Functional Analysis of Recombinant Human Serum Albumin Domains for Pharmaceutical Applications

PURPOSE: Functional analysis of the three recombinant human serum albumin (rHSA) domains and their potential as stand-alone proteins for use as drug delivery protein carriers. METHODS: Protein structure was examined by fluorescence and CD spectroscopy. Ligand binding was estimated by ultrafiltration. Antioxidant activity was estimated by measuring the quenching of dihydrorhodamine 123. Esterase-like activity and enolase-like activity were estimated from the rate of hydrolysis of p-nitrophenyl acetate and conversion of dihydrotestosterone from the 3-keto to 3-enol form, respectively. The domains of human serum albumin (HSA) were radiolabeled with 111In to evaluate their pharmacokinetics. RESULTS: The ligand binding ability of subsites Ia and Ib could not be detected in domain II. However, the binding of ligands to subsite Ic and site II were preserved in domain II and domain III, respectively. Domain III retained about 45% of its esterase-like activity, and weaker esterase-like activity was also observed in domain I. All domains showed low enolase-like activity in a pH 7.4 phosphate buffer, but domain II had higher activity in a pH 9.2 carbonate buffer. Domain I exhibited antioxidant activity comparable to that of rHSA. All three of the 111In-radiolabeled domains were rapidly eliminated from HSA, with high accumulation in the kidneys. CONCLUSION: Domain I of HSA has great potential for further development as a drug delivery protein carrier, due to its favorable properties and the presence of a free cysteine residue.     

Therapeutic index of methotrexate depends on circadian cycling of tumour necrosis factor‐α in collagen‐induced arthritic rats and mice

Objectives Rheumatoid arthritis is an autoimmune disorder of unknown aetiology. Morning stiffness, a characteristic feature of rheumatoid arthritis, shows a 24‐h rhythm. Noticing this rhythm, we hypothesized the presence of a similar rhythm for a rheumatoid arthritis indicator, in addition to dosing‐time dependency of the anti‐rheumatic effect of methotrexate in arthritis induced by collagen in rats and mice, which reflect the symptomatology of rheumatoid arthritis patients. Methods To measure tumour necrosis factor (TNF)‐α concentration, blood was taken at different times (2, 6, 10, 14, 18 or 22 h after the light was turned on (HALO)) in collagen‐induced arthritic mice. Methotrexate was administered at two different dosing times based on these findings to estimate arthritis. Key findings The arthritis score was significantly lower in the 22 HALO‐treated group than in the control and 10 HALO‐treated groups in collagen‐induced arthritic rats and mice. Plasma TNF‐α concentrations showed obvious 24‐h rhythms, with higher levels at light phase and lower levels at dark phase after rheumatoid arthritis crisis. Arthritis was relieved after administration of methotrexate during the dark phase in synchronization with the 24‐h rhythm. Conclusions Our findings suggest that choosing an optimal dosing time associated with the 24‐h cycling of TNF‐α could lead to effective treatment of rheumatoid arthritis by methotrexate.     

Development of an entirely plasmid-based reverse genetics system for 12-segmented double-stranded RNA viruses

The family Reoviridae is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family Reoviridae, the genera Cardoreovirus, Phytoreovirus, Seadornavirus, Mycoreovirus, and Coltivirus contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for Reoviridae viruses such as Orthoreovirus, Orbivirus, Cypovirus, and Rotavirus, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for Reoviridae viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus Coltivirus, family Reoviridae), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an N-glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus Coltivirus but also the replication machinery of the family Reoviridae.

