Plasma Anti-factor Xa Activity Research Articles

Glass bead-activated clotting time (gb-ACT) for monitoring the anticoagulation effects in elderly patients with high risk of venous thromboembolism (VTE)

ABSTRACT Background To find a new bedside method to monitor the anticoagulation effects of low-molecular-weight heparins (LMWHs) in patients with a high risk of venous thromboembolism (VTE). Research design and methods A total of 32 hospitalized patients (aged ≥60 years) who were at high risk of VTE were assigned to receive subcutaneous LMWH for 5 to 14 days. Plasma anti-factor Xa (anti-Xa) activity was conducted by a chromogenic method, and the glass bead-activated whole blood clotting time (gb-ACT) value was obtained by a Sonoclot Analyzer. Results A correlation between the gb-ACT values and the anti-Xa levels was suggested (R = 0.447, p = 0.002), and it was stronger in the older group aged 80 years above (R = 0.467, p = 0.008) and in the group of patients with an eGFR of 30 ~ 60 mL/min (R = 0.565, p = 0.005). The area under the curve (AUC) for gb-ACT by receiver operating characteristic (ROC) curve evaluation was 0.725 (p = 0.011), and the gb-ACT >282.5s provided a sensitivity of 60% and specificity of 74% for anti-Xa >0.800 IU/ml. Conclusions The gb-ACT values detected by a Sonoclot Analyzer could act as a novel bedside method in the monitoring of LMWH anticoagulation.

Patients' Plasma Activity of Heparin, low-Molecular-Weight Heparin or no Anticoagulants on Urine Based DOAC Test Strips.

DOAC Dipstick determines specifically the presence and absence of direct oral anticoagulants (DOACs) from patients’ urine samples and handmade test strips performed as well as the commercial version. To compare plasma activity (chromogenic substrate assays) from plasma samples with results from urine samples (DOAC test strips) of patients treated with heparin, low-molecular weight heparin (LMWH) and without anticoagulation. Plasma anti-factor Xa (aXa) activity was determined by Coamatic chromogenic substrate assay and compared to the presence of anticoagulants in urine by DOAC test strips. Patients were treated for least 5 days and samples were taken 4 hrs after administration in comparison to no treatment with an anticoagulant (n = 42). A total of 100 patients were included treated with heparin (n = 29), LMWH nadroparin (n = 29) or no anticoagulants (n = 42). Plasma aXa levels of patients treated with heparin (2 × 7.500 IU daily subcutaneously, 12 male, age 67.4 ± 11.5 years) were 0,18 IU/ml ± 0,15 IU/ml (mean, standard deviation), with LMWH (1 × 3000 IU daily subcutaneously, 15 male, age 64.2 ± 14.1 years) 0,17 IU/ml ± 0,16 IU/l, and with no anticoagulants (28 male, age 64.2 ± 15.6 years) 0,02 IU/ml ± 0.01 IU/ml. All factor Xa and thrombin inhibitor pad results of test strips were negative. We conclude that DOAC Dipstick has a high probability of not detecting heparin and LMWH in patients on treatment as well as in urine samples of patients not treated with an anticoagulant.

606 Oral anticoagulants in fragile patients with percutaneous endoscopic gastrostomy and atrial fibrillation: the Origami study

Abstract Aims Randomized trials support the safety and efficacy of direct oral anticoagulants (DOACs) vs. vitamin K antagonists (VKA) in patients with nonvalvular atrial fibrillation (AF), leading to increased use of these compounds. Crushed forms of DOACs have shown to be reliable, but evidence supporting percutaneous endoscopic gastrostomy (PEG) delivery is lacking. PEG is a long-term option for enteral food and drug delivery in patients unable to maintain oral intake, bypassing the risks and disadvantages of parenteral nutrition. We investigate the safety and efficacy of Edoxaban administered via PEG in patients with atrial fibrillation and a clinical indication for a long-term anticoagulation. Methods and results In this prospective, single centre observational study, 12 PEG-treated patients with indication to anticoagulation will receive edoxaban via PEG and will be followed-up to 6 months. Plasma anti-Factor Xa activity and edoxaban concentrations will be assessed. Thromboembolic (ischaemic stroke, systemic embolism, venous thromboembolism) and bleeding events (Bleeding Academic Research Consortium and Thrombolysis in Myocardial Infarction) will be recorded at 1 and 6 months. A retrospective analysis of 5 AF cases undergoing PEG-implantation at our Institution, who received edoxaban via PEG, showed plasma anti-FXa levels at steady state of 146 ± 15 ng/ml, without major adverse event at a mean follow-up of 6 months. Conclusion We prospectively investigate PEG-administration of edoxaban in PEG-treated patients requiring long term anticoagulation. Our preliminary retrospective data support this route of DOAC administration.

