PH27178A - A pharmaceutical composition including basic placenta angiogenic factor and heparin - Google Patents

Description

a2717% ,

The present invention relates to the ine duction of slaveted levels of endogenous timsus plas- minogan activator (tPA). Specificelly, the present - invention relates to the induction of thesa elevated tPA levels through the administration of & basic placenta angiogenic fector (PAF).

Tissua plasminogen activator rocently has , heen studied ss a potential therapeutic agent for the treatment of myocardial infarctions snd certain other blood clotting disorders. In particular, it has been postulated that tPA will dissolve the blood clots which occur in the coronary arteriss during a myo- cardial infarction, thus ra-opening the coronary arteries end re-sstablishing blood circulation to the portions of the haart muscle which otherwise would have bean damaged during the heart attack,

So However, there are certaln drawbacks to the " usa’ of tPA ss ® therapeutic sgant. In particular, ‘tigsus plasminogen activator has an axtremaly short half life snd no system has yest been idantified or developed which is cepeble of susteining tha elavated tPA levels for the time needed to dissolva not only the clots present in the coronary arteries at the time of the infarction but also the clots which may remain circulating after the infarction and could

, form emboli in pther portions of tha body. These awboli might be responsible for various complice- oo tiona, including gtroke.

To overcome this problem of achieving sug- tained, glevated tPA lgvals in ths circulatory asyatam, the present jnventors have discovarsd that the administration of baslc placenta angiogenic fan- tor (PAF) will incresse endoganous tPA levels. The - basic PAF may be administered over a pariod of img, such as in the form of an intravenous drip, and will cause the circulsting tPA lgvels to remain alavated at least for the duration of its administration.

SUMMARY DF THE INVENTION

An object of the prasant invention is to pro- vida a method for causing elavatsed levels of sndoge-

Lo | "gus, circulating tPA through the administration of ‘a tharapautic agente. Another object of the present invention 1s to provide a mathod for the treatment of myocardial jnfarctions end other blood-clotting disorders by administration of ® tharapsutlc agant gapable of increasing the andoganous, circulating tPA levels.

Additional objects and adventages of tha in~- vantion will be sat forth in part in the description -d-

, which follows, and in part will be obvious from the dascription or may be learned from practice of the fnvention., The objects and adventagas may be realized end attained by means of the instrumentali- tigs and combinations particularly pointad out in the appsnded claima.

To achieve the cbjects and In gccordance with the purposes of the prasent invention, a mathod is disclosed which causes slevated circulating levels of gndogencus or natural ly-produced tPA. This method comprisas administering basic plecenta angioganic factor at a level and for a time sufficient to csusae an alsvation in circulatory tPA levels. It is in- tended that these lsvels remain slevated for 24-28 hours. In addition, the basic PAF may be adminis tered in conjunction with heparin, which, ss ax- plained mora fully harainbelow, will increase its , efficacy.

Co Moreover, the present invantion further achieves tha sbove objects by satting forth a method for the treatment of myocardial {nfarcticns involve ing at least a partiel dissolution of a portion of the blood clots formed in thas coronary grtaries OT in other blood-carrying vessels which comprises! (a) administering basic placants angiogenic - lh -

, factor at a dosaga gufficlant to result in a conti- nuing elevated, circulating tPA lavael capable thare- by of increasing the patential for plasminogen scti- vation; and (b) at least pertiaslly dissolving & portion of the blood clots present in blnod-carrying vessels by exposure to the increased plasmin levele induced by the eleveted tPA level. Another embodiment af thie invention includes the additional step (c) of pre- venting re-occlusion of ths blopd-carrying vagsels by cantinued exposure to the elevated tPA levals.

It is to be understood that both the foregaing - general dascription and the following detailad des- - criptlon ere axamplary and axplenatory only and are not restrictive of the invention, ess claimed.

DESCRIPTION OF THE PREFERRED EMBODIMENT

) - he Reference will now be made in detail to the ‘presently preferred smbodiments of the invention, which, together with the following examples, serve to axplain the principlas of the invantione.

As noted sbovae, the prasent invantion relates to a method of atimulating tha production of ale- vated levels of endogenous tPA in animales, humens ) and cultured cella. Thia method involvas, in part,

, the administration of basic placenta angiogenic factor (PAF). This basic PAF is also known Ba

PGF basic or basic FGF. Besic PA has been pre- viously described and methods for its isolation and recambinant-DNA methods for its production hava been providad in tuo United Stetes patent epplicationa.

These epplications arse U.S. Patent Application Sarial

No. 809,873, filed December 17, 1986, by Moscatelll - gt al. sntitlsd "Human Placenta Angiogenic Fecter

Capsble of Stimulating Capillary Endothelial Cell

Proteasa Synthesis, DNA Synthesis and Migration” and

United States Patent Application Sarisl No. 888,554, filed on July 16, 1986, by Moscatelli st sl., entitled "Human Placenta Anglogenic Factor Capable of Sti- mulating Capillary Endothelial Cell Protease Byn- thesis, DNA Synthesls and Migration." Both of thess a United Statas Patant Applications ara specifically

Co rcorporated herein by reference. Tha compositions ' ‘described by thase pstent applications will be re- ferred to hereinafter as "besic PAF"

In this method, basic PAF 1B sdministered at a dosege sufficient to result in en slevation of circulating tPA lavela. Thess elevated tPA levels sre capable of inducing glevated pleamin lavels.

The exposura of clots in blood-carrying vessals to

, the elevated plasmin levels caused by thess ela- vated tPA levels rasults in at least partiel disse- lution of the clots.

