Hirohiko Shimizu

Development of Poly(ethylene glycol) Conjugated Lactoferrin for Oral Administration

Lactoferrin (LF) is an iron-binding glycoprotein that possesses multifunctional biological activities. Recent reports from clinical trials suggest that LF is potentially effective as a therapeutic protein against cancer and gangrene. However, pharmaceutical proteins such as LF are unstable in vivo. Therefore, to improve stability, we developed mono-PEGylated bovine LF (20k-PEG-bLf) with branched 20 kDa (2 x 10 kDa) poly(ethylene glycol) (PEG). We examined in vitro activities such as iron binding, IL-6 cell based assay, and resistance to a proteolytic enzyme in artificial gastric fluid. The 20k-PEG-bLf protein was fully active in iron binding and exhibited 69.6 +/- 2.9% (mean +/- S.E., n = 6) of the original anti-inflammatory activity. The proteolytic half-life increased 2-fold over that of unmodified LF. In vivo pharmacokinetic analyses were performed to examine absorption from the intestinal epithelium and serum clearance. Direct administration of 20k-PEG-bLf (30 mg/kg) into rat stomachs demonstrated that the amount of absorption from the intestinal tract increased approximately 10-fold relative to unmodified LF. Intravenous injection of the protein (1 mg/kg) revealed that 20k-PEG-bLf prolongs serum half-life by approximately 5.4-fold, and that the area under the curve (AUC) was increased approximately 9.2-fold compared to that of unmodified LF. PEGylation improved the physical and pharmacokinetic properties of bovine LF. This is the first report on the use of bioconjugation of LF for the development of a promising oral pharmaceutical agent.

Characterization of four different mammalian-cell-derived recombinant human interferon-β1s

In order to evaluate the structural identification of various recombinant human interferon-beta 1s, the recombinant proteins were produced in four different mammalian cells (human PC12 and PC8 lung adenocarcinoma cells, Chinese hamster ovary cells and mouse C127 cells) and characterized. Each mammalian-cell-derived recombinant human interferon-beta 1 represented a single band of 23 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, the same molecular mass as fibroblast-derived natural human interferon-beta 1. Specific activities, amino acid compositions, amino-terminal sequences, peptide maps on C18 reversed-phase high-performance liquid chromatography and circular dichroic spectra of recombinant proteins were in good agreement with natural ones. On the other hand, the patterns of isoelectric focusing were different between mammalian-cell-derived recombinant human interferon-beta 1s and natural human interferon-beta 1. Sugar composition analysis revealed that the recombinant protein from Chinese hamster ovary cells has a similar sugar composition to that of natural protein and the other recombinant proteins have increased amounts of galactose and glucosamine in comparison to the natural protein. Furthermore, there is no galactosamine in the natural protein, while small amounts of galactosamine were detected in the oligosaccharides released from PC8- and C127-derived recombinant proteins by N-glycanase. These results indicate that mammalian-cell-derived recombinant human interferon-beta 1s have identical polypeptides to those of natural human interferon-beta 1 but their carbohydrate moieties, including unusual N-linked oligosaccharides, are individually different from natural ones and depend on the host cell.

Stability of human interferon-ß1: oligomeric human interferon-ß1 is inactive but is reactivated by monomerization

Human interferon-s1 is extremely stable in a low ionic strength solution of pH 2 such as 10 mM HCl at 37°C. However, the presence of 0.15 M NaCl led to a remarkable loss of antiviral activity. The molecular-sieve high-performance liquid chromatography revealed that, whereas completely active human interferon-s1 eluted as a 25 kDa species (monomeric form), the inactivated preparation eluted primarily as a 90 kDa species (oligomeric form). The specific activity (units per protein) of the oligomeric form was approx. 10% of that of the monomeric form. This observation shows that oligomeric human interferon-s1 is apparently in an inactive form. When the oligomeric eluate was resolved by polyacrylamide gel containing sodium dodecyl sulphate (SDS), it appeared to be monomeric under non-reducing conditions. Monomerization of the oligomeric human interferon-s1 by treatment with 1% SDS, fully regenerated its antiviral activity. These results suggest that the inactivation of the human interferon-s1 preparation was caused by its oligomerization via hydrophobic interactions without the formation of intermolecular disulphide bonds. These oligomers can be dissociated by SDS to restore biological activity.

