Recombinant human factor VIIa and a factor VIIa-analogue reduces heparin and low molecular weight heparin (LMWH)-induced bleeding in rats

Summary.  Background:  Heparin and low molecular weight heparin (LMWH) are widely used for prevention and treatment of thromboemobolic events, but may occasionally cause uncontrollable bleeding. Heparin can readily be antagonized with protamine, but this is less effective against LMWH. Objectives:  To test the effects of rFVIIa or an analogue of rFVIIa, NN1731, on heparin- and LMWH-induced bleeding in rats. Methods:  Initially the doses of heparin and tinzaparin (a LMWH) were determined by dose-titration. Following pretreatment with heparin or tinzaparin in rats, tail-transection was performed, and the effect of rFVIIa and NN1731 on the bleeding was observed. Results:  rFVIIa (5, 10 and 20 mg kg⁻¹) reduced bleeding time and blood loss caused by heparin- and tinzaparin-induced bleeding, using doses of 200 IU kg⁻¹ ( n  = 8) and 500 IU kg⁻¹ ( n  = 9), respectively. Similarly, 10 mg kg⁻¹ NN1731 significantly reduced both heparin- and tinzaparin-induced bleeding to the normal level. Following severe anticoagulation with 1800 IU kg⁻¹ tinzaparin, 10 mg kg⁻¹ NN1731 reduced and normalized the bleeding, while the effect of 20 mg kg⁻¹ rFVIIa failed to reach statistical significance. These data are consistent with the hypothesis that rFVIIa/NN1731 are capable of generating sufficient thrombin locally on the surface of activated platelets to induce hemostasis in the presence of heparin/LMWH. Conclusions:  This study suggests that rFVIIa and NN1731 may have the potential to control bleedings caused by heparin or LMWH.     

rFVIIa and a new enhanced rFVIIa-analogue, NN1731, reduce bleeding in clopidogrel-treated and in thrombocytopenic rats

Summary.  Background: The pharmacological effect of rFVIIa occurs at the surface of activated platelets by enhancing thrombin generation at the site of vascular damage. It is therefore important to study the effects of rFVIIa in platelet-related bleeding situations. We examined the effect of rFVIIa and an rFVIIa-analogue, NN1731, on clopidogrel-induced and thrombocytopenic bleeding in rats. Methods and results: Clopidogrel [10 mg kg⁻¹; per oral (p.o.)] severely inhibited platelet aggregation and increased blood loss after tail-transection four hours after administration. Treatment with rFVIIa (5, 10, 20 mg kg⁻¹) or NN1731 (1, 5, 10 mg  kg⁻¹), administered five minutes after induction of bleeding, reduced blood loss significantly and dose-dependently. NN1731 had an increased hemostatic potential compared with rFVIIa, reducing blood loss to the control level, whereas this was not even achieved with the highest dose of rFVIIa. Antibody-induced thrombocytopenia reduced platelet numbers by more than 90% and increased the blood loss after tail-transection. Treatment with 10 and 20 mg kg⁻¹ rFVIIa significantly reduced blood loss, whereas 10 mg kg⁻¹ NN1731 reduced the bleeding to control levels. Conclusions: The hemostatic effect of rFVIIa and NN1731 was demonstrated in thrombocytopenic and clopidogrel-treated rats, showing efficacy in situations with decreased platelet number or functionality. Our findings are consistent with the hypothesis that rFVIIa/NN1731 contribute to hemostasis by thrombin generation even in situations with platelet disorders. Furthermore, NN1731 demonstrated a higher hemostatic potential than rFVIIa.     

Acenocoumarol therapy in pediatric patients

Summary.  To determine guidelines for administering and monitoring acenocoumarol therapy in children, 93 patients (median 5.1 years, range: 0.2–18 years) were prospectively evaluated over a 33-month period. The loading doses used were: <1 year, 0.20 mg kg⁻¹; >1–5 years, 0.09 mg kg⁻¹; 6–10 years, 0.07 mg kg⁻¹; 11–18 years, 0.06 mg kg⁻¹. In this study, the loading dose and the dose to achieve and maintain target therapeutic range (TTR) for acenocoumarol are age-dependent, with infants having the highest and teenagers having the lowest requirements. The use of a different loading dose according to age has allowed most of the children (80%) in all the age groups to achieve TTR in less than 1 week. No patients had serious bleeding or thrombotic complications. We conclude that there is an age-dependent response to acenocoumarol in pediatric patients. The implementation of an age-adjusted loading dose regimen reduces the length of hospitalization required to achieve effective anticoagulant therapy.     

