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Chemotherapy for peritoneal dissemination is poorly effective owing to limited drug transfer from the blood to the intraperitoneal (i.p.) compartment after intravenous (i.v.) administration. i.p. chemotherapy has been investigated to improve drug delivery to tumors; however, the efficacy continues to be debated. As anticancer drugs have low molecular weight and are rapidly excreted through the peritoneal blood vessels, maintaining the i.p. concentration as high as expected is a challenge. In this study, we examined whether i.p. administration is an efficient route of administration of high-molecular-weight immune checkpoint inhibitors (ICIs) for the treatment of peritoneal dissemination using a model of peritoneal disseminated carcinoma. After i.p. administration, the amount of anti-PD-L1 antibody transferred into i.p. tumors increased by approximately eight folds compared to that after i.v. administration. Intratumoral distribution analysis revealed that anti-PD-L1 antibodies were delivered directly from the i.p. space to the surface of tumor tissue, and that they deeply penetrated the tumor tissues after i.p. administration; in contrast, after i.v. administration, anti-PD-L1 antibodies were only distributed around blood vessels in tumor tissues via the enhanced permeability and retention (EPR) effect. Owing to the enhanced delivery, the therapeutic efficacy of anti-PD-L1 antibody in the peritoneal dissemination models was also improved after i.p. administration compared to that after i.v. administration. This is the first study to clearly demonstrate an EPR-independent delivery of ICIs to i.p. tumors by which ICIs were delivered in a massive amount to the tumor tissue via direct penetration after i.p. administration.

A cellular assay for evaluating the binding and internalization of biologics using primary human liver sinusoidal endothelial cells (LSEC) is not readily available, since human LSEC generally lose their receptor expression and internalization activity during the purifying processes and cell culturing. Here, we propose a novel cell-based assay using human liver non-parenchymal cells (NPC) as an alternative method using LSEC. To identify the LSEC population, NPC were stained with CD31 and CD45, and analyzed by flow cytometry. The expression of Fc gamma receptor IIB (FcγRIIB), one of the LSEC markers was detected in the CD31-positive and the CD45-negative fractions. The concentration-dependent binding and internalization of the anti-FcγRIIB antibody was also quantified in the LSEC fraction in human NPC. Saturated binding and internalization curves were obtained for the anti-FcγRIIB antibody. In the case of the negative control antibody, however, binding and internalization were negligible. The findings reported here indicate that cell-based assays using fresh human liver NPC will be useful for evaluating the binding and internalization of biologics as well as for determining pharmacokinetic parameters.

Lipid nanoparticles (LNPs) are one of the most successful technologies in mRNA delivery. While the liver has been the most frequent target for LNP delivery of mRNA, technologies for delivering mRNA molecules to extrahepatic tissues are also important. Herein, we report on the development of a LNP that targets secondary lymphoid tissues. We designed new types of alcohol-soluble phosphatidylserine (PS) derivatives as materials which target immune cells, and then incorporated them into LNPs using a microfluidic technique with a high degree of scalability and reproducibility. The resulting LNP that contained the synthesized PS delivered mRNA to the spleen much more efficiently compared to a control LNP. A sub-organ analysis revealed that the PS-loaded LNP was extensively taken up by tissue-resident macrophages in the red pulp and the marginal zone of the spleen. Thus, the PS-loaded LNP reported in this study would be a promising strategy for clinical applications that involve delivering mRNA to the spleen. This article is protected by copyright. All rights reserved.

