cases the whole organ. The early organ
dysfunction is considered to originate from injuries of endothelial cells resulting in
inadequate oxygen and substrate delivery as well as reduced waste product clearance.
30 Beyond transplantation injuries, resulting from ischemia and reperfusion~ these are a well
recognized clinical problems in, for example, myocardial infarction and the following
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thrombolytic treatment. As disclosed in Laboratory Investigation, 1996, Vol. 74, No. 1, p.
86 (J Kajstura et al.), both myocardial ischemia and hypoxia can induce cell death, such
as programmed cell death (apoptosis) in the heart following myocardial infarction which
may lead to massive loss of cells and further organ damages.
S In the liver, the infl~mm:~tory response to ischernia and reperfusion is suggested to
be primarily mediated by resident macrophages, the Kupffer cells, while the heart in such a
situation suffers from invading immune competent cells which might cause persistent
.
mJunes.
It would consequently be highly desirable to find a suitable therapy to preserve or
10 improve the endothelial cell function and 11iminish the infl~mm~tory response of the
immunt~ competent cells during and after the mentioned complications, as well as form a
protection against cell death.
In response to ischemia/reperfusion and infl~lnm~tory mediators, endothelial andimmune competent cells produce oxygen free radicals which exert a detrimental metabolic
15 load on exposed cells termed oxidative stress. The oxidative stress precipitates severe
damages to biological molecules, especially to DNA, lipids and proteins. The protection
against oxidative stress and hence the salvage of tissues and organs might be achieved only
partially by supplying antioxidants and ensuring an adequate level of antioxidant enzymes.
It would therefore also be desirable to be able to provide a therapy which also is useful for
20 improving the protection of cells against damages origin~ting from oxidative stress.
In the International patent application WO 92/15546 certain osmolytes, such as
taurine, which are capable of crossing the blood brain barrier, are suggested in the
protection of cells being at risk to be damaged from lactic acidosis from oxygendeficiency. In this publication, however, the osmolyte exert its beneficial effect by
25 providing buffering action and not by directly acting on specific cells in order to modulate
their response to the disorderly event. Furthermore, the osmolyte taurine has been
suggested to have certain beneficial effects to heart in 3apanese Circ. Journ. 1992, Vol. 56,
p. 95 (J Azuma et al.) following congestive heart failure. It is concluded that taurine
possibly contributes to a regulation of the myocardial calcium uptake and thus may
30 increase the myocardial activity.
-
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According to the present invention, it has been surprisingly found that certain osmolytes,
such as betaine and taurine, have a powerful capacity to maintain the cellular integrity in
specific cells, and thereby the organ function, subjected to a depletion of oxygen in an
anoxia model or oxidative stress, as demonstrated in an isolated, perfused liver. The
5 present invention shows that selected osmolytes can be employed as important regulators
of endothelial and immune competent cell function. The osmolytes have a capacity to
protect these cell types or to affect such cells to modulate their response to the mentioned
complications and thereby m~int:~ining the function of vital organs challenged by
pathologic events, such as an inadequate blood supply.
The failing liver is an early event in sepsis and accompanied by raised enzyme
leakage from the liver, for example lactate dehydrogenase (LDH) which indicates a
compromised cellular integrity. As a sign of an adequate treatment, the hepatic function
and enzyme leakage is restored to near normal levels within days. This course ofpathological events and the impact of a successful treatment, reflects the clinical
15 importance of the marked decrease in LDH leakage in response to osmolyte treatment
following anoxia, as will be described in the present invention.
Consequently, it is an object of the present invention to preserve and
improve the endothelial cell function and ~limini.~h the infl~mm:~tory response of the
immune competent cells by a supplementation of an effective amount of certain osmolytic
20 agents. It is also an object of the present invention to, by means of an osmolyte therapy, to
improve the capacity of the tissue to resist oxidative stress, in order to prevent and treat
damages resulting from such a condition and thereby improve the possibility of organ
protection and rescue.