In addition, reoccluslon of the blood-carrying vessels may also be prevented by the slsvated tPA levels.

The results obtained by this method, i.e., at lsast pertisl dissolution of clots, are belisvad tn be obtained in part es a re- sult of certain properties of the basic P/F. 3 Basic PAF, isolated in a purified form or . manuf ecturad through a racombinent-DNA method accord- ing to the procedures sat forth in the Moscatelll at.al. applications, supra, posssssas at least thres particular properties.

Thase include (1) the ability tn cause cell migration, (2) the sbility, in the pre- gence nf serum, to cause csll division (the mitogenic proparty), snd (3) the ghility to induce the synthe- ; gis of protssses by capillary endothelial calls. “ oo **" Tha prasant inventors have discovered en : "additional proparty of basic PAF.

Specifically, when contected with most types of sndothelial calls, . low doses of basic PAF cause the endothelial cells - ta produce, smong other protesses, tissue plasmino- gen sctivator.

Tissue plasminogen activator hss been shown to be produced in vitre in accordanca with this method with a tima~lsg of several hours,

, and will be able to be produced in vivo by admi- nistration of PAF in the manner described more fully hereinbelow.

In addition, ths presant investigators heve discovered that large doses of PAF adminigtared to rabbits elicits a rapid increase jn circulating levels of tPA. This increase is so rapld that it cannot be sccounted for by ds novo gynthaesis but ra- oo flacts a changas in the distribution of existing tPA from compartments of the vescular bed into the plasma,

The administration of basic PA alone to 8 human or animal by a mathod designed to bring the pF into contect with endothelial cells rasults in the production of tPA and is thus ambodiad within tha scope of the present inventian. Howevar, it hes bean noted in some situations that administration

BB of basic PAF es tha sola active $ngredient in a hharmaceutical preparation will stimulate not only tra production but also cell migration and mitoge~ nesls, perticulerly in an arsa denuded of live endo- thelisl cells by a myocardial infarction, This additional result is also dessirsblae because the migretion and division of new andothelisl cells in to the srea of the clot could restora andothallal monolayer in areas in tha occluded vessels whera

, gndothelial cells have died.

In addition, whan combined with heparin, basic PAF retains tPA inducing properties but loses ita mitogenic properties. Thus, administration of =a i 5 pharmacauticel preparation containing both basic PAF and heparin as active ingredients results in a pra- paration which ia contemplated for uss in alterns- tive embodiments of this invention. oo Moreover, it is believed that the same effect, i.8., the blocking of only the mitogenic proparties of basic PAF, may be achieved by combination of the basic PAF with various heparin fragmants. Thass haparin fragments ars among those which sara capeble of binding to the basic PAF,

It is postulated that the elimination of these properties of basic PAF dus to the presance of heparin

Co or heparin fragments might indicate that the pro- "tain which comprisss the basic PAF popsesses at least tuo functions, one of which is responsible for pro- tessa production and another of which possesses the mitogenic end migratory activity. Thus, it is postulated that haperin ia capshle of inactivating the mitogenic function without affecting the proteese- production function.

It is possible that thase functions ars ime . - 9 -

Cen :

, parted by discrate and uwpparoble partlung pf the

PAF molecule. In this cese, it is also anvisionad that the mathad of the prasent jnventlion cpuld be practiced Ly administering a pharmaceutical nrepara- tion whose active gngradient consists of tha portion of the basic PF molecule which possesses an actlve proteasa production fFunation bub which does not poaness an active mitogenic function.

The active composition of the verious prbodi- ments of the present jnvantion is preferably adninis- tered in a 1iquid forme Houwouer , other gdministre- tion farms, gush ag an gphalent mist, ars also envi- aiongd. The preferred carrier 1s 2 physiologic galing golution, but it io contemplated that other pharmacou tical ly-accontanie 1iquid carriers may alan be uead. In One ambodiment, it is preferred that the oe . , Jiquid carrier for tho basic PIF contain a sprotein

Do stabilizer." proferably, the protein gtobilizer is albumin oT heparin. A particularly preferred gto bilizer is plasma ohtained from the patient who 18 to recelve tha hasic PIF.

The baalc pF may ba formulated snta a phar~ maceutical composition by combination of the basic

PF with 8 11iquid carrisr as described shove. Pro- tein stabilizers and heparin may be includsd in the

, initisl formulation or may be added to the prepara- tion immediately prior to administration to tha patient. (nce the pharmaceutical preparation has been formulated, it why be stored frozen or as a dehy- drated or lyophilized powder in sterile vials. It is preferrad that a protein stahilizer be added to the pharmaceutical preparntion prior to dehydration or lyaphilization. Freferred storage is frozen at least -2070. 1% is tn be noted that it in preferrsd that the Lasic FF ia both administered end storad in a formulation that hos a physiological pH. Tt is pre- sently believed that storage and aumirnistration at a high pH, i.e., greater than 10, or at a low pH, i,8., less than f+, is undesirable.

N It is presently preferred to administer the " therspautic composition containing bacle PAF via an intravenous route. A praferred administration route includes the storage of basic PAF at -20° C. in sterile vials, either in the presence of heparin or without. If without haparin, the heparin is added immediately subsaquent to thewing and prior to administration to the patisnte In this prafarred method, the frozen basic PAF is thowed immediately

, prior to administration to the patient. Upon thau- ing, 8 volume auf ficient tO quapend the hasic PIF, usually 1 ml, nf the patient's plasma 1g added to the basic pig. Thin plasma will soprve poth to sus- pand the nasie AF and tn sUPPLY protein which will atubilize the therapeutic materiale

The desired rouse of hasic DiE may pa odninls- tored by bolus or Ly glam drip nither method 3o- tended tO greats 2 rede termined concentration nf the active ingeedinnd in thn pakinnt's Llood supply. The gpenific doan 19 calculated according to the body weight of the patients Tt is anted that the maln= tenance of cirouloting cuncantrations of PF of 1laa8 than Deo nanngramns (ng) per ml of plasma may not he on effective therapy while the prolonged maintenance of circulating 1pvals in axcess oF 5 microgram (ug) he por fl of ploawna WBY have undesirable gide effects.

