A new low molecular weight heparin derivative. In vitro and in vivo studies

THROMBOSIS RESEARCH 31; 611-621, 1983 0049-3848/83 $3.00 + .OO Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.

A NEW LOW MOLECULAR WEIGHT HEPARIN DERIVATIVE In vitro and in vivo studies Martine Aiach' , Anne Michaud', Jean-Luc Balian', M. Lefebvre3, M. Waler', J. Fourtillan3. 1. Laboratoire d'hematologie (Pr M. Leclerc) - Faculte de pharmacie de l'universite Paris XI - Rue J.B. Clement - 92290 CHATENAY (France). 2. Departement de recherche clinique - Laboratoire Pharmuka - 35, quai du Moulin-de-Cage - B.P. 158 - 92231 GENNEVILLIERS Cedex (France). 3. C.E.R.O.M. - 4, rue des Gaillards - 86000 POITIERS (France). Received 30.5.83; Accepted in original form Editor H.C. Hemker

7.6.83 by

ABSTRACT We present here the in vitro and in vivo evaluation of a new heparin low molecular weight (LMW) derivative that can be prepared on an industrial scale. The in vitro anti activated factor Xa (anti Xa) activity was 67 per cent that of standard heparin while antithrombin and anticoagulant activities were respectively limited to 16 and 10 per cent. The same discrepancy between anti Xa and anticoagulant activity was found after intravenous (IV) or subcutaneous (SC) injection to ten healthy volunteers. From the kinetic of the anti Xa activity disappearance it can be assumed that the mechanism of elimination of LMW heparin is not strongly different from that of traditional heparin. INTRODUCTION Heparin is widely distributed in animal tissues ; this glycosaminoglycan has a potent antithrombotic action and exhibits a large polydispersion in its molecular weight (1,2,3,4). Heparin interacts with coagulation mainly by accelerating the inhibition rate of clotting proteases by the natural inhibitor antithrombin III (AT III) (5). Its major disadvantage is to impair normal hemostasis (6,7). Their exists a molecular weight dependency of the anticoagulant activity of heparin, the higher molecular weight component showing the higher activity (8,9,10,11,12). LMW heparin derivatives have been recently proposed as antithrombotic drugs. Their ability to prevent venous thrombosis extension has been shown in several experimental models (13,14,15). LMW compounds can selecKey words : LMW heparin - In vitro activity - Pharmacokinetic.

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tively enhance the inhibition of some proteases, F Xa, F XIIa, kallikrein, but have weak effect on thrombin and F IXa inhibition (12,16,17). This results in a reduced prolongation of global clotting tests (14,15). Thus lower incidence of hemorrhagic complications can be theoritically expected from using LMW heparin derivatives in clinical situations. The purpose of this study was to determine the in vitro and in vivo activity of a LMW heparin derivative obtained by chemical depolymerization and available on an industrial scale. Kinetic studiesoftheanti Xa, antithrombin, and anticoagulant activitieswereperformed after either IV or SC injection in ten healthy volunteers. MATERIAL AND METHODS LMW heparin (code number PK 10 169) was purchased by the research department of Pharmuka Laboratories (Gennevilliers - France). The starting material was a benzylic ester of heparin. PK 10 169 was obtained by a partial and controlled depolymerization. Molecular weight of the fragments obtained ranged from 4,000 to 6,000. Heparin was the third international standard (1 mg = 173 IU). - Laboratory _________ _procedures --------Plasma anti Xa and antithrombin activities were evaluated with an amidolytic assay using a discrete analyzer "Isamat" (ISA-Biologie - Cachan France) as previously described (18). Chromogenic substrates S 2238, S 2337 and bovine F Xa were from FlowKabi (Puteaux - France). Purified human thrombin was from Stago (Asnieres France). Briefly 12.5 ~1 of plasma sample were added to 250 ~1 of thrombin diluted in 0.05 M Tris, 7.5 lo- 1 M Na2 EDTA, NaCl 0.175 M, pH 8.4. The mixture was incubated for 30 set at 30" C. The amount of residual protease was evaluated by adding S 2238 to a final concentration ofO.lmM 1-l. The initial velocity of the substrate hydrolysis was recorded by measuring the increase in optical density (A OD/min). The concentration of thrombin in the reaction mixture was adjusted in order to give a value of 0.3 to 0.4 OD units per minute. ??

??

Anti Xa activity was measured according to the same procedure, except that 25 ~1 of sample was added to 250 ~1 of diluted F Xa. The final concentration of the substrate S 2337 in the mixture was 0.131&l 1-l. ??

