Safety, Tolerability, and Pharmacodynamics of Intrathecal Injection of Recombinant Human HGF (KP-100) in Subjects With Amyotrophic Lateral Sclerosis: A Phase I Trial.

Hepatocyte growth factor is an endogenous pleiotropic factor shown to act as a potent neuroprotectant against disease progression in animal models of amyotrophic lateral sclerosis, which is a devastating, adult-onset motor neuron disease. To evaluate the safety, tolerability, and pharmacokinetics of recombinant 5-residue-deleted human hepatocyte growth factor (KP-100) injected intrathecally through an implantable catheter connected to a subcutaneous port, we conducted a first-in-human phase I trial of intrathecal KP-100 in 15 Japanese patients with amyotrophic lateral sclerosis. The regimen was a single injection of 3 escalating doses (0.2, 0.6, and 2.0 mg/body) in 9 subjects followed by 2 doses (0.6 and 2.0 mg/body) repeated 5 times at 1-week intervals in 6 subjects (3 subjects/group). With single-dose administration, the mean half-life of KP-100 in the cerebrospinal fluid was 1.2 to 1.4 days, with its maximum concentration increasing in a dose-dependent manner. With multiple-dose administration, the trough KP-100 concentrations in the cerebrospinal fluid generally remained constant for any dose, despite multiple dosing. There were no deaths, serious adverse events, or device malfunctions leading to discontinuation. In all subjects, plasma KP-100 concentrations were <1 ng/mL, or below the lower limit of detection at all time points of measurement. Anti-KP-100 antibody was not detected in the cerebrospinal fluid or plasma specimens from any of the subjects throughout the KP-100 dosing period. These results suggest that KP-100, as well as the device used to administer it, is safe and tolerable. A phase II trial is warranted in patients with various central nervous system diseases such as amyotrophic lateral sclerosis.

Pharmacokinetics and pharmacodynamics following intravenous administration of recombinant human hepatocyte growth factor in rats with renal injury.

BACKGROUND/AIM: Hepatocyte growth factor (HGF) plays a role in the regeneration and protection of the kidney, but little information is available concerning the pharmacokinetics of therapeutic treatment with HGF. In this study, HGF was administered after the onset of renal injury, and pharmacokinetic analysis was performed simultaneously with an efficacious dose. METHODS: For the study of pharmacodynamics, recombinant human HGF was intravenously administered to rats with glycerol-induced acute kidney injury (AKI). In the pharmacokinetic study, rats subjected to glycerol injection or renal ischemia-reperfusion were used as models of AKI, and rats subjected to 5/6 nephrectomy were used as models of chronic kidney disease (CKD). RESULTS: After intravenous administration of HGF at doses of 0.5-2.0 mg/kg, the elevation of blood urea nitrogen was suppressed, indicating that HGF had a pharmacodynamic effect. However, no significant difference was seen in the pharmacokinetic parameters such as clearance, distribution volume and half-life between the normal, AKI and CKD groups. CONCLUSION: The intravenous administration of HGF after the onset of renal dysfunction exerted a pharmacological effect on AKI, and renal injury did not affect the clearance of plasma HGF. This unaffected profile may serve as a base for the safety of HGF during therapeutic administration.

Pharmacokinetic modeling of hepatocyte growth factor in experimental animals and humans.

Hepatocyte growth factor (HGF) is under development for treatment of renal failure. This study was designed to clarify changes in HGF pharmacokinetics in renal failure and to establish a pharmacokinetic model applicable to single and repeated doses. The plasma concentration profile in mice with glycerol-induced acute renal failure was similar to that in normal mice, indicating a minimal contribution of kidney to systemic clearance of HGF. Nevertheless, accumulation of fluorescein-4-isocyanate-labeled HGF in renal tubules in both cases suggests the occurrence of efficient endocytosis of HGF in kidney. A pharmacokinetic model including plasma and liver compartments was constructed, incorporating both high- and low-affinity receptors for association and subsequent endocytosis of HGF because HGF is eliminated via specific receptor c-Met and heparin-like substance. The model well explained the plasma concentration profiles at all doses examined after bolus injection in animals and humans, and those during infusion in rodents. It includes externalization of receptors, which is negatively regulated by HGF, and can explain the gradual increase in trough concentration during repeated dosing in monkeys. Overall pharmacokinetic profiles of HGF are governed by at least two receptors and are well described by this pharmacokinetic model, which should assist in safe management of clinical trials.

Recombinant human hepatocyte growth factor (HGF), but not rat HGF, elicits glomerular injury and albuminuria in normal rats via an immune complex-dependent mechanism.

1. Hepatocyte growth factor (HGF) has the therapeutic potential to improve renal fibrosis and proteinuria in rodents with chronic kidney disease. In contrast, long-term administration of human HGF to normal rats reportedly elicits proteinuria. Thus, the role of HGF during proteinuria remains contentious. The aim of the present study was to demonstrate that human HGF is antigenic to rodents and that immune complex formation causes proteinuria. 2. We administered either human or rat HGF to normal rats for 28 days. Albuminuria was evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The renal phenotypes of the two HGF treatments were examined using histological techniques. 3. Administration of human HGF (1 mg/kg per day, i.v.) to rats led to severe albuminuria and glomerular hypertrophy in association with increased blood levels of anti-human HGF IgG and IgG deposition in mesangial areas. Furthermore, an immune complex between human HGF and anti-human HGF IgG stimulated the production of proteinuric cytokines (including transforming growth factor-ß) in rat cultured mesangial cells. In contrast, treatment of healthy rats with rat HGF for 4 weeks caused neither mesangial IgG deposition nor elevated anti-HGF IgG in the blood. Overall, rat HGF did not provoke albuminuria. 4. We conclude that human HGF produces pseudotoxic effects in normal rat kidneys via an immune complex-mediated pathway, whereas syngenic HGF is safe due to less deposition of glomerular IgG. Our results affirm the safety of the repeated use of syngenic HGF for the treatment of chronic organ diseases, such as renal fibrosis and liver cirrhosis.

