Note: Descriptions are shown in the official language in which they were submitted.
:~25~
-- 2
BACKGROUND OF THE INVENTION
The present invention relates to a lipoprotein
adsorbent for use in extracorporeal circulation treatment
in order to remove harm-ful lipoproteins in blood,
especially low density lipoproteins (hereinafter referred
to as "LDL") and very low density lipoproteins
(hereinafter referred to as "VLDL"), from blood, plasma
or serum by selectively adsorbing the lipoproteins and a
process for preparing thereof.
It has been known that LDL and VLDL among
lipoproteins present in blood, contain a large amount
of cholesterol and cause arteriosclerosis. In hyper-
cholesterolemia such as familial hyperlipemia LDL shows
several times higher values than those observed in normal
conditions and causes coronary arteriosclerosis.
Although regimen or medication with probucol or
cholestyramine have been employed for treatment of
hypercholesterolemia such as familial hyperlipemia, they
show only limited effect and have a fear of unfavorable
side effects. Hitherto, familial hyperlipemia, in
particular, can be effectively treated only by the so
called plasma exchange therapy, where the plasma in the
body of the patient is separated and exchanged with
normal plasma or replacing fluid containing albumin. As
is well known, however, the plasma exchange therapy has
various defects, i.e.
(1) it needs tG employ expensive fresh plasma
or plasma fractions,
(2) it removes not only harmful components but
also effective ones, and
(3) it has a danger to lead to infection by
hepatitis viruses and the like.
A method for removing harmful components in
blood by using a membrance has been adopted in order to
overcome the above-mentioned defects. However, the
method still has defects. For example, it does not have
a sufficient selectivity and needs to supplement a part
of proteins in plasma which are removed concurrently with
~'~
125~
~ 3
the removal of harmful components.
Also, for the same purpose, a method using an
immune adsorbent, in which an antibody is immobilized,
has been employed. Though selectivity in the method is
almost satisfactory, there exist many problems such as
difficulty for obtaining the antibody, a high price of
the antibody, difficulty for sterilizing adsorbent and
poor stability of the adsorbent when preserving.
Furthermore, there has been adopted an adsorbent
based on the principle of the affinity chromatography,
wherein a compound having an affinity for har~ful
components tsuch compound is hereinafter referred to as
"ligand") is immobilized. The adsorbent has a good
selectivity and the ligand employed is not too expensive.
However, it is required to lower the cost in order to
use in extracorporeal circulation treatmer.t in large
quantities. ~ince the adsorbent based on the principle
of the affinity chromatography has a carrier ~ade of a
soft gel such as agarose, it provides a poor flow rate of
body fluids and frequently produces cloggings.
At a small cost, there has been known a
lipoprotein adsorbent (by Maaskant, N. et al.) which is
obtained by cross-linking polyvinyl sulfate (ester of
polyvinyl alcohol and sulfuric acid) in an aqueous
solution by applying ~ ray, which makes the resultant
insoluble in water. However, in the method, where a
porous gel which is made water-insoluble by cross-
linking a water-soluble polymer previously converted to
sulfate is obtained, the amount of the sulfuric acid
residue decreases to a great extent as the cross-linking
reaction proceeds and a solvent which can be employed in
the cross-linking reaction is substantially restricted to
water due to hydrophile property of the sulfuric acid
residue in the polymer converted to sulfate, which results
in a great restriction on the method of cross-linking
which can be employed. Furthermore, there is a problem
such as great difficulty in beads formation.
The adsorbent used in hemoperfusion or plasma
'716
-- 4
perfusion therapy employing extracorporeal circulation
(so-called plasmapheresis) is required to have enough
mechanical strength (pressure durability~ so that it can
provide a large flow rate. The gel prepared as mentioned
above, however, contains a polymer having an essentially
high degree of hydrophile property and thus it cannot be
a hard gel even though a water-insoluble gel is formed by
the cross-linking and the like, which results in the gel
being improper for use in extracorporeal circulation to
cause consolidation.
The object of the present invention is to
provide a sa~e and low-cost adsorbent for use in
extracorporeal circulation treatment, which can
selectively remove LDL and VLDL, by preparing a
water-insoluble porous hard gel, followed by direct
sulfation.
SUMMARY OF THE INVENTION
In accordance with the present invention, there
can be provided a lipoprotein adsorbent for use in
extracorporeal circulation treatment which is made of a
water-insoluble porous hard gel which has an exclusion
limit value from 106 to 109 measured by using globular
proteins and comprises a polymer having hydroxy group in
at least a part of the molecule, at least a part of
hydroxy groups on the surface of said gel being sulfated.