The family Reoviridae is a nonenveloped virus group classified into 15 genera. These viruses have double-stranded (ds) RNA genomes with 9 to 12 segments. This family includes several important pathogens in both humans and animals. Mammalian orthoreovirus (MRV) and Nelson Bay orthoreovirus (NBV), which belong to the genus Orthoreovirus, have genomes consisting of 10 segments of dsRNA. MRV is an experimental model for studies of Reoviridae virus replication and pathogenesis. NBV, classified into the fusogenic subgroup of this genus, is associated with acute respiratory tract infections in humans (1–3). The fusogenic orthoreoviruses encode a unique fusion-associated small transmembrane (FAST) protein associated with cell–cell fusion and viral pathogenesis (4–6). Bluetongue virus (BTV) and African horse sickness virus (AHSV) belong to the genus Orbivirus, have genomes with 10 segments of dsRNA, and cause severe diseases in domestic animals (7, 8). Rotavirus (RV, genus Rotavirus) has a genome with 11 segments of dsRNA and causes severe diarrhea in young children. RV infection is responsible for 128,500 deaths per year worldwide, predominately in developing countries (9). Colorado tick fever virus (CTFV), an arthropod-borne virus transmitted by ticks and belonging to the genus Coltivirus, has a dsRNA genome comprising 12 segments. CTFV causes a variety of symptoms in humans, including abrupt fever, chills, headache, myalgia, and abdominal pain (10, 11). Within the genus Coltivirus (in addition to CTFV), Eyach virus, Shelly Headland virus, Kundal virus, and Tarumizu tick virus (TarTV) have been isolated from, or detected in, ticks in Europe, Australia, India, and Japan, respectively (12–17). Taï Forest reovirus was isolated from free-tailed bats in Côte d’Ivoire (18), and Lishui pangolin virus was detected in pangolins in China (19). These reports suggest that coltiviruses are distributed in multiple species worldwide. However, the molecular mechanism underlying the propagation and pathogenesis of these viruses remains largely unknown.Reverse genetics systems are powerful tools used to study many aspects of viral biology and virus–host interactions and also provide an opportunity to generate recombinant viruses that can be used for vaccines or as viral vectors. This technology has been used for several viruses in the family Reoviridae. In the genus Orthoreovirus, an entirely plasmid-based reverse genetics system was developed for MRV in 2007 (20). This is the first example of engineering recombinant Reoviridae viruses entirely from cloned complementary DNAs (cDNAs). This system was established by cotransfection of cloned cDNAs representing 10 MRV gene segments, each flanked by the T7 promoter and hepatitis delta virus (HDV) ribozyme, into cells expressing T7 RNA polymerase. Subsequently, a reverse genetics system for NBV, belonging to a fusogenic reovirus group, was established based on the MRV rescue system (21). In the genus Orbivirus, RNA- and DNA-based reverse genetics systems were developed for BTV, AHSV, and Epizootic hemorrhagic disease virus (22–25). Although development of a reverse genetics system for the genus Rotavirus has lagged behind those for the genera Orthoreovirus and Orbivirus, our group recently developed the first plasmid-based reverse genetics system for RV by cotransfecting plasmids encoding unique heterogeneous viral proteins, NBV FAST, and vaccinia virus capping enzyme as rescue enhancers along with the 11 RV T7 promoter-based rescue plasmids (26). In addition, reverse genetics systems were also developed for Bombyx mori cypovirus and Dendrolimus punctatus cypovirus (genus Cypovirus), which are insect pathogens and have a genome consisting of 10 segments of dsRNA (27, 28). Since reverse genetics systems were developed for Reoviridae viruses with genomes containing 10 or 11 segments of dsRNA, studies of the family Reoviridae have advanced markedly. However, to date, no reverse genetics system has been established for Reoviridae viruses with 12 genome segments.In this study, we isolated a TarTV strain from a raccoon dog postmortem. Using this TarTV strain, we established an entire plasmid-based reverse genetics system and rescued VP12 mutant viruses and a HiBiT-tagged reporter virus. This system is a useful tool to generate recombinant coltiviruses with 12 dsRNA genome segments.     

Combined treatment with dipeptidyl peptidase 4 (DPP4) inhibitor sitagliptin and elemental diets reduced indomethacin-induced intestinal injury in rats via the increase of mucosal glucagon-like peptide-2 concentration

