Pharmacology 4:152-162 (1970)

Anti-adhesive drugs Thrombosis Platelet aggregation Red cell aggregation Peripheral circulation

2-Methyl- 2-tert.butyl-ketolactone, an Anti-adhesive Drug Preventing Platelet and Red Cell Aggregation
H. I. BICHER 1
Department of Anatomy, Medical University of South Carolina, Charleston, SC

The causal participation of blood cell aggregation in thrombus formation has been described by several investigators under clinical [3] and experimental [4, 14, 15] conditions. Of the blood cells involved, special attention has been given to the erythrocytes and platelets. These cells seem to possess the ability to start and accelerate the chain of events that leads to organic vessel occlusion. Knisely [12] and Bioch [3] observed long ago the pathological significance of intravascular red ceil aggregation (sludge). BorgstrSrn [6] was able to demonstrate a direct etiological relation between experimentally induced sludge and venous thrombosis in the rabbit. In previous work [1] we suggested that intravascular red cell aggregation may be a factor in the induction of anoxic myocardial damage. The importance of platelet aggregation for hemostasis and for the pathogenesis of thrombotic processes is,~well established [4, 18]. The adherence of platelets to each other and to other surfaces is considered a fundamental step in the formation of plugs and thrombi [14]. The purpose of this paper is to assess the potential therapeutic value of a group of drugs that share the common property of preventing both red cell and platelet aggregation in vitro and in vivo. Six different methods have been devised or adopted to evaluate this action on a quantitative basis, and with their aid, a new chemical compound, 2-Methyl-2-tert. butyl-ketolactone, subsequentWork done as partial fulfillment for a Ph.D. Thesis, Tel Aviv University.
Received: January 27, 1970.

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15 3

ly referred to as substance 86, has been compared with the already known compounds and developed as a prototype for this kind of potential anti-thrombotic drug. Materials and Methods
ADP 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 Methyl R~ tert.-butyl Fig. 1. The chemical structure of substance 86.

A. Platelets 1. 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 antecubital vein in the experiments carried out with human platelets, or from the femoral artery through a polyethylene canula in nembutal anesthetized cats. Sodium citrate 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 test tube containing 0.1 cc of the added solutions (solvents tested drug). The tube 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 Professor M. Cais for scientific discussions related to this paper and the supply of substance 86.

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

blood only test tubes, and those containing increasing concentrations of the tested 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 further platelet adhesiveness, and a further drop in the number of remaining platelets, Here again, the difference between test tubes containing ADP alone, or ADP plus 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 stick together the formed elements of blood when agglutinated. By this method, the pressure required to force blood at a constant rate through a screen with multiple 20 micron-square pores is measured. When the blood elements are separated they pass through the pores without any appreciable resistance. An increase in pressure indicates an increase in the number of aggregated cells in the blood. Swank demonstrated that the addition of ADP to the tested blood induced a sharp rise in the SFP, that is probably dependent on an increased platelet aggregation, although other cells probably participated in this action. In order to clarify the effect of the studied compounds on platelets as a simplified system, ADP was added at a concentration of 1 ~gJcc to PRP (platelet rich plasma), and then increasing doses of the tested drug were added at successive runs. A decrease in the SFP indicates prevention of the ADP induced platelet aggregation.. (c) The ADP Induced Platelet Aggregation Time Test This method is based upon the determination of the time required for macroscopical platelet aggregation to appear after the addition of adenosine diphosphate 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 hemolysis test tube. The test tube is shaken continuously until macroscopic platelet aggregates appear. This aggregation starts as fine platelet conglomerates that converge into big masses of platelets (Snowstorm like phenomenon). The first 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. Erythrocytes Long ago Fahreus [9] postulated that increased red cell aggregation decreases the suspension stability of blood, thus providing abnormally high erythrocyte sedimentation rates (ESI~). The ability of the tested compounds to decrease sedimentation rates was used for a quantitative determination of their anti-red cell aggregation properties in the in oitro and in oioo experiments. Direct microscopic observation of erythrocyte aggregation, in the living microcirculation or under suitable in oitro conditions, provided additional evidence of the ability of the investigated drugs to break up red cell aggregates.

