Publications by authors named "Akane Morita"

30 Publications

  • Page 1 of 1

Impairment of endothelium-dependent vasodilator function of retinal blood vessels in adult rats with a history of retinopathy of prematurity.

J Pharmacol Sci 2021 Aug 7;146(4):233-243. Epub 2021 May 7.

Department of Molecular Pharmacology, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address:

Retinopathy of prematurity (ROP) is a proliferative retinal vascular disease, initiated by delayed retinal vascular growth after premature birth. In the majority of cases, ROP resolves spontaneously; however, a history of ROP may increase the risk of long-term visual problems. In this study, we evaluated the endothelial function of retinal blood vessels in adult rats with a history of ROP. ROP was induced in rats by subcutaneous injection of a vascular endothelial growth factor receptor tyrosine kinase inhibitor (KRN633) on postnatal day (P) 7 and P8. On P56, vasodilator responses to acetylcholine, GSK1016790A (an activator of transient receptor potential vanilloid 4 channels), NOR3 (a nitric oxide [NO] donor), and salbutamol (a β-adrenoceptor agonist) were assessed. Compared to age-matched controls, retinal vasodilator responses to acetylcholine and GSK1016790A were attenuated in P56 rats with a history of ROP. No attenuation of acetylcholine-induced retinal vasodilator response was observed under inhibition of NO synthase. Retinal vasodilator responses to NOR3 and salbutamol were unaffected. These results suggest that the production of and/or release of NO is impaired in retinal blood vessels in adult rats with a history of ROP. A history of ROP might increase the risk of impaired retinal circulation in adulthood.
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http://dx.doi.org/10.1016/j.jphs.2021.04.008DOI Listing
August 2021

Metformin Protects against NMDA-Induced Retinal Injury through the MEK/ERK Signaling Pathway in Rats.

Int J Mol Sci 2021 Apr 23;22(9). Epub 2021 Apr 23.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo 108-8641, Japan.

Metformin, an anti-hyperglycemic drug of the biguanide class, exerts positive effects in several non-diabetes-related diseases. In this study, we aimed to examine the protective effects of metformin against -methyl-D-aspartic acid (NMDA)-induced excitotoxic retinal damage in rats and determine the mechanisms of its protective effects. Male Sprague-Dawley rats (7 to 9 weeks old) were used in this study. Following intravitreal injection of NMDA (200 nmol/eye), the number of neuronal cells in the ganglion cell layer and parvalbumin-positive amacrine cells decreased, whereas the number of CD45-positive leukocytes and Iba1-positive microglia increased. Metformin attenuated these NMDA-induced responses. The neuroprotective effect of metformin was abolished by compound C, an inhibitor of AMP-activated protein kinase (AMPK). The AMPK activator, AICAR, exerted a neuroprotective effect in NMDA-induced retinal injury. The MEK1/2 inhibitor, U0126, reduced the neuroprotective effect of metformin. These results suggest that metformin protects against NMDA-induced retinal neurotoxicity through activation of the AMPK and MEK/extracellular signal-regulated kinase (ERK) signaling pathways. This neuroprotective effect could be partially attributable to the inhibitory effects on inflammatory responses.
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http://dx.doi.org/10.3390/ijms22094439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8123037PMC
April 2021

Pharmacological depletion of retinal neurons prevents vertical angiogenic sprouting without affecting the superficial vascular plexus.

Dev Dyn 2021 Apr 2;250(4):497-512. Epub 2020 Nov 2.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan.

Background: In mice, a tri-layered (superficial, intermediate, and deep) vascular structure is formed in the retina during the third postnatal week. Short-term treatment of newborn mice with vascular endothelial growth factor (VEGF) receptor inhibitors delays the formation of superficial vascular plexus and this allows us to investigate the developmental process of superficial and deep vascular plexuses at the same time. Using this model, we examined the effect of pharmacological depletion of retinal neurons on the formation of superficial and deep vascular plexuses.

