Preclinical safety and pharmacokinetics of recombinant human factor XIII.

Factor XIII (FXIII) is a thrombin-activated protransglutaminase responsible for fibrin clot stabilization and longevity. Deficiency in FXIII is associated with diffuse bleeding and wound-healing disorders in humans. This report summarizes results from several studies conducted in adult cynomolgus monkeys (M. fascicularis) to evaluate the safety and pharmacokinetics of recombinant human factor XIII A(2) dimer (rFXIII). Intravenous slow bolus injection of rFXIII resulted in the expected formation of the heterotetramer rA(2)cnB(2), prolonged circulating half-life (5-7 days), and increased plasma transglutaminase activity. Recombinant FXIII was well tolerated as a single dose up to 20 mg/kg rFXIII (2840 U/kg), as repeated daily doses up to 6 mg/kg (852 U/kg) for 14 days, and as 3 repeated doses of 8 mg/kg (1136 U/kg) separated by 14 days. Overt toxicity occurred after a single intravenous injection of = 22.5 mg/kg rFXIII (3150 U/kg), or with 2 doses of = 12.5 mg/kg (1775 U/kg) administered within 72 hours. The rFXIII-mediated toxicity was expressed as an acute systemic occlusive coagulopathy. Evaluation of plasma samples from dosed animals demonstrated formation of cross-linked fibrin/fibrinogen oligomers and higher-order protein aggregates, which are hypothesized to be responsible for the observed vessel occlusion and associated embolic sequelae. These results demonstrate that rFXIII-mediated toxicity results from exaggerated pharmacological activity of the molecule at supraphysiological concentrations. The absence of observed toxicological effect with repeated intravenous doses up to 8 mg/kg (1136 U/kg) was used to support an initial clinical dose range of 0.014 to 0.35 mg/kg (2-50 U/kg).

Platelet-derived growth factor C induces liver fibrosis, steatosis, and hepatocellular carcinoma.

Members of the platelet-derived growth factor (PDGF) ligand family are known to play important roles in wound healing and fibrotic disease. We show that both transient and stable expression of PDGF-C results in the development of liver fibrosis consisting of the deposition of collagen in a pericellular and perivenular pattern that resembles human alcoholic and nonalcoholic fatty liver disease. Fibrosis in PDGF-C transgenic mice, as demonstrated by staining and hydroxyproline content, is preceded by activation and proliferation of hepatic stellate cells, as shown by collagen, alpha-smooth muscle actin and glial fibrillary acidic protein staining and between 8 and 12 months of age is followed by the development of liver adenomas and hepatocellular carcinomas. The hepatic expression of a number of known profibrotic genes, including type beta1 TGF, PDGF receptors alpha and beta, and tissue inhibitors of matrix metalloproteinases-1 and -2, increased by 4 weeks of age. Increased PDGF receptor alpha and beta protein levels were associated with activation of extracellular regulated kinase-1 and -2 and protein kinase B. At 9 months of age, PDGF-C transgenic mice had enlarged livers associated with increased fibrosis, steatosis, cell dysplasia, and hepatocellular carcinomas. These studies indicate that hepatic expression of PDGF-C induces a number of profibrotic pathways, suggesting that this growth factor may act as an initiator of fibrosis. Moreover, PDGF-C transgenic mice represent a unique model for the study of hepatic fibrosis progressing to tumorigenesis.

Exogenous PDGF-D is a potent mesangial cell mitogen and causes a severe mesangial proliferative glomerulopathy.

The PDGF family consists of at least four members, PDGF-A, -B, -C, and -D. All of the PDGF isoforms bind and signal through two known receptors, PDGF receptor-alpha and PDGF receptor-beta, which are constitutively expressed in the kidney and are upregulated in specific diseases. It is well established that PDGF-B plays a pivotal role in the mediation of glomerular mesangial cell proliferation. However, little is known of the roles of the recently discovered PDGF-C and -D in mediating renal injury. In this study, adenovirus constructs encoding PDGF-B, -C, and -D were injected into mice. Mice with high circulating levels of PDGF-D developed a severe mesangial proliferative glomerulopathy, characterized by enlarged glomeruli and a striking increase in glomerular cellularity. The PDGF-B-overexpressing mice had a milder proliferative glomerulopathy, whereas the mice overexpressing PDGF-C and those that received adenovirus alone showed no measurable response. Mitogenicity of PDGF-D and -B for mesangial cells was confirmed in vitro. These findings emphasize the importance of engagement of PDGF receptor-beta in transducing mesangial cell proliferation and demonstrate that PDGF-D is a major mediator of mesangial cell proliferation. Finally, this approach has resulted in a unique and potentially valuable model of mesangial proliferative glomerulopathy and its resolution.