ORal anticoagulants In fraGile patients with percutAneous endoscopic gastrostoMy and atrIal fibrillation: the (ORIGAMI) study.

The ORal anticoagulants In fraGile patients with percutAneous endoscopic gastrostoMy and atrIal fibrillation (ORIGAMI) study investigates the safety and efficacy of Edoxaban administered via PEG in patients with atrial fibrillation and a clinical indication for a long-term anticoagulation. In this prospective, single-centre observational study, 12 PEG-treated patients with indication to anticoagulation will receive edoxaban via PEG and will be followed up to 6 months. Plasma antifactor Xa activity and edoxaban concentrations will be assessed. Thromboembolic (ischaemic stroke, systemic embolism, venous thromboembolism) and bleeding events (Bleeding Academic Research Consortium and Thrombolysis in Myocardial Infarction) will be recorded at 1 and 6 months. A retrospective analysis of five atrial fibrillation cases undergoing PEG implantation at our Institution who received edoxaban via PEG showed plasma anti-FXa levels at a steady state of 146 ± 15 ng/ml, without major adverse event at a mean follow-up of 6 months. ORIGAMI prospectively investigates PEG-administration of edoxaban in PEG-treated patients requiring long-term anticoagulation. Our preliminary retrospective data support this route of DOAC administration. NCT04271293.

Plasma levels of enoxaparin oligosaccharides, antifactor-Xa and thrombin generation in patients undergoing haemodialysis.

: Low molecular weight heparins are used during haemodialysis for thromboprophylaxis of the dialysis circuit, with plasma antifactor-Xa (anti-Xa) activity used as a surrogate measure for effective anticoagulation. However, this pharmacokinetic parameter does not always correlate with pharmacodynamic effects in patients. The aim of this study was to investigate the relationship between actual plasma levels of the low molecular weight heparins enoxaparin, anti-Xa activity, and global coagulation measurement of thrombin generation during haemodialysis. Blood was analysed from 16 adult patients with end-stage kidney disease at 0, 2, 4 h, and at completion of 31 dialysis sessions where single fixed doses of 20 (n = 3), 40 (n = 16), 60 (n = 6), or 80 (n = 6) mg of enoxaparin (equating to 0.23-1.07 mg/kg) were used as thromboprophylaxis. Plasma enoxaparin oligosaccharides [degree of polymerization (dp)6-dp16] were measured by high-performance size exclusion chromatography, anti-Xa activity by colourimetric assay, and thrombin generation by calibrated automated thrombogram. Plasma enoxaparin fragments were undetectable at the beginning of each dialysis, peaked at 2 h to levels that correlated with dose (r = 0.68, P < 0.001) then remained relatively stable. In contrast, therapeutic anti-Xa levels achieved at 2 h in 18 cases (58%) quickly dropped to only six cases (19%) at the end of dialysis, by which time thrombin generation had also recovered in 81% of patients. Statistical modelling revealed a threshold value of anti-Xa at 0.53 IU/ml that supressed thrombin generation to 15.28% of baseline (P < 0.001). Despite loss of anticoagulant activity in the majority of patients, plasma levels of enoxaparin oligosaccharides remained detectable and relatively unchanged throughout dialysis.

Endothelial Protective Properties of Low Molecular Weight Heparin (LMWH) Tinzaparin in Combination with Statin: A Novel Potential Therapeutic Tool in Preventing Cancer Metastasis?

