Note: Descriptions are shown in the official language in which they were submitted.
~1VO 94122885 215 9 6 4 9 PCT/CA94/00161
A~COAGULANTCOMPOUNDS
F~LDOFINVEN~ON
This invention relates to novel pharmaceutical compositions for use as blood
antico~ nt~. More particularly this invention relates to pharmaceutical compositions
which inhibit Factor IXa in the blood coagulation cascade, and to compositions which
may be used to render material surfaces anticoagulative, such as prosthetic implant
surfaces and artificial devices and blood carrying devices such as tubing, prosthetic heart
valves, and extracorporeal devices such as renal dialysis m~chines.
BACKGROUNDOFI~VEN~ON
Heparin and polysaccharide derivatives thereof have long been used for selective
anticoagulation activity both in vivo and in vitro. Heparin is a sul&te-cont~ining
polys~crh~ride which can be extracted from bovine and porcine lung and intestinal
mucosa. Heparin is not, however, a pure compound but is a mixture of polysaccharides
with a continuous distribution of molecular weights in the range 1,500 to 30,000 daltons.
The activity thereof is somewhat variable, depending on the source and molecular weight.
This can cause problems, such as risk of bleeding complications, because different
patients react very differently to a given dosage. Heparin is not orally active and must
be given parenterally. While several synthetic oligo- and polysaccharides based on the
structure of heparin have been described in the literature there remains a need for a
simple, relatively low molecular weight, synthetic saccharide, which may be orally active,
for use as an anticoagulant pharmaceutical and as a coating for prostheses, tubing, glass
and sirnilar sur&ces which come into contact with blood so as to reduce thrombotic
problems. Vejay Nair, in U.S. patent 4,021,544, describes a series of sulfated maltoses
and oligos~cch~rides of the maltose series having a formula:
SUB~ 11 ~ ~ITE S~IEET
wo 94,22885 21$9 PCT/CA94/00161
CH20SO--3M
\ /(~OSO-3M 1/\ n
OSO- M+
where M is hydrogen or a salt of an alkali metal, alkaline earth metal, ammonium,
tri(loweralkyl)amine (Cl-C6), piperidine, pyrazine, alkanolamine (Cl-C6) and
cycloalkanolamine (C3-C6); and n is 2-10, which are used to inhibit the complement
system of warm blooded ~nim~lc. "Complements" refer to a complex group of proteins
in blood and other body fluids that, working together with antibodies or other factors,
play an important role as mediators of immune, allergic, immunochemical and/or
immllnopathological reactions. Complement inhibitors can be used therapeutically for
such non-immunologic diseases as paroxysmal nocturnal haemoglobinuria and hereditary
angio-neurotic edema. While activation of the complement system may also accelerate
blood clotting, there is no evidence that inhibition thereof would have any anticoagulant
activity and, indeed, the opposite is probably the case. In this series of compounds,
however, the anomeric hydroxyl group is not blocked but may be sulfated. It has now
been found that slllf~ted and sulfonated oligosac~h~rides preferably having blocked
anomeric centers, surprisingly exhibit anticoagulant properties specific to inhibition of
Factor IXa.
OBJECr OF INVENTION
It is, therefore, one object of the present invention to provide novel compounds
having anticoagulant activity both in vivo and in vitro.
Another object of this invention is to provide a composition and method for
producing antithrombotic surfaces for implanted prostheses, extracorporeal devices,
laboratory equipment and the like.
SUE~ 1TE SHEET
~o 94/2288s 2 1 S 9 B g ~ PCT/CA94/0016
BRIEF STATEMENT OF INVENTION
By one aspect of this invention there is provided a disaccharide cont~ining at least
one sulfur based anion, which exhibits antithrombotic properties specific to inh ibition of
Factor IXa.
By another aspect of this invention there is provided an anticoagulant preparation
comprising a pharmaceutically effective amount of a disaccharide containing at least one
sulfur based anion, which exhibits antithrombotic properties specific to inhibition of
Factor IXa, and a pharmaceutically acceptable carrier therefor.
