D. Hoppensteadt

Comparative Study on the in vitro and in vivo Activities of Seven Low-Molecular-Weight Heparins

Different low-molecular-weight (LMW) heparins are produced by fractionation, enzymatic and chemical methods. Although the manufacturers assigned molecular weights of these agents are similar (around 5,000 daltons), they exhibit considerable molecular structural heterogeneity due to the variations in the manufacturing process. In vitro standardization of these LMW heparins produces highly variable results due to the variability of assay specificity. In a comparative study with seven LMW heparins, differences were found in molecular weight distribution, antifactor Xa activity, antifactor IIa activity, Heptest activity, USP potency, platelet interactions, protamine and platelet factor 4 neutralization and charge density ratios. Relative antithrombotic actions varied between the seven agents and the ratio of the intravenous:subcutaneous effects were inconsistent with expected results based on in vitro properties or observed ex vivo effects. These products were not bioequivalent in equigravimetric or equipharmacopeial dosages (antifactor Xa, USP). Although the intravenous bioavailability was proportional to the potency for an individual agent, the subcutaneous bioavailability of the same agent showed differences from that of the intravenous regimen. In addition, significant differences between the bleeding profiles of these agents were noted in both intravenous and subcutaneous routes. The bleeding profiles did not correlate well with the relative proportion of the antifactor Xa component. However, some relationship to the bleeding effect was observed with the antifactor IIa and activated-partial-thromboplastin-time activities. These LMW heparins produced different effects on platelets as studied in the heparin-induced thrombocytopenia and platelet activation systems. These observations suggest that the individual composition of each LMW heparin determines its in vivo behavior which may account for the different safety/efficacy ratios observed in clinical trials.

Biochemical and Pharmacologic Inequivalence of Low Molecular Weight Heparins

LMW heparin fractions obtained from various sources must not be considered as bioequivalent in both the in vitro and in vivo responses. Because of compositional variations, these agents exhibit individual behavior and should be considered as distinct drugs whose safety and efficacy profile must be determined separately. Currently, there is no valid LMW heparin standard available, however, different LMW heparins can be profiled in identical test systems. It is erroneous to assume that most LMW heparins will behave in a similar fashion in terms of safety and efficacy. The need for defined tests to characterized these agents is evident and efforts to profile their actions should be made at both the basic and applied levels. Needless to say, the true efficacy of LMW heparins can only be validated in well-designed randomized clinical trials. Optimization of LMW heparins in preclinical pharmacologic studies, as reported here, is a crucial factor in the development of these agents. The superior clinical efficacy/safety performance of some of the LMW heparins in contrast to other LMW heparins is a result of extensive preclinical pharmacologic investigations undertaken to optimize the therapeutic index of these agents. Such optimization studies have not been conducted during the development of many LMW heparins, resulting in decreased efficacy and undue complications.

Comparative antithrombotic and hemorrhagic effects of dermatan sulfate, heparin sulfate and heparin

Dermatan sulfate and heparan sulfate are currently under development as potential antithrombotic drugs. In our studies we have evaluated the relative antithrombotic and bleeding effects of these two agents in comparison to heparin, the commonly used anticoagulant. In a rabbit model of stasis thrombosis, a 500 micrograms/kg IV dose of dermatan or heparan produced 50-60% inhibition of induced in vivo thrombosis. At 750 micrograms/kg, both agents produced greater than 75% inhibition of thrombosis. Ex vivo measurement of plasma samples obtained from these animals demonstrated variable clotting effects at the lower dose and a proportional increase in the clotting activity at the higher dose. No anti-Xa or anti-IIa activity was observed in any sample. In contrast, animals treated with only 100 micrograms/kg heparin showed complete inhibition of induced thrombosis with significant anti-Xa and anti-IIa activities as well as prolongation of the clotting assays (APTT, TT and HeptestR). In the hemorrhagic studies utilizing a rabbit ear blood loss model, a 5.0 mg/kg IV dose of dermatan or heparan produced much less blood loss than heparin. On a gravimetric basis, dermatan and heparan were 10 fold less hemorrhagic than heparin. These results indicate that the relative contribution of plasmatic and cellular sites to the mediation of the antithrombotic action of heparin, dermatan and heparan differ. Although the antithrombotic dosages of dermatan and heparan are higher than heparin, due to the different mechanisms of action of each agent, a better safety index may be provided by dermatan and heparan than heparin.