Introduction8.2 million people die from cancer worldwide every year. LMWH prolongs survival in newly-diagnosed, limited stage lung cancer. Interestingly, trials recruiting patients with other cancers have also demonstrated improved survival in patients with long life-expectancy but not in advanced malignancy, suggesting that the benefit may be mediated by metastasis prevention. LMWHs inhibit metastasis in vivo but may cause bleeding complications when used at effective doses. Consequently, LMWH is unlikely to be used for metastasis prevention unless bleeding side-effects can be urgently addressed. Enhanced endothelial barrier (EB) permeability is a hallmark of metastasis and permits the extravasation of circulating tumour cells into the tissues of the target organ.AimsTo characterise LMWH Tinzaparin-mediated EB protection and underlying mechanisms and to optimise its cytoprotective properties in a manner that may reduce the associated bleeding risk.MethodsEA.hy926 endothelial monolayers were exposed to LMWH (including a novel LMWH formulation comprised of a fraction of LMWH tinzaparin consisting of short polysaccharide chains, mean MW 2.8KDa), simvastatin (a lipid-lowering agent shown to exhibit EB protective properties) and either metastasis-associated agonists (thrombin & VEGF) or calcein blue-AM labelled metastatic tumour cells (DU145, prostate carcinoma). EB function was assessed through the measurement of the migration of an Evans blue-conjugated albumin solution/labelled tumour cells through the monolayers. The role of PAR-1 signalling in modulating permeability was assessed by flow cytometry and permeability assays using an anti-PAR-1 cleavage site antibody and PAR-1 activating/inhibitory peptides. Endothelial myosin light chain-2 diphosphorylation status (MLC-2; the key determinant of endothelial cell actin cytoskeleton contraction) was determined by SDS-PAGE and western blotting. The anticoagulant activity of the LMWHs was determined by calibrated automated thrombography (CAT) and plasma anti-factor Xa activity assays.ResultsTinzaparin treatment prior to thrombin (IIa) and VEGF exposure significantly attenuated agonist-induced EB permeability in a concentration-dependent manner. This cytoprotective effect was observed within a clinically relevant concentration range (0-2IU/mL). Incubation of endothelial monolayers with LMWH also significantly reduced the migration of metastatic carcinoma cells (fluorescence of culture media containing labelled migrated tumour cells reduced to 73.9±5.7% of baseline; p<0.01).IIa-induced permeability was found to be entirely mediated through PAR-1 cleavage. IIa-induced permeability was lost in the presence of an anti-PAR-1 receptor antibody and a PAR-1 signalling inhibitory peptide. Tinzaparin did not alter EA.hy926 PAR-1 expression and did not inhibit IIa-mediated PAR-1 cleavage but reduced IIa-induced MLC-2 phosphorylation and attenuated PAR-1 activating peptide-induced permeability, suggesting that the protective effect of LMWH is not mediated through an inhibition of IIa proteolytic activity (and is therefore independent of its anticoagulant activity).The 2.8KDa LMWH fraction exhibited diminished anticoagulant activity relative to Tinzaparin but retained EB protective properties and attenuated tumour cell trans-migration (at a concentration equivalent to 0.5IU/mL, tumour cell migration was reduced to 76.4±4.7% of baseline and IIa-induced albumin permeability was attenuated to 66.9±8.9%).Remarkably, the cytoprotective properties of Tinzaparin were significantly enhanced when combined with a statin. The combination of a sub-anticoagulant concentration of Tinzaparin (0.1IU/mL) and a clinically relevant concentration of simvastatin (20nM), attenuated EB permeability to a significantly greater extent that that induced by each agent alone (IIa-induced EB permeability was reduced to 7.9±0.2% of baseline (p<0.05)).ConclusionLMWH enhances the barrier function of endothelium in vitro. This effect is not linked to its anticoagulant activity. Strategies which permit using either non-anticoagulant LMWH or sub-anticoagulant LMWH concentrations in combination with other barrier-protective agents may represent a means of delivering this potentially anti-metastatic activity of LMWH to patients without conferring a bleeding risk. DisclosuresMaguire:Actelion UK: Research Funding. Ní Áinle:Actelion UK: Research Funding.