Effect of colchiceine and ursodeoxycholic acid on hepatocyte and erythrocyte membranes and liver histology in experimentally induced carbon tetrachloride cirrhosis in rats

Colchiceine and ursodeoxycholic acid (UDCA) are drugs currently in use as therapy for different types of liver damage. We evaluated their ability to reverse the damage induced by carbon tetrachloride (CCl₄) in rats. Six groups were analysed: (1) CCl₄ (0.4 g kg⁻¹, i.p., three times a week) for 13 weeks; (2) CCl₄ for 8 weeks followed by colchiceine (60 μg kg⁻¹) + CCl₄ for 5 weeks; (3) CCl₄ for 8 weeks and thereafter UDCA (25 mg kg⁻¹) + CCl₄ for 5 weeks. Groups 4, 5 and 6 were appropriate controls of colchiceine, UDCA and vehicles respectively. Na⁺,K⁺- and Ca²⁺-ATPase activities and the cholesterol–phospholipid (CH/PL) ratio from erythrocyte and hepatocyte membranes were quantified. Membrane enzymatic activities and CH/PL ratios were affected more in group 1 than groups 2 and 3. We concluded that colchiceine and UDCA were effective drugs in this model of liver damage.     

Effect of atorvastatin on microRNA 221 / 222 expression in endothelial progenitor cells obtained from patients with coronary artery disease

Background  Endothelial progenitor cells (EPCs) play an important role in the maintenance of vascular integrity. Lipid lowering therapy (LLT) with statins may contribute to biologically relevant activities including the proliferation of endothelial cells. The physiological role of microRNA (miR)-221/222, a newly discovered class of small RNA, is closely linked to the proliferation of endothelial cells. We therefore investigated whether LLT with statins might affect miR-221/222 expression in EPCs obtained from patients with coronary artery disease (CAD).
Materials and methods  This study included 44 patients with stable CAD and 22 subjects without CAD (non-CAD). Patients with CAD were randomized to 12 months of LLT with atorvastatin (10 mg day⁻¹) or pravastatin (10 mg day⁻¹). EPCs were obtained from peripheral blood at baseline and after 12 months of statin therapy. Levels of miR-221/222 in EPCs were measured by real-time RT-PCR.
Results  Levels of miR-221/222 were significantly higher in the CAD group than in the non-CAD group ( P  < 0·01). Levels of miR-221/222 were weakly negatively correlated with EPC number in the CAD group. After 12 months of therapy, changes in lipid profiles were greater in the atorvastatin group than in the pravastatin group. LLT with atorvastatin markedly increased EPC numbers and decreased miR-221/222 levels (all P  < 0·05), whereas LLT with pravastatin did not change EPC numbers or miR-221/222 levels.
Conclusions  This study demonstrates that LLT with atorvastatin increases EPC numbers and decreases miR-221/222 levels in patients with CAD, possibly contributing to the beneficial effects of LLT with atorvastatin in this disorder.     

Anti-inflammatory effects of 5-HT3 receptor antagonist, tropisetron on experimental colitis in rats

