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.

[Determination of recombinant hirudin in urine of rat by spectrophotometric method].

To develop a spectrophotometric method for determining the concentration of recombinant hirudin (rH) in urine of rats. rH concentration was determined based on the rH inhibility to thrombin which hydrolyzed the Chromozym TH TH chromogenic substrate to form the specific pNA absorbed at 405 nm. The standard rH in rat urine was determined by the spectrophotometric method at concentration of 6.25 to 75 ng x mL(-1) with day and intra-day RSD < 10%, method recoveries of > 95% and the dilution recoveries of > 93%. The rH samples of rat urines which iv dose of 0.5, 1.0, and 2.0 mg x kg(-1) were collected and analyzed by the CSA method. Their cumulative excretion rH at 0-12 hr were (116.850 +/- 57.160), (235.544 +/- 39.375) and (474.986 +/- 85.426) microg x kg(-1). The calculated cumulative excretion rate of three doses is about 23% which indicates that the rH was eliminated in the way of a linear kinetics in rats. The rH content in rat urine could be measured by the spectrophotometric method accurately, reliably and sensitively for the rH urinary excretion dynamics study.

Online preconcentration of recombinant Arg-Gly-Asp-hirudin using dynamic pH junction for analysis in human urine samples by capillary electrophoresis-mass spectrometry.

A sensitive and effort-saving method was established and validated for the quantitative determination of recombinant Arg-Gly-Asp-hirudin (rRGD-hirudin) in human urine samples. The assay was performed on a uncoated fused silica capillary of 70 cm x 50 microm I.D. and a positive voltage of 30 kV was applied. The sample was injected under pressure of 50 mbar for 300 s and the temperature of capillary was kept 25 degrees C. Sheath liquid consisting of 30% methanol and 70% of 0.1% formic acid aqueous solution flowing at 7 microL/min was supplied to the CE-electrospray interface. Utilizing the dynamic pH junction technique, a lower limit of quantitation of approximately 35 nM was achieved (concentration coefficiency was about 100-fold) without complex sample preprocessing procedure. CE-MS conditions and parameters were also optimized to obtain better performance. The method has been successfully applied in clinical research of rRGD-hirudin.

Pharmacokinetics and pharmacology of hirulog-like peptide.

Hirulog-like peptide (HLP), a new thrombin peptide inhibitor, effectively reduces neointimal formation or restenosis in rat and rabbit vascular injury models. The present study investigated the pharmacokinetics and pharmacology of HLP in Sprague-Dawley (SD) rats. The input response of HLP in rats was studied using radioisotopic tracing method. Male SD rats were intravenously injected with a single dose of I-HLP (3.2, 6.4, or 12.8 mg/kg) for pharmacokinetic analysis. The concentration-time curve of I-HLP following bolus injection fitted a 3-compartment model. The half-life of HLP in rats was between 25 and 31 min following 3.2 to 12.8 mg/kg of bolus injection. Radioactivity of I-HLP was detected in all tested tissues and was most abundant in kidneys or stomachs. Blood pressure, respiratory frequency, and heart rates were not significantly altered during continuous intravenous infusion with saline or 1.6 to 3.2 mg/kg/h of HLP for 4 h. Bleeding time and activated partial thromboplastin time were significantly prolonged in rats infused with HLP compared to vehicle. ADP-induced platelet aggregation was significantly reduced in the HLP-treated groups compared with controls. The results suggest that HLP possesses first-order kinetic characteristics. HLP is secreted mainly through kidneys. Beside its anticoagulant activity, no other adverse effect was detected in SD rats receiving HLP.

Characterization of the postglomerular renal metabolism of lepirudin in healthy volunteers.

INTRODUCTION: The anticoagulant r-hirudin lepirudin is eliminated exclusively via the kidneys. We examined the C-terminal amino acid degradation of lepirudin by the proximal kidney tubulus cells in humans as well as the antithrombotic efficacy of the metabolites and quantified the metabolite portions. MATERIALS AND METHODS: In vitro metabolites of lepirudin were produced by adding 250 microg lepirudin to urine of three healthy volunteers and a concentration of 100 ml fresh urine to 1.5 ml and subsequent separation by high performance liquid chromatography. Anticoagulant activities of the mass spectrometrically identified metabolites were measured by ecarin clotting time and protein determination with bicinchoninic acid. In 10 healthy volunteers 1 mg lepirudin was administered intravenously, urine was collected during the following 2 h. The urine amount containing 50 microg lepirudin measured by ecarin clotting time was enzyme-inactivated and measured analogously to the in vitro samples. RESULTS: The in vitro generated metabolites were shortened amino acid by amino acid at the C-terminal end, up to five amino acids. Their anticoagulant activity was reduced to 92.6% (M64), 80.1% (M63) and 74.4% (M60,61,62) in comparison to lepirudin. Lepirudin (57.9 +/- 8.6%) was eliminated unchanged via the kidneys. Identical to the in vitro situation metabolite fragments were built in the distribution M64 = 8.1 +/- 5.7%, M63 = 21.1 +/- 6.5%, and M60,61,62 = 12.9 +/- 4.5%. CONCLUSIONS: Lepirudin is metabolized spontaneously in more than 10-fold concentrated urine. Metabolization of lepirudin takes place in the proximal tubulus cells as well. In vitro, the degradation takes place amino acid by amino acid, but in vivo even dipeptides and perhaps tripeptides are degraded.

