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Pharmacodynamic Effects of Low Molecular Weight Heparin in Obese Subjects Following Subcutaneous Administration of 75 IU/Kg on Plasma Tissue Factor Pathway Inhibitor and Nitric Oxide

Posted on: Wednesday, 10 August 2005, 03:01 CDT

Aim. Pharmacodynamic effects of the low molecular weight heparin tinzaparin on plasma levels of tissue factor pathway inhibitor (TFPI) and nitric oxide (NO) were compared in obese subjects, as well as in normal healthy controls.

Methods. Obese (n=13) patients received a single 75 IU/kg SC injection (the deep vein thrombosis prophylaxis dose) of tinzaparin. Blood samples were obtained pre- and postadministration of drug and at different intervals over 24 h and assayed for total TFPI and NO stable metabolites (nitrates and nitrites) plasma levels, using a specific immunoassay and calorimetric methods.

Results. Mean maximum plasma TFPI levels approached 150-230 ng/ ml at the 0.8 h and up to 5 h posttinzaparin dose compared to basal TFPI levels of 35-90 ng/mL. Plasma TFPI levels were still about 2- fold above basal levels at 12 h and fell to basal levels at 16 h after tinzaparin dose. Basal plasma levels of NO, but not TFPI, were significantly lower (P<0.01) in obese patients compared to controls. Similar TFPI (3-fold above basal at peak) and NO pharmacodynamic profiles for tinzaparin at 75 anti-Xa IU/kg were demonstrated in obese and in normal healthy subjects. Plasma NO (nitrate + nitrite) showed a lag time of about 5-6 h posttinzaparin followed by a steady increase with a peak at 12-15 h and slow decline with a significant residual level at 24 h in obese and healthy subjects.

Conclusion. Data suggest a normal responsiveness of vascular endothelial cells and other cellular compartments to tinzaparin with regard to the pharmacodynamic profiles of plasma TFPI and NO in obese subjects.

[Int Angiol 2005;24:40-2]

Key words: Heparins - Heparin, low molecular weight - Tissue factor pathway inhibitor - Nitric oxide - Obesity - Tinzaparin - Pharmacodynamics.

Heparin and low molecular weight heparins (LMWHs) have multiple pharmacologic activities.1 Although early studies focused on their anti-Xa and anti-IIa properties, LMWHs have other effects, which include stimulation of the release of tissue factor pathway inhibitor (TFPI) and nitric oxide (NO).

TFPI may have a number of therapeutic benefits, including reduction in thrombus formation and anti-inflammatory and antineoplastic effects.1,2 The release of TFPI may be responsible, in part, for the pharmacologic effects of heparin and LMWHs. Although an increase in level of TFPI in response to LMWHs, such as tinzaparin, has been established,3 there are few studies of the effects of LMWHs on TFPI levels in various disease states. In diabetic patients without multiple complications, for example, an increase in levels of TFPI appears to be dependent on obesity and age rather than on diabetes alone.4

NO, also known as endothelium-derived relaxing factor, has multiple physiologic effects.5, 6 Dis eases associated with abnormal NO production include hypercholesterolemia, hypertension, diabetes, atherosclerosis, septic shock, ischemia/reperfusion injury (stroke), carcinogenesis, and woundhealing disorders.7, 8 Heparin and its improved version LMWH are known to have polypharmacological actions at various levels. Earlier studies focused on the anti-Xa and anti- IIa pharmacodynamics for the different LMWHs. This current investigation examined the pharmacodynamic effects of tinzaparin beyond its anti-Xa and anti-IIa effects.

We examined the effects of the LMWH tinzaparin on plasma levels of TFPI and NO in obese subjects as compared to normal healthy human subjects.

TABLE I.-Characteristics of the different patient groups.

Materials and methods

Subjects and treatment groups

The characteristics of the different patient groups are listed in Table I. Pharmacodynamic study examined weight-based dosing of tinzaparin in heavy-weight/obese subjects. Single doses (175 and 75 IU/kg) were administered subcutaneously (SC) to 37 healthy heavyweight subjects (101-165 kg; 26-61 kg/m^sup 2^). AUA and Amax values of anti-Xa and anti-IIa activities were consistent over these body weight and body mass index (BMI) ranges, indicating that tinzaparin pharmacodynamics are not influenced by body weight or BMI. The range of AUA and Amax values in the study population overlapped that of historical control normal-weight subjects (<100 kg), indicating that weight-adjusted tinzaparin dosing yields a predictable response regardless of body weight or BMI.9

Sample collection and assays

Blood samples were obtained pre- and postadministration of tinzaparin and at different intervals over 24 h. Via specific immunoassay and calorimetry, respectively, samples were evaluated for plasma levels of total TFPI and NO-stable metabolites (nitrates and nitrites).

Measurement of plasma TFPI antigen

Plasma was isolated from sodium citrate-treated blood samples. Plasma samples were analyzed in duplicate for total TFPI antigen with a sensitive and specific ELISA (Total TFPI ELISA kit #849; American Diagnostica, Greenwich, CT, USA). Plasma samples were incubated in microtest wells coated with a rabbit antihuman TFPI polyclonal antibody. TFPI was detected with a biotinylated monoclonal antibody specific for Kunitz domain 1 of TFPI and conjugated to streptavidin-conjugated horseradish peroxidase. After reaction with tetramethylbenzidine, sulfuric acid was added to develop the final color. The absorbances of samples were read at 450 nM and compared against a standard calibration curve developed with purified TFPI (American Diagnostica). The inter- and intrasample variation ranged from 2% to 4% for this assay.

