Individual thrombin generation and spontaneous bleeding rate during personalized prophylaxis with Nuwiq® (human-cl rhFVIII) in previously treated patients with severe haemophilia A.

INTRODUCTION: For individuals with haemophilia A, prophylaxis with factor VIII (FVIII) is typically directed towards trough activity >1 IU/dL; however, some patients still experience spontaneous bleeding events (sBEs). AIM: Aims were to evaluate relationships of endogenous thrombin potential (ETP) and FVIII:C with occurrence of clinical bleeding. METHODS: GENA-21 was a prospective, open-label, phase IIIb study investigating the safety and efficacy of Nuwiq® (human-cl rhFVIII) in previously treated adults with severe haemophilia A. The study included a 72-hour pharmacokinetic (PK) evaluation phase and a 6-month personalized prophylaxis phase in which treatment was guided by PK parameters. This subanalysis assessed FVIII:C by one-stage assay and ETP by thrombin generation assay in blood samples. RESULTS: Baseline mean ETP was lower in the 7 patients who experienced sBEs during personalized prophylaxis versus 25 who did not (n = 32 with data from PK phase and prophylaxis phase; P = .0002). During personalized prophylaxis (n = 49), only patients with lower median trough ETP experienced sBEs (8/49 patients; ROC AUC = 0.9421; P < .0001); there was no significant relationship for FVIII:C in predicting sBEs (ROC AUC = 0.5838; P = .4750). Directly following infusion of human-cl rhFVIII, ETP was lower in patients who experienced sBEs versus those who did not (P = .0002), whereas FVIII:C did not differ significantly between these groups. CONCLUSIONS: In adults with severe haemophilia A and reduced thrombin generation, increased frequency of spontaneous bleeding was observed irrespective of trough FVIII levels. Thus, personalized prophylaxis should take into account variables other than FVIII:C. Large prospective trials are needed to verify ETP as a marker for spontaneous bleeding.

Long-term tolerability, immunogenicity and efficacy of Nuwiq® (human-cl rhFVIII) in children with severe haemophilia A.

INTRODUCTION: Nuwiq® (human-cl rhFVIII, simoctocog alfa) is a 4th generation recombinant human FVIII, without chemical modification or fusion with any other protein, produced in a human cell line. AIM/METHODS: This study (GENA-13) was an extension of the GENA-03 study in which previously treated children aged 2-12 years with severe haemophilia A received Nuwiq® prophylaxis for ≥6 months. GENA-13 examined long-term tolerability, immunogenicity and efficacy of Nuwiq® prophylaxis in children. RESULTS: Of 59 patients enrolled in GENA-03, 49 continued Nuwiq® prophylaxis in GENA-13 for a median (range) of 30.0 (9.5-52.0) months. No patient withdrew due to drug-related adverse events or developed inhibitors. Only 2 of 20 518 infusions were associated with possibly related adverse events (dyspnoea, fever). The estimated annualized bleeding rate (ABR) was 0.67 (95% CI: 0.44, 1.02) for spontaneous and 2.88 (95% CI: 1.86, 4.46) for all bleeds. Younger children (2-5 years) had lower ABRs than children aged 6-12 years. Annualized bleeding rates were reduced in GENA-13 vs GENA-03, especially for spontaneous bleeds in younger children (71% reduction; ABR ratio 0.29 [95% CI: 0.11, 0.74]). Nuwiq® efficacy was rated as excellent/good in the treatment of 83.0% of 305 evaluated breakthrough bleeds. Surgical prophylaxis with Nuwiq® was rated as excellent for all 17 assessed procedures. CONCLUSION: Long-term treatment with Nuwiq® for the prevention of bleeds in children with severe haemophilia A was well tolerated, effective and reduced spontaneous bleeding by up to 70% compared with GENA-03.

Immunogenicity, efficacy and safety of Nuwiq® (human-cl rhFVIII) in previously untreated patients with severe haemophilia A-Interim results from the NuProtect Study.