BackgroundWith the increased use of immune checkpoint inhibitors (ICIs), side effects and toxicity are a great concern. Anaphylaxis has been identified as a potential adverse event induced by ICIs. Anaphylaxis is a life-threatening medical emergency. However, the mechanisms and factors that can potentially influence the incidence and severity of anaphylaxis in patients with cancer remain unclear.MethodsHealthy, murine colon 26, CT26, breast 4T1, EMT6, and renal RENCA tumor-bearing mice were treated with an anti-PD-L1 antibody (clone 10F.9G2). Symptoms of anaphylaxis were evaluated along with body temperature and mortality. The amounts of antidrug antibody and platelet-activating factor (PAF) in the blood were quantified via ELISA and liquid chromatography-mass spectrometry (LC-MS/MS). Immune cells were analyzed and isolated using a flow cytometer and magnetic-activated cell sorting, respectively.ResultsRepeated administration of the anti-PD-L1 antibody 10F.9G2 to tumor-bearing mice caused fatal anaphylaxis, depending on the type of tumor model. After administration, antidrug immunoglobulin G (IgG), but not IgE antibodies, were produced, and PAF was released as a chemical mediator during anaphylaxis, indicating that anaphylaxis was caused by an IgG-dependent pathway. Anaphylaxis induced by 10F.9G2 was treated with a PAF receptor antagonist. We identified that neutrophils and macrophages were PAF-producing effector cells during anaphylaxis, and the tumor-bearing models with increased numbers of neutrophils and macrophages showed lethal anaphylaxis after treatment with 10F.9G2. Depletion of both neutrophils and macrophages using clodronate liposomes prevented anaphylaxis in tumor-bearing mice.ConclusionsThus, increased numbers of neutrophils and macrophages associated with cancer progression may be risk factors for anaphylaxis. These findings may provide useful insights into the mechanism of anaphylaxis following the administration of immune checkpoint inhibitors in human subjects.

Toxoplasma gondii affects the health of humans and livestock and causes severe illness in the fetus and immunocompromised individuals. Because of the high incidence and severe consequences of T. gondii infection, a safe and suitable vaccine is needed. We found that lipid nanoparticles (LNPs) consisting of a series of functional materials prepared with vitamin E, such as SS-cleavable and pH-activated lipid-like materials (ssPalmE), were a safe and efficient way to develop next-generation DNA vaccines. In this study, we prepared ssPalmE-LNP to encapsulate pCpG-free-T. gondii dense granule protein 15 DNA (ssPalmE-LNPTgGRA15). Following a challenge infection with avirulent PLK strain of T. gondii, the mice immunized with ssPalmE-LNPTgGRA15 had a significantly higher survival rate and lower clinical scores compared with unimmunized and ssPalmE-LNPnon-coding-immunized mice. Immunization of mice with the ssPalmE-LNPTgGRA15 led to a significantly higher production of specific IgG1 and IG2c antibodies compared with unimmunized and ssPalmE-LNPnon-coding-immunized mice, while there was no statistically significant difference in the concentration of serum interferon-gamma at the acute stage of the infection. These findings indicate that ssPalmE-LNP is an effective cargo for the transportation of DNA vaccines for protozoan infections. To explore the mechanism of protective immunity induced by ssPalmE-LNPTgGRA15, further immunological study is needed in the future.

Systemically administered lipid nanoparticles (LNPs) are complexed with Apolipoprotein E (ApoE) in the bloodstream, and the complex is subsequently largely taken up by hepatocytes. Based on a previous report showing that, like blood, lymph fluid also contains ApoE, and that LECs, in turn, expresses a low density-lipoprotein receptor (LDLR), which is the receptor responsible for the ApoE-bound LNP, we hypothesized that subcutaneously administered LNPs would be taken up by LECs via an ApoE-LDLR pathway. Our in vitro studies using immortal LECs that we established in a previous study showed that LEC indeed took up LNPs in an ApoE-dependent manner. We then reported on the development of LNPs that target the lymphatic endothelium for in vivo siRNA delivery after subcutaneous administration. The key to success for in vivo LEC targeting is that the surface needs to be modified with a high density of polyethylene glycol (PEG)-conjugated lipids with short acyl chains (C14). The LNPs were drained into the lymphatic system, and then accumulated in lymphatic endothelial cells in an ApoE-dependent manner, most likely after the release of the PEG-lipid. Subcutaneous administration of optimized LNPs containing encapsulated siRNA against VEGFR3, a marker of LECs, significantly inhibited the expression of VEGFR3. These findings are the first report of a simple straightforward strategy for targeting lymphatic endothelial cells by using ionizable lipid-formulated LNPs.

