Note: Descriptions are shown in the official language in which they were submitted.
CA 02476446 2004-08-16
WO 031068212 PCTIEP03101020
Use of inhibitors of the sodium/hydrogen exchanger for the treatment of
thrombotic and inflammatory disorders
The invention relates to the use of inhibitors of the cellular sodium/hydrogen
exchanger in human and veterinary medicine for the prevention and treatment
of acute or chronic diseases caused by elevated levels of von Willebrand
factor
in the blood. The inhibitors can therefore be employed for the treatment of
thrombotic and inflammatory disorders.
Inhibitors of the sodium/hydrogen exhanger (NHE) have in recent years been
characterized in numerous preclinical studies as substances which are suitable
in
a superior manner in cases of cardiac hypopertusion for protecting the cardiac
tissue which is endangered by the acute onset of the ischemic event from
death. Protection of cardiac tissue by NHE inhibitors encompasses all degrees
of
harm caused by the hypoperfusion, starting from cardiac arrhythmias via
hypercontraction of the myocardium and temporary loss of function up to death
of cardiac tissue and the permanent damage associated therewith.
The mechanism of action of NHE inhibitors which is important in the acute
ischemic event comprises their reduction of the enhanced influx of sodium ions
which arises in acutely hypoperfused tissue due to activation of the NHE as a
consequence of intracellular acidification. This delays the situation of
tissue
sodium overload. Since there is coupling of sodium and calcium ion transport
in
cardiac tissue, this prevents the life-threatening calcium overload of heart
cells.
It is also known that NHE inhibitors provide protection to the central nervous
system (CNS), such agents protecting the CNS, in a similar way to the heart,
against acute ischemic states. These states are caused by acute hypoperfusion
and thus by a deficient supply of nutrients, oxygen or minerals. Such ischemic
harm to the CNS is particularly pronounced in cases of central infarctions
such as
stroke. Thus, as expected, no protective effects of NHE inhibitors against
these
acute events were observable where blood flow was normal and healthy,
because there was no acute onset of ischemic harm to cardiac tissue or CNS
tissue.
CA 02476446 2004-08-16
2
Numerous classes of substances which intervene in the interplay of coagulation
factors and thus cause cessation of the coagulation cascade are described in
the prior art. Likewise, numerous action principles which do not suppress
thrombus formation, but cause the dissolution (lysis) of thrombi which have
already formed, have been developed. Some of these action principles, which
intervene at a wide variety of junction points in said cascade, have been
introduced into therapy to prevent thrombogenesis, such as derivatives of the
vitamin K group (phylloquinones), factor VIII and factor IX products, platelet
aggregation inhibitors such as acetylsalicylic acid, dipyridamole and
ticlopidine,
anticoagulants such as heparins or heparinoids.
The blood coagulation cascade can be divided mechanistically into two
pathways as depicted in the following diagram, namely into an intrinsic and an
extrinsic route, the two of which finally meet in the activation of factor X
and the
resulting generation of thrombin and subsequently of fibrin:
Intrinsic Extrinsic
XII --~ Xlla VII + TF
1
xl ~ xla
1
Ix ---~ Ixa
X --~ Xa Platelet Aggregation
1
Prothrombin ---~ Thrombin
1
Fibrinogen ----~ Fibrin
Scheme 1: blood coagulation cascade
It is important in the therapeutic use of such blood coagulation inhibitors
that the
inhibition of coagulation achieved is not too strong or complete, which would
CA 02476446 2004-08-16
inhibit the formation of microthrombi and microcoagulations which are vital
and
which must take place at the microtraumata which are continually happening.
Only imprecise adjustment of the degree of inhibition of coagulation is
possible
as a result of differences in the response of the particular individual at a
particular time, and the degree must be carefully monitored where possible. If
these many small coagulation processes which ure Nermanently taking place
are inhibited there is a high risk of extensive hemorrhage (hemophilia).
