`Amselem
`
`[54] THERAPEUTIC COMPOSITION HAVING
`AN INHIBITING ACTIVITY ON BLOOD
`PLATE AGGREGATION
`
`[75] Inventor: Armand Amselem, Toulouse, France
`.
`[73] Assignee: Centre d’Etudes pour I’Industrie
`Pharmaceu?que, Toulouse’ France
`
`[21] Appl. No.: 671,496
`_
`[22] Flled:
`
`Mar- 29, 1976
`
`[30]
`
`Foreign Application priority Data
`
`Apr. 18,
`
`France .............................. ..
`
`[51] Int. Cl.2 .......................................... .. A61K 31/625
`[52] US. Cl. ................ ..
`.
`.......... .. 424/232
`[58] Field of Search .............................. .. 424/230, 232
`
`[11]
`[45]
`
`4,080,447
`Mar. 21, 1978
`
`[56]
`
`7,303,503
`
`References Cited
`N PATENT DOCUM NTS
`F0
`REIG
`E
`2/1973
`
`France.
`
`OTHER PUBLICATIONS
`Chem. Abst. (1) 77 — 14689 H, (1972).
`Chem- Abst- (2)73 - 11899341, (1973)
`Primary Examiner-Stanley J. Friedman
`Attorney, Agent, or Firm-—Young & Thompson
`[57]
`ABSTRACT
`This invention relates to a therapeutic composition
`comprising, as active ingredient, a combination of 5-(2
`ch1oro-benzyl)-4,5,6,7-tetrahydro-thieno[3,2-c]-pyri
`dine and acetylsalicylic acid, the components of the
`combination being both present in the free state or as a
`pharmacologically acceptable Salt.
`composition is
`useful both preventively and curatively for the treat
`ment of thrombo-embolic diseases.
`
`8 Claims, No Drawings
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1015, p. 1 of 5
`
`
`
`1
`
`4,080,447
`
`2
`II PHARMACOLOGICAL INVESTIGATION
`Said investigation related to the inhibiting activity on
`blood plate aggregation of the composition of this in
`vention.
`1. Experimental procedure
`Wistar strain rats, having a body weight of not less
`than 400 g, are fasted 24 hours prior to the test, while
`being maintained under slight anesthesia with diethyl
`ether. A jugular vein is exposed after incision of the skin
`and dissection of the subcutaneous tissues.
`The venous puncture is effected by means of a 5 ml
`Plas'tipak B.D. type syringe with a BD 19 G l l/4 nee
`dle and containing 0.32 ml sodium citrate solution.
`Blood is taken until the 3 ml mark is attained.
`Mixing is insured by tumbling over the syringe 3
`times in succession and the citrate containing blood is
`immediately transferred to a polyethylene or silicone
`glass hemolysis tube.
`A ?rst centrifugation is effected at 100 g during 30
`minutes; it gives a platelet-rich plasma (P.R.P.) a por
`tion of which is collected. A second centrifugation is
`effected at 600 g during 20 minutes; it provides a plasma
`having a low platelet content (P.P.P.)
`The platelet concentrations are determined in the
`P.R.P. and the P.P.P. by means of a Coulter type appa
`ratus. A suitable mixture of both plasmas provides a
`plasma containing 600,000i-20,000 platelets per mm3;
`the plasma thus reconstituted will be used in all aggre
`gation determinations.
`1.1. Determination of ADP-induced blood plate ag
`gregation
`0.4 ml plasma are placed in a siliconized tube pro
`vided with a bar magnet, also siliconized. The tube is
`introduced in a Bryston type aggregometer connected
`to an optical density variation recorder.
`The thermostat-controlled bath is maintained at 37° C
`and the magnetic stirrer is set at 1100 r.p.m.
`When, after a suitable period of time, the base line
`representing plasma transmission has reached a stable
`value, 0.05 ml of a solution containing 10 PM A.D.P.
`(adenosine diphosphate) are added. Platelet aggregation
`induces an increase of light transmission; this is fol
`lowed by a decrease during the disaggregation stage
`and, thus, the maximum optical density variation re
`corded characterizes the extent of the aggregation.
`1.2. Determination of collagen-induced blood plate
`aggregation
`-
`The above procedure is used, except that the A.D.P
`solution is substituted with 0.05 ml of a collagen suspen
`sion (E420 : 0.27) (bovine tendon extract). The resulting
`aggregation is characterized by the value of the initial
`velocity of the phenomenon, measured by the slope, at
`the beginning of the curve, expressed as AD.O./mn.