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 or malignant diseases was used for the preliminary evaluation of the screened compounds. Four Westergreen sedimentation rate tubes filled with blood according to the normal clinical procedure were used. Two of the ESR tubes served as controls. The tested compounds were added at a concentration of 0.5 mg/cc to the other two. Active compounds completely inhibited the sedimentation rate at this concentration. (e) Erythrocyte Sedimentation Rate (ESR) Thorsen and Hint Method. Quantitative Studies This procedure, first published by Thorsen and Hint in 1950 [21], provides for a simple quantitative estimation of the erythrocyte aggregation properties of colloids or plasma. It has been adapted for the determination of the antiaggregation properties of drugs, as a screening method that lends itself to the numerical comparison of the relative strength of action of the different compounds. The method is based on the measurement of the ESR in Westergreen tubes, using a series of dilutions of plasma or artificial colloids. One part of washed human red cells is suspended in 2 parts of each dilution of the suspension fluid consisting of progressive dilutions of the tested colloid, diluted in saline. In this way a fixed hematocrit value is obtained. The log of the one hour sedimentation rates is plotted against the log of the colloid concentrations in the respective tubes. The points lie in straight lines with nearly the same slopes. The points where lines cut the abscissae give the concentration of colloid which causes a sedimentation rate of 1 mm!h. This parameter, called the critical point by Thorsen and Hint, is dependent on the colloid concentrations in individual tubes and can, therefore, be considered as a quantitative measure for their erythrocyte aggregation power. When the colloid concentration is lower than critical, the erythrocyte aggregation disappears; when the concentration is raised aggregation and sedimentation rate rise very rapidly. For the determination of the anti-aggregation properties of 86 and related compounds, a fixed aggregation force was chosen, giving a sedimentation rate of 40 mm/h (in the present experiments this was obtained by using dextran, MW150,000, at 1Uo concentration). Increased concentration of the tested substances were added, and the inhibition of aggregation as represented by the decrease in the sedimentation rate, was measured as percentages of the control.

In vivo Experiments
In oivo experiments were performed for all three platelet procedures a, b, c, on nembutal anesthetized cats and dogs, after a~terial, venular and tracheal cannulation. Blood samples were obtained from the artery while all solutions were injected into the vein. Polyethylene cannulae were used.

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

The in vivo anti-red cell aggregation power of the tested compounds was determined by two methods: 1. Erythrocyte Sedimentation Rate was performed according to the Westergreen technique, as previously described (d). The experiments were conducted on nembutalized cats. High erythrocyte sedimentation rates were found usually in these animals, or were induced by the intravenous injection of high viscous dectran, MW 150 000, 1 g/kg. (f) Vital Microscopy of the Peripheral Circulation Microscopic observations of the microcirculation were performed on the transilluminated omentum of the cat. Using a Zeiss dissection microscope, the observations were made at magnification power of X180 and never continued for more than 10 rain at a time. Subsequent observations were made occasionally, but not more than twice in the same animal. Intravascular red cell aggregation was induced by injecting high molecular weight dextran intravenously at a concentration of 1 gjkg. 2. Mice Acute Toxicity LDz0 of 86 and related compound on mice was determined according to the method described by Reed and Muench f17], using ten animals for each dose level. Experimental Design

Phenylbutazone, chloropromazine, and 86 were tested in parallel for their activity 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. Substance 86 was also tested in vivo according to the described procedures.

Results Substance 86 prevented platelet aggregation and adhesiveness in vitro at a dose of 2,000/zg/cc (tables I, II, and III) and red cell aggregation at a counteraction of 250/~g/cc (table III). Phenylbutazone was about twice as active as 86 and chloropromazine about 10 times more active. The only drug that could be used in oioo at effective doses was 86, because of its low toxicity (LDs0:2,000 mg/kg, I.P., mice). At a dose of 200 mg/kg i.v., it prevented platelet adhesiveness and aggregation in all methods tested and also inhibited the erythrocyte sedimentation rate for a period of 2 to 4 h (table IV). Microscopic observations of the microcirculation after infusion of high molecular weight dextran revealed a slow and stagnant circulation of blood. The red cells were aggregated into big masses

An Anti-adhesive Drug Preventing Platelet and Red Cell Aggregation 157 Table 1. Rolling tube (platelet adhesiveness to glass). In vitro experiments
Relative potency of action - drugs tested: 86, phenylbutazone, chloropromazine Drug Normal human blood Dose (~g/cc) Adhesiveness ~o of prevention Dose preventing approx. 40% adhesiveness (~g/cc)

86

2,000 1,000 500 100

2,000 1,000 500 100

34 30 2~ 1
62 44 30 24

2,000

Phenylbutazone

1,000

Chloropromazine

200 100 50

71 47 25

100

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

Drug Dose %of prevention Mean effective dose (approx. 55% prevention)

86

2,000 1,000 500

60 25 1

2,000

Phenylbu[azone

2,000 1,000 500 200 100 50

75 56 36 68 51 22

1,000

Chloropromazine

100

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Table llI. Relative potency of 86, phenylbutazone and chloropromazine as anti-adhesive drugs. In vitro experiments
Test: ESR (Thorsen-Hint) Mean effectiYe doses (gg/cc) ADP induced platelet adhesiveness tes~ Rolling tube ADP platelet aggregation time SFP

86 Phenylbutazone Chloropromazine

250 100 10

2,000 1,000 200

2,000 1,000 50

2,000 250 20

2,000 1,000 100

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

Drug tested: 86. Dose: 200 mgjkg i.v. Mean ESR 4 5 % Prevention St. dev. St. er.

