Organ glycosaminoglycan distribution after intravenous and oral administration in rats


Marchi, E.; Barbanti, M.; Milani, R.; Breccia, A.; Fini, A.; Gattavecchia, E.

Seminars in Thrombosis and Hemostasis 20(3): 297-300

1994


GAGs were purified from urine of dogs after intranasal administration of 40 mg/kg ITF 1300. The electrophoretic patterns of urine GAGs in acidic buffer showed the presence of heparin together with chondroitins, heparan sulfate, and hyaluronic acid. The heparin present in urines was purified using chondroitinase ABC, and its purity was tested by electrophoresis in acidic buffer. The sample obtained was characterized by 13C-NMR, showing the same characteristic signals of the heparin starting material.

SEMINARS
IN
THROMBOSIS
AND
HEMOSTASIS—VOLUME
20,
NO.
3,
1994
Organ
Glycosaminoglycan
Distribution
After
Intravenous
and
Oral
Administration
in
Rats
EGIDIO
MARCHI,
Ph.D.,
MARIA
BARBANTI,
Ph.D.,
RITA
MILANI,
Ph.D.,
ALBERTO
BRECCIA,
Ph.D.,
ADAMO
FINI,
Ph.D.,
and
ENRICO
GATTAVECCHIA,
Ph.D.
Glycosaminoglycans
(GAGs)
are
a
class
of
natural
products
extracted
from
animal
organs
(intestine,
lungs,
liver,
etc)
that
have
many
biologic
activities.
1
'
2
Molecu-
lar
mass,
charge
density,
and
chemical
structure
strongly
influence
not
only
the
biologic
activity,
but
3-6
probably
also
the
pharmacokinetic
behavior
of
GAGs.
7
Several
authors
have
measured
the
anticoagulant
pharmacodynamic
effects
of
heparin
activated
partial
thromboplastin
time
(APTT),
anti-Factor
Xa
(AXa)
to
explain
its
antithrombotic
effects,
but
the
results
were
not
satisfactory.
8
In
fact,
the
anticoagulant
effects,
after
in-
travenous
or
subcutaneous
heparin
administration,
rap-
idly
(in
a
dose
dependent
way)
disappear
while
the
an-
tithrombotic
activity
is
still
detectable.
Therefore,
what
significance
can
be
attributed
to
heparin
pharmacokinet-
ics
if
the
compound
rapidly
disappears
from
the
blood,
and
the
remaining
low
levels
produce
no
anticoagulant
effect,
when
the
antithrombotic
effect
is
still
present?
Some
authors
have
demonstrated
that
the
disappear-
ance
of
heparin
from
plasma
depended
on
a
saturable
mechanism
after
a
low-dose
administration,
and
an
un-
saturable
mechanism
predominated
when
heparin
was
administered
at
high
doses.
7
'
9
Other
authors
have
demon-
strated
a
selective
binding
of
heparin
to
human
endothe-
lial
cells
in
vitro
and
in
vivo.
10,11
Therefore,
the
fast
clearance
of
heparin
from
the
blood
may
be
related
to
a
widespread
cellular
system,
corresponding
to
a
saturable
mechanism.
If
this
hypothesis
is
true
for
intravenous
and
subcu-
taneous
administration,
why
could
it
not
be
possible
From
the
Alfa
Wassermann
S.p.A.,
Bologna
and
Facolta
di
Far-
macia,
Univerita
degli
Studi
di
Bologna,
Bologna,
Italy.
Reprint
requests:
Dr.
Marchi,
Head
of
R&D
Department,
Alfa
Wassermann
S.p.A.,
Via
Ragazzi
del
'99,
5,
40133
Bologna,
Italy.
when
one
administers
heparin
or
other
GAGs
orally?
Jacques
et
al.
12
demonstrated
a
high
concentration
of
heparin
on
the
endothelium
and
a
low
concentration
in
plasma
in
rats.