The present invention demonstrates that other~vise metabolically inert osmolytes25 have a high potency in protecting organs or tissues from such damages and dysfunctions
resulting from ischemia and reperfusion, hypoxia or oxidative stress.
Description of the invention
The present invention is related to the use of an effective amount of an osmolyte in
the preparation of a therapeutic agent capable treating or preventing complications
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resulting from ischemia, hypoxia or oxidative stress by affecting cells which produce
m~-liators of such complications. Such cells may have an active part in the immune system
and typically include, but are not strictly limited to, immune competent cells, endothelial
- cells and hepatocytes. In particular, this type of cells are protected to maintain their
5 regular metabolic function or are affected to modulate their response to the complications
of ischemia, hypoxia and oxidative stress, in order to m~int~ining the function of vital
organs challenged from pathologic events, such an inadequate blood supply. Thesecomplications typically can involve phenomena as cell death examplified by programrned
cell death (apoptosis) and necrosis, as well as an increase in the activity of inducible nitric
10 oxide synthase (iNOS). The ischemic or hypoxic conditions typically origin from a
situation where the ordinary blood flow of substrates to an organ or a tissue is interrupted
or reduced, so the regular metabolism is altered. Such situations can occur in connection
to a large variety of traumatic events, such as myocardial infarction, bypass surgery of the
heart or other organs or organ transplantation.
It is also an important aspect of the present invention to use effective
amounts osmolytes in the manufacture of a preparation that is capable of preventing
complications which can arrive from ischemia, hypoxia or oxidative stress for patients who
are identified to be at high risk for acquiring such a complication. The present invention
also serves as a cytoprotective therapy by increasing a correct cellular hydration in
20 response to stress. In particular patients suffering from identified vascular dysfunctions,
such as those suffering from the effects or diabetes or who are expecting additional
surgery or therapy can benefit from a therapy with selected osmolytes according to the
present invention in connection with their regular therapy.
The osmolytes are defined as agents used by the cells to regulate the level of
25 hydration by a specific transport mechanism through the cellular membranes. Such agents
traditionally have been considered biologically inert, except for their function as substrates
in metabolic pathways. In the context of the present invention, the osmolytes are defined
as agents that are used in the regulation of the cellular hydration with the additional
capacity to protect organs against injuries resulting from ischemia, hypoxia and oxidative
30 stress. In addition, such osmolytes are useful for the preservation of the organ function at
abnormal temperatures (hypothermia) induced during preservation prior to the
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WO 97/38685 PCT/EP97/01861
transplantation. The osmolytes are preferably selected from a group consisting of polyols,
amino acids and methylamines which are endogenously occurring in the body for
regulating the individual cellular volume and osmolarity after exposure to osmotic
variations and other stimuli related to the immune defense, as explained in our co-pending
Swedish patent application 9601395-8.
According to the present invention, it is especially preferred to use amino acidosmolytes, methylamine osmolytes, such as taurine and betaine and certain polyols, such as
myo-inositol, but the skilled person could be able to identify other individual osmolytes
capable of acting as an osmolarity regulating agent for specific cells of an elected organ or
of a certain tissue and such compounds will also be conceivable to use within the context
of the present invention. The osmolytes can be administered as salts or as precursors, such
as alkyl esters of osmolytes or osmolytes in oligopeptides, capable of being released at
their functional cellular target. All such a l nini.stration forms especially selected for
delivering the osmolyte to the cells, therefore are parts of the present invention.
Alternatively, biological precursors to osmolytes can be a-lmini.~tered when suitable, as is
examplified by a supplement of choline as a precursor to betaine. As an example, choline
can be converted to betaine by hepatocytes for transport to the Kupffer cells of the liver
where it may exert the mentioned effects. Choline can however not be converted to
betaine by the Kupffer cells.
According to the present invention it is possible to add one or several constituents
capable of contributing to a prevention of the impairing effects resulting from the ischemic
or hypoxic conditions. Examples of such compounds are for example, found among
certain amino acids, their precursors and derivatives, such as alpha-ketoglutarate as
disclosed in WO 95/34301 (Pharmacia AB) which hereby is incorporated as a reference.