BRIEF DESCRIP~ON OF DRAWI~GS
Figure 1 is a sketch illustrating the blood coagulation cascade;
Figure 2 is a sketch similar to Figure 1 but showing the blood coagulation cascade
in a simplified form;
Figure 3 is a graph showing A~l-r prolongation at various concentrations of test
compounds in plasma.
Figure 4 is a graph illustrating Factor IXa clotting time with increasing
concentrations of selected inhibitors;
Figure 5 is a graph illustrating Factor X activation versus concentration of
inhibitor #3;
Figure 6 is a graph illustrating Factor X intrinsic fluorescence versus concentration
of inhibitor #3;
Figure 7 is a graph illustrating Factor IXa fluorescence versus concentration of
inhibitor #3;
Figure 8 is a graph illustrating Factor X activation and intrinsic fluorescence
versus concentration of inhibitor #3; and
SUBSTITUTE SHEET
W O 94122885 ~ 1 5 9 6- 4 9 PCT/CA94/00161
Figure 9 is a graph illustrating Factor X activation and Factor Ixa intrinsic
fluorescence versus concentration of inhibitor #3.
DETAILED DESCRIFrION OF PREFERRED EMBODIMENTS
Sulfated compounds of the present invention fall into two groups namely non-
reducing ~ ccll~rides having a general formula:
H, Rl
O
' 2 ~ ~ ) ~ O R
~ J~
H,R2 H,R2
where Rl is H or (CH20SO-3 M+)n, n is an integer from 1-6,
R2 iS OSO-3 M+ and M is selected from NH4, trialkylammoniurn, cyclic
ammonium, alkali metals and ~Ik~line earth metals,
and R is
H, R
O
' 2 ~ O R
~I J~
H,R2 H,R2
/\
H-Rl ~ ~ CH20SO--3M
'l (
H,R2 H,R2
and reducing (~ cch~rides having a blocked anomeric center of the type
SUBS 111 IJTE SHEET
WO 94122885 ~ t PCT/CA94100161
- Illustrative, but not limiting, examples of compounds within these general
formulae include:
(a) Sucrose octasulfate sodium salt:
CH2 OSO -3 Na
~ 0\~
Na -03 so\~
H o5o-3 Na +
Na 03 SOCH~/ \
\~ 3 1~/cH2oso-3Na+
3 Na H
(b) Trehalose octasulfate sodium salt:
CH 2OSO- 3Na
O\
E3Na+ 03 SO
S
SUE~ UTE SHEET
PCT/CA94/00161
CH20SO -3Na
Na -O3SO\
H Na -O 3 SO
and (c) Methyl ,~-D-lactoside hept~ lf~te sodium salt:
CH20SO-3Na CH20SO-3Na
Na -O3 SO/É \ H/I \OCH3
SO- N + ~ SO N + Hl~/
H 3 H OSO-3Na
Example 1
Preparation of sucrose octasulfate ammonium salt.
2-Picoline was fractionally distilled to remove the water azeotrope and the
collected "dry" picoline was dried over CaH2 and re-distilled. The solvent was stored in
a sealed flask until used.
SUBSTlTlJTE SHEET
2~ 6 ~ 9
A dry 1-L three-necked flask, fitted with a sealed mechanical stirrer, a 50-mL
vented dloppillg funnel, and a stopper, was charged with dry 2-picoline (92 mL). The
flask was pIaced in an oil bath held at 45C and brisk stirring was begun. The dropping
funnel was charged with chlorosulfonic acid (16 mL, 240.7 mrnol) and the top of the
funnel was fitted with a calcium chloride drying tube. The chlorosulfonic acid was
carefully dripped into the 2-picoline over 1.5 h. The bath temperature was raised to
60C, and powdered sucrose (10.0 g, 29.2 mmol) was added, rinsing the sides of the joint
and the flask clean with a small amount of dry 2-picoline from a dry pipette. Stirring
was continued for 1-1.5 hours more, to afford a viscous purple solution.