In accordance with the present invention, there can also
be provided a process for preparing a lipoprotein
adsorbent for use in extracorporeal circulation
treatment, which is hard and has a great adsorbing
capacity as well as an excellent selectivity, by forming
a water-insoluble porous hard gel which has an exclusion
limit value from l0~ to 109 measured by using globular
proteins and comprises a polymer having hydroxy group in
at least a part of the molecule followed by direct
conversion of hydroxy groups on the surface of said gel
to sulfates.
lZ~Z~
-- 5
sRIEF DESCRIPTION OF THE DRAWING
Fig. 1 is a graph showing a relation between a
flow rate and a pressure drop obtained in Reference
Example~
DETAILED DESC~IPTION OF THE INVENTION
. . _
In the present invention, a water-insoluble
porous hard gel of a polymer haivng hydroxy group in at
least a part of the molecule is employed.
The above-mentioned water-insoluble porous hard
gel of a polvmer having hydroxy group in at least a part
of the molecule may be a crystalline polymer, or it may
be any polymer which is inherently water-insoluble by
nature or made water-insoluble by cross-linking.
Non-limitative examples of the polymer having
hydroxy group in at least a part of the molecule are,
for instance, a polymer having an unit of the formula:
-~CH2-CH~- in at least a part of the molecule such as
OH
polyvinyl alcohol or hydrolysate of a copolymer of
ethylene and vinyl acetate; a polymer having a unit of the
formula:
CIH3
~CH2 1
Cl=O
O-CH2-CH2-OH
such as polyhydroxyethyl methacrylate, a copolymer
containing hydroxyethyl methacrylate and the like; and
polysaccharides such as cellulose, cellulose derivatives
having hydroxy group including hydroxyethyl cellulose,
agarose and dextran. In the above-mentioned examples, a
polymer having a unit of the formula: -~CH2-CH~- in at
OH
least a part of the molecule or polysaccharides is
particularly preferable.
Hydroxy group in the polymer may be derived
from a monomer capable of forming a polymer as in case of
1~5~
-- 6
polyvinyl alcohol or polyhydroxyethyl methacrylate, or it
may be introduced in the polymer by a chemical
modification of the polymer, or it may be derived from
the cross-linking agent which is used for forming an
insoluble gel.
Non-limitative exmaples of the above-mentioned
cross-linking agent, having hydroxy group or forming
hydroxy group in the reaction, are typically polyvalent
unsaturated compounds having hydroxy group such as
pentaerythritol dimethacrylate, diallylidene
pentaerythritol and glycerol dimethacrylate, or compounds
having oxirane ring such as epichlorohydrin, butanediol
diglycidyl ether and glycidyl methacrylate.
As previously mentioned, the corss-linking can
be carried out during polymerization, after
polymerization, or both during and after polymerization
in order to obtain the water-insoluble polymer. The
water-insoluble gel used in the present invent on may be
made water-insoluble by any type of the cross-linking
such as cross-linking during polymerization, cross-linking
after polymerization or cross-linking during and after
polymerization as mentioned above. Further, the
water-insoluble gel used in the present invention is
required to be a hard gel.
The term "hard gel" herein referrs to a gel
which is less swelled with a solvent and less deformed by
a pressure than a soft gel such as dextran, agarose or
acrylamide. A hard gel and a soft gel can be
distinguished from each other in the following manner:
i.e. as shown in the Reference Example herein below, when
a relation between a flow rate and a pressure drop is
determined by passing an aqueous liquid through a
cylindrical column uniformly packed with a gel, a hard
gel shows an almost linear relationship while a soft gel
shows a non-linear relationship. In case of a soft gel,
a gel is deformed and consolidated over a certain point
of pressure and therefore a flow rate does not increase
any more. In the present invention, a gel having the
lZ5~6
above linear relationship at least by 0.3 kg/cm2 is
referred to as "hard gel".
A hard gel is formed by various methods, any of
which can be employed in the presen~ invention.
In some case, only a soft gel is obtained when
a water-insoluble gel is formed solely from a polymer
having hydroxy group since, in general, such polymer
exhibits a high degree of hydrophile property In such
case, however, a hard gel can be formed by employing the
polymer having hydroxy group in combination with another
polymer which but does not necessarily have hydroxy
group but can form a hard gel.
