Preclinical Pharmacokinetics Evaluation of Anti-heparin-binding EGF-like Growth Factor (HB-EGF) Monoclonal Antibody Using Cynomolgus Monkeys via (89)Zr-immuno-PET Study and the Determination of Drug Concentrations in Serum and Cerebrospinal Fluid.

PURPOSE: Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family and is an important therapeutic target in some types of human cancers. KHK2866 is a humanized anti-HB-EGF monoclonal antibody IgG that neutralizes HB-EGF activity by inhibiting the binding of HB-EGF to its receptors. The phase I study of KHK2866 was discontinued because of neuropsychiatric toxicity. In this study, the pharmacokinetics of KHK2866 was evaluated by (89)Zr-immuno-PET study and the determination of drug concentrations in serum and cerebrospinal fluid using cynomolgus monkeys was performed in order to predict neurotoxicity in a reverse-translational manner. METHODS: KHK2866 was radiolabeled with (89)Zr for preclinical evaluations in normal cynomolgus monkeys and its distribution was analyzed. Furthermore, as a separate study, KHK2866 concentrations in serum and cerebrospinal fluid were determined after administration of a single dose. RESULTS: PET studies with monkeys revealed (89)Zr-KHK2866 accumulation in the liver, spleen and joints of multiple parts, but not in brain. In addition, the pharmacokinetic analyses in serum and CSF demonstrated a low penetration of KHK2866 into the brain. CONCLUSIONS: These studies indicate the difficulty of prediction for neuropsychiatric toxicity of monoclonal antibodies in human by means of pharmacokinetic evaluations using cynomolgus monkeys.

Three Cases of Acute Calcific Retropharyngeal Tendinitis.

Acute calcific retropharyngeal tendinitis is an inflammation of the longus coli muscle characterized by the acute onset of neck pain, swallowing pain, and limitations of neck movement. Although symptoms subside spontaneously within one to two weeks, many cases are treated with antibiotics because clinical outcomes are similar to a severe infection of the retropharyngeal area such as a retropharyngeal abscess. We report herein on 3 cases of acute calcific retropharyngeal tendinitis. The first and second cases were hospitalized, had many examinations and were diagnosed retrospectively. The third patient was treated as an outpatient after a CT scan. Typical CT imaging shows prevertebral soft-tissue swelling without ring enhancement, and amorphous calcification just anterior to the atlanto-axial joint, allowing us early diagnosis.

Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells.

Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs(flox/flox);mb1(cre/)(+):Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.

Adaptation of a duck influenza A virus in quail.

Quail are thought to serve as intermediate hosts of influenza A viruses between aquatic birds and terrestrial birds, such as chickens, due to their high susceptibility to aquatic-bird viruses, which then adapt to replicate efficiently in their new hosts. However, does replication of aquatic-bird influenza viruses in quail similarly result in their efficient replication in humans? Using sialic acid-galactose linkage-specific lectins, we found both avian (sialic acid-α2-3-galactose [Siaα2-3Gal] linkages on sialyloligosaccharides)--and human (Siaα2-6Gal)-type receptors on the tracheal cells of quail, consistent with previous reports. We also passaged a duck H3N2 virus in quail 19 times. Sequence analysis revealed that eight mutations accumulated in hemagglutinin (HA) during these passages. Interestingly, many of the altered HA amino acids found in the adapted virus are present in human seasonal viruses, but not in duck viruses. We also found that stepwise stalk deletion of neuraminidase occurred during passages, resulting in reduced neuraminidase function. Despite some hemagglutinin mutations near the receptor binding pocket, appreciable changes in receptor specificity were not detected. However, reverse-genetics-generated viruses that possessed the hemagglutinin and neuraminidase of the quail-passaged virus replicated significantly better than the virus possessing the parent HA and neuraminidase in normal human bronchial epithelial cells, whereas no significant difference in replication between the two viruses was observed in duck cells. Further, the quail-passaged but not the original duck virus replicated in human bronchial epithelial cells. These data indicate that quail can serve as intermediate hosts for aquatic-bird influenza viruses to be transmitted to humans.

Avian flu: influenza virus receptors in the human airway.

Although more than 100 people have been infected by H5N1 influenza A viruses, human-to-human transmission is rare. What are the molecular barriers limiting human-to-human transmission? Here we demonstrate an anatomical difference in the distribution in the human airway of the different binding molecules preferred by the avian and human influenza viruses. The respective molecules are sialic acid linked to galactose by an alpha-2,3 linkage (SAalpha2,3Gal) and by an alpha-2,6 linkage (SAalpha2,6Gal). Our findings may provide a rational explanation for why H5N1 viruses at present rarely infect and spread between humans although they can replicate efficiently in the lungs.

