Eita Sasaki

A Novel Mouse Model for Phenytoin-Induced Liver Injury: Involvement of Immune-Related Factors and P450-Mediated Metabolism

Drug-induced liver injury is an important issue for drug development and clinical drug therapy; however, in most cases, it is difficult to predict or prevent these reactions due to a lack of suitable animal models and the unknown mechanisms of action. Phenytoin (DPH) is an anticonvulsant drug that is widely used for the treatment of epilepsy. Some patients who are administered DPH will suffer symptoms of drug-induced liver injury characterized by hepatic necrosis. DPH-induced liver injury occurs in 1 in 1000 or 1 in 10 000 patients. Clinically, 75% of patients who develop liver injury develop a fever and 63% develop a rash. In this study, we established a mouse model for DPH-induced liver injury and analyzed the mechanisms for hepatotoxicity in the presence of immune-related or inflammation-related factors and metabolic activation. Female C57BL/6 mice were administered DPH for 5 days in combination with L-buthionine-S,R-sulfoximine. Then, the plasma alanine aminotransferase (ALT) levels were increased, hepatic lesions were observed during the histological evaluations, the hepatic glutathione levels were significantly reduced, and the oxidative stress marker levels were significantly increased. The inhibition of cytochrome P450-dependent oxidative metabolism significantly suppressed the elevated plasma ALT levels and depleted hepatic glutathione. Among the innate immune factors, the hepatic mRNA levels of NACHT, LRR, pyrin domain-containing protein 3, interleukin-1β, and damage-associated molecular patterns were significantly increased. Prostaglandin E₁ treatment ameliorated the hepatic injury caused by DPH. In conclusion, cytochrome P450-dependent metabolic activation followed by the stimulation of the innate immune responses is involved in DPH-induced liver injury.

Involvement of oxidative stress and immune- and inflammation-related factors in azathioprine-induced liver injury

Drug-induced liver injury (DILI) is a growing concern in the fields of drug development and clinical drug therapy because numerous drugs have been linked to hepatotoxicity. However, it is difficult to predict DILI in humans due to the lack of experimental animal models. Although azathioprine (AZA), which is a widely used immunosuppressive drug, is generally well tolerated, a small number of patients prescribed AZA develop severe hepatitis. However, the mechanism underlying this process has not yet been elucidated. In this study, we developed a mouse model of AZA-induced liver injury and investigated the mechanisms responsible for the hepatotoxicity of AZA. Female BALB/c mice were orally administered AZA. After AZA administration, the plasma levels of alanine aminotransferase and aspartate aminotransferase were increased, and liver damage was confirmed through a histological evaluation. In addition, the hepatic glutathione levels and superoxide dismutase activity were significantly decreased. The plasma levels of reactive oxygen species were significantly increased during the early phase of AZA-induced liver injury, and the hepatic mRNA levels of immune- and inflammation-related factors were also significantly changed. In conclusion, oxidative stress and the subsequently activated immune- and inflammation-related factors are involved in AZA-induced liver injury.

Carbamazepine-Induced Liver Injury Requires CYP3A-Mediated Metabolism and Glutathione Depletion in Rats

Carbamazepine (CBZ) is widely used as an antiepileptic agent and causes rare but severe liver injury in humans. It has been generally recognized that reactive metabolites formed via the metabolic activation reaction contribute to the onset of liver injuries by several drugs. However, the role of CBZ metabolism in the development of liver injury is not fully understood. In this study, we developed a novel rat model of CBZ-induced liver injury and attempted to elucidate the associated mechanisms by focusing on the metabolism of CBZ. The repeated administration of CBZ for 5 days in combination with l-buthionine sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor, resulted in increases in the plasma alanine aminotransferase (ALT) levels and centrilobular necrosis in the liver that were observed in various degrees. The CBZ and 2-hydroxy-CBZ concentrations in the plasma after the last CBZ administration were lower in the rats with high plasma ALT levels compared with those with normal plasma ALT levels, showing the possibility that the further metabolism of CBZ and/or 2-hydroxy-CBZ is associated with the liver injury. Although a single administration of CBZ did not affect the plasma ALT levels, even when cotreated with BSO, pretreatment with dexamethasone, a CYP3A inducer, increased the plasma ALT levels. In addition, the rats cotreated with troleandomycin or ketoconazole, CYP3A inhibitors, suppressed the increased plasma ALT levels. In conclusion, reactive metabolite(s) of CBZ produced by CYP3A under the GSH-depleted condition might be involved in the development of liver injury in rats.

A novel vaccinological evaluation of intranasal vaccine and adjuvant safety for preclinical tests.

