Note: Descriptions are shown in the official language in which they were submitted.
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ANGIOTENSIN II RECEPTOR BLOCKERS FOR PREVENTING THE DEVELOPMENT OR PROGRESSION
OF MICROVASCULAR DISEASE DUE TO DIABETES
The present invention relates to the field of inhibitors of angiotensin II
receptor
blockers and particularly~addresses their use in diabetes to prevent the
development
or progression of microvascular disease (i.e. disease involving small blood
vessels)
affecting eyes (diabetic retinopathy) and kidneys (diabetic nephropathy).
Diabetes is a disorder in which the body is unable to metabolize carbohydrates
(e.g.,
food starches, sugars; cellulose) properly. The disease is characterized
by~excessive
amounts of sugar in the blood (hyperglycemia) and urine, inadequate production
and/or utilization of insulin, and by thirst, hunger, and loss of weight.
Diabetes is
affecting about 2% of the population. Of these 10-18% are insulin dependant
(type 1 )
diabetics and the remainder non insulin dependant (type 2) diabetics.
Retinopathy is damage to the;retina caused..by microvascular changes. Diabetic
retinopathy is a specific ~microvascular complication of both typel and type 2
diabetes. The prevalence of retinopathy is strongly linked to the duration of
diabetes.
After 20 years of diabetes nearly all patients with type 1 diabetes and over
60% of
patients with type 2 diabetes have some degree of retinopathy. A diabetic is
25 times
more likely to go blind than a person in the general population. Upton a fifth
of newly
diagnosed diabetics have been found to have some retinopathy. Additionally,
retinopathy develops earlier~and is more severe in diabetics with elevated
systolic
blood pressure levels. On average, a careful eye examination reveals mild
retinal
abnormalities about seven years after the onset of diabetes, but the damage
that
threatens vision usually does not occur until much later. Diabetic retinopathy
is the
most common cause of blindness in the working age population in many
countries.
In the early phases of retinopathy, weakening of the small blood vessels in
the retina
produces bulges in the vessels (microaneurysms) and leakage of fluid
(exudates)
and blood (hemorrhages). Proliferative retinopathy, a later stage of the
disease,
involves the growth of fragile new blood vessels on the retina and into the
vitreous, a
jelly-like substance inside the eyeball. These vessels can rupture and release
blood
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into the vitreous, which causes blurred vision or temporary blindness. The
scar tissue
that may subsequently develop can pull on the retina and cause retinal
detachment,
which may lead to permanent vision loss. Macular edema-swelling due to fluid
accumulating around the macular, the part of the retina most crucial for fine
vision,
may also occur. If proliferative retinopathy is left untreated, about half of
those who
have it will become blind within five years, compared to just 5% of those who
receive
treatment.
The condition can be treated with laser photocoagulation, if it is detected
early.
Additionally, reduction in hyperglycemia at any time in the course of diabetes
will
result in a significant decrease in the long-term incidence and progression of
retinopathy and in the development~of visual loss. In the EUCLID study, the
° .
- ,,
angiotensin converting enzyme~(ACE) inhibitor lisinopril reduced the risk of .
~,; ...:
progression of retinopathy by approximately 50%, and also significantly
reduce~:the.~
.. a .~ _: , , , , ,... ,.:z, :. .
~~15 .risk. of:progression to proliferative retinopathy. However, in the
EUCLID Study
f~ , ;.. , .
retinopathy was not a primary eridpoint and the study was~not sufficiently
powered for
eye-related outcomes. Preventing the development or progression of the
condition
has the potential to save vision at a relatively low cost compared to the
costs
associated with a loss of vision. Thus, it is an object of the present
invention to
provide further means which contribute to the prevention of the development or
progression of diabetic retinopathy.
Nephropathy is the deterioration of the kidneys. Diabetic nephropathy is a
specific
microvascular complication of both typel and type 2 diabetes. Type 1 diabetes
is
more likely to lead to the final stage of nephropathy called end-stage renal
disease
(ESRD). There are five stages of diabetic nephropathy, the fifth stage is
ESRD.