An important aspect of the present invention is to use therapeutically effectiveamounts of an osmolyte and a thrombolytic agent in combination for the manufacture of
an agent capable of treating complications resulting from ischemia, hypoxia or oxidative
stress. Such an agent will be especially useful for treating complications in relation to
myocardial infarction wherein the thrombolytic agent with a capacity to induce Iysis of
blood clots, or the procedure of percutaneous transluminal coronary angioplasty (PTCA)
is combined with osmolytes to minimize the risk of coronary and vascular damages and
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W 097/38685 PCT~EP97/01861
restenosis. It is the intention that conventionally employed agents with thrombolytic
activity such as streptokinase shall be used in combination with osmolytes of the types
described above.
- The present invention is also related to a composition comprising an effective
S amount of the mentioned osmolytes for administration to an organ or a tissue being
subjected, or at the risk of being subjected, to an insufficient supply of substrates
necessary for maintaining the normal metabolic function together with a pharmacologically
acceptable carrier. Such compositions are especially suitable for being supplied to the
heart in connection with its interruption from a regular blood flow for example for treating
myocardial infarction, during coronary bypass surgery or transplantation. Such
compositions can further comprise agents as incorporated in conventional preservation
solutions or cardioplegic agents, such as Plegisol(E~' (Abbott Laboratories), St. Thomas
solution or the University of Wisconsin solution or other preservative agents or energy
substrates as suggested in WO 95/34301.
For the treatment of myocardial infarction, the compositions can preferably as
mentioned be combined with a conventional thrombolytic agent, such as streptokinase.
The thrombolytic agent can be added to the osmolytic composition, or administered
separately in a predetermined manner. The inventive compositions can also be included in
blood cardioplegia and in solutions useful as blood substitutes.
The compositions according to the present invention are also useful as solutions for
the preservation of organs illtellupted from their regular blood flow in combination with
conventional preservative agents.
It is also a part of the present invention to provide compositions for the treatment of
patients suffering from diabetes or such post-traumatic patients dependent on an insulin
therapy, comprising an effective amount insulin in a conventional dosage form together
with a therapeutically effective amount of at least one of the selected osmolytes, as
mentioned above. Such a composition can be in the form of an injectible preparation or an
otherwise ~lmini~terable dosage form of a conventional insulin in an effective amount,
either directly mixed with osmolytes, or with the osmolyte preparation separately
administerable in the as a part of kit, to be self administered by the patient in the
connection with the insulin therapy.
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W O 97/38685 PCTAEP97101861
Effective amounts of the osmolytes in the inventive compositions shall, suitably after
~-lmini.~tration, provide between about 50 ~M up to about 10 mM of osmolyte
concentration in the fluid supplied to the organ or the tissue, preferably between a
concentration of about 0.1 mM up to about 1-2 mM and most preferably about 0.5 mM.
5 An especially effective composition has been shown to comprise betaine and taurine at a
total concentration of about 0.2 mM.
Detailed and exemplifying description of the invention
Fig. 1 shows an anoxic model on a perfused liver, wherein lactate dehydrogenase
(LDH) in the effluent is used as a marker on cellular impairments is plotted against the
perfusion time for control and the incorporation of 0.1 mM and I mM of betaine in the
perfusion solution of 385 mosmA, respectively.
Fig. 2 shows the effect of ambient osmolality on mRNA levels for the betaine
15 transporter (BGT-1), the taurine transporter (TAUT), the myo-inositol transporter
(SMIT) and GAPD~ in the rat liver endothelial cells. Char~ges in osmolality wereperformed by appn)pliate addition/removal of sodium chloride. The mRNA levels were
determined by Northern blot analysis.
Fig. 3 shows the time-dependent induction of BGT-1 (betaine transporting protein)
20 and TAUT (taurine transporting protein) and SMIT (the myo-inositol transporter) mRNA-
levels in rat Kupffer cells. The Kupffer cells were exposed to LPS ( 1 llg/ml) in
normoosmotic (305 mosmol/l) or hyperosmotic (405 mosmol/l) media for the time periods
indicated and mRNA levels for BGT- 1, TAUT, SMIT and glyceraldehydephosphate
dehydrogenase (GAPDH) as a standard were determined by Northern blot analysis.