Background  There is a pressing need for research that will lead to the development of new therapeutic approaches for treating inflammatory bowel disease (IBD). The aim of this study was to investigate the effects of tropisetron, a 5-Hydroxytryptamine (5-HT)-3 receptor antagonist with anti-inflammatory properties in a model of experimental colitis in rat.
Materials and methods  Acetic acid model of colitis in rats was used. Colitis was induced by intracolonal instillation of 4% (v/v) acetic acid. One hour after induction of colitis, intraperitoneal (IP) or intrarectal (IR) tropisetron (2 mg kg⁻¹, either route) or dexamethasone (1 mg kg⁻¹, either route) was administered. The severity of colitis was assessed 24 h later using macroscopic and microscopic changes of damaged colon, measurement of inflammatory cytokines interleukin-1β, interleukin-6 and tumour necrosis factor-α levels and oxidative stress markers myeloperoxidase (MPO) and malondialdehyde (MDA) in colonic tissues.
Results  Tropisetron decreased colonic macroscopic and microscopic damage scores. This was associated with significant reduction in both neutrophil infiltration indicated by decreased colonic MPO activity and lipid peroxidation measured by MDA content, as well as a decreased colonic inflammatory cytokines. IR tropisetron decreased colonic damage that was associated with decreased neutrophil infiltration, lipid peroxidation and colonic inflammatory cytokines. Beneficial effects of tropisetron were lower than those of dexamethasone. No significant differences were observed between IP and IR administration with the exception of MDA level more diminished by IP tropisetron and dexamethasone.
Conclusions  Tropisetron exert beneficial effects in experimental rat colitis and therefore might be useful in the treatment of IBD.     

Long-lasting antithrombotic effects of a single dose of human recombinant, active site-blocked factor VII: insights into possible mechanism(s) of action

Summary.  Tissue factor (TF) is important in initiating intravascular thrombosis. We demonstrated that active-site blocked factor VII (FVIIai) inhibits intravascular thrombosis for at least 6 h following a single injection, despite FVIIai plasma half-life was ∼45 min. The aims of the present study were: (a) to determine the duration of the antithrombotic effects of a single injection of FVIIai; and (b) to assess whether FVIIai prolonged effects can be explained by a slow dissociation rate from TF in the arterial wall. Cyclic flow variations (CFVs), obtained in stenotic rabbit carotid arteries with endothelial injury, were abolished by either FVIIai (100 µg kg⁻¹ min⁻¹ for 10 min) or hirudin (1 mg kg⁻¹). After CFVs were abolished, carotid blood flow velocity was recorded continuously for 24 h. CFVs restored in all hirudin-treated animals after 2.1 ± 0.3 h, while they restored in only four of nine FVIIai-treated rabbits in 10.1 ± 2.2 h. Five animals in this group did not show restoration of CFVs up to 24 h. Immunohistochemistry revealed that FVIIai was still bound to the arterial wall 24 h following its administration, despite at this time FVIIai plasma levels were undetectable. Prothrombin time and partial thromboplastin time did not change significantly. FVIIai exerts potent, long-lasting antithrombotic effects without affecting systemic hemostatic parameters; a possible mechanism is a slow dissociation rate of FVIIai from TF. These proprieties make FVIIai particularly attractive as an antithrombotic intervention.     

Measurement of post-absorptive glucose kinetics in non-insulin-dependent diabetic patients: methodological aspects

Post-absorptive glucose metabolism was studied in non-insulin-dependent diabetes mellitus (NIDDM) patients and normal subjects using dideuterated glucose as tracer. From the progressive fall in blood glucose levels and the increase in isotopic enrichment, the post-absorptive situation could not be regarded as a steady state for glucose metabolism, and non-steady-state approximations had therefore to be applied. However, this did not alter significantly the results in the 10 NIDDM patients studied. Significantly higher values of endogenous glucose production (EGP) were obtained (178.1 ± 24.0 mg m⁻² min⁻¹ vs. 80.2 ± 14.4; P  < 0.01) if the tracer priming dose was not adapted to the degree of hyperglycaemia. Valid measurements could be made after only 1 h isotopic equilibration time if an appropriately matched priming dose was employed. Methodologically acceptable values for EGP in the 10 NIDDM patients did not differ significantly from those of 10 normal control subjects (80.2 ± 14.4 mg m⁻² min⁻¹ vs. 85.6 ± 3.9; not significant). The post-absorptive hyperglycaemia in these patients was assumed to stem essentially from a defect in peripheral glucose uptake.     