Results: Neuronal cell loss induced by an intravitreal injection of N-methyl-d-aspartic acid on postnatal day (P) 8 delayed vascular development in the deep layer but not in the superficial layer in mice treated with KRN633, a VEGF receptor inhibitor, on P0 and P1. In KRN633-treated mice, neuronal cell loss decreased the number of vertical sprouts originating from the superficial plexus without affecting the number of angiogenic sprouts growing in front. Neuronal cell loss did not impair networks of fibronectin and astrocytes in the superficial layer.

Conclusions: Our results suggest that inner retinal neurons play a crucial role in forming the deep vascular plexus by directing the sprouts from the superficial blood vessels to the deep layer.
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http://dx.doi.org/10.1002/dvdy.263DOI Listing
April 2021

The process of revascularization in the neonatal mouse retina following short-term blockade of vascular endothelial growth factor receptors.

Cell Tissue Res 2020 Dec 8;382(3):529-549. Epub 2020 Sep 8.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.

Misdirected vascular growth frequently occurs in the neovascular diseases in the retina. However, the mechanisms are still not fully understood. In the present study, we created capillary-free zones in the central and peripheral retinas in neonatal mice by pharmacological blockade of vascular endothelial growth factor (VEGF) signaling. Using this model, we investigated the process and mechanisms of revascularization in the central and peripheral avascular areas. After the completion of a 2-day treatment with the VEGF receptor tyrosine kinase inhibitor KRN633 on postnatal day (P) 4 and P5, revascularization started on P8 in the central avascular area where capillaries had been dropped out. The expression levels of VEGF were higher in the peripheral than in the central avascular area. However, the expansion of the vasculature in the peripheral avascular retina remained suppressed until revascularization had been completed in the central avascular area. Additionally, we found disorganized endothelial cell division, misdirected blood vessels with irregular diameters, and abnormal fibronectin networks at the border of the vascular front and the avascular retina. In the central avascular area, a slight amount of fibronectin as non-vascular component re-formed to provide a scaffold for revascularization. Mechanistic analysis revealed that higher levels of VEGF attenuated the migratory response of endothelial cells without decreasing the proliferative activity. These results suggest that the presence of concentration range of VEGF, which enhances both migration and proliferation of the endothelial cells, and the structurally normal fibronectin network contribute to determine the proper direction of angiogenesis.
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http://dx.doi.org/10.1007/s00441-020-03276-9DOI Listing
December 2020

Abnormal Vascular Phenotypes Associated with the Timing of Interruption of Retinal Vascular Development in Rats.

Biol Pharm Bull 2020 ;43(5):859-863

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences.

Pathological angiogenesis is a leading cause of blindness in several retinal diseases. The key driving factor inducing pathological angiogenesis is the pronounced hypoxia leading to a marked, increased production of vascular endothelial growth factor (VEGF). The aim of this study was to determine whether the abnormal vascular growth occurs in a manner dependent on the degree of the vascular defects. Vascular defects of two different degrees were created in the retina by subcutaneously treating neonatal rats with the VEGF receptor (VEGFR) tyrosine kinase inhibitor KRN633 on postnatal day (P) 4 and P5 (P4/5) or P7 and P8 (P7/8). The structure of the retinal vasculature changes was examined immunohistochemically. Prevention of vascular growth and regression of some preformed capillaries were observed on the next day, after completion of each treatment (i.e., P6 and P9). The vascular regrowth occurred as a result of eliminating the inhibitory effect on the VEGFR signaling pathway. KRN633 (P4/5)-treated rats exhibited a retinal vasculature with aggressive intravitreal neovascularization on P21. On the other hand, the appearance of tortuous arteries is a representative vascular pathological feature in retinas of KRN633 (P7/8)-treated groups. These results suggest that an interruption of the retinal vascular development at different time points induces different vascular pathological features in the retina. Pharmacological agents targeting the VEGF signaling pathway are useful for creating an abnormal retinal vasculature with various pathological features in order to evaluate the efficacy of anti-angiogenic compounds.
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http://dx.doi.org/10.1248/bpb.b19-01065DOI Listing
January 2021

[Expression changes in microRNA in the retina of retinal degenerative diseases].

