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(Circulation. 1995;92:2819-2824.)
© 1995 American Heart Association, Inc.

Articles

Prolonged Antithrombin Activity of Low-Molecular-Weight Heparins

Clinical Implications for the Treatment of Thromboembolic Diseases

Presented in part during the 66th Scientific Sessions of the American Heart Association, Atlanta, Ga, November 8-11, 1993.

Giancarlo Agnelli; Alfonso Iorio; Cinzia Renga; Enrico Boschetti; Giuseppe G. Nenci; Frederick A. Ofosu; Jack Hirsh

From the Istituto di Medicina Interna e di Medicina Vascolare, Università di Perugia, Italy; and the Hamilton Civic Hospital Research Centre, McMaster University, Hamilton, Ontario, Canada.

Correspondence to Giancarlo Agnelli, MD, Istituto di Medicina Interna e di Medicina Vascolare, Università di Perugia, via Enrico dal Pozzo, 06122 Perugia, Italy.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Background The mechanism for the efficacy of once- or twice-daily subcutaneous injections of low-molecular-weight heparins (LMWHs) for the treatment of venous thromboembolism has been difficult to explain. The confusion exists because the observation from experimental studies that the antithrombin activity of LMWHs is necessary for their antithrombotic effect is inconsistent with the reported short half-life of the antithrombin activity of LMWHs. Previous pharmacokinetic studies were performed with lower doses of LMWHs than have been used in contemporary trials, and antithrombin activity was assessed with the barely sensitive chromogenic assay.

Methods and Results We performed a pharmacokinetic study to compare the relative half-lives of prophylactic and therapeutic doses of LMWHs assessing antithrombin activity with both the chromogenic and a more sensitive assay (plasma thrombin neutralization assay). An eight-way cross-over randomized study in healthy volunteers was performed. Enoxaparin (20 and 40 mg and 1 and 2 mg/kg) and nadroparin (7500 and 10 000 ICU and 225 and 450 ICU/kg) were administered subcutaneously. The maximal peak activity for aPTT ratio was 1.7. A dose-dependent peak activity was found for both antifactor Xa and antithrombin activities. Disappearance time of these activities after the highest dose of both LMWHs was longer than 16 hours. Overall mean antifactor Xa activity half-life was 4.6 hours. Overall mean antithrombin activity half-life was longer than 4 hours.

Conclusions Our results provide an explanation for the effectiveness of LMWHs administered either once or twice daily. High and sustained plasma antithrombin activity is achieved when LMWHs are administered in therapeutic doses used in contemporary trials with only a moderate prolongation of the aPTT.

Key Words: low-molecular-weight heparins • thrombin • thromboembolic diseases

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
There is evidence from a number of clinical trials^{1 2 3 4 5 6 7 8 9} and from two overview analyses^{10 11} that LMWHs are effective in the treatment of venous thrombosis. In the two largest trials, both of which showed strong trends for increased efficacy of LMWHs over unfractionated heparin, the LMWH was given subcutaneously either once or twice daily.^{8 9} These therapeutic regimens are under evaluation in patients with unstable angina, and similar daily dosages have been evaluated in myocardial infarction.^{12 13} The mechanism for the efficacy of these subcutaneous regimens of LMWHs for the treatment of venous thrombosis has been difficult to explain.¹⁴ Results of in vitro and animal studies indicate that the antithrombin activity of LMWHs is necessary for their antithrombotic effect,^{15 16 17} a notion that is inconsistent with the reported very short half-life of the antithrombin activity of LMWHs.^{18 19 20 21} Previous pharmacokinetic studies were performed with lower doses of LMWHs than have been used in the successful treatment trials of DVT.^{18 19 22} Therefore, the results of these pharmacokinetic studies with low doses of LMWHs might not be applicable to the clearance of higher doses of LMWHs.