In this case, formation of a hard gel can be
carried out, for example, by mixing more than two kinds
of polymer, or by coating a polymer having hydroxy group
on the surface of a hard gel which is previously
prepared. However, the present invention is not limited
to these method.
The term "porous" in the present invention
means that a gel has a porosity of not less than 20 % and
a specific surface of not less than 3 m2/g.
A water-insoluble porous hard gel of a polymer
having hydroxy group in at least a part of the molecule
used in the present invention is required, in the first
place, to have continuous pores with a large diameter so
that LDL and VLDL, which are macromolecules having
molecular weight of at least not less than 1 x 106, can
easily enter in the pores to be adsorbed.
For measuring the pore size, there are various
kinds of methods, among which mercury porosimetry is most
frequently employed. In case of a hydrophilic gel, an
exclusion limit is usually adopted as a measure of the
pore size.
The term "exclusion limit" in the present
invention means, as described in the literature such as
"Jikken Kosoku Ekitai Chromatography (Experimental High
Speed Liquid Chromatography)", Hiroyuki Hatano and
Toshihiko Hanai, published by Kabushiki Kaisha Kagaku
1~3~7
-- 8
Dojin, the minimum molecular weight of the molecule which
cannot permeate into a pore, i.e. which is excluded, in a
gel permeation chromatography.
It is known that a value of an exclusion limit
varies depending on a kind of the substances employed,
among which an exclusion limit value with such molecules
as globular proteins, dextran and polyethylene glycol has
been quite investigated, whereas that with lipoproteins
has been hardly investigated. Thus, in the present
invention, a value of an exclusion limit measured by
using globular proteins and/or viruses, which are
regarded as the most similar substances to the
lipoproteins, is suitably employed.
As the result of the investigation of the
present inventors, using a variety of water-insoluble
porous gels having a different value of an exclusion
limit, it is unexpectedly shown that a gel having an
exclusion limit value of about l x 106, which is smaller
than the molecular weight of LDL and VLDL, can adsorb LDL
and VLDL to some extent and that a gel having a larger
pore size does not always exhibit an increased capacity
of adsorbing but, conversely, it is observed that an
adsorbing capacity of such gel decreases or proteins other
than LDL and VLDL are likely to be adsorbed, which means
there exist an optimum range of a pore si2e. That is, it
is found that a water-insoluble porous hard gel having
an exclusion limit of less than l x 106 can hardly adsorb
LDL and VLDL and is not suited for practical use, whereas
a water-insoluble porous hard gel having an exclusion
limit of one million to several millions, which is nearly
the molecular weight of LDL and VLDL, can adsorb LDL and
VLDL to some extent. Subsequently, it is observed that
an amount of an adsorbed LDL and VLDL increases with the
increase of an exclusion limit, by and by reaching its
top, and it extremely decreases when an exclusion limit
is over l x 108 because in the region, a content of a
polymer having hydroxy group in at least a part of the
molecule, which makes up a water-insoluble porous hard
7:~6
g
gel, per volume of a gel is lowered and consequently an
amount of hydroxy group per volume of gel is also
lowered, thus a sufficient amount of sulfuric acid
residue cannot be introduced in the polymer. Therefore,
an exclusion limit of a water-insoluble porous hard gel
employed in the present invention is 106 to 108,
preferably 3 x 106 to 7 x 107.
With respect to a porous structure of a water-
insoluble porous hard gel used in the present invention, a
structure uniformly having pores at any part of the gel
is more preferable than a structure having pores only on
the surface of the gel for the purpose of adsorbing a
larger amount of LDL and VLDL to be adsorbed.
A shape of a water-insoluble porous hard gel
used in the present invention can be optionally selected
from shapes such as particle, fiber, sheet and hollow
fiber. When a water-insoluble porous hard gel with a
shape of particle is used, the particle size is
preferably 1 to 5000 ~m.
There can be sulfated at least a part of
hydroxy group in a water-insoluble porous hard gel by
various methods such as, for instance, a method of
reacting a water-insoluble porous hard gel having hydroxy
group with chlorosulfonic acid or sulfuric anhydride in
the presence of pyridine or N,N-dimethylformamide and a
method of directly reacting hydroxy group with sulfuric
acid in a solvent such as N,N-dimethylformamide. Though
any method can be employed for sulfation of hydroxy
group, sulfation is preferably carried out under
anhydrous or nearly anhydrous conditions since such
conditions can improve the efficiency of sulfation.
Since a water-insoluble porous hard gel of the
present invention is converted to sulfate by the
above-mentioned method, hydroxy group mainly on the
surface of a water-insoluble porous hard gel is directly
sulfated.
