txa2 receptor antagonist

Prostaglandins

and

Medicine

3:

279-290,

g,ll-AZO-lj-OXA-Is-HYDROXYPROSTANOIC SYNTHETASE

INHIBITOR

Sheung-Tsam College

of

School;

and

University

Kam

and

Pharmacy; Jonathan of

AND

A

Earl M.

Minnesota,

ACID: PGH2/TXA2

Philip

1979

S. W.

RECEPTOR

Portoghese, Dunham,

Gerrard,

A

Dept.

Minneapolis,

of

THROMBOXANE

ANTAGONIST

Dept.

Dept.

POTENT

of

of

Medicinal

Pharmacology,

Pediatrics;

Minnesota

55455,

Medical

Chemistry, Medical School,

USA

ABSTRACT 9,11-Azo-13-oxa-15-hydroxyprostanoic acid (AOHP) is shown to be a potent inhibitor of thromboxane synthetase with an IC50 at about lO+jM. It also blocked the agonist actions of 9,11-epoxymethanoPGH2 (IC50 9 x 10m7M) and TxA2 (IC50 2.4 x 10V6M) on human plateletrich plasma indicating that it also is a PGH2/TxA2 receptor blocker.

INTRODUCTION Thromboxane A2 (TxA2) is one of the major metabolites of arachidonic acid in human platelets (1) and is an extremely potent inducer of platelet aggregation (2). Since TxA2 is considered to be one of the important endogenous factors associated with the initiation of platelet aggregation and hence thrombosis (3), logical approaches to the development of antithrombotic agents would involve the selective inhibition of TxA2 biosynthesis and the blockage of TxA2 receptors in human platelets. There are a number of selective TxA2 synthetase inhibitors (4-7) but there are relatively few TxA2 receptor antagonists (8-10). We report on the biological activity of a new prostaglandin endoperoxide analogue, 9,11-azo-13-oxa-15-hydroxyprostanoic acid (AOHP) which exhibits properties of TxA2 receptor blockade and TxA2 synthetase inhibition.

bH AOH

P

279

MATERIALS AND METHODS Chernrcals and Reagents. [l- 14CJArachidonic acid (55.5 uCi/ mol) was obtained from New England Nuclear, Boston, Massachusetts. Fully characterized [l-14Clprostaglandin H2 (11) (1 uCi/ 11101) was a gift from Dr. G. Graff, Department of Pharmacology, University of Minnesota. Prostaglandins E2, F2 , D2, 6-keto-PGFl and 9,11-epoxymethanoPGH2 (U44609) were gifts from Dr. J. E. Pike of the Upjohn Company. 9,11-Azo-13-oxa-15-hydroxyprostanoic acid (AOHP) was handled in ethanol solution. Platelet Aggregation Studies. Platelet-rich plasma (PRP), aspirinated PRP (100 ug aspirin/ml of PRP) and wasned platelets were Platelet-rich plasma prepared according to Gerrard et al. (12). was incubated with different concentrations of AOHP at 37.5'C for 1.5 minutes berore being added to a measuring cuvette which contained a standard quantity of prostanoid aggregating agents. Aggregation (or inhibition of agyregation) was monitored for five minutes on a Payton dual channel aggregometer. For TxA2-induced agyregation, the TxA2 was generated by mixing suspended platelet microsomes (0.1 mL, 1.9 mg protein/m&) with After stirring different amounts of PGH2 at 37.5"C in a cuvette. for 15 seconas, aspirinated PRP (0.9 mL) was added and the aggregation Boiled microsonres were used for control experiments. was monitored. Enzymes. Aspirin-treated human platelet microsomes and pig aorta microsomes were prepared according to Kulkarni (13) and Moncada et al. (14) and consisted of 3.8 mg and 8.5 mg of protein per mL of microsomal mixture respectively (15). Metabolism Studies of [l-l4C]Arachidonic Acid by Platelets. Wasned human platelets (2 mL, 2 x 10y platelets/mL) were incubated for 1.5 minute with AOHP (0.2 i~mol, final concentration 10e4M) and then 3.28 nmol each of [l-14C]arachidonic acid and unlabeled arachidonic acid was added. Aggregation was monitored for five The metabolites were extracted and esterified according minutes. The fatty acid esters were developed on to Gerrard et al. (12). TLC (silica gel) with water-saturated lo:5 iso-octane-ethyl acetate. The plate was scanned for radioactivity on a Berthold LB 2760 TLC scanner and the area of the plate which corresponded to TxB2 and other prostanoid metabolites (Rf 0.03) (12) was scraped and the silica gel was extracted with 1:l metnanol-ether. After removal of the organic solvent, the residue was transferred to a TLC plate and developed with 97:2 ether-methanol. The prostaglandin standards were visualized by application of lU% phosphomolybdic acid in EtOH. A control also was performed in the absence of AOHP.