The sentinel lymph node (LN) is the first LN to which lymph fluid flows from tumor tissue. We identified the key parameters of liposomes (LPs) that affect their accumulation in regional (primary) LNs with minimum leakage to its connecting (secondary) LNs by a comprehensive analysis of the LN-to-LN trafficking of LPs with various surface charges and various sizes. We used a lymphatic flow-modified (LFM) mouse that allows for the chronological analysis of inguinal (primary) LN-to-axillary (secondary) LN at the body surface. As a result, the anionic medium-sized LPs (130 nm on average) exhibited the highest accumulation in the primary LNs. A mechanism-based analysis revealed that CD169-positive macrophages in LNs were the dominant cell population that captures anionic LPs. Sentinel LN imaging was also performed by the intratumoral injection of fluorescent medium-sized anionic LPs using a breast cancer orthotopic model. In comparison with the typically used contrast agent indocyanine green, the anionic LPs were detected in sentinel LNs with a high sensitivity. Additionally, the co-injection of hyaluronidase significantly improved the sensitivity of detection of the fluorescent LPs in sentinel LNs. In conclusion, medium-sized anionic LPs combined with hyaluronidase represents a potent strategy for investigating sentinel LNs., Graphical Abstract, Comprehensive analysis on lymphatic trafficking of liposomes with various physicochemical properties revealed that anionic 130-nm liposomes preferably retained in the lymph node at which they first reached. This retention allowed for detection of sentinel lymph node with cancer models. Moreover, extracellular matrix degradation by hyaluronidase improved detection sensitivity.

Blood-brain barrier (BBB)-permeable middle- or macromolecules (middle/macromolecules) have recently attracted significant attention as new drug delivery carriers into the human brain via receptor-mediated transcytosis (RMT). During the development process of such carriers, it is necessary to thoroughly evaluate their human BBB permeability levels. In such evaluations, our recently established human immortalized cell-based multicellular spheroidal BBB models (hiMCS-BBB models) have shown high potential. However, the specifics of those capabilities have yet to be elucidated. Therefore, in this study, we characterize the ability of the hiMCS-BBB models to evaluate RMT-mediated BBB penetration properties of middle/macromolecules. More specifically, we began by validating transferrin receptor (TfR)-mediated RMT functionalities using transferrin in the hiMCS-BBB models and then examined the BBB permeability levels of MEM189 antibodies (known BBB-permeable anti-TfR antibodies). The obtained results showed that, as with the case of transferrin, temperature-dependent uptake of MEM189 antibodies was observed in the hiMCS-BBB models, and the extent of that uptake increased in a time-dependent manner until reaching a plateau after around 2 h. To further expand the evaluation applicability of the models, we also examined the BBB permeability levels of the recently developed SLS cyclic peptide and observed that peptide uptake was also temperature-dependent. To summarize, our results show that the hiMCS-BBB models possess the ability to evaluate the RMT-mediated BBB-permeable properties of antibodies and peptides and thus have the potential to provide valuable tools for use in the exploration and identification of middle/macromolecules showing excellent BBB permeability levels, thereby contributing powerfully to the development of new drug delivery carriers for transporting drugs into the human brain.

An autopsy of a patient in Japan with coronavirus disease indicated pneumonia lung pathology, manifested as diffuse alveolar damage. We detected severe acute respiratory syndrome coronavirus 2 antigen in alveolar epithelial cells and macrophages. Coronavirus disease is essentially a lower respiratory tract disease characterized by direct viral injury of alveolar epithelial cells.

Benzbromarone, a uricosuric drug, has the potential to cause serious hepatotoxicity. Several studies have shown the formation of reactive metabolites of benzbromarone and their association with hepatotoxicity in mice. However, it is unknown whether those reactive metabolites are generated in humans in vivo. In the present study, we firstly investigated the pharmacokinetic profiles of benzbromarone in chimeric TK-NOG mice transplanted with human hepatocytes (humanized-liver mice) and then investigated whether reactive metabolites could be generated. The area under the plasma concentration-time curve ratio of benzbromarone and its major metabolites (benzbromarone: 1'-hydroxy benzbromarone: 6-hydroxy benzbromarone) in humanized-liver mice was 1: 1.2: 0.7, which was similar to that reported in humans. In addition, glutathione conjugates and their further metabolites derived from the epoxidation of the benzofuran ring and 1',6-dihydroxylation of benzbromarone were detected in the livers, urine and plasma. Furthermore, their peak intensities in mass spectrometry showed markedly higher levels compared with those of TK-NOG mice. These results suggested that the metabolic profiles of benzbromarone in humanized-liver mice were similar to those in humans and that the reactive metabolites detected in humanized-liver mice could be generated and are associated with the benzbromarone-induced hepatotoxicity in humans.