The gut incretin glucagon-like peptide-1 (GLP-1) and the intestinotropic hormone GLP-2 are released from enteroendocrine L cells in response to ingested nutrients. Treatment with an exogenous GLP-2 analogue increases intestinal villous mass and prevents intestinal injury. Since GLP-2 is rapidly degraded by dipeptidyl peptidase 4 (DPP4), DPP4 inhibition may be an effective treatment for intestinal ulcers. We measured mRNA expression and DPP enzymatic activity in intestinal segments. Mucosal DPP activity and GLP concentrations were measured after administration of the DPP4 inhibitor sitagliptin (STG). Small intestinal ulcers were induced by indomethacin (IM) injection. STG was given before IM treatment, or orally administered after IM treatment with or without an elemental diet (ED). DPP4 mRNA expression and enzymatic activity were high in the jejunum and ileum. STG dose-dependently suppressed ileal mucosal enzyme activity. Treatment with STG prior to IM reduced small intestinal ulcer scores. Combined treatment with STG and ED accelerated intestinal ulcer healing, accompanied by increased mucosal GLP-2 concentrations. The reduction of ulcers by ED and STG was reversed by co-administration of the GLP-2 receptor antagonist. DPP4 inhibition combined with luminal nutrients, which up-regulate mucosal concentrations of GLP-2, may be an effective therapy for the treatment of small intestinal ulcers.     

Long-term effect of lamivudine treatment on the incidence of hepatocellular carcinoma in patients with hepatitis B virus infection

Background

Nucleotide analogues have recently been approved for the treatment of patients with hepatitis B virus (HBV) infection. However, it is still controversial whether the decrease of HBV-DNA amount induced by treatment with nucleotide analogues can reduce the risk of hepatocellular carcinoma (HCC) development in HBV patients.

Methods

A total of 293 HBV patients without HCC who were treated with lamivudine (LAM) were enrolled in a multicenter trial. The incidence of HCC was examined after the start of LAM therapy, and the risk factors for liver carcinogenesis were analyzed. The mean follow-up period was 67.6?±?27.4?months.

Results

On multivariate analysis for HCC development in all patients, age ≥50?years, platelet count <14.0?×?10⁴/mm³, cirrhosis, and median HBV-DNA levels of ≥4.0?log copies/ml during LAM treatment were significant risk factors. The cumulative carcinogenesis rate at 5?years was 3% in patients with chronic hepatitis and 30% in those with cirrhosis. For the chronic hepatitis patients, the log-rank test showed the significant risk factors related to HCC development to be age ≥50?years, platelet count <14.0?×?10⁴/mm³, and hepatitis B e antigen negativity, but median HBV-DNA levels of <4.0?log copies/ml (maintained viral response, MVR) did not significantly suppress the development of HCC. In cirrhosis patients, however, the attainment of MVR during LAM treatment was revealed to reduce the risk of HCC development.

Conclusions

These results suggest that the incidence of HCC in HBV patients with cirrhosis can be reduced in those with an MVR induced by consecutive LAM treatment.     

Antimicrobial activity of SM-17466, a novel carbapenem antibiotic with potent activity against methicillin-resistant Staphylococcus aureus.

The in vitro and in vivo antibacterial activities of SM-17466, a new 1 beta-methyl carbapenem, were evaluated against a wide range of clinical bacterial isoaltes and compared with the activities of meropenem, imipenem, vancomycin, and arbekacin. SM-17466 had a broad spectrum of action against gram-positive bacteria, showing especially potent activity against methicillin-resistant staphylococci. The MICs of SM-17466, meropenem, imipenem, vancomycin, and arbekacin at which 90% of clinical isolates of methicillin-resistant Staphylococcus aureus were inhibited were 3.13, 50, 100, 1.56, and 3.13 micrograms/ml, respectively. This activity of SM-17466 was almost equivalent to those of the antibiotics used for the treatment of infections caused by this organism. SM-17466 also showed bactericidal activity against methicillin-resistant S. aureus. In contrast, SM-17466 was less active against gram-negative bacteria, especially against Pseudomonas aeruginosa, compared with the other carbapenems; however, of the carbapenems, SM-17466 exhibited the highest activity against Haemophilus influenzae and Bacteriodes fragilis. SM-17466, at a 50% inhibitory concentration of less than 1 microgram/ml, bound to penicillin-binding proteins 1 to 4 in methicillin-susceptible S. aureus and also had good binding to penicillin-binding protein 2' in a methicillin-resistant strain (50% inhibitory concentration, 5.9 micrograms/ml). This high affinity, which was 10 and 20 times greater than those for meropenem and imipenem, respectively, was reflected in the potent activity of SM-17466 against methicillin-resistant S. aureus. SM-17466 demonstrated excellent in vivo efficacy against methicillin-susceptible and -resistant S. aureus strains in a mouse peritoneal infection model: the efficacy of SM-17466 against methicillin-resistant strains was equal to or one-third that of vancomycin. This activity was comparable to the in vitro activity of SM-17466. The subcutaneous injection of SM-17466 in mice revealed that the half-life of SM-17466 in serum was about 18 min, intermediate between those of vancomycin and arbekacin and 1.5-fold that of imipenem-cilastatin. SM-17466 was resistant to hydrolysis by swine renal dehydropeptidase I, to an extent comparable to the resistance shown by meropenem.     