The mixture was cooled to room temperature over -1 h, and then the flask was
immersed in an ice-water bath. Stirring became difficult, but was continued as 1/2
concentrated aqueous ammonium hydroxide solution (70 mL) was added. At this point,
a drop of the clear purple solution placed on moist Ph paper produced the green color
of Ph ~8. Ethanol (95% v/v, 250 Ml) was added portionwise to the stirred solution, to
produce a tan solid precipitate. The suspension was stirred vigorously for 30 min. The
precipitate was collected by filtration, washed sequentially with 95~o ethanol (2 x 50 Ml),
acetone (50 Ml), and ether (50 Ml). The product was left in a vacuum desiccator
overnight, to remove traces of 2-picoline. Crude recovery was 35.43 g.
The tan powder was dissolved in water (60 Ml). The solution was decolorized and
filtered; the filtrate was added to a separatory funnel, and the Ph was adjusted to 8.5
with concentrated aqueous ammonium hydroxide. A S00-Ml Erlenmeyer flask was fitted
with a mechanical stirrer, and ethanol (300 Ml) was added. The product solution was
slowly dripped into the vigorously stirred ethanol over 2 h. A white precipitate was
SUB~ JTE Sl 5EET
WO 94/22885 2 15 9 6 4 9 PCT/CA94100161
formed, which was collected by filtration, washed with absolute ethanol, and then with
acetone. The product was dried in vacuo, yield 27.5 g (84%); mp 155-158C (dec.).
Example 2
Preparation of sucrose octasulfate sodium salt (Lazaridis method)
Sucrose oct~clllf~te ammonium salt (10 g) prepared in Example 1 was dissolved
in water (50 Ml), and the Ph was raised to 9.0 with 20~o w/v aqueous sodium hydroxide.
Ethanol (95%) was added to the solution with vigorous mechanical stirring, until an oily
precipitate was formed. Scratching of the precipitate eventually caused solidification, and
the solid was collected by filtration. This was dissolved in a minimllm amount of water,
the solution was filtered, and the Ph of the filtrate was adjusted to 9 with 20~o sodium
hydroxide. Alcohol was added, with vigorous stirring, to form a fine powder. When
addition was complete, the suspension was stirred for several hours more. The product
was collected by filtration, washed sequentially with absolute alcohol, acetone, and ether,
and dried in vacuo overnight over sodium hydroxide. Yield of white powder, 8.96 g
(87%); mp 159-166C (dec.); MW 1158.
Example 3
Preparation of trehalose octasulfate sodium salt
Using the apparatus described in Example 1, chlorosulfonic acid (17.9 Ml, 269
mmol) was added dropwise to 2-picoline (90 Ml) at 45C, over 1.25 h. The temperature
was raised to 60C, and powdered and dried ~, ~-trehalose monohydrate (11.00 g, 29.1
mmol) was added. Stirring was continued for 1.5 h. The mixture was then cooled on an
ice-water bath. Water (150 Ml) was added cautiously, followed by barium hydroxide
octahydrate (48.14 g, 152.6 mmol). The rrlixture was stirred for 1 h. The suspension was
poured into centrifuge flasks, and centrifuged at ~2000 rpm for 15 min. The supernatant
SUBSTITIJTE SHEET
~0 94/22885 2t?~ ; PCT/CA94/00161
was dec~nted, the solids were re-suspended in a small am of water, and centrifuged
again. The supernatant was added to the first supernatant, and the deep red-purple
liquid was vigorously stirred mechanically while 95% ethanol (400 Ml) was slowly added
dropwise. This procedure caused an oily material to separate; stirring overnight caused
this oil to solidify. The solid was collected by filtration, and washed with 95~o ethanol,
and then with ether. I~e powdery material was allowed to stand in vacuo to remove
traces of picoline. The crude barium salt weighed 34.15 g.
This barium salt was dissolved in water (100 Ml), and the solution was decolorized
by the addition of Norit~ charcoal (S g). The solution was filtered through Celite~, and
the filtrate was diluted to a volume of 750 Ml. This solution was passed down a 2 cm
x 90 cm column cont~inin~ Amberlite~ IR-120(Na+). The column was flushed with
water (500 Ml) and the combined eluate was concentrated to a volume of 300 mL on the
rotary evaporator, m~int~inin~ a bath temperature of <40C. This solution was freeze-
dried to provide a fluffy white powder, 20.0 g (59%); MW 1164.