The current status and history of AFLAS.

The Asian federation of laboratory animal science associations (AFLAS) was established on November 29, 2003, and will celebrate its 20th anniversary in 2023. During this time, the number of AFLAS member associations and societies increased from six founders to eleven, and eight AFLAS congresses and 19 council meetings were held. In addition, the education and training system of laboratory animal science and technology funding program to support the activities of AFLAS member associations or societies started in 2015. Unfortunately, the COVID-19 pandemic had a great impact on the activities of AFLAS, and the 10th Congress which was scheduled to be held in Thailand in 2021 had to be canceled. AFLAS must have its members work together to overcome this difficult situation and further develop.

Toll-like receptor pre-stimulation protects mice against lethal infection with highly pathogenic influenza viruses.

Since the beginning of the 20th century, humans have experienced four influenza pandemics, including the devastating 1918 'Spanish influenza'. Moreover, H5N1 highly pathogenic avian influenza (HPAI) viruses are currently spreading worldwide, although they are not yet efficiently transmitted among humans. While the threat of a global pandemic involving a highly pathogenic influenza virus strain looms large, our mechanisms to address such a catastrophe remain limited. Here, we show that pre-stimulation of Toll-like receptors (TLRs) 2 and 4 increased resistance against influenza viruses known to induce high pathogenicity in animal models. Our data emphasize the complexity of the host response against different influenza viruses, and suggest that TLR agonists might be utilized to protect against lethality associated with highly pathogenic influenza virus infection in humans.

Metabolism of mono- and dichloro-dibenzo-p-dioxins by Phanerochaete chrysosporium cytochromes P450.

The white-rot fungus Phanerochaete chrysosporium possesses biodegradative capabilities of polychlorinated dibenzo-p-dioxins (PCDDs). One hundred twenty yeast clones expressing individual P450s of P. chrysosporum (PcCYPs), generated in our previous efforts, were screened for transformation of dioxin, and 40 positive clones were obtained. Of these clones, six clones showed metabolism of 2-chloro-dibenzo-p-dioxin, and a microsomal PcCYP designated as PcCYP11a3 showed much higher activity than any other PcCYPs. The turnover numbers of hydroxylation activities of PcCYP11a3 toward 1-MCDD (58 min(-1)) and 2-MCDD (13 min(-1)) are more than 200 times higher than those of previously reported PcCYP65a2. In addition, PcCYP11a3 catalyzes hydroxylation of 2,3-dichlorodibenzo-p-dioxin. To our best knowledge, PcCYP11a3 has the highest activity toward PCDDs among the known CYPs derived from microorganisms. Although PcCYP11a3 showed no detectable activity toward 2,7-dichloro-dibenzop-dioxin and 2,3,7-trichloro-dibenzo-p-dioxin, PcCYP11a3 is promising as a template whose activity would be enhanced by site-directed mutagenesis.

Randomized, Ascending Dose, Phase 2 Study of KHK4083, an Anti-OX40 Monoclonal Antibody, in Moderately Active Ulcerative Colitis.

Background: OX40 (CD134) plays a role in the maintenance of late T-cell proliferation and survival. KHK4083 is a monoclonal antibody directed against OX40. We aimed to assess the safety and preliminary efficacy of KHK4083 in patients with moderately active ulcerative colitis (UC). Methods: In this multicenter, double-blind, parallel-group, phase 2 study, patients with moderately active UC patients were randomized to ascending doses of intravenous KHK4083 (1, 3, or 10 mg/kg) or placebo every 2 weeks for 12 weeks. The primary endpoint was safety. The primary efficacy end point was the change from baseline in mean modified Mayo endoscopy subscore at week 12. Treatment with KHK4083 or placebo was continued every 4 weeks for up to 52 weeks in responders. Results: Long-term treatment with KHK4083 was well tolerated, with treatment-related adverse events being predominantly transient mild-to-moderate infusion-related reactions. Exploratory analysis of biopsy samples showed the virtually complete elimination of OX40+ cells in colon mucosa after 12 weeks of KHK4083 treatment. There were no significant differences between any of the randomized KHK4083 dose groups and placebo for the mean change in Mayo endoscopy subscore from baseline to week 12. Conclusions: KHK4083 can be safely administered intravenously at doses up to 10 mg/kg every 2 or 4 weeks for up to 52 weeks. Proof of pharmacodynamic action was confirmed by depletion of the elevated levels of the OX40+ cells associated with UC at all tested doses. Clinical response and mucosal healing (endoscopic improvement) in this population was not correlated with ablation of OX40+ T cells.