INTRODUCTION: Nuwiq® (Human-cl rhFVIII) is a fourth generation recombinant FVIII, produced in a human cell line, without chemical modification or protein fusion. No inhibitors developed in studies with Nuwiq® in 201 previously treated patients with haemophilia A (HA). The immunogenicity, efficacy and safety of Nuwiq® in previously untreated patients (PUPs) with severe HA are being assessed in the ongoing NuProtect study. METHODS: The study, conducted across 38 centres worldwide, is evaluating 110 true PUPs of all ages and ethnicities enrolled for study up to 100 exposure days (EDs) or 5 years maximum. The primary objective is to assess the immunogenicity of Nuwiq® (inhibitor activity ≥0.6 BU) using the Nijmegen-modified Bethesda assay at a central laboratory. RESULTS: Data for 66 PUPs with ≥20 EDs from a preplanned interim analysis were analysed. High-titre (HT) inhibitors developed in 8 of 66 patients after a median of 11.5 EDs (range 6-24). Five patients developed low-titre inhibitors (4 transient). The cumulative incidence (95% confidence interval) was 12.8% (4.5%, 21.2%) for HT inhibitors and 20.8% (10.7%, 31.0%) for all inhibitors. During inhibitor-free periods, median annualized bleeding rates during prophylaxis were 0 for spontaneous bleeds and 2.40 for all bleeds. Efficacy was rated as "excellent" or "good" in treating 91.8% of bleeds. Efficacy of surgical prophylaxis was "excellent" or "good" for 8 (89%) procedures and "moderate" for 1 (11%). No tolerability concerns were evident. CONCLUSION: These interim data show a cumulative incidence of 12.8% for HT inhibitors and convincing efficacy and tolerability in PUPs treated with Nuwiq® .

Pharmacokinetics, clot strength and safety of a new fibrinogen concentrate: randomized comparison with active control in congenital fibrinogen deficiency.

Essentials Congenital afibrinogenemia causes a potentially life-threatening bleeding and clotting tendency. Two human fibrinogen concentrates (HFCs) were compared in a randomized pharmacokinetic study. Bioequivalence was not shown for AUCnorm , which was significantly larger for the new HFC. Increases in clot strength were comparable, and no thromboses or deaths occurred in the study. SUMMARY: Background Human fibrinogen concentrate (HFC) corrects fibrinogen deficiency in congenital a-/hypofibrinogenemia. Objectives To assess pharmacokinetics (PK), effects on thromboelastometry maximum clot firmness (MCF), and safety of a new double virus-inactivated/eliminated, highly purified HFC vs. active control. Patients/Methods In this multinational, randomized, phase II, open-label, crossover study in 22 congenital afibrinogenemia patients aged ≥ 12 years, 70 mg kg-1 of new HFC (FIBRYGA, Octapharma AG) or control (Haemocomplettan® P/RiaSTAP™, CSL Behring GmbH) were administered, followed by crossover to the other concentrate. Fibrinogen activity, PK and MCF in plasma were assessed. Results The concentrates were not bioequivalent for the primary endpoint, AUCnorm (mean ratio, 1.196; 90% confidence interval [CI], 1.117, 1.281). Remaining PK parameters (Cmaxnorm , IVR, t1/2 , MRT) reflected bioequivalence between concentrates, except for clearance (mean ratio, 0.836; 90% CI, 0.781, 0.895) and Vss (mean ratio, 0.886; 90% CI, 0.791, 0.994). Mean AUCnorm was significantly larger for the new HFC (1.62 ± 0.45 vs. 1.38 ± 0.47 h kg g L-1  mg-1 , P = 0.0001) and mean clearance was significantly slower (0.665 ± 0.197 vs. 0.804 ± 0.255 mL h-1  kg-1 , P = 0.0002). Mean MCF increased from 0 mm to 9.68 mm (new HFC) and 10.00 mm (control) 1-hour post-infusion (mean difference, -0.32 mm; 95% CI, -1.70, 1.07, n.s.). No deaths, thromboses, viral seroconversions or serious related adverse events occurred. Conclusions Bioequivalence was not demonstrated for AUCnorm , clearance and Vss . Larger AUCnorm and slower clearance were observed for the new HFC. Remaining pharmacokinetic parameters reflected bioequivalence to control. Safety profiles and increases in clot strength were comparable between concentrates.