Time after in~ ection

ESR (mm/hour) rest No. 1 2 3

0 1 3 5 15 60 120 240

70 1 3 5 24 42 46 62

45 1 3 8 18 24 35 39

60 1 3 6 26 35 45 55

65 1 3 10 25 31 39 45

70 1 5 15 35 45 60 62

62 1 34 88 256 35 41 52

98 94 86 59 44 36 15

0 0.9 3.9 6.2 8.4 9.6 10.3

0 0.4 1.8 2.7 3.4 4.3 4.6

(sludge) with spaces of cell free plasma separating them (plasma skimming). 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 diseerned (fig. 2). After the intravenous administration of 86 200 mg/kg, the circulation became faster, red cell aggregates separated, and the plasma skimming disappeared, especially in the arterioles. Venular sludge 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 provides an accurate tool for the easy and quick quantitative evaluation

An Anti-adhesive Drug Preventing Platelet and Red Cell Aggregation 159

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. Note plasma skimming at arrows. B. 15 min after the injection of 86, 200 mg/kg i.v. - same area. Plasma skimming disappeared red cell flow in continuous.

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

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

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

Discussion When screening a series of compounds for their anti-adhesive action on blood cells, not only their relative potency of action should be considered, but also their toxicity, both as regards the LDs0 and hemolytic effect on blood cells. This second aspect is particularly important since this type of drug tends to increase blood cell fragility. Evaluation of hemolytic potential should be a prerequisite to in vioo testing. Substance 86 shows a margin of 1:30 in this respect (effective concentration 1 mg/cc, hemolytic concentration 30 mg/cc). The therapeutic index of this substance is 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 preventing both red cell and platelet aggregation indicate that this property of preventing red cells from sticking to each other is more generalized and affects not only the blood platelets. OBrien [16] described the ability of certain antimalarials, local anesthetics and imipramine derivatives, to inhibit the ADP induced platelet aggregation. Constantine [8] reported that histamine possesses a similar effect and that this property is also shared by some antihistamines and anti-inflammatory compounds. OBrien proposed the name of anti-adhesive drugs to include all chemicals sharing this type of activity. The anti-adhesive drugs have been reported to induce platelet swelling also probably by inhibiting a phosphoprotein kinase [11]. They also affect the active uptake of serotonin by platelets, but not the passive histamine absorption [13]. These results again suggest an interference with the functions of the platelet membrane. Our results demonstrated that the anti-adhesive drugs should also be considered as anti-sludge, and that it may be possible to develop a compound that will be sufficiently non-toxic and active enough to prevent both types of blood cell aggregation i~ vivo. Substance 86 should be considered as a prototype for this type of

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

potential anti-thrombotic compound. Even if its activity is still not enough for further clinical testing (200 mg/kg will be too high a dose for use in the human) it can be assumed, in view of the fact that some of the compounds tested were ten times more active in vitro, that further screening will lead to the development of an usable drug.
Summary
A new type of potential anti-thrombotic drug is described. The antiadhesive drugs, able to prevent intravascular platelet and red cell aggregation and improve peripheral circulation by an effect at the level of the blood cell membranes, are proven to be effective both in vitro and in oioo. The methodology for their screening and evaluation is reported. Substance 86 (2-Methyl2-tert. butyl-ketolactone), the first non-toxic compound potent enough as to allow in vioo use, is considered as a prototype for this kind of drugs. More potent compounds, though, will be needed to be found before substances suitable for human use will be ready for clinical trials.

References
1. 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 man and 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.: A comparison of the surfaces of human erythrocytes from health and disease by.in oivo light microscopy and in vitro 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 vein thrombi following tissue injury. Acta chir. scand. Suppl. 247 (1959). 7. Castaneda, A. R.; Bernstein, E.; Bangstadt, F. and Varco, R. L.: Effect of PVP, 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 by histamine. Nature, Lond. 207:91 (1965). 9. Fahreus, R.: The suspension stability of the blood. Acta reed. scand. 55: 1 (1921).

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I0. Gaardner, A.; Jonsen, J.; Leland, S.; Hellem, A. and Owren, P. A.: Adenosine diphosphate 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 and serotonine 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 of adenine nucleotides on thrombus formation, platelet count and blood coagulation. Exp. molec. Path. 5:43 (1966). 15. Nordog, A. and Ravick, T. 0.: Some effects of adrenaline on rat platelets in vitro and in viuo. Scand. J. clin. Lab. Invest. 17: Suppl. 84:151 (1964). 16. OBrien, J. R.: Platelet aggregation. Part II. Some results from a new method 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 in haemorrhagic disease and thrombosis. Scand. J. Haemat. 3:5 (1965). 19. Swank, R. L.: Adhesiveness of platelets and leukocytes during acute exsanguination. Amer. J. Physiol. 202:261 (1962). 20. Swank, R. L.; Roth, J. and Jansen, J.: Screen filtration pressure method and adhesiveness and aggregation of blood cells. J. appl. Physiol. 19:340 (1964). 21. Taub, W. and Cats, M.: The synthesis of ketolactone with potential pharmacodynamic properties. Bull. Res. Counc. Israel 11~: 18 (1962). 22. Wright, H. P.: Changes in the adhesiveness of blood platelets following parturition and surgical operation. J. Path. Bac. 55:461 (1942).

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