Acute effects of retinoids on fibrinolysis before and after venous occlusion in healthy humans

THROMBOSIS RESEARCH 50; 175-179, 1988 0049-3848188 $3.00 + .OO Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.

ACUTE EFFECTS OF RETlNOIDS ON FIBRINOLYSIS BEFORE AND AFTER VENOUS OCCLUSION IN HEALTHY HUMANS H.Bounameauxl, G.ReberZ, Y.Bounameaux3 1 Unit of Angiology. Department of Medicine and 2 Unit of Hemostasis, University Hospital of Geneva, CH-1211-Geneva 4, Switzerland and J Clinical Research Division, F.Hoffmann-La Roche 6 Co., CH-4002 Basle, Switzerland (Received 18.12.1987; Accepted in revised form 25.1.1988 by Editor M. Verstraete)

ABSTHACT The effects of acitretin and etretinate on the fibrinolytic system have been investigated in a double-blind, placebo--controlled cross-over study in twelve healthy volunteers. Euglobulin fibrinolytic activity, euglobulin lysis tissue-type plasminogen clot time, activator (t-PA) activity and antigen concentration in plasma were measured before and after 10 min of venous occlusion (VO). three hours after ingestion of the compounds. The plasminogen activator inhibitor (PAI) activity was also measured in each session before fibrinolysis stimulation. None of the parameters studied was significantly affected by the two retinoids in comparison with placebo. Our study demonstrates that a single oral dose of 100 mg of etretinate or acitretin, investigated 3 hours after drug administration does not significantly influence the fibrinolytic system in vivo in humans. This observation does not exclude an effect of retinoids after prolonged intake. INTRODUCTION The vascular endothelium plays a key role in the in vivo regulation of fibrinolysis via mechanisms which are not completely understood (1). Pharmacological intervention to modulate these

Key words: etretinate, plasminogen activator.

acitretin,

retinoids, fibrinolysis,

175

tissue

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regulatory mechanisms might be of potential therapeutic interest. Different retinoids have been shown to stimulate the synthesis and release of tissue-type plasminogen activator (t-PA) by cultured bovine endothelial cells (2) and human synovial fibroblasts (3). We therefore, studied the fibrinolytic system before and after stimulation by venous occlusion following ingestion of retinoids in healthy volunteers. The trial was designed as a double-blind, placebo-controlled cross-over study comparing two retinoids (4) etretinate and its active metabolite acitretin in twelve male, healthy subjects. METHODS Studv desian: Twelve healthy male non-smokers (mean age + SD, 34 + 2.5 years) not having taken any drug during the last seven days entered the trial after giving informed, written consent. The volunteers ingested with 100 ml of milk under the control of the investigator placebo, etretinate (Tigason@, F.Hoffmann-La Roche Switzerland) (100 mg) or acitretin (100 mg) on & Co., Basle, three sessions separated from each other by at least 96 hours. The sequence of the three sessions was randomized. All capsules were of identical appearance. Three hours after drug intake the underwent a first clean venipuncture using a volunteers then tourniquet kept in place for less than thirty seconds; venous occlusion (VO) was carried out at the contralateral arm for 10 min at 100 mmHg (5). During the last minute of VO blood was sampled again from a forearm vein of the occluded arm. VO was carried out alternatively on the left and the right arm in order to increase the interval between two procedures on the same arm. The study was performed according to the Declaration protocol was approved by the Ethical of Helsinki and the Committee of our institution. assays : Blood collected in plastic tubes Fibrinolvsis was 0.01 mol/l) sodium citrate (final concentration containing v/v) which was immediately transferred on ice. Plasma was (l/10, obtained by centrifugation at 4OC at 2’300 g for 15 min and stored in aliquots at -80°C until assayed. Part of the plasma was acidified with acetate buffer 1.0 M (pH 3.9) (l/l, v/v) before freezing. The following measurements were performed: t-PA antigen (t-PA:Ag) using the ELISA 1) on titrated plasma: described by Holvoet et a1.(6) and materials kindly provided (Leuven, Belgium); t-PA activity by P. Holvoet and D. Collen (on the acidified samples) and plasminogen activator inhibitor activity (PA1 activity) using the CoA set t-PA/PA1 from Kabi Sweden) after the method of Chmielewska et Vitrum (Stockholm, al. (7); the euglobulin lysis time 2) on plasma euglobulin fractions: euglobulin fibrinolytic activity (ELT) (8) and the (EFA) All assays were performed using the fibrin plate method (9). in duplicate of triplicate. Hematocrit

was also

measured

in blood

taken

before

and after

VO.