Efficacy and safety of Nuwiq® (human-cl rhFVIII) in patients with severe haemophilia A undergoing surgical procedures.

INTRODUCTION: Haemophilia A patients are at a high risk of excess bleeding during surgeries. The aim of haemostatic therapy during the perioperative period is to normalize FVIII level perioperatively and postoperatively to maintain normal haemostasis until wound healing is complete. AIMS/METHODS: To examine the efficacy of Nuwiq® (simoctocog alfa, human-cl rhFVIII), a 4th generation recombinant FVIII produced in a human cell line, for surgical prophylaxis in patients with severe haemophilia A. This analysis assessed the efficacy of Nuwiq® during surgical procedures and in the postoperative period in seven clinical studies of previously treated patients (PTPs) with severe haemophilia A. RESULTS: Thirty-six patients, aged 3-55 years, received surgical prophylaxis with Nuwiq® for 60 surgeries (28 major and 32 minor). Efficacy was evaluated for 52 surgeries (25 major and 27 minor). The success rate of Nuwiq® treatment was 98.1% (51 of 52 evaluated surgeries); haemostatic efficacy was assessed as "excellent" or "good" in all but one major surgery (assessed as "moderate"). The number of infusions ranged from 1 to 19 for minor surgeries and from 3 to 76 for major surgeries. The median (range) daily doses were 42.0 (28.2-100.9) IU kg-1 for minor surgeries and 69.3 (43.3-135.6) IU kg-1 for major surgeries. There were no serious treatment-related adverse events, and none of the patients developed FVIII inhibitors. CONCLUSIONS: The results of this pooled analysis show that Nuwiq® was efficacious in maintaining haemostasis during and after major and minor surgical procedures in PTPs with severe haemophilia A.

PK-guided personalized prophylaxis with Nuwiq® (human-cl rhFVIII) in adults with severe haemophilia A.

INTRODUCTION: Nuwiq® (human-cl rhFVIII) is a 4th generation recombinant human FVIII, without chemical modification or protein fusion, produced in a human cell-line. AIMS/METHODS: This study (NuPreviq) was a prospective, open-label, multicentre, phase IIIb study of the efficacy and safety of personalized prophylaxis with Nuwiq® in 66 previously treated adults with severe haemophilia A. NuPreviq had three phases: (i) a 72-h pharmacokinetic (PK) phase; (ii) a 1-3 month standard prophylaxis phase; and (iii) a 6-month personalized prophylaxis phase. The personalized prophylaxis regimen was based on individual PK modelling for each patient according to whether their PK profile most closely fitted a two- or one-compartment model (NuPreviq approach). In cases of uncertainty, a noncompartment model was applied. RESULTS: The median dosing interval during personalized prophylaxis was 3.5 days, with 57% of patients on ≤2 weekly dosing. Mean annualized bleeding rates during personalized prophylaxis were 1.45 (median [interquartile range, IQR]: 0 [0, 1.9]) for all bleeds, 0.79 (median [IQR]: 0 [0, 0]) for spontaneous bleeds, and 0.91 (median [IQR]: 0 [0, 0]) for joint bleeds. During personalized prophylaxis, 83.1% of patients were spontaneous bleed-free. Compared with standard prophylaxis, median weekly prophylaxis dose was reduced by 7.2% from 100.0 to 92.8 IU kg-1 during the last 2 months of personalized prophylaxis. There were no FVIII inhibitors or treatment-related serious or severe adverse events. CONCLUSION: PK-guided personalized prophylaxis with Nuwiq® provided bleeding protection and enabled the dosing interval to be extended to twice weekly or less in many patients and an overall dose reduction.

Prophylaxis vs. on-demand treatment with Nuwiq(®) (Human-cl rhFVIII) in adults with severe haemophilia A.

