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Some novel inhibitors of platelet aggregation: Acute toxicity in mice and its relationship to in vitro efficacy and toxicity
14,356-363(1990)
FUNDAMENTALANDAPPLlEDTOXlCOLOCiY
Some Novel Inhibitors of Platelet Aggregation: Acute Toxicity in Mice and Its Relationship to in Vitro Efficacy and Toxicity II. Nipecotoylaminoalkane and Nipecotoylpiperazine Congeners
W.H.
LAWRENCE,A.LASSLO,'
J.E. TURNER,*Z.FENG,ANDS.
E. BOND
Department ofMedicinal Chemistry, College of Pharmacy, and *Department of Biologic and Diagnostic Sciences, College of Dentistry, The Health Science Center, University of Tennessee, Memphis, Tennessee 38163
Received May 9, 1989; accepted August 22, 1989 Some Novel Inhibitors of Platelet Aggregation: Acute Toxicity in Mice and Its Relationship to in Vitro Efficacy and Toxicity. II. Nipecotoylaminoalkane and Nipecotoylpiperazine Congeners. LAWRENCE, W.H., LASSLO, A.,TuRNER,J. E..FENG,Z., AND BoND,S.E.(I~~O). Fundam. Appl. Toxicol. 14, 356-363. Four closely related nipecotoyl congeners are employed as molecular probes to evaluate the effects of systematic molecular changes upon lethal potency of the compounds. The in vivo toxicities, effected by changes in molecular structure, are compared to their in vitro concentrations inhibiting ADP-induced aggregation and epinephrine-induced primary aggregation of human blood platelets and their toxicities to mouse fibroblasts (L-929 cells) in culture. To assist in the selection of compounds which offer the greatest promise as therapeutic agents for further evaluation and to guide future development of optimal molecular structures, a ratio of acute ip LD50 (pmol/kg) [T,] to concentration inhibiting 50% ADP-induced platelet aggregation (pmol/liter) [A] is calculated for each compound. These ratios range from 2.4 1 to 24.92 for the four compounds included in this study. o 1990 Society ofToxicology.
Inappropriate intravascular platelet aggregation and thrombus formation pose significant health risks in a number of clinical conditions. Contact of blood with synthetic polymeric materials is one such situation which results in platelet adhesion and activation to produce thrombus formation. In an effort to develop better and more specific antithrombotic agents, a number of novel platelet aggregation inhibitors have been designed and synthesized in our laboratories and evaluated for in vitro platelet aggregation inhibitory activity (Lasslo et al., 1983, 1986). Lass10 et al. (1984; Lasslo, 1984) envisioned these compounds to inhibit platelet aggregation by interacting with anionic phos’ To whom reprint requests should be addressed. 0272-0590/90 $3.00 Copyright 0 1990 by the Society of Toxicology. All rights ofreproduction in any form reserved.
pholipids to stabilize membrane complexes of the dense tubular system and other storage sites which sequester calcium in platelets, thereby impeding or blocking mobilization of additional Ca’+. This would, in effect, elevate the threshold required for triggering or sustaining platelet aggregation (Lass10 et al., 1984).
Previously, we determined the in vivo acute toxicity in mice for a group of five carbamoylpiperidine congeners and related the lethal potencies of the compounds to their in vitro potency to inhibit ADP-induced human platelet aggregation and to reduce L-cell growth in culture (Lawrence et al., 1988b). One of these compounds, a,a’-bis[3-(N,Ndiethylcarbamoyl)piperidino]-p-xylene dihydrobromide (GT-12), yielded a ratio of 73
356
PLATELET
u-i51
AGGREGATION
Opa”
!J”’
ring
analog
~c-N~-cql -HI
357
INHIBITORS H3c\N-; \
CH2 -H2C
m IH. H3CFH2)~CH2
CH2(CH2)gCH3
BPAT
‘0 IH*
*HI ; CH2W2)&H3
- 117
/
H3C W BPAT
+ bls [4(CH 2, ]
7 2 )a CH2
- 161
+ bla [4(CH 1, ]
t
f
CH2(CH2),CH3 HgCW&H2 BPAT
- 143
FIG. 1. Structural similarities and differences between the nipecotoyl congeners. The net molecular change between compounds is indicated on the arrow.
when acute LD50 (pmol/kg) in male mice was compared to Ia @mol/liter) to inhibit ADP-induced human platelet aggregation in vitro (Lawrence et al., 1988b). Subsequently, this compound was found to exert a marked inhibition of ADP-stimulated platelet aggregation in platelet-rich plasma prepared from blood drawn from treated beagle dogs (Lawrence et al., 1988a) and treated male baboons (Kelly et al., 1989). Kelly et al. (1989) also found that platelets from GT-12-treated baboons were much less sensitive to collageninduced aggregation and were less prone to be implicated in thrombus formation. The present investigation was undertaken to examine, in vivo, the acute lethal potency of four closely related nipecotoyl congeners (Fig. 1) in mice and to relate this activity to (1) chemical structure, (2) in vitro activity inhibiting ADP-induced aggregation of human platelets, (3) potency to inhibit epinephrineinduced primary aggregation of human platelets in vitro, and (4) inhibition of L-cell growth in culture. Because of rather limited
aqueous solubility of these compounds, they were suspended in a 1% methylcellulose aqueous solution for ip administration to mice. The systematic structural changes in these molecules were designed for the use of these compounds as molecular probes in evaluating the influence of these gradual alterations upon in vitro inhibition of platelet aggregation, in vitro cellular toxicity, and in vivo toxicity. MATERIALS
AND
METHODS
Test compounds and suspensions. The compounds used in this study and their designations are as follows: BPAT- I 17, N,N’-bis( 1-decylnipecotoyl)piperazine dihydriodide; BPAT-143, N,N’-bis( l-hexylnipecotoyl)piperazine dihydriodide; BPAT- 16 I, 1,2-bis[N-( ldecylnipecotoyl)-N-methylaminolethane dihydriodide; and BPAT-163, 1,2-bis[N-( 1-hexylnipecotoyl)-N-methylaminolethane dihydriodide (see Fig. 2). Syntheses and analyses of these compounds have been reported by Lasslo and colleagues (Lass.10et nl., 1983; Petrusewicz et al., 1989). Due to their limited aqueous solubility, and in order to maintain a uniform vehicle for their administration,
358
LAWRENCE
ET AL
R, = -(CH,),
-CH,
=
BPAT
- 163
[NW.
= 734.691
R,
- CH 3 =
BPAT
- 161
[M.W.
= 846.801
= - (CH,),
I?, - CH, R 2 = -(CH,),
-CH,
=
BPAT
- 143
[M.W.
= 732.571
R, = -(CH,),
-CH,
=
BPAT
- 117
[M.W.