Many
authors
have
concluded
that
GAGs
were
not
absorbed
because
it
was
impossible
to
detect
any
AXa
or
APTT
activity
in
humans
after
oral
adminis-
tration.
13
Because
of
the
slow
passage
of
GAGs
through
the
intestinal
wall
after
oral
administration,
the
cellular
system
probably
never
becomes
saturated;
therefore,
one
could
have
a
preferential
binding
of
the
nondesulfated
GAGs
to
the
cells
(endothelial,
reticuloendothelial,
he-
matic),
and,
consequently,
the
low
plasma
concentra-
tions
do
not
induce
any
anticoagulant
activity
(APTT,
AXa,
thrombin
time
[TT]).
To
verify
the
GAG—cell
interaction
and
the
hypoth-
esis
that
the
GAG
affinity
for
the
endothelial
cells
was
due
to
the
degree
of
sulfation
of
GAG,
we
carried
out
some
experiments
in
rats,
utilizing
a
radiolabeled
heparin
fraction
and
a
radiolabeled
desulfated
heparin
derivative
(Table
1).
MATERIALS
AND
METHODS
Materials
Fast-moving
heparin
fraction
was
extracted
from
Sulodexide.
14
N-desulfated-N-acethylated
heparin
(NDNA)
(Suleparoide
14
)
was
extracted
from
Hemovasal
(Manetti,
Roberts)
(Table
1).
Fast-moving
heparin
was
desulfated,
using
the
method
of
Levy
and
Petracek,
15
to
obtain
about
2%
of
free
NH
2
on
the
glucosamine
monomer.
NDNA
hep-
arin
derivative,
containing
about
3%
of
free
NH
2
,
was
used.
Both
compounds
were
treated
with
SHPP
reagent
and
the
derivatives
iodinated
with
125
1
in
accordance
with
Copyright
©
1994
by
Thieme
Medical
Publishers,
Inc.,
381
Park
Avenue
South,
New
York,
NY
10016.
All
rights
reserved.
297
298
SEMINARS
IN
THROMBOSIS
AND
HEMOSTASIS—VOLUME
20,
NO.
3,
1994
TABLE
1.
Physiochemical
and
Biochemical
Characteristics
of
Glycosaminoglycans
APTT
AXa
lUlmg
lUlmg
S0
3
/COOH
MW
Fast
moving
fraction
(from
<50
---
--
-90
1.9
0.1
7500
sulodexide)
N-Desulfated-N-acetylated
<10
<10
1.3
±
0.1
8300
heparin
(commercial
heparin)
Prevention
Model
Thrombolytic
Model
(therapeutic)
-
i.v.
injection
(compound)
vena
cava
ligature
10'
-
vena
cava
ligature
6h
2h
i.v.
injection
(compound)
-
thrombus
collection
2h
thrombus
collection
the
procedure
of
Dawes
and
Pepper.
16
The
specific
radio-
activity
125
1
was
82.3
µCi/mg
for
fast-moving
heparin
and
103.2
R.Ci/mg
for
NDNA.
24
h
370C
(drying)
24
h
370
C
(drying)
dried
weight
of
the
thrombus
Methods
The
compounds
were
administered
to
rats
by
intra-
venous
injection
(vena
cava)
or
by
intraduodenal
admin-
istration,
because
the
iodinated
compounds
were
not
sta-
ble
in
the
acidic
medium
of
the
stomach
(Table
2).
At
15,
30,
and
120
minutes,
blood,
aorta,
vena
cava,
liver,
kidney,
and
lung
were
removed
and
the
radioactivity
determined
with
a
gamma
multichannel
spectrophotome-
ter
utilizing
sodium
iodide.
The
in
vivo
preventive
an-
tithrombotic
activity
of
both
compounds
was
determined
after
intravenous
and
oral
administration
by
the
Reyers
mode1,
17
and
the
thrombolytic
activity
was
determined
only
after
intravenous
injection
in
accordance
with
a
modified
Reyers
model'
s
(Fig.
