0024_2-methyl-2-tert.butyl-ketolactone, an anti-adhesive drug.pdf

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Pharmacology 4:152-162 (1970) Anti-adhesive drugsThrombosisPlatelet aggregationRed cell aggregationPeripheral circulation

2-Methyl- 2-tert.butyl-ketolactone,an A nti-adhesive Dru g Preventing Plateletand Red C ell Aggregation

H I B I C H E R 1Department of Anatomy, Medical University of South Carolina, Charleston, SC

The causal participation of blood cell aggregation in thrombusformation has been described by several investigators under clini-cal [3] and experimental [4, 14, 15] conditions. Of the blood cellsinvolved, special attention has been given to the erythrocytes andplatelets. These cells seem to possess the ability to start andaccelerate the chain of events that leads to organic vessel occlusion.

Knisely [12] and Bioch [3] observed long ago the pathologicalsignificance of intravascular red ceil aggregation (sludge). Borg-strSrn [6] was able to demonstrate a direct etiological relation be-tween experimentally induced sludge and venous thrombosis in therabbit. In previous work [1] we suggested that intravascular redcell aggregation may be a factor in the induction of anoxic myo-cardial damage.

The importance of platelet aggregation for hemostasis and forthe pathogenesis of thrombotic processes is,~well established [4, 18].The adherence of platelets to each other and to other surfaces isconsidered a fundamental step in the formation of plugs andthrombi [14].The purpose of this paper is to assess the potential therapeuticvalue of a group of drugs that share the common property of pre-venting both red cell and platelet aggregation in vitro and in vivo.Six different methods have been devised or adopted to evaluatethis action on a quantitative basis, and with their aid, a newchemical compound, 2-Methyl-2-tert. butyl-ketolactone, subsequent-

Work done as partial fulfillment for a Ph.D. Thesis, Tel Aviv University.Received: January 27, 1970.


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Bicher 15 3

ly referred to as substance 86, has been compared with the al-ready known compounds and developed as a prototype for thiskind of potential anti-thrombotic drug.

Materials and MethodsADP Sigma Chemical Corp., USA.High molecular weight dextran (Rheomacrodex, M.V. 40,000), Pharmacia,

Upsala, Sweden.Phenylbutazone (Butazolidine) Geigy, Basel, Switzerland.Chloropromazine, Specia, Paris.86 and derivatives of the same ketolactone were synthesized according

to the method described by Taub ~ et al. [21] (fig. 1).

o~R /\O O

Substance 86 = R1 MethylR~ tert.-butyl

Fig. 1. The chemical structure of substance 86.

A. Platelets1. The Testing o[ Anti-platelet Adhesioeness Properties of Chemical Compounds(a) The Rolling Tube Platelet Adhesiveness Test

This method is a modification of the procedure described by Wright [22]for clinical investigation.

Blood is obtained with a plastic syringe by venipuncture of the antecubitalvein in the experiments carried out with human platelets, or from the femoralartery through a polyethylene canula in nembutal anesthetized cats. Sodiumcitrate 3.8Uo, in a proportion of 1 : 10 is used as anticoagulant.

0.4 cc of blood are placed in a 10 cc, 1.2 cm diameter non-siliconized testtube containing 0.1 cc of the added solutions (solvents tested drug). Thetube is then slowly rotated on its longer axis, 16 times/min, for 2 min.

The adhesive platelets adhere to the wall of the test tube. Those remaining~n the blood are counted. The difference between platelet counts of control,

~ The author expresses his gratitude to Professor W. Taub and ProfessorM. Cais for scientific discussions related to this paper and the supply ofsubstance 86.


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154 Bicher, 2-Methyl-2-tert. butyl-ketolactone

blood only test tubes, and those containing increasing concentrations of thetested drug indicates the percentage of platelet adhesiveness prevention.

A second set of test tubes is run as described, but adenosine diphosphate(ADP) is added to them at a concentration of 0.5 ~tg/cc. This induces furtherplatelet adhesiveness, and a further drop in the number of remaining platelets,Here again, the difference between test tubes containing ADP alone, or ADPplus the tested drug indicates the percentage of adhesiveness prevention.(b) Screen Filtration Pressure

The screen filtration pressure (SFP) has been described by Swank [19, 20]as a suitable procedure for the determination of the forces that tend to sticktogether the formed elements of blood when agglutinated.