AT III assay was performed with an automated amidolytic assay using a kit "AT Prest" (Stago) and the Isamat. Activated partial thromboplastin time (APTT) was evaluated using reagent from General Diagnostics (Rueil-Malmaison - France) and the automated coagulometer KC 10 (A.H.S. - Cergy-Pontoise - France). - -----_______ In vivo study Ten healthy male and female volunteers participated the study. Informed consent was obtained from all of them. Physical examination and biological study were normal. They refrained from medications 8 days before and through Out the study. The main physical features are listed in table I. Single doses (25 mg in 1 ml by IV route and 75 mg in 0.3 ml by SC route) were administrated. In a first period, ten subjects were randomized to receive either IV or

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SC injection. In a second period, the treatments were reversed with a wash out period of 13 days. In any cases, the subjects had fasted 12 hours before and 3 hours after the injection.

Age /ears

Body weight kg

Height cm

1

24

82

180

M

2

25

61

169

F

3

22

50

165

F

4

25

72

185

M

5

25

64

172

M

6

26

80

184

M

7

24

60

170

M

8

23

58

167

M

9

25

75

185

M

10

24

67

170

M

Subjects

Sexe

PK I( 169 IV SC

Table I Main physical features in the ten healthy volunteers The injections were made at 8 am. Blood samples were drawn from each subjects immediatly before IV injection (To), then 2, 5, 10, 15, 20, 30, 45 minutes, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12 hours after To ; immediately before SC injection, then 30 minutes, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 24 hours after. Blood samples were collected in 0.11 M trisodium citrate (1:9). Platelet poor plasma was obtained by centrifugation at 15" C, 2,500 g during 20 minutes, within 30 minutes following puncture. Plasma was frozen at - 20' C and analyzed within three months. Pooled normal plasma was obtained in the same conditions from thirty healthy blood donors. The results were calculated in international units by comparing the activity of the sample to that of normal plasma added with known amount of the 3rd international standard. The levels of biological activity after injection were analyzed using an HP 100 computer system. The peak activity (Amax) and the time for peak value (Tmax) were noted. The apparent half-life (T l/2) was obtained by regression analysis in the last phase in which the points seemed to be on a straight line in a semi-logarithmic scale. - ---_-----^--In vitro study Pooled normal plasma frozen in 1 ml aliquot was added with either the standard heparin or PK 10 169. Final concentrations of 0, 2, 4, 6, 8 ug per ml (PK 10 169) or 0, 1, 2, 3, 4, 5 Pg per ml (heparin) were chosen in order to be in the same ranges of activity.

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It was found in ten repeated experiments that the F Xa or thrombin inhibition, expressed as a ratio between the activity provoked by PK 10 169 (or heparin) and the physiological activity, best fit with a linear function of concentration. The slope of the regression (a) line expressed the enhancement of F Xa or thrombin inhibition provoked by 1 pg PK 10 169 or 1 119heparin added to 1 ml plasma. The ratio between the (a) values found for PK 10 169 (aPK) and the (a) value found for standard heparin (ah) was used as a comparison between the two compounds (fig. 1). F Xa inhibition plasma alone

plasma t PK 10 169 or heparin enhanced activity

physiological activity 1

1 V

VO

v - vo Vo

= enhancement of the physiological activity by PK 10 169 or heparin.

v - vo =a Vo aPK

(C)

C = PK 10 169 (PK) or heparin (h) concentration in plasma.

= relative activity of the 2 compounds.

ah Fig. 1 Rationale for comparing heparin and PK 10 169 in vitro anti Xa activity RESULTS - _____________ In vitro study Based on the calculation method detailed in "material and methods", 1 mg PK 10 169 had the same anti Xa enhancing activity as 0.67 mg standard heparin. In term of European Pharmacopeia, this is equivalent to 113 IU (fig. 2). The slope of the regression line (ap = 0.054) obtained by plotting the increase in anti Xa activity against the kK 10 169 concentration in plasma can be used as an absolute value for biological activity : 1 Pg PK 10 169 added to 1 ml human plasma provokes a 5.4 per cent enhancement of the natural anti Xa activity. The reproductibility of the method calculated from ten separate determinations was good (mean aPK value : 0.054 with a standard deviation of 0.0047 ; coefficient of variation = 8.7 per cent). The same procedure was used for evaluating the antithrombin activity (fig. 3) : 1 pg PK 10 169 had the same enhancing potency as 0.16 pg heparin (in term of pharmacopeial unit : 28 Ill).