INTRODUCTION: Haemophilia A is treated with FVIII, either prophylactically or on demand. Prophylaxis is the gold standard in children and evidence is accumulating in adults. AIMS/METHODS: The aim of this analysis was to compare prophylaxis vs. on-demand treatment with Nuwiq(®) (Human-cl rhFVIII), a new-generation rFVIII expressed in a human cell line, in previously treated patients (PTPs) with severe haemophilia A. Data were analysed from two similarly designed, multinational, prospective, open-label studies with similar inclusion and exclusion criteria and comparable patient demographics. Human-cl rhFVIII was administered either prophylactically in a study of 32 adults or on-demand in a study of 22 patients (20 adults and two adolescents). RESULTS: Patients treated prophylactically experienced 36 bleeds compared with 997 bleeds in patients treated on-demand (mean observation periods: 180 and 335 days respectively). Based on a negative binomial regression model, annualized bleeding rate (ABR) during prophylaxis was 2.30 (95% CI: 1.54, 3.44) compared with 57.74 (95% CI: 43.36, 76.91) during on-demand treatment, which equates to a 96% lower ABR during prophylaxis. 'Excellent' or 'good' efficacy in the treatment of bleeds was achieved with Human-cl rhFVIII in 100% of 28 evaluated bleeds during the prophylaxis study and 94.5% of 985 evaluated bleeds during the on-demand study. No inhibitors, treatment-related serious adverse events or severe adverse events were recorded during prophylaxis or or-demand treatment. CONCLUSIONS: Prophylaxis with Human-cl rhFVIII reduces recurrent bleeding in adult PTPs with severe haemophilia A and adds further supportive evidence for the benefits of prophylaxis in adults.

Novel, human cell line-derived recombinant factor VIII (human-cl rhFVIII; Nuwiq® ) in adults with severe haemophilia A: efficacy and safety.

INTRODUCTION: Nuwiq® [human cell line-derived recombinant factor VIII (human-cl rhFVIII)] is a new generation rFVIII protein, without chemical modification or fusion to any other protein, produced in a human cell line. AIM/METHODS: This prospective, open-label, multinational phase III study assessed the efficacy and safety of human-cl rhFVIII in 32 adult previously treated patients (PTPs) with severe haemophilia A during standard prophylaxis for ≥6 months and ≥50 exposure days. Efficacy in treating bleeds and during surgical prophylaxis was also assessed. RESULTS: Prophylactic efficacy, based on mean monthly bleeding rate, was rated as 'excellent' or 'good' in 97% of patients for all bleeds and in 100% of patients for spontaneous bleeds. Mean (SD) annualized bleeding rate was 2.28 (3.73) [median = 0.9] for all bleeds, 1.16 (2.57) [median = 0] for spontaneous bleeds and 1.00 (1.79) [median = 0] for traumatic bleeds. There were no bleeds in 50% of patients and there were no major, life-threatening bleeds. Efficacy was 'excellent' or 'good' in treating 28 (100%) of 28 bleeds. Overall efficacy was rated as 'excellent' during four surgical procedures (three major, one minor) and 'moderate' during one major surgery. Incremental in vivo recovery (IVR) data were comparable with the one-stage and chromogenic assays. IVR was >2.0% per IU kg-1 for all measurements and stable over 6 months. No patients developed FVIII inhibitors and there were no treatment-related serious or severe adverse events. CONCLUSION: These results in adult PTPs indicate that human-cl rhFVIII is effective for the prevention and treatment of bleeds in adults with severe haemophilia A.

First prospective report on immune tolerance in poor risk haemophilia A inhibitor patients with a single factor VIII/von Willebrand factor concentrate in an observational immune tolerance induction study.