By this method, the pressure required to force blood at a constant ratethrough a screen with multiple 20 micron-square pores is measured. Whenthe blood elements are separated they pass through the pores without anyappreciable resistance. An increase in pressure indicates an increase in thenumber of aggregated cells in the blood.

Swank demonstrated that the addition of ADP to the tested blood induceda sharp rise in the SFP, that is probably dependent on an increased plateletaggregation, although other cells probably participated in this action.

In order to clarify the effect of the studied compounds on platelets as asimplified system, ADP was added at a concentration of 1 ~gJcc to PRP (plateletrich plasma), and then increasing doses of the tested drug were added atsuccessive runs. A decrease in the SFP indicates prevention of the ADP in-duced platelet aggregation..(c) The ADP Induced Platelet Aggregation Time Test

This method is based upon the determination of the time required formacroscopical platelet aggregation to appear after the addition of adenosinediphosphate to platelet rich plasma in a non-siliconized test tube.

ADP at a concentration of 0.5 ~g was added to 0.2 ml PRP in an hemolysistest tube. The test tube is shaken continuously until macroscopic plateletaggregates appear. This aggregation starts as fine platelet conglomerates thatconverge into big masses of platelets (Snowstorm like phenomenon). Thefirst phase is recorded as one plus and the second as two plus aggregation.The tested substance is added to the reaction mixture 2 rain before the ADP(dissolved in 0.02 cc of tris buffer).

B. ErythrocytesLong ago Fahreus [9] postulated that increased red cell aggregation de-

creases the suspension stability of blood, thus providing abnormally high ery-throcyte sedimentation rates (ESI~). The ability of the tested compounds todecrease sedimentation rates was used for a quantitative determination oftheir anti-red cell aggregation properties in the in oitro and in oioo experiments.Direct microscopic observation of erythrocyte aggregation, in the living micro-circulation or under suitable in oitro conditions, provided additional evidenceof the ability of the investigated drugs to break up red cell aggregates.


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An Anti-adhesive Drug Preventing Platelet and Red Cell Aggregation 155

In vitro Experiments(d) Erythrocyte Sedimentation Rate (ESR) Westergreen

High ESR blood obtained from patients suffering from infections ormalignant diseases was used for the preliminary evaluation of the screenedcompounds. Four Westergreen sedimentation rate tubes filled with bloodaccording to the normal clinical procedure were used. Two of the ESR tubesserved as controls. The tested compounds were added at a concentration of0.5 mg/cc to the other two. Active compounds completely inhibited the sedi-mentation rate at this concentration.

(e) Erythrocyte Sedimentation Rate (ESR) Thorsen and Hint Method. Quanti-tative Studies

This procedure, first published by Thorsen and Hint in 1950 [21], providesfor a simple quantitative estimation of the erythrocyte aggregation propertiesof colloids or plasma. It has been adapted for the determination of the anti-aggregation properties of drugs, as a screening method that lends itself tothe numerical comparison of the relative strength of action of the differentcompounds.

The method is based on the measurement of the ESR in Westergreentubes, using a series of dilutions of plasma or artificial colloids. One part ofwashed human red cells is suspended in 2 parts of each dilution of the sus-pension fluid consisting of progressive dilutions of the tested colloid, dilutedin saline. In this way a fixed hematocrit value is obtained.

The log of the one hour sedimentation rates is plotted against the logof the colloid concentrations in the respective tubes. The points lie in straightlines with nearly the same slopes. The points where lines cut the abscissaegive the concentration of colloid which causes a sedimentation rate of 1 mm h.This parameter, called the critical point by Thorsen and Hint is dependenton the colloid concentrations in individual tubes and can, therefore, be con-sidered as a quantitative measure for their erythrocyte aggregation power.When the colloid concentration is lower than critical, the erythrocyte aggre-gation disappears; when the concentration is raised aggregation and sedi-mentation rate rise very rapidly.

For the determination of the anti-aggregation properties of 86 and relatedcompounds, a fixed aggregation force was chosen, giving a sedimentation rateof 40 mm/h (in the present experiments this was obtained by using dextran,MW150,000, at 1Uo concentration). Increased concentration of the testedsubstances were added, and the inhibition of aggregation as represented by thedecrease in the sedimentation rate, was measured as percentages of the control.