An amount of the introduced sulfuric acid
residue is preferably 0.1 ~mol to 10 mmol, more
1~5~7~6
-- 10
preferably 10 ~mol to 1 mmol per 1 m~ of a water-insoluble
porous hard gel used in the present invention. When the
amount is less than 0.1 ~mol, a sufficient adsorbing
capacity cannot be obtained. When the amount exceeds 10
mmol, nonspecific adsorption, especially an adsorption of
fibrinogen increases and a pH change of the body fluids
may be caused, which make the gel unsuitable for a
practical usage.
The adsorbent according to the present
invention can be used for treatment in various ways.
Most simply, the adsorbent of the present
invention can be used as follows: i.e. blood is
introduced outside the body of the patient to be put in a
blood bag, with which the adsorben-t of the present
invention is mixed to adsorb LDL and VLDL, followed by
removing the adsorbent through filter, the blood treated
in this way being put back to the body of the patient.
Though the method does not need an intricate apparatus,
it has defects such that an amount of the blood treated
at one time is small, it takes much time for treatment
and an operation in the method is somewhat troublesome.
In another method, a column is packed with the
adsorbent of the present invention, which is incorporated
into an extracorporeal circulation circuit with
circulation of the blood, wherein either whole blood is
directly circulated or only plasma separated from the
blood is passed through the column. Though the adsorbent
of the present invention can be used in both of the
above-mentioned methods, it is preferably used in the
latter method as mentioned above.
Selectivity and efficiency of the removal of
LDL and VLDL can be improved by adding, when using the
adsorbent of the present invention, polyvalent metallic
ion to blood or plasma to be treated. The examples of
polyvalent metallic ion to be used for this purpose is,
for instance, alkaline-earth metal ions such as calcium
ion, magnesium ion, barium ion and strontium ion, ion of
Group III of the Periodic Table such as aluminum ion, ion
12C~Z7~
of Group VII of the Periodic Table such as manganese ion
and ion of Group VIII of the Periodic Table such as
cobalt ion.
By using the adsorbent of the present
invention, LDL and VLDL can be selectively and
effectively removed from the body fluids of the patientO
Further, the adsorbent of the present invention can be
prepared in a lower cost than the adsorbent based on the
principle of the affinity chromatography, in which a
relatively expensive ligand is employed.
The present invention is more specifically
described and explained by the following Reference
Example, Examples and Comparative Examples. It is to be
understood that the present invention is not limited to
the Reference Example, Examples and Comparative Examples
and various changes and modifications can be made without
departing from the scope and spirit of the present
invention.
Reference Exam~le
A relation between a flow rate and a pressure
drop is determined by passing water by means of a
peristaitic pump through cylindrical glass columns
e~uipped at both ends with filters having a pore size of
15 ~m (inner diameter: 9 mm, column length: 150 mm), in
which an agarose gel (Biogel A5m made by Biorad Co.,
particle size: 50 to 100 mesh) and hard gels made of a
polymer (Toyopearl HW 65 made by Toyo Soda Manufacturing
Co., Ltd., particle size: 50 to 100 ~m, and Cellulofine
GC-700 made by Chisso Corporation, particle size: 45 to
105 ~m) are filled respectively. The results were shown
in Fig. 1.
As shown in Fig. 1, an increase of a flow rate
is nearly proportional to that of a pressure in case of
hard gels made of a polymer, whereas in case of an
agarose gel, consolidation occurrs and a flow rate does
not increase even if a pressure increases.
* Trade Mark
lZ~Z71f~
- 12
Example_l
There was dried 10 mQ of cross-linked
polyacrylate gel (Toyopearl HW 75, exclusion limit of
proteins: 5 x 106, particle size: 50 to 100 ~m), which
was a hard gel having pores at any part thereof, by a
critical point drying method in ethanol. The resultant
dried gel was suspended in 10 mQ of N,N-dimethylformamide
sufficiently dehydrated and the suspension was cooled
with ice, to which 1 mQ of chlorosulfonic acid was added
dropwise under stirring, the stirring being continued for
10 minutes after the dropwise addition was co~pleted.
After completion of the reaction, the reaction mixture
was neutralized with 10 % aqueous solution of sodium
hydroxide, the gel being filtered and washed with a great
excess of water to give a water-insoluble porous hard gel
on whose surface 0.4 mmol/mQ of sulfuric acid residue was
introduced.