Progress from one stage to the next can take many years, with 23 years being
the
average length of time to reach stage five. Diabetes is the most common cause
of
ESRD accounting for more than 40 percent of cases in the US.
Treatment for diabetic nephropathy attempts to manage and slow the progression
of
the disease. Aggressive blood pressure control is by far the most important
factor in
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protecting kidney function. Angiotensin-converting enzyme inhibitors are
considered
to provide the best protection for~the kidneys. According to the RENAAL.Study
(Brenner et al, The New England Journal of Medicine 345:861-869, 2001) the
angiotensin II receptor blocker losartan might offer similar protection, but
concerns
have been raised regarding to both the patient population as well as the
outcome
measures. Due to these methodological flaws and the incomplete data in the
study
the question of the effectiveness and safety of this treatment in diabetic
nephropathy
remains unanswered (Fisman et al, Cardiovascular Diabetology 1:2, 2002). From
the
data of the similar IDNT study (Lewis et al, The New England Journal of
Medicine
345:851-860, 2001) it has been concluded that the angiotensin-II-receptor
blocker
irbesartan.is effective in protecting against the progression of nephropathy
due to
Type II diabetes. Yet, preventing the development or progression of the
condition has
the potential rto save kidney function Thus, it .is another object of the
present ~;
invention to provide further means viihich: contribute to.the prevention' of
the
1:5: .development or~ progression of. diabetic: nephropathy,
Angiotensin II plays a major role in pathophysiology, especially as the most
potent
blood pressure increasing agent in humans. Angiotensin II receptor blockers,
particularly blockers of the type 1 receptor, are used for treating elevated
blood
pressure and congestive heart failure in a mammal. Examples of angiotensin II
receptor blockers (also called angiotensin II antagonists) are described in
EP-A-253310 , EP-A-323841, EP-A-324377, EP-A-420237, EP-A-43983,
EP-A-459136, EP-A-475206, EP-A-502314, EP-A-504888, EP-A-514198,
WO 91/14679, WO 93/20816, US 4,355,040 and US 4,880,804. Specific angiotensin
II receptor blockers are sartans such as candesartan, eprosartan, irbesartan,
losartan, olmesartan, tasosartan, telmisartan or valsartan.
The ongoing Diabetic Retinopathy Candesartan Trials (DIRECT) program has been
established to determine whether AT1-receptor blockade with candesartan can
prevent the incidence and progression of diabetic retinopathy. This program
involves
normotensive or treated hypertensive individuals and will assess the potential
of an
AT1-receptor blocker to protect against the pathological changes in the eye
following
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diabetes. (Sjolie and~Chaturvedi, Journal of Human Hypertension (August 2002)
16
Suppl, pages 42-46).
In the context of the present invention the effect of angiotensin II receptor
blockers on
the development or progression of retinopathy is determined in a cell culture
system
avoiding extensive clinical trials. The system allows to determine, whether or
not a
selected or potential angiotensin II inhibitor is effective in the prevention
of the
development or progression of retinopathy.
Vessels of the microvasculature are composed of only two types of cells,
endothelial
cells and pericytes. Pericytes regulate the growth of co-cultured endothelial
cells and
serve a pivotal role in the maintenance of microvascular. homeostasis. For
instance
they:preserve the ability of co-cultured .endothelial= cells.,to produce
prostacycli.n~and
to~ protect them against.lipid-peroxide-inducedr.injuryr:,Pericyte loss and
dysfunction:
are characteristic histopathological hallmarks::~obse~r~ed:in the early phase
of.;diabetic
retinopathy.