In vivo ExperimentsIn oivo experiments were performed for all three platelet procedures a,

b, c, on nembutal anesthetized cats and dogs, after a~terial, venular and trachealcannulation. Blood samples were obtained from the artery while all solutionswere injected into the vein. Polyethylene cannulae were used.


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1 5 6 Bicher, 2-Methyl-2-tert. butyl-ketolactone

The in vivo anti-red cell aggregation power of the tested compounds wasdetermined by two methods:

1. Erythrocyte Sedimentation Rate was performed according to the Wester-green technique, as previously described (d). The experiments were conductedon nembutalized cats. High erythrocyte sedimentation rates were foundusually in these animals, or were induced by the intravenous injection of highviscous dectran, MW 150 000, 1 g/kg.(f) Vital Microscopy of the Peripheral Circulation

Microscopic observations of the microcirculation were performed on thetransilluminated omentum of the cat. Using a Zeiss dissection microscope,the observations were made at magnification power of X180 and never con-tinued for more than 10 rain at a time. Subsequent observations were madeoccasionally, but not more than twice in the same animal.

Intravascular red cell aggregation was induced by injecting high mole-cular weight dextran intravenously at a concentration of 1 gjkg.2. Mice Acute Toxicity

LDz0 of 86 and related compound on mice was determined according tothe method described by Reed and Muench f17], using ten animals for eachdose level.

Experimental DesignPhenylbutazone, chloropromazine, and 86 were tested in parallel for theiractivity to prevent platelet and red cell aggregation in vitro, according to the

methods a, b, c, d and e and their relative effectiveness was compared. Sub-stance 86 was also tested in vivo according to the described procedures.

ResultsSubstance 86 prevented platelet aggregation and adhesiveness

in vitro at a dose of 2,000/zg/cc (tables I, II, and III) and red cellaggregation at a counteraction of 250/~g/cc (table III). Phenyl-butazone was about twice as active as 86 and chloropromazineabout 10 times more active.

The only drug that could be used in oioo at effective doses was86, because of its low toxicity (LDs0:2,000 mg/kg, I.P., mice). Ata dose of 200 mg/kg i.v., it prevented platelet adhesiveness andaggregation in all methods tested and also inhibited the erythro-cyte sedimentation rate for a period of 2 to 4 h (table IV).

Microscopic observations of the microcirculation after infusionof high molecular weight dextran revealed a slow and stagnantcirculation of blood. The red cells were aggregated into big masses


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Table 1. Rolling tube (platelet adhesiveness to glass). In vitro experimentsRelative potency of action - drugs tested: 86, phenylbutazone, chloropromazineDrug Normal human blood

Dose A dhesivene ss Dose preventing(~g/cc) ~o of preve ntion approx. 40adhesiveness(~g/cc)

86 2,000 34 2,0001,000 3050 0 2~10 0 1

Phenylbutazone 2,000 62 1,0001,000 44

50 0 3010 0 24

Chloropromazine 20 0 71 10 010 0 4750 25

Table II. Screen filtration pressure (SFP). In vitro experiments

Drug Dose %of pre- Mean effec-vention tive dose(approx. 55%prevention)

86 2,000 60 2,0001,000 25

50 0 1Phenylbu[azone 2,000 75 1,0001,000 56

500 36Chloropromazine 20 0 68 100

10 0 5150 22


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158 Bicher, 2-Methyl-2-tert. butyl-ketolactoneTable llI Relative potency of 86, phenylbutazone and chloropromazine as

anti-adhesive drugs. In vitro experiments

Test: ESR Mean effectiYe doses (gg/cc)(Thorsen-Hint) ADP induced Rolling tube ADP platelet SFPplatelet adhe- aggregationsiveness tes~ time

86 250 2,000 2,000 2,000 2,000Phenylbutazone 100 1,000 1,000 250 1,000Chloropromazine 10 20 0 50 20 100

Table IV Erythrocyte sedimentation rate (ESR). In viuo experiments - Cat

Drug tested: 86 . Dose: 200 mgjkg i.v.