Enteric‐formulated lactoferrin was more effectively transported into blood circulation from gastrointestinal tract in adult rats

We have previously demonstrated that intestinally infused bovine lactoferrin (bLF) is transported into the blood circulation via the lymphatic pathway, not via the portal circulation. Therefore, in the present study, we further investigated whether intragastrically infused enteric‐formulated bLF (EF‐bLF) was more efficiently absorbed than bLF from the intestine in adult rats. The rats were randomly divided into three groups: 30 and 300 mg kg−1 non‐enteric‐formulated bLF (non‐EF‐bLF) groups and a 30 mg kg−1 EF‐bLF group. Thoracic lymph was collected from a thoracic lymph duct under general anaesthesia. Bovine lactoferrin was infused into the stomach or duodenal lumen via a needle for a period of over 1 min in a volume of 1 ml kg−1. The bLF transported into the lymph was assayed quantitatively by double‐antibody enzyme‐linked immunosorbent assay (ELISA). Following the intragastric administration of bLF, the three groups showed almost the same lymph flow, but the bLF concentration in the lymph fluid in the EF‐bLF group increased significantly and peaked 3 h after administration. With intraduodenal administration, the bLF concentration in the lymph fluid of the higher non‐EF‐bLF group was significantly higher than those of the other groups. The amount of absorbed bLF in the EF‐bLF group was, however, about 10 times higher than that in the lower non‐EF‐bLF group, when it was administered intragastrically. These data show that enteric‐formulated bLF is less susceptible to gastric pepsin and is more efficiently absorbed from the intestine than is non‐enteric‐formulated bLF.

Milk-derived lactoferrin may block tolerance to morphine analgesia

Lactoferrin (LF) is a multifunctional protein that is widely found in milk, blood, and other biological fluids. In the present study, we investigated the possibility that LF may block a tolerance to morphine-induced analgesia in the mouse. The nociceptive effect of bovine milk-derived LF (bLF) was estimated in the mouse tail-flick test. Although an intraperitoneal (100 mg/kg) or an oral (300 mg/kg) administration of bLF did not show remarkable analgesia, a combination with intraperitoneal administration of morphine (3 mg/kg) strikingly enhanced morphine-induced analgesia. Moreover, repeated administration of morphine at doses of 3 mg/kg (ip) or 5 mg/kg (ip) caused a tolerance to the morphine on the 5th or 7th day, respectively. In contrast, the combination of bLF (100 mg/kg, ip) with morphine (3 mg/kg, ip) retarded the development of tolerance to the 9th day, although bLF did not show any effect on the mice that had obtained tolerance to morphine. Furthermore, the potentiative effect of bLF was partially blocked by pre-treatment with N(G)-nitro-L-arginine methyl ester (L-NAME), a nonselective nitric oxide synthase (NOS) inhibitor, and completely blocked by 7-nitroindazole (7-NI), a selective neuronal NOS (nNOS) inhibitor. Methylene blue (MB), a guanylate cyclase (GC) inhibitor, also dose-dependently prevented the potentiative effect of bLF. These results suggest that bLF selectively activates nNOS and then accelerates NO production. The increased NO in turn modulates the GC activity and finally enhances the endogenous opioid system via cyclic guanosine monophosphate production. We conclude that bLF may block the development of tolerance to morphine in mice, possibly via the selective activation of nNOS.

Functional expression of human leukocyte elastase (HLE)/medullasin in eukaryotic cells

We have cloned a full length cDNA for human leukocyte elastase (HLE, EC 3.4.21.37)/medullasin from the cDNA library of human leukemic cell line, ML3. Recombinant plasmid for the expression of HLE cDNA in eukaryotic cells was constructed in which HLE cDNA was fused in a frame to a leader sequence of human interleukin-2 (IL-2). COS-1 cells, transfected with the plasmid, secreted fusion protein consists of N-terminal 8 amino acid (aa) residues of human IL-2 and 238 aa residues of HLE. As the fusion protein was designed to be connected through lysine residue, elastase activity was generated after digestion of the fusion protein with lysyl-endopeptidase.