Validity of the Newly Established Low-Molecular-Weight Heparin Standard in Cross-Referencing Low-Molecular-Weight Heparins

Four low-molecular-weight-heparin (LMWH) preparations have recently become available for clinical usage. Some ten preparations are currently under preclinical development. The safety/efficacy profile of each of these LMWHs is now known to be quite distinct from one and other. In order to minimize the variations in the pharmacologic responses and to cross-reference different products on a common scale, the WHO has recently introduced a LMWH standard. This standard is a nitrous-acid-depolymerized product provided by KabiVitrum (Stockholm, Sweden) with a specific physiochemical and biological profile. Although the in vitro characteristics of this standard are comparable to other LMWH, the in vivo behavior of this reference and the parent product, Fragmin, are quite different in comparison to other products. In a well-defined uniform biochemical and pharmacologic screening, seven LMWH were compared at WHO reference-adjusted potency using pharmacologically valid dose-response curves. Cross-referencing of various LMWHs against the WHO standard in terms of anti-Xa activity resulted in a marked elevation of the anti-IIa component of some of the LMWHs. Establishment of this WHO standard stipulated that the clinical performance of cross-referenced LMWHs would be equivalent to one another and variation in potency could be minimized. Our data suggest that utilizing the WHO, standard individual potency (anti-Xa)-adjusted LMWHs exhibit distinct safety/efficacy profiles which were not related in their observed in vitro potency. Each LMWH has distinct multiparametric molecular and biochemical characteristics which determine the pharmacologic activities of individual agents. Thus cross-referencing of LMWHs by one in vitro assay is of questionable value as it may result in serious dosimetric errors, resulting in serious thrombotic and bleeding complications.

In vitro studies on thrombin generation in citrated, r-hirudinized and heparinized whole blood

The generation of thrombin in citrated, r-hirudinized and heparinized whole blood (final concentrations 10, 25, 50 micrograms/ml) was studied in the presence or absence of various activators. Whole blood from healthy volunteers was activated either by glass or dextran sulfate (contact activation; CA) as well as by thromboplastin/calcium chloride (extrinsic activation; EA) and by ellagic acid/cephaloplastin/calcium chloride (intrinsic activation; IA) and incubated 10 (CA, EA, IA) and 30 (CA) min at 37 degrees C. After the incubation period plasma levels of prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin III complex (TAT) were measured utilizing ELISA methodology. After EA or IA no blood clotting occurred in all r-hirudinized samples and in heparinized blood at the highest concentration used. Despite inhibition of clotting F1 + 2 levels were increased both in r-hirudinized and in heparinized blood. However, in blood anticoagulated with heparin F1 + 2 levels after EA, IA and especially after CA were markedly lower than in r-hirudinized blood. Furthermore, in r-hirudinized blood an increase of TAT levels was found. The enhancement of F1 + 2 and TAT levels by r-hirudin was concentration-dependent and was lower in the higher r-hirudin concentration. The results indicate that in r-hirudinized whole blood significant amounts of thrombin can be generated which may elicit thrombin-mediated feedback mechanisms.

The thrombolytic potency of LMW-heparin compared to urokinase in a rabbit jugular vein clot lysis model.

Since the beginning of the clinical use of low molecular weight (LMW) heparins their thrombolytic or profibrinolytic potency has been a matter of controversial discussions. Regarding this problem, the aim of our study was to test a LMW-heparin (Sandoparin) in an in vivo model comparing its lytic activity to unfractionated heparin and urokinase at different doses. For this purpose a newly developed short-term rabbit jugular vein clot lysis model was developed. Urokinase infused at doses of 3300, 6600 and 10,000 U/kg to control animals for one hour showed a clear dose-dependent clot lysis. Test animals were injected with a bolus of 0.5 mg/kg of LMW-heparin followed by a constant infusion of either 0.5, 1.0 or 2.0 mg/kg for one hour. A similar dose-dependent effect was observed for LMW-heparin as for urokinase. Unfractionated heparin did not exhibit a dose-dependent lytic activity in this model. No lysis was found in rabbits treated with saline. These findings suggest that the LMW-heparin tested exhibits a dose-dependent in vivo lytic activity which can be compared to clinically effective doses of urokinase, and that this activity is not present with unfractionated heparin.

Influence of recombinant hirudin and unfractionated heparin on thrombin and factor Xa generation in extrinsic and intrinsic activated systems

Using biochemically defined conditions on a fast kinetic centrifugal analyzer the effect of recombinant hirudin (rH) and unfractionated heparin (UH) on thrombin and factor Xa generation was investigated. Diluted fibrinogen deficient human plasma was incubated with increasing concentrations of the anticoagulants and protease generation was initiated either by extrinsic (EA; thromboplastin/calcium chloride) or intrinsic (IA; ellagic acid/cephaloplastin/calcium chloride) activation of the coagulation process. Generation of thrombin or factor Xa was measured continuously by amidolytic assays using the specific chromogenic substrates Spectrozyme TH and Spectrozyme FXa. By means of calibration curves for thrombin and factor Xa the IC50 values for the inhibition of the proteases were calculated. It was found that rH and UH were nearly equally effective in inhibiting both the thrombin and factor Xa formation after IA, whereas in EA system rH produced a stronger inhibition on thrombin generation than UH, which in general showed a more pronounced effect after intrinsic than after extrinsic activation. The results suggest that, with regards to thrombin and factor Xa generation, rH does not exhibit a much higher activity than UH. This may be an expression that thrombin-mediated positive feedback-reactions are not influenced by rH as strongly as expected when using a highly specific and selective thrombin inhibitor. Furthermore, it can be concluded that protease generation assays may be useful in the characterization of anticoagulants/antithrombotics.