The method according to the present invention allows to screen for angiotensin
11
receptor blockers, and in particular for angiotensin ll receptor blockers
which prevent
the development or progression of diabetic retinopathy or nephropathy. It
comprises
(a) treating pericyte tissue culture cells with or without angiotensin II in
the presence
or absence of a potential angiotensin II receptor blocker compound,
{b) measuring the amount of intracellularly generated reactive oxygen species,
and
(c) identifying the compounds, which inhibit the intracellular generation of
reactive
oxygen species induced by the presence of angiotensin II in the culture
medium.
The cell culture system used is based on pericytes isolated~from mammalian
retina
such as bovine retina. The cells are maintained in commercially available cell
culture
media such as Dulbecco's Eagle's medium usually supplemented with fetal bovine
serum. The term reactive oxygen species comprises molecules like hydrogen
peroxide, ions like the hypochlorite ion, radicals like the hydroxyl radical
which is the
most reactive of them all, and the superoxide anion which is both ion and
radical. An
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important aspect of the method is the finding that the intracellular
generation of
reactive oxygen species in pericytes increases in a dose-dependent manner
after
treating the cultured cells with angiotensin II. Simultaneously DNA synthesis
as
measured by the incorporation of [3H]thymidine in pericytes is decreased,
whereas
mRNAs for vascular permeability factor (VEGF), a specific mitogen to
endothelial
cells involved in the pathogenesis of proliferative diabetic retinopathy, and
platelet -
derived growth factor-B (PDGF-B), a potent mitogen and chemoattractant for
microvascular endothelial cells and glial cells in the retina, are increased.
Angiotensin II is a trigger of high blood pressure known as a major risk
factor for
diabetic retinopathy and nephropathy. Reactive oxygen species damage other
molecules and,.thus, the cell structures of which they are part. Generally
cells use a
variety of defenses.against theharmful effects of reactive oxygen species
including
small molecules:with antioxidative properties such as,.~alplia-tocopherol
(vitamin E),
uric acid, andvitamin C or the two.'enzymes superoXide .elismuf~aae and
catalase.
Adding-additional amounts of antioxidants like N-acetylcysteiri (NAC) during
the
treatment of pericytes with angiotensin II reverses the increase in the
generation of
reactive oxygen species induced by the presence of angiotensin II.
Due to these findings, compounds which are devoid of antioxidative properties
will
prevent the development or progression of diabetic retinopathy or nephropathy,
if
they are capable of inhibiting in pericyte cell cultures the intracellular
generation of
reactive oxygen species induced by the presence of angiotensin II in the cell
culture
medium. Thus, compounds can be screened by treating pericytes 1-48 hours,
preferably 24 hours with or without angiotensin II in the presence or absence
of such
a compound. Following treatment the generation of reactive oxygen species is
measured. Using this screening method the angiotensin II receptor blockers
such as
telmisartan were found to inhibit in pericyte cell cultures the increase in
the
generation of reactive oxygen species induced by angiotensin II, whereas
treatment
with the receptor blocker alone did not affect the generation of reactive
oxygen
species. Thus, activation of angiotensin II receptor signaling in pericytes
contributes
to the pathogenesis of diabetic microvascular disease and antagonizing
angiotensin
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II with compounds such as telmisartan prevent the development or progression
of
diseases such as 'diabetic retinopathy by attenuating pericyte loss and
dysfunction.
As a consequence of these results the present invention teaches a method of
preventing the development or progression of microvascular disease due to
diabetes
such diabetic retinopathy or nephropathy comprising administering to an
individual in
need thereof a pharmaceutically effective amount of an angiotensin Il receptor
blocker. The angiotensin II receptor blockers can be used for the production
of a
pharmaceutical composition to prevent the development or progression of
microvascular disease due to diabetes in an individual in need thereof.
Preferred examples of angiotensin II receptor blockers are candesartan,
eprosartan,
irbesartan, losartan, .olmesartan, telmisartan or valsartan, but
any=receptor:blocker
can be used which iscapable of inhibiting ~in, pericyte cell cultures.ahe,
increase in the
1,5~: ~ generation of reactive oxygen species induced by angiotensiw
~I:~~Individuals
'vconsidered to be in need of such a treatment are affected by one or more
risk factors
of diabetic retinopathy. Examples of such risk factors are diabetes, elevated
blood
glucose level, proteinuria, elevated blood urea nitrogen, elevated blood
creatinine,
microalbuminuria or systemic hypertension.
