Phase separation in phosphatidylcholine bilayers as a predictor of inhibition of blood platelet aggregation by amantadines

273

Biochimica et Biophysica Acta, 4 6 7 ( 1 9 7 7 ) 2 7 3 - - 2 7 9 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 77730

PHASE SEPARATION IN P H O S P H A T I D Y L C H O L I N E BILAYERS AS A P R E D I C T O R OF INHIBITION OF BLOOD PLATELET A G G R E G A T I O N BY AMANTADINES

R O B E R T W. C O L M A N a, J A Y A K U C H I B H O T L A a, M A H E N D R A K. J A I N b,c a n d R O G E R K. M U R R A Y , Jr. c

a Coagulation Unit, Hematology-Oncology Section, Department of Medicine, Hospital of the University of Pennsylvania, Philadelph&, Pa. 19104, b Division of Health Sciences, University of Delaware, Newark, Del. 19711 and e Department of Chemistry, University of Delaware, Newark, Del. 19711 (U.S.A.) (Received January 20th, 1977)

Summary The ability of eleven amantadine derivatives to induce phase separation in dipalmitoyl phosphatidylcholine bilayers was studied by differential scanning calorimetry. The relative p o t e n c y varied with the shape and size of the hydrocarbon cage. These agents also markedly inhibited blood platelet aggregation. The relative potencies of these c o m p o u n d s to induce phase separation showed a significant correlation (r = 0.70) with their platelet inhibitory activity suggesting that their pharmacologic action may be at the level of the platelet membrane. The effective concentration of the parent c o m p o n e n t amantadine is similar to its pharmacologic concentration suggesting its use as an anti-platelet drug.

Introduction

Arterial thrombosis occurs in response to a disruption of vascular endothelium which results in the exposure of subendothelial components of the vessel wall to blood platelets [1]. The conclusion that blood platelets are involved in thrombotic vascular disease follows from the observations that: (a) platelets show enhanced sensitivity to aggregating agents in type II hyperlipoproteinemia [2], myocardial infarction and stroke [3,4], coronary atherosclerosis [5], and diabetes [6]; (b) platelet microaggregates circulate in peripheral vascular disease and transient cerebrovascular ischemia [7]; and (c)platelet survival deAbbreviations: HHW r, half-height width~ IDs0, concentration of t h e a m a n t a d i n e derivative that by 50%.

inhibits p l a t e l e t a g g r e g a t i o n

274 creases in coronary disease [8], hyperlipoproteinemia, and cerebrovascular disease [9]. A variety of pharmacologic agents have been employed to modify platelet behavior in vivo [10] and in vitro [11]. Some drugs like aspirin and non-steroidal anti-inflammatory agents appear to act by inhibiting platelet cyclo-oxygenase [12], an enzyme required for the synthesis of prostaglandin cyclic endoperoxides [13,14] and t h r o m b o x a n e A2 [14]. However, the actions of several lipid-soluble drugs, including antihistamines (e.g. cyproheptadine [15]), hypolipemic agents (e.g. clofibrate [16] and halofenate [17]), and agents active on the central nervous system (e.g. chlorpromazine [18]), appear to be localized at the platelet membrane. However, direct evidence for membrane modification by these drugs has n o t been reported. The initial adhesion of blood platelets to the vessel wall and the aggregation of platelets to each other result in the formation of a platelet thrombus which later is reinforced by fibrin [19]. Consequently, inhibitors of platelet aggregation are of considerable interest as they offer the potential for treating or preventing arterial thrombosis. We now wish to present results which suggest that differential scanning calorimetry of doped lipid bilayers can be employed to predict the relative potencies of platelet inhibiting drugs whose action is localized at the platelet membrane. Methods and Results Amantadine [1 ], a lipophilic quasi-spherical c o m p o u n d , which has been shown to be effective in preventing influenza A2 [20] and ameliorating the symptoms of Parkinson's disease [21], enters all cell membranes including those of the nervous system [20]. The structural formulae of the eleven amantadine derivatives whose hydrochloride salts were employed in this study are given in Figs. l a , l b and lc. Platelets for the aggregation studies were prepared from venous blood drawn from fasting blood donors who had abstained from all medication for at least 2 weeks prior to venesection. Venous blood was collected through siliconized needles into plastic syringes and then anticoagulated with sodium citrate (final concentration 0.013 M). Platelet-rich plasma with a platelet count of 350 000 -+ 50 000 was prepared by centrifugation of the samples at 170 g for 10 min at 23°C. The remaining blood was centrifuged at 12 000 X g for 4 min at 23°C to

(a)

(b)

R1

(c) NH 2 //

R3 R1

CH 3

CH 3 CN 3

Fig.

