CA2033140A1 - Thrombolytically acting combined preparation - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A thrombolytically acting agent comprises a combination of a non-fibrin-binding form of the thrombolytic agent v-PA and a fibrin-binding plasminogen activator.

Description

2~33~

T~ROMBOI,Y~A~I.Y AC~N~ C~INlb~ION ~ I02~

~ro~ ~1~ :
~is app~ica~ion iæ z~ ~nt~nu~tion in pa~t o~ U.~.
Applia~tlon Seri~l No. 07/479, 427, i~iled ~ebruary 1~, 19~0, c~rxs~ponding to P~dex~l R~public o~ ~:erm~ny Applicatiotl~ P 3~ 04 5~0, of P`sbruary 13, 198~ and P 3~ 17 ~4~r o~ M~y 30, 198~, who~e ~ntire di~ lo~ure~
~re h~reby inco~pox~t~d by re~rf:ncs herein.

Thi~ i n~en~ion relate~3 to n~aw agQnt~ with ~hro~olytic actio~.
~rh~ombQ~ oacur by ~h~ ~ormation o~ ~loo~ ~o~ in ?od ve~ . VenoUa thr~bos~, ln~l~din~ lun~
eml:oli~ms, and a~t~rial ~htoml~o~o~, lncludin~ a~u~a hlyo~ar~ial in~ar~tion~, are dlst~ngui~h~l. Lung ~mbolisms ~nd ~nyoaardial inP~rc;~tion ~re liiE~-th~e~tenlng oacJurx~n~, whi~h re~ e media~l e~rgen~y lnte~v~ntion .
Be~id~ er~n~ inva~iv~ ~nethod~, in ret:~l3nt y~r~, ~nzymatia th~oml:301y~s w:ith pl~mino~n ao~iv~to~s h~v~
slain~d ~c~eptanc~ ~ th~r~y ;~or ~r~erial Andlv~nou~
th~ ubR~C~ n~ d ~ ~hrQm1o~1yti~
a~nt~, ~onv~r-t pla~minog~n, the in~ctiv~ p~o~nzyrn~ o~
tl~e ~ibrinoly~ia ~y~t~m in ~he blooca, ~nto th~ ~atlv~
2~ prot~olytlc ~nzy~ p~a~min. Pl~ n, in t~l~n, di~olvo~
the ~ibrou~ ma~rlal ~lbrln, an ~s~entlal ~ompon~nt o~ a blood ~lot, whi~h l~a~ ~o ~he r~ning ~P tha alo~d ve~e~ an~ ~o ~he r-3~0ns~itu~ion o~ the b~ood ~low~ ~ut pla~mir~ relAtiv~ly ur~ap~al~i~ pro~ e~ i.e. ' once ~ q:~ 3 ~