Dose determination of fondaparinux in healthy cats

To establish practical doses and administration frequencies of fondaparinux for cats that would approximate human therapeutic peak and trough plasma anti-factor Xa activities for thromboprophylaxis (TP) and thrombosis treatment (TT) protocols. 6 healthy adult purpose-bred cats. Dosage protocols for TP and TT were selected on the basis of a single compartment pharmacokinetic model incorporating data from humans but modified to account for the higher body weight-normalized cardiac output of cats. Fondaparinux was administered at 0.06 mg/kg, SC, every 12 hours (TP) for 7 days in one session, and 0.20 mg/kg, SC, every 12 hours (TT) for 7 days in another, with a minimum of 1 week separating the sessions. Plasma anti-factor Xa activity was measured before fondaparinux administration (day 1) and at 2 (peak) and 12 (trough) hours after drug administration on days 1 and 7. Platelet aggregation and thromobelastographic (TEG) parameters were also measured 2 hours after drug administration on day 7. Peak plasma anti-factor Xa activities on day 7 for TP (median, 0.59 mg/L; range, 0.36 to 0.77 mg/L) and TT (median, 1.66 mg/L; range, 1.52 to 2.00 mg/L) protocols were within therapeutic ranges for humans. However, only the TP protocol achieved trough anti-factor Xa activity considered therapeutic in humans (median, 0.19 mg/L; range, 0.00 to 0.37 mg/L) on day 7. There were significant changes in the TEG parameters at peak for the TT protocol, suggesting a hypocoagulable state. No significant changes in platelet aggregation were evident for either protocol. A fondaparinux dosage of 0.06 or 0.20 mg/kg, SC, every 12 hours, was sufficient to achieve a peak plasma anti-factor Xa activity in cats that has been deemed therapeutic in humans. This study provided preliminary data necessary to perform fondaparinux dose-determination and clinical efficacy studies.

Inhalable Liposomes of Low Molecular Weight Heparin for the Treatment of Venous Thromboembolism

This study tests the feasibility of inhalable pegylated liposomal formulations of low molecular weight heparin (LMWH) for treatment of two clinical manifestations of vascular thromboembolism: deep vein thrombosis (DVT) and pulmonary embolism (PE). Conventional distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and long-circulating pegylated (DSPE-PEG-2000 and DSPE-PEG-5000) liposomes were prepared by hydration method. Formulations were evaluated for particle size, entrapment efficiency, stability, pulmonary absorption, anticoagulant, and thrombolytic effects in rats. Pulmonary absorption was monitored by measuring plasma antifactor Xa activity; anticoagulant and thrombolytic effects were studied by measuring reduction in thrombus weight and amount of dissolved radioactive clot in the blood, respectively. Pegylated liposomal were smaller and showed greater drug entrapment efficiency than conventional liposomes. All formulations produced an increase in pulmonary absorption and circulation time of LMWH upon first dosing. Three repeated dosings of conventional liposomes resulted in decreased half-life and bioavailability; no changes in these parameters were observed with pegylated liposomes. PEG-2000 liposomes were effective in reducing thrombus weight when administered every 48 h over 8 days. In terms of thrombolytic effects and dosing frequency, PEG-2000 liposomes administered via the pulmonary route at a dose of 100 U/kg were as effective as 50 U/kg LMWH administered subcutaneously. This paper suggests that inhalable pegylated liposomes of LMWH could be a potential noninvasive approach for DVT and PE treatment.

Low-Molecular-Weight Heparin Dosage in Newborn Foals

Heparin is used in humans as prophylaxis of hypercoagulable states and disseminated intravascular coagulation (DIC). However, babies need a higher heparin dose than do adults. Septic neonate foals are at high risk of hypercoagulable state and DIC, and there is limited objective information about heparin dose for equine neonates. To assess whether neonate foals require higher dosages of low-molecular-weight heparin (LMWH) than adults. Eighteen healthy and 11 septic neonate foals. Experimental and clinical studies. Firstly, healthy foals were randomly distributed in 2 groups, 1 receiving 50 IU/kg SC of dalteparin and the 2nd group receiving 100 IU/kg SC of dalteparin, once daily for 3 days. Blood samples were collected before and 3, 6, 27, and 51 hours after the 1st LMWH administration. Plasma antifactor-Xa activity was measured, together with hemostatic and hematologic parameters used to assess the risk of bleeding. Subsequently, septic foals were treated blindly either with placebo (saline) or 100 IU/kg of dalteparin for 3 days. Plasma antifactor-Xa activity and other hemostatic parameters were determined before and after treatment. Plasma antifactor-Xa activity in healthy foals was below prophylactic activity when using the adult dosage (50 IU/kg), whereas prophylactic activities were achieved when using the double dosage (100 IU/kg). No hemorrhagic events and erythrocyte-related complications were observed with either dosage. In the clinical study, only 4/6 septic foals had plasma antifactor-Xa activity adequate for prophylaxis. Equine neonates require higher dosages of LMWH compared with adults to reach prophylactic heparinemia.