Plasma NO levels

NO in plasma was measured according to the same principle like the measurement of soluble adhesion molecules using ELISA that measure nitrate and nitrite, the NO stable metabolites.

Statistical analyses

All data are expressed as mean standard deviation (SD). Statistical significance for data presented was analyzed by Student's t test or ANOVA. Differences were considered statistically significant when P<0.05.

Results

Plasma levels of TFPI

Mean maximum plasma levels of TFPI approached 150 to 230 ng/mL at 0.8 h and up to 5 h posttinzaparin, compared with basal TFPI levels of 35 to 90 ng/mL. The plasma levels of TFPI remained elevated at approximately 2-fold above basal levels at 12 h postdose. At 16 h after administration of the dose of tinzaparin, the plasma levels of TFPI fell to basal levels (Figure 1).

Plasma levels of NO (nitrate and nitrite)

Administration of tinzaparin caused an increase in plasma levels of NO in obese subjects. The pattern of the effect of tinzaparin on NO included a lag time of about 5 to 6 h posttinzaparin followed by a steady increase. A peak occurred at 12 to 15 h; there was a slow decline with a significant residual level of NO at 24 h (Figure 1). Basal plasma levels of NO, but not TFPI, were significantly lower (P<0.01) in obese patients compared with healthy subjects of normal weight (Table I).

Figure 1.-Effects of tinzaparin at deep-vein thrombosis dose (175 IU/kg, SC, q.d.) on plasma levels of tissue factor pathway inhibitor (TFPI) and nitric oxide levels in healthy human subjects.

Discussion and conclusions

Administration of a single SC dose of 75 IU/kg of tinzaparin resulted in an approximately 3-fold increase in TFPI plasma levels in obese subjects. SC tinzaparin dosing in heavy or obese patients is appropriate on body weight alone; the dose need not be capped at a maximal absolute dose.9

A similar pharmacodynamic profile of plasma NO increase was shown in all 3 groups. The administration of tinzaparin caused an increase in plasma levels of NO that peaked 12 to 15 h after administration.

The administration of a single, prophylactic dose of tinzaparin resulted in an increase in levels of NO, which occurred later than the increase in levels of TFPI. The data suggest normal response of vascular endothelial cells and other cellular compartments to the LMWH with regard to the pharmacodynamic profiles of plasma TFPI and NO in obese subjects.

Received October 15, 2004. Accepted for publication December 29, 2004.

References

1. Mousa S. The low molecular weight heparin, tinzaparin, in thrombosis and beyond. Cardiovasc Drug Rev 2002;20:199-216.

2. Mousa S, Kaiser B. Tissue factor pathway inhibitor in thrombosis and beyond: role of heparin. Drugs of the Future 2004;29:751-66.

3. Mousa SA, Bozarth J, Barrett JS. Pharmacodynamic properties of the low molecular weight heparin, tinzaparin: effect of molecular weight distribution on plasma tissue factor pathway inhibitor in healthy human subjects. J Clin Pharmacol 2003;43:727-34.

4. Vambergue A, Rugeri L, Gaveriaux V, Devos P, Martin A, Fermon C el al. Factor VII, tissue factor pathway inhibitor, and monocyte tissue factor in diabetes mellitus: influence of type of diabetes, obesity index, and age. Thromb Res 2001;101:367-75.

5. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327:524-6.

6. Murohara T, Asahara T. Nitric oxide and angiogenesis in cardiovascular disease. Antioxid Redox Signal 2002;4:825-31.

7. Bohlen HG, Nase GP. Obesity lowers hyperglycemic threshold for impaired in vivo endothelial nitric oxide function. Am J Physiol Heart Circ Physiol 2002;283:H391-7.

8. Williams IL, Wheatcroft SB, Shah AM, Kearney MT. Obesity, atherosclerosis and the vascular endothelium: mechanisms of r\educed nitric oxide bioavailability in obese humans. Int J Obes Relat Metab Disord 2002;26:754-64.

9. Hainer JW, Sherrard DJ, Swan SK, Barrett JS, Assaid CA, Fossler MJ et al. Intravenous and subcutaneous weightbased dosing of the low molecular weight heparin tinzaparin (Innohep) in end-stage renal disease patients undergoing chronic hemodialysis. Am J Kidney Dis 2002;40:5318.

S. A. MOUSA, K. JOHANSEN

Albany College of Pharmacy and Pharmaceutical Research Institute, Albany, NY, USA

Leo Pharmaceuticals, Ballerup, Denmark

Address reprint requests to: S. A. Mousa, PhD, MBA, FACC, FACB, Pharmaceutical Research Institute, 106 New Scotland Avenue, Albany, NY 12208, USA. E-mail: mousas@acp.edu

Copyright Edizioni Minerva Medica Mar 2005

Source: International Angiology

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