`2. Results
`2.1. Collagen-induced aggregation (initial aggrega
`tion velocity AD.O./mn)
`' Three groups of animals were used, each group com
`prising 20 rats.
`_
`The ?rst group was administered 100 mg/kg ticlopi
`dine, by gastric intubation; the second group was ad
`ministered 100 mg/kg aspirin, under the same experi
`mental conditions; and the third group was adminis
`tered 100 mg tichlopidine + 100 mg aspirin.
`- The experimental results obtained are given in Table
`I.
`
`THERAPEUTIC COMPOSITION HAVING AN
`INHIBITING ACTIVITY ON BLOOD PLATE
`AGGREGATION
`This invention relates to a therapeutic composition
`having an inhibiting activity on blood plate aggregation
`comprising, as active ingredient, a combination of 5-(2
`chloro-benzyl)-4,5,6,7-tetrahydro-thieno[3,2-c]-pyri
`dine (or ticlopidine) and acetyl salicylic acid (or aspi
`rin), the components of the combination being both
`present in the free state or as a pharmacologically ac
`ceptable salt.
`Ticlopidine was previously described in French pa
`tent application N" P.V. 73,03503, ?led Feb. 1st, 1973; it
`has the following structural formula:
`
`LII
`
`10
`
`N-CH,
`
`Cl
`
`20
`
`25
`
`Ti clopidine, and its pharmacologically acceptable
`salts, have pharmacologically useful inhibiting proper
`ties on blood plate aggregation.
`Acetylsalicylic acid and its non-toxic salts have long
`been known for their antalgic, anti-in?ammatory and
`antithermic pharmacological properties. Recently, it
`was found that aspirin is capable of modifying blood
`plate aggregation by inhibiting same, preventing the
`formation of blood-plate aggregates which are responsi
`ble for arterial thromboses.
`~
`According to the invention, it has now been found
`that, in the presence of acetylsalicylic acid, ticlopidine
`has a markedly superior hemodynamic activity, both
`qualitatively and quantitatively.
`This action is apparent from the results of the phar
`40
`macological and clinical investigation reported below
`together with the toxicological investigation.
`
`30
`
`35
`
`I. TOXICOLOGICAL INVESTIGATION
`45
`Said investigation related to:
`(1) The acute toxicity of ticlopidine, of aspirin and of
`the combination of this invention: in white Swiss strain
`mice, the LDSO/ 72 hours was found to be 2.3 g/ kg in the
`case of ticlopidine, 1.1 g/kg in the case of aspirin, and
`1.8 g/kg in the case of the combination of this invention
`in a ratio of l g ticlopidine for 2 g aspirin; in white
`Wistar strain rats, the LDSO/ 72 hours was found to be in
`excess of 4 g/kg in the case of ticlopidine, 1.5 g/kg in
`the case of aspirin and 2.7 g/kg in the case of the combi
`nation of this invention in a ratio of l g ticlopidine for 2
`g aspirin;
`I
`(2) The chronic toxicity;
`(3) The. delayed toxicity;
`(4 ) The local and systemic tolerance. -'
`It is found that the composition of this invention is
`well tolerated, under the conditions used for the experi
`ments in animals, whether on oral, parenteral or rectal
`administration, without causing any local or systemic
`intolerance reactions. Similarly, investigation of the
`acute toxicity of the composition provided no evidence
`of synergistic toxicity but, on the contrary, a decrease of
`the LDSO.
`I
`
`65
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1015, p. 2 of 5
`
`
`
`4,080,447
`
`TABLE I
`After treatment
`2 hours
`3 hours
`velo-
`velo-
`city
`city
`0.005
`0.008
`
`I%
`96.7
`
`1%
`94.5
`
`1 hour‘
`velo-
`city
`0.110
`
`q 1%
`26.7
`
`Velocity
`before
`treatment
`(control)
`0.150
`
`0.170
`
`0.035
`
`74.9
`
`0.008
`
`95.3
`
`0.015
`
`89.4
`
`0.030
`
`4 hours
`
`veo
`city
`0.015
`
`1%
`90.0
`
`82.4
`
`Treat-
`ment
`Ticlo-
`pidine
`Aspirin
`Ticlo
`pidine
`+
`Aspirin
`‘1% = percent inhibition
`
`0.160
`
`0.003
`
`98.1
`
`0.002
`
`.