= 844.781
FIG. 2. Basic structure of the nipecotoyl congeners. (I) The nipecotoylaminoalkane the nipecotoylpiperazine congeners. all compounds were suspended in 1% methylcellulose (Methocel A4M, Dow Chemical Co., Midland, MI) solution for administration to mice. The concentration of the initial suspension was such that it provided the high dose for the mice, after which it was serially diluted with 1% methylcellulose solution to yield appropriate concentrations for subsequent dose levels. Although we had no reason to suspect that these compounds were not stable in aqueous suspension, all suspensions were freshly prepared just prior to dosing mice, and all mice were dosed within 2-3 hr ofpreparing the initial suspension. Suspensions were prepared with a nonaerating stirrer (Model S25 with a 25A attachment, Glas-Col, Terre Haute, IN) to minimize dosing errors from air entrapped in the suspensions. Treated controls received an equivalent volume (10 ml/kg) of I % methylcellulose solution; untreated controls were also maintained. Animals. Young adult male and female ICR mice, 25 + 5 g [Hsd:(ICR)BR], from the barrier-sustained colonies of Harlan Sprague Dawley, Inc. (Indianapolis, IN) were used in these studies, Upon receipt, mice were acclimatized to our facilities for 6 days or more prior to use. Mice were housed in a temperature-controlled environment at 23.3 + 1.1“C (74 -t 2°F) and a relative humidity of 50 f lo%, with 12-hr light/dark cycles. They had ac-
congeners; and (II)
cessto Rodent Lab Chow (Purina 500 1, Ralston-Purina Co., St. Louis, MO) and fresh tap water ad libitum. Procedures. Acute toxicity studies were conducted in two stages. First, LD50 values were approximated employing two male and two female mice at each of not less than 5 and up to 10 dose levels. These preliminary results did not show any trend with respect to sex-related differences in lethality and therefore, due to limited quantities of some compounds, the more extensive determinations were performed in male mice only. In all cases, 14day mortality data were used to calculate the acute LD50 values. The method of Weil (1952) was used for the first stage and, for the second stage, Cornfield and Mantel’s (1950) modification of Karl&s method was employed. Mice were dosed ip with 10 ml/kg of the appropriate suspension to provide the desired dose. Animals were observed about 2-3 hr after dosing for signs of the compounds’ activity and/or toxicity, after which they were returned to the animal quarters where they were checked daily for 2 weeks. After 2 weeks, all surviving mice were terminated by CO2 inhalation. Specimens of the heart, kidneys, liver, lungs, adrenals, and any suspicious tissues were removed from two or more mice which received one or more of the higher doses (equal to or greater than the LD50) and preserved in 10% buffered formalin for
PLATELET
AGGREGATION
359
INHIBITORS
TABLE I ACUTELD~O(~~%CONFIDENCEINTERVAL),
ip,ICR MICE:COMPOUNDSSUSPENDEDIN 1% METHYLCELLULOSE
Compound
Sex
Preliminary (2/group)
10 mice/group
BPAT-117
M F M F M F M F
150 (75-300) mg/kg 150 (106-212)mg/kg 300 (2 12-424) mg/kg 357” mg/kg 45 (27-73) mg/kg 37.5 (26.5-53) mg/kg 212 (150-300)mg/kg 150 (106-212)mg/kg
26 1(226-302) mg/kg
BPAT-143 BPAT- 16 1 BPAT-163
322 (289-357) mg/kg 49 (43-57) mg/kg 2 19 ( 184-262) mg/kg
n There were no deaths in groups receiving 300 mg/kg or less,while both mice died in the groups receiving 424 and 600 mg/kg.
histological evaluation. The tissues were routinely processed into hematoxylin and eosin (H 8r E) stained slides for light microscopic evaluation.
RESULTS The ip acute LD50 values for these compounds and their 95% confidence intervals are presented in Table 1. Unlike the previous study of carbamoylpiperidine congeners in which mice generally died promptly (< 1 hr) or survived the 14day observation period (Lawrence et al., 1988b), these compounds, except BPAT- 163, tended to produce deaths for several days after dosing. There were no deaths, however, in the concurrent vehicle-treated controls. For example, 29 of the 70 mice dosed with BPAT117 died, none of these died within about 2 hr after dosing, 3 died within 24 hr, 22 died in 2-4 days, and 4 more died by the 5th to 8th days. This pattern was also fairly typical for BPAT- 143 and BPAT- 16 1. The mortality profile for BPAT-163, on the other hand, was different. A total of 24 of the 60 mice dosed with BPAT- 163 died; 22 of these died within 15 min to - 1 hr after dosing. The two delayed deaths occurred on the 7th day in a group that received 150 mg/kg, less than the LD50.
Postdosing signs exhibited by mice were dose-related and tended to be more prominent in those mice receiving doses approximately equal to or exceeding the LDSOs. The predominant effects observed were signs of hypoactivity, sedation, and somnolence (e.g., from CNS depression). However, these were often preceeded by sporadic episodes of muscular hyperexcitability (twitches, tremors, or jumping movements). These mice tended to walk unsteadily and stiff-legged, with an occasional jump or brief episode of muscle twitching or tremor. BPAT-163 exhibited a greater tendency toward spasticity than the other three compounds, but sedation and somnolence characteristically intervened. Many of the mice receiving these nipecotoyl congeners exhibited prolonged (for several days) signs of abnormality, e.g., lethargy, ruffled fur, squinted eyes, etc., which was in marked contrast to the rapid recovery of mice treated with the carbamoylpiperidine congenets in the earlier study (Lawrence et al., 1988b). Some of the mice that died promptly were necropsied and examined for abnormalities, especially internal bleeding, but no compound-related abnormalities were observed. Except for BPAT- 163, the mice dying promptly generally received doses which were much greater than the LD50.
360
LAWRENCE
ET AL.
TABLE 2 AC-IIV~TY
Structural change”
TRENDS
vs STRUCTURAL
MODIFICATIONS
Male ICR mice, ip
In vitro aggregation
Cell culture
Toxicity: LD50, rmol/kg
Activity: IaSO, rmol/liter*
Toxicity: ICGSO, pmol/literb
Basic structure I Hexyltodecyl(l63/161) Close ring: Basic structure I to II Hexyl(163/143) Decyl(161/117) Basic structure II Hexyl to decyl ( 143/l 17)
t
298 to 58
4
18to24
t
26 to 2
f:
298 to 440 58 to 309
t
18to55 24 to 12
i
26 to 29 2 to 3
t
440 to 309
f
55 to 12
t
29to3
n See Fig. 2. ’ Data from Lasslo et al. (1986).