1. (a) Structure

of seven

amantadine

derivatives:

(1)

R 1 = NH2,

R 2 = H; (2) R 1 = NHCH3,

(3) R 1 = NHC2Hs, R 2 = H; (4) R 1 = NH-cyclic-C6H1 l , R 2 = H; (5) R 1 = CH2NH2, CH(CH3)NH 2, R 2 = H; (7) R 1 = H, R 2 = NH 2. (b) Structure oftrimethylamantadine of three homoamantadine R3 = NH2, R 1 =R 1 =R

derivatives: 2 =H.

(9)

R 1 = NH2,

R 2 = R 3 = H; (10)

R 2 = NH2,

R 2 = H;

R 2 = H; (6) R 1 = [8]. (c) Structure R 1 = R 3 = Hi (11)

275 produce platelet-poor plasma which had less than 1000 platetets per pl. Platelet aggregation was studied with an aggregometer according to a modification [3] of the method of Born [22]. As aggregation occurs, the a m o u n t of light scattering decreases and more light is transmitted. The extent of aggregation was determined b y the percent difference in light transmission between plateletrich and platelet-poor plasma after 15 min of stirring at 1200 rev./min and 37°C. The sensitivity of the sample to ADP (Sigma Chemical Co., St. Louis, Mo.) was determined at the lowest concentration of the agent which would produce a "second wave" response [2]. This threshold concentration was found to show a high correlation (r = 0.95) with the threshold determined by the use of serotonin release [23]. It was found in preliminary studies that if the final concentration of methanol exceeded 0.5 M then platelet aggregation was inhibited. Therefore no more than 5 pl of methanol containing the amantadine derivative was added to 500 pl of platelet-rich plasma (final concentration of methanol 0.25 M) before being incubated at 37°C for 10 min witho u t stirring. The threshold concentration of ADP was then added and the percent aggregation of platelets for a given concentration of the amantadine derivative was compared to the percent aggregation of platelets with the solvent, methanol (see Fig. 2). By carrying out analogous experiments for a number of concentrations of the drug, the concentration of the amantadine derivative that inhibited platelet aggregation by 50% (i.e. IDs0) could readily be determined (see Fig. 3). Unsonicated multilamellar liposomes containing D L-1,2-dipalmitoyl phosphatidylcholine (Sigma Chemical Co.) and amantadine derivatives were prepared for the differential scanning calorimetry experiments according to the procedures employed for similar studies with other variously substituted amantadines [24]. A known volume of a methanolic solution of an amantadine derivative was evaporated to dryness in a small test tube. An aliquot of liposomes was tranferred onto the film of amantadine residue and the mixture was vigorously

100.0

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~_

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37.5

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1

2

3 Time (min)

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Fig. 2. Platelet-rich p l a s m a ( 5 0 0 pl) was i n c u b a t e d 1 0 rain at 3 7 ° C w i t h ( t o p ) 2 . 2 5 pl of a m e t h a n o l solut i o n of a m a n t a d i n e ( s t r u c t u r e 1) (final c o n c e n t r a t i o n of s t r u c t u r e 1, 2.79 raM), or w i t h ( m i d d l e ) 1.3 pl of this s o l u t i o n (final c o n c h , o f s t r u c t u r e 1, 161 raM), o r w i t h ( l o w e r ) 3 ttl o f m e t h a n o l ( n o d r u g ) . P l a t e l e t a g g r e g a t i o n was p e r f o r m e d as d e s c r i b e d in the t e x t .

276

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/

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S 50-

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No drug

5

0.40

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315

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Amantadine (10 raM)

~

,u

i

38 40 42 Temp. (°C)

Fig. 3. By m e a n s o f the e x p e r i m e n t a l p r o c e d u r e e m p l o y e d in Fig. 2, the p e r c e n t i n h i b i t i o n o f p l a t e l e t a g g r e g a t i o n i n d u c e d b y various c o n c e n t r a t i o n s o f s t r u c t u r e 1 was d e t e r m i n e d and thus the c o n c e n t r a t i o n o f s t r u c t u r e 1 required to inhibit P l a t e l e t a g g r e g a t i o n b y 50% ( I D 5 0 ) was o b t a i n e d . Fig. 4. D i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y profiles o f pure d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e l i p o s o m e s ( 7 0 raM) and o f s u c h l i p o s o m e s d o p e d w i t h s t r u c t u r e 1 ( 1 0 . 6 7 m M ) . T h e h a l f - h e i g h t w i d t h s (HHW') are noted.