Lymphatic endothelial cells (LECs) that form lymphatic vessels play a pivotal role in immune regulation. It was recently reported that LECs suppress the antigen-dependent anti-tumor immunity in cancer tissues. Thus, regulating the function of LECs is a promising strategy for cancer therapy. The objective of this study was to develop a method for the selective delivery of small interfering RNA (siRNA) to LECs. For this purpose, the siRNA was formulated into nanoparticles (LNPs) to prevent them from being degraded in body fluids and to facilitate their penetration of the cell membrane. A breakthrough technology for achieving this is ONPATTRO®, a world's first siRNA drug. Since LNPs are taken up by hepatocytes relatively well via low-density lipoprotein receptors, most of the LNP systems that have been developed so far target hepatocytes. In this study, we report on the development of a new method for the rapid and convenient method for modifying LNPs with antibodies using the CLick reaction on the Interface of the nanoParticle (CLIP). The CLIP approach was faster and more versatile than the conventional method using amide coupling. As a demonstration, we report on the LEC-targeted siRNA delivery by using antibody-modified LNPs both in vitro and in vivo. The method used for the modification of LNPs is highly promising and has the potential for expanding the LNP-based delivery of nucleic acids in the future.

Metastasis of cancer cells to lymph nodes (LN) is a common modality of metastasis in clinical settings, but the mechanisms involved in lymphatic metastasis remain unclear compared to hematogenous metastasis to bones and the brain. To elucidate the molecular mechanisms responsible for melanoma LN metastasis, we first generated LN metastasis-prone melanoma cells (C8161F2) by the sequential in vivo transplantation of parental melanoma cells (C8161F0). Although the in vitro/in vivo proliferative potential of these melanoma cells were similar, the metastatic potential of the C8161F2 for LNs was significantly enhanced. We then conducted a proteomics analysis to identify the proteins and pathways that contribute to LN metastasis. We identified six proteins (three: up-regulated and three: down-regulated) whose expressions were statistically significantly different by more than 2-fold in the two cell groups. Some of these genes are responsible for the activation of the transforming growth factor-β (TGF-β)-related pathway, a well-known inducer of epithelial-mesenchymal transition (EMT). In addition, a gene ontology analysis revealed that the enhanced cell-cell adhesion appears to be involved in lymphatic metastasis. In conclusion, we established highly lymphatic metastatic melanoma cells, which would be valuable for studies of the molecular mechanisms responsible for lymphatic metastasis.

The sentinel lymph node (SLN) is the first lymph node into which lymphatic fluid from tumor tissues flows. The development of a highly sensitive probe for detecting SLNs is desired for the lymph node dissection through intraoperative biopsy. We have previously shown that anionic liposomes tend to accumulate in lymph nodes and that macrophage uptake of liposomes contributes to their accumulation. In the present study, we found that among anionic lipids, phosphatidylserine (PS)-containing liposomes were substantially taken up by macrophages. We identified a new lipid composition to improve the SNL-selectivity of liposome accumulation based on Design-of-Experiment. The optimized PS-containing particles were more selectively accumulate to SLN lymph nodes than existing imaging agents indocyanine green. These results indicate the effectiveness of PS-containing anionic particles in SLN imaging.

The second messenger 2′3′-cyclic-GMP-AMP (cGAMP) is thought to be transmitted from brain carcinomas to astrocytes via gap junctions, which functions to promote metastasis in the brain parenchyma. In the current study, we established a method to introduce cGAMP into astrocytes, which simulates the state of astrocytes that have been invaded by cGAMP around tumors. Astrocytes incorporating cGAMP were analyzed by metabolomics, which demonstrated that cGAMP increased glutamate production and astrocyte secretion. The same trend was observed for γ-aminobutyric acid (GABA). Conversely, glutamine production and secretion were decreased by cGAMP treatment. Due to the fundamental role of astrocytes in regulation of the glutamine–glutamate cycle, such metabolic changes may represent a potential mechanism and therapeutic target for alteration of the central nervous system (CNS) environment and the malignant transformation of brain carcinomas.