Nihon Yakurigaku Zasshi 2020 ;155(2):81-86

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences.

Because visual information accounts for 80-90% of sensory information that we get from our circumstance, loss of vision seriously diminishes our quality of life. According to a recent epidemiological study, glaucoma is the first, and retinitis pigmentosa (RP) is the second leading causes of acquired blindness in Japan. Degeneration of the retinal ganglion cells (RGC) and photoreceptor cells causes glaucoma and RP, respectively. Intraocular pressure-lowering therapy is an only effective treatment for glaucoma, and the agents that protect RGC directly against glaucomatous injury have not been available yet. In addition, there is no effective treatment for RP at present. microRNAs are a class of small, endogenous, non-coding RNAs comprised of approximately 20 nucleotides. It has been clarified that microRNAs reduces the stability of the target mRNAs and/or repress the translation of the target genes. A single microRNA can affect the transcription of multiple mRNAs, and almost 30% of human genes are thought to be regulated by microRNAs. Therefore, it has been considered that the expression changes of microRNAs are possible to cause various diseases, such as cancer and neurodegenerative diseases. Recently, the expression changes in microRNAs have been reported in the retina of experimental model animals for glaucoma and RP. The expressional changes of microRNAs are suggested to be related with development and progression of glaucoma and RP. Here, we will discuss about the relationship between the expressional changes of microRNAs and neuronal cell death in glaucoma and RP.
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http://dx.doi.org/10.1254/fpj.19121DOI Listing
March 2020

Role of transient receptor potential vanilloid subtype 4 in the regulation of azoymethane/dextran sulphate sodium-induced colitis-associated cancer in mice.

Eur J Pharmacol 2020 Jan 10;867:172853. Epub 2019 Dec 10.

Division of Pathological Sciences, Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Kyoto, 6078414, Japan.

Ca-permeable ion channels, such as transient receptor channels, are one of the potential therapeutic targets in cancer. Transient receptor potential vanilloid subtype 4 (TRPV4) is a nonselective cation channel associated with cancer progression. This study investigates the roles of TRPV4 in the pathogenesis of colitis-associated cancer (CAC) in mice. The role of TRPV4 was examined in azoxymethane (AOM)/dextran sulphate sodium (DSS)-induced murine CAC model. The formation of colon tumours induced by AOM/DSS treatment was significantly attenuated in TRPV4-deficient mice (TRPV4KO). TRPV4 was co-localised with markers of angiogenesis and macrophages. AOM/DSS treatment upregulated the expression of CD105, vascular endothelial growth factor receptor 2, and TRPV4 in wildtype, but the upregulation of CD105 was significantly attenuated in TRPV4KO. Bone marrow chimera experiments indicated that TRPV4, expressed in both vascular endothelial cells and bone marrow-derived macrophages, played a significant role in colitis-associated tumorigenesis. There was no significant difference in the population of hematopoietic cells, neutrophils, and monocytes between untreated and AOM/DSS-treated WT and TRPV4KO on flow cytometric analysis. TRPV4 activation by a selective agonist induced TNF-α and CXCL2 release in macrophages. Furthermore, TRPV4 activation enhanced the proliferation of human umbilical vein endothelial cells. These results suggest that TRPV4 expressed in neovascular endothelial cells and bone marrow-derived macrophages contributes to the progression of CAC in mice.
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http://dx.doi.org/10.1016/j.ejphar.2019.172853DOI Listing
January 2020

Changes in components of the neurovascular unit in the retina in a rat model of retinopathy of prematurity.

Cell Tissue Res 2020 Mar 2;379(3):473-486. Epub 2019 Dec 2.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.