LL | Accordingly, 1 is prefarred thal doses garly in the therapy De administered in a holus guch that circulating jevels of piF reach an initial lavels af 1-2 micrograms per ml of plasma followed by Hosa designed to keep tho circulating 1avel of PHF at oT above approx imately 50 nanograms per ml ol plasmBe

The time between administration of the bolus and commencement of the maintenance dogas is dependant

, . on the half-life of PAF in the circulation. It is expscted that the tnelusion of heparin or heparin fragmante in the pharmaceutical composition will affect this paramatser.

Further rafinament of the calculations neces- sary to determina the appropriete dosage for treat- ment involving each of the above-mentioned formula- ; ~ tions are routainly mads by thoss of ordinary skill wo in thes ert and sre within the gmbit of tasks routine- ly performed by them without undue experimentation, gspscially in light of ths dosaga information end as- : says disclosed harein. These dosages may be ascer- toined through use of the sstablishad assays for de- termining dosages utilized in conjunction with appropriate dose-response data.

It should slso be noted that the basic PAF formulations described herain may be used for vete- "rier applications, with the dosage ranges haing "the same as those specified sbove for humans.

It is understood that tha application of teachings of the present fnvention to a specific: problem ar anvironment will be within the capabilli- ties of ona having ordinery skill in the art in light of the teachings conteined herein. Examples of the products of the prasant invention and rspra-

nn , sentative processes for their preperation and use appear in the following examples.

EXAMPLE 1 - Induction of Tissue Plasminogen Active- tor Production in Human Foreskin Capil- lary Endothalial (WFCE) Bells hy an

Angiogenic Factor from a Hepatoma Soni- cata.

Isolation of human foreskin cspillsry sndo- thelial (HFCE) cells - Nsonatal foraskins were ob- tained directly after circumcision in the neonatal nursery. Esch praparation wes incubated in Dule- becco's Modif ied Eagles Minimal Essential {iMad ium (DMEM) (obtained from Flow Laboratories (Medium Cat.

No. # 10-331-22) pe 68 (1985) with penicillin (100

U/ml) and streptomycin (1 mg/ml) for 15 min, and the

Co dermal tiasus was excised from the epidermis and

O ‘minced using curved scissors.

CL | Tha tissus was digested with 0.75% (w/v) col- lagenasa (Wonthington) in phosphata-buf fered aaline (PBS) containing 0.5% (w/v) bovine serum albumin (BSA) for 20 min. at room temperature. Medium w 1th garum was added to stop the digestion.

Tha digasted tissue was gently gapirated with a 10 ml pipette and passed through a Nites 110 micron mash’ nylon-covered funnel which allowad small - 1h -

, aggregates of cells to pass hut retained larger pisces of tissue. Tha filtered material was pal leted end gently resuspended in 3 ml of cultura mne- dium consisting of 20% (v/v) heat-inectivated pooled human serum, 30% (v/v) medium conditioned by mouse gsrcoma 180 cells, 50 ug/ml of endothelial cell growth supplement (ECGS) (Collsborstive Research), penicillin (10 U/ml), and atreptomycin (100 ug/ml) {n DMEM (HFCE maintenance medium). .

Human serum samples wers obtained from a hepa- titis testing laboratory. Serum samples from healthy donors taken for routine screening were poolsd end sub jected to centrifugation at 10,000 rpm in a Sore vall GSA rotor to remove cells. Uhen heat-inacti- vated sarum wes desired, the s arum wes incubated at 56°C. for 30 min. The serum wes then filtered. through & Nalgene 0.45 um filter and, 1f necessary, " attired at 4°C. until use. - - A 1.5% (w/v) solution of gelatin (Eastman

Kodak) in PBS was prepared snd autoclaved. Ali quots of the gelatin solution wera added to tissue cultura dishes ssveral hours bafors sasding the calls and allowed to incubats at room temperature. The splution was sspirated snd the dishes were weshed with POS to remove excess gelatine

,

The Mitex filtered materisl, resuspended in culture medium with inactivated human sarum, was plated onto a 60 nm gelatin-coated petrl dish and cells ware asllowsd to attach overnight. The sure

S Pace was thoroughly washed with PBS and fresh medium was added every other day thereafter. buring isola~ tion, viasbla endotheliel cells were racognized as clusters of spproximately 3-10 cellswith cherac- teristic morphology which was easily distinguishable lo from fibroblests, Conteminating fTibrablasts, whare=- aver visible, were mechanically scraped from the dish under direct microscopic observation using a thin glass probe prepared by drawing e gless pastaur pipette through a flems to produce 8 beaded tip : (approximately 0,1 mm). This technigue was carried put with the tissue culture dish on the stage of a

Wild inverted phase-contrast microscope in a laminar oy flow hood. All cells st the periphery of the dish, le. out of visual range, were removed by scraping with a silicone spatula. The medium wes then changed twice to remove floating cells. This process wes repaated occasionally as needed.