Fig. 4 shows an anoxic model on perfused liver similar to the one shown in
Fig. 1, wherein the LDH release is measured in the effluent after perfusion with solutions
of 385 mosmM enriched with 0.100 mM betaine, 0.100 mM betaine + 0.100 mM taurine.
Fig. 5 shows a similar anoxic model as in Fig. 1, wherein PGE2 (prostaglandin E2)
levels are measured in the effluent during anoxia and reperfusion with a 385 mosmM
solution which has been provided with 0.100 mM betaine and I rnM betaine, respectively.
. ..
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Fig. 6 shows a model for inducing oxidative stress, wherein a rat liver is exposed to
a solution of 0.2 rnM t-butylhydroperoxide (t-BOOH) and perfusion with a 305 mosmM
solution without and with I mM betaine. The protective effect of l mM betaine in the
perfusate is determined as LDH release in the effluent.
Fig. 7A shows the modulation of the CD95 ligand mRNA expression (a mediator for
apoptosis) in rat Kupffer cells in response to LPS challenge ( l ug/ml for 6h). In
experiments shown in bars I and 2, the cells were not incubated with LPS. In experiments
shown in bars 2 and 4, 5 mmol/l betaine was added 30 min before and throughout the
whole 6 h measurement period. Total RNA was extracted, reverse transcribed and
quantified by using PCR technique. Results are expressed as the ratio of number of CD95
ligand transcripts obtained with the indicated primers to the numbers of rat hypoxanthine-
guanine phsophoribyltransferase (HPRT) transcripts.
Fig. 7B shows the same experiment as in Fig 7A performed with rat sinusoidal
endothelial cells.
Fig. 8 shows the influence of betaine on the transporters for betaine and taurine
(BGT-1 and TAUT) and on inducible nitric oxide synthase mRNA levels in RAW 264.7mouse macrophages during hyperosmolarity. The macrophages were exposed to LPS (1llg/ml) for 6 hours in the presence or absence of 0.1 or 5 mmoVl betaine. The mRNA
levels of the transporters and iNOS were determined by Northern blot analysis.
Material and methods
Isolation and culture of Kupffer cells
Kupffer cells from male Wistar rats of 300-400 g body weight raised in the localinstitute for laboratory animals were isolated by collagenase-pronase perfusion and
separated by a single Nycodenz gradient and centrifugal elutriation. Cells were cultured in
RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) for
48 h. The experiments were performed during the following 24 h using Krebs-Henseleit
hydrogen carbonate buffer (pH 7.4) containing l0 mM glucose and 1% FCS. At that time
the cultures consisted of more than 99% Kupffer cells as demonstrated by their
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morphological appearance and their ability to phagocytose l,um Latex particles, which is
not observed in cultured endothelial cells. The osmolarity was varied by changing the NaCI
concentration. The viability of Kupffer cells was more than 95% as assessed by trypan blue
exclusion. Kupffer cell volume was measured by flow resistance cytometry using a Casy I
5 cell counter and analyzer system (Scharfe Systeme, Reutlingen, Germany). In
normoosmotic medium, the average Kupffer cell volume was 724 + 24 fl (7 different
preparations). Protein content was 0.039 + 0.009 mg per 106 cells (n=7). Assuming a
water content of 80% of whole Kupffer cell volume, a mean intracellular water space of
14,9 IlUmg protein is estimated. Vialibility of the incubations was routinely tested by
10 lactate dehydrogenase (LDH) release at the end of the incubation. 12-24 h hyperosmotic
(405 mosmolA) or a hypoosmotic (205 mosmol/l) exposure was without effect on LDHrelease.