Inhalable Lactose-Based Dry Powder Formulations of Low Molecular Weight Heparin

Currently low molecular weight heparin (LMWH) is administered as subcutaneous injection. This study sought to investigate the feasibility of LMWH as an inhalable dry powder (DPI) formulation and evaluate the interaction of the drug with lactose when used as a carrier. The study also compares the extent of pulmonary absorption of LMWH administered as a dry powder with that administered as an aerosolized aqueous solution. The formulations were prepared by mixing LMWH in an aqueous solution of lactose followed by lyophilization of the resulting solution. The lyophilized preparation was then ground and sieved. Physical characterization of the formulations was performed by Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), scanning electron microscopy (SEM), particle size analysis, and determination of aerodynamic diameter. For in vivo studies, formulations were administered to anesthetized rats, and drug absorption was monitored by measuring plasma antifactor Xa activity. In the FTIR scan, all characteristic peaks of lactose and LMWH were observed, suggesting that there was no strong interaction between lactose and LMWH. Although the aerodynamic diameter of the formulation (DPI-2) that was sieved through 170- and 230-mesh screens was similar to that of the formulation (DPI-1) sieved through 120- and 170-mesh screens, the particle sizes of the two formulations were significantly different. Dry powder formulations of LMWH were better absorbed compared to an inhalable solution of LMWH. One of the dry powder formulations (DPI-2) produced an almost 1.5-fold increase in the relative bioavailability (41.6%) compared to the liquid formulation of LMWH (32.5%). Overall, the data presented here suggest that lactose does not adversely affect the physical-chemical characteristics of the drug, and that lactose can be used as a carrier for pulmonary delivery of LMWH.

Alkanoylsucroses in nasal delivery of low molecular weight heparins: in-vivo absorption and reversibility studies in rats.

The efficacy of alkanoylsucroses in enhancing nasal absorption of low molecular weight heparin (LMWH) and the time span of action of these agents on the nasal membrane has been investigated. In this regard, LMWH formulated with alkanoylsucroses was administered nasally to anaesthetized male Sprague-Dawley rats and the absorption of LMWH was determined by measuring plasma antifactor Xa activity. The duration of action of these agents at the site of administration was investigated by an in-vivo reversibility study. The potency and efficacy of dodecanoylsucrose was compared with that of sodium glycocholate. Alkanoylsucroses used in this study include dodecanoylsucrose, decanoylsucrose and octanoylsucrose. These agents enhance nasal absorption of enoxaparin in a dose-dependent and chain-length-dependent manner. Of the agents tested, dodecanoylsucrose was found to be the most potent in enhancing nasal absorption of LMWH. The bioavailability of enoxaparin formulated with alkanoylsucroses was increased by several folds compared with enoxaparin formulated in saline. The reversibility study with dodecanoylsucrose showed that the effect of alkanoylsucroses faded away with time and the duration of action of this agent at the site of administration was 120-140 min. Dodecanoylsucrose was found to be twice as potent as sodium glycocholate. Overall, the nasal absorption of LMWH was effectively enhanced by co-administration of alkanoylsucroses and the effect of alkanoylsucroses on nasal epithelium was found to be reversible. The potency of these agents depends on their hydrophobic chain lengths.

Modulation of gastrointestinal permeability of low-molecular-weight heparin by L-arginine: in-vivo and in-vitro evaluation

