raphy of the airiirie fraction indicated that the saiiw amines were present as in dog urine. Chroinatography of the acid fraction indicated that the saiiip arid ~va. preseiit. Acknowledgment.-The autliors gratefully ackiiowl-
edge the assistance of other members of the research group at A. H. Robins Co.. in particular, that of 1)). I3eriiard I‘ranko and X s s .Josephiiic. Garhcr for thr :iiiimal wurk a i d Dr. Nerndoii ,Jeiikins for syntlmis 01 coiiipouiids.
A Simple Test Tube Arrangement for Screening Fibrinolytic A4ctivityof Synthetic Organic Compounds1
It has previously been .> a irrant-in-aid of the \\ 5nrnmg Heart 4ssociation. N 1 on Iiaulla “C‘lieinistri r i f llliornbolvsls Iiilman I ibrino7\ me?. Charips C ’Thomas P , ~ h l i s h e r ,Springheid, I11 1961, p
Dugni, “byntiiesii of Organ!< ( orngounds W‘hicli Induce I iliiinol esis, Boulder Colo 1961 14) I\. N. \ o n Iiaulla and K. I . < m i l l’roc. hoc L r p l l Bzoi V e d . , 106,
510 (1961). ii I
atid (for a relatcJ group of coiiipouiids) urea (110 L ~ C t i i r i l y) < t liiourea < clt Iiyliii~ea < allyl-2-thioui*c~a .\h a working hypothr4h it was assiuned that hydrot iopy c~ouldbc the coiiiiiioii d(>iioiiiiriatorfor t h e activity of t hcse colnpol1Ild~. I l l oldcl. to 1est the h y p o t h otliw coinpouiid* with hiioivti hydrotropic activity qiiitc different Ytructiirc- 11CY(’ .;titdid Large asyiiii i i o t r ic* hydrotropic anioiis lil1(~ L’.1-diiiietliylberizeiir~wlfoiiatc and 2-naphttialeiic~ulfoliatebrouglit about a, ,?-fold iiiciease iii activity a\ coinpared to iii(~t1iaii~ liercm large asyniinet ric Iiydrot ropic cat ioiia 1 1 1 ~ ( ~ t t s t r~-y1-propyla1ii1ii01iiiii1i hroniide were cwiipl(1t(.I?ii1cffcctive.j Conipouiidh wit ti a carboxyl group at ( , l i d of the hydrophilic side cliaiii were slightly more active than thc oiies with sulf‘oiiic acid. Thercforc, :it presciit , our scwch for hettrr coiiipounds is coiicciiI iatccl on the f o r i i i c ~group. It has bec11i possible 11) vaiyiiig the s1riicturch iiirthor t o illcrease t tie activity i)y a factor of approsiiuately 40 arid to define SOIIIC atl(lit ional structural requiretuerit s for an active c.oii1poulld (for csan1ple Iliscvission).i 11 is lils~lyt1i:it i i i t cmivc scarell i i i u r c active coiiipouiids \vi11 (L\-c.ntually lead t o syiit het ic tliroiiibolytic drugs. l‘or t l i ( w studies, a yuaiititativc. l ~ u rather t elaborate testiiig proctdure has been lived previously.i ’I’hc iiivestigatioii\ \ v c w Iianipered by the lacl, ill (1uaiiiitativcJ tthztiiig ilicthods zuitablc for ~ C I and, iiiost iiiiportant, suitahle for the m e a d faniiliar with I he t echiiiqw of hlood coagulation aiid fibrinolysis. .I ne\\ , siiiipler nietliod, together n i t 11 ~oiiieinstructive cuiiiples oi 1 est results, is brirfly described iii the 1:sprriliiciital section. It IS hoped that the procedure n-ill c>riablcthe organic chenikt to participate in the quest for I hroinbolytic agents. t l i c b
Experimental Principle. -Huinaii pla5iii:i clot& : L ~ Csuspended :it 37” 1 1 1 I .2fter 24-hr. ~iii~uh,it~i~ii. iif :L compound to be tested
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TESTFOR FIBRINOLYTIC ACTIVITY
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the clots are inspected for the extent of lysis which has occurred. Material and Methods. Reagents.-(&) Human plaama is obtained by centrifuging outdated (older than 3 weeks) ACD bank blood (from any blood bank), separating the plasma, snd freezing it in lots at - 15' or lower; the p h m a must be frozen before it is ready for use; plssma from mimals cannot be used; (b) 0.5 M calrium chloride; and (e) hiilfered saline: 0.85% NaCl and barbital acetate buffers (1:4). Glassware.-(a) Vials, 1.85 ml., 9 X 30 mm., Kimax Nr. 60Y30/L; (b) test tubes, 13 X 100 mm. with rim, Kimax Nr. 45042; (e) stirring rads, 3 X 125 mm., Kimax Nr. 40500; (d) pipets, various aises; and (e) rubber stoppers from Becton snd Dirkinson vrtcutainer tubes, series 3204. The stoppers receive a well-centered hore and are slipped over the stirring rods in such a wty that the upper end of the rod protrudes 1 in. above the stopper. Apparatus-(a) All-ginss water bath thermostat set at 37"; (h) aluminum or stainless steel rack to suspend test tubes in water bath thermostat; (c) supporting stand9 for making eylindershaped clots around the stirring rods; and (d) a plastic bar (10.2 X 27.9 X 0.51 em.). The wpporting stand with v i d s i i i place (Figure 1) consists of a bottom plate (10.2 X 27.9 X 1.27 cm.),