The amount of receptor blocker used is dependent on the actual active
ingredient
and usually corresponds to the amount used to treat hypertension. The active
compounds can be administered orally, bucally, parenterally, by inhalation
spray,
rectally or topically, the oral administration being preferred. Parenteral
administration
may include subcutaneous, intravenous, intramuscular and intrasternal
injections and
infusion techniques.
The pharmaceutical composition for preventing the development or progression
of
diabetic retinopathy comprising a pharmaceutically effective amount of an
angiotensin II receptor blocker is primarily dependent on the way of
administration.
Dosage ranges include 0.5 to 500 mg/kg p.o., preferably 2 to 80 mg/kg p.o.,
and 3
mg/kg i.v.
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The active compounds can be orally administered in a wide variety of different
dosage forms, i.e. they may be formulated with various pharmaceutically
acceptable
inert carriers in the form of tablets, capsules, lozenges, troches, hard
candies,
powders, sprays, aqueous suspensions, elixirs, syrups, and the like. Such
carriers
include. solid diluents or fillers, sterile aqueous media and various non-
toxic organic
solvents, etc. Moreover, such oral pharmaceutical formulations can be suitably
sweetened and/or flavored by means of various agents of the type commonly
employed for such purposes. In general, the compounds of this invention are
present
in such oral dosage forms at concentration levels ranging from about 0.5% to
about
90% by weight of the total composition, in amounts which are sufficient to
provide the
desired unit dosages. Other suitable dosage forms for the compounds of this. -
.:
.invention include controlled release formulations and devices well known
to.~those
who practice in the art.
1':5:..
.For:purposes of oral administration, tablets containing various excipientS
such as
sodium citrate, calcium carbonate and calcium phosphate may be employed along
with various disintegrants such as starch and preferably potato or tapioca
starch,
alginic acid and certain complex silicate, together with binding agents such
as
polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating
agents
such as magnesium stearate, sodium lauryl sulfate and talc or compositions of
a
similar type may also be employed as fillers in soft and hard-filled gelatin
capsules;
. included lactose or milk sugar as well as high molecular weight polyethylene
glycols.
When aqueous suspensions and/or elixirs are desired for oral administration,
the
essential active ingredient therein may be combined with various sweetening or
flavoring agents, coloring matter or dyes and, if so desired, emulsifying
agents and/or
water, ethanol, propylene glycol, glycerin and various Like combinations
thereof.
For purposes of parenteral administration, solutions of the compounds in
sesame or
~ peanut oil or in aqueous propylene glycol may be employed, as well as
sterile
aqueous solutions of the corresponding pharmaceutically acceptable salts. Such
aqueous solutions should be suitably buffered if necessary, and the liquid
diluent
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rendered isotonic with sufficient saline or glucose. These particular aqueous
solutions are especially suitable for intravenous, intramuscular and sub-
cutaneous
injection purposes. In this connection, the sterile aqueous media employed are
readily obtained by standard techniques well known to those skilled in the
art. For
instance, distilled water is ordinarily used as the liquid diluent and the
final
preparation is passed through a suitable bacterial filter such as a sintered
glass filter
or a diatomaceousearth or unglazed porcelain filter. Preferred filters of this
type
include the Berkefeld, the Chamberland and the Asbestos Disk-Metal Seitz
filter,
wherein the fluid is sucked into a sterile container with the aid of a suction
pump. The
necessary steps should be taken throughout the preparation of these injectable
solutions to insure that the final products are obtained in a sterile
condition. For
purposes.~of transdermal administration, the dosage form of the particular
compound
or compounds~may include, by way of example, solutions, lotions, ointments,
creams,.
gels, suppositories, rate-limiting sustained release formulations and devices
therefor..;
Such dosage forms comprise the particular compound or compounds and may
include ethanol, water, penetration enhancer.~nd~inert carriers such as gel-
producingw
materials, mineral oil, emulsifying agents, benzyl alcohol and the like.
