[Contribution to the world of 3D cell products created by bio 3D printing technology and to the life science field].

We have been making 3D tissues consist of cells only, based on the corporate philosophy of "contributing to dramatic advances in medical care through the practical application of innovative 3D cell stacking technology." Currently, in the field of regenerative medicine, we are working toward obtaining approval from the Ministry of Health, Labor and Welfare and commercializing large artificial organs that are made from patients' own cells and have functions such as nerve regeneration, osteochondral regeneration, and blood vessels. On the other hand, this three-dimensional cell stacking technology can be extended to technology for culturing cells in an environment similar to the human body, and is expected to serve as a new methodology for evaluating the effects of new products in various fields on living organisms. Therefore, we are planning a business to provide developers of pharmaceuticals, foods, cosmetics, etc. with a small device called "Functional Cell Device (FCD)" that reproduces some of the functions of human organs outside the body. As the first step, we have developed a three-dimensional liver construct (3D mini-liver). The in vitro human liver model has a wide range of usage, such as evaluation of hepatotoxicity of drugs, elucidation of drug metabolism mechanism, and model of liver disease. In this report, we will outline it together with actual examples in regenerative medicine.

[Development of 3D cellular products based on unique AM technology, and its contribution to medical care].

Cyfuse Biomedical K.K. is a R&D venture company established in 2010 aiming at industrialization of its 3D cellular products for regenerative medicine based on innovative 3D cell stacking technology, and has newly listed on the Growth Market of the Tokyo Stock Exchange in December 2022. We are developing 3D cellular products consisted of only human cells through our unique platform technology created from the fusion of two disparate technologies; engineering and biology. Three pipelines aiming for approval as products for regenerative medicine have already advanced to the stage of human clinical trials, and are expected to implemented in society in the near future. In addition, we also have been developing functional cellular devices (FCD) using our core technology, the Kenzan method, and have begun marketing FCD as support tools that contribute to the development of new drugs. We will introduce in detail our contribution to the medical field that we are aiming for, with specific examples, also referring to the unique additive manufacturing (AM) technology that supports the realization of our cellular products as above.

Serum soluble triggering receptor expressed on myeloid cells-2 was not altered by rTMS in patients with treatment-resistant depression.

AIM: Repetitive transcranial magnetic stimulation (rTMS) is one of the most effective and minimally invasive treatments for treatment-resistant depression (TRD). However, the mechanism underlying the therapeutic effects of rTMS in patients with TRD remains unclear. In recent years, the pathogenesis of depression has been closely associated with chronic inflammation and microglia are believed to play an important role in chronic inflammation. Triggering receptor expressed on myeloid cells-2 (TREM2) plays an important role in microglial neuroinflammatory regulation. In this study, we investigated the changes in peripheral soluble TREM2 (sTREM2) before and after rTMS treatment in patients with TRD. METHODS: Twenty-six patients with TRD were enrolled in this frequency (10 Hz) rTMS study. Depressive symptoms, cognitive function, and serum sTREM2 concentrations were measured at baseline and the end of the 6-week rTMS treatment. RESULTS: This study showed that rTMS ameliorated depressive symptoms and partially improved cognitive dysfunction in TRD. However, rTMS treatment did not alter serum sTREM2 levels. CONCLUSIONS: This is the first sTREM2 study in patients with TRD who underwent rTMS treatment. These results suggest that serum sTREM2 may not be relevant for the mechanism underlying the therapeutic effect of rTMS in patients with TRD. Future studies should confirm the present findings using a larger patient sample and a sham rTMS procedure, as well as CSF sTREM2. Furthermore, a longitudinal study should be conducted to clarify the effects of rTMS on sTREM2 levels.

Effects of spatially filtered fearful faces and awareness on amygdala activity in adults with autism spectrum disorder: A magnetoencephalography study.

BACKGROUND: The amygdala is pivotal in emotional face processing. Spatial frequencies (SFs) of visual images are divided and processed via two visual pathways: low spatial frequency (LSF) information is conveyed by the magnocellular pathway, while the parvocellular pathway carries high spatial frequency information. We hypothesized that altered amygdala activity might underlie atypical social communication caused by changes in both conscious and non-conscious emotional face processing in the brain in individuals with autism spectrum disorder (ASD). METHOD: Eighteen adults with ASD and 18 typically developing (TD) peers participated in this study. Spatially filtered fearful- and neutral-expression faces and object stimuli were presented under supraliminal or subliminal conditions, and neuromagnetic responses in the amygdala were measured using 306-channel whole-head magnetoencephalography. RESULTS: The latency of the evoked responses at approximately 200 ms to unfiltered neutral face stimuli and object stimuli in the ASD group was shorter than that in the TD group in the unaware condition. Regarding emotional face processing, the evoked responses in the ASD group were larger than those in the TD group under the aware condition. The later positive shift during 200-500 ms (ARV) was larger than that in the TD group, regardless of awareness. Moreover, ARV to HSF face stimuli was larger than that to the other spatial filtered face stimuli in the aware condition. CONCLUSION: Regardless of awareness, ARV might reflect atypical face information processing in the ASD brain.