ApoAI-phosphatidylcholine infusion neutralizes the atherothrombotic effects of C-reactive protein in humans

Summary.  Background:  High-density lipoprotein (HDL) exerts a variety of anti-atherothrombotic functions, including a potent anti-inflammatory impact. In line, the direct pro-inflammatory effects of C-reactive protein (CRP) can be attenuated by HDL in vitro . Objective:  To evaluate whether this also holds true in humans, we assessed the ability of reconstituted HDL to neutralize CRP-mediated activation of coagulation and inflammation. Methods:  Fifteen healthy male volunteers received an infusion of recombinant human (rh)CRP (1.25 mg kg⁻¹ body weight). In eight of these volunteers, an infusion of human apoAI reconstituted with phosphatidylcholine (apoAI-PC; 80 mg kg⁻¹ body weight) preceded rhCRP infusion. Results:  Infusion of rhCRP alone elicited an inflammatory response and thrombin generation. In individuals who received apoAI-PC prior to rhCRP, these effects were abolished. Parallel tests in primary human endothelial cells showed that apoAI-PC preincubation with rhCRP abolished the CRP-mediated activation of inflammation as assessed by IL-6 release. Although we were able to show that rhCRP co-eluted with HDL after size-exclusion chromatography, plasmon surface resonance indicated the absence of a direct interaction between HDL and CRP. Conclusion:  Infusion of apoAI-PC prior to rhCRP in humans completely prevents the direct atherothrombotic effects of rhCRP. These findings imply that administration of apoAI-PC may offer benefit in patients with increased CRP.     

The hormone sensitive lipase gene in familial combined hyperlipidemia and insulin resistance

Backround Insulin resistance in the most common familial dyslipidemia, familial combined hyperlipidemia (FCHL), could be due to variations in the hormone sensitive lipase (HSL) gene.
Materials and methods The coding region of the HSL gene was screened with the single strand conformation polymorphism analysis in probands of 27 FCHL families with 228 members. In addition, the C-60G promoter substitution of the HSL gene was determined by the restriction fragment length polymorphism analysis in these subjects.
Results No variants in the coding region of the HSL gene were found and the allele frequencies of the C-60G promoter substitution and the silent variant (G3138A) in the 3' untranslated region did not differ between 110 control subjects and 27 probands with FCHL. However, in control women the C-60G substitution was associated with high body mass index [30·6 ± 0·9 kg m⁻² (mean ± SD) in subjects with the C/G genotype and 24·8 ± 4·6 in subjects with the C/C genotype, P  = 0·012], and in control men with high rates of insulin-stimulated whole body glucose uptake (70·1 ± 14·7 vs. 56·7 ± 14·2 µmol kg⁻¹ min⁻¹, P  = 0·014). In 228 FCHL family members this substitution was associated with high low-density lipoprotein cholesterol levels in men (4·51 ± 1·12 vs. 5·17 ± 1·28 mmol L⁻¹, P  = 0·049), but not in women.
Conclusions The HSL gene is not a major gene for FCHL. However, the − 60G allele of this gene may affect body weight, insulin sensitivity and serum cholesterol levels.     

A new approach to treatment of bleeding episodes in young hemophilia patients: a single bolus megadose of recombinant activated factor VII (NovoSeven®)

Summary.  Recombinant activated factor VII (rFVIIa, NovoSeven^®) represents an effective treatment for hemophilia patients with inhibitors, but no consensus as to the best dosing regimen exists. We assessed the efficacy and safety of a rFVIIa 'megadose' (300 µg kg⁻¹ bolus) as treatment for bleeds in three young inhibitor patients. Of 114 bleeds, 95 responded to a single dose. Pain relief was faster and therapy duration significantly shorter than with continuous infusion (CI) regimens or standard boluses (90 µg kg⁻¹ every 3 h). Rebleeding occurred in 9.6% of cases and 19/114 episodes required a second bolus injection. Although rFVIIa consumption per bleed (median: 300 µg kg⁻¹) was higher than with standard boluses (180–270 µg kg⁻¹), patients found single bolus administration more convenient than recurrent injections or CI. With two exceptions, no complications occurred within 3 h of treatment, despite high FVII:C levels (median: 27.4 U mL⁻¹; range: 19.8–54 U mL⁻¹). Treatment of bleeds with a rFVIIa megadose in young inhibitor patients is effective and well tolerated.     