Angiotensin II receptor blockers may be administered in a daily dosage of 10
mg (or
0.143 mg/kg, based on a person of 70 kg) to 500 mg (7.143 mg/kg) orally and of
about 20 mg (0.286 mg/kg) parenterally, preferably of 20 mg (0.286 mg/kg) to
100
mg (1.429 mg/kg) orally. Particularly preferred is an oral daily dosage of 40
mg
(0.571 mg/kg) to 80 mg (1.143 mg/kg) or specifically of about 80 mg (1.143
mg/kg).
Several angiotensin II receptor blockers are already on the market and can be
used
for administration, e.g. Approvel~, Atacand~, Blopress~, Cozaar~, Diovan~,
Karvea~, Lortaan~, Lorzaar~, Losaprex~, Micardis~, Neo-Lotan~ or Oscaar~, and
Tevete n~.
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Examples
All values were presented as means ~ standard errors (SE). Statistical
significance
was evaluated using Student's ttest for paired comparison; P<0.05 was
considered
significant.
Example 1 - Measurement of reactive oxygen species in pericytes
Pericytes were isolated from bovine retina and maintained in Dulbecco's
Eagle's
medium supplemented with 20% of fetal bovine serum (FBS) as described in
Yamagishi et al, Circulation 87:1969, 1993.
Angiotensin II treatment was carried out in medium containing 2% FBS.
Pericytes
were treated with or without 10-' or 10-6 M angiotensin II in the presence or
absence
of 10-' M telmisartan for 24 hours. Then the intracellular formation of
reactive oxygen
species was detected~as described in Yamagishi ~et al, A J Biol Chem
276:25096,
2001 by using the~fluorescent probe CM-H2DCFDA (Molecular Probes Inc, Eugene,
OR).
Angiotensin II increased intracellular generation of reactive oxygen species
in a
dose-dependent manner. 10-6 M angiotensin II resulted in an increase of about
1.3
fold. Telmisartan was found to completely inhibit the angiotensin II-induced
increase
in the generation of reactive oxygen species in pericytes, while telmisartan
alone did
not affect the generation.
Example 2 - Measurement of [3H]thymidine incorporation in pericytes
Pericytes were treated with or without 10-' M angiotensin in the presence or
absence
of 1 mM N-acetylcystein (NAC) for 24 hours, and then [3H]thymidine
incorporation in
cells was determined as described in Yamagishi et al, FEBS Lett 384:103, 1996.
Angiotensin II significantly inhibited DNA synthesis in pericytes. NAC
significantly
prevented the angiotensin II-induced decrease in DNA synthesis in pericytes.
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Example 3 - Quantitative reverse transcription PCR of VEGF m-RNA
Sequences and primers for detecting VEGF and ~i-actin mRNAs are described in
Yamagishi et al, J Biol Chem 272:8723, 1997.
Poly(A)+ RNAs were isolated from cells treated with or without 10-' M
angiotensin II in
the presence or absence of 10-' M telmisartan or 1 mM NAC for 4 hours, and
analyzed by quantitative reverse transcription PCR (RT-PCR) as described in
Yamagishi et al, Diabetologia 41:1435, 1998. The amounts of poly(A)+ RNA
templates (about 30ng) and cell cycle numbers for amplification (28 cycles for
VEGF
gene and 22 cycles for ~i-actin gene) were chosen in quantitative ranges were
reactions proceeded linearly, which had been determined by plotting signal
intensities as functions of the template amounts and cell cycle numbers as
described
in Yamagishi et al, J Biol Chem 277:20309, 2002.