We therefore performed a pharmacokinetic study in healthy volunteers to compare the relative half-lives of prophylactic and therapeutic doses of LMWHs by assessing both antithrombin and antifactor Xa activities. Because the levels of antithrombin activity achieved with prophylactic doses are just above the limit of detection of the chromogenic antithrombin assay, we also measured the plasma levels of antithrombin activity with a more sensitive assay (PTNA) based on the ability of LMWHs to catalyze thrombin inactivation by ATIII in nondiluted plasma.^{23 24}

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents
Human a-thrombin (1000 NIH units/mg) was purchased from Sigma Chemical Co. Recombinant hirudin (CGP 39393) was generously provided by CIBA-GEIGY Ltd, Biotechnology Department. aPTT, chromogenic antifactor Xa, and antithrombin assays were performed on ACL from Instrumentation Laboratory with DADE Actin-FS from Baxter Healthcare Corporation, Dade Division; Rotachrom HBPM/LMWH from Diagnostica Stago; and IL Test Heparin (IIa) from Instrumentation Laboratory, respectively. Thrombin/antithrombin-III complexes were determined by an ELISA using a commercial kit (Enzygnost TAT) from Behringwerke AG.

Study Population
Six healthy volunteers (five men and one woman) between 20 and 28 years old and weighing 53 to 72 kg were selected for the study. No other treatment was allowed 1 week before and during the study. A careful history was taken, and a complete physical examination was performed. Prestudy laboratory examination included full blood count, blood chemistry, urinalysis, and coagulation tests. An informed written consent was obtained from each volunteer. The protocol and the consent form were approved by the Ethical Committee of the Region Umbria, Italy.

Treatments
PK 10169 (Enoxaparin, 100 mg/mL) was obtained from Rhône-Poulenc Rorer. CY-216 (Nadroparin, 25 000 ICU/mL) was obtained from Sanofi-Winthrop. The study was planned as an eight-way randomized cross-over study. The following treatments were administered subcutaneously, 1 week apart: enoxaparin, 20 and 40 mg and 1 and 2 mg/kg; nadroparin, 7500 and 10 000 ICU and 225 and 450 ICU/kg. The two lowest doses of each agent were selected because they are used for the prophylaxis of DVT in moderate- and high-risk patients, respectively. The two highest doses of each agent were selected because they were used in the treatment of venous thrombosis when given as twice- or once-daily injections, respectively.

Plasma Sampling
With a 19-gauge butterfly needle regularly flushed with saline when left in situ, blood was collected from the antecubital vein of healthy volunteers into plastic syringes prefilled with 1:10 vol of 129 mmol/L (3.8 g/dL) trisodium citrate. The first 3 mL of blood was discarded to avoid stasis-induced activation or dilution by flushing saline. After thorough mixing with the anticoagulant, the red blood cells were sedimented by centrifugation at 2300 g for 15 minutes at 4°C, and the platelet-poor plasma was either assayed immediately or stored at -80°C. Plasma samples were drawn before and 30, 60, and 120 minutes after treatment injection and then every 2 hours up to 24 hours.

PTNA
For the PTNA,^{23 24} each sample was divided in two aliquots: one aliquot was used for assay of endogenous TAT complexes, and the other aliquot was used to assay the heparin-generated TAT complexes. Endogenous TAT complexes were measured directly in the first plasma aliquot. For the assay of heparin-generated TAT complexes, one vol containing 2 nmol/L human -thrombin solution in 0.05 mol/L Tris, 0.1 mol/L NaCl, 0.025% sodium azide, and 0.1% albumin were added to the second plasma aliquot. The mixture was incubated at 37°C for 15 seconds. The reaction was stopped by the addition of 2 vol of a 100 nmol/L recombinant hirudin aqueous solution, and then TAT complexes were assayed. The amount of heparin-generated TAT complexes was then calculated by subtraction from the total TAT complexes assayed in the second plasma aliquot the endogenous TAT complexes. The results were reported in micromoles per liter of TAT complexes.

Pharmacokinetic Analysis
Pharmacokinetic analysis was performed according to the method of Gibaldi and Perrier.²⁵ The half-lives of enoxaparin and nadroparin were calculated for each subject from the elimination curve of heparin activity by using the following formula: ln2/ß, where ß is the estimate constant of the exponential curve, fitted with a nonlinear weighted least-squares regression model (SAS-NLIN).²⁶ Consecutive values of the declining part of the curve generating significant estimates were included in the evaluation. The reciprocal of heparin concentration was introduced as a weight in the model because of the direct linear correlation between heparin concentration and its variance. Parameters were expressed as mean±SD of the six volunteers. Overall mean half-life was calculated as the general mean value of all estimated values of antifactor Xa or antithrombin activity. Ninety-five percent confidence intervals were also calculated.