Example 4
Preparation of methyl B-D-lactoside heptasulfate ammonium salt
Following a procedure similar to that used in Example 1, 2-picoline SO3 complex
was prepared at 45C from chlorosulfonic acid (15.4 mL, 232 mmol) and 2-picoline (990
rnL). The temperature was raised to 60C, and methyl ~-D lactoside (10.00 g, 28.06
mmol) was added. The mixture was stirred at 60C for lh before being cooled. The
solution was chilled in an ice-water bath, as half concentrated ammonium hydroxide
solution (75 mL) was added. Ethanol (95%, 500 mL) was added with vigorous stirring.
This procedure caused separation of an oily material, which was allowed to settle. The
red supernatant was decanted, and more ethanol was added to the residue, which was
SUB~ I l I UTE St~EET
WO 94/2288~ 2 15 9 6 4 9 PCT/CA94/00161
i i i, ~
ground up thoroughly. This procedure induced solidification, and the ethanolic
suspension of the product was stirred vigorously overnight. The crude ammonium salt
(35.1 g) was collected by filtration.
The salt was dissolved in water (60 Ml) and the solution was decolorized by
addition of Norit8' charcoal (1 g). The solution was filtered through Celite~, and the
filtrate was vigorously stirred as 95% ethanol (~200 Ml) was added. This procedure
caused separation of an oily white material. The supernatant was decanted, and the oily
material was triturated with ethanol and ground into a fine powder. The powder was
dried in vacuo over sodium hydroxide pellets for 2 days, yield 28.11 g (97%).
Example 5
Preparation of methyl ~-D-lactoside heptasulfate sodium salt
Methyl ,~-D-lactoside hept~l-lf~te ammonium salt (12 g, 11.6 mmol), as prepared
by Example 4, was dissolved in water (60 Ml), and the solution was filtered. The Ph of
the filtrate was adjusted to 8 with 20% w/v sodium hydroxide solution. While the
aqueous solution was being vigorously stirred, 95% ethanol (150 Ml) was added. This
procedure caused separation of an oily material. The supernatant was decanted, the oily
residue was dissolved in water (100 Ml) and the solution was freeze-dried. The resulting
powder was dissolved again in water (100 Ml) and the solution was freeze-dried. The
powdery product was dried 24 h in vacuo at 60C. Yield 10.41 g (84%); mp 167-174C
(dec.).
~0
SUBSTITUTE S~:ET
~NO 94/2~885 21 5 9 C: 4~ PCT/CA94/00161
Example 6
Screening tests for anticoagulation effects
(a) In Vitro Testing
The compositions produced according to Example 5, Example 3 and Example 2,
and sucrose (MW360) as a control, were design~ted compounds 1, 2, 3 and 4,
respectively for blind screening tests of their anticoagulation properties. Each compound
was added to human plasma at increasing concentrations and the Activated Partial
Thromboplastin Time (APl r), Prothrombin Time (PI) and Thrombin Clotting Time
(TCI) were measured using standard protocols (Figure 3). Compound 4 showed no
anticoagulant activity but in the case of compounds 1, 2 and 3 the TCI and PT remained
the same as the control, while the APIT showed a dose related response by prolongation
of APIT.
(b) In Vivo Studies
In vivo recovery and survival of anticoagulant activity
This study was designed to measure the prolongation of the API~ (activated
partial thromboplastin time) as well as the platelet and white blood cell (WBC) counts
in an animal model. A 4.5 kg normal male New Zealand white rabbit was selected for
this purpose. 50 mg, in 5 ml PBS, of sucrose octasulfate sodium salt (compound 3) was
infused into the right ear vein of an unaesthetized male NZW rabbit over a 40 second
period. In a second study, a male NZW rabbit was anesthetized and a stomach tube
placed so that the test solution, sucrose octasulfate at 200 mg/kg body weight, could be
?,~mini~tered in a 200 mg/ml water. The infusions were well tolerated. Blood samples
were drawn from the left ear vein via a 21 gauge butterfly needle immediately pre-
infusion and then at 1, 5, 10, 20, 46, 70, 123, 182 and 240 minutes post infusion (injected
SUB~;TITUTE SI~EET
wo 94l22885 215 9 6 ~ 9 PCT/CA94100161
samples) and at 10 minute intervals over 2 hours in the case of the orally a~1mini~tered
dose. The blood samples were collected into 1/lOth volume of buffered citrate and also
into pediatric EDTA tubes for cell counts. The citrated samples were centrifuged to
obtain platelet-poor plasma and frozen. Complete blood counts were performed on a
Baker System 9000 automated haemotology instrument. Cell counts were expressed as
percent of the pre-infusion value. APIT tests were performed m~nllally in glass tubes
as follows: 100 ~l plasma was put into a tube 37C, 100 ,ul API~ reagent (Organon
Teknika) was added and incubated for 3 minutes at 37C. 100 ,ul 25 mM CaCl2 was
then added and the time to clotting was then measured with a stopwatch. A standard
line was constructed by adding known amounts of test material to citrated normal rabbit
plasma. The prolongation of the APl'r (in seconds) was plotted against the
concentration of anticoagulant and the prolongation of each of the test samples was read
as apparent activity from this line.
