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.

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.

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.

Determination of antihypertensive peptides from an izumi shrimp hydrolysate.

The izumi shrimp (Plesionika izumiae Omori, 1971) is an unused resource which can be caught off the southern coast of Tokushima Prefecture. We have previously found that an izumi shrimp hydrolysate significantly inhibited the age-associated spontaneous increase in blood pressure in stroke-prone spontaneously hypertensive rats. In this present study, two angiotensin I-converting enzyme inhibitory peptides were isolated from an izumi shrimp hydrolysate by using high-performance liquid chromatography, and their amino acid sequences were determined to be Val-Trp-Tyr-His-Thr and Val-Trp. A single oral administration of synthetic Val-Trp-Tyr-His-Thr or Val-Trp significantly decreased the blood pressure in stroke-prone spontaneously hypertensive rats. The antigenicity and allergenicity of the izumi shrimp hydrolysate against BALB/c mice were very low. These results demonstrate that the angiotensin I-converting enzyme inhibitory peptides isolated from the izumi shrimp hydrolysate had an anti-hypertensive effect on rats.

Induction of hepatocyte growth factor by fucoidan and fucoidan-derived oligosaccharides.

Fucoidan, which is extracted from brown seaweed, is a complex sulphated polysaccharide that is mostly composed of L-fucose and sulphated ester groups. The structural and anionic characteristics of fucoidan are similar to those of heparin. Heparin stimulates production of hepatocyte growth factor (HGF), which has key roles in tissue regeneration. We have shown that fucoidan and fucoidan-derived oligosaccharides have similar ability to stimulate production of HGF as heparin and heparin-derived oligosaccharides. This induction of HGF by heparin or fucoidan and their oligosaccharide derivates occurs primarily at the level of translation, probably via the same mechanism. Fucoidan may thus be useful to protect tissues and organs from various injuries and diseases, via mechanisms involving HGF.

Stimulation of hepatocyte growth factor production by heparin-derived oligosaccharides.

We previously reported that heparin post-transcriptionally stimulates the production of hepatocyte growth factor (HGF). In this study, we addressed the size-dependency of heparin fragments on the HGF-inducing activity aiming to obtain fragments without antiblood coagulant activity. Heparin fragments, produced by digestion with heparinase, were size-fractionated and tested for HGF-inducing activity in cultured human fibroblasts. The HGF-inducing activity deceased with the reduction in oligosaccharide size. Decasaccharides exerted an activity comparable with undigested heparin, while smaller oligosaccharides showed lesser activities. The anticoagulant activity of heparin fragments also decreased with size and anticoagulant activity of decasaccharides was <13% that of undigested heparin. Further fractionation of decasaccharides by anion-exchange chromatography revealed that most of the decasaccharides had HGF-inducing activity and the extent of sulfation was roughly related to the activity. The lack of N-sulfation in heparin markedly reduced HGF-inducing activity, whereas 2-O-desulfation or 6-O-desulation had a lesser influence. Moreover, an N-sulfated disaccharide showed significant HGF-inducing activity, suggesting the involvement of N-sulfation in HGF-inducing activity. Because of the much reduced anticoagulant activity, potential applications of heparin-derived oligosaccharides such as decasaccharides is considerable as a therapeutic agent for many diseases.

Multiple biological responses are induced by glycosylation-deficient hepatocyte growth factor.

HGF (hepatocyte growth factor), a heterodimeric glycoprotein composed of alpha- and beta-chains, exerts biological activities through the c-Met receptor tyrosine kinase. The alpha-chain has three glycosylation sites, while the beta-chain has two; however, the role of sugar chains on HGF is still unknown. To address the significance of glycosylation of HGF, three different types of glycosylation-deficient HGFs, i.e. non-glycosylated in the alpha-chain, the beta-chain, and in both the alpha- and beta-chains, were respectively expressed in COS-7 cells and then purified from culture supernatants. Unexpectedly, glycosylation-deficient HGFs induced tyrosine phosphorylation of the c-Met receptor and subsequent phosphorylation of ERK (extracellular-signal-regulated kinase) and Akt in rat hepatocytes with the same potency as glycosylated HGF. Consistent with this, glycosylation-deficient HGFs strongly stimulated DNA synthesis of hepatocytes equal to glycosylated HGF. Likewise, glycosylation-deficient HGFs induced cell scattering and branching tubulogenesis in MDCK (Madin-Darby canine kidney) cells, and thus were indistinguishable from glycosylated HGF in biological activities. Glycosylation also did not affect stability, protease sensitivity and tissue distribution, although the plasma clearance of HGF was slightly prolonged by glycosylation deficiency. Glycosylation deficiency resulted in a decrease in post-transcriptional biosynthesis of HGF in the cells, whereas extracellularly secreted HGFs were efficiently activated to a two-chain form. These results indicate that glycosylation influences post-transcriptional biosynthesis of HGF, whereas biological activities and basic physicochemical characteristics are retained, even in completely non-glycosylated HGF. Hence, non-glycosylated HGF is promising as an alternative for glycosylated HGF in clinical applications.