Synthetic oligosaccharides can replace animal-sourced low–molecular weight heparins

A synthetic homogeneous low–molecular weight heparin provides a safe and effective therapeutic alternative. A reliable animal-free heparin drug Full-sized and low–molecular weight heparins are widely used to treat a variety of clotting disorders. Although low–molecular weight heparins are safer and more convenient to use than full-size heparin, they are still animal-derived products that present a risk of contamination and supply chain interruptions and are limited with respect to standardization and reversibility of anticoagulation. A method developed by Xu et al. offers a potential alternative to animal-sourced heparins in the form of a chemical synthesis process that can be scaled up to produce heparin dodecasaccharides with reversible activity in adequate quantities for potential therapeutic use. Low–molecular weight heparin (LMWH) is used clinically to treat clotting disorders. As an animal-sourced product, LMWH is a highly heterogeneous mixture, and its anticoagulant activity is not fully reversible by protamine. Furthermore, the reliability of the LMWH supply chain is a concern for regulatory agencies. We demonstrate the synthesis of heparin dodecasaccharides (12-mers) at the gram scale. In vitro experiments demonstrate that the anticoagulant activity of the 12-mers could be reversed using protamine. One of these, labeled as 12-mer-1, reduced the size of blood clots in the mouse model of deep vein thrombosis and attenuated circulating procoagulant markers in the mouse model of sickle cell disease. An ex vivo experiment demonstrates that the anticoagulant activity of 12-mer-1 could be reversed by protamine. 12-mer-1 was also examined in a nonhuman primate model to determine its pharmacodynamic parameters. A 7-day toxicity study in a rat model showed no toxic effects. The data suggest that a synthetic homogeneous oligosaccharide can replace animal-sourced LMWHs.

Comparative studies on the inhibitory spectrum of recombinant hirudin, DuP 714 and heparin on thrombin and factor Xa generation in biochemically defined systems

The effect of antithrombotic drugs on the generation of serine proteases was studied in a biochemically defined system in which the prothrombin complex concentrate Konyne provided the necessary coagulation factors in the absence of plasma. The amount of thrombin and factor Xa generation was measured with a chromogenic substrate on a microcentrifugal analyzer. Furthermore, the assay was modified by supplementation with either purified antithrombin III or factor V. The synthetic peptide Ac-(D)Phe-Pro-boroArg-OH (DuP 714) was shown to be the most effective inhibitor of thrombin and also had strong inhibitor actions against factor Xa generation. Recombinant hirudin (rH) was nearly as active as DuP 714 on thrombin generation, however, it was less effective on factor Xa generation. With rH no concentration-dependent inhibition of factor Xa generation was found, i.e. over a wide range of concentration it only produced a steady inhibition of about 40-50% without further increase. The addition of AT-III to the system did not influence the action of DuP 714 or rH, but it strongly increased the inhibitory effects of unfractionated heparin (PMH) as well as of a low molecular weight heparin (LMWH) on both thrombin and factor Xa generation. The addition of factor V to the assay system did not cause any changes in the activity of all agents on protease generation.

Inhibitory effects of TFPI variants on thrombin and factor Xa generation in fibrinogen-deficient human plasma.

Using a fast kinetic centrifugal analyzer, the inhibitory effects of glycosylated and unglycosylated full-length and truncated forms of TFPI on protease generation were studied in fibrinogen-deficient human plasma after extrinsic (EA) or intrinsic (IA) activation of coagulation. When the assay system was supplemented with increasing amounts of the TFPI variants the generation of both thrombin and factor Xa was inhibited in a concentration-dependent manner. Clear differences in the effectiveness of the TFPI variants were found. After EA, the unglycosylated full-length TFPI was most effective followed by the glycosylated full-length form. The C-terminal truncated TFPI showed the lowest inhibitory activity in this system. However, its efficiency increased several fold when coagulation was activated via the intrinsic pathway. Comparing the IC50 values after IA, the truncated TFPI was more effective than the unglycosylated full-length form and nearly as effective as the glycosylated full-length TFPI. After both EA and IA the thrombin generation inhibition by TFPI variants was more pronounced than the inhibition of factor Xa generation. The results show that chemical modifications of the TFPI structure can result in changes of TFPIs inhibitory properties to activated clotting factors leading to differences in protease generation inhibition.