280

Metabolism of [l-14C]Prostaglandin H, by Microsomal Preparations Suspended human platelet microsomes (0.5 mL, 0.38 mg protein/ml) were incubated with a standard quantity of AOHP at 37.5'C for 2 minutes and added to 0.005 pmol [l-14C]PGH2. Stirring was continued for 5 minutes. The microsomal mixture was acidified and extracted with EtOAc. The organic layers were evaporated to dryness and the residue was developed twice on TLC with 1% HOAc in EtOAc. Similar procedures were employed for the preparation of pig aorta microsomes (0.5 mL, 0.85 mg protein/mL) and the incubation with AOHP and [l-14C]PGH2 (0.005 pmol). The metabolites were separated on TLC by double development with water-saturated 11:5:2 EtOAc-isooctane-HOAc. RESULTS AND DISCUSSION Preliminary studies of AOHP showed that it does not induce platelet aggregation and does not inhibit ADP-induced aggregation of aspirinated platelet-rich plasma. However, AOHP (10W4M) is able to inhibit arachidonic acid (3.28 nmol/mL)-induced aggregation in washed platelets. The effect of AOHP on arachidonic acid metabolism in the washed platelets is shown in Figure 1. In contrast to the

A

B

0

IO

$a

E2

(Me)(Me)

TX

15 cm

f32

(Me)

Figure 1. Thin-layer radiochromatograms of products isolated after incubation of [l-l4Clarachidonic acid with washed human platelets in the absence (A) and presence (B) of AOHP (10B4M).

281

control, no TxB2 was formed in the presence of lo-*M AOHP, indicating that thromboxane synthetase was totally inhibited. The concomitant increase in the formation of PGE2 and PGFz~ which accompanied the inhibition is readily explicable because a selective blockade of thromboxane synthetase will divert the transformation of PGH2 to other prostaglandins. Altiiough it is not possible from these results to distinguish between enzymatic and nonenzymatic conversion of PGH2 to E2 and F2e, the increase in E2 and F2e formation suggests that endoperoxide formation from arachidonic acid is not inhibited by AOHP. Consequently, it appears that AOHP is not a cyclooxygenase inhibitor. Experiments were also performed to examine the direct effect of AOHP on thromboxane synthetase. [l-14C]Prostaglandin H2 was incubated with platelet microsomes in the presence of various concentrations of AOHP. The PGH2 metabolites were isolated and developed on TLC. The radiochromatograms are shown in Figure 2 together with a control. The control experiment shows only two major peaks which correspond to 12L-hydroxy-5,6,10-heptadecatrienoic acid In the presence of AOHP, the formation of TxB2 (HHT) and TxB2. was inhibited in a concentration-dependent manner. At a concentration of lo-6M of AOHP, the TxB2 formed is approximately half that of the control and consequently its ICso value is in the micromolar range. Accompanying the decrease of TxB2, the TLC radiochromatogram also shows an increase in the formation of PGF2c, PGE2, and PGD2 with The concomitant decrease in a concomitant decrease in HHT formation. the formation of HHT and TxB2 is in keeping with previous reports (16-18) of the parallel effects on these metabolites. In view of the potent antiaggregatory activity of prostacyclin, it was of interest to determine the effect of AOHP on the biosynthesis of this 1P4GH2metabolite; thus pig aorta microsomes were incubated with [l- C]PGH2 in the presence of AOHP and the metabolites were separated with TLC. Figure 3 shows radiochromatograms from incubations containing different concentrations of AOHP. The formation of the prostacyclin metabolite, 6-keto-PGFl,, appears to be only partially inhibited (
282

CONT

-4 10

M

-5 10 M

lo

-6

M AOHP

0

5 @@Q@ Fza

Figure 2. bation of experiment microsomes with those

E2

10

15 cm a

TxB2

D2 HHT

Radiochromatograms of the products isolated after incu[l-l*C]PGH2 With human platelet microsomes. The control and the experiment with lO'*M AOHP were duplicated using from separate donors and the results are consistant shown above.

