Impact of CytoSorb on kinetics of vancomycin and bivalirudin in critically ill patients.

CytoSorb is a promising tool to treat severe inflammatory status with multiple mechanisms in the acute care setting. Its effect on drugs is, however, poorly documented in vivo, although removal of small molecules might translate into decreased blood levels of life-saving medications. The aim of this study was to assess the impact of CytoSorb on vancomycin and bivalirudin clearance in a large population of critically ill patients. We performed a single-center analysis of CytoSorb treatments performed between January 2018 and March 2019 in critically ill patients admitted to our intensive care unit. A total of 109 CytoSorb treatments were performed in 89 patients. A decrease in lactate dehydrogenase (P = .007), troponin T (P = .022), and creatine phosphokinase (P = .013) was reported during treatment. Vancomycin dose required significant adjustments during treatment (P < .001), but no significant change was necessary after the first 3 days. Similarly, the requirements of bivalirudin significantly changed over days (P < .001), but no dose adjustment was needed after the first 3 days of treatment. No differences in terms of vancomycin and bivalirudin dose need was observed between patients on extracorporeal membrane oxygenation and those who were not (P = .6 and P = .6, respectively), between patients with and without continuous veno-venous hemofiltration (P = .9 and P = .9, respectively), and between CytoSorb responders or not (P = .4 and P = .7, respectively). CytoSorb is effective in mitigating the systemic inflammatory response and safe with respect to vancomycin and bivalirudin administration. These preliminary data further support the use of CytoSorb as adjunct therapy in critically ill patients.

Pharmacokinetic and Pharmacodynamic Modeling and Simulation Analysis of CTB-001, a Recently Developed Generic of Bivalirudin.

PURPOSE: CTB-001, a recently developed generic version of bivalirudin, an FDA-approved anticoagulant used for prophylaxis and treatment of cardiovascular diseases, has shown good efficacy and safety in clinical trials. We characterized the pharmacokinetics (PK) and pharmacodynamics (PD) of CTB-001 by modeling and simulation analysis. METHODS: PK/PD data were collected from a randomized, double-blind, placebo-controlled, single-dose, dose-escalation phase 1 study conducted in 24 healthy Korean male subjects. PK/PD analysis was conducted sequentially by nonlinear mixed-effects modeling implemented in NONMEM®. Monte-Carlo simulations were conducted for PK, activated partial thromboplastin time (aPTT), prothrombin time (PT), and thrombin time (TT). RESULTS: The CTB-101 PK was best described by a three-compartment linear model with a saturable binding peripheral compartment. All PD endpoints showed dose-response relationship, and their changes over time paralleled those of CTB-101 concentrations. A simple maximum effect model best described the aPTT, PT in INR, PT in seconds, and TT, whereas an inhibitory simple maximum effect model best described PT in percentages. The maximum duration of effect of CTB-001 on aPTT prolongation was 52.1 s. CONCLUSIONS: The modeling and simulation analysis well-characterized the PK and PD of CTB-001 in healthy Koreans, which will be valuable for identifying optimal dosing regimens of CBT-001.

Bivalirudin for Pediatric Procedural Anticoagulation: A Narrative Review.

Bivalirudin (Angiomax; The Medicines Company, Parsippany, NJ), a direct thrombin inhibitor, has found increasing utilization as a heparin alternative in the pediatric population, most commonly for the treatment of thrombosis secondary to heparin-induced thrombocytopenia. Due to the relative rarity of heparin-induced thrombocytopenia as well as the lack of Food and Drug Administration-approved indications in this age group, much of what is known regarding the pharmacokinetics and pharmacodynamics of bivalirudin in this population has been extrapolated from adult data. This narrative review will present recommendations regarding the use of bivalirudin for procedural anticoagulation in the pediatric population based on the published literature.

The effects of a hirudin/liposome complex on a diabetic nephropathy rat model.