Asynchronous neural oscillations associated with subliminal affective face priming in autism spectrum disorder.

Autism spectrum disorder (ASD) is characterized by social communicative disturbance. Social communication requires rapid processing and accurate cognition regarding others' emotional expressions. Previous electrophysiological studies have attempted to elucidate the processes underlying atypical face-specific N170 responses to emotional faces in ASD. The present study explored subliminal affective priming effects (SAPEs) on the N170 response and time-frequency analysis of intertrial phase coherence (ITPC) for the N170 in ASD. Fifteen participants [seven participants with ASD and eight typically developing (TD) controls] were recruited for the experiment. Event-related potentials were recorded with a 128-channel electroencephalography device while participants performed an emotional face judgment task. The results revealed enhanced N170 amplitude for supraliminal target-face stimuli when they were preceded by subliminal fearful-face stimuli, in both the ASD and TD groups. Interestingly, TD participants exhibited higher alpha-ITPC in the subliminal fearful-face priming condition in the right face-specific area in the N170 time window. In contrast, there were no significant differences in ITPC in any frequency bands between the subliminal fearful and neutral priming conditions in the ASD group. Asynchronous phase-locking neural activities in the face-specific area may underlie impaired nonconscious face processing in ASD, despite the presence of common features of SAPEs for the N170 component in both the ASD and TD groups.

Changes in the metabolites of cerebrospinal fluid induced by rTMS in treatment-resistant depression: A pilot study.

Repetitive transcranial magnetic stimulation (rTMS) improves depressive symptoms in treatment-resistant depression (TRD). This study aimed to analyze changes in cerebrospinal fluid (CSF) metabolites in patients with TRD after rTMS. Five patients with TRD were enrolled in a high frequency (10-Hz) rTMS study. The concentration of 72 CSF metabolites were measured at baseline and at the end of the 6-week rTMS treatment. rTMS significantly increased CSF niacinamide, kynurenine, and creatinine levels and significantly decreased CSF cystine levels, but not the levels of the other 68 CSF metabolites. This is the first CSF metabolomics study on patients with TRD who underwent rTMS.

Weighted Blind Source Separation Can Decompose the Frequency Mismatch Response by Deviant Concatenation: An MEG Study.

The mismatch response (MMR) is thought to be a neurophysiological measure of novel auditory detection that could serve as a translational biomarker of various neurological diseases. When recorded with electroencephalography (EEG) or magnetoencephalography (MEG), the MMR is traditionally extracted by subtracting the event-related potential/field (ERP/ERF) elicited in response to "deviant" sounds that occur randomly within a train of repetitive "standard" sounds. However, there are several problems with such a subtraction, which include increased noise and the neural adaptation problem. On the basis of the original theory underlying MMR (i.e., the memory-comparison process), the MMR should be present only in deviant epochs. Therefore, we proposed a novel method called weighted-BSS T/k, which uses only the deviant response to derive the MMR. Deviant concatenation and weight assignment are the primary procedures of weighted-BSS T/k, which maximize the benefits of time-delayed correlation. We hypothesized that this novel weighted-BSS T/k method highlights responses related to the detection of the deviant stimulus and is more sensitive than independent component analysis (ICA). To test this hypothesis and the validity and efficacy of the weighted-BSS T/k in comparison with ICA (infomax), we evaluated the methods in 12 healthy adults. Auditory stimuli were presented at a constant rate of 2 Hz. Frequency MMRs at a sensor level were obtained from the bilateral temporal lobes with the subtraction approach at 96-276 ms (the MMR time range), defined based on spatio-temporal cluster permutation analysis. In the application of the weighted-BSS T/k, the deviant responses were given a constant weight using a rectangular window on the MMR time range. The ERF elicited by the weighted deviant responses demonstrated one or a few dominant components representing the MMR that fitted well with that of the sensor space analysis using the conventional subtraction approach. In contrast, infomax or weighted-infomax revealed many minor or pseudo components as constituents of the MMR. Our single-trial, contrast-free approach may assist in using the MMR in basic and clinical research, and it opens a new and potentially useful way to analyze event-related MEG/EEG data.