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o.o: 5 o.s3

‘c

I

o

,: OA=

5

0.3-

0.2-

O-l-

I

,

0

1

5 10 PK 10 160 or heparln concentration

(pQ/ml)

Fig. 2 In vitro anti Xa activity : dose relation curve for anti Xa enhancing activity. * ODplasma - * ODplasma + :F itpi!qn

vertical axis :

-ah

* ODplasma 0 3rd international standard : mean value for 10 assays ?? PK.10 169 batch no 573 : mean value for 10 assays. ; -aPK = 0.082 r = 0.990 = 0.055 r = 0.997

r

0

I

1

5 10 PK 10 169 or hopwIn concentr~tlon

+Q/ml)

Fig. 3 In vitro antithrombin activity : dose relation curves for heparin and PK 10 169 -O-ah

= 0.234

same legend as Fig. 2 r = 0.997 ; "PK = 0.038

r = 0.997

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More difficult was the assessment of the anticoagulant activity. In ten separate experiments, we found natural logarithm (log e) of APTT to be a linear function of heparin or PK 10 169 concentrations. The comparison of the slopes of the regression lines allows an estimation of the anticoagulant activity : 1 mg PK 10 169 is equivalent to 0.107 mg standard heparin. However, the reproductibility of the slope value evaluation (aPK) in ten separate experiments was poor : m f SD = 0.045 f 0.0096 ; cv = 21 per cent (fig. 4).

‘;

220-

: :: 2 121-

49-

Fig. 4 In vitro anticoagulant activity : dose relation curve for APTT. : log e APTT (set) vertical axis horizontal axis : concentration of PK 10 169 or heparin in plasma (Pg/ml). 0 3rd international standard : mean value for 10 assays PK 10 169 batch no 3055118 : mean value for 10 assays -ah

= 0.460

r = 0.990

; --

apK = 0.049

r = 0.9999

- -------_---In vivo study Figure 5 depicts the decrease in biological activity after IV injection. Anti Xa activity followed a double course disappearance. The half-life mean 0.5 hours. The activities were indetectable in most subjects value was 2.8 after 4 hours. Thus being less accurately determined because of very low values, antithrombin activity disappearance seemed to follow anti Xa activity. APTT was almost normal during the whole experiment. Biological activity was measured during 24 hours after SC injection cmax and T l/2 obtai(Fig. 6). Table II compares the mean values for a, ned in anti Xa and antithrombin assay system : the discrepancy between the mean values observed in each assay for Tmax and T l/2 may be related to the lower values observed in the antithrombin assay (the standard deviation being very high). The ratio between anti Xa and antithrombin a mained below 1.3 time the pretreatment value during the

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Fig. 5 Biological activity of LMW heparin after a bolus IV injection (25 mg) vertical axis

: logarithmic scale. Anti Xa and antithrombin activities in IU/ml, APTT in seconds.

horizontal axis : time in hours. anti Xa (I), antithrombin (i), and APTT (I) assays : mean value standard deviation in 10 healthy subjects.

amax

Tmax

T l/2

U/ml

(hours)

(hours)

anti Xa assay

0.63 ?:0.15

3.25 ?r0.65

5.9 t 1.6

antithrombin assay

0.13 + 0.04

4.00 ?r1.30

7.7 f 5.0

Table II Kinetic parameters observed in ten healthy volunteers after SC injection of 75 mg PK 10 169 (mean value ?rstandard deviation).

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I

0

I

,

2

I

I

4

I

I

6

,

I

8

I

I

10

I

,

12

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:;

,

24 hour

Fig. 6 Biological activity of LMW heparin after a SC injection (75 mg) vertical axis

: logarithmic scale. Anti Xa and antithrombin activities in IU/ml, APTT in seconds.

horizontal axis : time in hours. anti Xa (I), antithrombin (a), and APTT (;) assays : mean value + standard deviation in 10 healthy subjects.

DISCUSSION The results presented here demonstrate that PK 10 169, a LMW heparin derivative, can enhance in vitro anti Xa activity at a rate which is 67 per cent that of heparin, while antithrombin and anticoagulant activities are very low. A similar high anti Xa to anticoagulant ratio was observed in vivo after SC or IV injection in ten healthy volunteers. Heparin and LMW heparin, being different molecular species, should not be compared in biological assays as the dose response curves are not parallel. This has previously been pointed out (14) or overcome by testing concentrations giving similar activities (15). We propose here to evaluate the activities of heparin and its derivative by comparing the slopes of the respective dose response curve in a standardized system. It was found that 1 vg per ml PK 10 169 provokes a 5.4 per cent increase in the plasma anti Xa activity ; 1 Pg per ml heparin results in a 8 per cent increase. In other words, 1 pg PK 10 169 has the same anti Xa activity as 0.67 pg heparin. These data could be expressed in term of pharmacopeial units (1 mg heparin = 173 IU) : PK 10 169 respective activities would be 113 IU in anti Xa assay, 28 IU in antithrombin