L-Arginine is the principal physiological precursor of nitric oxide (NO, a key neurotransmitter) that plays a versatile role in the physiology of the gastrointestinal tract. In this study, the efficacy of L-arginine in enhancing intestinal absorption of ardeparin, a low-molecular-weight heparin (LMWH) was investigated in Caco-2 cell monolayers and a rat model. Regional permeability studies using rat intestine were performed using a modified Ussing chamber. Cell viability in the presence of various concentrations of enhancer was determined by MTT assay. Furthermore, the eventual mucosal epithelial damage was histologically evaluated. LMWH formulated with L-arginine was administered orally to male Sprague-Dawley rats and the absorption of LMWH was determined by measuring plasma anti-factor Xa activity. Higher ardeparin in-vitro permeability (approximately 3 fold) compared with control was observed in the presence of 2% L-arginine. Regional permeability studies indicated predominant absorption in the colon region. Cell viability studies showed no significant cytotoxicity below 0.8% L-arginine. The oral bioavailability of ardeparin formulated with L-arginine (250 mg kg(-1)) was increased by approximately 2 fold compared with control. The formulation was well tolerated by the rats and no abnormal histopathological findings were observed in intestinal tissues of rats exposed to L-arginine. These results suggest that L-arginine may be useful in enhancing the intestinal absorption of LMWHs.

Cationic liposomes as carriers for aerosolized formulations of an anionic drug: Safety and efficacy study

This study tests the hypothesis that pegylated cationic liposomes are a viable carrier for inhalable formulations of low molecular weight heparin, an anionic drug. Cationic liposomal formulations of low molecular weight heparin were prepared by the hydration method using 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt), cholesterol and 1,2-distearoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]. The formulations were characterized for particle size, entrapment efficiency, pulmonary absorption and pharmacological efficacy. For absorption studies, the formulations were administered to anesthetized male Sprague–Dawley rats via the pulmonary route and drug absorption was monitored by measuring plasma anti-factor Xa activity. The pharmacological efficacy of the formulations was studied in rodent models of pulmonary embolism and deep vein thrombosis. The mean particle size of the liposomes was 104.8 ± 20.7 nm and the drug entrapment efficiency was 90.3 ± 0.1%. The half-life of the cationic liposomal formulation was 10.6 ± 0.2 h, a 2.2-fold increase compared to low molecular weight heparin formulated in saline, and the relative bioavailability was ∼73.4 ± 19.1% when compared to subcutaneously administered drug. A once-every-other-day inhaled dose of the formulation showed similar efficacy in reducing thrombus weight as a once-daily dose of subcutaneously administered drug. Likewise, cationic liposomal formulations administered via the pulmonary route 6 h prior to embolization in the lungs showed a thrombolytic effect comparable to that of low molecular weight heparin administered subcutaneously 2 h before embolization. Histological examination of lung tissue and measurement of injury markers in bronchoalveolar lavage fluid suggest that the formulations did not produce extensive damage. The results demonstrate that pegylated cationic liposomes could be a viable carrier for an inhalable formulation of low molecular weight heparin.

Safety of low-dose subcutaneous enoxaparin for the prevention of venous thromboembolism after primary intracerebral haemorrhage

Background The risks and benefits of low molecular weight heparins (LMWH) for the prevention of deep-vein thrombosis (DVT) and pulmonary embolism (PE) after intracerebral haemorrhage (ICH) have not been assessed. The few studies on this subject have revealed conflicting results. Methods We retrospectively evaluated whether subcutaneous enoxaparin (20 mg daily) reduced symptomatic venous complications or caused increased 3-month death rate. We included 407 patients who were admitted to a stroke unit and survived the first two days after onset of ICH. There were 232 patients who received anticoagulant treatment for the prevention of DVT and PE and 175 who did not. Results Despite the fact that the treated patients were in worse clinical condition at the start of the treatment, 3-month death rate was 19% among them compared to 21% among those not receiving anticoagulant therapy. Low-dose subcutaneous enoxaparin (20 mg once daily) induced a significant plasma anti-factor Xa activity 2-3 hours after administration (p = 0.018). Haematoma enlargements (33%) occurred in 9% and 7% of the treated and untreated patients, whereas symptomatic venous thromboembolic complications were observed in 3% and 2%, respectively. Conclusions We did not observe any increased mortality among ICH patients who survived the first 2 days after the onset of ICH and were thereafter treated with enoxaparin 20 mg daily relative to patients remaining untreated. A randomized trial of the effect of LMWH with a higher dose in prevention of venous thromboembolic complications would be indicated.