It has been reported that there exist five alternatively spliced products from
the single
VEGF gene. They are designated as VEGF,2,, VEGF,45, VEGFISS VEGF189; and
VEGF2os. Since Northern blot analysis can not clearly discrii-ninate these
five mRNA
products, we employed a more sensitive semi-quantitative RT-PCR technique as
described in Okamoto et al, FASEB J 16:1928, 2002. In these experiments, cDNA
products of 486 and 618 base pairs length are amplified from mRNAs for VEGFI2y
and VEGFisS, respectively. Angiotensin II significantly up-regulated these
secretory
forms of VEGF mRNA levels in pericytes. The VEGF mRNA level was about 1.5 fold
higher than the basal level when exposed to 10-' M angiotensin II. Telmisartan
and
NAC were found to completely inhibit the angiotensin II-induced up-regulation
of
VEGF mRNA levels in pericytes.
Example 4 - Molecular cloning of bovine PDGF-B partial cDNAs
Partial cDNAs for bovine PDGF-B were cloned using primer sequences designed
from the conserved amino acid sequences GELESL and NNRNVQ in human and
sheep PDGF-B. The upstream and downstream primers were
5'-GGCGAGCTGGAGAGCTT-3' and 5'-CTGCACGTTGCGGTTGT-3', respectively.
A 213-base pair RT-PCR product was amplified from 30 ng of bovine retinal
pericyte
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poly(A)+ RNA and cloned using the pGEM-T Easy Vector System according to the
manufacturer's instructions (Promega, Madison, WI, USA). Cloned PCR products
were sequenced by the chain termination method according to the manufacturer's
instructions (DNA Sequencing Kit, Applied Biosysterns, Foster, CA, USA). The
cloned bovine cDNA fragments showed strong sequence similarity with human and
sheep PDGF-B. Nucleotide identities were 91 % and 94°l°, amino
acid identities 91
and 96% with human and sheep PDGF-B, respectively.
Example 5 - Quantitative reverse transcription PCR of PDGF-B m-RNA
To investigate the effects of angiotensin 11 on PDGF-B gene expression in
cultured
retinal pericytes, poly(A)+ RNAs were isolated from cells treated with or
without
10-' M angiotensin II in the presence or absence of 10-' M telmisartan or 1 mM
NAC
for 4 hours, and analyzed by RT-PCR as described iri Yamagishi et al, Kidney
Int
:15 ~, 63:464, 20p3. The amounts of poly(A)+ RNA templates (about 30ng) and
cell cycle
numbers for amplification (28 cycles for PDGF-B gene and 22 cycles for ~i-
actin
gene) were chosen in quantitative ranges were reactions proceeded linearly,
which
had been determined by plotting signal intensities as functions of the
template
amounts and cell cycle numbers as described in Yamagishi et al, J Biol Chem
277:20309, 2002. Sequences of primers for detecting bovine ~i-actin.mRNAs were
the same as described in Okamoto et al, FASEB J 16:1928, 2002.
PDGF-B has been implicated in vascular proliferative retinopathies, and
hemizygous
rhodopsin promoter/PDGF-B transgenic mice were shown to exhibit proliferation
of
vascular cells, gliaf cells and retinal pigment epithelial cells resulting in
retinal
detachment. In the present experiment angiotensin II was found to
significantly up-
regulate PDGF-B mRNA levels in pericytes. When exposed to 10-' M angiotensin
II
the PDGF-B mRNA level was about 5-fold higher than the basal level.
Telmisartan or
NAC were found to significantly inhibit the angiotensin II induced up-
regulation of
PDGF mRNA levels. From this it is concluded that angiotensin II-type 1
receptor
interaction is involved in the pathogenesis of retinal detachment in
proliferative
diabetic retinopathy through overexpression of PDGF-B, and that antagonizing
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angiotensin II action by angiotensin II receptor blockers delays or even
prevents the
progression of diabetic retinopathy by. attenuating PDGF-B expression in vivo.