1).
In
the
preventive
model
the
compounds
were
admin-
istered
10
minutes
before
the
vena
cava
was
ligated,
whereas
in
the
thrombolytic
model
the
compounds
were
administered
6
hours
after
vena
cava
ligation.
-
dried
weight
of
the
thrombus
FIG.
1.
Experimental
model—Reyers
model.
showed
that
NDNA,
compared
with
fast-moving
hep-
arin,
lost
about
50%
of
the
selective
binding
to
endothe-
lium
and
organs.
If
our
hypothesis,
that
the
GAG—endo-
thelium
interaction
is
important
for
the
antithrombotic
activity
is
correct,
we
would
expect
the
NDNA
to
exert
a
lower
antithrombotic
activity
than
FMH.
We
utilized
Reyers
model
as
a
preventive
and
therapeutic
model.
The
results
are
summarized
in
Table
6.
Fast-moving
heparin
had
a
higher
antithrombotic
activity,
both
in
preventive
and
therapeutic
models.
Be-
cause
clot
formation
is
important
in
the
preventive
model,
it
was
foreseeable
that
NDNA
would
have
a
lower
TABLE
3.
Blood
and
Plasma
Radioactivity
after
Intravenous
Administration
(60
µCi/Rat)
(Bq/mL)*
Blood
Plasma
Time
(min)
RESULTS
AND
DISCUSSION
FMHt
NDNAt
FMH
NDNA
After
intravenous
administration
the
NDNA
com-
pound
(Table
3)
reached
a
blood
and
plasma
level
(Bq/
mL)
about
five
times
higher
than
those
of
FMH.
This
result
could
be
explained
by
the
enhanced
pharmacoki-
netic
behavior
of
NDNA,
or
by
its
reduced
capacity
to
bind
to
endothelial
cells,
because
of
its
lower
sulfation
degree.
Measurement
of
the
radioactivity
on
the
vena
cava,
aorta
(Table
4)
and
organs
(Table
5)
(Bq/mg)
15
5.848
29.970
14.872
51.800
30
4.358
18.870
7.486
28.860
120
889
7.030
1.494
12.210
*
Blood
drawn
at
15,
30,
and
120
minutes.
t
FMH:
fast-moving
heparin;
NDNA:
N-desulfated-N-acetylated
hep-
arin.
TABLE
4.
Vessel
Radioactivity
After
Intravenous
Administration
(60
µCi/Rat)
(Bq/mL)*
Vena
Cava
Aorta
TABLE
2.
Intravenous
and
Oral
Administration
of
1251
Glycosaminoglycan
in
Rat
(-300
g)
Time
(min)
FMH
NDNA
FMH
NDNA
µCilkg
Dose
mg/kg
Fast-moving
heparin
180
12.5
N-Desulfated-N-acetylated
heparin
180
12.5
15
80.1
42.5
60.5
32.9
30
44.8
24.0
42.4
34.0
120
48.9
16.6
29.1
22.6
*
See
footnotes
in
Table
3.
Kidney
Liver
Lung
Vena
Cava
Aorta
Time
(min)
FMH
NDNA
FMH
NDNA
FMH
NDNA
Time
(min.)
FMH
NDNA
FMH
NDNA
ORGAN
GLYCOSAMINOGLYCAN
DISTRIBUTION-MARCH!
ET
AL
299
TABLE
5.
Tissue
Radioactivity
after
Intravenous
Administration
(60
µCi/Rat)
(Bq/mL)*
TABLE
8.
Vessel
Radioactivity
After
Oral
Administration
(60
µCi/Rat)
(Bq/mL)
15
117.5
44.4
28.1
17.0
23.0
12.2
30
99.7
42.2
28.6
13.7
15.9
8.9
120
96.8
52.5
34.6
24.0
15.3
5.92
*
See
footnotes
in
Table
3.
potency
because
of
its
low
anticoagulant
activity
due
to
the
N-desulfation.