A novel time-delayed correlation method decomposes mismatch response without using subtraction.

The mismatch response (MMR) is thought to be a neurophysiological measure of novel auditory detection that could serve as a translational biomarker of various neurological diseases. When recorded with electroencephalography (EEG) or magnetoencephalography (MEG), the MMR is traditionally extracted by subtracting the event-related potential/field (ERP/ERF) elicited in response to "deviant" sounds that occur randomly within a train of repetitive "standard" sounds. To overcome the limitations of this subtraction procedure, we propose a novel method which we call weighted-BSST/k, which uses only the deviant response to derive the MMR. We hypothesized that this novel weighted-BSST/k method highlights responses related to the detection of the deviant stimulus and is more sensitive than independent component analysis (ICA). To test this hypothesis and the validity and efficacy of the weighted-BSST/k in comparison with ICA (infomax), we evaluated the methods in 12 healthy adults. Auditory stimuli were presented at a constant rate of 2 Hz. Frequency MMRs at a sensor level were obtained from the bilateral temporal lobes with the subtraction approach at 96-276 ms (the MMR time range), defined on the basis of spatio-temporal cluster permutation analysis. In the application of the weighted-BSST/k, the deviant responses were given a constant weight on the MMR time range. The ERF elicited by the weighted deviant responses demonstrated one or a few dominant components representing the MMR with a high signal-to-noise ratio and similar topography to that of the sensor space analysis using the subtraction approach. In contrast, infomax or weighted-infomax revealed many minor or pseudo components as constituents of the MMR. Our new approach may assist in using the MMR in basic and clinical research.Clinical Relevance-Our proposed method opens a new and potentially useful way to analyze event-related MEG/EEG data.

Biodistribution studies for cell therapy products: Current status and issues.

Information on the biodistribution (BD) of cell therapy products (CTPs) is essential for prediction and assessment of their efficacy and toxicity profiles in non-clinical and clinical studies. To conduct BD studies, it is necessary to understand regulatory requirements, implementation status, and analytical methods. This review aimed at surveying international and Japanese trends concerning the BD study for CTPs and the following subjects were investigated, which were considered particularly important: 1) comparison of guidelines to understand the regulatory status of BD studies in a global setting; 2) case studies of the BD study using databases to understand its current status in cell therapy; 3) case studies on quantitative polymerase chain reaction (qPCR) used primarily in non-clinical BD studies for CTPs; and 4) survey of imaging methods used for non-clinical and clinical BD studies. The results in this review will be a useful resource for implementing BD studies.

The changes in kynurenine metabolites induced by rTMS in treatment-resistant depression: A pilot study.

BACKGROUND: Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive brain stimulation technique that is considered a valuable and promising technique for improving depressive symptoms in treatment-resistant depression (TRD). However, the exact mechanism by which rTMS ameliorates depressive symptoms remains to be clarified. OBJECTIVE: The aim of the present study was to analyzed the changes in metabolites of patients with TRD in the rTMS treatment, especially focusing on the kynurenine (KYN) pathway. METHODS: Thirteen participants with TRD were enrolled in a high-frequency (10 Hz) rTMS study. Cognitive function, depressive symptoms and the concentration of plasma tryptophan (TRP) metabolites were measured at baseline and at the endpoint of rTMS treatment. RESULTS: rTMS treatment significantly improved depressive symptom scores and some subscales of cognitive dysfunction. The present study has demonstrated that rTMS treatment significantly increased plasma TRP levels and significantly decreased plasma serotonin levels, while plasma KYN and kynurenic acid level as well as KYN/TRP ratio remained unchanged. CONCLUSIONS: This is the first metabolomic study of patients with TRD undergoing rTMS treatment. To validate the present results, it is necessary to increase the number of cases including controls, use a sample of cerebrospinal fluid, and measure blood concentration over time in the course of rTMS treatment.

Gender differences in subliminal affective face priming: A high-density ERP study.