An impairment of cellular interactions between the elements of the neurovascular unit contributes to the onset and/or progression of retinal diseases. The present study aims to examine how elements of the neurovascular unit are altered in a rat model of retinopathy of prematurity (ROP). Neonatal rats were treated subcutaneously with the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor KRN633 (10 mg/kg) on postnatal day (P) 7 and P8 to induce ROP. Morphological assessments were performed of blood vessels, astrocytes and neuronal cells in the retina. Aggressive angiogenesis, tortuous arteries and enlarged veins were observed in the retinal vasculature of KRN633-treated (ROP) rats from P14 to P28, compared to age-matched control (vehicle-treated) animals. Morphological abnormalities in the retinal vasculature showed a tendency toward spontaneous recovery from P28 to P35 in ROP rats. Immunofluorescence staining for glial fibrillary acidic protein and Pax2 (astrocyte markers) revealed that morphological changes to and a reduction in the number of astrocytes occurred in ROP rats. The developmental cell death was slightly accelerated in ROP rats; however, no visible changes in the morphology of retinal layers were observed on P35. The abnormalities in astrocytes might contribute, at least in part, to the formation of abnormal retinal blood vessels and the pathogenesis of ROP.
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http://dx.doi.org/10.1007/s00441-019-03112-9DOI Listing
March 2020

Cellular Mechanisms of Angiogenesis in Neonatal Rat Models of Retinal Neurodegeneration.

Int J Mol Sci 2019 Sep 25;20(19). Epub 2019 Sep 25.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.

Νeuronal and glial cells play an important role in the development of vasculature in the retina. In this study, we investigated whether re-vascularization occurs in retinal neurodegenerative injury models. To induce retinal injury, -methyl-D-aspartic acid (NMDA, 200 nmol) or kainic acid (KA, 20 nmol) was injected into the vitreous chamber of the eye on postnatal day (P)7. Morphological changes in retinal neurons and vasculature were assessed on P14, P21, and P35. Prevention of vascular growth and regression of some capillaries were observed on P14 in retinas of NMDA- and KA-treated eyes. However, vascular growth and re-vascularization started on P21, and the retinal vascular network was established by P35 in retinas with neurodegenerative injuries. The re-vascularization was suppressed by a two-day treatment with KRN633, an inhibitor of VEGF receptor tyrosine kinase, on P21 and P22. Astrocytes and Müller cells expressed vascular endothelial growth factor (VEGF), and the distribution pattern of VEGF was almost the same between the control and the NMDA-induced retinal neurodegenerative injury model, except for the difference in the thickness of the inner retinal layer. During re-vascularization, angiogenic sprouts from pre-existing blood vessels were present along the network of fibronectins formed by astrocytes. These results suggest that glial cells contribute to angiogenesis in neonatal rat models of retinal neurodegeneration.
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http://dx.doi.org/10.3390/ijms20194759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801463PMC
September 2019

Attenuation of Retinal Endothelial Vasodilator Function in a Rat Model of Retinopathy of Prematurity.

Curr Eye Res 2019 12 18;44(12):1360-1368. Epub 2019 Jul 18.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan.

: Retinopathy of prematurity (ROP) is characterized by morphological abnormalities in retinal blood vessels, but how an episode of ROP affects vascular function remains to be fully elucidated. The purpose of the present study was to assess the distribution of pericyte/smooth muscle in retinal blood vessels and retinal vasodilator responses in a rat model of ROP.: ROP was induced in rats by the subcutaneous injection of the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor KRN633 (10 mg/kg) on postnatal day (P) 7 and P8. The distribution of pericyte/smooth muscle in retinal blood vessels was examined on P14 and P35 by immunohistochemistry. Retinal vasodilator responses were assessed on P35 by measuring the diameter of retinal arterioles in fundus images.: In retinas of KRN633-treated (ROP) rats, progressive angiogenesis, tortuous arteries, enlarged veins, and enhanced expression of α-smooth muscle actin in pericytes on capillaries and veins were observed on P14. These abnormalities in retinal vasculature showed a tendency to normalize by P35. Vasodilation of retinal arterioles induced by acetylcholine, an endothelium-dependent vasodilator, was smaller in P35 ROP rats than age-matched controls, whereas retinal vasodilator responses to the nitric oxide (NO) donor NOR3 were unaltered.: Phenotypic changes in pericytes occur in the ROP model rats and endothelium-dependent vasodilatory mechanisms in retinal blood vessels are impaired. The impaired vasodilator function may contribute to the progression and pathogenesis of ROP.
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http://dx.doi.org/10.1080/02713683.2019.1641825DOI Listing
December 2019