INTRODUCTION/BACKGROUND: Development of neutralizing inhibitors against factor VIII (FVIII) is a major complication of haemophilia A treatment. AIM: The ongoing, international, open-label, uncontrolled, observational immune tolerance induction (ObsITI) study evaluates ITI, the standard of care in patients with inhibitors. PATIENTS/METHODS: Forty-eight prospective patients in this interim analysis received a single plasma-derived, von Willebrand factor-stabilized, FVIII concentrate (pdFVIII/VWF) for ITI. According to recommended Bonn protocol, 'low responders' at ITI start (<5 BU) received 50-100 IU FVIII kg(-1) daily, or every other day; 'high responders' (≥5 BU) received 100 IU FVIII kg(-1) every 12 h. RESULTS: Forty of 48 patients (83.3%), had at least one risk factor for poor ITI-prognosis at ITI start (i.e. age ≥7 years, >2 years since inhibitor diagnosis, inhibitor titre ≥10 BU at the start of ITI, or prior ITI failure). Nonetheless, 34 patients (70.8%) achieved complete success, 3 (6.3%) partial success, 1 (2.1%) partial response; ITI failed in 10 patients (20.8%), all with poor prognosis factors. All six low responders achieved complete success. ITI outcome was significantly associated with inhibitor titre level at ITI start (P = 0.0068), number of poor prognosis factors for ITI success (P = 0.0187), monthly bleeding rate during ITI (P = 0.0005) and peak inhibitor titre during ITI (P = 0.0007). Twenty-two of 35 high responder patients (62.9%) with ≥1 poor prognosis factor achieved complete success. CONCLUSION: Treatment with a single pdFVIII/VWF concentrate, mainly according to the Bonn protocol, resulted in a high ITI success rate in haemophilia A patients with inhibitors and poor prognosis for ITI success.

Novel, human cell line-derived recombinant factor VIII (Human-cl rhFVIII, Nuwiq® ) in children with severe haemophilia A: efficacy, safety and pharmacokinetics.

INTRODUCTION: Nuwiq® (Human-cl rhFVIII) is a new-generation recombinant factor VIII (rFVIII) protein, without chemical modification or fusion to any other protein, produced in a human cell line. AIM/METHODS: This prospective, open-label, multinational phase III study assessed the efficacy and safety of Human-cl rhFVIII in 59 previously treated patients (PTPs) with severe haemophilia A aged 2-12 years (2-5 [N = 29]; 6-12 [N = 30]) during standard prophylaxis (≥50 exposure days and ≥6 months). Efficacy in treating breakthrough bleeds and during surgical prophylaxis was also assessed. RESULTS: An initial pharmacokinetic assessment (N = 13 per age subgroup) demonstrated comparable results with the one-stage and chromogenic assays. Mean (SD) half-life was 11.9 (5.4) and 13.1 (2.6) hours in children aged 2-5 years and 6-12 years respectively (one-stage assay). Prophylactic efficacy, based on mean monthly bleeding rate, was 'excellent' or 'good' in 91.5% of children for all bleeds and in 96.6% of children for spontaneous bleeds. Mean (SD) annualized bleeding rate was 4.12 (5.22) [median 1.9] for all bleeds, 1.50 (3.32) [median 0] for spontaneous bleeds and 2.34 (3.54) [median 1.57] for traumatic bleeds. There were no major, life-threatening bleeds. Efficacy was 'excellent' or 'good' in the treatment of 82.4% of breakthrough bleeds. Overall efficacy during five major surgeries was rated as 'excellent'. There were no FVIII inhibitors or treatment-related serious adverse events. CONCLUSION: These results in paediatric PTPs indicate that Human-cl rhFVIII is effective for the prevention and treatment of bleeds.

The first recombinant FVIII produced in human cells--an update on its clinical development programme.

The development of inhibitors and the need for frequent venous access for FVIII injection are major challenges in current haemophilia treatment. Presently available recombinant FVIII (rFVIII) products produced in hamster cell lines are associated with inhibitor formation in up to 32% of previously untreated patients. The new human cell line-derived recombinant human FVIII (Human-cl rhFVIII) protein is the first native, unmodified truly human rFVIII product produced in a human cell line without additive animal proteins. The aim of using a human cell line for the production of rFVIII is the avoidance of non-human epitopes on rFVIII, thereby potentially reducing the rate of inhibitor development, avoiding allergic reactions and allowing personalized prophylaxis with the chance of fewer infusions. Studies to date show that prophylaxis with Human-cl rhFVIII prevents 96% of bleeding events in adults with severe haemophilia A when compared to on-demand treatment. Available pharmacokinetic data with a mean half-life of 17.1 h allow personalized prophylaxis with the chance of fewer infusions. Studies in previously treated children and adults indicate that Human-cl rhFVIII is efficacious and safe in the prevention and treatment of bleeding episodes and that none of the treated patients developed inhibitors or allergic reactions thus far.