Generation of engineered recombinant hepatocyte growth factor cleaved and activated by Genenase I.

Hepatocyte growth factor (HGF) is biosynthesized as a biologically inactive, single-chain form (pro-HGF). Its activation is associated with cleavage at Arg494-Val495 into a two-chain mature form composed of disulfide-linked alpha- and beta-chains. Because serum is a major source of HGF activator (the predominant serine protease responsible for the processing of pro-HGF), serum-free production of recombinant, two-chain HGF had not been established. In this study, to enable serum-free production of two-chain HGF, we generated engineered human pro-HGFs that can be specifically cleaved and activated by Genenase I. Since Genenase I specifically cleaves the C-terminus of the His-Tyr sequence, which does not exist in human HGF, Arg494 (the C-terminus of the HGF alpha-chain) was replaced by His-Tyr, Ala-Ala-His-Tyr, Pro-Gly-His-Tyr, or Pro-Gly-Ala-Ala-His-Tyr. Genenase I cleaved engineered pro-HGFs specifically at the replaced amino acid sequences, forming a disulfide-linked two-chain form. The cleavage was most efficient in the case of the Pro-Gly-Ala-Ala-His-Tyr sequence, and cleaved HGFs displayed biological activities identical to those of wild-type HGF. Considering a potential medical application of HGF, the present technique is valuable because it enables the production of recombinant, two-chain HGF entirely without serum and extends the choice of host cells and organisms for recombinant production.

The bacterial transposon Tn7 causes premature polyadenylation of mRNA in eukaryotic organisms: TAGKO mutagenesis in filamentous fungi.

TAGKO is a Tn7-based transposition system for genome wide mutagenesis in filamentous fungi. The effects of transposon insertion on the expression of TAGKO alleles were examined in Magnaporthe grisea and Mycosphaerella graminicola. Northern analysis showed that stable, truncated transcripts were expressed in the TAGKO mutants. Mapping of the 3'-ends of TAGKO cDNAs revealed that they all contain Tn7 end sequences, regardless of the transposon orientation. Polyadenylation signals characteristic of eukaryotic genes, preceded by stop codons in all frames, are located in both ends of the bacterial transposon. Thus, TAGKO transcripts are prematurely polyadenylated, and truncated proteins are predicted to be translated in the fungal mutants. Depending on the extent of protein truncation, TAGKO mutations in HPD4 (encoding p-hydroxyphenylpyruvate dioxygenase) resulted in tyrosine sensitivity in the two fungi. Similarly, a particular M.grisea CBS1 (encoding cystathionine beta-synthase) TAGKO cDNA failed to complement cysteine auxotrophy in a yeast CBS mutant. TAGKO, therefore, represents a useful tool for in vivo study of truncated gene products in filamentous fungi.

Sequence analysis and functional characterization of the dialkylglycine decarboxylase gene DGD1 from Mycosphaerella graminicola.

Dialkylglycine decarboxylase is a pyridoxal phosphate-dependent enzyme in the aminotransferases class III group of enzymes. The enzyme is unique in terms of catalyzing both decarboxylation and transamination. Although the enzymatic activity is present in some bacteria and fungi, the biological role is unclear. We identified and disrupted the dialkylglycine decarboxylase-encoding gene DGD1 in the wheat blotch fungus Mycosphaerella graminicola by transposon-arrayed gene knockout. The DGD1 gene is highly similar to dialkylglycine decarboxylase from the soil bacterium Burkholderia cepacia. Phylogenetic analysis of various class III aminotransferases showed that dialkylglycine decarboxylases from bacteria and fungi are found in a distinct cluster. Functional analysis revealed that dgd1 disruption mutants display wild-type morphology and pathogenicity to wheat. The dgd1 mutants cannot utilize 2-methylalanine as a sole nitrogen source, as assessed by large-scale nutritional utilization analysis. This is the first description of a mutant phenotype of the fungal dialkylglycine decarboxylase gene.

Efficient gene identification and targeted gene disruption in the wheat blotch fungus Mycosphaerella graminicola using TAGKO.

TAGKO ( transposon- arrayed gene knock out) is a highly efficient method for gene discovery and gene function assignment in the rice blast fungus Magnaporthe grisea. Here, we report the application of genome-wide TAGKO to the wheat blotch fungus Mycosphaerella graminicola, including the successful development of electroporation-based transformation for this fungus. A M. graminicola genomic cosmid library was constructed and a pool of 250 cosmid clones was mutagenized by in vitro transposition. Sequence analysis identified 5,110 unique insertion events in the M. graminicola genome. Eleven transposon-tagged cosmid clones (TAGKO clones) were chosen and transformed into the wild-type strain by electroporation. Ten TAGKO clones out of 11 produced gene-specific mutants at a targeting frequency of 15-28%, significantly higher than that of conventional gene-disruption constructs. The remaining clone failed to produce viable mutants, thereby providing indirect evidence for the identification of an essential gene.