Liquid chromatography method for determination of bivalirudin in human plasma and urine using automated ortho-phthalaldehyde derivatization and fluorescence detection.

A high-performance liquid chromatographic (HPLC) method was developed using solid-phase extraction, o-phthalaldehyde (OPA) derivatization and fluorescence detection for the determination of the direct thrombin inhibitor bivalirudin in human plasma and urine. The use of this assay will facilitate the study of the pharmacodynamics of bivalirudin in studies of special patient populations. A C(18) bioanalytical column at a flow rate of 1 ml/min with an aqueous trifluoroacetic acid (0.1% TFA in deionized water, pH 2.2, v/v) mobile phase and methanol gradient was used. The assay demonstrated linearity from 3 to 20 microg/ml bivalirudin in plasma, with a detection limit of 1 microg/ml. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of bivalirudin in patients undergoing percutaneous coronary interventions (PCIs).

Characterization of C-terminal-truncated urinary metabolites of a recombinant hirudin in rats.

Although it has been reported that hirudin was excreted in urine mainly as its nonmetabolized form in humans, dogs, and rabbits, no report has been published about the molecular nature of urinary metabolites in rats. We found that nonmetabolized hirudin could not be detected in rat urine after its i.v. administration and that urinary metabolites of recombinant hirudin CX-397 consisted of at least the following six C-terminal-truncated peptides: CX-397(1-49), CX-397(1-50), CX-397(1-51), CX-397(1-52), CX-397(1-54), and CX-397(1-55), in the ratio of roughly 11, 51, 3, 11, 19, and 5%, respectively. In conclusion, the urinary metabolism of recombinant hirudin in rats is different from that in humans, dogs, and rabbits, suggesting that the handling of hirudin in rat kidney is unique among them.

Two-site immunoassay of recombinant hirudin based on two monoclonal antibodies.

Two monoclonal antibodies (mAbs), 10-2 and 10-5, both directed against recombinant hirudin variant 2-Lys47 (rHV2), were selected for their high affinity and epitopic specificities to develop a two-site immunoassay of rHV2. The mAb concentrations, incubation time, and temperature were optimized. The immunoassay has a detection limit for rHV2 of 45 ng/L in plasma and 30 ng/L in urine. The reactivity of the mAbs was tested against rHV2 and several forms of this protein truncated in the carboxyl terminus. The capture mAb 10-2 was found to be mainly directed against rHV2, whereas tracer mAb 10-5 was independent of the carboxyl-terminal region of the protein. This explains the high specificity of the immunoassay for the 65-amino acid form of hirudin.

Pharmacology of r-hirudin in renal impairment.

The pharmacokinetic properties of r-hirudin were studied in nine patients suffering from different degrees of renal insufficiency. To this end, r-hirudin was administered intravenously at dosages of 0.1 mg/kg. The elimination half-life t1/2 beta was determined in blood plasma and the cumulative r-hirudin excretion in urine was measured over 48 h. In healthy volunteers t1/2 beta was 0.9 +/- 0.2 h; the cumulative r-hirudin excretion in urine after 48 h amounted to 38 +/- 10% of the dose administered, most of this quantity was excreted during the first hours. In seven patients with chronic renal failure, t1/2 beta was 15 to 41 h; in three of these patients cumulative urinary r-hirudin excretion was increased to 70-80%, in four patients cumulative r-hirudin excretion in urine within 48 h amounted to 39 +/- 8%, but was delayed in time. In 2 bilaterally nephrectomized patients, t1/2 beta was 168 and 316 h, resp. The renal clearance of hirudin was significantly and linearly correlated with the creatinine clearance (r = 0.872). In all patients aPTT and bleeding time were only moderately prolonged. Because of the modified pharmacokinetic behaviour the administration of hirudin in patients with impaired renal function requires individually adjusted dosages or prolonged administration intervals.

Radioimmunoassay for the detection of active-site specific thrombin inhibitors in biological fluids. I. Assay characteristics and quantitation of recombinant hirudin.