To
the
contrary,
in
the
therapeutic
model,
in
which
the
thrombus
was
aged
for
6
hours
before
administration
of
the
compounds,
the
fibrinolytic
activity
18
was
more
important
than
the
anticoagulant
ac-
tivity.
In
this
model,
we
can
hypothesize
the
important
role
of
GAG-endothelial
cell
interaction
and
therefore
explain
the
lower
antithrombotic
and
thrombolytic
po-
tency
of
NDNA
that
showed
a
reduced
endothelial
affin-
ity.
Tables
7,
8,
9
summarize
the
results
obtained
after
oral
administration.
The
compound
was
administered
in
oil-in-water
emulsion
by
intraduodenal
injection.
In
this
experiment
NDNA
reached
blood
and
plasma
radioactiv-
ity
levels
that
were
about
five
times
higher
than
those
of
fast-moving
heparin.
If
we
consider
the
radioactivity
on
the
vessel,
the
results
showed
that
fast-moving
heparin
had
a
10-fold
greater
capacity
than
NDNA
to
bind
to
endothelial
cells.
Considering
that
the
total
endothelial
surface
in
humans
is
about
12
million
square
centimeters,
TABLE
6.
Antithrombotic
Activity
Prevention
Model
(ED
50
mglkg
iv)
Therapeutic
Model
Tt
Lt
(ED
50
mglkg
iv)
FMH
0.5
0.27
0.99
Heparin
0.4
0.2
1.4
NDNA
>>8
5
8
*
See
footnotes
in
Table
3.
t
T:
50%
of
rats
without
thrombi;
L:
50%
of
reduction
of
thrombus
weight
vs
control;
IV:
intravenous.
TABLE
7.
Blood
and
Plasma
Radioactivity
After
Oral
Administration
(60
µCi/Rat)
(Bq/mL)*
15
20.6
0.7
15.2
1.7
30
22.5
1.7
11.2
1.0
120
16.2
2.4
8.6
3.3
*
See
footnotes
in
Table
3.
in
comparison
with
only
6000
to
7000
mL
of
blood,
it
is
evident
that
GAGs
with
a
low
affinity
for
the
endothelial
cells
can
be
found
at
a
high
concentration
in
blood
or
plasma.
After
intravenous
administration
of
fast-moving
he-
parin
(a
more
sulfated
compound),
the
radioactivity
bound
to
the
vessel
was
only
four
times
higher
than
that
reached
after
oral
administration
of
the
same
amount
of
cold
and
radiolabeled
compound.
This
result
confirms
our
hypothesis
that,
after
oral
administration,
the
slower
step
is
the
intestinal
absorption,
whereas
the
endothelial
distribution
(saturation
mechanism)
is
the
faster
step;
therefore
it
is
possible
to
have
low
blood
and
high
endo-
thelial
concentration
of
GAGs.
We
obtained
similar
re-
sults
in
two
previous
studies
in
which we
demonstrated
that
fluorescinated
sulodexide,
a
natural
mixture
of
80%
fast-moving
heparin
and
20%
dermatan
sulfate
(only
the
fast-moving
heparin
fraction
was
fluorescinated),
was
distributed
in
plasma
and
in
vessels
19
and
on
the
throm-
bus
18
after
intravenous
and
oral
administration.
How-
ever,
the
fluorescence
in
vessel
and
thrombus
was
also
detectable
2
hours
after
drug
administration
when
the
plasma
fluorescence
and
the
anticoagulant
activity
were
no
longer
detectable.
CONCLUSIONS
Our
experimental
data
confirm
that
the
pharmaco-
distribution
of
GAGs
is
a
balance
between
cells
(endothe-
lial,
reticuloendothelial,
hematic)
and
blood
proteins.
This
balance
is
a
dynamic
mechanism
affected
by
GAG
metabolism
(desulfation)
and
elimination.
The
GAG
ca-
Blood
Plasma
TABLE
9.