BACKGROUND: Hirudin, an extract from Hirudo spp., is an anticoagulant used to treat a variety of renal diseases, including diabetic nephropathy (DN). Currently, hirudin has to be used at high dosages to treat DN because it poorly targets the kidneys, although at high dosages it can have severe side effects. Developing a targeted drug delivery system for hirudin, then, could boost its positive therapeutic effects while lowering the risk of side effects. Liposomes have been demonstrated to have significant renal targeting potential, but here we show that a hirudin-loaded liposome is an effective delivery method for patients with DN. METHOD: In this study, we prepared a hirudin/liposome complex and tested its efficacy by injecting it into a rat model. We then compared the renal accumulation of hirudin between complex-injected rat models and rat models that received injections of hirudin alone. We also investigated the mechanisms behind the complex's effects. RESULT: The hirudin/liposome complex increased the accumulation of hirudin in kidney tissues and relieved the renal injury in DN rat models. Moreover, the hirudin/liposome complex down-regulated the expression of TGF-ß1 and VEGF in the kidneys. CONCLUSION: We demonstrated that a hirudin/liposome complex can have a significant positive effect on DN. The mechanism may be that the complex inhibits the expression of VEGF and TGF-ß1.

Monitoring of argatroban and lepirudin anticoagulation in critically ill patients by conventional laboratory parameters and rotational thromboelastometry - a prospectively controlled randomized double-blind clinical trial.

BACKGROUND: Argatroban or lepirudin anticoagulation therapy in patients with heparin induced thrombocytopenia (HIT) or HIT suspect is typically monitored using the activated partial thromboplastin time (aPTT). Although aPTT correlates well with plasma levels of argatroban and lepirudin in healthy volunteers, it might not be the method of choice in critically ill patients. However, in-vivo data is lacking for this patient population. Therefore, we studied in vivo whether ROTEM or global clotting times would provide an alternative for monitoring the anticoagulant intensity effects in critically ill patients. METHODS: This study was part of the double-blind randomized trial "Argatroban versus Lepirudin in critically ill patients (ALicia)", which compared critically ill patients treated with argatroban or lepirudin. Following institutional review board approval and written informed consent, for this sub-study blood of 35 critically ill patients was analysed. Before as well as 12, 24, 48 and 72 h after initiation of argatroban or lepirudin infusion, blood was analysed for aPTT, aPTT ratios, thrombin time (TT), INTEM CT,INTEM CT ratios, EXTEM CT, EXTEM CT ratios and maximum clot firmness (MCF) and correlated with the corresponding plasma concentrations of the direct thrombin inhibitor. RESULTS: To reach a target aPTT of 1.5 to 2 times baseline, median [IQR] plasma concentrations of 0.35 [0.01-1.2] µg/ml argatroban and 0.17 [0.1-0.32] µg/ml lepirudin were required. For both drugs, there was no significant correlation between aPTT and aPTT ratios and plasma concentrations. INTEM CT, INTEM CT ratios, EXTEM CT, EXTEM CT ratios, TT and TT ratios correlated significantly with plasma concentrations of both drugs. Additionally, agreement between argatroban plasma levels and EXTEM CT and EXTEM CT ratios were superior to agreement between argatroban plasma levels and aPTT in the Bland Altman analysis. MCF remained unchanged during therapy with both drugs. CONCLUSION: In critically ill patients, TT and ROTEM parameters may provide better correlation to argatroban and lepirudin plasma concentrations than aPTT. TRIAL REGISTRATION: ClinicalTrials.gov , NCT00798525 , registered on 25 Nov 2008.

Anticoagulation in children: Making the most of little patients and little evidence.

Thrombotic complications are increasing at a steady and significant rate in children resulting in the more widespread use of anticoagulation in this population. Anticoagulant drugs in children can be divided into the standard agents (heparin, low molecular weight heparin, and vitamin K antagonists) and alternative agents (argatroban, bivalirudin, and fondaparinux). This review will compare and contrast the standard and alternative anticoagulants and suggest situations in which it may be appropriate to use argatroban, bivalirudin, and fondaparinux. Clearly, the standard anticoagulants all have significant shortcomings including variable pharmacokinetics, issues with therapeutic drug monitoring, frequency of administration, efficacy, and adverse effects. The alternative anticoagulants have properties which overcome these shortcomings and prospective clinical trial data are presented supporting the current and future use of these agents in place of the standard anticoagulants.

Neuroprotective activity of stiripentol with a possible involvement of voltage-dependent calcium and sodium channels.