Role of Neuron⁻Glia Signaling in Regulation of Retinal Vascular Tone in Rats.

Int J Mol Sci 2019 Apr 20;20(8). Epub 2019 Apr 20.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.

The interactions between neuronal, glial, and vascular cells play a key role in regulating blood flow in the retina. In the present study, we examined the role of the interactions between neuronal and glial cells in regulating the retinal vascular tone in rats upon stimulation of retinal neuronal cells by intravitreal injection of N-methyl-d-aspartic acid (NMDA). The retinal vascular response was assessed by measuring the diameter of the retinal arterioles in the in vivo fundus images. Intravitreal injection of NMDA produced retinal vasodilation that was significantly diminished following the pharmacological inhibition of nitric oxide (NO) synthase (nNOS), loss of inner retinal neurons, or intravitreal injection of glial toxins. Immunohistochemistry revealed the expression of nNOS in ganglion and calretinin-positive amacrine cells. Moreover, glial toxins significantly prevented the retinal vasodilator response induced by intravitreal injection of NOR3, an NO donor. Mechanistic analysis revealed that NO enhanced the production of vasodilatory prostanoids and epoxyeicosatrienoic acids in glial cells in a ryanodine receptor type 1-dependent manner, subsequently inducing the retinal vasodilator response. These results suggest that the NO released from stimulated neuronal cells acts as a key messenger in neuron-glia signaling, thereby causing neuronal activity-dependent and glial cell-mediated vasodilation in the retina.
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http://dx.doi.org/10.3390/ijms20081952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514555PMC
April 2019

Involvement of matrix metalloproteinases in capillary degeneration following NMDA-induced neurotoxicity in the neonatal rat retina.

Exp Eye Res 2019 05 16;182:101-108. Epub 2019 Mar 16.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan. Electronic address:

Interactions between neuronal cells and vascular cells in the retina are critical for maintaining retinal tissue homeostasis. Impairment of cellular interactions contributes to development and progression of retinal diseases. Previous studies demonstrated that neuronal cell damage leads to capillary degeneration in an N-methyl-D-aspartic acid (NMDA)-induced retinal degeneration model. However, the mechanisms underlying this phenomenon are not fully understood. In this study, we examined the possible role of matrix metalloproteinase (MMP)-9 in neuronal cell loss and capillary degeneration in NMDA-treated retinas of neonatal rats. Intravitreal injection of NMDA (50 or 200 nmol) was performed on postnatal day (P) 7 and morphological changes in retinal neurons and vasculature were examined on P14. The MMP inhibitor CP101537 (100 nmol) or vehicle (dimethyl sulfoxide) was intravitreally injected simultaneously with, or 2 days after, NMDA injection. CP101537 protected against neurovascular degeneration in a time-dependent manner as follows: 1) simultaneous injection of CP101537 with NMDA prevented morphological changes in retinal neurons induced by NMDA (50 nmol); and 2) reduction in capillary density and number of vertical sprouts induced by NMDA (200 nmol) was prevented when CP101537 was injected 2 days after NMDA injection. Gelatin zymography and western blot analyses indicated that activity and protein levels of MMP-9 were enhanced from 4 h to 2 days after NMDA injection. Increased activity and protein levels of MMP-9 were suppressed by MMP inhibitors (CP101537 and GM6001). In situ zymography revealed that MMP activity was enhanced throughout the retinal vasculature in NMDA-treated retinas. These results indicate that MMP-9 plays an important role in neurovascular degeneration in the injured retina. Inhibition of MMP-9 may be an effective strategy for preventing and reducing neurovascular degeneration.
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http://dx.doi.org/10.1016/j.exer.2019.03.005DOI Listing
May 2019

Anti-angiogenic effects of valproic acid in a mouse model of oxygen-induced retinopathy.