After the 1Cday observation period, surviving mice were terminated by CO* inhalation. No compound-related abnormalities were observed at necropsy. There were no apparent histopathological differences, in H & E slides, between randomly selected BPATtreated (Z or >LD50) mice and treated controls in tissues of the lung, heart, kidney, and adrenals. The most significant histologic alteration, in terms of possible toxicity, was fatty change of hepatocytes seen in some vehicle-treated controls and compound-treated animals. However, the etiology of fatty change is nonspecific and generally the condition is considered to be reversible. DISCUSSION An examination of the effects from systematic structural modifications of these nipecotoy1 congeners upon their biological activities reveals a number of interesting trends, as shown in Table 2. In general, replacing the 1,2-bis(methylamino)ethane moiety with a piperazine ring or decreasing lipophilicity by shortening the alkyl chains tends to decrease both in vivo and in vitro toxicity, although the magnitude of the effect is markedly different
in vivo compared to that in vitro. The potency of these compounds in inhibiting ADP-induced aggregation of human blood platelets in vitro is not consistently enhanced by either of these changes. Thus, the most interesting pattern to follow is the ratio of the acute in vivo toxicity and platelet aggregation-inhibitory potency (r,/A Ratio in Table 3). The 1,2 - bis[N- (1 - decylnipecotoyl) - Nmethylaminolethane compound (BPAT161) exerted the most potent lethal effect in mice. Reducing lipophilicity by shortening the bis-alkyl chains from decyl to hexyl (yielding BPAT- 163) strikingly reduced toxicity to mice without significantly altering its potency to inhibit ADP-induced platelet aggregation in vitro (Lass10 et al., 1986; Petrusewicz et al., 1989). This change increased the magnitude of the LD50/Ia50 ratio from 2.4 1 to,16.%‘(Table 3). When the 1,2-bis(methylamino)ethane moiety of BPAT- 16 1 is replaced with a piperazine ring (BPAT-117) mouse toxicity is markedly decreased (=BPAT-163), with an increase in in vitro platelet aggregation inhibitory potency. This structural change increased the ratio of LDSO/Ia50 from 2.41 to 24.92 (see Table 3), the highest values ob-
PLATELET
AGGREGATION TABLE
361
INHIBITORS 3
SUMMARY OF ACTIVITIES, MEANS, AND RATIOS ACUE
Compound BPAT-117 BPAT-143 BPAT- 161 BPAT-163
LD50 in mice ( mWkg) Wml
309.0 439.5 57.9 298.1
MO: Inhib. ADPinduced aggregation (wall liter) [A]”
NO: Inhib. Epi-induced plima~ aggregation (wmll liter)[Elb
ICG50: Inhib. cell growth (PmoV liter) [ICC]’
T&f ratio
TmIE ratio
12.4
11.9 78.5 25.8 63.3
3.3 28.5 2.1 26.1
24.92 8.03 2.41 16.56
25.97 5.60 2.24 4.71
54.1
24.0 18.0’
ratio
ratio
ICC/E ratio
93.64 15.42 27.57 11.42
0.27 0.52 0.09 I .45
0.28 0.36 0.08 0.4 I
T,/ICG
ICC/A
a 1)ata from Lass10 PI nl. (I 986). ’ I)ata from Petrusewicz ef al. (1989). ’ Petrusewicz et al. ( 1989) redetermined the Ia for this compound and found it to he 2 1.2 pmol/liter. If this new value is used, ratio values will be slightly altered but their rank order will not he affected.
sened for any of these four nipecotoyl congeners. Decreasing lipophilicity of BPAT- 117 by shortening the bis-alkyl chains from decyl to hexyl yields a compound (BPAT- 143) which exhibits even less in vivo toxicity than BPAT117, but the platelet aggregation inhibitory activity is disproportionately reduced. Consequently, this structural change decreases the magnitude of the LD50/Ia50 ratio from 24.92 to 8.03 for these two compounds (Table 3). In this case, the beneficial effect upon acute toxicity in mice elicited by the reduction of the lipophilic character of the molecule was more than offset by the lesser ability of the compound to inhibit ADP-induced platelet aggregation. Also, it is significant that BPAT-I 17 was subjected to an oral safety test in rats (data not presented). The compound was suspended in 1% methylcellulose and administered intragastrically to groups of 10 male Sprague-Dawley rats (250 f 50 g) at doses of 262,367,47 1, and 576 mg/kg. One rat in the high dose group (576 mg/kg or 682 pmol/kg) died on the 6th day, while the others survived the 1Cday observation period. No treatmentrelated abnormalities were observed from histological examination (H & E stained slides) of the adrenals, bowel, heart, kidney,
liver, lung, spleen, and stomach of animals surviving 14 days after treatment. Finally, when the 1,2-bis(methylamino)ethane moiety of BPAT-163 is replaced by a piperazine ring (BPAT-143) without a change in the N-alkyl substituents, there is a decrease in both acute in vivo toxicity and in vitro activity to inhibit platelet aggregation. Quantitatively, the change is greater in the inhibition of platelet aggregation activity than in mouse toxicity., Thus, the ratio of LD50/ Ia is reduced by about 50% (from 16.56 to 8.03) from this structural change. It is of interest to note, however, that the best balance between inhibition of ADP-induced platelet aggregation and acute toxicity in mice could not be attributed either to the piperazine ring or to the more hpophilic alkyl chains, per se, within these two miniseries. The most favorable toxicity to activity ratio was obtained with the piperazine ring and the more lipophilic (decyl) alkyl chains (BPAT117), while the second most favorable ratio was obtained from BPAT-163 which contains the 1,2-bis(methylamino)ethane-linking moiety and the less lipophilic (hexyl) alkyl chains. Petrusewicz et al. (1989) investigated the activity of these nipecotoyl congeners, as well as some carbamoylpiperidine congeners, to
362
LAWRENCE
inhibit epinephrine-induced primary aggregation of human platelets in vitro. They found BPAT-I 17 to be the most potent inhibitor of epinephrine-induced primary aggregation, followed by BPAT-161 > BPATI63 > BPAT-143. When these results are examined with respect to their in vivo toxicities to mice (acute LDSO/in vitro Ie50, see Table 3), BPAT- 117 provides the most favorable ratio, followed by BPAT-143 > BPAT- 163 > BPAT-16 1. Inhibition of ADP-induced aggregation by these compounds provides a slightly different ratio (acute LDSO/in vitro Ia50) rank order (BPAT-117 > BPAT163 > BPAT-143 > BPAT-16 1). Although BPAT- 16 1 is a potent inhibitor of both ADPinduced and epinephrine-induced platelet aggregation in vitro, its potent toxicity, both to mice in vivo and to cells in culture (Lass10 et al., 1986), ranks BPAT- 16 1 as the least favorable of these compounds when toxicity is also considered. It may be of interest to note that Petrusewicz et al. (1989) did not obtain 50% inhibition of epinephrine-induced primary aggregation of platelets with a,cu’-bis[3-(NJVdiethylcarbamoyl)piperidino]-p-xylene dihydrobromide (GT- 12). On the other hand, previous studies (Lasslo et al., 1986; Lawrence et al., 1988b) indicated GT-12 to be one of the most active inhibitors of ADP-induced platelet aggregation with the least in vivo toxicity, yielding an LD50/Ia50 ratio of 73.42 (Lawrence et al., 1988b). Folie et al. (1988) reported that GT- 12 was effective in whole human blood to inhibit platelet aggregation and thrombus rate of growth on collagen-coated glass in a parallel plate flow chamber, and Kelly et al. (1989) observed reduced platelet deposition and thrombus formation in test segments of Dacron vascular graft and collagen-coated tubing which were incorporated into an exteriorized femoral arteriovenous shunt of GT-12-treated normal male baboons. These findings clearly indicate that inhibition of ADP-induced platelet aggregation is a better model than inhibition of epineph-
ET AL. TABLE 4 RANKORDEROFCOMPOLJNDS
Compound GT-12” BPAT- 117 BPAT-163 G-l 10” BPAT- 143 G-l 12” BPAT-161 G-29” G-32”
T,,, /A
ratio
73.42 24.92 16.56 11.03 8.03 5.18 2.41 1.19 0.02
Note. Based upon the magnitude of ratios of in vivo toxicity in male, ICR mice (T,,,) [LD50, pmol/kg] and in vitro activity to inhibit ADP-induced human platelet aggregation (A) [Ia50, rmol/liter]. ’ From Lawrence et al. (1988b).