Positively charged polyethylenimines enhance nasal absorption of the negatively charged drug, low molecular weight heparin

This study tests the hypothesis that positively charged polyethylenimines (PEIs) enhance nasal absorption of low molecular weight heparin (LMWH) by reducing the negative surface charge of the drug molecule. Physical interactions between PEIs and LMWH were studied by Fourier transform infrared (FTIR) spectroscopy, particle size analysis, conductivity measurements, zeta potential analysis, and azure A assay. The efficacy of PEIs in enhancing nasal absorption of LMWH was studied by administering LMWH formulated with PEI into the nose of anesthetized rats and monitoring drug absorption by measuring plasma anti-factor Xa activity. The metabolic stability of LMWH was evaluated by incubating the drug in rat nasal mucosal homogenates. FTIR spectra of the LMWH–PEI formulation showed a shift in peak position compared to LMWH or PEI alone. Decreases in conductivity, zeta potential and the amount of free LMWH in the PEI–LMWH formulation, as revealed by azure A assay, suggest that PEIs possibly neutralize the negative surface charge of LMWH. The efficacy of PEI in enhancing the bioavailability of nasally administered LMWH can be ranked as PEI-1000 kDa ≥ PEI-750 kDa > PEI-25 kDa. When PEI-1000 kDa was used at a concentration of 0.25%, there was a 4-fold increase in both the absolute and relative bioavailabilities of LMWH compared to the control formulation. Overall, these results indicate that polyethylenimines can be used as potential carriers for nasally administered LMWHs.

Microscale isolation and analysis of heparin from plasma using an anion-exchange spin column

Heparin, a linear highly sulfated polysaccharide, is a member of the glycosaminoglycan (GAG)1 family [1,2]. Since the 1930s, heparin has been widely used as a clinical anticoagulant [3,4]. More recently, low-molecular weight heparin (LMWH) derivatives have come into widespread use as anticoagulant and antithrombotic drugs [5]. Although heparin’s clinical use predated the establishment of the US. Food and Drug Administration (FDA), the approval of LMWH required a number of pharmacological studies on these new drugs. The pharmacologically relevant concentration of heparin and LMWH in plasma is between 0.1 and 1.0 anti-factor XaU/ml, corresponding to approximately 1–10 µg/ml of drug [6]. Plasma is a complex mixture, making the direct analysis of heparin or LMWH at these concentrations exceedingly challenging. Thus, most of the pharmacological studies of heparin and LMWH have relied on bioassays such as anti-factor Xa activity to evaluate these agents, providing only pharmacodynamic data. This inability to obtain pharmacokinetic data on LMWH is a serious concern because the plasma anti-factor Xa activity of LMWH does not correlate well with its therapeutic efficacy [7].

Pulmonary Delivery of Low Molecular Weight Heparins

To investigate if pulmonary delivery of low molecular weight heparin (LMWH) formulated with tetradecyl-beta-maltoside (TDM) or dimethyl-beta-cyclodextrin (DMbetaCD) could be a feasible alternative to subcutaneous injections for the treatment of pulmonary embolism. The pulmonary absorption of two LMWHs and unfractionated heparin formulated with TDM or DMbetaCD was studied in cell culture and rodent model. The in vitro study was performed by measuring the transport of radiolabeled enoxaparin and mannitol across human bronchial epithelial cells (Calu-3) in the presence or absence of varying concentrations of TDM or DMbetaCD. The changes in transepithelial electrical resistance (TEER) and enoxaparin metabolic stability were also investigated using Calu-3 cells. In vivo absorption studies were performed by measuring plasma anti-factor Xa activity after pulmonary administration of enoxaparin, dalteparin, or unfractionated heparin to anesthetized rats. In vitro experiments conducted in Calu-3 cells suggest that the addition of TDM or DMbetaCD to the apical chamber results in a significant increase in 3H-enoxaparin and 14C-mannitol permeability and a decrease in TEER across the Calu-3 cell monolayer. Enoxaparin incubated in Calu-3 cell extracts was stable for 8 h. In vivo studies indicate that both TDM and DMbetaCD enhance pulmonary absorption of LMWH. However, TDM was found to be more potent than DMbetaCD in both in vitro transport and in vivo absorption studies. TDM and DMbetaCD enhance pulmonary absorption of LMWH both in vitro and in vivo, with TDM being more efficacious than DMbetaCD. Both agents increase drug transport by acting mainly on the membrane rather than interacting with the drug.