The GALA peptide (WEAALAEALAEALAEHLAEALAEALEALAA) was originally designed to induce the destabilization of endosomal membranes based on its ability to undergo a pH-dependent conformational change from a random coil to an α-helix. We recently found that liposomes modified with GALA peptide (GALA-LPs) extensively accumulate in lung endothelial cells (ECs) after intravenous injection. However, the uptake mechanism of GALA-LPs and their ability to reach alveolar epithelium was unclear. We report herein that GALA-LPs are internalized into ECs via a clathrin-mediated pathway. Surprisingly, GALA-LPs had the ability to pass lung ECs and reach other cells through transcytosis. GALA-LPs were taken up by >70% of lung ECs, while they also accumulated in ~30% of type I alveolar epithelium (ATI). GALA-modified gold nanoparticles were detected in ECs, in the basement membrane and in other cells such as ATI, type II alveolar epithelium (ATII) and alveolar macrophages. Consistent with this result, a significant gene knockdown was achieved in lung epithelium cells using GALA-LPs encapsulating anti-podoplanin siRNA. This indicates that GALA-LPs can be used as a carrier for delivering macromolecules to parenchymal as well as to endothelial cells in the lung. Although caveolae are commonly linked to the transendothelial transport of proteins and antibodies, our data indicate that clathrin-mediated endocytosis might also participate in the transcytosis process.

While the use of in vitro-transcribed mRNA (IVT-mRNA) in therapeutics is a rapidly expanding area, the transfection of the exogenous IVT-mRNA is accompanied by a risk of immune activation. This immunological defense mechanism suppresses cellular translation process and can reduce transfection efficiency to a considerable extent. In the present study, we investigated the in vitro effects of Integrated Stress Response Inhibitor (ISRIB), and dexamethasone, a steroidal anti-inflammatory drug, on the transfection activity of a lipid nanoparticle (LNP) that was composed of ionizable lipids and IVT-mRNA. In the case of transfection to mouse embryonic fibroblast (MEF) cells, ISRIB mainly enhanced the transfection activity at an early stage of transfection (0-6 h). In contrast, dexamethasone caused an increase in transfection activity at intermediate-late stages of transfection (4-48 h). We also investigated the in vivo effects of dexamethasone using an LNP on that the IVT-mRNA and lipid-conjugated dexamethasone (Dex-Pal) were co-loaded. The intravenous administration of the LNP successfully enhanced the protein expression in a mouse liver by up to 6.6-fold. Collectively, the co-delivery of an anti-inflammatory drug is a promising approach for enhancing transfection efficiency of IVT-mRNA.

In vitro blood-brain barrier (BBB) models are essential research tools for use in developing brain-targeted drugs and understanding the physiological and pathophysiological functions of the BBB. To develop BBB models with better functionalities, three-dimensional (3D) culture methods have gained significant attention as a promising approach. In this study, we report on the development of a human conditionally immortalized cell-based multicellular spheroidal BBB (hiMCS-BBB) model. After being seeded into non-attachment culture wells, HASTR/ci35 (astrocytes) and HBPC/ci37 cells (brain pericytes) self-assemble to form a spheroid core that is then covered with an outer monolayer of HBMEC/ci18 cells (brain microvascular endothelial cells). The results of immunocytochemistry showed the protein expression of several cellular junction and BBB-enriched transporter genes in HBMEC/ci18 cells of the spheroid model. The permeability assays showed that the hiMCS-BBB model exhibited barrier functions against the penetration of dextran (5 and 70 kDa) and rhodamine123 (a P-glycoprotein substrate) into the core. On the other hand, facilitation of 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose (2-NBDG; a fluorescent glucose analog) uptake was observed in the hiMCS-BBB model. Furthermore, tumor necrosis factor-alpha treatment elicited an inflammatory response in HBMEC/ci18 cells, thereby suggesting that BBB inflammation can be recapitulated in the hiMCS-BBB model. To summarize, we have developed an hiMCS-BBB model that possesses fundamental BBB properties, which can be expected to provide a useful and highly accessible experimental platform for accelerating various BBB studies.

Evaluation of the binding and uptake of an antibody in liver non-parenchymal cells (NPC), including liver sinusoidal endothelial cells, is important for revealing its pharmacokinetic (PK) behavior, since NPC has important roles in eliminating an antibody from the blood via the Fc fragment of IgG receptor IIB (FcγRIIB). However, there is currently no in vitro quantitative assay using NPC. This study reports on the development of a cell-based assay for evaluating the binding and uptake of such an antibody using liver NPC of mice and monkeys. In mice, the FcγRIIB-expressing cells were identified in the CD146-positive and CD45-negative fraction by flow cytometry. A titration assay was performed to determine the PK parameters, and the obtained parameter was comparable to that determined by the fitting of the in vivo PK. This approach was also extended to NPC from monkeys. The concentration-dependent binding and uptake was measured to determine the PK parameters using monkey NPC, the FcγRIIB-expressing fraction of which was identified by CD31 and CD45. The findings presented herein demonstrate that the in vitro liver NPC assay using flow cytometry is a useful tool to determine the binding and uptake of biologics and to predict the PK.