Corrigendum to: Randomized, Ascending Dose, Phase 2 Study of KHK4083, an Anti-OX40 Monoclonal Antibody, in Moderately Active Ulcerative Colitis.

[This corrects the article DOI: 10.1093/crocol/otaa049.].

Enzymatic properties of cytochrome P450 catalyzing 3'-hydroxylation of naringenin from the white-rot fungus Phanerochaete chrysosporium.

We cloned full-length cDNAs of more than 130 cytochrome P450s (P450s) derived from Phanerochaete chrysosporium, and successfully expressed 70 isoforms using a co-expression system of P. chrysosporium P450 and yeast NADPH-P450 reductase in Saccharomyces cerevisiae. Of these P450s, a microsomal P450 designated as PcCYP65a2 consists of 626 amino acid residues with a molecular mass of 68.3kDa. Sequence alignment of PcCYP65a2 and human CYP1A2 revealed a unique structure of PcCYP65a2. Functional analysis of PcCYP65a2 using the recombinant S. cerevisiae cells demonstrated that this P450 catalyzes 3'-hydroxylation of naringenin to yield eriodictyol, which has various biological and pharmacological properties. In addition, the recombinant S. cerevisiae cells expressing PcCYP65a2 metabolized such polyaromatic compounds as dibenzo-p-dioxin (DD), 2-monochloroDD, biphenyl, and naphthalene. These results suggest that PcCYP65a2 is practically useful for both bioconversion and bioremediation.

Inactivation of Escherichia coli endotoxin by soft hydrothermal processing.

Bacterial endotoxins, also known as lipopolysaccharides, are a fever-producing by-product of gram-negative bacteria commonly known as pyrogens. It is essential to remove endotoxins from parenteral preparations since they have multiple injurious biological activities. Because of their strong heat resistance (e.g., requiring dry-heat sterilization at 250 degrees C for 30 min) and the formation of various supramolecular aggregates, depyrogenation is more difficult than sterilization. We report here that soft hydrothermal processing, which has many advantages in safety and cost efficiency, is sufficient to assure complete depyrogenation by the inactivation of endotoxins. The endotoxin concentration in a sample was measured by using a chromogenic limulus method with an endotoxin-specific limulus reagent. The endotoxin concentration was calculated from a standard curve obtained using a serial dilution of a standard solution. We show that endotoxins were completely inactivated by soft hydrothermal processing at 130 degrees C for 60 min or at 140 degrees C for 30 min in the presence of a high steam saturation ratio or with a flow system. Moreover, it is easy to remove endotoxins from water by soft hydrothermal processing similarly at 130 degrees C for 60 min or at 140 degrees C for 30 min, without any requirement for ultrafiltration, nonselective adsorption with a hydrophobic adsorbent, or an anion exchanger. These findings indicate that soft hydrothermal processing, applied in the presence of a high steam saturation ratio or with a flow system, can inactivate endotoxins and may be useful for the depyrogenation of parenterals, including end products and medical devices that cannot be exposed to the high temperatures of dry heat treatments.

Mutant rat strain lacking D-amino-acid oxidase.

D-amino-acid oxidase (DAO) is known to be associated with schizophrenia. Since the expression of DAO gene had been reported to be very low in LEA rats, we examined LEA/SENDAI rats in detail. These rats did not have DAO activity, enzyme protein or mRNA encoding this enzyme. Sequencing of the 5'-upstream region of the DAO gene revealed the deletion of one triplet in the 15 TAA repeats approximately 700-bp upstream of the transcription start point. A 1.3-kb upstream fragment containing the TAA repeats and the transcription start point was inserted into a reporter vector and was transfected into COS-1, NRK-52E and CCL-PK1 cells. Although the fragments containing 15 or 14 repeats had high promoter activity, the fragment containing 13 repeats had very weak activity. Electrophoretic mobility-shift assays showed that the nuclear extracts from COS-1 and COS-7 cells had proteins that bound to the oligonucleotides containing the TAA repeats. These results suggest that the TAA repeats are important for expression of the DAO gene. The LEA/SENDAI rats lacking DAO would be a useful tool for the investigations aimed at the elucidation of the relationships between this flavoenzyme and schizophrenia.

Survey report on public awareness concerning the use of animals in scientific research in Japan.