Time after ESR (mm/hour) Mean % Pre- St. dev. St. er.in~ ec t ion E S R ventionrest No. 1 2 3 4 5

0 70 45 60 65 70 621 1 1 1 1 1 1 98 0 03 3 3 3 3 5 34 94 0. 9 0.45 5 8 6 10 15 88 86 3.9 1.8

15 24 18 26 25 35 25 6 59 6.2 2.760 42 24 35 31 45 35 44 8.4 3. 4

120 46 35 45 39 60 41 36 9.6 4. 3240 62 39 55 45 62 52 15 10.3 4. 6

(sludge) with spaces of cell free plasma separating them (plasmaskimming). The sludge formation was especially heavy in venules,but was present also in capillaries and arterioles. In many places,red cell impaetion of small posteapillary venules could be dis-eerned (fig. 2).After the intravenous administration of 86 200 mg/kg, thecirculation became faster, red cell aggregates separated, and theplasma skimming disappeared, especially in the arterioles. Venularsludge remained in most eases. This effect lasted for one hour,after which time observations were discontinued. The most strik:ing improvement occurred during the first 15 min after injection.Our modification of the Thorsen and Hint method providesan accurate tool for the easy and quick quantitative evaluation


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Fig 2 Prevention by 86 of sludge formation in oivo. Omentum microcircu-lation in a cat after bleeding and injection of high molecular weight dextran(for description of methods see text). A. Sludge in a venule and arteriole. Noteplasma skimming at arrows. B. 15 min after the injection of 86, 200 mg/kgi.v. - same area. Plasma skimming disappeared red cell flow in continuous.

of the anti-aggressive power of new chemical compounds. Theresults obtained, summarized in table III, show a good parallelism,both in respect to the type of activity and relative potency ofaction of the different drugs when tested using this method, or themethods previously described for platelet agglutination. The con-centrations of the drugs needed to disperse the red cell aggregates


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160 Bicher, 2-Methyl-2-tert. butyl-ketolactone

are less than those required to disperse platelets, probably becausethe forces holding the erythrocytes together (mainly a matter ofcoating or physical attraction [3, 7] are not as strong as thoselinking platelet aggregates (an active chemical process requiringenergy consumption [5, 10]).

DiscussionWhen screening a series of compounds for their anti-adhesiveaction on blood cells, not only their relative potency of action

should be considered, but also their toxicity, both as regards theLDs0 and hemolytic effect on blood cells. This second aspect isparticularly important since this type of drug tends to increaseblood cell fragility. Evaluation of hemolytic potential should bea prerequisite to in vioo testing. Substance 86 shows a margin of1:30 in this respect (effective concentration 1 mg/cc, hemolyticconcentration 30 mg/cc). The therapeutic index of this substanceis also satisfactory (1 : 10, ED~0 200 mg/kg, LD~0 2,000 mg/kg).

The fact that this compound acted in the same way in prevent-ing both red cell and platelet aggregation indicate that this prop-erty of preventing red cells from sticking to each other is moregeneralized and affects not only the blood platelets. O Brien [16]described the ability of certain antimalarials, local anestheticsand imipramine derivatives, to inhibit the ADP induced plateletaggregation. Constantine [8] reported that histamine possesses asimilar effect and that this property is also shared by some anti-histamines and anti-inflammatory compounds. O Brien proposedthe name of anti-adhesive drugs to include all chemicals sharingthis type of activity.

The anti-adhesive drugs have been reported to induce plateletswelling also probably by inhibiting a phosphoprotein kinase [11].They also affect the active uptake of serotonin by platelets, butnot the passive histamine absorption [13]. These results again sug-gest an interference with the functions of the platelet membrane.

Our results demonstrated that the anti-adhesive drugs shouldalso be considered as anti-sludge, and that it may be possible todevelop a compound that will be sufficiently non-toxic and activeenough to prevent both types of blood cell aggregation i~ vivoSubstance 86 should be considered as a prototype for this type of


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An Anti-adhesive Drug Preventing Platelet and Red Cell Aggregation 1 61

potential anti-thrombotic compound. Even if its activity is stillnot enough for further clinical testing 200 mg/kg will be too higha dose for use in the human) it can be assumed, in view of thefact that some of the comp ounds tested were ten times more activein vitro that further screening will lead to the development of anusable drug.

SummaryA new type of potential anti-thrombotic drug is described. The anti-

adhesive drugs, able to prevent intravascular platelet and red cell aggregationand improve peripheral circulation by an effect at the level of the blood cellmembranes, are proven to be effective both in vitro and in oioo. The metho-dology for their screening and evaluation is reported. Substance 86 (2-Methyl-2-tert. butyl-ketolactone), the first non-toxic compound potent enough as to allowin vioo use, is considered as a prototype for this kind of drugs. More potentcompounds, though, will be needed to be found before substances suitable forhuman use will be ready for clinical trials.