Vaccines are administered to healthy humans, including infants, so the safety and efficacy must be very high. Therefore, evaluating vaccine safety in preclinical and clinical studies, according to World Health Organization guidelines, is crucial for vaccine development and clinical use. A change in the route of administration is considered to alter a vaccines immunogenicity. Several adjuvants have also been developed and approved for use in vaccines. However, the addition of adjuvants to vaccines may cause unwanted immune responses, including facial nerve paralysis and narcolepsy. Therefore, a more accurate and comprehensive strategy must be used to develope next-generation vaccines for ensuring vaccine safety. Previously, we have developed a system with which to evaluate vaccine safety in rats using a systematic vaccinological approach and 20 marker genes. In this study, we developed a safety evaluation system for nasally administered influenza vaccines and adjuvanted influenza vaccines using these marker genes. Expression of these genes increased dose-dependent manner when mice were intranasally administered the toxicity reference vaccine. When the adjuvant CpG K3 or a CpG-K3-combined influenza vaccine was administered intranasally, marker gene expression increased in a CpG-K3-dose-dependent way. A histopathological analysis indicated that marker gene expression correlated with vaccine- or adjuvant-induced phenotypic changes in the lung and nasal mucosa. We believe that the marker genes expression analyses will be useful in preclinical testing, adjuvant development, and selecting the appropriate dose of adjuvant in nasal administration vaccines.

Impact of the SCF signaling pathway on leukemia stem cell-mediated ATL initiation and progression in an HBZ transgenic mouse model

Adult T-cell leukemia (ATL) is a malignant disease caused by human T-lymphotropic virus type 1. In aggressive ATL, the response to chemotherapy is extremely poor. We hypothesized that this poor response is due to the existence of chemotherapy-resistant cells, such as leukemic stem cells. Previously, we successfully identified an ATL stem cell (ATLSC) candidate as the c-kit+/CD38−/CD71− cells in an ATL mouse model using Tax transgenic mice. Here, with a new ATL mouse model using HBZ-transgenic mice, we further discovered that the functional ATLSC candidate, which commonly expresses c-kit, is drug-resistant and has the ability to initiate tumors and reconstitute lymphomatous cells. We characterized the ATLSCs as c-kit+/CD4−/CD8− cells and found that they have a similar gene expression profile as T cell progenitors. Additionally, we found that AP-1 gene family members, including Junb, Jund, and Fosb, were up-regulated in the ATLSC fraction. The results of an in vitro assay showed that ATLSCs cultured with cytokines known to promote stem cell expansion, such as stem cell factor (SCF), showed highly proliferative activity and maintained their stem cell fraction. Inhibition of c-kit–SCF signaling with the neutralizing antibody ACK2 affected ATLSC self-renewal and proliferation. Experiments in Sl/Sld mice, which have a mutation in the membrane-bound c-kit ligand, found that ATL development was completely blocked in these mice. These results clearly suggest that the c-kit–SCF signal plays a key role in ATLSC self-renewal and in ATL initiation and disease progression.

Pathogenetic analyses of carbamazepine-induced liver injury in F344 rats focused on immune- and inflammation-related factors

Drug-induced liver injury is one of the major reasons for a drug to be withdrawn postmarketing. Carbamazepine (CBZ), an anticonvulsant agent, has been reported rarely to cause liver failure in humans. We recently generated a rat model of CBZ-induced liver injury using F344 rats for five consecutive days of CBZ administration combined with a glutathione (GSH) depletor, L-buthionine S,R-sulfoximine, treatment. The involvement of metabolic activation was demonstrated in developing CBZ-induced liver injury, and a difference in metabolic activation reactions between mice and rats was indicated. In this study, we analyzed the pathogenetic mechanism of CBZ-induced liver injury, primarily focusing on immune- and inflammation-related factors using the rat model for CBZ-induced liver injury. After the last CBZ administration, plasma alanine aminotransfearase (ALT) levels were drastically increased. In the histopathological evaluation, time-dependent hepatocellular degeneration and necrosis were observed in the centrilobular region. Different from mice, although hepatic mRNA expression levels of inflammation-related genes were increased, T-helper cell-related genes were not predominantly changed in rats. The number of ED1- and ED2-positive macrophages was increased in injured centrilobular areas in the liver with CBZ-induced liver injury. Treatment with a Kupffer cell depletor, gadolinium chloride, prevented the elevation of plasma ALT levels and an increase in the hepatic mRNA expression levels of inflammation-related genes. Hepatic adenosine triphosphate (ATP) contents were significantly decreased 24 h after CBZ administration. Therefore, the Kupffer cells-mediated inflammation was predominant in the development of the CBZ-induced liver injury in rats.