`
`98.8
`
`0.003
`
`98.1
`
`0.005
`
`96.9
`
`2.2. A.D.P.-induced aggregation (ADO max =
`maximum aggregation)
`The same experimental procedure is used, group I
`being administered (gastric intubation) 100 mg ti
`clopidine/kg, group II being administered 100 mg as
`pirin/kg and group III being administered 100 mg ti
`: clopidine + 100 mg aspirin/kg.
`The results obtained are given in Table II. It is appar
`ent from said results that, whatever method is used .for
`the determination of blood-plate aggregation (with
`A.D.P. or with collagen), the composition of this inven
`tion exhibits potentiated inhibiting properties on blood
`plate aggregation, with respect to its individual compo
`nents; at the dosages used in the above tests, the synergy
`is predominantly apparent from the extension of the
`anti-aggregation effects: the anti-aggregation effects are
`superior to the sum of the activity inherent to ticlopi
`dine and of that inherent to aspirin.
`TABLE II
`
`15
`
`20
`
`25
`
`30
`
`The platelets contained in the supernatant from the
`?rst centrifugation are counted. Suitable dilution of the
`supernatant is effected with the P.P.P., to give a plasma
`containing from 200,000 to 300,000 platelets/mm3, this
`mixture representing the platelet-rich plasma (P.R.P.).
`1.1.4. Determination of the aggregation
`The determination is effected in an aggregometer
`[photometen the thermostatically controlled cells of
`which (37° C) contain a bar magnet stirred at constant
`speed (1100 r.p.m.)].
`In view of the optical density differences observed
`from one plasma to the other, it is necessary to calibrate
`the photometer between two terminals: one is obtained
`by adding 0.3 ml P.R.P. in a cell containing a bar mag
`net, and adding thereto a volume of buffer equal to the
`volume of solution which will contain the aggregation
`inducing agent. The other is obtained with 0.3 ml P.P.P.
`to which is added an identical volume of buffer. The
`determination is carried out with a cell containing 0.3'
`
`Before
`treat
`After treatment
`ment
`4 hours
`2 hours
`3 hours
`1 hour
`ADOmax
`(control) ADOmax I% ADOmax I% ADOmax 1% ADOmax I%
`
`0.165
`0.185
`
`0.115
`0.180
`
`30.3
`2.7
`
`0.080
`0.190
`
`51.5
`-2.7
`
`0.090
`0.178
`
`45.5
`3.8
`
`0.120
`0.183
`
`27.3
`1.1
`
`0.170
`
`0.070
`
`58.8
`
`0.060
`
`64.7
`
`0.065
`
`61.8
`
`0.080
`
`52.9
`
`Treat-
`ment
`Ticlo
`pidine _
`Aspirin
`Ticlo
`pidine
`+
`Aspirin
`
`III. CLINICAL TESTS
`The results obtained in laboratory animals made it
`possible to test the composition of this invention in
`humans.
`1. Experimental procedure
`1.1.‘ Determination of blood plate aggregation
`1.1.1. Principle
`The method comprises the in vitro determination of
`blood plate aggregation, in the presence of various ag
`gregation-inducing agents.
`'
`1.1.2. Collection of blood samples
`Blood is taken by direct venous puncture with a
`plate-bearing needle. Use of a syringe should be
`avoided. The blood is collected in a plastic tube, over
`3.8% sodium citrate, in a ratio of l/ 10.
`1.1.3. Preparation of a mixture having optimum plate
`let concentration
`-
`The blood is centrifuged at 1500 r.p.m. during 10
`minutes; the supernatant. is decanted by means of an
`unwettable device.
`The resulting sediment is again centrifuged at 3000
`r.p.m. during 30 minutes, to give a plasma having a low
`platelet content (P.P.P.).
`
`45
`
`50
`
`55
`
`60
`
`65
`
`ml P.R.P. heated to 37° C to which is added the solution
`of aggregation-inducing agent. Some authors use one
`volume of solution containing the aggregation-inducing
`agent in 0.1 ml, others, a much smaller volume.
`1.1.5. Aggregation-inducing agents used
`1.1.5.1. A.D.P.
`ADP is a physiological mediator involved in blood
`plate aggregation.
`.
`1.1.5.2. Collagen
`A veal tendon extract, collagen, is available in freeze
`dried condition. It will keep for about two months, after
`rehydration. To be diluted and incubated at thetime of
`use, at 37° C, during a period of time which varies with
`each batch.
`.'