A growing body of data has shown that recurrent epileptic seizures may be caused by an excessive release of the excitatory neurotransmitter glutamate in the brain. Glutamatergic overstimulation results in massive neuronal influxes of calcium and sodium through N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainic acid glutamate subtype receptors and also through voltage-gated calcium and sodium channels. These persistent and abnormal sodium and calcium entry points have deleterious consequences (neurotoxicity) for neuronal function. The therapeutic value of an antiepileptic drug would include not only control of seizure activity but also protection of neuronal tissue. The present study examines the in vitro neuroprotective effects of stiripentol, an antiepileptic compound with γ-aminobutyric acidergic properties, on neuronal-astroglial cultures from rat cerebral cortex exposed to oxygen-glucose deprivation (OGD) or to glutamate (40 µM for 20 min), two in vitro models of brain injury. In addition, the affinity of stiripentol for the different glutamate receptor subtypes and the interaction with the cell influx of Na(+) and of Ca(2+) enhanced by veratridine and NMDA, respectively, are assessed. Stiripentol (10-100 µM) included in the culture medium during OGD or with glutamate significantly increased the number of surviving neurons relative to controls. Stiripentol displayed no binding affinity for different subtypes of glutamate receptors (IC50 >100 µM) but significantly blocked the entry of Na(+) and Ca(2+) activated by veratridine and NMDA, respectively. These results suggest that Na(+) and Ca(2+) channels could contribute to the neuroprotective properties of sitiripentol.

Prediction of drug concentration-time profiles of therapeutic proteins in humans from animals.

The main objective of this work is to evaluate two methods to predict concentration-time profiles of therapeutic proteins in humans in a multi-compartment system using animal pharmacokinetic parameters. The prediction of concentration-time profiles in humans in a multi-compartment system was based on two proposed methods (volumes of distribution and pharmacokinetic constants) of Mordenti. Seven protein drugs (5 monoclonal antibodies, one epoetin, and one thrombin inhibitor) from the literature were chosen that were described by two-compartment model in both humans and animals. Two- compartment model parameters (CL, V(c), V(ss), V(ß), α, A, ß and B) at least from 3 animals were scaled to humans and then were used to predict plasma concentrations-time profiles in humans. The results showed that both the methods provided almost similar concentration-time profiles for most of the drugs. It appears that the proposed methods of Mordenti can be used for reasonably accurate prediction of concentration-time profiles of therapeutic proteins in humans.

Development and validation of a highly sensitive LC-MS/MS method for quantitation of bivalirudin in human plasma: application to a human pharmacokinetic study.

A sensitive, specific and simple LC-MS/MS method was developed for the identification and quantification of bivalirudin in human plasma using diazepam as an internal standard (IS). The API-4000 LC-MS/MS was operated under multiple-reaction monitoring mode using electrospray ionization. The sample preparation consisted of an easy protein precipitation sample pretreatment with methanol. Chromatographic separation was achieved on a Zorbax Eclipse plus C18 100 × 2.1 mm column with a mobile phase of water-methanol-0.1% formic acid. The analytes were detected with a triple quadrupole Quantum Access with positive ionization. Ions monitored in the multiple-reaction monitoring mode were m/z 1091 → 650 for bivalirudin (at 2.70 min) and m/z 285 → 193 for diazepam (at 3.85 min). The developed method was validated in human plasma with a lower limit of quantitation of 20 µg/L for bivalirudin. A linear response function was established for the range of concentrations 20-10,000 µg/L (r > 0.998) for bivalirudin. The intra- and inter-day precision values for bivalirudin met the acceptance criteria as per US Food and Drug Administration guidelines. Bivalirudin was stable in the battery of stability studies, viz. bench-top, freeze-thaw cycles and long-term stability. The developed assay method was applied to an intravenous administration study in humans.

[Preparation of freeze-dried powder of recombinate hirudin-2 nanoparticle for nasal delivery and permeability through nasal membrane in vitro].