Colonias of sndothalial cells were salected weeks of culture using lerge cloning rings end the following trypsinization tachniques. Cells wers

2nnK , washed with PBS and incubated with 0.25% (w/v) hog pancreas trypsin (ICN) in D.4 M NeCl, 0.005 M KC1, 0.025 M Tpia-HCl, pH 7.4, 0,002 1 EOTA for savaral minutes. The trypsinization was monitored by phrase contrast microscopy. When the cells becams rounded and detached from the dish (spproximataly 3 min), the trypsinization was stopped by the addition of gqual or greater volumes of medium with serum

Thareaf ter, the cells wers maintained ss deacribad shove.

The cnlls were subsequently subcultured on 35 mm gelatin-coated patri dishes at a dilution of like

Preparation of hepatoma gonicate - Cells from a human hepatoma call lina, GK HEP=1 cells (Accession No. HTB52, American Type Culturs Col=

Tn lection (ATCC), Rockvills, Maryland), were grown * to'gonfluence in 150 mm dishes in DMEM with 5% : fatal calf serum (FCS). The cells ware then washed twice with ice-cold PBS and scraped from the dish with a silicones spatula. The cella were pooled and goniceted for a total of 3 min, on ice. Tha soni- cate was then clerified by centrifugation at 40,000 ’ rpm in a Backman T4150 rotor for 1 hr. at LC. The supernatant was aliquoted and stored at -70°C. until

, use

Tetra decanoyl phorbol acetate (TPA) treet- mant of cella - Cells were grown to confluence in

HFCE maintenance medium in 35 mm gelstin-coated dishage. Culturss wers preincubated {n DMEM contain- ing 15% human agerum (1.8. without ECGS oT 5-180 cell conditioned medium) for 24 hr. They were than incubated in DMEM containing 15% human saTum with the addition of 2 X ld M TRA for 18 hr. TPA=-CON- taining medium wag prepared fresh by diluting a 2 x 10~% M gtack solution sn 100% EtOH into the med lum and usad ymmadiatelye. For the measurement of plasmi~ nogen activator (pA) In conditioned medium, TPA was added tO gerum-free medium and the incubation end: collection wera parformed as described.

Hapatoma sonicate trestmant of cells ~- Cells oo wara grown to confluence in HFCE maintenance mad ium in 35 mm gelatin-coated dishes, Cultures wers pre- sncubated in DMEM containing 15% human garum (1.8. without ECGS oT 5-180 cell conditioned mad ium) for ol hr. They were then incubaterd in OMEM containing - 15% human SeTum with the sddition of 10% hapatoms sonicate In pas (Final concantration 0.1 mg/ml) for 18 hr. Hepatoma gonicate was prepared as dagcribed above and thawed ymmadiately before USRe

277K ,

Plasminogen activator assay = Plasminogen activetor was assayed by ths 1251 ¢ibrin plate me thod described by Unkeless gt ale in Je Expe Mad. 137: 85-111 (1973), apecifically incorporated here- in hy refarence. The =sssays ware performed in 96 well Linbro trays with 3 cn’ surface arsa/wslle

Each well wes coated with 30 ug of plasminogen-fras 125; fibrinogen with @ specific activity of approxi mately 2000 cpm/uge. The platas wera dried for 72 hr. at 37°¢c, Fibrinogen vas converted to insoluble fibrin by the addition of medium containing 2.5% serum as a source of thrombin, After a 3 hr. incu- bation, the wells wera washed twice with water and gtarad at 4°Cc. until use.

Copditioned medium and Triton X~100 deter gent axtracts of cells were prepared and assayad gspantislly as described by Grass st.Ble. in J. Cell nv ‘Biol. 95. 974-981 (1982), specifically incorporated

BE herein hy raefarance. Serum-frae conditioned medium was harvestad and cellulsr dabris waa removed by centrifugation et 2000 rpm in en IEC cantrifuge with a 284 rotor for 2 min. Monolayers wers washed twice with POS, and tha tells were scraped from the dish in 250 ul of 0.5% Triton in 0.1 M Tris-HCl, pH 8.1 using a ailicone spetula. Cell nucle} wers

, removed by low-speed centrifugation at 700 rpm for 10 min. Somples wera stored at -20%C. until use.

Miquots of cell extracts (1 ug) er condi- tioned medium (25 ul) were added to duplicate wells in 0.5 ml of 0.1 11 Tris-HCl buffer, pH 8.1, contain- ing 4 ug of purified OF P-Treatad human plasminogen (preporad from humon plasma by the method of Deutsch and Mertz, as described in Scisnce 170: 1095-1D% (1970), specifically incorporated hersin by raference) i 10 and 0.025% BSA as carrier protein. The assay was in- ! cubated in a humidified atmosphere at 37°C. One- hundred ul aliquots were removed from duplicate wells at ? hr. end soluble 1257 fibrin degradation products were counted in a Packard gamma scintillation counter.

Results are expresgad os a parcent of the total re- lsaschle counts as measured By the addition of tryp- pin to duplicate wells in gach essay. Standard t curves ware prepared in each assay by measuring the activities of a stendard renga of urokinasas samples.

Semples wera tested for plasminogen-independent pro=- tesss activity by the omission of plasminogen fram the incubatlon buffer.