Exam~le 2
A method described in Example of Japanese
Unexamined Patent Publication No. 12656/1983 was
employed, i.e. a uniform mixture of 100 g of vinyl
acetate, 24.1 g of triallyl isocyanurate, 124 g of ethyl
acetate, 124 g of heptane, 3.1 g of polyvinyl acetate
25 (degree of polymerization: 500) and 3.1 g of 2,2'-
azobisisobutyronitrile, and 400 mQ of water in which 1
by weight of polyvinyl alcohol, 0.05 % by weight of
sodium dihydrogenphosphate 2H2O and 1.5 % by weight of
disodium hydrogenphosphate-l2H2O were dissolved were
charged in flask. After sufficient stirring, a
suspension polymerization was carried out by stirring the
mixture for 18 hours at 56.5C, further for 5 hours at
75C to give a granular copolymer, which was then
filtered, washed with water, extracted with acetone and
subjected to an ester interchange reaction for 18 hours
at 40C in a solvent of 46.5 g of sodium hydroxide and 2
Q of methanol. There was dried 10 mQ of the thus
obtained water-insoluble porous hard gel having vinyl
l~SZ~7i6
- 13
alcohol as a main constitutional unit (exclusion limit:
about 1.8 x 106, average particle size: 150 ~m) by a
critical point drying method in acetone. The resultant
dried gel was suspended in 10 mQ of N,N-dimethylformamide
sufficiently dehydrated and the suspension was cooled
with ice, to which 1 mQ of chlorosulfonic acid was added
dropwise under stirring, the stirring being continued for
10 minutes after the dropwise addition was completed.
After completion of the reaction, the reaction mixture
was neutralized with 10 % aqueous solution of sodium
hydroxide, the gel being filtered and washed sufficiently
with water to give a water-insoluble porous hard gel on
whose surface 0.8 mmol/mQ of sulfuric acid residue was
introduced.
Examples 3 to 4 and Comparative Example 1
There was added 6 mQ of plasma obtained from
the patient suffering from a familial hyperlipemia to 1
mQ of each gel in a test tube prepared in Examples 1
and 2 respectively and the resultant mixture was
incubated under stirring for 2 hours at 37C (Examples 3
and 4). An amount of LDL, VLDL, HDL cholesterol and
fibrinogen in each supernatant was determined. The
results were shown in Table 1.
An amount of LDL, VLDL, HDL cholesterol and
fibrinogen in case that the adsorbent was not added was
also determined. The result was shown in Table 1
(Comparative Example 1).
~25~7
a)
o~ ~ ~ ~ CO
~ ~ o
Q E~ t`
._, _
w o
C~
~ ~ ~ a~
~D O
co ~r
~ ~ ~`~ In CO
,
E~
~0 a)
~ . ~
~ ,~ a) ~
~3 ~ Ll o O O O
C ~--
Q ~¢ 0 0
O
.
s:: ~ ~ ~ ~
o ~ a) ~3 a) a) a) ~ aJ
~ Q ~
o o ~ n~ Q, Ll r~l Q~ O
~ ~q O C~ E~ 0 ~4 ~ Z
E~ ~ ~ ~ X ~ L~ X
~ W
lZ~2~7~;
-- l; --
~ s shown in Table 1, both LDL and VLDL were
adsorbed whereas HDL cholesterol and fibrinogen were
hardly adsorbed by using the adsorbent according to the
present invention.
ExamDle 5
There was dried 10 mQ of a porous cellulose gel
(CK gel A-3 made by Chisso Corporation, exclusion limit
of globular proteins: 5 x 107, particle size: 45 to 105
~m) by a critical point drying method in ethanol. The
resultant dried gel was suspended in 10 mQ of pyridine
sufficiently dehydrated and the suspension was cooled
with ice, to which 2 mQ of chlorosulfonic acid was added
dropwise under stirring, the stirring being continued for
10 minutes after the dropwise addition was completed.
After completion of the reaction, the gel was filtered
and washed successively with pyridine and water to give a
cellulose gel on whose surface was introduced sulfuric
acid residue in an amount shown in Table 2.
Example 6
There was dried 10 mQ of a porous cellulose gel
(Cellulofine GCL-2000 made by Chisso Corporation,
exclusion limit of globular proteins: 3 x 107, particle
size: 45 to 105 ~m, a cross-linked gel) by a critical
point drying method in ethanol. The resultant dried
gel was suspended in 10 mQ of pyridine sufficiently
dehydrated and the suspension was cooled with ice, to
which 2 m~ of chlorosulfonic acid was added dropwise
under stirring, the stirring being continued for 10
minutes after the dropwise addition was completed. After
completion of the reaction, the gel was filtered and
washed successively with pyridine and water to give a
cellulose gel on whose surface was introduced sulfuric
acid residue in an amount shown in Table 2.