Preventive effects of interleukin 1 β for ACNU-induced myelosuppression in malignant brain tumors: the experimental and preliminary clinical studies

Summary The effect of recombinant human interleukin 1 (rHuIL-1 ) on myelosuppression induced by 3-[(4-amino-2-methyl-5-pyrimidynyl)methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU) was studied. In in vivo study using BALB/c mice, pretreatment with 1 µ g/mouse of rHuIL-1 as a single intraperitoneal (i.p.) injection had a significant preventive effect on thrombocytopenia as well as granulocytopenia induced by ACNU at an intravenous dose of 60 mg/kg. Facilitated recovery by rHuIL-1 administered seven days after injection of high-dose ACNU was also observed. Experimental combination immunochemotherapy with high-dose ACNU and rHuIL-1 was performed in nude mice inoculated with human glioblastoma subcutaneously. The elongation of the survival time of the tumor bearing nude mice was also observed in combined use of high dose ACNU with rHuIL-1 . Seven patients with malignant brain tumors received intravenous 2.5-3 mg/kg ACNU. All patients were subcutaneously injected with 2 × 10⁴-U or more rHuIL-1 twice a week or daily. The mean nadir of leukocyte, granulocyte, and thrombocyte counts of the 7 patients received 2.5-3 mg/kg ACNU were significantly higher than in matched historical controls. In combination with rHuIL-1 , it may be possible to use chemotherapeutic agents at a relatively high dose.     

Control of Mechanical Properties of Thermoplastic Polyurethane Elastomers by Restriction of Crystallization of Soft Segment

Mechanical properties of thermoplastic polyurethane elastomers based on either polyether or polycarbonate (PC)-glycols, 4,4’-dipheylmethane diisocyanate (1,1’-methylenebis(4-isocyanatobenzene)), 1,4-butanediol, were controlled by restriction of crystallization of polymer glycols. For the polyether glycol based-polyurethane elastomers (PUEs), poly(oxytetramethylene) glycol (PTMG), and PTMG incorporating dimethyl groups (PTG-X) and methyl side groups (PTG-L) were employed as a polymer glycol. For the PC-glycol, the randomly copolymerized PC-glycols with hexamethylene (C₆) and tetramethylene (C₄) units between carbonate groups with various composition ratios (C₄/C₆ = 0/100, 50/50, 70/30 and 90/10) were employed. The degree of microphase separation and mechanical properties of both the PUEs were investigated using differential scanning calorimetry, dynamic viscoelastic property measurements and tensile testing. Mechanical properties could be controlled by changing the molar ratio of two different monomer components.     

Relationship of hypotension and cerebral vasospasm in patients with aneurysmal subarachnoid hemorrhage

OBJECTIVES: Changes in systemic arterial blood pressure and the degree of cerebral vasospasm were investigated in 125 patients with aneurysmal subarachnoid hemorrhage.METHODS: Systemic arterial blood pressure was measured every 2 hours in each patient for a period of more than 2 weeks, and a fall in systemic blood pressure (FBP) was defined as a decrease of >40 mmHg of systolic blood pressure between two consecutive measurements.RESULTS: A total of 91 FBPs occurred in 52 (41.6%) of 125 patients despite specific post-operative management to prevent hypovolemia. Five (5.5%) of the 91 FBPs occurred just before the onset of symptomatic vasospasm. Symptomatic vasospasm was observed in 36 (69.2%) of 52 patients with FBP and in 32 (43.8%) of 73 patients without FBP (p<0.01, chi-squared test). A hypodense area on computed tomographic scans in association with cerebral vasospasm was observed in 25 (48.1%) of 52 patients with FBP and in 21 (28.8%) of 73 patients without FBP (p<0.05).DISCUSSION: We conclude that FBP might result from delayed cerebral vasospasm and/or brain dysfunction owing to subarachnoid hemorrhage itself.