Protein determinations - Protein determina- tions of cell extracts wers made by the Blorad

Coomassie Blue staining technique, using bovine

, gerum albumin 08 a gtandarde plasminogen activator production by HEFCE galls - PRA 1pvels were measured in both the call 8x= yracts (ce11-associated) and, ln some casas, 8eru™ free conditioned medium of confluent cultures of

HFCE cells grown under yar ious conditionge PA javels were measured in wany gipferant jgplates of WCE cells, with the results of b jgplates ghown in FiQe 1, Cell cultures ware grown to confluence as des gribed aOVBe pefore initiating an axperiment, the cells were preincubated in DMEM containing only 15% human garumn for 24 NWT. This was nacaesary tacauss the presente pf ECGH in the growth mad ium elevated jguels of basaling PA activity in untreated culturase

The high hasal levels of PA resulted in 8 ducreas® in the cnlculated atimulation py TPA and hapatom@ ; ne sonicate to approximately p-fold. A 24 hTe prain- cubation in the gence of both cas and 5-180 cell conditioned mad ium 1pwerad the pagelline PA gotivitye

TherefoTres at the gtart of the experiments tha cul- furs medium wad changed yo DMEM containing 15% human garum with OF without TPA BE 2% 1077 M or hepatom® gonicate at 0.1 mg/ml The culture? ware sncubated averniohts and the naxt day the conditioned mad lum was temoved, and cell axtrects ware prepersd ag des~ ani , cribed above, The PA activity in 1 microgram of cell protein ues routinely measured as described above.

Tha cancentrations of both TPA and hepatoma gsonicate tested were chosen because they were ahoum to glvs maximal atimulation of PA in Novine Cepillary

Endothelisl (BCE) cell cultures 88 dascribed gy Gross gt al. in Je Cell Ginl., supra; end Gross, et al. proc. Natl. Aead. Scle a0: 2623-2627 (1963), speci- fically incorporated herein by reference. Doae-ras= nonee assays showed that TPA at 2 x 10~7 M and hepe- toma nonlcate ot 0.1 mg/ml gave optimal stimulation in the

HFCE cells.

Figura 1 shous the PA levels maoasurad in gx tracts of cultured HEFCE cells, In pach isnlate tested, PA lavels in untreatsd cultures Ware rels- tively lou compared to GCE cells. Both TPA and hapa= , tomo gonicate produced an pnhancemant of PA activity © over untreatsd control cultures tn avery isolate tested, The degres of stimulation of PA activity veried betuean different isolates hut was sluays five- to fiftesn- fold above the basal levels of! un= treated cultures for both TPA and crude hepatoma sonicate-trastad cells. Tha raason for this verie- tion 1s unknown, The inoraasad fibrinolytic sotivity

, nas plasminogen-dependant; in the sbssncae of plagmi- nogen, no ectivity was seen. BCE cells contained re- * lativaly high levels of PA in untreated cultures end responded to treatment with TPA with increesed levels of PA,

The reaults of this gxperiment ahowed that

HWFCE cella respond to both TPA and an angiogenic fector present in the hepatoma sonicate with ine cresses in cell-associated fA activity. The present investigators have damonstrotad that ths human hapa- toma calls, 5K HEP=l, produce an angiogenic factor that is equivalent to P/F Bs characterized in the two U.3. Patent applications of Mogcatalli st al. discussed above. It 1s concluded that tha effect of the hepatoma cell sonicate on the induction of PA in HFCE cells could be fully substituted by purified

PAF « exwirie_2 Identification Of The PA Produced hy

HFCE Cells In Rasponsa to stimuletion OF

Hepatoma Cell Sonicats na Tissue Type PA 35,~cystaine labelling of cell cultures -

Celle were grown to confluence in stenderd maintse nance medium. Control cultures were alsa grown to confluence in standard medium. Thase included RAPMI=

, 7272, a human melanoma cell line known to produce high levels of tPA, as described by ni jkin, D.C. and

Collen, D., J. Biol. Chem. 256: 7035-7041 (1981), and human embyronic lung cells, a call strain knoun to produce high levels of uPA and described by Rifkin in J. Cell Phys. 97: 421-427 (1978), specifically incorporoted herein by reference. Cultures wera pre= tneubated with DMEM containing 15% human serum (i.s.,

ECGS and 5-160 conditioned medium wers removed) for 24 hr. The cells were treatasd fer 16 hr. with or without TPA or hapatoma sonicata in DMEM containing 15% human serum, The cells were than preincubated in

DMEM without cysteine for 2 hr. Finally, the con- fluent cultures of HFCE cells were radio lsballed for 5 hr with 35 cysteine (50 uCi/ml) in DMEM without gystaine containing 2% (v/v) dialyzed ponlad human sarum,

TERRE Imnunoprecipitetion - Cell extracts were prepared far immunoprecipitation by a mndification of the method described by Stanley, J. R. et al. in Cell 24: 097-903 (1981), specifically incorporated harein by refarance. Conditioned madium was harvested from the dishes and clerified by centrifugation at 2000 rpm for 5 min. The cell monolayers were washed 3 times with cold PBS, lysed in 250 ul of AIPA buffer - 2h -

, (0.05 M Tris-HCl, pH 742, containing 1% Triton X-100, 1% sodium devxycholats,, 0.1% sodium dodecylsulfate (SDS), De15 M Mall, 1 mM EDTA, 2 mM PMSF), screped from the bottom of the dish with & silicones spetula, 1aft on ice for 10 min, and clarified by centrifuge tion at 10,000 x g for 10 min. BBefore specific immunoprecipitation, saeliples were prashsorbed using non-immune rabblt serum and protein A-Sepharose to raduce nonspecific binding. Fiftean ul of normal rabbit sarum wera incubated with the samples in 8 to- tal of 1 ml in RIPA buffer overnight at 4°C. with mix- ing. Twenty ul of packed protein A-Sepharose beads ware addad with mixing for 90 min. at 4°c, Tha pel- lets wore collected by centrifugation in an Eppen- dorf microfuge, and the gupsrnatants were sub jected to specific immunopracipltation. Immunoprecipita- tion was performed using gaturating amounts (20 ul) " HF rabbit antisnrum to human urokinase plasminogen '" ‘getivator (uPA), or to human ten, for 16 hr. at uc.