The world-first success of lipid nanoparticle (LNP)-based siRNA therapeutics (ONPATTRO®) promises to accelerate developments in siRNA therapeutics/gene therapy using LNP-type drug delivery systems (DDS). In this study, we explore the optimal composition of an LNP containing a self-degradable material (ssPalmO-Phe) for the delivery of oligonucleotides. siRNA or antisense oligonucleotides (ASO) were encapsulated in LNP with different lipid compositions. The hepatic knockdown efficiency of the target genes and liver toxicity were evaluated. The optimal compositions for the siRNA were different from those for ASO, and different from those for mRNA that were reported in a previous study. Extracellular stability, endosomal escape and cellular uptake appear to be the key processes for the successful delivery of mRNA, siRNA and ASO, respectively. Moreover, the compositions of the LNPs likely contribute to their toxicity. The lipid composition of the LNP needs to be optimized depending on the type of nucleic acids under consideration if the applications of LNPs are to be further expanded.

We herein report 2 cases of gastric cancer treated by S-1 and oxaliplatin combination therapy before later undergoing gastrectomy. The pathological results of both cases demonstrated complete response. Case 1 had a giant tumor which was suspected to have invaded the pancreas. Case 2 was associated with extensive lymph node metastasis. Based on the findings of these 2 cases, preoperative chemotherapy with S-1 and oxaliplatin for advanced gastric cancer shows sufficient efficacy.

It is important to predict drug-drug interactions via inhibition of intestinal cytochrome P450 3A (CYP3A) which is a determinant of bioavailability of orally administered CYP3A substrates. However, inhibitory effects of macrolide antibiotics on CYP3A-mediated metabolism are not entirely identical between humans and rodents.We investigated the effects of macrolide antibiotics, clarithromycin and erythromycin, on in vitro and in vivo metabolism of triazolam, a CYP3A substrate, in CYP3A-humanised mice generated by using a mouse artificial chromosome vector carrying a human CYP3A gene.Metabolic activities of triazolam were inhibited by macrolide antibiotics in liver and intestine microsomes of CYP3A-humanised mice.The area under the plasma concentration-time curve ratios of 4-hydroxytriazolam to triazolam after oral dosing of triazolam were significantly decreased by multiple administration of macrolide antibiotics. The plasma concentrations ratios of α-hydroxytriazolam and 4-hydroxytriazolam to triazolam in portal blood were significantly decreased by multiple administration of clarithromycin in CYP3A-humanised mice.These results suggest that intestinal CYP3A activity was inhibited by macrolide antibiotics in CYP3A-humanised mice in vitro and in vivo. The plasma concentrations of triazolam and its metabolites in the portal blood of CYP3A-humanised mice would be useful for direct evaluation of intestinal CYP3A-mediated drug-drug interactions. It is important to predict drug-drug interactions via inhibition of intestinal cytochrome P450 3A (CYP3A) which is a determinant of bioavailability of orally administered CYP3A substrates. However, inhibitory effects of macrolide antibiotics on CYP3A-mediated metabolism are not entirely identical between humans and rodents. We investigated the effects of macrolide antibiotics, clarithromycin and erythromycin, on in vitro and in vivo metabolism of triazolam, a CYP3A substrate, in CYP3A-humanised mice generated by using a mouse artificial chromosome vector carrying a human CYP3A gene. Metabolic activities of triazolam were inhibited by macrolide antibiotics in liver and intestine microsomes of CYP3A-humanised mice. The area under the plasma concentration-time curve ratios of 4-hydroxytriazolam to triazolam after oral dosing of triazolam were significantly decreased by multiple administration of macrolide antibiotics. The plasma concentrations ratios of α-hydroxytriazolam and 4-hydroxytriazolam to triazolam in portal blood were significantly decreased by multiple administration of clarithromycin in CYP3A-humanised mice. These results suggest that intestinal CYP3A activity was inhibited by macrolide antibiotics in CYP3A-humanised mice in vitro and in vivo. The plasma concentrations of triazolam and its metabolites in the portal blood of CYP3A-humanised mice would be useful for direct evaluation of intestinal CYP3A-mediated drug-drug interactions.