We conducted a survey on 3,096 members of the public in order to find out about public awareness concerning the use of animals in scientific research in Japan and statistically analyzed the results. Regarding the necessity of experiments, research, and educational activities using animals, 55-62% respondents answered that "development of medicine and medical technology for human beings", "development of medicine and medical technology for animals", "practical training at medical schools", and "practical training at schools of veterinary medicine" were necessary, while 9-12% respondents answered that they were not necessary. These results showed that the Japanese public can dispassionately accept that animal experiments are necessary. Regarding the image of animal experiments, 50-70% respondents also supported animal experiments aimed at "advances in science and medicine", "securing of human health and safety", and "pursuit of economic interest". On the other hand, when faced with questions that featured emotional language, a majority (51-57% of people) felt that "animal experiments are painful and cruel acts" and that "experimental animals are to be pitied". This survey showed that the majority of the Japanese public can accept the necessity of animal experiments, but experts and researchers involved in animal experiments should consider seriously the large number of respondents that agreed with emotive descriptions of animal research.

An anti-ASCT2 monoclonal antibody suppresses gastric cancer growth by inducing oxidative stress and antibody dependent cellular toxicity in preclinical models.

Glutamine is a major nutrient for cancer cells during rapid proliferation. Alanine-serine-cysteine (ASC) transporter 2 (ASCT2; SLC1A5) mediates glutamine uptake in a variety of cancer cells. We previously reported that KM8094, a novel anti-ASCT2 humanized monoclonal antibody, possesses anti-tumor efficacy in gastric cancer patient-derived xenografts. The aim of this study was to investigate the molecular mechanism underlying the effect of KM8094 and to further substantiate the preclinical feasibility of using KM8094 as a potential therapeutic agent against gastric cancer. First, ASCT2 was found to be highly expressed in cancer tissues derived from gastric cancer patients by an immunohistochemical analysis. Next, we performed in vitro studies using multiple gastric cancer cell lines and observed that several gastric cancer cells expressing ASCT2 showed glutamine-dependent cell growth, which was repressed by KM8094. We found that KM8094 inhibited the glutamine uptake, leading to the reduction of glutathione (GSH) level and the elevation of oxidative stress. KM8094 suppressed the cell cycle progression and increased the apoptosis. Furthermore, KM8094 exerted antibody dependent cellular cytotoxicity (ADCC) against human gastric cancer cells in vitro. Finally, in vivo studies revealed that KM8094 suppressed tumor growth in several gastric cancer xenografts. This effect was enhanced by docetaxel, one of the agents commonly used in gastric cancer therapy. Thus, our findings suggest that KM8094 is a potential new therapeutic agent for gastric cancer expressing ASCT2, which blocks the cellular glutamine metabolism and possesses ADCC activity.

Anti-tumor efficacy evaluation of a novel monoclonal antibody targeting neutral amino acid transporter ASCT2 using patient-derived xenograft mouse models of gastric cancer.

ASC amino acid transporter 2 (ASCT2), also known as solute linked carrier family 1 member A5 (SLC1A5) is a Na+-dependent glutamine/neutral amino acid transporter. ASCT2 acts as a high-affinity transporter of L-glutamine (Gln) and has been reported to be up-regulated in a variety of cancerous tissues including stomach, liver, and kidney. In this study, we evaluated anti-tumor efficacy of a novel anti-ASCT2 humanized monoclonal antibody, KM8094, which has a neutralizing activity against glutamine uptake, as a therapeutic antibody against gastric cancer and explored clinical predictive biomarker candidates by utilizing patient-derived xenograft (PDX) mouse models. Anti-tumor efficacy studies revealed that some of the PDX models used were responsive to KM8094 and the others were not. Interestingly, we observed a correlation between anti-tumor efficacy and low antigen expression as well as low basal levels of glutamine uptake, suggesting ASCT2 expression level could be a potential predictive biomarker for KM8094. We then further explored predictive biomarker candidates by multi-omics analysis on gastric cancer PDX mouse models. As a result, a few potential candidates such as TFF2, MUC13, and ANG were selected by gene expression and DNA methylation array analyses. In addition, metabolomics analysis revealed clear differences in intracellular energy status and redox status between responsive and non-responsive PDX models.

Restoration of copper metabolism and rescue of hepatic abnormalities in LEC rats, an animal model of Wilson disease, by expression of human ATP7B gene.