Twenty ul of packed protein N-Gepharose beads were added for 90 min, at 4%. and immuna complexes on peads ware palleted by centrifugation. The pellets were extensively washed with RIPA buffer (five washes of one ml each) and H,0 (twice), and boiled for 2 minutes in reducing sample buffer containing 5% P= - 25 = nl 1

, marcaptogthanol and applied to 5.16% gradient 5D0S5- polyacrylamide nels as described by lapmmli, U.K. in Nature 277: 600-685 (1970), specifically incor- porated hernin by reference. Immediately after slnctrophorasis, the gnls were fixed and processed for fluorogrophy. gpS-polynsrylanids gel elsctrophoreals - GDBe polyacrylamide gel plontrophoresis wes prtrformed in a slob gel apparatus uning the discontinuous buffer system of Leemmll, SUprf. The separating gnl can- glstard of a linear 5 to 16% zcrylaomide grodient; gtacking gels wera 3% acrylamide. Protein semples wore mixed with agqual volumes of 2X sample buffer to a final concentration of 0,0625 0 Tris-tiCl, 10% gly- gerol, 2% DS, 0.001% Bromophanal Blur, pH 6.8, and 8% o_merceptor thanol and boiled for 2 min. The fol lowing proteins were used as mnlecular weight ,. sghandards: p-.galactosidase My, = 130,000), phospho® : rylass A (My = 90,000), bovine serum albumin (My = 88,000), aldolase (M = 43,000), soybean trypsin in hibitor (My = 20,000}, and lsctalbumin (M. = 14,200)

Gels were fixed and stained using the silver stein method of Wray et ale, Pnal. 8ipchem. 118: 197-20 (1981).

Flurography - 5DS-polyscrylamide gals of 355.

, cyasteino-labelled proteins were processed scoording to the procedure of Bonner and Laskay as described tn Fur. J. Diochome 46: 83-80 (1974), specifically incorporated herrin hy refaranca. After PPO=-DMSD im- pregnatlon, the dried gels ware expnged to praflashad kodak XAR=5 film at -70°C. for 2 weeks.

Lo Characterization of 2A py immunogprecipitation -

Two types of PA have been described: tissue-type PA

Co (tPA) and urokinase-typas PA (uP). Each FA has 8 characteristic molecular weight in 505=~ polyacryle

Lo amide gels: tPA approximately G6 daltong, uPA 50K dsltons.

Endothelial cells have basen thnught ta be a aource of ti°A. It has bean shown to be produced by endothelial calls cultured from human umbilical vein (Levin, £. G., I'roce Natl. /cad, Sci. 80: 6804-6808 (1983) and bovine aorta (Levine and Loskutoff, Je . Oell Binl. 93: 631-635 (1982), Howsver, thease cella were obtained from large vessels. Since the vest majority of the andothelium is comprised of micro- vessel calls, thay may be an important source of tPA, lMoscatelli, D. A., Je Call Alochaem, 303 19-29 (1986) characterized PA made by bovine capillery en- dothelial cells under unstimulated conditions as well ss after stimulation with TFA or hepatoma soni-

, cate. Using both immunoprecipitation techniques and biochemicel assays, he showed the presence of tPA. Tissue-typa FA was {dentified as a hroad band of fibrinolytically active material of Me spproxi- mately 66K to 93K daltons. After langthy labelling and incubation periods, a minor amount of uPA was identified. Cell extracts and conditioned medium were also prepared from cultures treated with TPA or hepatoma sonicate. It was shown that there was a quantitative and not gualitative change In the PA species produced. Levin and Loskutoff, (1982) supra have also hoswun thet bovine sortic gndothalisl cells produce both types of PA, whose molecular waighte are in agreement with those deseribad hy Moscatelli.

Based on thesa results, thers smems ta be little difference in the type of PA produced by bevine capillary endothelial cells and bovine aortic ando-

Bh thelial calls, The circulating tPA is proposed to be derived from both large vassel end microvassel endotheliasl cells, although the surface area of the microvasculature is much larger end thoss cella may be tha major source of tPA.

To characterize the typa of PA produced by human microvesssl endothelial cells, HFCE cells were grown to confluence undar standard maintanance condi

JIE

, tions. They were then trestad with or without tPA or hepatoma sonicate. Sixteen hours after treatment, tha cells were rardinlabelled for 5 hr, wlth 35. cysteine at 50 uli/ml in tha presence of tPA or hepa- toma aonicate as described above. Conditioned medium was harvested and cell extracts were prepared and sub jected to specific immunoprac ipitation with anti- gerum to tPA. Samples were first praabsorbed with normal rsbibit serum. Immunoprecipitates wera sub-

Jected to 5DS-polyncrylamide gel electrophoresis end fluorngraphy as described. fis previously noted by the fibrin-plate assay, thars appears to be little PA in untreated HFCE cule tures as scen by only a faint band with an My in the range of 66K daltons. The bend was not present in the immunoprecipitate obtained with preimmune sarum.

However, tPA is easily dotected in the immunopreci- pitates of TPh-treated cultures as 8 broad band at ‘the molecular weight range of the NPM1-7272 standard (66K-93K). There is also an increase in tha amount of immunoprecipitateble tPA in the hepatoma sonicate- treated cultures. Thus tPA ig present only in low amounts ln the cell extracts of untreated HFCE cells.