OBJECTIVE: To optimize the freeze-dried powder preparation technology of recombinate hirudin-2 (rHV2) nanoparticle which has bio-adhesive characteristic for nasal delivery, also to investigate its stability and permeability through nasal membrane in vitro. METHODS: Taking the appearance, rediffusion of nanoparticle and rHV2 encapsulation efficiency as the evaluation indexes. Cryoprotector, the preparative technique and the effect of illumination and high temperature factors on its stability for rHV2 freeze-dried powder were investigated. Using Fraze diffusion cell technique, the permeability of rHV2 across rabbit nasal mucous membrane in chitosan solution, chitosan nanoparticle, and nanoparticle frozen-dried powder were compared with that in normal saline solution. RESULTS: The optimized preparation of rHV2 nanoparticle freeze-dried powder was as follows: 5% trehalose and glucose (1:1) was used as cryoprotector, nanoparticle solution was freezed for 24 h in vacuum frozen-dryer after being pre-freezed for 24 h. The content of rHV2 in the freeze-dried powder was 1.1 ug/mg. Illumination had little effect on the appearance, rediffusion and encapsulation efficiency of the rHV2 freeze-dried powder. High temperature could obviously influence the appearance of nanoparticle freeze-dried powder. The permeability coefficient (P) of nanoparticle was 5 times more than that in chictonson solution. It was indicated that chitosan nanoparticle has effect on increasing the permeability of rHV2. CONCLUSION: The freeze-dried powder of chitosan nanoparticle can be a good nasal preparation of rHV2.

Population pharmacokinetics and pharmacodynamics of bivalirudin in young healthy Chinese volunteers.

AIM: To investigate the population pharmacokinetics (PK) and pharmacodynamics (PD) of bivalirudin, a synthetic bivalent direct thrombin inhibitor, in young healthy Chinese subjects. METHODS: Thirty-six young healthy volunteers were randomly assigned into 4 groups received bivalirudin 0.5 mg/kg, 0.75 mg/kg, and 1.05 mg/kg intravenous bolus, 0.75 mg/kg intravenous bolus followed by 1.75 mg/kg intravenous infusion per hour for 4 h. Blood samples were collected to measure bivalirudin plasma concentration and activated clotting time (ACT). Population PK-PD analysis was performed using the nonlinear mixed-effects model software NONMEM. The final models were validated with bootstrap and prediction-corrected visual predictive check (pcVPC) approaches. RESULTS: The final PK model was a two-compartment model without covariates. The typical PK population values of clearance (CL), apparent distribution volume of the central-compartment (V(1)), inter-compartmental clearance (Q) and apparent distribution volume of the peripheral compartment (V(2)) were 0.323 L·h(-1)·kg(-1), 0.086 L/kg, 0.0957 L·h(-1)·kg(-1), and 0.0554 L/kg, respectively. The inter-individual variabilities of these parameters were 14.8%, 24.2%, fixed to 0% and 15.6%, respectively. The final PK-PD model was a sigmoid E(max) model without the Hill coefficient. In this model, a covariate, red blood cell count (RBC(*)), had a significant effect on the EC(50) value. The typical PD population values of maximum effect (E(max)), EC(50), baseline ACT value (E(0)) and the coefficient of RBC(*) on EC(50) were 318 s, 2.44 mg/L, 134 s and 1.70, respectively. The inter-individual variabilities of E(max), EC(50), and E(0) were 6.80%, 46.4%, and 4.10%, respectively. CONCLUSION: Population PK-PD models of bivalirudin in healthy young Chinese subjects have been developed, which may provide a reference for future use of bivalirudin in China.

[Comparative in vitro study of anticoagulant activity of RA36 DNA aptamer in human, rabbit, and rat blood plasma].

DNA aptamer RA36 with a molecular weight of 10000 is direct-acting anticoagulant whose efficacy is lower than that of recombinant hirudin and unfractionated heparin (UFH) in blood clotting time (BCT), activated blood recalcification time (ABRT), recalcification time (RT), prothrombin time (PT), and activated partial thromboplastin time (APTT) tests. The anticoagulant effect of RA36 is comparable with that of UFH in the thrombin time (TT) test, but is lower than the effect of recombinant hirudin. Analysis of the blood and plasma anticoagulant activity during intravenous bolus administration of aptamer RA36 in rabbits and rats is based on the use ABRT (in blood case) and APTT/RT (in plasma case) tests. The range of doses for evaluation of pharmacodynamic parameters of RA36 during intravenous bolus administration in rabbits and rats is 3 - 34 mg/kg and 1 - 27 mg/kg, respectively. Accordingly, designed dose range for humans is 1 -29 mg/kg.

Pediatric catheterization laboratory anticoagulation with bivalirudin.