for~d in thQ blo~ des~:roy~3 the pro~eolytlc c: omponl3nt~ in the l~lood whlch ~re a}: ~301utely ~ n~
for an intact h~m~;ta~ . g ., i~ ri~oq~n) And thu po~ibly in~uc:e~ dançJ~r of bleedin~ ri~3ks.
Th~ ~irst generatioh thrombolytic:: ~g~nt~;, 5trep~0kina5e a~d uro~cina~a, are ~;U~3tana~ ~hich, ~nce inj ected into the cir~ula~ion I ~y~tem~tlcally convert pla!3minoyen into pla min and induce ~ ~y~te~n~tic pro~eoly~i~. Throm~oly8i~ therapiee with the~e ~ub?it~nces are, there~o~, often accompani~d by bleeding c~mplication~, The ~tbrin-~peoifi :: thron~bolysis, d~loped a~ A re~ult, in whiah the r~com~ nant pla3minc~gen acti~tor~ o~ the ~ ue typ~, called t PA, ~r~ u~ed, should l~ad ou~ o~ ~:hi~ dile~a. t-PA ha~ only a slight af~inity ~or pla~mino~en in th~ cir~ulatory By~tem~ But in the pr~s~n~e oP th6!! fibrou~ melteri~l ribrin, wi~h whi~h it ~acts on ~p~ciPi~ ~inding ~iteQ, this ~ffinity i~ inc:r~ ed ~y ~ multiple, whlan re~ults in pla~min ~or~n~tion in ~ thrombu~ ~ur~ace. Thi3 oorlaept wa~ abl~ t~ rerified ln ~tr~ and in anlmal experim~nt~tion t~ts, but the allnical ~tudles ~howed thAt larg~ amounlt~ o~ t-PA ~re ne~e~ry to cau~e ~ rapid ;olu~ion o~ a coron~L~y thrombo~
Howe~rer, wh~n ~u~h high dv~s o~ t-PA are in~u~ed~
tl~er~ r~ult~, 6imllar to the aa~e~ o~ ~trept~kln~e and uxo}cinase, a ~;y~tamatic prot~olysis ~::ormected With aL
r~alative bl~3~ding Xi~k~ To~l~y ~her~ i~ t;alk o~ ~nly A
relaki~rR ~ibrin ~p~ci~ialty of t,he t-PA~ Irhe rea~orl :eor thi~ i5 ~In ~ nt1al p~o~erty 0~ ~ha t-Pl~ Thi~ mo~ aul~
i~ ~ p~ot~ , Wh~ h llnd~3r ~vc~r~le aontiit;ion5 ~high 6In~yme~ ~onC~ tr~tion, long 2xpo~ure tim~, high ~ub~trat~a o~n~en~r~tion, op~lmal pH, arl~ lon m~diun~) co~Qrt~
pl~in~g~n ln'co pla~min ~v~n ~n ~h~ Ab~nae of ~lbrln.
All ~h~ con~litlon~ ~r~ me~ in pr~nt ~lini~ tandard 35 th~r~py Wi~h t-PA.
In th~3 s~arch Pc~r ~or~ ~paai~lc pla~minvgRn ~ativ~ s~ whiah m~et the crite~rion o~ ~ibrin - ~ ., . . ,. - . ; ~ . . . . : . , :

. : ; : . , ~' : . . : , 2~

speci.icity, a n~w, n~tur~l fibrinolytiaally aat~vç3 substanc~3 wi~h th~ d.Q~ na~ion v-P~ ~a~ DSPA :~rom Desmoclu~ Salivnry Pla~mino~erl Ac:tl~a~or) h~ n ~und.
See, e~., Buropean Paten~ Appl~c~tion ~a~liqa~ No.
0,383,417, and ecauiYalent U.~. Patent Appl~aation ~rial No. 07/47~,4~7 o~ February 13, 1~0, ~hc~e etltlx~
di~:alo~ures arç~ incorporated by r~ferenoe herein.

~hi~ inv~nti~n rel~ta~ to a ~:hron~olyti~lly a~ti~re agent a~mpri~ing the nvr~ rin-birlding lower mol~cular wai~ht form~; of DSPA b~ v PA,-~) and ~SPA ~na (~r-PA~) with mole~ular waigh~E; of 4~i,000 and 42,5)00 (~urope~n Patent Appli~:at~n Pul~lic~tion No. 0,38~,417 and IJ~.
Applicatlon Serial No~ ~79, 4Z7~ and a ~ibrin-bindlng pl~minogen activator O
Suitable ~ fibr~n-bindin~ pl~minogen activa~or~
are, e.g., prouroklna~ u-Pa), t~Pa, and the hi~her m~lscul~r weigh~ ~orm o~ th~ thro~olyti~ agen~ ~PA
tl)SPA alphal ~Vu-pa-~1) an~ r)SPA alpha2 ~v-PA-~2) ~ }SuropR~n Patent Applic:ation Pub~ atlon 21O. 0j383,417, ~nd U.S.
Ap~lloation ~erial No. 47~, 427~, with ~ mol~culAr w6~igh~
~f 52, oO~.
~h~3 m~le~ula~ weigh~ given here repre~en~ morQ
accurate value~ th~n tho~e repor~ad ln the m~ntioned prior ~pplication~ for the ~ame ~ub~tanc~ ut ~h~ ~
~ubs~anoe~ ar~ the ~ame.
Th~ thromboly~ lly ~ lv~ ingr~dierl~ v-PA ( ~PA~
. r~pr~s~nta a t~ew, r~atur~lly oc:currin~ pl~ino~n ao~lv~tor w~ich dis~olv~ bloo~ olots in the hum~n ~ody 3 0 and t}lU~ uit~bl~ ~or ~r~a~m~n~ oX , ~, . g ., myo~rdial in~arotion (Europ~An P~nt ~pliaati~n ~bliaatlon N~.
0,383,417 and U.S~ Appliaation ~orial N~. 47~,4:27). It 1~ ~ound ln th~ in all ~p~ie~ o~ ba~ h~
~nu~ in sm~ll corlce:ntration~ and i~
~xpre~ ed :~rom th~ c~ oE the ~ rary gl~nd~ o:~ thi~
anin~al ~enu~ at~ o~ t~l~ genu~ ~e~modus ~p~ mpri~e , , , ~

, '. . ~ : ~ , ', ,, . :

: . . . . .