Culture medium RPMI 1640 (without phenol red) and fetal calf serum (FCS) were from
Biochrom (Berlin, Germany)
Isolation and culture of endothelial cells
Endothelial cells of male Wistar rats were isolated according to the collagenase-
pronase method and centrifugal elutriation technique, as described for the Kupffer cells.
20 Isolated endothelial cells were incubated the first day for 4 hours in the ~p~opliate culture
medium adjusted to the desired osmolarity (205, 255, 305 or 405 mosmolA). The cells
were harvested following incubation and used for mRNA analysis. The cell viability was
routinely iested by determination of enzyme leakage, 4 hours of a hyperosmotic (405
mosmA) or a hypoosmotic incubation was without effect on viability.
Liver perfusion
Livers of Wistar rats (100-150 gram body mass) were perfused in situ as described
in Eur. J. Biochem., 1989, Vol. 181, p. 709-716, in the physiological antegrade direction
30 (from portal to hepatic vein) in an open recirculating system. The perfusion medium used
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W O 97/3868~ PCT~EP97/01861
11
was bicarbonate buffered Krebs-Henseleit saline medium (equlibrated with 02/C02 95:5
by volume). Anoxia was introduced by interrupting the supply oxygenated buffer
Cell and organ integrity was measured as release of lactate dehydrogenase (LDH) in
the liver effluent. The determination of the LDH content was performed according to a
5 routine spectrophotometric technique and expressed as milliunits/gram liver and minute.
Northern blot analysis
Total RNA from near-confluent culture plates of Kupffer cells and endothelial cells
10 were isolated by using guanidinethiocyanate solution. RNA samples were electrophoresed
in a 0.8% agarose/3% formaldehyde and then blotted onto Hybond-N nylon membraneswith 20X SSC (3 M NaCI, 0.3 M sodium citrate). After brief rinsing with water and UV-
cro.~slinking (Hoefer UV-crosslinker 500), the membranes were inspected under UVillumination to determine RNA integrity and location of the 28S and 1 8S rRNA bands.
Blots were then subjected to a 3 h-prehybridization at 43 jC in 50% deionized forrnamide,
in sodium phosphate buffer (0.25 M, pH 7.2), containing 0.25 M NaCI, 1 mM EDTA, 100
mg/m~ salmon sperm DNA and 7% SDS. Hybridization was carried out in the same
solution with approx. 106 cpm/ml (a-32P)dCTP-labeled random primed BGT-I, TAUT
and GAPDH cDNA probes. Membranes were washed three times in 2x SSC/0. 1% SDS
20 and twice in sodium phosphate buffer (25 mM, pH 7.2)/EDTA (I mM)/I % SDS. Blots
were then exposed to Kodak AR X-omat film at 70~C with intensifying screens and
analysed with PDI densitometry scanning (Pharmacia, Freiburg, Germany).
Statistics
Values are expresses as mean S.E.M (n= number of preparations).
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W O 97/38685 12 PCT/EP97/01861
Discussion of the results
As shown in Fig. l, hypoxia resulted in a marked increase in LDH release
demonstrating a deteriorating cell and organ integrity and function. The described cell and
5 tissue damage was characterized by an early injury, evident during hypoxia challenge
recognized by an escalating LDH release and a late injury when normoxia was reinstituted
(reperfusion injury). In a dose dependent manner, treatment with O. l mM and l mM
betaine solution was determined to ~limini.sh or even abolish the injury during and
following hypoxia.
Fig. 2 and Fig. 3 show that mRNA for the betaine transport protein, BGT- I, the
taurine transport protein, TAUT and the myo-inositol transporter SMIT, were expressed
both in endothelial cells and Kupffer cells. The endothelial cells were strongly dependent
on ambient osmolarity (Fig. 2) which demonstrates that osmolytes are important
components in the regulation of cellular function in both immune competent cells and the
15 endothelial cells of the vasculature. Moreover, in endothelial cells TAUT tended to be
more intensively expressed than BGT-1 in response to the 4 hours of exposure to
hyperosmolarity. In Kupffer cells, there was a time dependent increase in BGT- l and
TAUT mRNA expression, see Fig. 3. These findings shows that the composition of
osmolytes, used according to the present invention, can be tailored to optimize therapeutic
20 efficacy with respect to a target cell type, as well as the timing of the therapeutic
intervention.