The predicted 100% recovery for the amount infused (4.3 x 10-5 moles infused)
40 ml/kg x 4.5kg
was 239 ~M. As can be seen, the measured activity in the 1 minute sample was 130~m
which is an actual recovery of about 555~o, and a half life of approximately 5 rninutes.
Immediately post infusion the platelet count was low, but recovered within 5
minutes. There was no apparent change in the WBC count.
SU~ UTE SHEET
~VO 94122885 ?1 $~ 6 ~ g PCT/CA94/00161
The results are tabulated below:
Table 1
l~me AP1rP Sec plts x 109/L VVBC x 109/L [cmpd #3]
(manual) P~ r
pre 2Sr 2T 0 326 (100%) 7.6 (100%) 0
1' 34~ 3T 9 125 (38%) 8.2 (108%) 130~M
5' 30~ 32" S~ 264 (81%) 7.5 (98%) 75~M
10' 28~ 28~ 3 310 (95%) 7.4 (97%) 35aM
20' 24~ 23~ -1 288 (88%) 7.2 (95%)
46' 28~ 27~ 3 300 (92%) 6.7 (88%)
70' 26~ 25~ 1 291 (89%) 6.5 (86%)
123' 27" 26~ 2 352 (108%) 7.6 (100%)
182 27 28 2
240' 27" 29~ 2
Table 2
Oral Dosing Experiments
Sucrose Oct~c-llf~te 200 mg/kg body weight
Results mean (x) of 2 studies
x APIT PROLONG- x PLATELET CT
TIME (SEC) ATION (SEC) x 109/L
Pre 7 min 30.5 - 298
Pre 6 min 28.5 0
OR~L DOSE GIVEN AT ZERO TIME
Post 15 min 34.0 5.5 233
Post 30 min 33.5 5.0 249
Post 45 min 34.5 6.0 248
Post 60 min 35.0 6.5 263
Post 75 min 34.0 5.5 265
Post 90 min 32.0* 3.5 40û*
* N = 1 (1 specimen clotted)
13
SU~S~ JTE SHEET
WO 94122885 ?,~s964 PCTICA94/00161
Conclusion
A 5-6 second prolongation of APTI` observed 15 min after oral ingestion that was
sustained for 90 minutes.
It will be appreciated by reference to Figure 2 that the AP IT is simply a measure
of blood co~ tion in the overall blood coagulation cascade and no inferences as to
where the inhibitor is active can be drawn. The PT, however, is a measure of the
activation of Factor X to Factor Xa (prothrombinase) and activation of prothrombin to
thrombin via the extrinsic pathway, and the TCI` is a measure of the conversion of
fibrinogen to fibrin by thrombin alone. It can be deduced that as there is no increase
in Pr or TCI`, which measures the extrinsic pathway and the conversion of fibrinogen,
respectively, the antico~ nt activity of the test compounds is confined to the inhibition
of one or more components of the intrinsic pathway, i.e defined by the activation of
Factor XII to XIIa, and/or XI to XIa and/or IX to IXa, and subsequent action of IXa.
These tests do not, however, establish precisely where in the intrinsic pathway that
the anticoagulation effect occurs. It was, therefore, necessary to conduct a further series
of tests to determine what was being affected in the intrinsic pathway. The further tests,
described below in Examples 7, 8, and 9, involved (a) plasma systems using activated
blood Factors XIa, Xa and XIa and (b) a purified components approach.