Inhibited neutrophil functions in patients treated with nifedipine but not with verapamil or diltiazem

Neutrophil functions were studied in patients receiving calcium channel blockers: nifedipine, diltiazem or verapamil. Neutrophils from patients treated with nifedipine showed a significantly lower superoxide generation stimulated by phorbol myristate acetate (PMA) (50 ng mL⁻¹), opsonized zymosan (1 mg mL⁻¹) or formyl-methionyl-leucyl-phenylalanine (FMLP) (10⁻⁷  m ), whereas superoxide generation by neutrophils of patients receiving diltiazem or verapamil showed only a slight and insignificant reduction compared with controls. Similarly, chemotaxis towards 10⁻⁷  m FMLP and phagocytosis were significantly lower in patients receiving nifedipine compared with controls and were only slightly reduced in patients receiving diltiazem or verapamil. Nifedipine was the most efficient drug in inhibiting the rise in intracellular calcium ion concentration ([Ca²⁺]_i) when added in vitro and in neutrophils of patients receiving this drug, whereas verapamil had no significant effect. The correlation between the inhibitory effect of nifedipine on neutrophil function and the elevation of [Ca²⁺]_i suggests that nifedipine inhibits neutrophil functions through its effect on [Ca²⁺]_i. However, it is not the sole mechanism as superoxide generation induced by PMA, an agent that does not induce a rise in [Ca²⁺]_i, is also inhibited. The unique effect of nifedipine in reducing neutrophil functions in vivo suggests its clinical implications concerning response to acute ischaemic myocardial events.     

Tumour-specific induction of immune complexes: DCP-IgM in hepatocellular carcinoma

Background   In the sera of liver, colorectal and prostate cancer patients, several biomarkers may be detected as IgM immune complexes. To determine whether the presence of immune complexes was correlated to an increase of IgMs, we measured the IgM content in the sera of patients with hepatocellular carcinoma (HCC) and cirrhosis, and evaluated the occurrence of des-gamma-carboxy prothrombin (DCP) as immune complexes ( DCP-IgM ) compared to the levels of DCP and alpha-fetoprotein (AFP).
Patients and methods   Serum samples from 31 patients with cirrhosis, 33 untreated HCC patients diagnosed by ultrasound, computed tomography and/or magnetic resonance and confirmed by histopathology, when indicated, and 30 healthy controls were analysed. Concentrations of IgM and DCP-IgM were determined by ELISAs.
Results   Circulating IgM in patients with HCC (median level = 1·79 mg mL⁻¹) and cirrhosis (1·09 mg mL⁻¹) were not significantly different ( P  = 0·1376) while DCP-IgM were significantly higher in HCC patients (median level = 2171·2 AU mL⁻¹) than in those with cirrhosis (1152 AU mL⁻¹, P  = 0·0047). No correlation was found between DCP-IgM and IgM in HCC ( r  = 0·227) and cirrhosis patients ( r  = 0·475). DPC-IgM was positive in 55% (18/33) of HCC patients and in 26% (8/31) of cirrhosis patients compared to 39% and 26% for DCP and 48% and 13% for AFP. DCP-IgM, DCP and AFP tests had 100% specificity in healthy controls.
Conclusions   DCP-IgM in HCC patients was not associated with an increase in IgM concentration. DCP-IgM was more frequently detected in HCC patients than DCP and AFP, strengthening the diagnostic role of IgM immune complexes for liver cancer.     

Vasoactive substances in early dumping syndrome: effects of dumping provocation with and without octreotide

In patients after gastric surgery, early dumping symptoms can be provoked by oral glucose challenge. Octreotide effectively prevents the occurrence of dumping symptoms. We have studied plasma renin activity (PRA), aldosterone and atrial natriuretic peptide (ANP) concentrations in nine patients with early dumping, 10 surgical control subjects and nine healthy control subjects after an oral glucose challenge preceded by either placebo or 25 μg of octreotide subcutaneously (s.c.). In the dumping group, basal PRA was signifi-cantly ( P  < 0.01) higher (3.9 ± 0.6 μg L⁻¹ h⁻¹) than in either surgical or healthy control subjects (1.1 ± 0.3 μg L⁻¹ h⁻¹ and 1.1 ± 0.2 μg L⁻¹ h⁻¹ respectively) and showed a significant rise after glucose ingestion to 5.4 ± 0.9 μg L⁻¹ h⁻¹ that did not occur in control subjects. Aldosterone concentration showed a concomitant rise. In dumping patients, plasma ANP decreased after glucose ingestion from 31 ± 6 ng L⁻¹ to 21 ± 5 ng L⁻¹ ( P  < 0.05). This decrease did not occur in control subjects. Early dumping is associated with an activation of the renin–aldosterone axis and a decrease in plasma ANP, reflecting a hypovolaemic state. Octreotide prevents the occurrence of these changes.     