`1.1.6. Analysis of the resulting curves
`1.1.6.1. ADP
`Different ADP concentrations are used. At low con
`centrations (0.1-1 11M), ADP induces a reversible ag
`gregation. At intermediate concentrations (l-3 uM),
`there may be observed an aggregation induced by the
`added ADP (exogenous ADP) followed by partial dis
`aggregation and by a secondary reaggregation, the lat
`ter being due to the release of ADP contained in these
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1015, p. 3 of 5
`
`
`
`4,080,447
`6
`5
`on ADP-induced aggregation and nil in the test with
`platelets (endogenous'ADP): this is the so-called “dou-'
`collagen,
`ble-wave” phenomenon.
`»
`is receptive to the inhibiting activity of the composi
`At high ADP concentrations (3-10 11M), a single
`tion of this invention, said activity being much higher in
`aggregation wave is observed, due to the mer-gence of
`5 the case of ADP and resulting in total inhibition of the
`the two preceding phenomena.
`.
`collagen-induced aggregation test.
`The standard procedure involves preparing different
`"
`'
`ADP concentrations (0.5, l, 2, 5 and 10 11M). The phe-
`IV‘ THERAPEUTIC APPLICATIONS
`nomenon is characterized by the maximum transmission
`In view of its outstanding inhibiting activity on blood
`obtained during aggregation, the apparatus being set in
`such a manner that 100% corresponds to P.P.P. and 0% 10 plate aggregation, the composition of this invention is
`to P.R.P.
`applicable to all diseases which produce a pathological
`'
`:
`1
`1.1.6.2. Collagen
`modi?cation of blood plate aggregation, such as throm
`A single concentration is used. A latency stage exists,
`bo-embolic diseases, for example. Therefore, the com
`obtained on aggregation, after which the platelets re-
`position of this invention may be used both preventively
`lease ADP and induce blood plate aggregation. The 15 and curatively.
`.
`following are determined:
`V. PHARMACEUTICAL FORMULATION
`01(mn) : latency time on aggregation
`In the above applications, the composition is prefera
`03(mn) = half-aggregation time
`bly administered by the oral or the rectal route, the
`A = maximum percent aggregation
`20 active ingredients being combined with the carriers and
`I
`1.2. Investigative procedure:
`excipients suitable for such routes of administration.
`Mr. Pierre D .
`. . was administered, twice daily, dur-
`Generally, the aspirin/ticlopidine weight ratio, in the
`ing 7 successive days, two 250 mg aspirin tablets; the
`composition of this invention, is greater than 1 and
`following week, he was administered daily, during 7
`preferably comprised between 2 and 4.
`successive days, the composition of this invention as
`Each unit dose may advantageously contain
`tablets containing each 250 mg aspirin and 125 mg ti- 25
`0.050-0.7 g ticlopidine and 0.100-0.9 g aspirin, the daily
`clopidine. In other words, Mr. D .
`. . was administered,
`dosage regimen varying from 0.050 g to 2 g ticlopidine
`during the ?rst week, 1 g aspirin per day and, during the
`and from 0.100 g to 4 g aspirin.
`second week of treatment, 1 g aspirin + 0.500 g ticlopi-
`Non-limiting Examples of pharmaceutical formula
`dine per'day. Mr. Jean R .
`.
`. was treated in an identical
`manner, the test materials, however, being administered 30 tions of the composition of this invention are given
`in the reverse order. Thus, Mr R .
`.
`. was administered,
`below.
`during the ?rst week, 0.500 g ticlopidine per day and,
`during the second week of treatment, 1 g aspirin +
`0.500 g t1clop1d1ne per day.
`.
`1.3. Results
`The results of the blood-plate aggregation tests are
`tabulated in following Table III.
`TABLE III
`Percent aggregation
`Test with ADP.
`Test with collagen
`0.5,i1v1 111M ZuM 5;.LM 1011M
`0,
`0, ‘
`A
`20
`70
`80
`83
`81
`1.2
`1.9
`68
`28
`54
`69
`74 ,
`78 '
`1.8
`3.3
`60
`
`l - Tablets
`
`Ticlopidine
`35 Aspirin
`te
`l?aile starcht
`T?inesmm S em
`
`0.150 g
`0300 g
`gaggg g
`0:050?
`
`.
`
`.
`
`.
`
`Determi-
`nation
`Time
`Time 0
`+ 7 days
`
`Subject
`
`Mr.
`Pierre D..
`
`Mr
`Jean R..
