Effects of subcutaneously administered dermatan sulfate (MF 701) on the coagulation and fibrinolytic parameters of healthy volunteers

THROMBOSIS RESEARCH62;663-672,1991 0049-3848/91 $3.00+ .OOPrinted in theUSA. Copyright(c) 1991PergamonPress pk. All rights reserved.

EFFECTS OF SUBCUTANBOUSLY ADMINISTERED DERMATAN SULFATK (MF 701) ON TBE COAGULATION AND FIBRINOLYTIC PARAMETERS OF BEALTBY VOLUNTEERS.

A. Tripodi, M. Moia, B. Bottasso, P.M. Tenconi, F. Gianese, P.M. Mannucci

A. Bianchi Bonomi Hemophilia and Thrombosis Center and Institute Internal Medicine, IRCCS Maggiore Hospital and University of Of Milan, Italy (Received 19.1 .1991; accepted in revised form 12.3.1991 by Editor S. Coccheri)

Abstract

Eight healthy volunteers were given single subcutaneous doses of dermatan sulfate (DS, 100, 200 and 400 mg), heparin (5,000 IU) and placebo in random order. Wash-out between treatments was 1 10 days. Serial blood samples were taken before and up to 24 hours after treatment to measure coagulation and fibrinolytic parameters. Thrombin generation was significantly inhibited by DS and heparin compared to placebo. The effect of DS was doseitpendent. Peak inhibition after 200 mg DS was comparable to that of 5,000 IU heparin, but lasted longer. A small, bordeline significant prolongation of APTT was observed after 400 mg DS and heparin. The changes in PA1 and fibrinolytic activities were those of the circadian variation. No changes were seen in the other parameters tested. In conclusion, single S.C. doses of DS (200, or 400 mg) inhibit ex vivo thrombin generation equally or more than 5,000 IU heparin and for a longer time. The effect of both treatments on fibrinolysis is negligible.

INTRODUCTION

sulfate belongs to the family Dermatan (DS) glycosaminoglycans (GAGS), acid mucopolysaccarides with stro:; electronegative charges due to the presence of sulfate groups (1). Of the biological activities of these molecules, the most important is the ability to inhibiting the intrinsic pathway of family, heparin coagulation. Of the GAG has been blood KEY WORDS: Dermatan sulfate; heparin; pharmacodynamics; coagulation; fibrinolysis

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successfully used over the years for the prophylaxis and treatment of thromboembolic diseases (2). However, its clinical use is somewhat limited by the risk of bleeding (3, 4). The search for new antithrombotic agents with a more favourable antithrombotic to hemorrhagic ratio than that of heparin has recently brought attention to DS. Preliminary experiments demonstrated that when DS was added to human plasma at high concentrations, it prolonged the activated partial thromboplastin time (APTT) without potentiating antithrombin III suggesting it has a different mechanism of action than that (51, of heparin. Subsequently it was demonstrated that DS greatly potentiates the inhibitory activity of heparin cofactor II, a plasma inhibitor of thrombin, but not of factor Xa or other serine proteases (6). Experimental studies of venous thrombosis animal models have shown that DS ’ effective i:tithrombotic agent with a low hemorrhagic po&tiz? (7-12). The human pharmacokinetics of DS have recently been studied by different assay methods especially developed for this purpose (13-15). With this as background, we elected to compare the effects of single increasing doses of DS and a standard prophylactic dose of unfractionated heparin, both administered subcutaneously (s.c.), wide range of coagulation and fibrinolytic parameters of EEalFhy volunteers. A placebo-controlled, randomized cross-over design was used. Special attention was devoted to thrombin since it important in inhibition, appears highly the antithrombotic efficacy of both heparin and non-heparin GAGS (16, 17). MATERIALS,

SUBJECTS

ARD

MRTHODS

Materials. Sterile 0.5 ml ampoules containing 100 mg MF 701 DS (batch 077) or saline solution (placebo) were provided by was Mediolanum Farmaceutici S.p.A., Milano, Italy. MF 701 extracted from pig intestinal mucosa, contained 7% S and 31.9% uranic acid, and had a sulfate/carboxylate ratio of 1.33. The using declared anticoagulant activity (USP) was < 5 U/mg unfractionated heparin as a standard. Calcium heparin (5,000 Ill/O.2ml ampoule) was also provided by Mediolanum Farmaceutici. Subjects and study desiqn. Eight healthy volunteers (3 women and 5 men) were enrolled in the study after giving informed and written consent. Their age range was 26-38 years; body weights 51 to 80 kg. They took no other drugs that affect hemostasis in the week before or during the course of the study. Each volunteer was given five S.C. injections in a randomized sequence: 100, 200 and 400 mg DS, 5,000 IU calcium heparin and 1 ml saline. The highest DS dose was divided into two 1 ml injections, one on each side of the abdominal wall. The wash-out periods were 1 10 days. Blood samples were taken immediately before and 1.5, 3, 6, 12 and 24 hours after each treatment and anticoagulated with 3.8% trisodium citrate (9 parts of blood and 1 part of anticoagulant). Blood was immediately centrifuged for 15 minutes at 3,500 rpm in a refrigerated centrifuge. Plasma samples were distributed in