283

CONTROL

10

10

10

-4

-6

-8

M

M

M AOHP -I+

Figure 3. Radiochromatograms of the products isolated from incubation of [l-l*C]PGH, with pig aorta microsomes. The control experiment and the experiment with 10e4M AOHP were duplicated using microsomes from a separate animal and the results are consistent with those shown above.

284

The receptors which are blocked may be those that recognize the endoperoxides or TxA2. When the concentration ofU44609 was increased from 5.7 x lO-'M to 1.4 x 10e6M (Figure 41, the concentration of AOHP required to inhibit platelet aggregation also increased accordingly, as observed by a shift of the inhibition curve to the higher concentration of AOHP. The parallel nature of the two inhibition curves suggests that AOHP is a competitive inhibitor of U44609. Accordingly, AOHP also might act as a competitive antagonist of PGH2(G2) receptors and/or TxA2 receptors.

Log

[AOW]

(M)

Figure 4. Inhibition of U44609-induced aggregation by AOHP in human PRP. Aggregation induced by 5.7 x l@-'M U44609 (A) and 1.4 x 10e6M U44609 (B) in the presence of AOHP were expressed as a percentage of the maximum aggregation obtained by treating the plasma with 1.4 x 10-6M u44609. The values shown are the mean f standard error for the curve fi (N=3). Each point in the curve B (N=l) represents the average of two determinations. The value of N signifies the number of individual donors from which the data was obtained.

285

Experiments also were performed to evaluate the antagonistic activity of AOHP on TxA2-induced aggregation. Exogenous Tti2 was prepared by incubation of subaggregatory doses of PGH2 with fresh human platelet microsomes (20). Thus, PGH2 was incubated with human platelet microsomes (0.1 mL, 0.19 mg protein) for 15 seconds followed by addition of aspirinated PRP. Aggregation obtained by this means is mainly due to the effect of TxA2 (21). The ICso values of AOHP for TxA2-induced aggregation were determined to be 1.7 x 10m5M (N=2) with 0.93 nanomole of PGH ; 2.4 x 10e6M (N=l) with 0.46 nanomole of PGH2 (Figure 5). In &oth cases, the TxA2 generated from PGH2 and platelet microsomes was sufficient to induce irreversible platelet aggregation. The IC50 values of AOHP in theantagonism of platelet aggregation induced by different aggregating agentsare summarized in Table I.

100

z F a

t3

tr”

50

t3 t3

a

-4

-5

-6

Log

[AOHP]

WI)

Figure 5. Inhibition of TxA2-induced aggregation by AOHP in aspirinated human PEP. Aggregation induced by TxA2 generated from (A) 9.3 x 10-7M PGH2 or (B) 4.6 x 1O-7M PGH2 + platelet microsomes was expressed as a percentage of the maximum aggregation obtained by treating the plasma with 1.4 x 10m6M PGH2. Data shown are the average of two determinations.

286

Table

I.

Summary of IC50a values for AOHP in human PP.P.

Approximate AOHP IC50 (M)

Aggregating agent (concentration, M)

Nb -

9 x 10-7

u44609

(5.7 x 10-7)

3

1.4 x 10-5

PGH2

(1.4 x 10-6)

4

2.8 x lo+

PGH2C

(1.4 x lo+

3

1.7 x 10-5

TxA2=

(from 9.3 x 10s7M PGH2)

2

2.4 x lo+

TxA2

(from 4.6 x 10m7M PGH2)

1

aThe ICso is the concentration of AOHP required to inhibit the baggregation in PHP by 50%. Number of concentration-inhibition curves obtained from individual cblood donors used in the determination of IC50 values. Conducted in aspirinated PRP.

The selective inhibition of TxA2 biosynthesis by AOHP and its receptor antagonism of TxA2/PGH2 suggests that it has potential as a pharmacologic tool and is an important lead for the development of antithrombotic agents.