`
`+ 14 days
`Time 0
`+ 7 days
`
`+ 14 days
`
`10
`12
`14
`
`8
`
`21
`31
`24
`
`15
`
`33
`46
`30
`
`2O
`
`50
`70
`49
`
`36
`
`53 _ no aggregation
`74
`1.4
`2.1
`60
`1.4
`2.1
`
`61
`58
`
`44
`
`no aggregation
`
`agnes1um stearate
`
`.
`
`.
`
`.
`
`.
`
`.
`
`L
`.
`zimé?m tablets
`The following facts are apparent from the above
`Ticlopidine
`results: Mr. Pierre D .
`.
`. who was administered, during
`11:15pm".
`the ?rst week, aspirin only and, during the second
`Talc
`week, the compos1t1on of th1s 1nvent1on:
`is sparingly receptive to the inhibiting activity of 55 Shellac _
`.
`.
`.
`.
`Gum arable
`asp1r1n, because the test results show there 1s practically
`Granulated Sugar
`no signi?cant variation of ADP-induced aggregation
`Titanium dioxide
`and there is moderate inhibition of_'collagen-induced
`30:33:22,311” S
`aggregat1on, relatlng to the aggregatlon kinetics,
`Ticlopidine hydrochloride
`is highly receptive to the inhibiting activity of the_.60 Aspirin
`composition of this invention which has a clear effect
`Egnesium swarm
`on ADP-mduced aggregatlon and WhlCh produces a
`4-Dosage-bag
`collapse of collagen-induced aggregation.
`Ticlopidine
`Mr. Jean R . who was administered, during the ?rst
`#2111“
`week, ticlopidine only and, during the second week, the 65 Magnesium stearate
`
`composition of this invention: _ is sparingly receptive to the inhibiting activity of
`233??“
`t1clop1d1ne, sald inhibiting being moderately signi?cant
`Semi-synthetic glycerides, q.s. to make
`
`0.050 g
`
`0.100
`g
`0.100 g
`0100 g
`0.020 g
`‘mes
`0.010 g
`0050 g
`traces
`"aces
`0150 g
`0100a
`8:8}82
`
`0.200 g
`813(5):; 2
`0.200 g
`ggggg
`3 g for 1
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1015, p. 4 of 5
`
`
`
`4,080,447
`
`7
`-continued
`
`suppository (adult)
`
`8
`in a chemical form selected from the free compound
`and a pharmacologically acceptable salt thereof.
`4. Therapeutic composition having an inhibiting ac
`tivity on blood plate aggregation comprising, an active
`ingredient, a combination of 5-(2-chlorobenzyl)-4,5,6,7
`tetrahydro-thieno-[3,2-c]-pyridine, hereinafter called
`ticlopidine, and acetylsalicylic acid, hereinafter called
`aspirin, in unit dosage form, each unit does containing
`0.050-0.7 g ticlopidine and 0.100-0.9 g aspirin, the
`weight ratio of aspirin to ticlopidine being greater than
`one and up to four.
`5. Therapeutic composition as claimed in claim 4,
`wherein the active ingredient is combined with a phar
`macologically acceptable carrier.
`6. Therapeutic composition as claimed in claim 4,
`wherein each component of the combination is present
`in a chemical form selected from the free compound
`and a pharmacologically acceptable salt thereof.
`7. Therapeutic composition as claimed in claim 1,
`wherein said weight ratio is about 1:1.
`8. Therapeutic composition as claimed in claim 1,
`wherein said weight ratio is about 1:25.
`*
`*
`* * 1*
`
`Having now described our invention what We claim
`as new and desire to secure by Letters Patent is:
`1. Therapeutic composition having. an inhibiting ac
`tivity on blood plate aggregation comprising, as active
`ingredient, a combination of 5-(2-chloro-benzyl)
`4,5,6,7-tetrahydro-thieno-[3,2-c]-pyridine,
`hereinafter
`called ticlopidine, and acetylsalicylic acid, hereinafter
`called aspirin, formulated for the daily administration of
`0.050-2 g ticlopidine and 0100-4 g aspirin, the weight
`ratio of aspirin to ticlopidine being greater than one and
`up to four.
`2. Therapeutic composition as claimed in claim 1,
`wherein the active ingredient is combined with a phar
`macologically acceptable carrier.
`3. Therapeutic composition as claimed in claim 1,
`wherein each component of the combination is present
`
`20
`
`25
`
`30
`
`35
`
`45
`
`v50
`
`55
`
`65
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1015, p. 5 of 5
`
`
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