The disadvantage of the known therapeutic agents available on the market
which intervene as inhibitors in the coagulation event is therefore the high
risk of
bleeding complications. The risk of life-threatening hemorrhage exists
especially
during high-dose thrombolysis therapy, e.g. during therapy of acute myocardial
infarction or pulmonary embolism. There is thus an urgent need for therapeutic
agents which do not entail a risk of increased tendency to bleeding despite
overdosage.
Many of the known anticoagulant substances act by exerting an effect on the
blood platelets, the thrombocytes, and inhibiting their function or inhibiting
their
activation. The endothelium also evidently plays a central part in the
coagulation event. Thus, for example, the von Willebrand factor (vWF) which is
necessary for coagulation is produced for the most part in endothelial cells
and
is secreted by them permanently (constitutively) into the circulating blood in
order to ensure the necessary coagulation processes in the blood. A
considerable part of the produced vWF is stored in cytoplasmic granules,
called
Weibel-Palade bodies, and released as required through stimulation of
endothelial cells. If endothelial cells are unable to produce vWF and deliver
it to
the blood, the result is the well known genetic vWF-dependent disease, von
Willebrand-Jurgens syndrome, which is characterized by hemorrhages which
can scarcely be stopped.
It is only in recent years that disorders caused by elevated concentrations of
vWF
in the blood, thus inducing, for example, an increased tendency to blood
coagulation and inflammatory processes, have become known. Thus,
Kamphuisen et al, demonstrate on the basis of a large number of studies in
their
publication "Elevated factor VIII levels and the risk of thrombosis"
(Arterioscler.
Thromb. Vasc. Biol. 21 (5):731-738 (2001 )) that there is a significant
association
CA 02476446 2004-08-16
4
between elevated vWF levels in the blood and an increased rate of thrombotic
disorders. Factor VIII forms with vWF a complex as necessary precondition for
blood coagulation. It has been possible to establish that high levels of von
Willebrand factor and (vWF) and of vWF-bound factor VIII in the blood
represent
a clear thrombosis risk factor. However, antithrombotic agents which
antagonize
the stabilizing binding of vWF to fac~or VIII may also be disadvantageous
because, in the event of overdosage, substantial inhibition of blood
coagulation
and dangerous tendencies to bleeding must be expected.
In the attempt to find effective compounds for the treatment of acute or
chronic
diseases caused by elevated levels of von Willebrand factor in the blood, it
has
now been found that the compounds employed according to the invention
inhibit the release of von Willebrand factor from endothelial cells. The
compounds of the invention inhibit the massive pH-dependent release of vWF
which accumulates during ischemia.
Whereas the secretion takes place normally and constitutively at the normal pH
of blood which is known to be about 7.4, and part of the vWF is stored in
Weibel-
Palade bodies, it has now been found that there is a delay and reduction in
the
release of vWF as the pH falls. Exocytosis of the Weibel-Palade bodies in
which
the vWF is packaged is increasingly inhibited as the pH declines. Thus, under
acidotic conditions, there is a significant increase in Weibel-Palade bodies
and
thus extensive accumulation of vWF in the endothelial cell, and a reduced
constitutive and stimulated vWF secretion. This can be visualized by staining
procedures and demonstrated by quantitative measurements of vWF in the
supernatant. Such acidotic states with significant pH reductions below 7
occur,
for example, in cases of tissue ischemia. At the instant of realkalinization
and
endothelial cell stimulation, which corresponds to the reperfusion state,
within
seconds exocytosis takes place, and thus emptying of the Weibel-Palade bodies
(WPB), thus leading to massive release of the prothrombotic risk factor.
Besides vWF, the Weibel-Palade bodies also store the transmembrane protein
P-selectin (Wagner, D.D. 1993, Thromb. Haemost., 70:105-110).
P-Selectin is located in the vesicle membrane and, after vesicle fusion
(exocytosis), is incorporated into the plasma membrane of the endothelial
cell.