Effect of vascular injury on inhibition of venous thrombosis with ZK-807834, a direct inhibitor of factor Xa

Summary.  Inhibition of factor Xa with the small molecule inhibitor ZK-807834 (Mr 527 Da, K_i 0.11 nM) attenuates progression of thrombosis, but the ED₅₀ is substantially lower for venous compared with arterial thrombosis in experimental animals. To determine whether this reflects differences in the extent of vascular injury, we compared the dose–response of ZK-807834 for inhibition of venous thrombosis induced with a cotton thread and copper wire device in the presence and absence of balloon catheter-induced injury to the vena cava in rabbits. ZK-807834 administration over 2 h (total dosages of 0.0023–2.3 µmol kg⁻¹, n  = 6/group) resulted in dose-dependent reductions in clot weight compared with vehicle controls, but the ED₅₀ was 0.03 µmol kg⁻¹ for non-injured veins and 0.42 µmol kg⁻¹ for injured veins. We conclude that vascular injury invokes a tissue factor-mediated response that increases the dose requirements for inhibition of venous thrombosis with ZK-807834.     

Lack of effect of norepinephrine on cranial haemodynamics and headache in healthy volunteers

Stress is a provoking factor for both tension-type headache and migraine attacks. In the present single-blind study, we investigated if stress induced by norepinephrine (NE) could elicit delayed headache in 10 healthy subjects and recorded the cranial arterial responses. NE at a dose of 0.025 µg kg⁻¹ min⁻¹ or placebo was infused for 90 min and the headache was followed for 14 h. Blood flow velocity in the middle cerebral artery (measured with transcranial Doppler) and diameters of the temporal artery and the radial artery (measured with ultrasound) were followed for 2 h. There were no changes in these arterial parameters after NE. In both treatment groups three subjects developed delayed headaches. Thus, stress by NE infusion did not result in delayed headache.     

Effect of obesity on serum amiodarone concentration in Japanese patients: population pharmacokinetic investigation by multiple trough screen analysis

Objective:  To evaluate the influence of obesity on pharmacokinetics of amiodarone (AMD) using Non-Linear Mixed Effects Modelling ( nonmem ) in Japanese patients treated with oral therapy.
Method:  Serum concentrations of AMD were determined by high performance liquid chromatography. One hundred and fifty-one trough concentrations from 23 patients receiving repetitive oral AMD were collected. Body mass index (BMI) and body fat percentage were measured.
Results:  Estimates generated using nonmem indicated that the clearance of AMD was influenced by BMI, age and daily dosage of AMD. The final pharmacokinetic model was CL (L/h) = 0·16 · TBW · 0·53^AGE ≥ 65 · 0·78^BMI ≥ 25 · DD^0·51, V _d (L) = 10·2 · TBW, where CL is total body clearance, TBW is total body weight (kg), DD (mg/kg/day) is daily dosage of AMD, AGE (years) ≥65 = 1 for patient was 65 years old or over and 0 otherwise, BMI (kg/m²) ≥25 = 1 for patient was 25 kg/m² or over and 0 otherwise and V _d is apparent volume of distribution. The clearance of AMD decreased significantly by 22·3% with a BMI higher than 25 kg/m². The clearance of AMD also decreased significantly by 46·9% when patient age was more than 65 years.
Conclusion:  Population pharmacokinetic analysis confirms that obesity affects the pharmacokinetics of AMD.     