~ ~3 3 ~

all bat Itype~ o:e th~ Americarl aon~inent. The g~nu~
~odu~ o~ Centr~ rlcs arld MeX tao w~sl ex2lmin~d e~pecially well.
~he invention furth~3r r~lat~ tR pharmal::eUtit:~hl S agent~ on the b~ 3 o the ahotte ~escribe~ combin~lon og non-f~rln bindins~ and flbrin-bindlng pla~m~nogen aativator~, a~ w~ th~3 u~ual auxili~ry ~gent~ and vshlale~
Th~3 f il: rinolytia potency of v-PA ( DSPA), in c~raparison With th~ u~U~l pl~minogen a~lvators~ 1 ~tr~3ngthened ~till ~or~ by the above~de~arib~d comblnation uf i~ibrin~ lnd~ng ~nd non-l~ibr~ n-bindmg pl~Ls~noç~en actl~rator~.
Either or both ~ th~ non-i~r~n-~nta~ning low molecular weight :EOI:`m9 0~ v-PA can ~e combined with the~
~lhrin~bindlng pl~mino~ tivatorO ~ t~l amounta ~f hoth ~he f ib~in-bindirlg and ~on-f ibrin-b~ndlng ~omponent~ ar~ ~ e . g ., ~or d~ily par~ ral aclmini~tra~ion to humans, 5-500 mg, pre~rably 20-200 mg. The r~iQ ~
non-~flbrin-b~nding componen~ to fibrin~indin~ a~p~n~nt ~an vary ~rc~m 1/100 to ~00/1, pre~erably 1/10 to 10/1.
~en~rally, ~h~ two typ~ ~$ ag~ re ~imult~neou~ly admin~ ~ter~d, pr~3ferably in 1;he ~a:ne do~e unit, but ~an al~o ~ ~dl~lni~tared ~equen~ially in eal~h~r 2 5 order a~ long as b~th are bio~v~i~ ab~Q ~ ~he ~!:a~Q l;lm~ .
Adminl~tration i~ typicall~ analogou~ ~o ~hat ~or t~PA~
~he preferred rout~ ~ a~mini~ration i~ in~r~v~nou~ly.
Without f~rther ~labor~ n, it i~ h~lieved that one ~kill~d in ~he art aan, u~in~ the prG3~ding ds~crip~tion, 3U utillz~ th~ pras~n~ lnv~n~ion to itl~ ~eUlle!8t: ~x~ant- Th~
~ollowi~g pre~rred ~p~ ia ~mbodi~en~s ar~, ~here;eo~e, ~o b~3 ~on~tru~d a~ merely illu~ativa anA 21~ ll~ltakive Or ~.h~ re~maln~er o~ the dl~lo~ur~ ln ~ny way wh~t~oov~r .
Tn th2 fo~egc)ing and in ~h~ Pollowing ex~ 38, Zl 3 5 t~mperaturs~ ~re ~t i~or~h uncorr~acked ln degre~
Cel~ t ~nd, unl~6 oth~rwi~ indi~at~ 11 p~r~ arld p~arc~3n~ag~ are by we~ght.