J Pharmacol Sci 2018 Nov 14;138(3):203-208. Epub 2018 Oct 14.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address:

Pathological retinal angiogenesis contributes to the pathogenesis of several ocular diseases. Valproic acid, a widely used antiepileptic drug, exerts anti-angiogenic effects by inhibiting histone deacetylase (HDAC). Herein, we investigated the effects of valproic acid and vorinostat, a HDAC inhibitor, on pathological retinal angiogenesis in mice with oxygen-induced retinopathy (OIR). OIR was induced in neonatal mice by exposure to 80% oxygen from postnatal day (P) 7 to P10 and to atmospheric oxygen from P10 to P15. Mice were subcutaneously injected with valproic acid, vorinostat, or vehicle once a day from P10 to P14. At P15, retinal neovascular tufts and vascular growth in the central avascular zone were observed in mice with OIR. Additionally, immunoreactivity for phosphorylated ribosomal protein S6 (pS6), an indicator of mammalian target of rapamycin (mTOR) activity, was detected in the neovascular tufts. Both valproic acid and vorinostat reduced the formation of retinal neovascular tuft without affecting vascular growth in the central avascular zone. Valproic acid reduced the pS6 immunoreactivity in neovascular tufts. Given that vascular endothelial growth factor (VEGF) activates mTOR-dependent pathways in proliferating endothelial cells of the neonatal mouse retina, these results suggest that valproic acid suppresses pathological retinal angiogenesis by interrupting VEGF-mTOR pathways.
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http://dx.doi.org/10.1016/j.jphs.2018.10.004DOI Listing
November 2018

Establishment of an abnormal vascular patterning model in the mouse retina.

J Pharmacol Sci 2018 Apr 14;136(4):177-188. Epub 2018 Mar 14.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address:

Abnormalities in retinal blood vessels and neuronal function persist in eyes undergoing retinopathy of prematurity. In this study, we examined morphological and functional changes in retinal blood vessels and neurons in mice that had undergone short-term interruption of retinal vascular development through inhibition of vascular endothelial growth factor (VEGF) signaling. In mice treated with the VEGF receptor tyrosine kinase inhibitor KRN633 on postnatal day (P) 0 and 1, the vascular density in the retinal surface increased by P12, but development of deep retinal vascular plexus and choroidal vasculature was delayed until P14. Overall retinal morphology was mostly normal in KRN633-treated mice during the observation period (∼P28), with the exception of P8 and P14. On P28, abnormalities in retinal vascular patterns were evident, but electroretinogram and retinal blood perfusion were within the normal range. Abnormal architecture of retinal vasculature disturbs retinal hemodynamics; therefore, mice treated postnatally with VEGF receptor inhibitors could serve as an animal model for studying the regulatory mechanism of local retinal blood flow and the effect of persistent abnormal retinal vascular patterns on the risk of onset of retinal ischemia.
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http://dx.doi.org/10.1016/j.jphs.2018.03.002DOI Listing
April 2018

Transient phenotypic changes in endothelial cells and pericytes in neonatal mouse retina following short-term blockade of vascular endothelial growth factor receptors.

Dev Dyn 2018 05 5;247(5):699-711. Epub 2018 Jan 5.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan.

Background: A short-term interruption of vascular development causes structural abnormalities in retinal vasculature. However, the detailed changes in vascular components (endothelial cells, pericytes, and basement membranes) remain to be fully determined. The present study aimed to provide a detailed description of morphological changes in vascular components following a short-term interruption of retinal vascular development in mice.

