Phase I/II Study of Intrathecal Administration of Recombinant Human Hepatocyte Growth Factor in Patients with Acute Spinal Cord Injury: A Double-Blind, Randomized Clinical Trial of Safety and Efficacy.

Spinal cord injury (SCI) is an abrupt traumatic injury that leads to permanent functional loss, and no practical treatment is available. We have developed pharmaceutical recombinant human hepatocyte growth factor (KP-100), and its efficacy for SCI has been verified using animal models. The purpose of this study was to evaluate the safety and efficacy of intrathecal KP-100 administration for SCI patients in the acute phase. This investigation was a multi-center, randomized, double-blind study. Subjects with modified Frankel grade A/B1/B2 at 72 h after SCI were included. KP-100 was administered intrathecally. Subjects were followed up for 168 days after the first administration. Outcomes were evaluated using American Spinal Injury Association (ASIA) scores and subjected to analysis of covariance. Our results demonstrated that the subjects did not show any serious adverse events caused by KP-100. Forty-three subjects underwent neurological function testing (26 in KP-100 group; 17 in placebo group), which revealed that KP-100 contributed to motor improvement at Days 140 (p = 0.050) and 168 (p = 0.079). In the subset of subjects with Frankel grade A, the proportions of subjects who gained at least 1 point on their lower-extremity motor scores were 33.3% (5/15) and 6.3% (1/16) in the KP-100 and placebo groups, respectively (p = 0.083). Therefore, KP-100 has the potential to be useful and beneficial for SCI patients during the acute phase. However, this was a phase I/II trial and did not definitely address the question of efficacy; a larger phase III trial would be required to assess the efficacy.

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.

Dissociation of c-Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species.

Hepatocyte growth factor (HGF) is essential for embryogenesis, tissue regeneration and tumour malignancy through the activation of its receptor, c-Met. We previously demonstrated that HGF α-chain hairpin-loop, K1 domain and ß-chain are required for c-Met signalling. The sequential phosphorylation of tyrosine residues, from c-Met kinase domain to multidocking regions, is required for HGF-signalling transduction. Herein, we provide evidence that the disconcerted activation of c-Met tyrosine regions fails to induce biological functions. When human cells were incubated with 'mouse HGF', kinase domain activation (i.e. phospho-Tyr-1230/34/35) became evident, but the multidocking site (i.e. Tyr-1349) was not phosphorylated, resulting in unsuccessful induction of migration and mitogenesis. The binding ability of mouse HGF α-chain, or of ß-chain, to human c-Met was lower than that of human HGF, as evidenced by HGF-chimera assay. Notably, only four amino acid positions in HGF α-chain hairpin-loop and K1 domain and six positions in ß-chain differed between human HGF and mouse HGF. The human-specific amino acids (such as Gln-95 in hairpin-loop, Arg-134 in K1 domain and Cys-561 in ß-chain) may be important for accurate c-Met assembly and signalling transduction.

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 amino-terminus of mouse DNA methyltransferase 1 forms an independent domain and binds to DNA with the sequence involving PCNA binding motif.

DNA methylation patterns in genome are maintained during replication by a DNA methyltransferase Dnmt1. Mouse Dnmt1 is a 180 kDa protein comprising the N-terminal regulatory domain, which covers 2/3 of the molecule, and the rest C-terminal catalytic domain. In the present study, we demonstrated that the limited digestion of full-length Dnmt1 with different proteases produced a common N-terminal fragment, which migrated along with Dnmt1 (1-248) in SDS-polyacrylamide gel electrophoresis. Digestion of the N-terminal domains larger than Dnmt1 (1-248) with chymotrypsin again produced the fragment identical to the size of Dnmt1 (1-248). These results indicate that the N-terminal domain of 1-248 forms an independent domain. This N-terminal domain showed DNA binding activity, and the responsible sequence was narrowed to the 79 amino acid residues involving the proliferating cell nuclear antigen (PCNA) binding motif. The DNA binding activity did not distinguish between DNA methylated and non-methylated states, but preferred to bind to the minor groove of AT-rich sequence. The DNA binding activity of the N-terminal domain competed with the PCNA binding. We propose that DNA binding activity of the N-terminal domain contributes to the localization of Dnmt1 to AT-rich sequence such as Line 1, satellite, and the promoter of tissue-specific silent genes.