Statistical Analysis
Statistical evaluation of goodness-of-fit for regression was performed with the F test and the Akaike's information criterion²⁷ (comparing monoexponential and biexponential regression models). Linear regression analysis was adopted to test the effect of the dose on the pharmacokinetic parameters.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The A_max values (mean±SD) for aPTT (as ratio to basal value) were 1.25±0.04 and 1.58±0.10 for 225 and 450 ICU/kg of nadroparin, respectively, and 1.32±0.12 and 1.67±0.13 for 1 and 2 mg/kg of enoxaparin, respectively. Corresponding t_max values (mean±SD) were 4.00±1.41, 5.50±1.22, 2.50±1.23, and 4.00±1.26 hours.

The time course of plasma antifactor Xa and antithrombin activities after injection of the two higher doses of nadroparin and enoxaparin is shown in Fig 1. Pharmacokinetic parameters for antifactor Xa activity and for both chromogenic and PTNA-measured antithrombin activity are shown in Tables 1, 2, and 3. At the four doses evaluated in the present study, nadroparin and enoxaparin showed an exponential profile of the decreasing phase of the activity-time curves for both antifactor Xa and antithrombin activities.

View larger version (45K): [in this window] [in a new window]   Figure 1. Plots of time course of antifactor Xa activity and chromogenic and PTNA-measured antithrombin activity after nadroparin (225 and 450 ICU/kg) and enoxaparin (1 and 2 mg/kg). Mean values and 95% confidence intervals for the six volunteers are shown at each sampling time.

View this table: [in this window] [in a new window]   Table 1. Pharmacokinetic Parameters Based on Chromogenic Anti-Xa Activity

View this table: [in this window] [in a new window]   Table 2. Pharmacokinetic Parameters Based on Chromogenic Antithrombin Activity

View this table: [in this window] [in a new window]   Table 3. Pharmacokinetic Parameters Based on PTNA Antithrombin Activity

Overall mean antifactor Xa activity half-life was 4.6 hours. No significant correlation between dose and half-life was found at regression analysis, suggesting a dose-independent clearance mechanism(s) (Table 1). Half-life evaluation of the chromogenic antithrombin activity was possible only for high doses of nadroparin and enoxaparin (Table 2). Overall mean half-life was 3.8 hours, but a positive correlation between dose and half-life was found. Pharmacokinetic analysis of the PTNA-measured antithrombin activity was possible for all doses of nadroparin and the three higher doses of enoxaparin. Plasma half-life after these doses was 6.3 hours (overall mean value), without any significant dose effect (Table 3).

Dose dependence of A_max and t_d was shown for both agents and for antifactor Xa and antithrombin activities (Fig 2). A high degree of correlation between dose and A_max and t_d was found for both antifactor Xa and antithrombin activity (range, 0.54 to 0.90), and all regression analyses were highly significant (P<.0005). After subcutaneous injection of the highest doses of nadroparin and enoxaparin, antifactor Xa activity remained detectable up to 19 and 20 hours respectively, whereas PTNA-measured antithrombin activity was found up to 18 and 17 hours, respectively.

View larger version (22K): [in this window] [in a new window]   Figure 2. Anti-factor Xa (top), chromogenic antithrombin (middle), and PTNA-measured antithrombin (bottom) activity peak (left) and disappearance time (right) are plotted versus heparin dose (for nadroparin, 1 unit of the x axis is equal to 3.1(103 ICU; for enoxaparin, it is equal to 13.6 mg). Mean values and 95% confidence intervals are shown along with the linear regression line. All regression analyses were highly significant (P<5x10-4), and correlation coefficients ranged from .54 to .9.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We performed a pharmacokinetic study to compare the relative half-lives of prophylactic and therapeutic doses of LMWHs. Antifactor IIa activity was assessed both with the chromogenic assay and with a more sensitive assay based on the ability of LMWHs to catalyze the inactivation of thrombin by ATIII in nondiluted plasma (PTNA). Pharmacokinetic parameters of antifactor Xa activity were consistent with those reported in the literature,^{19 20 28} whereas our results are the first pharmacokinetic analysis of the antithrombin activity after therapeutic doses of LMWHs.