rine-induced primary platelet aggregation as a predictor for platelet response to material contact-induced platelet aggregation. An examination of in vivo toxicity in mice and in vitro toxicity to cells in culture (see Table 3) indicates that lengthening the bis-alkyl substituents which increases lipophilicity, from C6 to CIO, increases toxicity in both systems. However, exchange of the 1,Zbis (methylamino)ethane moiety for a piperazine ring reduces toxicity in both test systems [although the ICGSOs for the hexyl-substituted compounds (BPAT-143 and BPAT163) are essentially the same: 28.5 pmol/liter vs 26.1 pmol/liter]. On the other hand, the decyl-substituted nipecotoylpiperazine compound (BPAT-117) has virtuahy the same LD50 as the hexyl-substituted nipecotoylmethylaminoethane congener (BPAT- 163) (309 pmol/kg vs 298 pmol/kg), which is in marked contrast to their activities in cell culture (ICGSO = 3.3 pmol/liter vs 26.1 pmol/ liter, respectively). Thus, in cell culture, the toxicity of these compounds appears to be most closely linked with lipophilicity of the alkyl chains, whereas the piperazine-linked molecules are less toxic in mice than their 1,2-bis(methylamino)ethane analogs. Com-
PLATELET
AGGREGATION
parisons of in vivo to in vitro toxicities ofthese compounds range from 11 to 94 (see T,/ICG Ratio, Table 3). In conclusion, Table 4 shows a ranking of the nipecotoyl and carbamoylpiperidine congeners in decreasing order of their “estimated margin of safety,” based upon in vivo toxicity to mice and in vitro potency in inhibiting ADP-induced platelet aggregation (LD50, pmol/kg divided by Ia50, pmol/liter). This approach may be useful in selecting those compounds which offer the greatest probability of being suitable for possible therapeutic application. In utilizing this ranking system, however, one must also recognize potential pitfalls of combining such in vivo and in vitro data. For example, the compound’s potency to inhibit platelet aggregation could be markedly different in vivo than it was in plateletrich plasma in vitro; the compound may concentrate in nonactive tissues in vivo which the in vitro evaluation did not include; it could be metabolized/excreted in vivo more rapidly than anticipated; or subsequent studies may reveal unacceptable toxic effects which were not observed in the acute toxicity studies. Despite such possible! problems, this system of ranking helps establish priorities for further evaluation and guides future synthetic efforts in the direction of compounds which promise the best balance between antiplatelet activity and toxicity. ACKNOWLEDGMENTS This work was supported by NIH Grant ROl-HL22236 from the National Heart, Lung and Blood Institutes, and by the Gustavus and Louise Pfeiffer Research Foundation. Their fiscal support of these studies is sincerely appreciated.
REFERENCES CORNFIELD, J., AND MANTEL, N. (1950). Some new aspects of the application of maximum likelihood to the
363
INHIBITORS
calculation of the dosage response curve. Amer. Stat. 45, 181-210. FOLIE, B. J., MCINTIRE, L. V., AND LASSLO, A. (1988). Effects of a novel antiplatelet agent in mural thrombogenesis on collagen-coated glass. Blood 72,1393- 1400. Assoc. J.
KELLY,
A. B., MARZEC,
U. M.. HANSON,
S. R., LASSLO.
A., AND HARKER, L. A. (1989). Inhibition of thrombus formation in viva by the antiplatelet agent, GT- 12. Thromb. Haemostasis. 62,428. [Abstract] LASSLO, A. (1984). Blood platelet function, medicinal agents and other chemical entities. Fed. Proc. 43, 1382-1389. LASSLO, A., DILLINGHAM, E. O., MCCASTLAIN, J. C., CARTER-BURKS, G., QUINTANA, R. P., JOHNSON, R. W., AND NAYLOR, J. L. (1986). Interrelationships between the chemical structure of synthetic entities, their platelet aggregation inhibitory potency and their cellular toxicity, based on in vitro experiments. Med. Prog. Technol. 11, 109- I2 1. LASSLO,A., QUINTANA, R. P.. DUGDALE. M., JOHNSON, R. W., AND NAYLOR. J. L. (1983). Development of novel surface-active compounds for prophylaxis against and treatment of thromboembolic complications. ASAIO J. 6,47-59. LASSLO, A., QUINTANA, R. P., JOHNSON, R. W., NAYLOR, J. L., AND DUGDALE, M. (1984). Relationship between the chemical constitution of aggregation inhibitors and human blood platelet response profile. Biochim.
Biophys.
Acta 772,84-92.
LAWRENCE, W. H., GOLLAMUDI, R., DILLINGHAM, E. O., CARTER-BURKS, G., TISDELLE, P. A., AND LASSLO, A. (1988a). Preliminary in vivo studies on the platelet aggregation inhibitory activity of a,a’-bis[3(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide in beagle dogs. J. Pharm. Sci. 77, 464465.
LAWRENCE, W. H., TISDELLE, P. A., TURNER, J. E., DILLINGHAM, E. O., GOLLAMUDI, R., CARTER-BURKS, G., AND LASSLO, A. (1988b). Some novel inhibitors of platelet aggregation: Acute toxicity in mice and its relationship to in vitro activity and toxicity. Fundum. Appl.
Toxicol.
10,499-505.