Thera is an incresse in thse amount of tPA in TPA- or hapatoma gonicate-traated cultures as Judged by - 29 w=

, immunopraecipitation with antiserum raised sgalnst the

Tissue-~type PA was alao smmunoprecipltated from the conditioned medium of untreated, Thh~treated, and hepatoma sonicate-treated WFCE cells.

HFG mell conditioned wadium from untraated culturss contains little or no discernible tPA a8 measured hy ianunoprecipitetion. Howevnr, tPA is geen os a broad band in the molecular weight range of 66% ta 93K daltons in tmmunoprecipltated material from TRA-traanted HFCE call conditioned medium, Hepa toma sanicate alan produced an increasa in tha smount of immunopracipitatable tPA in the conditionad medium of HFCE culturas.

The tA immunnprecipitated from both tha cell extracts ang conditionsd media showed tha presence of a broad band corresponding to an Me of mproximate- ky 66K to 93K. The broad range is similar to that : obtained hy Moscatelld supra for BCE cells, Levin and Loskutoff, J. Cell Blol. Sb: 631-636 (1982) for bovine sortic endothelial cells, and Lavin E. Be,

Proc. Natl. Acad. Sci. 80: 6804-6808 (1983) for HUVE cells. Thase high moleculer welght forms of tPA hava been shown to he due to complexes formed between the thn and an inhibitor of FA which is slso produced by

, ths HUVE cells (Levin, 1963 supra) amd bovine aortic andotheliol cells (Loskutoff, at al., Proce Natl. fend. Sci. 80: 2956-2960 (1983)). It is likely that the high molecular weight formg of tPA geen here are also enzymo-inhibitor complaxof.

Imnunopreripttation of HFCE cell exiracts end conditioned medium weg 2lsn performed using antiserum prepared against nrokinage-type PA (uP), No radio- lehelled proteins were specifically immupnoprecipi- tated from either the cell extracts or conditioned med ium of either untreated or tranted cultures, Human ’ embryonic lung cells, known to produce urokinase, were sub jected to {mmunoprecipitation as a zantrol.

The rasulis were confirmed by fibrin auto- graphy sccording to tha method of OGranelli-Piperno and Reich, J. Exp. Med. 143: 203.224 (1978), specifi- cally incorporated herein by rnferenca. Aliquots of ,. cmrll extracts and conditioned medlum of both stimulated and unstimilated HFOE cell cultures were sub jected to 5DS-pnlyacrylamnide gel electrophornsis and laid over plesninogen-cantalning fibrin-sgar uels. Lysis zonas were observed only at themnleculor weight range of tPA, in the range of 66K tn 93% deltons. No lysis was seen at lower molscular weights, even with pro- longed incubation times.

,

Therefore, it appenrs that t0A is produced by human endothelial cells ir culture in low amounts.

Stimulation of the calls by either TA or hepatoma ponicate resulted in an increase in FA in hoth tha call axtrant and conditioned medium,

Urakinane-typa PA activity in HPCE cells could not be detected elthar by immunoprecipitation or hy biochemical assays of PA activity in fihrin-agar gela.

EXAMPLE 3 The effact of heparin on tigsue plesminogan activator (tPA) stimulation in bovine capil lary endothelial (BCE) cells by humen pla~ cental angiogenic fectar (hPAF).

Oovine capillary endothelial (BCE) cells werd japlated from the bovine sdrenal cortex and grown Bs described previously by Gross et al., 8UpPI8, apaci- fically incorporated herein by reference. The calls ware grown in alpha modified minimal essential medium oo NEM) supplemented with 10% (v/v) celf serum and anti- " blotics (penicillin 10 U/ml and streptomycin, 100 ug/ ml). GOefore assay, cells ware passaged with trypsin-

EDTA as described in Example 1 onto 35 mm dishes end allowed to grow to confluency.

Human placantal angiogenic factor was jisplated as described in the two U.S. Patent spplications of

Mascetelli et al. discussed above with the following

, modification. After elution from haeparin-Sspherosa, the active fractions ware dialyzed against U.2 mM / NaCl, 20 mh HES pH 6.0, clarified by cantrifugation at 100,000 g Tor GO min end loaded on a FPLC-mono 5 polumn equilibrated with the same huffer. The active protein was eluted with 8 gradient of 0.2 to 0.7 M

NaCl in 20 mM MES, pH 6.0. The active froctiona wers determined by bio assay on HCE cells ea described pre- ; viously by foscatelli pt al. 1n Proc. Natle Aca. Sci. lo a3: 2091-2095 (1986), specifically incorporated harain by reference.

Plasminogen Actlivetor Assay - Confluent cultures of HCE cells that had been maintained for at least two days in alpha MEM in 5% calf sarum were changed to fresh medium containing different amounts of baslc

PAF, as determined by protein assay, in the abgance or presence of heparin (50 mg/ml). Heparin, porcine “ intastinal mucosa, grade II, 176 units/mg, was pur- chased from Sigma (St. Louis). After incubation at 37° for 16 hours in @ humidified stmosphere of 10% CO, 90% air, the cell layers wera washed twice with cold ne phosphate-buffersed salina (PBS) pH 7.5 and wers ax- trected with 0.5% (v/v) Tritan X-100 in 0.1 M sodium phosphate pH 8.1 and the cell extracts esssysd for plasminogen sctivator (PA) amctivity as described in

,

Example 1.

In the sbsence of heperin, en increase in tha lavala of PA in cell extracts was seen at doses of basic PAF of 0.3 ng/ml and 10 ng/ml. The EDgq, the half maximel responses wes seen at approximately 3 ng/ ml. This corresponds to en increase from epproximate- ly 4 units of urokinass activity to spproximatsly 30 munits of urokinass activity. In the presence of 50 ug/ml heparin, thers wes 8 similar increase in the levels of PA with increasing levels of basic PAF starting at 0.3 ng/ml and continuing to 30 ng/ml.