Pharmacokinetics and safety of fibrinogen concentrate.

BACKGROUND: Although fibrinogen concentrate has been available for the treatment of congenital fibrinogen deficiency for years, knowledge of its pharmacokinetics comes from only two small studies. OBJECTIVES: To assess the pharmacokinetic (PK) profile, clot integrity and safety of fibrinogen concentrate (human) (FCH) in patients with afibrinogenemia. PATIENTS AND METHODS: A multinational, prospective, open-label, uncontrolled study of patients with afibrinogenemia > or = 6 years of age was conducted in the USA and Italy. Plasma was collected before and after infusion for PK analyses and evaluation by rotational thromboelastometry of maximum clot firmness (MCF) to assess clot integrity. Safety was assessed on the basis of adverse events and laboratory parameters. RESULTS: After a single dose of 70 mg kg(-1) body weight (b.w.) FCH in 14 patients, median incremental in vivo recovery was a 1.7 mg dL(-1) increase per mg kg(-1) b.w., and median levels were 1.3 g L(-1) for fibrinogen activity and antigen 1 h after infusion. Median half-life (t(1/2)) was 77.1 h for fibrinogen activity and 88.0 h for antigen. Plasma recovery in children < 16 years old was similar to that in adults aged 16 to < 65 years, but the t(1/2) and area under the curve were decreased, with an increased steady-state volume and clearance. MCF increased by a mean of 8.9 mm from baseline to 1 h after infusion of FCH (P < 0.0001). All four adverse events reported were mild, and none was serious or related to study drug. CONCLUSIONS: These PK findings confirm a rapid increase in plasma fibrinogen levels after infusion with FCH. Together with the clot integrity and safety data and published data on efficacy, the results support the idea that FCH substitution can restore hemostasis with a good safety profile.

Prothrombin complex concentrate (Beriplex P/N) for emergency anticoagulation reversal: a prospective multinational clinical trial.

BACKGROUND: Prothrombin complex concentrate (PCC) can substantially shorten the time needed to reverse antivitamin K oral anticoagulant therapy (OAT). OBJECTIVES. To determine the effectiveness and safety of emergency OAT reversal by a balanced pasteurized nanofiltered PCC (Beriplex P/N) containing coagulation factors II, VII, IX, and X, and anticoagulant proteins C and S. PATIENTS AND METHODS: Patients receiving OAT were eligible for this prospective multinational study if their International Normalized Ratio (INR) exceeded 2 and they required either an emergency surgical or urgent invasive diagnostic intervention or INR normalization due to acute bleeding. Stratified 25, 35, or 50 IU kg(-1) PCC doses were infused based on initial INR. Study endpoints included INR normalization (

Caring for the critically ill patient. High-dose antithrombin III in severe sepsis: a randomized controlled trial.