Results: Two-day treatment of neonatal mice with the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor KRN633 (10 mg/kg, subcutaneously) on postnatal day (P)0 and P1 (P0/1) and P4 and P5 (P4/5) induced different degrees and patterns of impairment of retinal vascular development. Three days after completion of the treatment, the delayed radial vascular growth occurred in P0/1 group mice, whereas in P4/5 group mice, revascularization preferentially occurred in the central avascular area, and radial vascular growth remained suppressed by P10. Differences in α-smooth muscle actin expression in pericytes were noted in the processes between normal vascular formation and vascular regrowth. The changes in vascular cells were associated with the hypoxia-induced enhancement of VEGF expression in the superficial retinal layer.

Conclusions: These findings suggest that the phenotype of vascular cells is altered following a short-term interruption of vascular development in the retina. Developmental Dynamics 247:699-711, 2018. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dvdy.24614DOI Listing
May 2018

Mammalian Target of Rapamycin (mTOR) as a Potential Therapeutic Target in Pathological Ocular Angiogenesis.

Biol Pharm Bull 2017 ;40(12):2045-2049

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences.

Pathological ocular angiogenesis is a causative factor of retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. Vascular endothelial growth factor (VEGF) plays an important role in pathological angiogenesis, and anti-VEGF agents have been used to treat the ocular diseases that are driven by pathological angiogenesis. However, adverse effects associated with the blockade of VEGF signaling, including impairments of normal retinal vascular growth and retinal function, were suggested. Therefore, the development of a safe, effective strategy to prevent pathological ocular angiogenesis is needed. Recent studies have demonstrated that inhibitors of the mammalian target of rapamycin (mTOR) target proliferating endothelial cells within the retinal vasculature. Here, we review the potential of targeting the mTOR pathway to treat pathological ocular angiogenesis.
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http://dx.doi.org/10.1248/bpb.b17-00475DOI Listing
July 2018

Role of Glial Cells in μ-Opioid Receptor-Mediated Vasodilation in the Rat Retina.

Curr Eye Res 2018 03 27;43(3):350-356. Epub 2017 Nov 27.

a Department of Molecular Pharmacology , Kitasato University School of Pharmaceutical Sciences , Tokyo Japan.

Purpose: Our recent study demonstrated that herkinorin, a non-opioid μ-receptor agonist derived from salvinorin A, dilates retinal arterioles through stimulation of μ-opioid receptors in rats. Activation of neuronal nitric oxide (NO) synthase and the presence of ganglion cells in the retina appear to be crucial for inducing μ-opioid receptor-mediated retinal vasodilation. In the present study, we examined the role of the interaction between neurons and glia in the retinal vasodilator mechanism involving μ-opioid receptors in rats.

Materials And Methods: The localization of μ-opioid receptors and neuronal NO synthase (nNOS) in the rat retina was examined using immunohistochemistry. The retinal vascular responses were evaluated by measuring the diameter of retinal arterioles in in vivo fundus images. Both systemic blood pressure and heart rate were continuously recorded.

Results: Immunoreactivity of μ-opioid receptors was found in ganglion cells and astrocytes, while that of nNOS was detected in ganglion cells and amacrine cells. Herkinorin increased retinal arteriolar diameter without significantly changing mean blood pressure and heart rate. The retinal vasodilator response to herkinorin was significantly attenuated by treatment with glial toxins (fluorocitrate and disialoganglioside-GD1b). The glial toxins markedly prevented vasodilation induced by intravitreal injection, but not by intravenous infusion, of NOR3, an NO donor.

Conclusion: These results suggest that retinal glial cells play an important role in the μ-opioid receptor-mediated retinal vasodilation in rats. Stimulation of μ-opioid receptors on retinal ganglion cells may affect the activity of glial cells, thereby changing retinal vascular tone.
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http://dx.doi.org/10.1080/02713683.2017.1403631DOI Listing
March 2018

A delay in vascularization induces abnormal astrocyte proliferation and migration in the mouse retina.