Example 7
Activated Factor clottin~ times
Respective aliquots of human blood plasma were mL~ced, in vitro, with 15 ~lg ml
of purified bovine Factor XIa (Enzyme Research Labs Inc.), or 300 ng/ml of purified
human Factor IXa (Enzyme Research Labs Inc.), or 9 ng/ml purified human Factor Xa
14
SUBS~ITUTE SHFET
94/22885 !; d`',~ 1 S9 C~ 9 PCT/CA94/00161
(Queen's University, Kingston). All concentrations refer to the final concentrations
achieved in the assay. Each aliquot was then divided into four smaller aliquots and 0,
40, 90 and 180 ~LM of each of compounds 1~ (as defined in Example 6) added and the
clotting time was determined. No anticoagulant effect was observed with respect to
Factor XIa or Factor Xa, but a marked effect was noted with respect to compounds 1-3
in plasma to which Factor IXa had been added. This effect is illustrated in Figure 4.
Example 8
Prothrombinase activity usin~ DAPA
This assay is described by Nesheim et al. in Biochemistry 18:996-1003 (1979) and
was designed to measure prothrombin activating potential of prothrombinase (see Figure
1) contained within highly purified components. Activation of prothrombin was
monitored with a freshly prepared fluorescent thrombin inhibitor DAPA. Reaction
ules consisted of purified bovine prothrombin (1.4 ,uM) in tris-buffered saline (T~.S)
pH 7.4, consisting of 2 mM CaCl2 and 3 ~LM DAPA at ambient temperature. Purified
bovine factor Va was added to 5 nM final, phospholipid vesicles (PCPS = 75%
phosphatidylcholine + 255'o phosphatidylserine) added to 10 ,uM final and the
antico~ nt compounds 1-4 to be tested were added to a final concentration of 100
,uM. The reaction was started with the addition of purified bovine factor Xa to a final
concentration of 5 nM.
No appreciable inhibition of prothrombinase (as defined by Figure 1) was seen
with any of these compounds.
S~IBSTITUTE SHEE~
WO 94/22885 ~ 5 9 6 4 9 PCT/CA94/00161
Example 9
Tenase activity measured chromogenically
This assay was performed to determine Factor X activating potential of an
activating enzyme complex con,plisillg highly purified components. Reaction mixtures
consisted of 0.5 ~M purified human factor X in HEPES buffered saline (HBS) pH 7.4,
cont~inin~ S mM CaCl2, 10 ,uM PCPS vesicles and 0.2 mM chromogenic substrate S-
2222. To 0.7 ml of this ll~ix lul e at ambient temperature was added purified recombinant
F.VIII(rF.VIII) to a final concentration of 2.5 nM followed by thrombin (1 nM final to
activate the rF.VIII). The anticoagulant test compounds were added in a 2 ~1 volume
and the reaction initiated with the addition of purified human F.lXa ( 1.4 nM final). The
cleavage of S-2222 by F.Xa was monitored at 405 nM and the slopes plotted vs the
concentration of inhibitor.
Half m~im~l inhibition was obtained at a concentration of 2 ,ILM with compound
3 as shown in Figure 5. No inhibition was seen with compound 4 (sucrose).
Example 10
Intrinsic fluorescence measurements
Binding of the anticoagulant compounds to and/or induction of conformational
changes in the individual components of the tenase complex (see Figure 1) was analyzed
by changes in intrinsic fluorescence. Samples at 22C were excited at a wavelength of
280 nm at a slit width of 1 nrn and emission was measured at 340 nm. The buffer used
was HEPES-buffered saline (HBS) pH 7.4 containing 5 mM CaCI2 and 0.01% Tween~-
80 and was filtered through a 0.2 ~ filter. Purified proteins were tested as follows:
recombinant F.VIII at a concentration of 55 nM, purified F.X at a concentration of 600
nM and purified F.IXa~ at a concentration of 200 nM. Concentrated solutions of the
16
SUBS 11 l UTE SHEET
NO 94/22885 , ! ' ' 21 S PCT/CA94/00161
test compounds were prepared in a solution cont~ining the protein to be tested. Aliquots
of these solutions were added to a cuvette cont~ining a solution of the test protein at the
same concentration. After each addition, intrinsic fluorescence of the protein was
measured. The inclusion of the protein in the concentrated solution elimin~ted the need
to correct for dilution of the protein during the titration and thereby allowed highly
accurate measurements of small relative changes in fluorescence. Titrations of
anticoagulants covered the concentration range of 0.1 to 30 ~M. The change in intrinsic
fluorescence induced by the addition of anticoagulant was expressed as a ratio relative
to initial fluorescence intensity obtained in the absence of the compound.