Antithrombotic
Activity
After
Oral
Administration
(Prevention
Model)
Time
(min)
FMH
NDNA
FMH
NDNA
15
169
851
211
1.628
30
196
1.073
230
1.739
120
386
1.221
467
2.072
*
See
footnotes
in
Table
3.
*
Fifty
percent
reduction
of
thrombus
weight
vs
control.
ED50*
Compound
(mglkg)
Fast-moving
heparin
17
N-Desulfated-N-acetylated
heparin
>100
300
SEMINARS
IN
THROMBOSIS
AND
HEMOSTASIS—VOLUME
20,
NO.
3,
1994
pacity
to
bind
cells
is
strictly
related
to
its
structure,
molecular
mass,
and
degree
of
sulfation.
A
less
sulfated
GAG
provokes
high
blood
levels
because
of
its
lower
cell
affinity.
Because
the
GAG
absorption
velocity
is
slow
after
oral
administration,
the
GAG
distribution
occurs
on
the
cells
and
therefore
the
blood
levels
of
the
compound
are
too
low
to
produce
a
measurable
anticoagulant
effect
(APTT,
AXa);
the
effect
on
coagulation
becomes
mea-
surable
when
the
oral
dose
is
increased.
Oral
GAG
ab-
sorption
could
be
improved
by
increasing
the
absorption
velocity
with
a
suitable
technological
formulation.
To
explore
the
global
antithrombotic
effect
of
GAGs,
we
must
consider
not
only
the
anticoagulant
ef-
fect,
but
also
the
GAG—cell
interaction
that
is
beyond
the
pharmacokinetic
parameter
measurements.
The
anticoag-
ulant
pharmacodynamic
effect
is
probably
not
strictly
related
to
pharmacokinetic
parameters
because
the
GAG—cell
interaction
can
increase
the
endogenous
GAGs
synthesis
and
release,
and
endogenous
GAGs
could
affect
the
global
anticoagulant
pharmacodynamic
activity.
Considering
the
importance
of
the
GAG—cell
inter-
action,
as
demonstrated
from
many
authors,
on:
(1)
mod-
ulation
of
the
fibrinolytic
system;
(2)
cell
proliferation;
(3)
inhibition
of
the
synthesis
and
release
of
procoagulant
factors;
(4)
synthesis
and
release
of
endogenous
GAGs;
(5)
endothelium
permeability;
and
(6)
inactivation
of
the
thrombin
bound
to
the
cell,
we
must
conclude
that
it
would
be
more
suitable
to
consider
GAG
distribution
than
GAG
blood
pharmacokinetics.
It
would
be
interest-
ing
to
find
an
unequivocal
correlation
between
blood
levels
and
endothelial
GAG
concentration
after
intrave-
nous
or
oral
administration.
Acknowledgment.
We
thank
Dr.
Ferri
(Pharmaceutical
Department,
University
of
Bologna)
for
the
radioactivity
mea-
surements.
REFERENCES
1.
Hardingham
TE:
Structure
and
biosynthesis
of
proteoglycans.
Rheumatology
10:143-183,
1986.
2.
Hascall
VC:
Functions
of
the
Proteoglycans.
John
Wiley
&
Sons,
Chichester,
U.K.,
1986.
3.
Hubbard
AR,
CA
Jennings,
TW
Barrowcliffe:
Anticoagulant
properties
in
vitro
of
heparan
sulfates.
Thromb
Res
35:567-576,
1984.
4.
Lindhardt
RJ,
A
Hakim,
JA
Liu,
D
Hoppensteadt,
G
Mascellani,
P
Bianchini,
J
Fareed:
Structural
features
of
dermatan
sulfate
and
their
relationship
to
anticoagulant
and
antithrombotic
activities.
Biochem
Pharmacol
42:1609-1619,
1991.
5.