Hepatic abnormalities in Long-Evans Cinnamon (LEC) rats, an animal model of Wilson disease (WD), were restored by the expression of the human ATP7B cDNA under the control of CAG promoter. Expression of ATP7B transcript and protein in the liver of the transgenic rats resulted in the restoration of biosynthesis of holoceruloplasmin and biliary copper excretion. Meanwhile, transgenic rats showed striking improvements in their hepatic abnormalities, i.e., rescue from fulminant hepatitis, late onset of hepatic cholangiofibrosis, suppression of hepatocellular carcinoma and much improved survival rates. Moreover, dramatic decreases were noted both in the levels of hepatic copper and iron in transgenic rats before the occurrence of hepatitis. These results indicated that the human ATP7B product compensated for the deficiency of the endogenous rattus protein and did function in intrahepatic copper transport by secreting copper into the plasma via incorporation into ceruloplasmin and by the excretion of copper into the bile, and that ATP7B is critical to hepatic dysfunctions in WD. This first successful transgenic rescue has important implications for the gene therapy of WD.

Experimental eradication of pinworms (Syphacia obvelata and Aspiculuris tetraptera) from mice colonies using ivermectin.

A spray administration of ivermectin was evaluated for the treatment of pinworm infection in mice. In this study, a spray of 0.1% ivermectin injectable solution over the entire cage once a week, for three consecutive weeks (one cycle treatment), was effective in eradicating both Syphacia obvelata and Aspiculuris tetraptera from mice under experimental conditions. In addition, no acute toxicity was observed in 105 mothers or 687 neonates treated with ivermectin, indicating that ivermectin does not affect murine reproduction. Finally, we attempted to eradicate pinworms from infected mice in our institute using this method. Two cycles of treatment were administered, with a two-week pause between cycles, resulting in complete eradication for at least one year. Treating mouse colonies with spray ivermectin is inexpensive, safe, requires very little labor and is very effective at eradicating pinworms from mice.

Effect of antigen-dependent clearance on pharmacokinetics of anti-heparin-binding EGF-like growth factor (HB-EGF) monoclonal antibody.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family and is an important therapeutic target in some types of human cancers. KM3566 is a mouse anti-HB-EGF monoclonal antibody that neutralizes HB-EGF activity by inhibiting the binding of HB-EGF to its receptors. Based on the results of our pharmacokinetics study, a humanized derivative antibody, KHK2866, is rapidly cleared from serum and shows nonlinear pharmacokinetics in cynomolgus monkeys. In this study, we examined the antigen-dependent clearance of an anti-HB-EGF monoclonal antibody in vivo and in vitro in order to pharmacokinetically explain the rapid elimination of KHK2866. We revealed tumor size-dependent clearance of KM3566 in in vivo studies and obtained good fits between the observed and simulated concentrations of KM3566 based on the two-compartment with a saturable route of clearance model. Furthermore, in vivo imaging analyses demonstrated tumor-specific distribution of KM3566. We then confirmed rapid internalization and distribution to lysosome of KM3566 at a cellular level. Moreover, we revealed that the amounts of HB-EGF on cell surface membrane were maintained even while HB-EGF was internalized with KM3566. Recycled or newly synthesized HB-EGF, therefore, may contribute to a consecutive clearance of KM3566, which could explain a rapid clearance from serum. These data suggested that the rapid elimination in pharmacokinetics of KM3566 is due to antigen-dependent clearance. Given that its antigen is expressed in a wide range of normal tissue, it is estimated that the rapid elimination of KHK2866 from cynomolgus monkey serum is caused by antigen-dependent clearance.

Visible, safe and certain endotracheal intubation using endoscope system and inhalation anesthesia for rats.

Anesthesia strongly influences laboratory animals, and it can also greatly affect the experimental data. Rats rank only second to mice in the number used in research fields, such as organ transplantation, regenerative medicine and imaging. Therefore, appropriate and effective anesthesia, including the protocol of the endotracheal intubation and inhalation anesthesia, is crucial. Hence, we evaluated these methods in this study. Twelve Wistar rats were intraperitoneally injected with M/M/B: 0.3/4/5, comprising of medetomidine, midazolam and butorphanol at a dose of 0.3 mg/kg + 4.0 mg/kg + 5.0 mg/kg body weight/rat, respectively. An endotracheal tube was then intubated into the trachea. After intubation, the rats were connected to the inhalation anesthesia circuit using isoflurane, and vital signs were measured until 30 min after connection. All intubations were successfully finished within 1 min, and the values of the vital signs were normal and stable. In addition, histopathological observation of the trachea and lungs showed no trauma. These results suggest that this visible endotracheal intubation method is simple, reliable, safe and favorable with regard to the rats' welfare.