Anticoagulant compound 3 caused no change with rF.VIII, a decrease in intrinsic
fluorescence with F.X (Figure 6) and an increase with FIXa (Figure 7). The change in
F.X did not saturate with increasing concentrations of compound 3 and the concentration
of compound 3 required to produce half-m~xim~l change in intrinsic fluorescence was
much higher than the concentration of compound 3 that produced half-m~im~31
anticoagulant activity in the tenase assay (Figure 8). The increases in intrinsic
fluorescence induced by compound 3 on F.IXa,~ were saturable, with the concentration
to produce half-m~rim~l response virtually identical to that for the anticoagulant activity
(Figure 9). No changes were induced by compound 4. Compounds 1 and 2 were
subsequently assayed and found to have very similar values to those obtained for 3.
Oral ~tlministration
Thus far the invention has been described primarily with reference to therapeutic
compositions for ~lminictration to a patient parenterally. The pharmaceutical
composition may be ~tlmini~tered parenterally in the form of a sterile aqueous solution
SUBS~ITUTE S~iEET
215g64~`
W O 94/22885 `~ CT/CA94/00161
or isotonic saline. However, oral ~lmini~tration~ in a pharmaceutically acceptable carrier
such as lactose or calcium carbonate, is also possible, as described in Example 6.
It will be appreciated by those skilled in the art that the demonstrated
anticoagulant activity of the compounds of this invention as assessed by AP~ suggests
that they will have antithrombotic activity as this test is used traditionally to monitor
proven antico~ nt activity of therapeutics such as heparin. The range of increase of
API~ associated with the therapeutic benefit of heparin has been established and the
dose of the compounds required to achieve an equivalent increase in API r has also
been established (see Figure 3). Therefore, assuming that the effect on APl~ by the
claimed compounds, compared with that of heparin, tr~ncl~tes into an equivalent
antithrombotic activity, the dosage of the compounds of this invention can be readily
calculated on a weight per kilogram of body weight basis designed to elevate the APTI
into the therapeutic range, given that for heparin an AP~T of 55-70 seconds can be
achieved at a dosage rate of 0.2 - 0.3 units heparin/ml plasma.
The invention does, however, also contemplate the treatment of surfaces which
come into contact with blood in vivo, extracorporeally or in vitro so as to render them
anticoagulative. It will be appreciated that in recent years silicones and silicone rubbers,
in addition to many other materials, have been used to m~nllf~ct~lre many medical
devices, such as implants and equipment to transport or transfer blood, i.e. tubing, in
which blood comes into contact with the silicone surface. Silicones are inert to human
tissue and the body is able to tolerate silicone materials without undue adverse effects.
However, blood tends to coagulate when in contact with silicone surfaces and this has
been seen as a serious shortcoming to the extended use of such materials. Heparin
coated surfaces have been suggested in the past to overcome this shortcorning, but there
18
~;UB~ UTE SHEET
9.`:
~O 94/22885 - PCTICA94/00161
is considerable difficulty in bonding heparin to a silicone surface. One of the few
effective methods involves exposing the heparin-coated surface to ionizing radiation.
Another method, described in U.S. patent 3,508,959, involves sulfonating a partially cured
silicone rubber by coating the rubber with an organosilane or organosiloxane and curing
the rubber so as to sulfonate the surface thereof. In accordance with the present
invention silicone and other surfaces, such as metals, glass and plastics materials with
which blood comes into contact, may be activated by reacting the surface with a chemical
linking agent, to which a selected compound of the present invention is then chemically
attached.
lg
SUB~ I I I UTE S~EET