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This means that every Weibel-Palade body exocytosis leads not only to
increased vWF release but also to increased P-selectin expression in the
endothelial cell membrane. The examples show vWF secretion (quantitive
measurement by ELISA) during acidosis and during subsequent repertusion. In
5 parallel, these quantitative measurements are confirmed by
immunofluorescence data G~1 the',~'eibci-Palade bodies. The measured vWF is
thus not only a marker of increased (increase in vWF secretion) or reduced
(decrease in vWF secretion) tendency to thrombosis (via increase in platelet
aggregation), but also a direct marker of increased or reduced P-selectin
expression in the endothelial cell membrane. P-Selectin serves as anchor for
leukocytes and thus the initial inflammatory reaction (Vestweber, D., Blanks,
J.E.
1999, Physiol. Rev., 79:181-213; Issekutz, A.C., Issekutz, T.B. 2002, J.
Immunol.,
168:1934-1939). The pathophysiological significance is wide-ranging and
confirmed for ischemia/reperfusion disorders, thromboses and arteriosclerosis
(Massberg, S., et al., 1998, Blood, 92:507-515;
Kita, T., et al., 2001, Ann. N. Y. Acad. Sci., 947:199-205). Besides the
significance of
P-selectin as marker of inflammation and initiator of inflammation, it plays
an
essential part in the process of cancer dissemination (Varki, A., Varki, N.M.
2001,
Braz. J. Med. Biol. Res. 34:711-717) and during various inflammations of
joints
(arthritis) (Veihelmann, A. et al, 1999, Microcirculation, 6: 281-290;
Mclnnes, I.B., et
al., 2001, J. Immunol., 167:4075-4082). Thus the mode of action of the
substances
described here may also find use as therapeutic agent for all the
abovementioned P-selectin-associated disorders.
The invention therefore relates to the use of inhibitors of the
sodium/hydrogen
exchanger for producing medicaments for the prophylaxis and therapy of acute
and chronic diseases caused by elevated levels of von Willebrand factor in
blood.
The invention further relates to the use of at least one of the following
compounds
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6
N\ 'NH2
H3C '~-
NH2
N~NHZ
U O NH2
O; S
O
/ N NH2
O O NH2
N
N\ N H 2
,SO v
O O H
2
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O
O N
~N
N\ NH2
O:S
O NH
2
\/o
N\ 'NH2
'l~
O NHZ
O N~NH2
w NH2
~O ~N
~O
N
N~ NHZ
O NH2
O
~N~N
H ~ I
N
NH
~ NH
O N =/
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O
NH2
N
N H2
N O
CH3
CI NH2
N
N H2
N~ O
or
F
N~N~~N H2
N H2
and/or all stereoisomeric forms of the abovementioned compounds and/or
mixtures of these forms in any ratio, and/or of the physiologically tolerated
salts
of the abovementioned compounds for producing a medicament for the
prophylaxis and therapy of acute or chronic diseases caused by elevated levels
of von Willebrand factor in the blood and/or increased expression of P-
selectin.
The invention further relates to the use of cariporide
H-03S-C H3
H C- N~N Hz
'l3
NH2
for producing a medicament for the prophylaxis and therapy of acute or
chronic diseases which are caused by elevated levels of von Willebrand factor
in
the blood and/or increased expression of P-selectin.
CA 02476446 2004-08-16
9
The abovementioned compounds are known and can be prepared as
described, for example, in EP 0 416 499, EP 0 556 673, EP 0 589 336, EP 0 b22
356,
EP 0 b99 66b, EP 0 708 088, EP 0 719 766, EP 0 726 254, EP 0 787 728, EP 0 972
767,
DE 19529612, DE 19601303, WO 99 00379 or T.Kawamoto et al., Potent and
selective Inhibition of the hur roan Na+i I-1+ exchanger isoform NHE1 by a
novel
aminoguanidine derivative T-162559, Eur.J. Pharmacol. 420 (2001), 1-8.
Where the abovementioned compounds allow diastereoisomeric or
enantiomeric forms and result as mixtures thereof in the chosen synthesis,
separation into the pure stereoisomers takes place either by chromatography on
an optionally chiral support material or, if the racemic abovementioned
compounds are able to form salts, by fractional crystallization of the
diastereomeric salts formed with an optically active base or acid as aid.