BACKGROUND: Pediatric physicians regularly face the problem of uncertain procedural anticoagulation in children, especially in neonates. We sought to evaluate the safety, plasma concentration (pharmacokinetics, PK), pharmacodynamics (PD), and dosing guidelines of bivalirudin when used as a procedural anticoagulant in pediatric percutaneous intravascular procedures. METHODS AND RESULTS: Pediatric subjects undergoing percutaneous intravascular procedures for congenital heart disease were enrolled and received the current weight-based dose used in percutaneous coronary interventions (0.75 mg/kg bolus, 1.75 mg/kg/hr infusion). Blood samples for PK/PD analyses were drawn, and safety was evaluated by monitoring bleeding and thrombosis events. A total of 110 patients (11 neonates, 33 infants, 32 young children, and 34 older children) were enrolled; 106 patients received the protocol dose. The PK/PD response of bivalirudin was predictable and behaved in a manner similar to that in adults. Weight-normalized bivalirudin clearance rates were more rapid in neonates and decreased with increasing age. Bivalirudin concentrations were slightly lower in neonates, with a trend to an increase with age. Activating clotting time response was consistent with adult studies and prolonged in all age groups, and there was reasonable correlation between activating clotting time and bivalirudin plasma concentrations across all age groups. There were few major bleeding (2 of 110, 1.8%) or thrombotic events (9 of 110, 8.2%) reported. CONCLUSIONS: PK/PD response of bivalirudin in the pediatric population is predictable and behaves in a manner similar to that in adults. Using adult dosing, bivalirudin safely provided the expected anticoagulant effect in the pediatric population undergoing intravascular procedures for congenital heart disease.

Pharmacokinetics, pharmacodynamics, tolerability and safety of single doses of bivalirudin in healthy chinese subjects.

The objectives of the present study were to assess pharmacokinetics, pharmacodynamics, tolerability and safety of intravenous administration of bivalirudin, a direct thrombin inhibitor, in healthy Chinese subjects. 48 subjects were equally divided into 4 groups (0.5 mg/kg, 0.75 mg/kg, 1.05 mg/kg intravenous bolus, and 0.75 mg/kg intravenous bolus followed by an infusion of 1.75 mg/kg per hour for 4 h) by a randomized, single-blind and placebo-controlled (bivalirudin groups: n=9/group; placebo groups: n=3/group) design. The safety observations showed that bivalirudin was well tolerated in the studied dose range, all adverse events were mild in severity. The half-life of bivalirudin was approximately 0.57 h (34 min), exposure increased in a dose-dependent manner. In group receiving a 0.75 mg/kg intravenous bolus followed by 1.75 mg/kg per hour infusion for 4 h, bivalirudin concentrations remained at 5000-5500 µg/l within the 4 h infusion period, which was similar to the reported data of Caucasian patients and can provide the desired anticoagulant effects. There was a strong correlation between bivalirudin concentration and anticoagulant effect. A Sigmoid model was used to fit the pharmacodynamic parameters activated clotting time (ACT), activated partial thromboplastin time (APTT) and prothrombin time (PT) and bivalirudin concentrations. The findings of this study suggest that the same dosing regimens of bivalirudin may be administered to Chinese and Caucasian patients. Ongoing and future studies in large populations may add further information.

Thrombosis prophylaxis in critically ill patients.

Incidence of deep vein thrombosis in critically ill patients depends on the underlying disease but may be as high as 60%. The Surviving Sepsis Campaign clearly recommends administering anticoagulation in the absence of specific contraindications in patients with severe sepsis or septic shock. The article discusses risk factor for thromboembolic events in critical illness as well as means of non-pharmacologic and pharmacologic thrombosis prophylaxis. Peripheral vasoconstriction, edema, shock, and administration of catecholamines may reduce the bioavailability and efficacy of subcutaneous administration of low molecular weight heparin. This article further elaborates on the problem and pathophysiology of heparin resistance. Continuous intravenous administration of new anticoagulants may be a promising alternative to indirect anticoagulants. Severity of illness and SAPS II-score determine dosing of the direct thrombin inhibitor argatroban which needs to be about 10-times lower than in patients without critical illness.

A phase I, single and continuous dose administration study on the safety, tolerability, and pharmacokinetics of neorudin, a novel recombinant anticoagulant protein, in healthy subjects.