At the lowest doses of haperin tasted, evan in the shsence of basic PA, there was an incraasa in the base levels of PA production, Howavar, there was no substantial changs in the EDgp nor was thare any sig~ nificent difference in the amount of atimilation in the prasence of heparin once background levels wers ., subtracted when compared to the stimulation seen in ' the sbsence of heparin.

Therefore, ths inventors have concluded that heparin does not block the ability of basic PAF to gtimulate PA activity in BCE cells, This contrasts with the reports of heparin blocking the mitogenic. effect of bovinas besic fibroblast growth fector, = molecules which is 98% homologous to banic PAF, oo - 34 -

,

Bovine basic fibroblest growth factor is reported to have virtually no mitogenic activity in tha pre- gence of haperin when tested on severel different types of andothelial calle. Massoglia, gt Ble

J. Cell Physiol. 27: 121-136 (1986).

EXAMPLE Lb Induction of Circulating Concentrations of Tissue Plasminogen Activator by Intre- vanous Administrstion of Placental Anglo- genic Factor } | Rabbits weighing 3.5 = 5.0 kg wera anesthetized with a combination of ketamine and xylazine during the course of the experimants. Placental angiogenic factor (PA) was adminiaterad in a voluma af 0.5 1.0 ml by injection into an ear vein. Arterisl blood samples wers takan from the opposite ear.

Blood samples were collacted immediately prior to administration of tha peptida snd at various time " pints thersafter. In a typical assay, blood samples ' ‘would be collected at 30 seconds intervals from the time of injsction to 5 minutes following injection.

Additional blood samples at approximately 10 minutes post-injection were routinely taken, Inhibitors of tissue plasminogan activetor end of plasminogan wers prevented from associating with thelr target pusteasas by ecidification of the blood semples immediately

, upon collection using a modification of tha proto- col described by B. Wiman, Be, gt _8l. in Clinica

Chimica Nota 127: 279-288 (1983), specificelly in- gorporated harein by reference. Briefly, a 0.5 ml pemple of blood was mixed immadistely upon drawing with O.4 ml of 1.0 M acetats buffered to pH 3.9 with

NgOH, end 0.05 ml of 3,2% w/v trisodium citrate.

Semples ware centrifuged to separate cells from plasma and 0.1 ml of plesma was added to en sdditional 0.11 ml of acetata buffer end 0.1 ml of 0.12 M Tris buffer pH 8.7. This solution was then incubated at 37°c. for 20 minutes to inactivate inhibitors of plesmin and plasminogen gctivator. Tusnty microliters oo of this solution was used in subsaquent plasminogen activator BABAYB.

Tissus plasminogen activator wes measured gssantially ss described by Ranby M., 8t 8l., in . Thrombosis Reaaarch 27: 743-749 (1982), specifically - incorporated herein by refersnce. Tha chramagenic plasmin subatrate 5-225). was rapleced in this mssay by benorlsucyl-hexshydrotyrosyl-lysine-para-nitro- anilide, (Spactrozyme Ply from Americen Diagnostica

Inc., Greenwich, CT). acidified and heat treated plasma samples were diluted 1:40 in 0.12 M Tris buf- for pH 8.7 containing purified plasminogen and the

, chromogenic plasmin substrate. The resection was . initlatad by the addition of des A fibrin and conti- nued at 37°C for 1-20 hours depending on ths ssnsi- tivity required. Fibrin and plasminogen depandance of the resction ware criteria for tPA activity.

Using this essay protocol, the in vivo effect of human PAF was tester in rebbits., A singles dose of 0.6 mg in phosphate bufferad sslines was givan,

Circulating levels of tPA rose in responses to thia doses to approximately twice thas unatimulated level within 3 minutes end remained elevated for saveral minutes tharsafter. This demonstretes that PAF can act in viva to elevate circulating levels of tPA,

Further, human PAF was, on a molar basis, mora active in this regard than alther deg~amino-D-arginine®- vasopressin (DDAVP) or bradykinin. Control rabbit were injected with phosphate-bufferad saline and a stowed no increase in eirculating tPA levala. - This demonstration that PAF 1s interacting with target cells in the vascular bed and promoting a biological responsa is consistent with tha cone tention that the peptide is blologically active in the circulation st least long enough to stimulate its natural receptor. Furthermoras, thes in vitre data that capillary endothelial celle have a second, sus-

, tained response in which tPA synthesis and secra- tion is induced and continues even after the stl- mulus (PAF) is removed. Therefore, it is expected that the prassent in vivo demonstration of tPA release in responses to PAF predicts the ability of PAF to mediate the second longer term responss As well. The second response does not require the continued pre- sence of PAF but is sensitive to the dose end length of the time that P/F ia maintained in the circula- tion.

It will be epparent to those skilled in the art that verious modifications and variations can ba made ta the processes and products of the praesent 1n- vantion. Thus, it is intended that the present in- vention cover these modifications and variations of this invention provided thay come within the scope of the appended claims and their squivalentse.

Claims (1)

1. WHAT I5 CLAIMED IS: 1, A pharmaceutical composition comprising basic placenta anglogenic factor PAF and heparin in a pharmaeceutically-accepteble carrier wherein the heparin is present in an amount sufficient to ste- bilize the PAF.

   DAVID A.

   MOSCATELLI DANIEL B.

   RIWKEN ' ! DAVID F.

   CARMICHAEL Inventors ! \ ! cL yo