Studies on aldolases I. Amino acid composition and subunit structure of rabbit-muscle aldolase

Abstract 1. 1. In order to obtain precise information about the chemical nature of rabbit-muscle aldolase (fructose-1,6-diphosphate d -glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13), the amino acid composition has been determined. By assuming the molecular weight of the performic acid-oxidized aldolase to be 143000, it is shown that the oxidized aldolase contains 1320 amino acid residues per molecule. 2. 2. Tryptic peptides of the oxidized aldolase have been separated on a Dowex 50-X2 column and the amino acid composition and yield of 9 of them have been determined. The yields of 8 out of the 9 peptides analyzed were 2.1–2.7 moles/mole of aldolase. Consideration of these data leads to the conclusion that the aldolase molecule probably consists of 3 subunits which are chemically identical, at least to a considerable extent. If this is so, each subunit should contain 440 amino acid residues, and the 8 peptides mentioned above would account for about one-fifth of the whole molecule.

Relationship between peroxisome proliferator-activated receptor-γ activation and the ameliorative effects of ascochlorin derivatives on type II diabetes

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a crucial factor in the development of insulin resistance associated with type II diabetes. We previously found that 4-O-carboxymethyl ascochlorin, a derivative of ascochlorin, ameliorates diabetes and activates PPAR-γ. Here, we compared the relationship between the amelioration of type II diabetes in db/db mice lacking leptin receptor, and PPAR-γ activation by 4-O-carboxymethyl-ascochlorin, as well as by 4-O-methyl-ascochlorin, a derivative that does not activate PPAR-γ. Administration of these compounds significantly reduces blood glucose in a dose-dependent manner, whereas blood cholesterol is significantly elevated in 4-O-carboxymethyl-ascochlorin-treated mice but is significantly decreased in 4-O-methyl-ascochlorin-treated mice. Pioglitazone, a potent PPAR-γ agonist with a thiazolidinedione structure, reduces glucose but elevates cholesterol blood levels. These results suggest that ascochlorin derivatives ameliorate diabetes through a mechanism that is probably independent of PPAR-γ activation, although PPAR-γ activation could be partially involved in the ameliorative effect in certain derivatives.

Constitutive Long-Term Production of Recombinant Human Interferon-Gamma by Transformed Mouse C127 Cells Cultured in Serum Free Medium

Transformed mouse C127 cells in which human interferon-gamma cDNA was inserted were grown well in a microcarrier culture system. The cells were maintained for more than two months even in a serum free condition without cell proliferation and continued to produce a high level of recombinant human interferon-gamma (rHuIFN-r). The maintenance of the cells grown on the microcarriers was fairly prolonged by adding 0, 1% bovine serum albumin to the serum free medium. The flow-cytometrical analysis for cell cycle showed more than 85% of these maintained cells remained in G0/G1 phase, differing from the case of the growing cells. However, we confirmed the copy-numbers of rHuIFN-r cDNA were safely retained even in the maintained cells for a long period. The rHuIFN-r preparation obtained in the serum free condition was also glycosylated protein similar to natural HuIFN-r derived from human periferal blood lymphocytes.

Low molecular weight peptide inhibitors of medullasin: purification and structure

Two low molecular weight peptide inhibitors of medullasin were isolated from human bone marrow cells. Determination of their amino acid composition and amino acid sequence revealed that one inhibitor was composed of 36 amino acid residues and the other 34 amino acid residues which are identical with the C-terminal portions (Formula; see text) of the beta-chain of human hemoglobin. These two peptides when synthesized also showed the same degree of inhibitory effect on medullasin activity as the natural products. Neither the N-terminal portion of the inhibitor, composed of 21 amino residues, nor the C-terminal peptide, composed of 20 amino acids, inhibited medullasin activity. Medullasin was inhibited reversibly and non-competitively against by these inhibitors and was the most effectively inhibited serine protease among several tested.