PETRUSEWICZ, J., LASSLO, A., CARTER-BURKS, G., GOLLAMUDI, R., DILLINGHAM, E. O., AND BOND, S. E. ( 1989). Relationships between chemical structure and inhibition of epinephrine-induced human blood platelet aggregation. Biochim. Biophys. Acta 983,16 I166. WEIL, C. S. (1952). Tables for convenient calculation of median-effective dose (LD50 or ED50) and instructions in their use. Biometrics 8,249-263.
FUNDAMENTALANDAPPLlEDTOXlCOLOCiY
Some Novel Inhibitors of Platelet Aggregation: Acute Toxicity in Mice and Its Relationship to in Vitro Efficacy and Toxicity II. Nipecotoylaminoalkane and Nipecotoylpiperazine Congeners
W.H.
LAWRENCE,A.LASSLO,'
J.E. TURNER,*Z.FENG,ANDS.
E. BOND
Department ofMedicinal Chemistry, College of Pharmacy, and *Department of Biologic and Diagnostic Sciences, College of Dentistry, The Health Science Center, University of Tennessee, Memphis, Tennessee 38163
Received May 9, 1989; accepted August 22, 1989 Some Novel Inhibitors of Platelet Aggregation: Acute Toxicity in Mice and Its Relationship to in Vitro Efficacy and Toxicity. II. Nipecotoylaminoalkane and Nipecotoylpiperazine Congeners. LAWRENCE, W.H., LASSLO, A.,TuRNER,J. E..FENG,Z., AND BoND,S.E.(I~~O). Fundam. Appl. Toxicol. 14, 356-363. Four closely related nipecotoyl congeners are employed as molecular probes to evaluate the effects of systematic molecular changes upon lethal potency of the compounds. The in vivo toxicities, effected by changes in molecular structure, are compared to their in vitro concentrations inhibiting ADP-induced aggregation and epinephrine-induced primary aggregation of human blood platelets and their toxicities to mouse fibroblasts (L-929 cells) in culture. To assist in the selection of compounds which offer the greatest promise as therapeutic agents for further evaluation and to guide future development of optimal molecular structures, a ratio of acute ip LD50 (pmol/kg) [T,] to concentration inhibiting 50% ADP-induced platelet aggregation (pmol/liter) [A] is calculated for each compound. These ratios range from 2.4 1 to 24.92 for the four compounds included in this study. o 1990 Society ofToxicology.
Inappropriate intravascular platelet aggregation and thrombus formation pose significant health risks in a number of clinical conditions. Contact of blood with synthetic polymeric materials is one such situation which results in platelet adhesion and activation to produce thrombus formation. In an effort to develop better and more specific antithrombotic agents, a number of novel platelet aggregation inhibitors have been designed and synthesized in our laboratories and evaluated for in vitro platelet aggregation inhibitory activity (Lasslo et al., 1983, 1986). Lass10 et al. (1984; Lasslo, 1984) envisioned these compounds to inhibit platelet aggregation by interacting with anionic phos’ To whom reprint requests should be addressed. 0272-0590/90 $3.00 Copyright 0 1990 by the Society of Toxicology. All rights ofreproduction in any form reserved.
pholipids to stabilize membrane complexes of the dense tubular system and other storage sites which sequester calcium in platelets, thereby impeding or blocking mobilization of additional Ca’+. This would, in effect, elevate the threshold required for triggering or sustaining platelet aggregation (Lass10 et al., 1984).
Previously, we determined the in vivo acute toxicity in mice for a group of five carbamoylpiperidine congeners and related the lethal potencies of the compounds to their in vitro potency to inhibit ADP-induced human platelet aggregation and to reduce L-cell growth in culture (Lawrence et al., 1988b). One of these compounds, a,a’-bis[3-(N,Ndiethylcarbamoyl)piperidino]-p-xylene dihydrobromide (GT-12), yielded a ratio of 73
356
PLATELET
u-i51
AGGREGATION
Opa”
!J”’
ring
analog
~c-N~-cql -HI
357
INHIBITORS H3c\N-; \
CH2 -H2C
m IH. H3CFH2)~CH2
CH2(CH2)gCH3
BPAT
‘0 IH*
*HI ; CH2W2)&H3
- 117
/
H3C W BPAT
+ bls [4(CH 2, ]
7 2 )a CH2
- 161
+ bla [4(CH 1, ]
t
f
CH2(CH2),CH3 HgCW&H2 BPAT
- 143
FIG. 1. Structural similarities and differences between the nipecotoyl congeners. The net molecular change between compounds is indicated on the arrow.
when acute LD50 (pmol/kg) in male mice was compared to Ia @mol/liter) to inhibit ADP-induced human platelet aggregation in vitro (Lawrence et al., 1988b). Subsequently, this compound was found to exert a marked inhibition of ADP-stimulated platelet aggregation in platelet-rich plasma prepared from blood drawn from treated beagle dogs (Lawrence et al., 1988a) and treated male baboons (Kelly et al., 1989). Kelly et al. (1989) also found that platelets from GT-12-treated baboons were much less sensitive to collageninduced aggregation and were less prone to be implicated in thrombus formation. The present investigation was undertaken to examine, in vivo, the acute lethal potency of four closely related nipecotoyl congeners (Fig. 1) in mice and to relate this activity to (1) chemical structure, (2) in vitro activity inhibiting ADP-induced aggregation of human platelets, (3) potency to inhibit epinephrineinduced primary aggregation of human platelets in vitro, and (4) inhibition of L-cell growth in culture. Because of rather limited
aqueous solubility of these compounds, they were suspended in a 1% methylcellulose aqueous solution for ip administration to mice. The systematic structural changes in these molecules were designed for the use of these compounds as molecular probes in evaluating the influence of these gradual alterations upon in vitro inhibition of platelet aggregation, in vitro cellular toxicity, and in vivo toxicity. MATERIALS
AND
METHODS
Test compounds and suspensions. The compounds used in this study and their designations are as follows: BPAT- I 17, N,N’-bis( 1-decylnipecotoyl)piperazine dihydriodide; BPAT-143, N,N’-bis( l-hexylnipecotoyl)piperazine dihydriodide; BPAT- 16 I, 1,2-bis[N-( ldecylnipecotoyl)-N-methylaminolethane dihydriodide; and BPAT-163, 1,2-bis[N-( 1-hexylnipecotoyl)-N-methylaminolethane dihydriodide (see Fig. 2). Syntheses and analyses of these compounds have been reported by Lasslo and colleagues (Lass.10et nl., 1983; Petrusewicz et al., 1989). Due to their limited aqueous solubility, and in order to maintain a uniform vehicle for their administration,
358
LAWRENCE
ET AL
R, = -(CH,),
-CH,
=
BPAT
- 163
[NW.
= 734.691
R,
- CH 3 =
BPAT
- 161
[M.W.
= 846.801
= - (CH,),
I?, - CH, R 2 = -(CH,),
-CH,
=
BPAT
- 143
[M.W.