INTRODUCTION: Subliminal affective priming effects (SAPEs) refer to the phenomenon by which the presentation of an affective prime stimulus influences the subsequent affective evaluation of a target stimulus. Previous studies have reported that unconsciously processed stimuli affect behavioral performance more than consciously processed stimuli. However, the impact of SAPEs on the face-specific N170 component is unclear. We studied how SAPEs for fearful faces affected the N170 for subsequent supraliminal target faces using event-related potentials (ERPs). METHODS: Japanese adults (n = 44, 20 females) participated in this study. Subliminal prime faces (neutral or fearful) were presented for 17 ms, followed by a backward mask for 283 ms and 800 ms target faces (neutral, emotionally ambiguous, or fearful). 128-channel ERPs were recorded while participants judged the expression of target faces as neutral or fearful. Response rates and response times were also measured for assessing behavioral alterations. RESULTS: Although the behavioral results revealed no evidence of SAPEs, we found gender-related SAPEs in right N170 amplitude. Specifically, female participants exhibited enhanced right N170 amplitude for emotionally neutral faces primed by fearful faces, while male participants exhibited decreased N170 amplitude in fearful prime trials with fearful target faces. Male participants exhibited significant correlations between N170 amplitude and behavioral response time in the fearful prime-neutral target condition. CONCLUSIONS: Our ERP results suggest the existence of a gender difference in target-face processing preceded by subliminally presented face stimuli in the right occipito-temporal region.

Different hemispheric specialization for face/word recognition: A high-density ERP study with hemifield visual stimulation.

INTRODUCTION: The right fusiform face area (FFA) is important for face recognition, whereas the left visual word fusiform area (VWFA) is critical for word processing. Nevertheless, the early stages of unconscious and conscious face and word processing have not been studied systematically. MATERIALS AND METHODS: To explore hemispheric differences for face and word recognition, we manipulated the visual field (left vs. right) and stimulus duration (subliminal [17 ms] versus supraliminal [300 ms]). We recorded P100 and N170 peaks with high-density ERPs in response to faces/objects or Japanese words/scrambled words in 18 healthy young subjects. RESULTS: Contralateral P100 was larger than ipsilateral P100 for all stimulus types in the supraliminal, but not subliminal condition. The face- and word-N170s were not evoked in the subliminal condition. The N170 amplitude for the supraliminal face stimuli was significantly larger than that for the objects, and right hemispheric specialization was found for face recognition, irrespective of stimulus visual hemifield. Conversely, the supraliminal word-N170 amplitude was not significantly modulated by stimulus type, visual field, or hemisphere. CONCLUSIONS: These results suggest that visual awareness is crucial for face and word recognition. Our study using hemifield stimulus presentation further demonstrates the robust right FFA for face recognition but not the left VWFA for word recognition in the Japanese brain.

Changes in interleukin-1 beta induced by rTMS are significantly correlated with partial improvement of cognitive dysfunction in treatment-resistant depression: a pilot study.

The impairment experienced by many individuals with depression is closely related to the cognitive symptoms of the disorder. Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive brain stimulation method that provides a promising technique for improving cognitive symptoms in treatment-resistant depression (TRD). It has recently been demonstrated that TRD is associated with increased inflammatory process. In the present study, we investigated whether a relationship exists between changes in cognitive function and those in inflammatory cytokines before and after rTMS treatment. Eleven patients with TRD were enrolled in a high-frequency (10 Hz) rTMS study. Cognitive function, depressive symptoms and serum concentration of inflammatory cytokines (interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α) were measured at baseline and at the endpoint of rTMS treatment. rTMS treatment significantly improved depressive symptom scores and some subscales of cognitive dysfunction. The present study has demonstrated that partial changes in cognitive function and changes in IL-1ß were significantly correlated. The partial improvement of cognitive dysfunction by rTMS in the present study might be attributable to the reduction of peripheral IL-1ß levels. The present results should be replicated for verification in future studies.

Abnormal Frequency Mismatch Negativity in Early Psychosis Outpatient Subjects.

Background. Abnormalities of mismatch negativity (MMN), an event-related potential, indexing preattentive mechanisms, are consistently reported in schizophrenia (SZ). MMN abnormalities elicited to different deviant types have been recently shown to distinguish among patients according to length of their illness as well as inpatient versus outpatient status, and to be modulated by premorbid IQ. The objective of this study was to evaluate the MMN elicited by both frequency and duration deviant stimuli in patients with early schizophrenia (EP) recruited from an outpatient clinic in Boston, Massachusetts. Methods. Twenty-two healthy controls (HC) and 22 age-, handedness-, and gender-matched EP were tested using a frequency and duration MMN paradigm. Clinical data were also collected. Results. Frequency MMN amplitude but not duration MMN was significantly reduced in EP relative to HC subjects (P = .015). Conclusions. These results indicate that in this sample of early psychosis outpatient group, reductions in frequency MMN but not in duration MMN index clinical status. The relationship between age at first hospitalization and MMN frequency and duration amplitude and latency indicates that neurodevelopmental stage, auditory function, and clinical status are tightly linked.