Examples of suitable chiral stationary phases for separation of enantiomers by
thin-layer or column chromatography are modified silica gel supports (so-
called
Pirkle phases) and high molecular weight carbohydrates such as
triacetylcellulose. Gas chromatographic methods on chiral stationary phases
can also be used for analytical purposes after appropriate derivatization
known
to the skilled worker. To separate enantiomers of the racemic carboxylic
acids,
diastereomeric salts differing in solubility are formed using an optically
active,
usually commercially available, base such as (-)-nicotine, (+)- and
(-)-phenylethylamine,quinine bases, L-lysine or L- and D-arginine, the less
soluble
component is isolated as solid, the more soluble diastereomer is deposited
from
the mother liquor, and the pure enantiomers are obtained from the
diastereomeric salts obtained in this way. It is possible in the same way in
principle to convert the racemic compounds of the formula I containing a basic
group such as an amino group with optically active acids such as (+)-camphor-
10-sulfonic acid, D- and L-tartaric acid, D- and L- lactic acid and (+) and
(-)-mandelic acid into the pure enantiomers. Chiral compounds containing
alcohol or amine functions can also be converted with appropriately activated
or, where appropriate, N-protected enantiopure amino acids into the
corresponding esters or amides, or conversely convert chiral carboxylic acids
with carboxyl-protected enantiopure amino acids into the amides or with
enantiopure hydroxy carboxylic acids such as lactic acid into the
corresponding
CA 02476446 2004-08-16
chiral esters. The chirality of the amino acid or alcohol residue produced in
enantiopure form can then be utilized for separating the isomers by carrying
out
a separation of the diastereomers which are now present by crystallization or
chromatography on suitable stationary phases and then eliminating the
5 included chiral moiety by suitable methods.
Acidic or basic products of the abovementioned compounds can exist in the
form of their salts or in free form. Preference is given to pharmacologically
suitable salts, e.g. alkali metal or alkaline earth metal salts, or
hydrochlorides,
10 hydrobromides, sulfates, hemisulfates, all possible phosphates, and salts
of amino
acids, natural bases or carboxylic acids.
Physiologically tolerated salts are prepared from the abovementioned
compounds able to form salts, including the stereoisomeric forms thereof, in a
manner known per se. The carboxylic acids and hydroxamic acids form with
basic reagents such as hydroxides, carbonates, bicarbonates, alcoholates and
ammonia or organic bases, for example trimethyl- or triethylamine,
ethanolamine or triethanolamine or else basic amino acids, for example lysine,
ornithine or arginine, stable alkali metal, alkaline earth metal or optionally
substituted ammonium salts. Where the abovementioned compounds have
basic groups, stable acid addition salts can also be prepared with strong
acids.
Suitable for this purpose are both inorganic and organic acids, such as
hydrochloric, hydrobromic, sulfuric, phosphoric, methanesulfonic,
benzenesulfonic, p-toluenesulfonic, 4-bromobenzenesulfonic,
cyclohexylsulfamic, trifluoromethylsulfonic, acetic, oxalic, tartaric,
succinic or
trifluoroacetic acid. Methanesulfonic acid salts of the abovementioned
compounds are particularly preferred.
Owing to the pharmacological properties, the abovementioned compounds
ace suitable for the prophylaxis and therapy of acute or chronic diseases
which
are caused by elevated levels of von Willebrand factor in the blood and/or
increased expression of P-selectin.
These include thrombotic disorders provoked by ischemic states with subsequent
reperfusion; such as thromboses in acute myocardial, mesenteric or else
cerebral
CA 02476446 2004-08-16
11
infarction; thrombotic disorders occurring during or after surgical
operations;
pulmonary embolisms; deep vein thromboses as occur at an increased rate after
prolonged restriction of blood flow, especially in the lower extremities, for
example after prolonged lying or sitting, and imflammatory disorders as occur
during ischemia and subsequent reperfusion, during vasculitis (e.g. associated
with autoimmune disease or connective tissue disease).
These also include disorders which are caused by increased expression of
P-selectin, such as incipient inflammatory reactions; but also prophylaxis and
treatment of arteriosclerosis; and prophylaxis and treatment of cancer; also
inflammation of joints and arthritic disorders such as rheumatoid arthritis.