Fig. 4 shows that a co-:~lmini~tration of taurine and betaine during anoxia leads to a
reduced leakage of LDH from the Kupffer cells, when compared to a supplementation of
betaine only, or a standard solution of 385 mosmM. These results demonstrates a
25 possibility of obtaining an improved, or even a synergistic, organ protection by combining
different selected osmolytes.
It has also been demonstrated, see Fig. 5, that the decrease in LDH following
betaine treatment of the liver is accompanied by a reduced liver production of eicosanoids,
as represented in this experiment by the cyclooxygenase product prostaglandin E230 (PGE2). This indicates a general capacity of selected osmolytes to suppress the activity of
cells capable of prostaglandin synthesis, such as macrophages and Iymphocytes. The
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W O 97/38685 PCT~P97/01861
l3
activation of immune competent cells, evident for example following ischemia/reperfusion
is a major contributor to the esc~l~ting cell injury and necrosis in a process which can
extend to days and months after the ischemic or hypoxic event. Accordingly, the
downregulation of stim~ ted immune cells, achieved according to the parallel Swedish
5 patent application, provides further support for a protective effect of selected osmolytes in
the above described course of pathological events. A supplementation of osmolytes wil3
consequently suppress the macrophage activity which can be triggered by an ischemic or
hypoxic event which otherwise could lead to a rupture of vascular plaques leading to
thrombosis and an even more serious organ or tissues damages resulting from occlusions
of vessel lumens, see e.g. The Lancet, 1996, Vol. 347, pag. 305-306, P Weisberg et al.
Fig. 7A and 7B demonstrates the capacity of osmolytes in protection of apoptosis,
whereas Fig. 8 shows that osmolytes are effective in downregulating inducible nitric oxide
synthase (iNOS). As iNOS is a m~ tor of complications following ischemia, hypoxia and
oxidative stress, these results support the utility of osmolytes in the treatment of reducing
15 complications resulting from said stress situations.
Furthermore, a supplementation of selected osmolytes, according to the present
invention, to patients identified as being at risk of acquiring life-threatening coronary
syndromes of unstable angina and myocardial infarction, precipitated by the rupture of
cardiovascular plaques will be of benefit, since such a therapy will selectively modulate the
20 activity of macrophages on the plaques. The inventive osmolyte therapy, thus
demonstrates a considerable potential for supplying to such at risk patients who expect
complementary surgery or therapy.
This concludes that the present invention has contributing potential, in terms of
treating, but also in preventing damages resulting from ischemia and subsequent
25 reperfusion by a capacity in stabilizing vascular plaques.
A further aspect of preventing life threatening coronary syndromes by the inventive
osmolyte therapy concerns patients suffering from pathologically raised levels of
circulating metabolites capable of exerting osmotic stress on the vasculature, exemplified
by raised levels of circulating glucose in the diabetic state. As demonstrated in the
30 experiments shown in Fig. 2 endothelial cells subjected to osmotic stress express osmolyte
transporting proteins and thereby susceptibility to osmolyte therapy for their normalization
,
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W O 97/38685 PCTrEP97/01861 - -
of their cellular hydration and function. Hence, osmolytes have a potential in preventing
vascular dysfunctions leading to impairrnents of the blood flow, vascular dysfunction and
related diseases in the diabetic patient, for example by being ~rlmini.~tered in connection
with conventional insulin therapy as a preventive therapy for cardiovascular or other
5 vascular diseases in the diabetic state.
The benef1cial effect of osmolytes on the tissue capacity for scavenging oxygen free
radicals serves as a mf cll~ni~tic basis for the described improvement of tolerance to
oxidative stress as shown in Fig. 6 . The extent of damages from oxidative stress, also
10 resulting from reperfusion, can consequently be reduced therapy of supplying selected
osmolytes.
. , .