Improvement Of Frontal Lobe Dysfunction And White Matter Integrity By rTMS In Treatment-Resistant Depression.

AIM: The impairment experienced by many individuals with depression is closely related to the cognitive symptoms of the disorder. Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive brain stimulation method providing a promising technique for improving cognitive symptoms in treatment-resistant depression (TRD). In the present study, we investigated whether a relationship exists between improvements in frontal lobe dysfunction induced by rTMS and improvement of white matter integrity revealed by diffusion tensor imaging (DTI) in TRD patients receiving rTMS treatment. METHODS: A total of 12 patients with TRD were enrolled in a high-frequency (10 Hz) rTMS study (August 2013-January 2019). Frontal lobe function and depressive symptoms were assessed at baseline and at the endpoint of rTMS treatment. Fractional anisotropy (FA), as a measure of white matter integrity obtained from DTI, was investigated using a region-of-interest (ROI) approach. RESULTS: rTMS treatment significantly improved depressive symptom scores and some subscales of frontal lobe dysfunction. Category scores in the Word Fluency Test and scores on part 3 of the Color Stroop Test were improved independently of the improvement of depressive symptoms. In the ROI analysis, none of the FA increases in any region were correlated with improvement of any frontal lobe function (n = 12). CONCLUSION: Although rTMS resulted in partial improvement of frontal lobe dysfunction as well as white matter integrity, we found no correlation between improved frontal lobe dysfunction and improved white matter integrity in TRD patients.

Monaural 40-Hz auditory steady-state magnetic responses can be useful for identifying epileptic focus in mesial temporal lobe epilepsy.

OBJECTIVE: Patients with mesial temporal lobe epilepsy (mTLE) often exhibit central auditory processing (CAP) dysfunction. Monaural 40-Hz auditory steady-state magnetic responses (ASSRs) were recorded to explore the pathophysiology of mTLE. METHODS: Eighteen left mTLE patients, 11 right mTLE patients and 16 healthy controls (HCs) were examined. Monaural clicks were presented at a rate of 40 Hz. Phase-locking factor (PLF) and power values were analyzed within bilateral Heschl's gyri. RESULTS: Monaural 40-Hz ASSR demonstrated temporal frequency dynamics in both PLF and power data. Symmetrical hemispheric contralaterality was revealed in HCs. However, predominant contralaterality was absent in mTLE patients. Specifically, right mTLE patients exhibited a lack of contralaterality in response to left ear but not right ear stimulation, and vice versa in left mTLE patients. CONCLUSION: This is the first study to use monaural 40-Hz ASSR with unilateral mTLE patients to clarify the relationship between CAP and epileptic focus. CAP dysfunction was characterized by a lack of contralaterality corresponding to epileptic focus. SIGNIFICANCE: Monaural 40-Hz ASSR can provide useful information for localizing epileptic focus in mTLE patients.

Stimulation of alpha2-adrenergic receptors impairs influenza virus infection.

Influenza A viruses cause seasonal epidemics and occasional pandemics. The emergence of viruses resistant to neuraminidase (NA) inhibitors and M2 ion channel inhibitors underlines the need for alternate anti-influenza drugs with novel mechanisms of action. Here, we report the discovery of a host factor as a potential target of anti-influenza drugs. By using cell-based virus replication screening of a chemical library and several additional assays, we identified clonidine as a new anti-influenza agent in vitro. We found that clonidine, which is an agonist of the alpha2-adrenergic receptor (α2-AR), has an inhibitory effect on the replication of various influenza virus strains. α2-AR is a Gi-type G protein-coupled receptor that reduces intracellular cyclic AMP (cAMP) levels. In-depth analysis showed that stimulation of α2-ARs leads to impairment of influenza virus replication and that α2-AR agonists inhibit the virus assembly step, likely via a cAMP-mediated pathway. Although clonidine administration did not reduce lung virus titers or prevent body weight loss, it did suppress lung edema and improve survival in a murine lethal infection model. Clonidine may thus protect against lung damage caused by influenza virus infection. Our results identify α2-AR-mediated signaling as a key pathway to exploit in the development of anti-influenza agents.