= 732.571
R, = -(CH,),
-CH,
=
BPAT
- 117
[M.W.
= 844.781
FIG. 2. Basic structure of the nipecotoyl congeners. (I) The nipecotoylaminoalkane the nipecotoylpiperazine congeners. all compounds were suspended in 1% methylcellulose (Methocel A4M, Dow Chemical Co., Midland, MI) solution for administration to mice. The concentration of the initial suspension was such that it provided the high dose for the mice, after which it was serially diluted with 1% methylcellulose solution to yield appropriate concentrations for subsequent dose levels. Although we had no reason to suspect that these compounds were not stable in aqueous suspension, all suspensions were freshly prepared just prior to dosing mice, and all mice were dosed within 2-3 hr ofpreparing the initial suspension. Suspensions were prepared with a nonaerating stirrer (Model S25 with a 25A attachment, Glas-Col, Terre Haute, IN) to minimize dosing errors from air entrapped in the suspensions. Treated controls received an equivalent volume (10 ml/kg) of I % methylcellulose solution; untreated controls were also maintained. Animals. Young adult male and female ICR mice, 25 + 5 g [Hsd:(ICR)BR], from the barrier-sustained colonies of Harlan Sprague Dawley, Inc. (Indianapolis, IN) were used in these studies, Upon receipt, mice were acclimatized to our facilities for 6 days or more prior to use. Mice were housed in a temperature-controlled environment at 23.3 + 1.1“C (74 -t 2°F) and a relative humidity of 50 f lo%, with 12-hr light/dark cycles. They had ac-
congeners; and (II)
cessto Rodent Lab Chow (Purina 500 1, Ralston-Purina Co., St. Louis, MO) and fresh tap water ad libitum. Procedures. Acute toxicity studies were conducted in two stages. First, LD50 values were approximated employing two male and two female mice at each of not less than 5 and up to 10 dose levels. These preliminary results did not show any trend with respect to sex-related differences in lethality and therefore, due to limited quantities of some compounds, the more extensive determinations were performed in male mice only. In all cases, 14day mortality data were used to calculate the acute LD50 values. The method of Weil (1952) was used for the first stage and, for the second stage, Cornfield and Mantel’s (1950) modification of Karl&s method was employed. Mice were dosed ip with 10 ml/kg of the appropriate suspension to provide the desired dose. Animals were observed about 2-3 hr after dosing for signs of the compounds’ activity and/or toxicity, after which they were returned to the animal quarters where they were checked daily for 2 weeks. After 2 weeks, all surviving mice were terminated by CO2 inhalation. Specimens of the heart, kidneys, liver, lungs, adrenals, and any suspicious tissues were removed from two or more mice which received one or more of the higher doses (equal to or greater than the LD50) and preserved in 10% buffered formalin for
PLATELET
AGGREGATION
359
INHIBITORS
TABLE I ACUTELD~O(~~%CONFIDENCEINTERVAL),
ip,ICR MICE:COMPOUNDSSUSPENDEDIN 1% METHYLCELLULOSE
Compound
Sex
Preliminary (2/group)
10 mice/group
BPAT-117
M F M F M F M F
150 (75-300) mg/kg 150 (106-212)mg/kg 300 (2 12-424) mg/kg 357” mg/kg 45 (27-73) mg/kg 37.5 (26.5-53) mg/kg 212 (150-300)mg/kg 150 (106-212)mg/kg
26 1(226-302) mg/kg
BPAT-143 BPAT- 16 1 BPAT-163
322 (289-357) mg/kg 49 (43-57) mg/kg 2 19 ( 184-262) mg/kg
n There were no deaths in groups receiving 300 mg/kg or less,while both mice died in the groups receiving 424 and 600 mg/kg.
histological evaluation. The tissues were routinely processed into hematoxylin and eosin (H 8r E) stained slides for light microscopic evaluation.
RESULTS The ip acute LD50 values for these compounds and their 95% confidence intervals are presented in Table 1. Unlike the previous study of carbamoylpiperidine congeners in which mice generally died promptly (< 1 hr) or survived the 14day observation period (Lawrence et al., 1988b), these compounds, except BPAT- 163, tended to produce deaths for several days after dosing. There were no deaths, however, in the concurrent vehicle-treated controls. For example, 29 of the 70 mice dosed with BPAT117 died, none of these died within about 2 hr after dosing, 3 died within 24 hr, 22 died in 2-4 days, and 4 more died by the 5th to 8th days. This pattern was also fairly typical for BPAT- 143 and BPAT- 16 1. The mortality profile for BPAT-163, on the other hand, was different. A total of 24 of the 60 mice dosed with BPAT- 163 died; 22 of these died within 15 min to - 1 hr after dosing. The two delayed deaths occurred on the 7th day in a group that received 150 mg/kg, less than the LD50.
Postdosing signs exhibited by mice were dose-related and tended to be more prominent in those mice receiving doses approximately equal to or exceeding the LDSOs. The predominant effects observed were signs of hypoactivity, sedation, and somnolence (e.g., from CNS depression). However, these were often preceeded by sporadic episodes of muscular hyperexcitability (twitches, tremors, or jumping movements). These mice tended to walk unsteadily and stiff-legged, with an occasional jump or brief episode of muscle twitching or tremor. BPAT-163 exhibited a greater tendency toward spasticity than the other three compounds, but sedation and somnolence characteristically intervened. Many of the mice receiving these nipecotoyl congeners exhibited prolonged (for several days) signs of abnormality, e.g., lethargy, ruffled fur, squinted eyes, etc., which was in marked contrast to the rapid recovery of mice treated with the carbamoylpiperidine congenets in the earlier study (Lawrence et al., 1988b). Some of the mice that died promptly were necropsied and examined for abnormalities, especially internal bleeding, but no compound-related abnormalities were observed. Except for BPAT- 163, the mice dying promptly generally received doses which were much greater than the LD50.
360
LAWRENCE
ET AL.
TABLE 2 AC-IIV~TY
Structural change”
TRENDS
vs STRUCTURAL
MODIFICATIONS
Male ICR mice, ip
In vitro aggregation
Cell culture
Toxicity: LD50, rmol/kg
Activity: IaSO, rmol/liter*
Toxicity: ICGSO, pmol/literb
Basic structure I Hexyltodecyl(l63/161) Close ring: Basic structure I to II Hexyl(163/143) Decyl(161/117) Basic structure II Hexyl to decyl ( 143/l 17)
t
298 to 58
4
18to24
t
26 to 2
f:
298 to 440 58 to 309
t
18to55 24 to 12
i
26 to 29 2 to 3
t
440 to 309
f
55 to 12
t
29to3
n See Fig. 2. ’ Data from Lasslo et al. (1986).