Administration of the medicaments of the invention can take place by oral,
inhalational, rectal or transdermal administration or by subcutaneous,
intraarticular, intraperitoneal or intravenous injection. Oral administration
is
preferred.
The invention also relates to a process for producing a medicament, which
comprises converting at least one of the abovementioned compounds with a
pharmaceutically suitable and physiologically tolerated carrier and, where
appropriate, ofiher suitable active ingredients, additives or excipients into
a
suitable dosage form.
The abovementioned compounds are mixed with the additives suitable for this
purpose, such as carriers, stabilizers or inert diluents, and converted by
conventional methods into suitable dosage forms such as tablets, coated
tablets, two-piece capsules, aqueous, alcoholic or oily suspensions or aqueous
or
oily solutions. Examples of inert carriers which can be used are gum arabic,
magnesia, magnesium carbonate, potassium phosphate, lactose, glucose or
starch, especially corn starch. Preparation can moreover take place both as
dry
and as wet granules. Examples of suitable oily carriers or solvents are
vegetable
or animal oils, such as sunflower oil or fish liver oil.
For subcutaneous, intraperitoneal or intravenous administration, the active
compounds are converted into solution, suspension or emulsion if desired with
the substances suitable for this purpose, such as solubilizers, emulsifiers or
other
CA 02476446 2004-08-16
12
excipients. Examples of suitable solvents are physiological saline or
alcohols, e.g.
ethanol, propanol, glycerol, as well as sugar solutions such as glucose or
mannitol solutions, or else a mixture of the various solvents mentioned.
Also used are conventional aids such as carriers, disintegrants, binders,
coating
agents, swelling agents, glidants or lubricants; flavcrings, sweeteners and
solubilizers. Excipients which are frequently used and which may be mentioned
are magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars,
talc, milk protein, gelatin, starch, cellulose and derivatives thereof, animal
and
vegetable oils such as fish liver oil, sunflower, peanut or sesame oil,
polyethylene
glycol and solvents such as, for example, sterile water and monohydric and
polyhydric alcohols such as glycerol.
The abovementioned compounds are preferably produced and administered
as pharmaceutical products in dosage units, where one unit contains as active
ingredient a defined dose of the compound of the formula I. They can for this
purpose be administered orally in doses of from 0.01 mg/kg/day to 25.0
mg/kg/day, preferably 0.01 mg/kg/day to 5.0 mg/kg/day or parenterally in
doses of from 0.001 mg/kg/day to 5 mg/kg/day, preferably 0.001 mg/kg/day to
2.5 mg/kg/day. The dosage may also be increased in severe cases. However,
lower doses also suffice in many cases. These data relate to an adult weighing
about 75 kg.
The abovementioned compounds can be employed alone or in combination
with anticoagulant, platelet aggregation-inhibiting or fibrinolytic agents.
Coadministration can take place, for example, with factor Xa inhibitors,
standard
heparin, low molecular weight heparins such as enoxaparin, dalteparin,
certroparin, parnaparin or tinzaparin, direct thrombin inhibitors such as
hirudin,
aspirin, fibrinogen receptor antagonists, streptokinase, urokinase and/or
tissue
plasminogen activator (tPA).
It is known that the inhibitors of the sodium/hydrogen exchanger affect
platelet
aggregation and have an adhesion-inhibiting effect (see Rosskopf, Dieter, J.
Thromb. Thrombolysis (1999), 8(1), 15-23; or Nieuwland, Rienk; Akkerman,
Jan-Willem Nicolaas. Adv. Mol. Cell Biol. (1997), 18(Platelet), 353-366.
CA 02476446 2004-08-16
13
In contrast to the previously described effects on the aggregation of blood
platelets, the abovementioned compounds also show inhibition of excessive
release of von Willebrand factor. This novel antithrombotic action principle
differs
from the previously disclosed antithrombotic action principles in a crucial
and
advantageous manner in that
a) it acts only in ischemic tissue in the subsequent repertusion phase,
whereas
other cells not affected by the ischemia (preischemic) remain completely
unaffected, and
b) there is no need to worry about any of the dangerous hemorrhagic
complications during the lysis therapy.