CONTEXT: Activation of the coagulation system and depletion of endogenous anticoagulants are frequently found in patients with severe sepsis and septic shock. Diffuse microthrombus formation may induce organ dysfunction and lead to excess mortality in septic shock. Antithrombin III may provide protection from multiorgan failure and improve survival in severely ill patients. OBJECTIVE: To determine if high-dose antithrombin III (administered within 6 hours of onset) would provide a survival advantage in patients with severe sepsis and septic shock. DESIGN AND SETTING: Double-blind, placebo-controlled, multicenter phase 3 clinical trial in patients with severe sepsis (the KyberSept Trial) was conducted from March 1997 through January 2000. PATIENTS: A total of 2314 adult patients were randomized into 2 equal groups of 1157 to receive either intravenous antithrombin III (30 000 IU in total over 4 days) or a placebo (1% human albumin). MAIN OUTCOME MEASURE: All-cause mortality 28 days after initiation of study medication. RESULTS: Overall mortality at 28 days in the antithrombin III treatment group was 38.9% vs 38.7% in the placebo group (P =.94). Secondary end points, including mortality at 56 and 90 days and survival time in the intensive care unit, did not differ between the antithrombin III and placebo groups. In the subgroup of patients who did not receive concomitant heparin during the 4-day treatment phase (n = 698), the 28-day mortality was nonsignificantly lower in the antithrombin III group (37.8%) than in the placebo group (43.6%) (P =.08). This trend became significant after 90 days (n = 686; 44.9% for antithrombin III group vs 52.5% for placebo group; P =.03). In patients receiving antithrombin III and concomitant heparin, a significantly increased bleeding incidence was observed (23.8% for antithrombin III group vs 13.5% for placebo group; P<.001). CONCLUSIONS: High-dose antithrombin III therapy had no effect on 28-day all-cause mortality in adult patients with severe sepsis and septic shock when administered within 6 hours after the onset. High-dose antithrombin III was associated with an increased risk of hemorrhage when administered with heparin. There was some evidence to suggest a treatment benefit of antithrombin III in the subgroup of patients not receiving concomitant heparin.

Antithrombin III in patients with severe sepsis: a pharmacokinetic study.

OBJECTIVES: To evaluate the safety, pharmacokinetics, and the practicability of two different antithrombin III (AT III) high-dose regimens in patients with severe sepsis. DESIGN: Prospective, open, randomized, 2 parallel groups, multinational clinical trial. SETTING: Eleven academic medical center intensive care units (ICU) in Austria, Belgium, Denmark, Germany, Norway and Sweden. PATIENTS: Thirty-three patients with severe sepsis who received standard supportive care and antimicrobial therapy, in addition to the administration of AT III. INTERVENTIONS: Patients received an intravenous loading dose of 6,000 IU AT III followed by either intermittent bolus infusions of 1,000 IU AT III every 4 h or a continuous infusion of 250 IU AT III/h for 4 days, resulting in a total dose for both dosage regimens of 30,000 IU AT III. MEASUREMENTS: All patients were evaluated for safety and all but one for pharmacokinetics. RESULTS AND CONCLUSIONS: The administration of AT III was safe and well tolerated. The overall 28-day all-cause mortality was 30% (43% intermittent bolus infusions; 21% continuous infusion). The mean probability of dying according to the SAPS II was 48%. The difference in mortality between both groups was within the range of chance. AT III plasma levels were elevated from low baseline levels to above 120% soon after onset of AT III therapy and remained at these levels for the treatment phase of 4 days. Functional and immunologic levels of AT III corresponded very well. With an overall median volume of distribution of 4.5 l (range: 2.4-6.5 l), AT III only moderately extended beyond plasma. The overall median elimination half-life was 18.6 h (range: 5.1-37.4). Overall, median response was 1.75% per IU/kg (range: 1.14-2.8). The variability of elimination parameters was quite noteworthy (CV = 41-59%), whereas distribution-related parameters showed a moderate variability (CV = 24%). In spite of this variability, both high-dose IV regimens reliably provided AT III levels above 120% for all but one patient. An increased mortality was observed for patients with a distribution volume exceeding 4.5 l (or a response < 1.7% per IU/kg). AT III distribution volumes above 4.5 l might indicate a capillary leak phenomenon. The continuous infusion regimen was slightly preferred by the investigators with regard to practicability.

Automated analytical systems for drug development studies. Part IV. A microdialysis system to study the partitioning of lomefloxacin across an erythrocyte membrane in vitro.

An automated system utilizing microdialysis sampling, intermittent dosing, and liquid chromatographic analysis was assembled in order to study the partitioning of lomefloxacin, a fluoroquinolone antimicrobial, into human erythrocytes in vitro. The apparent erythrocyte:buffer partition coefficient was found to be approximately 2.0 with this system and by a manual method. The value was concentration-dependent; lower partition coefficients were observed at lomefloxacin concentrations less than 1 microgram ml-1. At all concentrations, values obtained by microdialysis were statistically indistinguishable from those obtained by a conventional manual method. The results indicate that erythrocyte partition coefficients can be measured successfully with the microdialysis system. Furthermore, microdialysis sampling eliminates the tedious methodology associated with traditional erythrocyte partitioning studies, including sample clean-up. Due to automated intermittent dosing and on-line LC analysis, the system operates unattended.