Connectopathy in Autism Spectrum Disorders: A Review of Evidence from Visual Evoked Potentials and Diffusion Magnetic Resonance Imaging.

Individuals with autism spectrum disorder (ASD) show superior performance in processing fine details; however, they often exhibit impairments of gestalt face, global motion perception, and visual attention as well as core social deficits. Increasing evidence has suggested that social deficits in ASD arise from abnormal functional and structural connectivities between and within distributed cortical networks that are recruited during social information processing. Because the human visual system is characterized by a set of parallel, hierarchical, multistage network systems, we hypothesized that the altered connectivity of visual networks contributes to social cognition impairment in ASD. In the present review, we focused on studies of altered connectivity of visual and attention networks in ASD using visual evoked potentials (VEPs), event-related potentials (ERPs), and diffusion tensor imaging (DTI). A series of VEP, ERP, and DTI studies conducted in our laboratory have demonstrated complex alterations (impairment and enhancement) of visual and attention networks in ASD. Recent data have suggested that the atypical visual perception observed in ASD is caused by altered connectivity within parallel visual pathways and attention networks, thereby contributing to the impaired social communication observed in ASD. Therefore, we conclude that the underlying pathophysiological mechanism of ASD constitutes a "connectopathy."

Enhanced Fine-Form Perception Does Not Contribute to Gestalt Face Perception in Autism Spectrum Disorder.

Individuals with autism spectrum disorder (ASD) show superior performance in processing fine detail, but often exhibit impaired gestalt face perception. The ventral visual stream from the primary visual cortex (V1) to the fusiform gyrus (V4) plays an important role in form (including faces) and color perception. The aim of this study was to investigate how the ventral stream is functionally altered in ASD. Visual evoked potentials were recorded in high-functioning ASD adults (n = 14) and typically developing (TD) adults (n = 14). We used three types of visual stimuli as follows: isoluminant chromatic (red/green, RG) gratings, high-contrast achromatic (black/white, BW) gratings with high spatial frequency (HSF, 5.3 cycles/degree), and face (neutral, happy, and angry faces) stimuli. Compared with TD controls, ASD adults exhibited longer N1 latency for RG, shorter N1 latency for BW, and shorter P1 latency, but prolonged N170 latency, for face stimuli. Moreover, a greater difference in latency between P1 and N170, or between N1 for BW and N170 (i.e., the prolongation of cortico-cortical conduction time between V1 and V4) was observed in ASD adults. These findings indicate that ASD adults have enhanced fine-form (local HSF) processing, but impaired color processing at V1. In addition, they exhibit impaired gestalt face processing due to deficits in integration of multiple local HSF facial information at V4. Thus, altered ventral stream function may contribute to abnormal social processing in ASD.

Lack of cross-resistance to FF-10501, an inhibitor of inosine-5'-monophosphate dehydrogenase, in azacitidine-resistant cell lines selected from SKM-1 and MOLM-13 leukemia cell lines.

Resistance to azacitidine is a major issue in the treatments of myelodysplastic syndrome and acute myeloid leukemia, and previous studies suggest that changes in drug metabolism are involved in the resistance. Therefore, drugs with mechanisms resistant or alternative to such metabolic changes have been desired for the treatment of resistant disease. We generated azacitidine-resistant cells derived from SKM-1 and MOLM-13 leukemia cell lines in vitro, analyzed the mechanisms, and examined the impact on the efficacy of other antimetabolic drugs. It appeared that the cell growth-inhibitory effect of azacitidine, expression levels of uridine-cytidine kinase 2, and the concentrations of azacitidine triphosphate were remarkably decreased in the resistant cells compared with those in parent cells. These results were consistent with previous observations that azacitidine resistance is derived from metabolic changes. Cross-resistance of greater than 10-fold (shift in IC50 value) was observed in azacitidine-resistant cells for decitabine and for cytarabine, but not for gemcitabine or the inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitors FF-10501 and mycophenolate mofetil (cross-resistance to 5-fluorouracil was cell line dependent). The IMPDH inhibitors maintained their cell growth-inhibitory activities in the azacitidine-resistant cell lines, in which the levels of adenine phosphoribosyltransferase (which converts FF-10501 to its active form, FF-10501 ribosylmonophosphate [FF-10501RMP]), FF-10501RMP, and the target enzyme, IMPDH, were equivalent to those in the parent cell lines. These results suggest that an IMPDH inhibitor such as FF-10501 could be an alternative therapeutic treatment for leukemia patients with acquired resistance to azacitidine.