The invention is explained in more detail by means of examples below.
The following examples demonstrated the effects of an extracellular acidosis
(pHex = 6.4) and the effects of the abovementioned compounds of the invention
on the intracellular pH (pH~ and the release of von-Willebrand factor (vWF).
All
the examples were carried out with human umbilical vein endothelial cells
(HUVEC). These comprise primary cell cultures isolated from the umbilical
vein.
For the following examples, the cells were cultivated either on gelatinized
glass
plates (measurement of the intracellular proton concentration) or on cell
culture
plates (12-well culture plates, Falcon, New Jersey, USA; measurement of vWF
release) after the first passage.
CA 02476446 2004-08-16
14
Example l:
Measurement of the intracellular pH
To measure the intracellular proton concentration (pHa, the HUVECs were
loaded with the pH-sensitive fluorescent dye BCECF-AM (2',7'-
bis(carboxyefihyl)-
5(6)-carboxyfluorescein). A Deltascan spectrofluorometer (PTI, Hamburg) was
employed for the subsequent fluorescence measurement. This measuring system
consists essentially of a UV light source, a monochromator, a photon detector
and the Felix and Oscar software packages (PTI, Hamburg) for controlling the
system via a computer. After alternate excitation with the wavelengths 439.5
nm
(pH-independent) and 490 nm (pH-sensitive), the ratio of the measured
emissions
of the BCECF (ratio) was reported and the pH was found after a calibration.
The
measuring cell is designed so that the parameters of temperature and carbon
dioxide partial pressure in the system are controlled during continuous
perfusion.
For the repertusion simulation, the experimental conditions were set at
37°C and
a carbon dioxide partial pressure of 5~ or 10~ by gassing the system and
perfusate.
In the experiment there was initially preincubation with sodium bicarbonate
buffer pHex 6.4 for 60 minutes in order to simulate respiratory metabolic
acidosis.
The initiation pertusion was then changed to sodium bicarbonate buffer of
pH 7.4 with lO,uM histamine as repertusion simulation.
These control experiments were compared with an experiment in which the NHE
inhibitor cariporide was added in a concentration of lO,uM to the reperfusion
buffer.
The results of several experiments have been summarized in Tables 1 and 2.
Table 1: Intracellular pH during extracellular acidosis (pH, (acidosis)) of at
least
15 minutes and under control conditions (Co).
Table 1:
pH; (Acidosis) 6.53 0.02 (mean SEM)
pH; (Co) 7.23 0.02 (mean SEM)
amn m n m mt_.n iVUW .7 t.ICVIUIIVI I IIUI11 Ifle llleQfl
CA 02476446 2004-08-16
Extracellular acidosis leads to intracellular acidification which persists
during the
acidosis. The intracellular acidotic pH is virtually identical to the
extracellular pH
(applied extracellular acidosis pHex = 6.4).
5
Table 2: Repertusion with experimental buffer containing cariporide (HOE) and
control buffer (Co). The initial rates of increase in the pH, values was found
after
60 minutes of acidosis from the measurements during the first 30 seconds after
reperfusion.
Table 2:
Rate of pH increase (0 pH / min)
Individual experiments Mean SEM
0.97
1.04
Co 0.89 0.97 0.04
0.88
1.07
0.30
0.24
HOE 0.23 0.27 0.02
0.34
0.24
When the extracellular pH changed from 6.4 to 7.4 there was a reduction by a
factor of 3.6 in the rate of increase in intracellular pH compared with the
control.
Thus, it is possible by using cariporide during reperfusion to reduce
significantly
the rate of realkalinization.
Example 2
Measurement of vWF release after repertusion
The measurements were carried out in a Heraeus Heracell incubator. This made
it possible to calculate the umbilical vein endothelial cells under controlled
CA 02476446 2004-08-16
16
physiological conditions (temperature 37°C, relative humidity 100, pC02
constant at 5~) and to ensure rapid change of different cell culture media.