A 19F NMR study of lomefloxacin in human erythrocytes and its interaction with hemoglobin.

19F NMR spectroscopy of a model fluoroquinolone, lomefloxacin, in an erythrocyte suspension showed separate resonances for the intra- and extra-cellular compartments. The intra-cellular peak revealed significant line broadening of the fluorine signals of lomefloxacin. Line broadening also occurred in the presence of oxyhemoglobin (HbO2), hematin, globin and iron. This evidence indicated that lomefloxacin interacted with these compounds; however, ultrafiltration experiments indicated that there was only weak binding (5%) of lomefloxacin to HbO2. 19F and 31P NMR spectroscopy revealed that lomefloxacin may compete with 2,3-diphosphoglycerate for its binding site on HbO2. An apparent partition coefficient of 1.90 +/- 0.15 was observed for lomefloxacin in human erythrocytes, utilizing LC analysis.

Localization of platelet-derived growth factor receptor subunit expression in chronic venous leg ulcers.

Cellular responses to platelet-derived growth factor, which affects all phases of the wound healing process, are dependent on the interaction of the growth factor with its cell surface receptors. Recently, we have shown that the platelet-derived growth factor-receptor was not expressed in uninjured human skin. In acute human wounds healing by secondary intention, both platelet-derived growth factor-receptor subunits were coordinately expressed, whereas no expression was found after reepithelialization at day 47. Even though impaired wound healing may be due to uncoordinated expression or the failure to express platelet-derived growth factor-receptor subunits, little is known regarding their expression in chronic ulcers. We studied the localization of platelet-derived growth factor-receptor expression in chronic venous leg ulcers of 15 patients with a median age of 73 years. Cryostat sections of biopsy specimens were immunostained with the use of antibodies against the alpha- and the beta-platelet-derived growth factor subunits. RNA was extracted from biopsy specimens and subjected to Northern blot analysis with the use of oligolabeled complementary DNA for the platelet-derived growth factor-receptor. Platelet-derived growth factor-receptor alpha- and beta-subunit expression was found in fibroblast-like cells within the wound bed and in cells beneath the epidermis of the wound edge. Platelet-derived growth factor-receptor beta-subunit expression was detected in endothelial cells of the vessels, in the granulation tissue, and the wound edge, whereas platelet-derived growth factor-receptor alpha-subunit was not expressed in endothelial cells of the uninjured skin. This finding suggests that the platelet-derived growth factor alpha-subunit may be involved in vessel formation during tissue repair. Both platelet-derived growth factor-receptor subunits were expressed at the messenger RNA level indicating that the synthesis is at least partly regulated at a pretranslational level. As the cellular responsiveness to growth factors depends on their specific receptors, our finding that both platelet-derived growth factor-receptor subunits are expressed in chronic venous ulcers substantiates the concept of therapeutic trials with recombinant platelet-derived growth factor.

The effect of protein binding on ivermectin uptake by bovine brain microvessel endothelial cells.

The effect of albumin binding on ivermectin uptake and transfer across the endothelial component of the blood-brain barrier (BBB) was determined with an in vitro model comprised of bovine brain microvessel endothelial cell (BMEC) monolayers. Cellular uptake of ivermectin was limited in the absence of albumin and 90% inhibited in the presence of 10% albumin. Cell membrane association of ivermectin, as followed by fluorescent probe labelling, was observed only at high (micromolar) concentrations of the drug. Membrane association was about 75% inhibited in the presence of albumin. Similarly, transfer across BMEC monolayers was restricted, equivalent to that of BBB impermeant markers. Unlike the uptake studies, however, albumin had little effect on the transfer of ivermectin across BMEC monolayers. These results support recent in vivo findings on the distribution of ivermectin into the brain and suggest that ivermectin has only a limited affinity for the endothelial component of the normal BBB.