shaken with two 3-mm glass beads on a Vortex mixer under a nitrogen atmosphere at 45--50°C for 5 min. The sample was then allowed to stand for 24-48 h before being employed for the differential scanning calorimetry experiment. For comparison, some samples were prepared by premixing the lipid and amantadine derivative in a solvent and then generating the liposomes from a mixed film. The differential scanning calorimetry profiles of the liposomes prepared by either method were identical. All differential scanning calorimetry samples were 75 mM in dipalmitoyl phosphatidylcholine, 50 mM in KC1, 5 mM in Tris buffer, and were prepared at pH 7.4. The samples were analyzed using a Perkin-Elmer DSC-1B calorimeter operating at a sensitivity of 1 mcal and a scanning rate of 1.25 ° C/min towards increasing temperature. The phase transition profile for pure dipalmitoyl phosphatidylcholine liposomes shows a sharp symmetrical transition commencing (Tc) at 41.0°C which is essentially complete in 1.6°C (Fig. 4). It was found for all of the cage amines in this study that liposomes doped with them showed another sharp transition at a lower temperature. As the concentration of the amantadine was increased, the area of the parent peak decreased and the area of the new peak increased. Thus, as the concentration of the amantadine derivative is increased, Tc decreases and the combined half-height width (HHW')of the two transitions increases (see Fig. 5). None of the amantadine derivatives had a phase transition by itself in the temperature regions studied. In order to compare the relative potencies of the amantadine derivatives, we have defined an arbitrary constant, HHW'100, which is the concentration of drug at which the HHW' of the phase transition profile of pure dipalmitoyl phosphatidylcholine

277

~

100 9O c 8C g

~ 7c

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0

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2

i

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Fig. 5. The concentration-dependent HHW r curve for structure 1 from which the concentration HHW t of pure dipalmitoyl phosphatidylcholine liposomes doubles (HHW~ o0) was determined.

at which

F i g . 6. P l o t o f I D 5 0 vs. H H W ~ 0 0 f o r e l e v e n a m a n t a d i n e derivatives. The equation of the line by linear regression a n a l y s i s o f t h e d a t a is y = 0 . 3 8 + O . 1 8 x w i t h r = 0 . 7 0 a n d P <: 0 . 0 2 .

obtained

liposomes has been increased by 100%. This parameter offers certain advantages over measurement of either integrated peak areas or Te. While at low solute concentrations the total peak area of the transition is fairly constant, it is not possible to resolve the individual peaks. On the other hand, at moderately high solute concentration the onset or midpoint of the transition cannot be adequately defined and/or accurately measured. By contrast, HHW' can be easily determined over a wide concentration range and it can be employed in the analysis of the several types of transition profiles that are induced by a variety of solutes [25]. In Fig. 6 the HHW',00 values obtained from the differential scanning calorimetry curves for the 11 amantadine derivatives examined are plotted against the IDs0 values for these compounds from the platelet aggregation studies. The correlation coefficient (r) is 0.70 (P < 0.02). Discussion The differential scanning calorimetry results obtained for the amantadine derivatives are consistent with our earlier observations that the position and orientation of a sclute within the lipid bilayer are critical factors in determining its relative potency [24]. For a given substituent, the shape and size of the hydrocarbon cage are exceedingly important [24]. This point is well illustrated by the trimethyl-substituted amantadine (Fig. 1 [8]) and the homoamantadines (Fig. 1 [9--11]) which are all significantly more potent than the parent amantadine (Fig. 1 [1]). The results further suggest that in the presence of amantadine derivatives an additive induced phase separation occurs in the bilayer in which

278 parent and modified phases independently coexist. Since these compounds generate in the bilayer a phase with a lower transition temperature, the fluidity of the d o p e d bilayer only increases in certain regions. There is a significant relationship between the relative potencies of various amantadines in inhibiting platelet aggregation in vitro and the abilities of these c o m p o u n d s in inducing phase separation in dipalmitoyl phosphatidylcholine bilayers. It has recently been shown that cholesterol incorporation into blood platelets increases the microviscosity of the platelet membrane [26], increases the sensitivity of the platelets to ADP and epinephrine [23] and alters platelet cyclic AMP metabolism [27]. It may follow here that a drug-induced increase in the fluidity of the membrane and/or the formation of border domains between gel and liquid crystalline regions in the membrane lead to decreased platelet aggregation. However, it remains to be determined if drugs which alter the organization of the bilayer in a qualitatively different fashion [24,28] produce similar results. Since the clinical plasma concentration of amantadine is 0.5 mM [29] and comparable to the IDs0 concentration, and since it is virtually non-toxic [20], these results suggest that amantadine should be considered as an anti-platelet agent. Acknowledgements We gratefully acknowledge the technical assistance of Ms. Nora Wu. This work was supported in part at the Hospital of the University of Pennsylvania by N.I.H. grant HL-18827 and at the University of Delaware by the Delaware Institute of Medical Education and Research. R.K.M. is a Camille and Henry Dreyfus Teacher-Scholar Grant Recipient, 1976--1981. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

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