A sensitive radioimmunoassay (RIA) for the quantitation of recombinant (r) hirudin in biological fluids is described. Taking advantage of the highly specific hirudin-thrombin interaction, a monoclonal antibody to human alpha-thrombin was used to capture hirudin-thrombin complexes in a competitive binding assay. Quantitation of r.hirudin in buffer, plasma or urine at concentrations ranging from 0.17 to 20 ng/ml (1.7 x 10(-3) to 2 x 10(-2) antithrombin units/ml) was achieved. In the absence of competing unlabelled r.hirudin the assay also measured alpha-thrombin (from 2 x 10(-4) to 1 x 10(-2) NIH units/ml) in citrated or defibrinated human plasma. A series of peptides corresponding to the carboxyl-terminal region of hirudin and with varying anticoagulant activities did not displace 125I-r.hirudin in the RIA described, confirming published data that these hirudin fragments bind to a site distant to the catalytic site of thrombin. The assay was used to test hirudin clearance after bolus i.v. injections of 0.1 mg r.hirudin [Val1-Val2] into human volunteers. The plasma concentrations and elimination kinetics of r.hirudin were in good agreement with published data and a close correlation between hirudin plasma concentration and prolonged clotting time was observed.

Studies for revealing a possible sensitization to hirudin after repeated intravenous injections in baboons.

The immunogenic potential of natural and recombinant hirudin was investigated using baboons. Animals received 4 intravenous hirudin injections of 1,000 antithrombin units/kg at certain times (day 1, 3, 8 and 42) and were checked for an immune response. None of the tests performed (in vitro histamine-release, ELISA for detection of humoral hirudin-specific IgG and IgM antibodies by indirect ELISA, direct skin test) revealed any kind of sensitization to hirudin. Urinary excretion of hirudin was not diminished by successive injections, if diuresis was within normal ranges. In summary, these results confirm previous observations indicating the weak antigenic potential of hirudin. Therefore, immune reactions to hirudin in man during therapeutic measures requiring several exposures to the inhibitor should not to be expected under normal circumstances.

A hirudin catching ELISA for quantitating the anticoagulant in biological fluids.

A catching ELISA has been developed that permits a quantitation of the anticoagulant hirudin in buffer, urine and plasma. In plasma hirudin can be determined in concentrations ranging from 0.2 to 25 ng/ml (2.4 X 10(-3) to 0.3 AT-U/ml), in urine between 0.8 and 200 ng/ml (0.01 and 2.4 AT-U/ml). The enzyme immunoassay allows a rapid, sensitive and reproducible quantitation of hirudin, and can thus be used to assess the pharmacokinetics of the anticoagulant in patients after parenteral and/or topic administration.

Evidence for the identity of hirudin isolated after kidney passage with the starting material.

Natural, sulfated and recombinant, non-sulfated hirudin preparations re-isolated from urine after kidney passage were compared with the corresponding administered preparations by reversed-phase high-performance liquid chromatography, and by amino acid sequence and composition analysis. It could be demonstrated that the hirudins were excreted in unmodified form. Natural hirudin could be fractionated by the chromatographic system used into a large number of isoinhibitor forms, some of which were shown to differ from each other in their amino acid sequence.

Pharmacokinetics of 125I-hirudin in rats and dogs.

Hirudin was 125I-labelled using a modified chloramine-T method. 125I-hirudin proved to be a suitable marker in pharmacokinetic studies, if unchanged 125I-hirudin in body fluids was determined by means of a binding assay using immobilized thrombin. In rats and dogs a study was performed on the pharmacokinetic behaviour of hirudin following intravenous and subcutaneous injection, resp., or one-hour infusion and pharmacokinetic parameters were calculated.

Pharmacokinetic studies with recombinant hirudin in dogs.

The knowledge of the pharmacokinetics of recombinant hirudin is essential for potential clinical use of this selective thrombin inhibitor. For detailed information about absorption, distribution and elimination, pharmacokinetic studies with recombinant hirudin were carried out in dogs. The plasma concentration time curve after intravenous injection could be best described by an open two-compartment model with first order kinetics. The subcutaneous application of recombinant hirudin produced anticoagulantly effective blood level for a prolonged period of time. Recombinant hirudin is distributed into the extracellular space. This is also found in nephrectomized dogs. Recombinant hirudin is eliminated through the kidneys by glomerular filtration in active form with a half-life of 72 minutes.

Quantitative enzyme-linked immunosorbent assay (ELISA) for hirudin.

A competitive ELISA assay has been developed that permits reproducible quantitation of the anticoagulant hirudin in buffer and urine. Coupling of peroxidase and hirudin was performed with the heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio)-propionate. In both solvents the lower limit of sensitivity was 8 ng hirudin/ml (0.08 AT-U) while the upper limit was 7.7 micrograms/ml (78.45 AT-U).