After the 1Cday observation period, surviving mice were terminated by CO* inhalation. No compound-related abnormalities were observed at necropsy. There were no apparent histopathological differences, in H & E slides, between randomly selected BPATtreated (Z or >LD50) mice and treated controls in tissues of the lung, heart, kidney, and adrenals. The most significant histologic alteration, in terms of possible toxicity, was fatty change of hepatocytes seen in some vehicle-treated controls and compound-treated animals. However, the etiology of fatty change is nonspecific and generally the condition is considered to be reversible. DISCUSSION An examination of the effects from systematic structural modifications of these nipecotoy1 congeners upon their biological activities reveals a number of interesting trends, as shown in Table 2. In general, replacing the 1,2-bis(methylamino)ethane moiety with a piperazine ring or decreasing lipophilicity by shortening the alkyl chains tends to decrease both in vivo and in vitro toxicity, although the magnitude of the effect is markedly different
in vivo compared to that in vitro. The potency of these compounds in inhibiting ADP-induced aggregation of human blood platelets in vitro is not consistently enhanced by either of these changes. Thus, the most interesting pattern to follow is the ratio of the acute in vivo toxicity and platelet aggregation-inhibitory potency (r,/A Ratio in Table 3). The 1,2 - bis[N- (1 - decylnipecotoyl) - Nmethylaminolethane compound (BPAT161) exerted the most potent lethal effect in mice. Reducing lipophilicity by shortening the bis-alkyl chains from decyl to hexyl (yielding BPAT- 163) strikingly reduced toxicity to mice without significantly altering its potency to inhibit ADP-induced platelet aggregation in vitro (Lass10 et al., 1986; Petrusewicz et al., 1989). This change increased the magnitude of the LD50/Ia50 ratio from 2.4 1 to,16.%‘(Table 3). When the 1,2-bis(methylamino)ethane moiety of BPAT- 16 1 is replaced with a piperazine ring (BPAT-117) mouse toxicity is markedly decreased (=BPAT-163), with an increase in in vitro platelet aggregation inhibitory potency. This structural change increased the ratio of LDSO/Ia50 from 2.41 to 24.92 (see Table 3), the highest values ob-
PLATELET
AGGREGATION TABLE
361
INHIBITORS 3
SUMMARY OF ACTIVITIES, MEANS, AND RATIOS ACUE
Compound BPAT-117 BPAT-143 BPAT- 161 BPAT-163
LD50 in mice ( mWkg) Wml
309.0 439.5 57.9 298.1
MO: Inhib. ADPinduced aggregation (wall liter) [A]”
NO: Inhib. Epi-induced plima~ aggregation (wmll liter)[Elb
ICG50: Inhib. cell growth (PmoV liter) [ICC]’
T&f ratio
TmIE ratio
12.4
11.9 78.5 25.8 63.3
3.3 28.5 2.1 26.1
24.92 8.03 2.41 16.56
25.97 5.60 2.24 4.71
54.1
24.0 18.0’
ratio
ratio
ICC/E ratio
93.64 15.42 27.57 11.42
0.27 0.52 0.09 I .45
0.28 0.36 0.08 0.4 I
T,/ICG
ICC/A
a 1)ata from Lass10 PI nl. (I 986). ’ I)ata from Petrusewicz ef al. (1989). ’ Petrusewicz et al. ( 1989) redetermined the Ia for this compound and found it to he 2 1.2 pmol/liter. If this new value is used, ratio values will be slightly altered but their rank order will not he affected.
sened for any of these four nipecotoyl congeners. Decreasing lipophilicity of BPAT- 117 by shortening the bis-alkyl chains from decyl to hexyl yields a compound (BPAT- 143) which exhibits even less in vivo toxicity than BPAT117, but the platelet aggregation inhibitory activity is disproportionately reduced. Consequently, this structural change decreases the magnitude of the LD50/Ia50 ratio from 24.92 to 8.03 for these two compounds (Table 3). In this case, the beneficial effect upon acute toxicity in mice elicited by the reduction of the lipophilic character of the molecule was more than offset by the lesser ability of the compound to inhibit ADP-induced platelet aggregation. Also, it is significant that BPAT-I 17 was subjected to an oral safety test in rats (data not presented). The compound was suspended in 1% methylcellulose and administered intragastrically to groups of 10 male Sprague-Dawley rats (250 f 50 g) at doses of 262,367,47 1, and 576 mg/kg. One rat in the high dose group (576 mg/kg or 682 pmol/kg) died on the 6th day, while the others survived the 1Cday observation period. No treatmentrelated abnormalities were observed from histological examination (H & E stained slides) of the adrenals, bowel, heart, kidney,
liver, lung, spleen, and stomach of animals surviving 14 days after treatment. Finally, when the 1,2-bis(methylamino)ethane moiety of BPAT-163 is replaced by a piperazine ring (BPAT-143) without a change in the N-alkyl substituents, there is a decrease in both acute in vivo toxicity and in vitro activity to inhibit platelet aggregation. Quantitatively, the change is greater in the inhibition of platelet aggregation activity than in mouse toxicity., Thus, the ratio of LD50/ Ia is reduced by about 50% (from 16.56 to 8.03) from this structural change. It is of interest to note, however, that the best balance between inhibition of ADP-induced platelet aggregation and acute toxicity in mice could not be attributed either to the piperazine ring or to the more hpophilic alkyl chains, per se, within these two miniseries. The most favorable toxicity to activity ratio was obtained with the piperazine ring and the more lipophilic (decyl) alkyl chains (BPAT117), while the second most favorable ratio was obtained from BPAT-163 which contains the 1,2-bis(methylamino)ethane-linking moiety and the less lipophilic (hexyl) alkyl chains. Petrusewicz et al. (1989) investigated the activity of these nipecotoyl congeners, as well as some carbamoylpiperidine congeners, to
362
LAWRENCE
inhibit epinephrine-induced primary aggregation of human platelets in vitro. They found BPAT-I 17 to be the most potent inhibitor of epinephrine-induced primary aggregation, followed by BPAT-161 > BPATI63 > BPAT-143. When these results are examined with respect to their in vivo toxicities to mice (acute LDSO/in vitro Ie50, see Table 3), BPAT- 117 provides the most favorable ratio, followed by BPAT-143 > BPAT- 163 > BPAT-16 1. Inhibition of ADP-induced aggregation by these compounds provides a slightly different ratio (acute LDSO/in vitro Ia50) rank order (BPAT-117 > BPAT163 > BPAT-143 > BPAT-16 1). Although BPAT- 16 1 is a potent inhibitor of both ADPinduced and epinephrine-induced platelet aggregation in vitro, its potent toxicity, both to mice in vivo and to cells in culture (Lass10 et al., 1986), ranks BPAT- 16 1 as the least favorable of these compounds when toxicity is also considered. It may be of interest to note that Petrusewicz et al. (1989) did not obtain 50% inhibition of epinephrine-induced primary aggregation of platelets with a,cu’-bis[3-(NJVdiethylcarbamoyl)piperidino]-p-xylene dihydrobromide (GT- 12). On the other hand, previous studies (Lasslo et al., 1986; Lawrence et al., 1988b) indicated GT-12 to be one of the most active inhibitors of ADP-induced platelet aggregation with the least in vivo toxicity, yielding an LD50/Ia50 ratio of 73.42 (Lawrence et al., 1988b). Folie et al. (1988) reported that GT- 12 was effective in whole human blood to inhibit platelet aggregation and thrombus rate of growth on collagen-coated glass in a parallel plate flow chamber, and Kelly et al. (1989) observed reduced platelet deposition and thrombus formation in test segments of Dacron vascular graft and collagen-coated tubing which were incorporated into an exteriorized femoral arteriovenous shunt of GT-12-treated normal male baboons. These findings clearly indicate that inhibition of ADP-induced platelet aggregation is a better model than inhibition of epineph-
ET AL. TABLE 4 RANKORDEROFCOMPOLJNDS
Compound GT-12” BPAT- 117 BPAT-163 G-l 10” BPAT- 143 G-l 12” BPAT-161 G-29” G-32”
T,,, /A
ratio
73.42 24.92 16.56 11.03 8.03 5.18 2.41 1.19 0.02
Note. Based upon the magnitude of ratios of in vivo toxicity in male, ICR mice (T,,,) [LD50, pmol/kg] and in vitro activity to inhibit ADP-induced human platelet aggregation (A) [Ia50, rmol/liter]. ’ From Lawrence et al. (1988b).
