Bioavailability and relative tissue distribution of [125I]-recombinant human thrombin following intravenous or subcutaneous administration to non-human primates.

BACKGROUND: Recombinant human thrombin (rhThrombin) is being developed as a general adjunct to hemostasis. Endogenous thrombin is rapidly inactivated by complex formation with antithrombin III and other inhibitors. It follows that these inhibitors will also inactivate any rhThrombin that reaches the systemic circulation. OBJECTIVES: Study goals were to determine the pharmacokinetic characteristics of [(125)I]-rhThrombin and [(125)I]-rhThrombin complexed to endogenous inhibitors, and the tissue distribution of rhThrombin-associated radioactivity in non-human primates. Hematology, serum chemistry and coagulation status were also monitored. METHODS: [(125)I]-rhThrombin was administered intravenously (i.v.; 3.5 U kg(-1)) or subcutaneously (s.c.; 350 U kg(-1)) to male cynomolgus monkeys. Plasma was analyzed for rhThrombin-associated radioactivity and non-compartmental analysis was used to determine the corresponding pharmacokinetic parameters. A size exclusion-high pressure liquid chromatography (SE-HPLC) method was used to quantitate rhThrombin complexes, non-complexed rhThrombin, and free [(125)I]. Whole-body gamma scintigraphy was used to follow radioactivity localization up to 72 h postdose. RESULTS: No adverse events were observed following [(125)I]-rhThrombin administration. The pharmacokinetic profile of rhThrombin-associated radioactivity following i.v. injection was multi-exponential with an initial half-life of approximately 10 min. Following both i.v. and s.c. dosing, the terminal half-life was approximately 15 h. SE-HPLC analysis revealed that rhThrombin was rapidly complexed to antithrombin III and other inhibitors in the systemic circulation following i.v. administration. Thus, rhThrombin-associated radioactivity in the blood was complexed and presumed inactive. [(125)I]-rhThrombin inhibitor complexes accumulated and were eliminated in the liver following both routes of administration. CONCLUSIONS: These data suggest that rhThrombin rapidly binds to endogenous inhibitors following either i.v. or s.c. administration.

Safety, pharmacokinetics, and immunogenicity of single-dose rFXIII administration to healthy volunteers.

BACKGROUND: Factor XIII (FXIII) is a transglutaminase that cross-links fibrin and other proteins to improve clot strength and resistance to fibrinolysis. Both congenital and acquired FXIII deficiency may result in a bleeding diathesis, and plasma-derived FXIII has been used to treat many of these clinical conditions. OBJECTIVES: A clinical study was designed and performed to evaluate the safety, pharmacokinetics, and immunogenicity of recombinant FXIII (rFXIII) administration to healthy adult volunteers. PATIENTS AND METHOD: Fifty healthy adult volunteers were enrolled in this randomized, double-blinded, placebo-controlled study. A single dose of rFXIII, ranging from 2 U kg(-1) to 50 U kg(-1), or placebo was administered. Safety was evaluated by capturing adverse events, clinical safety laboratory studies, and clinical score for deep venous thrombosis. Blood samples were taken for pharmacokinetic and immunogenicity analysis throughout the 28-day follow-up period. RESULTS: Recombinant FXIII was well tolerated, with no serious adverse events or dose-related toxicities. Following a single i.v. injection of 50 U kg(-1) rFXIII, the estimated terminal half-life was 270-320 h, the volume of distribution ranged from 40 to 75 mL kg(-1), and FXIII activity increased 1.77% per 1 U kg(-1) rFXIII administered. Increase in circulating A2B2 and decrease in free FXIII-B subunit indicate in vivo formation of FXIII heterotetramer. An immunogenic response to rFXIII or yeast, the production host, was not observed. CONCLUSIONS: Recombinant FXIII was well tolerated at doses of up to 50 U kg(-1) in healthy adult volunteers. The safety, pharmacological and immunological profile of rFXIII suggests it should be studied in patients with congenital FXIII deficiency as well as evaluated as a systemic hemostat in patients with acquired FXIII deficiency or hemorrhage.

High-pressure liquid chromatographic assay for the determination of thyrotropin-releasing hormone and its common metabolites in a physiological salt solution circulated through the isolated perfused rat lung.

A specific and sensitive assay for TRH (L-pyroglutamyl-L-histidyl-L-proline amide), 3H-TRH (L-proline 3,4-3H(N), histidyl-3-3H(N)), and four possible metabolites of TRH, present in the recirculated perfusate of an isolated perfused rat lung preparation, was developed. Unlike previous methods, the method developed does not require extraction of the analytes from the biological matrix. The crude sample was adjusted to a pH of 3.2 with concentrated trifluoroacetic acid and injected on to a PRP-1 (polystyrene divinylbenzene) column (10 microns, 25 cm x 4.6 mm i.d.). The mobile phase was 10% v/v acetonitrile and 90% v/v 0.75 g l-1 1-hepantanesulfonic acid in 0.004 M trifluoroacetic acid, adjusted to a pH of 2.4 with concentrated NaOH. The flow rate was 0.5 ml min-1 and the analytes were detected by UV absorption at a wavelength of 26 nm and by radiochemical detection utilizing a liquid scintillation counter. The nominal retention times for L-PRO, L-PRO-NH2, TRH, cyclo(HIS-PRO) and TRH-OH were 4.0 +/- 0.9, 10.0 +/- 0.2, 15.5 +/- 0.4, 19.2 +/- 0.5 and 25.3 +/- 0.5 min respectively. The assay performs well in terms of precision and accuracy as indicated by linear regression and intra-assay variability analysis.