The results of the present study demonstrate that high plasma heparin levels measured as either antifactor Xa and antithrombin are achieved when nadroparin or enoxaparin are administered subcutaneously in doses that are currently being used for the treatment of DVT and are under evaluation in patients with unstable angina and myocardial infarction.^{13 14} With once-daily doses of approximately 150 to 200 antifactor Xa units/kg, peak heparin levels of 1.5 to 2.0 units/mL measured as antifactor Xa and of 0.9 unit/mL measured as antithrombin are obtained. These high heparin levels are obtained with only a moderate prolongation of the aPTT. This finding confirms that the aPTT is quite insensitive to LMWHs and not suitable practically for clinical monitoring of the tested doses of nadroparin and enoxaparin. LMWHs have a more predictable anticoagulant response than unfractionated heparin, so they can be administered without laboratory monitoring.²⁹ Plasma heparin levels of more than 0.1 unit/mL as antifactor Xa activity and significantly more than preinjection value as PTNA-measured antithrombin activity remain detectable for up to 18 hours after a single subcutaneous injection. With twice-daily dosing of approximately 100 antifactor Xa units/kg peak plasma, levels of antifactor Xa of 0.85 to 1.0 unit/mL and of antithrombin of 0.5 unit/mL are obtained, and levels above 0.1 IU/mL of both antifactor Xa and antithrombin activities are observed 10 hours after injection. Based on these findings, it is not surprising that these LMWHs are at least as effective and probably more effective than unfractionated heparin for the treatment of venous thrombosis. The observed antifactor Xa levels with the twice-daily injections are approximately 2.5 times higher than those achieved with unfractionated heparin, whereas the plasma levels of antithrombin after LMWH is at the top level seen with unfractionated heparin.

Previous pharmacokinetic studies with LMWHs with lower doses of these agents reported a very short plasma half-life of antithrombin activity^{18 19 20 21} and therefore were inconsistent with the notion proposed from experimental studies that the antithrombin activity of LMWHs is critical for their antithrombotic effect. Our findings help to clarify this apparent discrepancy because they show that with high therapeutic doses, high levels of the antithrombin activity are achieved and sustained with both once- and twice-daily injections. The findings with the standard chromogenic assay were confirmed with a more sensitive assay that measures the ability of heparins (both LMWHs and unfractionated heparin) to catalyze the inhibition of thrombin by antithrombin III. The differences in the apparent plasma half-lives of antithrombin activities observed between the low doses used in prophylactic studies and the higher doses used in treatment studies have two possible explanations. They could be due to either a dose-dependent clearance of LMWHs or the relative insensitivity of the chromogenic antithrombin assay to the levels obtained with the low doses used in the previous pharmacokinetic studies. Our findings suggest that the insensitivity of the chromogenic assay to the low levels of antithrombin activity is the main reason for the discrepancy between the observed half-life with low doses and high doses of LMWHs. The results with the more sensitive PTNA suggest that the clearance of the antithrombin activity of LMWHs is dose independent, although the results with the chromogenic assay are consistent with some dose dependence for the clearance of the antithrombin activity of LMWHs. Therefore, the much shorter half-life of antithrombin of LMWHs reported in previous studies is likely to be caused by the inability of the chromogenic assay to detect the lower plasma levels seen with the lower doses.

In conclusion, our findings, along with the recently reported high bioavailability of antithrombin activity of LMWHs,^{30 31 32} provide an explanation for the effectiveness of LMWHs for the treatment of venous thrombosis. They also provide a promising pharmacological background for the use of LMWHs in the treatment of unstable angina and other arterial diseases. The relevance of our results to arterial thrombosis is under evaluation in clinical trials. The results of the present study demonstrate that high and sustained plasma heparin levels measured as either antifactor Xa and antithrombin activities are achieved when LMWHs are administered in doses used to treat DVT. LMWHs are attractive as antithrombotic agents because they can be administered in doses that achieve high antifactor Xa and antithrombin activities plasma levels. Actually, it is likely that unfractionated heparin would be equally effective if given in higher doses, but results of clinical trials indicate that any further increase in dose would be associated with risk of major bleeding.

	Selected Abbreviations and Acronyms

 

Amax = peak activity DVT = deep venous thrombosis ICU = Institute Choay Units LMWHs = low-molecular-weight heparins PTNA = plasma thrombin neutralization assay td = disappearance time tmax = time to peak activity TAT = thrombin-antithrombin complexes

Received January 24, 1995; revision received May 31, 1995; accepted June 23, 1995.

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