~, , Tl~ sntire di~closllr~s o~ all applisatiora~, p~t~3nt~
~nd publications, cit~d a)~ove and beîow, ~nd D~
corre~pondin~ Fe~r;ll R~pu}:~lic o~ G~.rmnny Appliç~ion P
~9 43 2~1.6, fil~d Decemb~r 22, 198~, are hereby ~n~orporatad by referenae.
D Y A 2~ P I. ~S
Fibrino~en ana pl~minogen are brought to coa~ulatlon in a P6~tri dl~;h by addition o~ thro}nbin.
t~oles o~ 3 ~m in dia~eter are punah~d in the r~ul~in~
~ibrin lay~ar, in whic:h ~ow v-PA i~ ~dded in dif~r~ t concentration~ and comlc inatis:~n~ o h mol~s~ular w~lght i~orms o~ th~ fibrin-~p~ai~i~ pl~s3~nog~n aativator v-PA;
ai~te~ inaubation at 37 ~ in a moi~t ch~ r, p~ dUc:~
concentration-dep~ndent ly~i~ h~lo~.
lg I~ the di~fl3r~n1; ~ole~ular we~gh~ PA for~ ar~
now cvmbined wi~h one 24nc)t}~er, ly~ halo~s ~r~ produced whic:h ar~ cl~arly gre~ter ~han the ly~i~ h~lo~ whioh w~u1d b~ ~2xpected ~rom a 8implel additi~re Q~ec~ By the c~nbination of high ~oleoul~r and low ~ol~oular w~ight v-O PA, a 6yne~giE;ti~ ef ~ct o~ ~h~ rinolysi~ 1~ cb~ained in Yitro. Thi~ ~ynergi~tia ef~ec~t i~ ~o ~ a~arib~ to the ~ac:t that th~ low~r ~ole~ular we~gh~ ~orr~ o v~
which lacks the ~ingP.r dom~in, can be l~e~r di~u~d in a fibrin clot ~y it a5~-~ibrin-binding property an~
the~ un~loc:k the blood cl~ y lt~ ~ibrinolyt~
aativity. The hlgh mol~cular w~ight~ Elhr~n-binding ~r-PA
varlant aan thu3 ~ompl~ly ly~e the already unbloc:Xed ~lbrin clot.
~alc~ul.a~ion~ o~ t.hQ re~:ult~ o~ thi~ Pibrirl pl~le~
te~t have 13h~wn that by the a~ination of ~he ~wv V-PA
~orm~, a clearly ~ynergi~ia e~ect is achieved ~r~nbaum, ~S.C. Clln. ~xp. Immunol. ~, 1~ 77~ .
Fr~m khe ~r~going de~crlption, on~ ~}clll~d in th~
ar~ ~n e~oily a~cRr~ain the ~ ntial char~ic~rl~ o~
this imre~ntion ~ind, without ~part;ing ~o~n ~he ~piri~ ~nd ~ OpQ th~r~of, ~in m~k~ variou3 ~ihan~e~ and modi~i4~tion~

: , . . .

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~J~ ' oi~ ~he invention to Adapt it to var~ ou~ u~g~ an~
::on~i~lQn~
The pr~ealng ~xampl~s CAII be repe~ed w~th ~imil2~r succes~ hy sube~i~u~ g th~ ~en~rlcally or spe~ ally de~icribed reactantæ ~n~/or op~3r~tin~ conditi~ o~ thi~:
1 nvent~.on ~or tho~3e u~ed ~n the pr~a~ding oxample~ .

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Claims (14)

1. A pharmaceutical composition comprising thromobolytically effective amounts of the non-fibrin-binding form of V-PA having a molecular weight of about 46,000 or about 42,000 or both said forms, and a fibrin-binding plasminogen activator.

2. A composition of claim 1, further comprising a pharmaceutically acceptable carrier.

3. A composition of claim 2, further comprising both of said forms of V-PA.

4. A composition of claims 3, wherein said fibrin-binding plasminogen activator is t-PA, the form of V-PA
having a molecular weight of about 52,000, or sou-PA.

5. A composition of claim 4, wherein said fibrin-binding plasminogen activator is the form of V-PA having a molecular weight of about 52,000.

6. A composition of claim 5, wherein said fibrin-binding plasminogen activator is t-PA.

7. A composition of claim 1 in the form of a single dose unit.

8. A method of achieving a thrombolytic effect comprising administering the non-fibrin-binding form of V-PA having a molecular weight of about 46,000 or about 42,000 or both said forms, and a fibrin-binding plasminogen activator.

9. A method of claim 8, comprising administering both of said 46,000 and 42,000 molecular weight forms.

10. A method of claim 9, wherein said fibrin-binding plasminogen activator is t-PA, the form of V-PA
having a molecular weight of about 52,000, or scu-PA.

11. A method of claim 9, wherein said fibrin-binding plasminogen activator is the form of V-PA having a molecular weight of about 52,000.

12. A method of claim 9, wherein said fibrin-binding plasminogen activator is t-PA.

13. A method of claim 9, wherein all active agents are administered in the form of a single dose unit.

14. A method of claim 9, wherein all active agents are administered simultaneously.