References1. Bicher, H. 1. and Beemer, A. M.: The role of sludge in the production of

experimental ischemic myocardial damage. Bibl. anat., vol. 9, p. 116 (Karger,Basel/New York 1967).2. Bloch, E. H.: Visual changes in the living mierovaseular system in manand experimental animals as they are related to thrombosis and embolism.Angiology 10:6 (1956).3. Bloch, E. H.; Powell, A.; Meryman, H. T.; Warner, L. and Kafig, E.: Acomparison of the surfaces of human erythrocytes from health and diseaseby.in oivo light microscopy and in vi t ro electron microscopy. Angiology 7:479 (1959).

4. Born, G. V. R. and Cross, M. J.: The aggregation of blood platelets. J.Physiol. 168:178 1963).5. Born, G. V. R. and Cross, M. J.: Effect of adenosine diphosphate in the

concentration of platelets in circulating blood. Nature, Lond. 197:174(1963).6. Brogstrom, S.; Gelin, L. E. and Zederfeldt, B.: The formation of veinthrombi following tissue injury. Acta chir. scand. Suppl. 247 (1959).

7. Castaneda, A. R.; Bernstein, E.; Bangstadt, F. and Varco, R. L.: Effect ofPVP, dextrose and dextran on red blood cell charge. Proc. III Europ. Conf.Microcirc. Israel J~ exp. Med. 11:128 (1964).

8. Constantine, J. W.: Inhibition of ADP induced platelet aggregation byhistamine. Nature, Lond. 207:91 (1965).9. Fahreus, R.: The suspension stability of the blood. Acta reed. scand. 55:1 (1921).


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16 2 BicherI0 . Gaardner, A.; Jonsen, J.; Leland, S.; Hellem, A. and Owren, P. A.: Adenosinediphosphate in red cells as a factor in the adhesiveness of human blood

platelets. Nature, Lond. 192:531 (1961).11. Judah, J. D.: Ciba foundation symposium on enzymes and drug action,

p. 339 (Churchill, London 1962).11. Judah, J. D.: Ciba foundation symposium on enzymes and drug action,

p. 339 (Churchill, London 1962).12. Kniselg, M. H.; Bloch, E. H.; Elliot, T. S. and Warner, L.: Sludged blood.Science 106:431 (1947).13. Markwardt, F.; Barthel, W. and Glusa E.: Changes in the histamine andserotonine content of blood platelets due to the effect of local anesthetics.Naunyn-Schmiedeberg Archiv 253-336 (1966).

14. Mustard, J. F.; Rowsell, H. C.; Lotz, F. and Hegardt, B.: The effect ofadenine nucleotides on thrombus formation, platelet count and bloodcoagulation. Exp. molec. Path. 5:43 (1966).

15. Nordog, A. and Ravick, T. 0.: Some effects of adrenaline on rat plateletsin vitro and in viuo. Scand. J. clin. Lab. Invest. 17: Suppl. 84:151 (1964).16. O Brien, J. R.: Platelet aggregation. Part II. Some results from a newmethod of study. J. clin. Path. 15:452 (1962).17. Reed, L. J. and Muench, H.: Biological methods for study of toxins. Amer.

J. Hyg. 27:493 1938).18. Rozenberg, M. C. and Firkin, B. G.: The rate of platelet aggregation inhaemorrhagic disease and thrombosis. Scand. J. Haemat. 3:5 (1965).

19. Swank, R. L.: Adhesiveness of platelets and leukocytes during acute ex-sanguination. Amer. J. Physiol. 202:261 (1962).20. Swank, R. L.; Roth, J. and Jansen, J.: Screen filtration pressure methodand adhesiveness and aggregation of blood cells. J. appl. Physiol. 19:340

(1964).21. Taub, W. and Cats M.: The synthesis of ketolactone with potential pharma-codynamic properties. Bull. Res. Counc. Israel 11~: 18 (1962).

22. Wright, H. P.: Changes in the adhesiveness of blood platelets followingparturition and surgical operation. J. Path. Bac. 55:461 (1942).

Authors address: H. I. Bicher, Department of Anatomy, Medical Universityof South Carolina, Charleston SC 29~01 (USA).

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