Said cells were initially incubated with acidotic medium (pH 6.4 composed of
the
ingredients: medium M199 w/Earle's & amino acids, w/L-glutamine, w/o
NaHC03, w/o Hepes + 0.0848 NaHC03 / I) or pH standard medium (pH 7.4
composed of the ingredients: medium M199 w/Earle's & amino acids,
w/L-glutamine, w/o NaHCO~, w/o Hepes + 2.2008 NaHC03 / I) for one, three or
48 hours. Before starting the reperfusion, samples of supernatant were taken
to
determine the vWF concentration under acidotic conditions (vWFa°~d~,~
and
control conditions (vWF~~. To simulate reperfusion, the medium was changed to
one with a pH of 7.4 (ingredients: medium M 199 w/Earle's & amino acids,
w/L-glutamine, w/o NaHC03, w/o Hepes + 2.200 g NaHC03 / I + lO,uM histamine)
to which the NHE inhibitor cariporide was added in a concentration of lO,uM.
Change to the same medium without corresponding addition of inhibitor served
as control.
The samples taken from the supernatant were used to determine the vWF
concentration. This was done by an ELISA method (enzyme-linked immuno
sorbent assay) using specific antibodies. The vWF content of standard human
plasma (Behring, Marburg) is calculated using an international standard (2'~
International Standard 87/718; National Institute for Biological Standards and
Control, London).
Table 3: vWF conzentration in the cell supernatant under acidotic (vWF~,~,~
and
under control conditions (vWF~~, measured after incubation for 15 minutes. The
vWF concentration under control conditions is set at 100.
Table 3.
vW F~o 100~~
vWFa~d~,s (constitutive) 46 1.1
VWFa~,ao~s (stimulated, histamine52 2.5~
50
NM)
The acidosis led to a distinct decrease in vWF secretion, both the
constitutive
secretion and the stimulated Weibei-Palade body secretion. The vWF secretion
was reduced by a factor of 2 compared with control cells during acidosis
CA 02476446 2004-08-16
17
(pHex = 6.4).
Table 4: vWF secretion was measured during a 10-minute repertusion time with
stimulation. The vWF secretion of the control cells (vWF~~ was set at 100. The
vWF concentration during the repertusion of preacidotic cells (vWF~,~ and the
vWF concentration durlrig reperfusion of preacidotic cells in the presence of
1 O,uM of cariporide (vWF,,~) have been indicated as values relative to the
control values. Control cells were incubated with cariporide (vWF~o+,~)
Table 4:
vWF~o 100
vWF~o+HOe 106 t 3.06
vWFa~,d~,s 193 t 8.0~
vWF,~ 139 16~
During the reperfusion there was a large increase in vWF secretion by a factor
of
2. Blockade of the NHE with cariporide reduces the increased vWF secretion by
almost 60~ and thus approaches the control values. Control cells incubated
with
cariporide (10 ~.M) showed no increase or decrease in vWF secretion.
The examples showed that extracellular acidosis as present for example during
ischemia led to an intracellular acidosis, resulting in reduced (constitutive
and
stimulated) vWF secretion and a reduced P-selectin expression. The subsequent
reperfusion and stimulation of the endothelial cells brought about rapid
intracellular realkalinization. There was a simultaneous great enhancement of
the
increased vWF secretion and increased P-selectin expression. A delay of the
realkalinization with cariporide reduced the increased vWF secretion and
P-selectin expression and thus the possible thrombosis and inflammatory
reactions. The examples showed that the intracellular pH is determined by the
extracellular pH. Secretion by the endothelial cells is in turn determined by
the
intracellular pH. It is thus possible, by inhibiting realkalinization, to
reduce greatly
the known endothelial cell activation during the reperfusion phase and the
worry, connected therewith, about rethrombosis (vWF secretion) and
inflammation. Incubation of healthy, non-acidotic control cells with
cariporide
showed no effect. This indicates a low potential for side effects and prevents
an
CA 02476446 2004-08-16
I8
excessive tendency to bleeding. The agent acts only where ischemia is present.