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coded plastic tubes, snap frozen and stored at -7O'C. The entire process from blood sampling to plasma storage lasted no more than one hour. All the assays (see below) were done in blind conditions at the end of the study, after thawing the frozen samples for 5 minutes at 37°C. Methods and reagents. The following measurements were obtained: prothrombin time (PT) (Manchester reagent, Laboratori Baldacci, Pisa, Italy); APTT (automated APTT, Organon Teknika, Durham N.C.); thrombin time (TT) (bovine thrombin from Boehringer, Mannheim, Germany). The results were expressed as ratios (subject/pooled normal plasma). The following assays were also carried out: fibrinogen assay (fibrin polymerization time, Boehringer), with results expressed as mg/dL; antithrombin III functional assay (heparin cofactor, Coatest Antithrombin, Kabi Diagnostica, Stockholm, Sweden); heparin cofactor II functional assay, as described by Tran and Duckert (18), with the chromogenic substrate S- 2238 (Kabi Diagnostica). The results of the last two assays were expressed as U/dL of a reference normal plasma pooled from 30 healthy donors (15 men and 15 women). The ex vivo thrombin generation test was performed as follows: 0.4 ml plasma were mixed with 0.4 ml of APTT reagent (Organon Teknika); after 2 minutes incubation at 37°C the mixture was recalcified by adding 0.4 ml of 25 mM calcium chloride and a stopwatch was started; the ensuing clot was then wrapped around a plastic rod and squeezed gently against the tube wall; 0.15 ml of the exuded fluid were then sampled at 1.5, 3 and 5 minute intervals and transferred into a tube which contained 0.4 ml 0.2 M Tris-HCl buffer, pH 8.25 and 0.3 ml of 0.74 mM thrombin chromogenic substrate S-2238, (Kabi Diagnostica) at 37'C. A second stopwatch was then started and the reaction stopped after 30 seconds by adding 0.3 ml of 50% acetic acid. The absorbance at 405 nm was then read against a blank and transformed into units/ml of generated thrombin by reading off a calibration curve of human thrombin (Fibrindex, Ortho Diagnostic Systems, Raritan, N.J.). The amount of thrombin generated during the three sampling procedures over the five minute incubation period was computed by calculating the area under the curve (generated thrombin vs. incubation time) and expressed as U x min/ml. Plasma euglobulin fibrinolytic activity was measured by the fibrin plate method (19) and results expressed as diameter (in millimeters) of the lysed area. Tissue plasminogen activator antigen was measured by an ELISA method (American (tPA) Diagnostica, New York, N.Y.) and results expressed as ng/ml. The activity of tPA inhibitor (PAI) was measured according to Verheijen (20), with reagents from Kabi Diagnostica, and results expressed as IU/ml. The following additional measurements were obtained for samples collected before and three hours after each treatment: plasminogen activity (Coatest Plasminogen, Kabi Diagnostica); antiplasmin activity (Coatest Antiplasmin, Kabi Diagnostica) and histidine-rich glycoprotein antigen (Behring, Scoppito, Italy). Routine hematology tests, including platelet counts, were performed pre-admission and 24 hours after each treatment. Statistical analyses. Homogeneity of baseline

(pre-treatment)

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values was tested by analysis of variance. To assess for differences between treatments the individual data for each of the performed measurements were plotted against the time of blood collection and the area under the curve (AUC) was then computed. For this, data were adjusted by subtracting the individual baseline levels and adding a fixed value corresponding to the greatest change against the general trend observed in any subject at any time point. Analysis of variance was then performed on the individual AUCs for each of the five treatments and the difference between them was rejected for p > 0.05. The observed maximum variation from baseline (Delta max) was also determined when appropriate. This approach is consistent with current recommendations for analysis of serial measurements in medical research (21). RESULTS Effects on coaqulation system. Figure 1 shows the effects of values. five treatments on the average APTT Small the prolongations of APTT over placebo values were observed 3-6 hours after treatment with both heparin and DS, but the betweentreatment difference was never statistically significant (P = 0.057). No significant between-treatment differences in PT, TT, were fibrinogen, antithrombin III and heparin cofactor II observed (not shown). ----A -0 --o 4 - -- *

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the mean ror eight subjects (SEM never exceeded 5% of the (subject/pooled mean). Results expressed as ratio normal plasma).

Figure 1. Effects of the five treatments on APTT. Each point

Figure 2 shows the effects of the five treatments on the ex vivo thrombin generation test. Baseline values for the five treatments did not significantly differ. Maximum inhibition of

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thrombin generation was observed at 3 hours after all four active treatments. Thrombin generation had reverted to baseline values 12 hours after heparin injection, but residual effects were still evident 24 hours after 200 and 400 mg doses of DS. The AUCs and Delta maxs derived from thrombin generation time-courses are shown in Table 1. The between-treatment difference in AUCs was highly significant (p < 0.001). One hundred mg DS and 5,000 IU heparin had equal cumulative effects on thrombin generation, as reflected by the respective AUCs; larger DS doses had proportionally greater effects. The peak effect of heparin (Delta max) was equal to that of 200 mg DS. 201

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2. Effects of the five treatments on the ex vivo thrombin generation test. Left panel: placebo and heparin; right panel: DS 100, 200 and 400 mg. Results expressed as means + SEM for eight subjects.