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I'igwe I.-St:~iid for p r d x t i o n ,>f pl:~mmclutii. Tlrc c h t s in the vials mid the inserted glass rods mound which they have been formed are seen i n front. The slots far retniiiing the rods are to he seen i n rear of the top plate. 0 1 1 the left side is a, clot inserted into the test solittion.
x 2i.9 x 0.63 cm.), and two supporting columns 3 7 - c m . diameter, 6.99 cm. long, with bosses on each end), all made of acrylic plastic. The bottom plate is drilled with twenty U.89-om. holes, 0.8 em. deep for holding the vials. The top plate has twenty corresponding 0.32cm. holes to closely approximate the diameter of the rods with 0.5-em. s10ta to provide an entry. Both plates have two larger corresponding holes for the bosses of the supporting columns. The stand allows the stirring rods to be placed firmly into the middle of the vials. A plastic bar put on the top plate (see Figure 1) holds the stirring rods in place. This arrangement keeps the rods in a perpendicular position which is an important feature for making suitahle clots. Testing for Fibrinolytic Activity. A. Preparation of Plasma Clots.-The frozen plasma was thawed and brought to 37". The following steps were carried out at room temperature. To 5 ml. of plasma. waa added 0.25 ml. of 0.5 M CaC12. The mixture was inverted once for thorough mixing. Next, 0.5 ml. of the recalcified plasma was quickly pipetted into each of the 10 vials which had been previously fitted into the holes of the bottom plate of the stand. Then the stirring rods carrying the stoppers were inserted through the corresponding slots in the upper plate into the center of the vial. Normally a solid clot formed within about 15 min. Thirty to forty-five minutes after addition of CaCL, vials and stirring rods were both carefully removed from the atand (use both hands). As the next step, the stirring rod was slowly pulled out of the vial together with the plasma clot which stuck firmly on its lower end. Care had to he taken that during this maneuver the clots were not squeezed (by tilting the atirring rod from its vertical axis). The stirring rods with rubber caps and clots were immediately inserted into the solution of compounds to be tested for fibrinolytic activity. The test salu-
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tion (2.5 ml.) had previonsly been pipetted into the test tubes which were hanging from the rack into the water bath thermostat. When the stopper was tightly in place closing the tube, the clot was suspended in abont the middle of the solution. It i s a& visable not to recalcify larger batches than 5 ml. of plasma. B. Preparation of Test Solution.-The compounds to be tested should preferably be Nx or K salts if applicable. They were dissolved in bsffered saline, pH 7.42. Some cumpounds not completely soluble in buffered saline were tested a8 suspensions in the same manner. If necessary, the p H wm adjusted after dissolution. \arir,ue roncentrations were tested such as0.5,0.4,0.3,0.2,0.1,0.075,0.05,0.025,andO01M.Thristhe optimal concentration for a h v i t y can be found. The use oi various concentrations is essential because a compomd might be inhihitory at higher vmicentmtim, effective at t.he middle range, and inexeetive rtt the lower ones. This feature is showii in Figure 2 with o-thymotic acid. Inhibition, activity, and ineffectiveness from left to right are elertrly seen. Figirre 2 shows also how unequivoral the positive reading is.