ExamDle 7
There was dried 10 m~ of CK gel A-3 by a
* Trade Mark
7~
- 16
critical point drying method in ethanol. The resultant
dried gel was suspended in 10 mQ of dimethylformamide
sufficiently dehydrated, to which 12 mQ of a solution of
4M N,N-dicyclohexylcarbodiimide/dimethylformamide was
added and the resultant was cooled with ice, to which 6
mQ of a solution of 2M sulfuric acid/dimethylformamide
was added dropwise under stirring, the stirring being
continued for 2 hours at 0C. After completion of the
reaction, the gel was filtered and washed successively
with dimethylformamide and water to give a cellulose gel
on whose surface was introduced sulfuric acid residue in
an amount shown in Table 2.
Example 8
There was dried 10 mQ of Cellulofine GCL-2000
by a critical point drying method in ethanol. The
resultant dried gel was suspended in 10 mQ of
dimethylformamide sufficiently dehydrated, to which
12 mQ of a solution of 4M N,N-dicyclohexylcarbodiimide/
dimethylformamide was added and the resultant was cooled
with ice, to which 6 m~ of a solution of 2M sulfuric
acid/dimethylformamide was added dropwise under stirring,
the stirring being continued for 2 hours at 0C. After
completion of the reaction, the gel was filtered and
washed successively with dimethylformamide and water to
give a cellulose gel on whose surface was introduced
sulfuric acid residue in an amount shown in Table 2.
Examples 9 to 10
The procedures of Example 5 were repeated
except that 6 mQ of chlorosulfonic acid and 8 mQ of
chlorosulfonic acid were used (Example 9 and Example 10
respec'ively) to give a cellulose gel on whose surface
was introduced sulfuric acid in an amount shown in
Table 2.
Comparative Example 2
The procedures of Example S were repeated
lZS~:71~
- 17
except that Cellulofine GC 700 (made by Chisso
Corporation, exclusion limit of globular proteins: 4 x
105, particle size: 45 to 105 ~m) was used as a cellulose
gel to give a cellulose gel on whose surface was
introduced sulfuric acid in an amount shown in Table 2.
Examples 11 to 16 and Comparative Examples 3 to 4
Each 1 m~ of gel prepared in Examples 5 to 10
and Comparative Example 2 respectively were put in a
test tube, to which 6 mQ of plasma obtained from the
patient suffering from a familial hyperlipemia was added
and the resultant was incubated for 2 hours at 37C under
stirring (Examples 11 to 16 and Comparative Example 3
respectively).
An amount of LDL, VLDL, HDL cholesterol and
fibrinogen was determined. The results were shown in
Table 2.
An amount of LDL, VLDL, HDL cholesterol and
fibrinogen in case that the gel was not added was also
determined (Comparative Example 3). The result was shown
in Table 2.
- lZ~ '71~
o ~ o ~ o o
c~ ~ ~ ~ ~ o 0 ~ ~
.
O
U~ o ~ o o ~ o o
~ S~ ~ ~ ~ ~ ~ ~ ~ ~
~ O --
C~
U~ CO o 1~ ~ U~
~ e ~ r co
W U~ D ~ Lr) O
C~ ~oo ~ t~ _J ~ ~
~ ~ ~ t~ ~o
E~ ~ a
o ~
s~ u~ D O
O h h ~ O O ~ ~ D ~ 1~1
~ ~ O O o o o o ~ O O
'cJ Il~
a) 1~
D ~ U~ nl
h
~ ~ c c c c c ~ c a~
~J C ~ C ~ ~ ~ '21 C
O Q O a) a~ a) ~J aJ O a) Q) O ~ a) O a) a) O Q) a) a) O t~
~ n ~ ~ D ~ ~ D ~ _I D ~ ~ Q ~ ~I D Ll ~1 D S~
a) o L~ , h ~ ~ a ~ Q, O
o~q o 3e o ~ o ~e o ~e o ~ o ~ O ~D~e æ
a) e ~
E-l 115~ Ll X ~ X~:1 h X ~ ~-1 X ~ ~I X ~1 ~I X ~ h O X
O ll~
-1~~ In ~
e
e e~ e ~
X o X o X
C~