Is laboratory monitoring of low-molecular-weight heparin therapy necessary? Yes

At the beginning of the clinical development of low-molecularweight heparin (LMWH) it was expected that they produce less anticoagulation and consequently less hemorrhages compared with unfractionated heparin (UFH). This has been demonstrated in several clinical trials for treatment of venous thromboembolism (VTE). This confirms, but does not exclude the lack of a benefit, to determine the anticoagulant effects of LMWH in certain groups of patients to avoid over- or underanticoagulation. All studies for treatment of thromboembolism with LMWH have been performed without determination of the anticoagulant effects of LMWH except for small and detailed clinical trials. Due to the lower binding of LMWH to plasma proteins and to cell surfaces, the plasma anti-factor (F)Xa activity generated by a given dose of LMWH is more predictable than for UFH. However, there is an unexpressed agreement of avoiding an unacceptable increase of the anticoagulant effect during LMWH therapy, increasing the hemorrhagic risk [1]. Some clinical trials indicate that the interpatient variability is less pronounced for LMWHs, leading to less frequent dose adjustments in a small proportion of patients for treatment of VTE compared with UFH [2,3]. Higher doses of LMWH lead to higher anti-FXa activities and to increased incidence of hemorrhagic events, indicating a clear association between plasma activities and side-effects [4].

Factor Xa-activated whole blood clotting time (Xa-ACT) for bedside monitoring of dalteparin anticoagulation during haemodialysis.

Low molecular weight heparins (LMWH) like dalteparin are increasingly used for anticoagulation during haemodialysis (HD). The available laboratory tests for monitoring LMWH anticoagulation are time-consuming and expensive, and the suitability of the conventional activated clotting time (ACT) is controversial. A simple and cheap bedside test would be useful. We studied the factor Xa-activated whole blood clotting time (Xa-ACT) in vitro and in vivo in nine patients undergoing chronic HD with i.v. dalteparin bolus anticoagulation and compared it with the conventional ACT. Plasma anti-factor Xa (antiXa) activity was determined with a chromogenic assay. Thrombin-antithrombin complexes were measured to detect coagulation activation. Xa-ACT and ACT were prolonged with rising dalteparin concentration. In vitro, both clotting times were strongly correlated with the antiXa levels (r = 0.94 and 0.89, respectively). Nevertheless, compared with the ACT, the Xa-ACT was considerably more sensitive to the LMWH in vitro (healthy blood: Xa-ACT 90 s/U vs ACT 26 s/U; uraemic blood: Xa-ACT 96 s/U vs ACT 31 s/U) as well as in vivo (Xa-ACT 81 s/U vs ACT 22 s/U) and reflected different intensities of anticoagulation. An initial dalteparin bolus of 80+/-11 U/kg body weight was able to prevent coagulation activation for up to 4 h of HD. For monitoring LMWH anticoagulation the Xa-ACT was superior to the conventional ACT in vitro as well as in vivo during HD. The Xa-ACT can be useful as a LMWH bedside test. The ACT was not sensitive enough to serve as a LMWH monitoring tool.

Chain Length-Dependent Effects of Alkylmaltosides on Nasal Absorption of Enoxaparin

The purpose of this study was to investigate whether the hydrophobic chain length of alkylmaltosides affects their efficacy as absorption promoters for nasally administered low-molecular-weight heparin and to study whether these agents enhance nasal absorption in a time-dependent manner without causing irreversible damage to the nasal epithelial membrane. For the nasal absorption studies, enoxaparin formulated with different alkylmaltosides was administered nasally to anesthetized rats and absorption of the drug was determined by measuring plasma anti-factor Xa activity. Reversibility studies were performed by administering enoxaparin at different time points after administration of alkylmaltosides. The AUC0–360 for plasma anti-factor Xa-time curves increased with the increase in alkylmaltoside concentration in the formulations. Absolute and relative bioavailability of enoxaparin were increased by two-fold when the alkyl chain length of maltosides was increased from 8 to 14 carbons. Alkylmaltosides therefore increase nasal absorption of enoxaparin in a dose- and chain length-dependent manner. Of the alkylmaltosides tested, tetradecylmaltoside is the most potent enhancer of nasal absorption of enoxaparin. Longer chain alkylmaltosides produce a more prolonged effect on nasal mucosa compared with those with shorter alkyl chain. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association