Monitoring biodistribution of glycoproteins with modified sugar chains.

Natural human interferon (hIFN)-gamma has mainly biantennary complex-type sugar chains. Previously, we successfully remodeled its sugar chain structure into: (a) highly branched types; or (b) highly sialylated types, by overexpression of: (a) N-acetylglucosaminyltransferase (GnT)-IV and/or GnT-V; or (b) sialyltransferases, in Chinese hamster ovary (CHO) cells. In addition, we prepared asialo hIFN-gammas by treatment with sialidase in vitro. In the present study, we assessed the bioactivity of remodeled hIFN-gamma in terms of antiviral activity, anticellular activity, and biodistribution. Structural changes to the sugar chains did not have a significant influence on the antiviral and anticellular activities of hIFN-gamma, although the attachment of the sugar chain itself affected both activities. However, the biodistribution differed significantly; the number of exposed galactose residues was the major determinant of the specific distribution to the liver and blood clearance rate of hIFN-gamma. This phenomenon was considered to be mediated by the hepatic asialoglycoprotein receptor (ASGP-R), and we showed a linear, not exponential, enhancement of the distribution to the liver with an increase in the number of exposed galactose residues. We also confirmed this tendency using fibroblast growth factor (FGF). Our observation is not the same as the "glycoside cluster effect." We thus provide important information on the character of modified recombinant glycoproteins.

Japanese citrus fruit (sudachi) juice is associated with increased bioavailability of calcium from whole small fish and suppressed bone resorption in rats.

Shirasuboshi (boiled and semi-dried whitebait) is a processed fish food that contains abundant calcium. It is eaten whole and commonly consumed in Japan. In this study, the effect of sudachi (Citrus sudachi) juice on calcium, magnesium and phosphorus bioavailability, and bone metabolism in rats was examined. After 14 d of diets low in calcium and phosphorus, male Sprague-Dawley rats were fed shirasuboshi diets containing dried shirasuboshi powder treated with 20% (S20) or 40% (S40) sudachi juice, or distilled water (C) (0.5% Ca; 0.3% P) for 14 d. The apparent absorptions and retentions of calcium, magnesium and phosphorus from shirasuboshi were determined. Bone formation was calculated by measuring serum osteocalcin, and bone resorption by measuring urinary pyridinoline and deoxypyridinoline. The apparent absorption and retention of calcium and magnesium in the S20 group were significantly higher than in the C and S40 groups. Although serum osteocalcin was not affected by the addition of sudachi juice, the urinary pyridinoline and deoxypyridinoline concentrations in the S40 group were significantly lower than in the C and S20 groups. Our results indicate that sudachi juice added to shirasuboshi was associated with increased calcium bioavailability and suppressed bone resorption in rats.

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.

Different reactivities of enzyme-linked immunosorbent assays for hepatocyte growth factor.

BACKGROUND: Hepatocyte growth factor (HGF) plays diverse roles in organ development, tissue regeneration, and tumor progression. Measurement of HGF concentrations in blood and tissues using enzyme-linked immunosorbent assay (ELISA) is a simple and easy way to understand the significance of HGF in tissue regeneration, pathophysiology, and diagnosis. METHODS: We evaluated 3 ELISA kits from different sources, referred to herein as kits A, B, and C for convenience. RESULTS: We found that the concentrations of human HGF determined using ELISA vary significantly depending on the source of the ELISA kit. Kits A and B detected both single-chain pro-HGF and 2-chain mature HGF, but kit C detected only 2-chain HGF. A difference in reactivity was also detected during analysis of plasma samples. When rat plasma collected 4 h after subcutaneous administration of human HGF was analyzed, the HGF concentration determined using kit B was remarkably higher than those obtained using kits A and C. Results of a biological assay and Western blot analysis indicated that kit B detects even degraded HGF, by which the HGF concentrations determined using kit B were significantly overestimated. CONCLUSIONS: This information serves as a guide for the selection of ELISA kits for human HGF.