ACKNOWLEDGEMENTS This work was supported in part by NIH research grant HL 16524 and HL 17871 and is abstracted in part from the Ph.D. thesis submitted by S. -T. Kam to the Graduate Faculty, University of Minnesota. The authors wish to thank Dr. G. Graff, Department of Pharmacology, University of Minnesota, for the [l-14C]PGH2 sample and Dr. J. E. Pike of the Upjohn Company for the prostaglandin standards.

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287

4) Samuelsson, M., M. Goldyne, E. Granstrom, M. Hamberg, S. Hammarstrom, and C. Malmsten. Prostaglandins and Thromboxanes. Ann. Rev. Biochem. -47:997. 1978. Inhibitors of Thromboxane 5) Diczfalusy, U., and S. Hammarstrom. Synthetase in Human Platelets. FEBS Iett. 82:107. 1977. 6) Gorman, R. R., G. L. Bundy, D. C. Peterson, F. F. Sun, 0. V. Miller, Inhibition of Human Platelet Thromboxane and F. A. Fitzpatrick. Synthetase by 9,11-Azoprosta-5,13-dienoic Acid. Proc. Nat. Acad. Sci., USA -74:4007. 1977. The Synthesis of 15-Deoxy-9,117) Bundy, G. L., and D. C. Peterson. (epoxyimino) prostaglandins. Tetrahedron Lett. 1:41. 1978. 8) MacIntyre, D. E. and J. L. Gordon. Descrimination between Platelet Prostaglandin Receptors and a Specific Antagonist of Bisenoic Prostaglandins. Throm. Res. -11:705. 1977. Trimethoquinol is a Potent 9) MacIntyre, D. E. and A. L. Willis. 63:361P. Prostaglandin Endoperoxide Antagonist. Br. J. Pharmac. 1978. 10) Fitzpatrick, F. A., G. L. Bundy, R. R. Gorman, and T. Honohan. 9,11-Epoxyiminoprosta-5,13_dienoic Acid is a Thromboxane A2 Nature 275:764. 1978. Antagonist in Human Platelets. 11)

Graff, G., J. H. Stephenson, D. B. Glass, M. K. Haddox, and N. D. Activation of Soluble Splenic Cell Guanylate Cyclase Goldberg. by Prostaglandin Endoperoxides and Fatty Acid Hydroperoxides. J. Biol. Chem. 253:7662. 1978.

12)

Gerrard, J. M., J. D. Peller, T. P. Krich, and J. G. White. Cyclic AMP and Platelet Prostaglandin Synthesis. Prostaglandins -14:39. 1977.

13) Kuljcarni, P. S. and K. E. Eakins. N-0164 Inhibits Generation of Thromboxane-A2-like Activity from Prostaglandin Endoperoxides by Human Platelet Microsomes. Prostaglandins -12:465. 1976. 14) Moncada, S., R. Gryglewski, S. Bunting, and J. R. Vane. An Enzyme Isolated from Arteries Transforms Prostaglandin EndoAggregation. Nature 263:663. 1976. 15) Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. -72:248. 1976. 16) Anderson, M. W., D. J. Crutchley, B. E. Tainer, and T. E. Eling. Kinetic Studies on the Conversion of Prostaglandin Endoperoxide PGH2 by Thromboxane Synthase. Prostaglandins 16:563. 1978.

288

Thromboxane Synthase from 17) Wlodawer, P. and S. Hammarstrom. Bovine Lung-Solubilization and Partial Purification. Biochem. Biophys. Res. Comm. -80:525. 1978. 18) Diczfalusy, V., P. Falardeau, and S. Hammarstrom. Conversion of Prostaglandin Endoperoxides to Cl7 -Hydroxy Acids Catalyzed by Human Thromboxane Synthase. FEBS Lett. -84:271. 1977. 19) Malmstem, C. Some Biological Effects of Prostaglandin Endoperoxide Analogs. Life Sciences -18:169. 1976. 20) Sun, F. F., J. P. Chapman, and J. C. McGuire. Prostaglandin Endoperoxide in Animal Tissues. 1055. 1977.

Metabolism of Prostaglandins -14:

21) Hamberg, M., J. Svensson, and B. Samuelsson. Thromboxanes: A New Group of Biologically Active compounds Derived from ProstaProc. Nat. Acad. Sci., USA -72:2994. 1975. glandin Endoperoxides.

289