Andersson
LO,
TW
Barrowcliffe,
E
Holmer,
EA
Johnson,
G
Sod-
erstrom:
Molecular
weight
dependency
of
the
heparin
potentiated
inhibition
of
thrombin
and
activated
factor
X.
Effect
of
heparin
neutralization
in
plasma.
Thromb
Res
15:531-541,
1979.
6.
Ofosu
FA,
MR
Buchanan,
N
Anvari,
LM
Smith,
MA
Bleychman:
Plasma
anticoagulant
mechanism
of
heparin,
heparan
sulfate
and
dermatan
sulfate.
Ann
NY
Acad
Sci
556:123-131,
1989.
7.
Caranobe
C,
A
Bonet,
AM
Gabaig,
D
Dupony,
P
Sie,
B
Boneu:
Disappearance
of
circulating
anti-Xa
activity
after
intravenous
in-
jection
of
standard
heparin
and
of
a
low
molecular
weight
heparin
(CY
216)
in
normal
and
nephrectomized
rabbits.
Thromb
Res
40:129-133,
1985.
8.
Barrowcliffe
TW:
LMW
Heparin:
Relationship
between
an-
tithrombotic
and
anticoagulant
effects.
In:
Barrowcliffe
TW
(Ed):
Heparin
and
Related
Polysaccharides.
Plenum
Press,
New
York,
1991,
pp
205-220.
9.
Boneu
B,
C
Caranobe,
AM
Gabaig,
D
Dupony,
P
Sie:
Evidence
for
a
saturable
mechanism
of
disappearance
of
standard
heparin
in
rabbits.
Thromb
Res
46:835-844,
1987.
10.
Von
Rijn
JLML,
M
Trillou,
J
Mardiguian,
G
Tobelen,
J
Caen:
Selective
binding
of
heparins
to
human
endothelial
cells.
Implica-
tions
for
pharmacokinetics.
Thromb
Res
45:211-222,
1987.
11.
Hiebert
LM,
LB
Jacques:
Heparin
uptake
on
endothelium.
Artery
2:26-37,
1976.
12.
Jacques
LB,
LM
Hiebert,
SM
Wice:
Prevention
of
thrombosis
by
oral
heparin
and
oral
dextran
sulphate.
Fibrinolysis
4
(Suppl
1):
89,
1985.
13.
Fisher
A,
T
Astrup:
On
administration
of
heparin
per
os.
Proc
Soc
Exp
Biol
Med
42:81-82,
1939.
14.
Callas
D,
D
Hoppensteadt,
W
Jeske,
0
Igbal,
J
Fareed:
Compara-
tive
pharmacologic
profile
of
a
glycosaminoglycan
mixture,
Sulo-
dexide
and
a
chemically
modified
heparin
derivative,
Suleparoide.
Semin
Thromb
Hemost
19:49-57,
1993.
15.
Levy
L,
FJ
Petracek:
Chemical
and
pharmacological
studies
of
N-desulfated
heparin.
Proc
Soc
Exp
Biol
Med
109:901-905,
1962.
16.
Dawes
J,
DS
Pepper:
Catabolism
of
low
dose
heparin
in
man.
Thromb
Res
14:845-860,
1979.
17.
Reyers
I,
L
Mussoni,
HB
Donati,
G
De
Gaetano:
Failure
of
aspirin
at
different
doses
to
modify
experimental
thrombosis
in
rats.
Thromb
Res
18:669-674,
1980.
18.
Barbanti
M,
S
Guizzardi,
F
Calanni,
E
Marchi,
M
Babbini:
An-
tithrombotic
and
thrombolytic
activity
of
Sulodexide
in
rats.
Int
J
Clin
Lab
Res
22:179-193,
1992.
19.
Cristofori
M,
R
Mastacchi,
M
Barbanti,
M
Sarret:
Pharmacoki-
netics
and
distribution
of
a
fluorescinated
glycosaminogly-
can,
Sulodexide,
in
rats.
Arzneimittelforschung
35:1513-1516,
1985.