rine-induced primary platelet aggregation as a predictor for platelet response to material contact-induced platelet aggregation. An examination of in vivo toxicity in mice and in vitro toxicity to cells in culture (see Table 3) indicates that lengthening the bis-alkyl substituents which increases lipophilicity, from C6 to CIO, increases toxicity in both systems. However, exchange of the 1,Zbis (methylamino)ethane moiety for a piperazine ring reduces toxicity in both test systems [although the ICGSOs for the hexyl-substituted compounds (BPAT-143 and BPAT163) are essentially the same: 28.5 pmol/liter vs 26.1 pmol/liter]. On the other hand, the decyl-substituted nipecotoylpiperazine compound (BPAT-117) has virtuahy the same LD50 as the hexyl-substituted nipecotoylmethylaminoethane congener (BPAT- 163) (309 pmol/kg vs 298 pmol/kg), which is in marked contrast to their activities in cell culture (ICGSO = 3.3 pmol/liter vs 26.1 pmol/ liter, respectively). Thus, in cell culture, the toxicity of these compounds appears to be most closely linked with lipophilicity of the alkyl chains, whereas the piperazine-linked molecules are less toxic in mice than their 1,2-bis(methylamino)ethane analogs. Com-
PLATELET
AGGREGATION
parisons of in vivo to in vitro toxicities ofthese compounds range from 11 to 94 (see T,/ICG Ratio, Table 3). In conclusion, Table 4 shows a ranking of the nipecotoyl and carbamoylpiperidine congeners in decreasing order of their “estimated margin of safety,” based upon in vivo toxicity to mice and in vitro potency in inhibiting ADP-induced platelet aggregation (LD50, pmol/kg divided by Ia50, pmol/liter). This approach may be useful in selecting those compounds which offer the greatest probability of being suitable for possible therapeutic application. In utilizing this ranking system, however, one must also recognize potential pitfalls of combining such in vivo and in vitro data. For example, the compound’s potency to inhibit platelet aggregation could be markedly different in vivo than it was in plateletrich plasma in vitro; the compound may concentrate in nonactive tissues in vivo which the in vitro evaluation did not include; it could be metabolized/excreted in vivo more rapidly than anticipated; or subsequent studies may reveal unacceptable toxic effects which were not observed in the acute toxicity studies. Despite such possible! problems, this system of ranking helps establish priorities for further evaluation and guides future synthetic efforts in the direction of compounds which promise the best balance between antiplatelet activity and toxicity. ACKNOWLEDGMENTS This work was supported by NIH Grant ROl-HL22236 from the National Heart, Lung and Blood Institutes, and by the Gustavus and Louise Pfeiffer Research Foundation. Their fiscal support of these studies is sincerely appreciated.
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363
INHIBITORS
calculation of the dosage response curve. Amer. Stat. 45, 181-210. FOLIE, B. J., MCINTIRE, L. V., AND LASSLO, A. (1988). Effects of a novel antiplatelet agent in mural thrombogenesis on collagen-coated glass. Blood 72,1393- 1400. Assoc. J.
KELLY,
A. B., MARZEC,
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S. R., LASSLO.
A., AND HARKER, L. A. (1989). Inhibition of thrombus formation in viva by the antiplatelet agent, GT- 12. Thromb. Haemostasis. 62,428. [Abstract] LASSLO, A. (1984). Blood platelet function, medicinal agents and other chemical entities. Fed. Proc. 43, 1382-1389. LASSLO, A., DILLINGHAM, E. O., MCCASTLAIN, J. C., CARTER-BURKS, G., QUINTANA, R. P., JOHNSON, R. W., AND NAYLOR, J. L. (1986). Interrelationships between the chemical structure of synthetic entities, their platelet aggregation inhibitory potency and their cellular toxicity, based on in vitro experiments. Med. Prog. Technol. 11, 109- I2 1. LASSLO,A., QUINTANA, R. P.. DUGDALE. M., JOHNSON, R. W., AND NAYLOR. J. L. (1983). Development of novel surface-active compounds for prophylaxis against and treatment of thromboembolic complications. ASAIO J. 6,47-59. LASSLO, A., QUINTANA, R. P., JOHNSON, R. W., NAYLOR, J. L., AND DUGDALE, M. (1984). Relationship between the chemical constitution of aggregation inhibitors and human blood platelet response profile. Biochim.
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LAWRENCE, W. H., TISDELLE, P. A., TURNER, J. E., DILLINGHAM, E. O., GOLLAMUDI, R., CARTER-BURKS, G., AND LASSLO, A. (1988b). Some novel inhibitors of platelet aggregation: Acute toxicity in mice and its relationship to in vitro activity and toxicity. Fundum. Appl.
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PETRUSEWICZ, J., LASSLO, A., CARTER-BURKS, G., GOLLAMUDI, R., DILLINGHAM, E. O., AND BOND, S. E. ( 1989). Relationships between chemical structure and inhibition of epinephrine-induced human blood platelet aggregation. Biochim. Biophys. Acta 983,16 I166. WEIL, C. S. (1952). Tables for convenient calculation of median-effective dose (LD50 or ED50) and instructions in their use. Biometrics 8,249-263.