TABLE 1. Areas under the curves (AUC) and maximum variations from baseline (Delta max) derived from individual time-courses of the thrombin qeneration test. Means L SEM Delta max AUC (% increase over placebo AUC) (U.min/ml) Treatment Placebo Heparin DS 100 mg DS 200 mg DS 400 mg

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Effects on the fibrinolytic system. Figure 3 shows the effects activity. The activity, as of five the measured by the fibrin plate, showed typical circadian variation, with no significant between-treatment difference. The highest activity was reached 6-12 hours after each treatment (which

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of the five treatments on fibrinolytic activity. Each point the mean for eight subjects (SEM never exceeded 10% of the mean). Results expressed as diameter (in millimeter) of lysed area.

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Figure 4 shows the effects of the five treatments on PAI activity, which also showed a marked circadian variation and no between-treatment differences. The pattern of PAI variation was inversely related to that of the fibrinolytic activity, with a marked decrease at 6-12 hours and reversion to pretreatment values after 24 hours. The pattern of changes for WA antigen was similar to that for PA1 (not shown). Antiplasmin, plasminogen and histidine-rich glycoprotein showed no within- or between-treatment differences (not shown). Side effects. All 8 volunteers completed the full treatment course without evidence of svstemic adverse reactions. All oosttreatment platelet counts r:mained within the normal range.* Two bruises were observed at injection sites, one after heparin and the other after 400 mg DS. A moderate burning sensation at DS injection sites was reported, probably due to the highly concentrated, hypertonic solution used for this particular study. DISCUSSION As a preliminary step toward the clinical assessment of DS as an antithrombotic agent, we studied its biological effects in healthy volunteers injected S.C. with three different doses (100, 200 and 400 mg) and compared the results with those obtained with heparin given at the single dosage used in the prophylaxis of deep vein thrombosis (5,000 IU). Despite being administered in doses up to 13 times higher than the heparin dose on a gravimetric basis, DS did not appreciably prolong the APTT and TT, which is consistent with previous findings in experimental ~;mai;) (7-10) and in humans given DS by extravascular routes . ;Iowever, we did find a significant, dose-related inhibition of ex-vivo thrombin generation after DS, also already observed in experimental animalstudies using other assay methods and conditions (7, 9). The time-course of thrombin inhibition induced by DS was different from that induced by heparin, the prolonged effect of DS being consistent with its s.c. phannacokinetic profile in man (14, 15). The apparent discrepancy between the inhibition of thrombin generation by DS and the lack of APTT and TT prolongation is probably due to the poor sensitivity of the clot APTT to minor changes in thrombin generation during formation, and to the inability of DS to inhibit the bovine thrombin used in our test (22). The ability of GAGS to potentiate thrombin inhibition seems to be crucial for the expression of their antithrombotic efficacy, regardless of whether or not they also potentiate factor Xa inhibition. It has been repeatedly demonstrated that the ex vivo inhibition of thrombin is a reliable index for estimating the experimental antithrombotic efficacy of both heparin and nonheparin GAGS (7, 16, 17). Therefore, the comparison of DS to a dose of heparin whose prophylactic efficacy is well established may be relevant to finding the dose-range for DS. Our data show that a dose of 100 to 200 mg DS is required to reproduce the thrombin inhibition of 5,000 IU heparin, depending on whether the cumulative or the peak effect is considered. Evidence provided by Abbadini et al (23) that DS induces

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release of plasminogen activator from endothelial cells in the perfused rat hindquarter prompted us to evaluate whether or not there were changes of fibrinolysis after DS administration. Under the conditions and at the doses we studied, DS had no effect. Our findings are substantially similar to those of Legnani et al. (24), who found that 100 or 200 mg DS caused little or no change of the fibrinolytic measurements in healthy volunteers provided the circadian variation of fibrinolysis was taken into account (25, 26). Differences in the species investigated and the experimental conditions employed may well account for the discrepancy of our results from those obtained with the rat hindquarter. Abbadini et al. (23) did not inject dermatan sulfate into the animals, but used a perfusion model in which the concentration of DS in the perfusion medium was 100 to 400 ug/ml. According to human pharmacokinetic data (13-15), the S.C. doses of DS employed in our study should have generated plasma concentrations not exceeding 3 ug/ml, i.e., one one-hundredth those obtained by Abbadini et al. In conclusion, we have demostrated that single S.C. doses of DS (100 to 400 mg) inhibit ex vivo thrombin generation in healthy volunteers equally or better than 5,000 IU heparin s.c., and for a longer time. The effects of both compounds on the other coagulation and fibrinolytic parameters are negligible. Our data provide a possible rational basis for choosing the range of DS doses to be tested in dose-finding clinical trials.

Acknowledgement: We wish to thank Dr. L. Merlini the statistical analysis.

who

performed

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