Elucidation of a novel epitope of a substrate-inducing monoclonal antibody against the serpin PAI-1

Summary.  Plasminogen activator inhibitor-1 (PAI-1) is the most important physiological inhibitor of plasminogen activators. Inhibition of PAI-1 constitutes a putative strategy for the prevention of cardiovascular disease. The monoclonal antibody MA-8H9D4 inhibits PAI-1 activity by inducing a substrate behavior in PAI-1. To identify the epitope, a rational approach was used to design various PAI-1 alanine mutants ( n  = 16) for evaluation of their affinity. PAI-1-R300A, PAI-1-Q303A and PAI-1-D305A had affinities for MA-8H9D4 of < 10⁵ M⁻¹, 2.0 × 10⁸ M⁻¹ and 2.5 × 10⁸ M⁻¹, respectively, whereas the affinity of wtPAI-1 is 3.3 × 10⁹ M⁻¹. The epitope on the axis of arginine 300, glutamine 303 and aspartic acid 305, located on the loop between α-helix I and β-strand 5A, demonstrates that MA-8H9D4 interferes with the final locking step in the serpin/proteinase interaction, thereby explaining its substrate inducing properties. The location of the epitope as well as the proposed mechanism of action is clearly different from that of other substrate inducing monoclonal antibodies against PAI-1. Elucidation of this novel epitope and the previously unidentified molecular mechanism opens new perspectives for the rational development of PAI-1-neutralizing compounds, as well as for the further exploration of synergistic effects between different PAI-1-inhibiting compounds.     

Safety and pharmacokinetics of a recombinant factor VIII with pegylated liposomes in severe hemophilia A

Summary.  Background:  BAY 79-4980 is a sucrose-formulated recombinant factor VIII (rFVIII-FS) combined with pegylated liposomes to prolong activity. Objectives:  To investigate the safety, tolerability, bioavailability, pharmacokinetics and pharmacodynamics of a single administration of BAY 79-4980 compared with standard rFVIII-FS in patients with severe hemophilia A. Methods:  This randomized, double-blind study consisted of two crossover substudies comparing two doses of liposomal rFVIII-FS with standard rFVIII-FS. Males (12–60 years) with severe hemophilia A received a single infusion of standard rFVIII-FS (35 IU kg⁻¹) followed by a single infusion of BAY 79-4980 (13 or 22 mg kg⁻¹ pegylated liposomes) or vice versa , with 12 observation days and a 2-day washout period between treatments. Results:  Twenty-six subjects were enrolled at two centers. No serious adverse events were reported. Transient increases in complement C3a, but not CH50, were seen in subjects receiving both the low- and high-liposome-dose BAY 79-4980. Mild transient elevations of total and low-density lipoprotein cholesterol were observed. There were no clinically significant differences in clotting or laboratory parameters or in pharmacokinetic behavior between BAY 79-4980 and standard rFVIII-FS. The number of subjects with spontaneous bleeds on days 1–14 postinfusion was low, and group comparisons were inconclusive. Conclusions:  Single-dose administration of BAY 79-4980 is well tolerated in patients with severe hemophilia A. Plasma pharmacokinetics of FVIII cannot explain the extended protection from bleeding observed previously with BAY 79-4980. Further studies of efficacy and long-term safety of chronic administration are planned.     

Assay of human erythrocyte sodium-dependent lithium efflux: the importance of timing of blood sampling

The activity of the human erythrocyte sodium–lithium countertransport (SLC) is stable over long periods in individuals. However, it is becoming increasingly evident that the transport system is susceptible to modulation, both acutely and chronically, by various factors. In this study, the authors observed temporal variation in SLC over a period of 10 h (08.00–18.00 hours) in healthy volunteers. SLC V _max was maximum (0.354 ± 0.051 mmol L⁻¹ cell⁻¹ h⁻¹; mean ± SE) at 'mid-day' and significantly higher than in the morning (0.291 ± 0.035 mmol L⁻¹ cell, h; P  < 0.010). Its value in the evening (0.316 ± 0.042 mmol L⁻¹ cell⁻¹ h⁻¹) was lower than at 'mid-day' ( P  < 0.045) but higher than in the morning ( P  < 0.037). These changes were not accompanied by any significant change in the affinity of the transporter for external sodium, K _m. Changes in SLC V _max did not correlate with the corresponding ones in either plasma cortisol or aldosterone. However, they correlated well with those in plasma renin activity, the correlation between mid-day and a.m. sets of values ( r  = 0.718; P  = 0.019) being better than that between mid-day and p.m. ( r  = 0.688; P  = 0.028). The authors conclude that changes in SLC occur during the day, and this need be taken into account in the planning and execution of studies involving determination of the activity of this transport system.