Figure 2.--\?ater bath ilrernrustiit, test t u l m wmlaining test sohition and clots after 24hr. inu~haLion. Using o-thymotir: acid, note partial clot dissolution, ( +), a t 0.03 ill; crwnplete a t 0.02 nud 0.01 M. dissolution,
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Evaluation of Results.-The results are read after 24-hr. ineubation. Complete lysis of the clot, 0.02 and 0.01 M in Figure 2 is partial, 0.03 M , as (+); and no lysis a8 recorded a8 (remaining concentrations). Complete dissolution of the clot cannot be mistaken: only a bare glass rod i s left; see Figure 2. There is occasionally a slight differenceof reactivity from plasma. to plasma which, however, does not effec.tthe basic information provided by the test. Comparing the results obtained with synthetic compounds with those obtained with enzymatic activators, the instability of the latter on incubation, and their susceptibility to plasma inhibitors haa to he kept in mind. A complete clot disaolutian within 24 hr. was not obtained with as much as 1000 units/ml. of strepti,. kinam in buffered saline, whereas 20-100 units brought about B partid dissolution. However, about 500-1M)O unitsjml. of streptokinase in plasma were required to induce a partial lysis, smaller amounts being ineffective. Urokinase, 50-100 unite (Ploug)/ml., in buffered saline induced a more or less complete lysis.
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Discussion The basic information obtained by the test cancerus priniarily the detection of the fibrinolysis-induciiig capacity of an organic synthetic compound but also shows the striking relationship between the structure of the compound and this capacity. Table I deinoiistrates such a structural relationship with some benzoic acid derivatives which have been studied with the new t,est. Benzoic acid is inactive; however, introductioii of an isopropyl group into the 4-position produces a rat.her active compound. Replacement of the isopropyl group by a t-but,yl group seems to enhance the activity further. Salicylic acid is inactive; introduction of a methyl group int,o Ihe 4-or 5-position results in a mildly active compound. o-Thymotic acid is very active, and swikhing of its carboxylic and hydroxylic group (2-hydroxycymene-3-carboxylic acid) reduces it,sactivity only moderately. If, however, the hydroxyl group of the o-thymotic acid is moved to the para position (p-thymotic acid), the activity is completely abolished. Another area of interest is halogenated benzoic acids. It has been shown previously that these are increasingly active in this order: F (no activity), CI, Br, and I, provided that the halogens are in para position. mtho-Halogenated compounds are ineffective, and the metu ones exhibit an intermediate activity.8 Table I shows the activity of the p-iodobenzoic acid as compared to the p-iodoethoxyethoxyacetic acid. Lengthening of the hydrophilic side chain has doubled the activity. Undoubtedly diligent and systematic variat,ioii of t,he molecules will create much more active coinpounds. To be suitable for eventual therapeut,ic use, a conipound must be able to produce clot lysis after being dissolved in human plasma (in order to duplicate the condition in the human body). With six compounds (see Table I) a comparison of the activity obtained after their dissolution in buffered saline and in human plasma has been made. By dissolution in plasma the activity of the compounds was slightly to moderat,ely reduced, but not lost. All compounds checked for this particular feature were inhibitory in higher concentrations. The active range varied from compound to compound. On a tentative basis, it is assumed that the compounds act as inhibitors. They might inhibit an antiactivator a t lower concentrations, thus freeing the activators(s) present in human plasma to induce a fibrinolytic reaction. At higher Concentrations, the compounds inhibit the fibrinolytic enzyme itself. Acknowledgment.-The technical assistance of M r . Gordon Ens is gratefully acknowledged.