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Blood was collected in light protected oxalated assavs: Retinoids Plasma obtained by centrifugation at 4OC at 2’300 g glass tubes. for 10 min was stored in black plastic tubes at -80°C until etretinate and acittetin were determined by a reversed phase using column switching and UV detection (R. Wyss, HPLC method, personal communication). Statistical analvsis: Wilcoxon matched-pairs test was used to compare the levels of the various components of the fibrinolytic system before and after VO. One-way analysis of variance was applied for the comparison between the drug regimens. All values are given as mean (SD). RESULTS The baseline levels (before VO) of ELT, EFA, t-PA activity, t-PA:Ag and PA1 (Table 1) were not statistically different three ingestion of hours after placebo, etretinate or acitretin. Following VO, ELT was significantly shorter (p < 0.005) and t-PA activity and t-PA:Ag significantly increased (all increases with p < 0.01) (Table 1). Effects

TABLE 1 of retinoids on the fibrinolvtic before and after venous occlusion

svstem

Before VO Treatment After VO* etretinate 22.7 (3.5) 11.6 (7.2) acitretin 20.8 8.9 (7.9) (3.8) placebo 22.8 (4.4) 10.6 (7.8) ---_-______________-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ etretinate EFA 0.2 1.9 (1.8) (0.5) acitretin 0 1.7 (1.8) (III/ml) 0 placebo 1.8 (1.7) --~-~~-~--~--~--~--_-~-~~--_-~_-~_~~-~_-~_~~-~~-~_t-PA:ACT etretinate 0.1 (0.4) 8.4 (10.2) acitretin 0.5 (0.6) 8.3 (10.2) (III/ml) 0.3 (0.5) placebo 7.2 (5.2) --__-__-_______-__-_~~~-~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6.2 (7.2) N.D. etretinate PA1 acitretin 7.8 (5.8) N.D. (U/ml) placebo 8.1 (13.3) N.D. ___-____-__-__-__-__-~-~~-~_~~~~~~~~~~~~~~~~~~~~~~~ 3.9 (2.7) 11.5 (7.3) t-PA:Ag etretinate acitretin 4.3 (3.0) 9.0 (6.3) (ng/ml) 4.1 (2.4) 9.7 (5.7) placebo _-_--_--_-~--_--_-~--~-~--~-~~~~~~~~~~~~~~~~~~~~~~~ etretinate 43.0 (1.7) 49.0 (2.0) Htc acitretin 42.3 (1.9) 48.5 (2.2) (0) placebo 43.1 (1.5) 48.7 (1.8) ELT (in TU)

was 12 for each Numbers in brackets are S.D. Number of values parameter; N.D.: got Determined; TU: Time @its: 1 TU represents above 6 hours were quoted as 25 TU; * All values 15 min. values after VO were significantly different from the values before, the differences between the three sessions were with p < 0.01; not significantly different for all parameters tested.

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The administration of 100 mg of etretinate resulted in plasma concentrations of about 1 pg/ml of etretinate and 0.3 ug/ml of acitretin three hours after ingestion (Table 2). Following 100 mg was approximately of etretin the plasma etretin concentration 0.8ug/ml whilst etretinate was almost not detectable (Table 2). TABLE 2 Plasma

levels of etretinate

and acitretin

in nq/ml (S.D.)

three hours after ingestion of the compounds

I I

_--_-----etretinatel acitretin

I

Ingested compounds etretinate) ------_-- I

acitretinl

placebo

-----__-

----_--_

1142 (698))

16 (36) I

2 (2.4)

277 (77) 1

760 (242))

1 (1.1)

DISCUSSION A stimulation of the plasminogen activator activity has been reported with a series of retinoids in cultured human synovial fibroblasts (3) and bovine endothelial cells (2). The stimulation of the fibroblasts was obtained at very low concentration6 of the compounds (ranging from 10-'&I for retinoic acid to lo-l1 for all-trans retinoic acid) and was first observed within 40 min under the experimental condition6 used (3). We therefore, studied the acute effects of two retinoids on several component6 of the fibrinolytic system in humans. Our data demonstrate that the two compounds tested, etretinate and acitretin, do not induce any significant change of the baseline level6 of ELT, EFA, t-PA and PAI nor of their response to venous activity, t-PA:Ag concentration6 following the occlusion. Although the plasma single oral doses used in this trial were similar to the steady state concentration6 obtained during chronic administration (4) our data do no exclude an effect of retinoids on the fibrinolytic system after prolonged intake. on the other hand, human endothelial cells in vivo might behave in a different manner a6 human synovial fibroblasts and bovine endothelial cells in culture. assistance of Ph. technical Acknowledaements: The excellent (Mrs. policlinic Minazio and of the nurse6 of the Medical F.Jassaud) is gratefully acknowledged. We thank Dr.R. Wyss, and v. Kuruppachery from F. Hoffmann-la Roche, Basle, responsible for retinoids dosage in plasma and for statistical analysis, respectively.

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(2)

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