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assay and 18 IU in APTT assay. These IU are widely used for heparin thus being extented to other antithrombotic compound. However, they should not be used for LMW derivatives and in assay system strongly differing from pharmacopeial methods. Similar difficulties were encountered in assessing the circulating activities and the pharmacokinetic features. The ex vivo samples were analyzed in two ways : the biological activities were expressed as amounts of F Xa or thrombin inhibited, then converted either in unit per ml (by comparing with a calibration curve made with heparin) or in Pg per ml (by comparing with a calibration curve made with PK 10 169). The latter system gave inconsistant results as the "concentrations" were different in the anti Xa assay (mean maximum activity 7.5 pg per ml) from those given by the antithrombin assay (4 pg per ml) (unpublished data). We prefered to use the unit system which clearly indicates that only biological activities could be evaluated. In our system 1 U is the anti Xa or antithrombin enhancing activity of 1 U standard heparin added to 1 ml normal plasma. One explanation for the inability of measuring concentrations would be that PK 10 169, being still an heterogenous compound, circulates in a molecular form differing from the initial preparation. The in vivo behaviour of the separate LMW chains has not been yet extensively studied. Experimental data show a similar uptake of heparin and LMW derivative by rat macrophages (19). For ethical reasons, a very low dose was used for the IV route as we had no information on the in vivo activity of the product in human being. Antithrombin and APTT values being mostly under the limit of sensitivity of the method could not be used for evaluating kinetic parameters. Fitting the decrease of anti Xa activity with a single exponential model led to calculate an apparent half-life of 2.8 + 0.5 hours for biological activity. However, a careful1 observation of the curves shows a biphasic kinetic : a first step decrease is followed by a slighly convexe disappearance curve. This has been predicted (20) and observed for heparin (22). This model is consistent with a first rapid binding of heparin (or LMW heparin) on a cellular compartment (for example vascular endothelial cells) followed by elimination in a saturable mechanism (20,21,22). More interesting in term of clinical applications are the results obtained after SC administration of 75 mg PK 10 169. APTT remained close to pretreatment values, confirming the low effect on global clotting test. However, the anti Xa activity was dramatically enhanced with maximum activity reaching 0.68 U per ml (mean value). Antithrombin activity was 4 to 5 time lower. This high anti Xa/anticoagulant activity ratio was observed following the injection of other LMW heparin derivative to man (14,23). The maximum anti Xa activity was observed 3 hours after the injection. Time for disappearance of half the biological activity was 6 hours. After 12 hours, the anti Xa activity was still 0.2 IU per ml, remaining 0.05 IU per ml after 24 hours. The ideal dosage would be one SC injection per day as proposed by Kakkar (23). The LMW heparin derivative PK 10 169 has been showed to be efficient in several experimental models including venous thrombosis (24) and extracorporeal circulation (25). This preliminary pharmacological data enable us to assume that PK 10 169 is of therapeutic interest. More information about the accumulation of the product after repeated doses, the dose dependance of the half-life, will help us to propose a therapeutic scheme which allows the maintenance of the desired circulating anti Xa activity.

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AKNOWLEDGEMENTS We wish to thank M.J. Deguingand for her skillful1 secretarial assistan-

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16. JORDAN, R.E., OOSTA, G.M., GARDNER, W.T., and ROSENGERG, R.D. The binding of low molecular weight heparin to hemostatic enzymes. J. biol. Chem., 255, 10073-10080, 1980. 17. CHOAY, J., LORMEAU, J.C. et PETITOU, M. Oligosaccharides de faible poids moleculaire presentant une activite inhibitrice du facteur Xa en milieu plasmatique. Ann. pharm. fr., -39, 37-44, 1981. 18. AIACH, M., LEON, M., MICHAUD, A., CAPRON, L. Adaptation of synthetic peptide substrate based assays on a discrete analyzer. to be published in Semin. Thromb. Haemost., 2 (30), 1983. 19. BLEIBERG, I., MACGREGOR, I. and ARONSON, M. Heparin receptors on mouse macrophages. Thromb. Res., -29, 53-61, 1983. 20. McAVOY, T.J. Pharmacokinetic modeling of heparin and its clinical implications. J. Pharmacokinet. Biopharm., 7, 331-354, 1979. 21. DAWES, J., and PEPPER, D.S. Catabolism of low-dose heparin in man. Thromb. Res., -14, 845-860, 1979. 22. DE SWART, C.A.M., NIJMEYER, B., ROELOFS, J.M.M., and SIXMA, J.J. Kinetics of intravenously administered heparin in normal humans. Blood, 60, 1251-1258, 1982. 23. KAKKAR, V.V., DJAZAVI, B., FOK, J., FLETCHER, M., SCULLY, M.F., WESTWICK, J. Low molecular weight heparin and prevention of post-operative deep vein thrombosis. Brit. Med. J., 284, 375-379, 1982. 24. FAREED J. Personnal communication. 25. AIACH, M., DREYFUS, G., MICHAUD, A., RELLAND, J., MURAWSKI, M., ATTAL, F., LECLERC, M., CARPENTIER, A. Low molecular weight (LMW) heparin derivatives in extracorporeal circulation (ECC). II. A blind randomized experiment comparing heparin and its LMW derivatives. (submitted).