The aim of this study was to evaluate the tolerability, safety, and pharmacokinetics of single and continuous dose administration of recombinant neorudin (EPR-hirudin, EH) by intravenous administration in healthy subjects, and to provide a safe dosage range for phase II clinical research. Forty-four subjects received EH as a single dose of between 0.2 and 2.0 mg/kg by intravenous bolus and drip infusion. In addition, 18 healthy subjects were randomly divided into three dose groups (0.15, 0.30, and 0.45 mg/kg/h) with 6 subjects in each group for the continuous administration trial. Single or continuous doses of neorudin were generally well tolerated by healthy adult subjects. There were no serious adverse events (SAEs), and all adverse events (AEs) were mild to moderate. Moreover, no subjects withdrew from the trial because of AEs. There were no clinically relevant changes in physical examination results, clinical chemistry, urinalysis, or vital signs. The incidence of adverse events was not significantly related to drug dose or systemic exposure. After single-dose and continuous administration, the serum EH concentration reached its peak at 5 min, and the exposure increased with the increase in the administered dose. The mean half-life (T1/2 ), clearance (Cl), and apparent volume of distribution (Vd) of EH ranged from 1.7 to 2.5 h, 123.9 to 179.7 ml/h/kg, and 402.7 to 615.2 ml/kg, respectively. The demonstrated safety, tolerability, and pharmacokinetic characteristics of EH can be used to guide rational drug dosing and choose therapeutic regimens in subsequent clinical studies. Clinical trial registration: Chinadrugtrials.org identifier: CTR20160444.

Nanoscaled polyion complex micelles for targeted delivery of recombinant hirudin to platelets based on cationic copolymer.

Polyion complex (PIC) micelles based on methoxy poly(ethylene glycol)-grafted-chitosan (mPEG-g-chitosan) and Arg-Gly-Asp conjugated poly(ethylene glycol)-grafted-chitosan (RGD-PEG-g-chitosan) were designed as carriers for platelet-targeted delivery of recombinant hirudin variant-2 (rHV2). The rHV2-loaded plain PIC micelles (mPIC micelles) and RGD conjugated PIC micelles (RGD-PIC micelles) were successfully prepared with mean size of 30.9 +/- 0.5 nm and 41.9 +/- 1.8 nm, and their encapsulation efficiencies were 76.90 +/- 0.84% and 81.08 +/- 0.85%, respectively. The pharmacokinetics experiments showed that the mean retention time (MRT) of rHV2 encapsulated in both kinds of micelles was significantly prolonged, especially for mPIC micelles. The confocal laser scanning microscopy intuitively proved the specific binding of RGD-PIC micelles to platelets. The efficacies of rHV2-loaded RGD-PIC micelles were greatly better than those of rHV2-loaded mPIC micelles and rHV2 solution in aspect of anticoagulation and inhibition of platelet aggregation. These results suggested platelet-targeting specificity of RGD-PIC micelles and that RGD-PIC micelles could potentially be used as a carrier for platelet-targeted delivery and long circulation of rHV2.

Alteration of pharmacokinetics of lepirudin caused by anti-lepirudin antibodies occurring after long-term subcutaneous treatment in a patient with recurrent VTE due to Behcets disease.

The direct thrombin inhibitor lepirudin is mainly applied in heparin-induced thrombocytopenia. We report here the case of a 37-year-old kurdish woman in whom Behcets disease was diagnosed in 1998 when she presented with a Budd Chiari syndrome (BCS) complicated by pulmonary embolism. Recurrent venous thromboembolism (VTE) occurred despite anticoagulant therapy with UFH, LMWH or phenprocoumon and various immunosuppressive therapy regimens. In 2001, when BCS recurred ultimately i.v. lepirudin was administered. When the patient improved and remained clinically stable lepirudin was applied subcutaneously. During long-term treatment with twice-daily 50 mg no further VTE was observed over the following years. Additionally, no bleeding complications occurred. In May 2005 anticoagulant therapy was switched to phenprocoumon. BCS reoccurred when INR values were suboptimal in February 2007, and lepirudin treatment was immediately restarted. After admission the patient received 50 mg b.i.d. lepirudin s.c. with plasma levels in the therapeutic range (0.5-1.0 mg / l). Over the following months, lepirudin levels repeatedly exceeded the upper limit of this range and the dosage was stepwise reduced. Finally, 20 mg b.i.d. were sufficient to obtain therapeutic levels. Renal function was normal, but lepirudin antibodies were present in high titer, as assessed by ELISA. We suppose that these antibodies reduce renal filtration of lepirudin thus leading to increased plasma levels. This case is an example for successful long-term therapeutic-dose anticoagulation with s.c. lepirudin in a patient with Behcets disease and recurrent VTE despite therapeutic anticoagulant therapy with LMWH or vitamin K antagonists. However, frequent measurement of lepirudin plasma levels is needed. If stepwise dose lowering is required over time, the presence of lepirudin antibodies should be considered.