In vitro marker gene expression analyses in human peripheral blood mononuclear cells: A tool to assess safety of influenza vaccines in humans

Abstract Vaccines are inoculated in healthy individuals from children to the elderly, and thus high levels of safety and consistency of vaccine quality in each lot must meet the required specifications by using preclinical and lot release testing. Because vaccines are inoculated into humans, recapitulation of biological reactions in humans should be considered for test methods. We have developed a new method to evaluate the safety of influenza vaccines using biomarker gene expression in mouse and rat models. Some biomarker genes are already known to be expressed in human lymphocytes, macrophages and dendritic cells; therefore, we considered some of these genes might be common biomarkers for human and mice to evaluate influenza vaccine safety. In this study, we used human peripheral blood mononuclear cells (PBMC) as a primary assessment tool to confirm the usefulness of potential marker genes in humans. Analysis of marker gene expression in PBMC revealed biomarker gene expressions were dose-relatedly increased in toxic reference influenza vaccine (RE)-stimulated PBMC. Although some marker genes showed increased expression in hemagglutinin split vaccine-stimulated PBMC, their expression levels were lower than that of RE in PBMC from two different donors. Many marker gene expressions correlated with chemokine production. Marker genes such as IRF7 were associated with other Type 1 interferon (IFN)-associated signals and were highly expressed in the CD304+ plasmacytoid dendritic cell (pDC) population. These results suggest PBMC and their marker genes may be useful for vaccine safety evaluation in humans.

Role of cytochrome P450-mediated metabolism and identification of novel thiol-conjugated metabolites in mice with phenytoin-induced liver injury

Phenytoin, 5,5-diphenylhydantoin (DPH), is widely used as an anticonvulsant agent. Severe hepatic injury rarely occurs in patients who received DPH. The development of liver injury is thought to be caused by reactive metabolites; however, the metabolites suggested to contribute to hepatotoxicity have not yet been detected in vivo and their effect on developing the liver injury is largely unknown. We recently demonstrated that DPH treatment decreased hepatic glutathione (GSH) contents, and GSH-depleted condition exacerbated DPH-induced liver injury in mice. The aim of the present study was to identify the reactive metabolite and to investigate the role of P450-mediated metabolisms in DPH-induced liver injury. We identified a novel GSH-conjugated (GS)-DPH, a conjugate of putative electrophilic arene oxide intermediate with GSH, in the bile of mice with DPH-induced liver injury. In plasma, cysteine- or N-acetylcysteine-conjugated DPH was detected, and these thiol conjugates levels were correlated with the plasma alanine aminotransferase (ALT) levels. These changes were significantly reduced by pretreatment with P450 inhibitor. Furthermore, the increases of hepatic P450 activities were in parallel with elevation of plasma thiol conjugates levels. These findings suggest that the arene oxide intermediate, which can be converted to thiol conjugates, is involved in DPH-induced liver injury.

Establishment of a novel safety assessment method for vaccine adjuvant development

Vaccines effectively prevent infectious diseases. Many types of vaccines against various pathogens that threaten humans are currently in widespread use. Recently, adjuvant adaptation has been attempted to activate innate immunity to enhance the effectiveness of vaccines. The effectiveness of adjuvants for vaccinations has been demonstrated in many animal models and clinical trials. Although a highly potent adjuvant tends to have high effectiveness, it also has the potential to increase the risk of side effects such as pain, edema, and fever. Indeed, highly effective adjuvants, such as poly(I:C), have not been clinically applied due to their high risks of toxicity in humans. Therefore, the task in the field of adjuvant development is to clinically apply highly effective and non- or low-toxic adjuvant-containing vaccines. To resolve this issue, it is essential to ensure a low risk of side effects and the high efficacy of an adjuvant in the early developmental phases. This review summarizes the theory and history of the current safety assessment methods for adjuvants, using the inactivated influenza vaccine as a model. Our novel method was developed as a system to judge the safety of a candidate compound using biomarkers identified by genomic technology and statistical tools. A systematic safety assessment tool for adjuvants would be of great use for predicting toxicity during novel adjuvant development, screening, and quality control.

Gene expression profiling toward the next generation safety control of influenza vaccines and adjuvants in Japan

Influenza becomes epidemic worldwide every year, and many individuals receive vaccination annually. Quality control relating to safety and potency of influenza vaccines is important to maintain public confidence. The safety of influenza vaccines has been assessed by clinical trials, and animal safety tests are performed to monitor the consistent quality between vaccines used for clinical trials and marketing; the biological responses in vaccinated animals are evaluated, including changes in body weight and white blood cell count. Animal safety tests have been contributing to the quality relating to the safety of influenza vaccines for decades, but improvements are needed. Although precise mechanisms involving biological changes in animal safety tests have not been fully elucidated, the application of cDNA microarray technology make it possible to reliably identify genes related to biological responses in vaccinated animals. From analysis of the expression profile of >10,000 genes of lung in animals treated with an inactivated whole virion influenza vaccine, we identified 17 marker genes whose expression patterns correlated well to changes in body weight and leukocyte count in vaccinated animals. In influenza HA vaccine-treated animals exhibiting subtle changes in biological responses, a robust expression pattern of marker genes was found. Furthermore, these marker genes could also be used in the evaluation of adjuvanted influenza vaccines. The expression profile of marker genes is expected to be an alternative indicator for safety control of various influenza vaccines conferring high sensitivity and short turnaround time. Thus, gene expression profiling may be a powerful tool for safety control of vaccines in the future.