A long-lasting, plasmin-activatable thrombin inhibitor aids clot lysis in vitro and does not promote bleeding in vivo.

The leech protein hirudin is a potent inhibitor of thrombin, but clinical use of recombinant hirudin is restricted by haemorrhagic risks, and complicated by hirudin's rapid clearance from the circulation. We previously employed albumin fusion to slow hirudin variant 3 (HV3) clearance. In this study, we hypothesized that reconfiguration of the chimera, appending human serum albumin (HSA) to the N-terminus of HV3, with an intervening plasmin cleavage site, would create a slowly cleared, plasmin-activatable HV3. Potential plasmin cleavage sites were screened by expression in Escherichia coli, interposed between glutathione sulfotransferase and HV3 domains. The most reactive sequence (GSGIYR-ITY) was recreated in C-terminally His-tagged albumin fusion protein HSACHV3, expressed in Pichia pastoris yeast and purified by nickel-chelate affinity chromatography. HSACHV3 showed no thrombin inhibitory activity in the absence of plasmin, but liberated active HV3 in a time- and concentration-dependent manner in its presence. In a discontinuous clot assay involving clot-bound thrombin, HSACHV3 assisted clot lysis by limiting clot extension in a tPA- and concentration-dependent manner. Similar results were obtained in plasma at higher concentrations of HSACHV3. The chimeric protein exhibited much slower clearance in mice than unfused HV3, and indistinguishable pharmacokinetics from unfused recombinant HSA. In a mouse tail transection bleeding model, doses of HSACHV3 identical to those of HV3 that elicited a four-fold increase in the volume of shed blood were without effect. Our results suggest that HSACHV3 is a fully latent, plasmin activatable, long-lasting hirudin, of potential benefit in thrombotic disorders resistant to natural or pharmacological clot lysis.

Safety, Pharmacokinetics and Pharmacodynamics of TNHH, a Novel Targeted Neutrophil-Inhibitory Hirulog Hybrid Glycoprotein, in Healthy Volunteers.

BACKGROUND: Targeted neutrophil inhibitory-hirulog (TNHH) is a novel hybrid glycoprotein that may be a potential drug candidate for acute ischaemic stroke. OBJECTIVE: The aim of this study was to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of TNHH in healthy volunteers and thereby determine the dose range for future clinical studies. METHODS: This randomized, placebo-controlled study was a single ascending dose design with dose levels of 0.05-1.8 mg/kg (n = 4-6 active, 2 placebos per cohort) in 68 participants. In the TNHH 0.2-1.8 mg/kg and control cohorts, pharmacokinetic and pharmacodynamic blood samples were collected over 168 h after intravenous (IV) administration. TNHH occupancy in peripheral blood neutrophils and blood coagulation were evaluated as the markers of target engagement. RESULTS: Two subjects withdrew from the trial before administration of the study treatment, 66 subjects are included in the data analysis. TNHH was well tolerated in all dose regimens. In total, five mild, self-limiting adverse events (AEs) were observed in 4 of the 66 study subjects. Dose-proportional increases in maximum plasma concentration (Cmax) and area under the curve (AUC0-t) of TNHH were observed. Traces of TNHH were excreted in urine. The elimination half-life (t½) ranged from 0.6 to 1.3 h in the eight groups with ascending dose levels. TNHH combined with CD11b/CD18 quickly achieved > 90% receptor occupancy in groups with doses above 0.2 mg/kg. The Cmax and AUC of binding TNHH with CD11b/CD18 increased with the dose. A significant prolongation with dose was observed on thrombin time (TT), and weak influences were observed on prothrombin time (PT) and activated partial thromboplastin time (APTT). CONCLUSION: TNHH was well-tolerated following IV infusion. The pharmacokinetic and pharmacodynamic characteristics of TNHH indicate that it merits clinical trials. It is recommended that the single dose of TNHH should be 1.0 mg/kg in future studies, and the expected effect may be achieved after 5-7 days of continuous administration. TRIAL REGISTRATION: The study is registered at http://www.chictr.org.cn as ChiCTR-TQR-14004752.