Dev Dyn 2017 03 3;246(3):186-200. Epub 2017 Feb 3.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan.

Background: Astrocytes migrate into the retina through the optic nerve head by means of the axons of retinal ganglion cells, and spread radially toward the peripheral retina. Endothelial cells migrate along the astrocyte cellular network to form the retinal surface vasculature. Here, we examined the effects of a delay in retinal vascularization on the migration and proliferation status of astrocytes in mice.

Results: A dose-dependent delay in retinal vascularization was observed in mice that had been treated with KRN633 (1-10 mg/kg), a VEGF receptor inhibitor, on the day of birth and on the following day. Delayed vascularization resulted in a delay in the astrocyte network formation, and an increase in astrocyte number in the optic nerve head and the vascular front. The increase in the number of astrocytes may be attributed to increased proliferation and delayed migration. These abnormalities in astrocyte behavior correlated with the degree of delay in retinal vascularization. The vascularization delay also led to retinal hypoxia, which subsequently stimulated VEGF leading to an increase in vascular density.

Conclusions: These findings suggest that a delay in normal vascularization leads to abnormal astrocyte behavior, which results in the formation of abnormal astrocyte and endothelial cell networks in the mouse retina. Developmental Dynamics 246:186-200, 2017. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dvdy.24484DOI Listing
March 2017

Exposure to high-concentration oxygen in the neonatal period induces abnormal retinal vascular patterning in mice.

Birth Defects Res B Dev Reprod Toxicol 2016 Dec 28;107(6):216-224. Epub 2016 Oct 28.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Minato-ku, Tokyo, Japan.

The interruption of vascular development could cause structural and functional abnormalities in tissues. We have previously reported that short-term treatment of newborn mice with vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitors induces abnormal retinal vascular growth and patterns. An exposure of neonatal mice to high-concentration oxygen disturbs normal retinal vascular development. The present study aimed to determine (1) whether vascular abnormalities are observed in the retina of newborn mice exposed to high concentrations of oxygen, and (2) how astrocyte network formation is affected following the exposure to hyperoxia. Newborn (postnatal day 0) mice were exposed to 75% oxygen for 48 or 96 hr. During hyperoxia exposure, VEGF expression decreased, and the onset of retinal vascularization was completely suppressed. After completion of the hyperoxic period, retinal vascularization occurred, but it was delayed in a hyperoxic exposure duration-dependent manner. In retinas of hyperoxia-exposed mice, dense capillary plexuses were found, and the number of arteries and veins decreased. The astrocyte network formation was slightly delayed under hyperoxic conditions, and the network became denser in retinas of mice with an episode of hyperoxia. Expression of VEGF levels in the avascular retina of mice that were exposed to hyperoxia was higher than that of control mice. These results suggest that short-term interruption of the onset of vascular development resulting from the reduction in VEGF signals induces abnormal vascular patterns in the mouse retina. The abnormalities in retinal astrocyte behavior might contribute to the formation of an abnormal retinal vascular growth.
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http://dx.doi.org/10.1002/bdrb.21187DOI Listing
December 2016

Treatment of newborn mice with inhibitors of vascular endothelial growth factor receptor tyrosine kinase induces abnormal retinal vascular patterning.

Biol Pharm Bull 2014 ;37(12):1986-9

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences.

We have previously reported that treatment of newborn mice with KRN633, a vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor, delayed retinal vascularization leading to abnormal retinal vascular growth and patterns. To determine whether similar abnormalities are observed in newborn mice treated with other VEGF receptor tyrosine kinase inhibitors, we administered axitinib to mice on the day of birth and on the following day. When compared with control pups, a significant delay in retinal vascularization was observed in pups treated with axitinib (5 mg/kg). Axitinib-treated pups had a very dense capillary network on postnatal day (P) 6 and fewer central arteries and veins on P8 and P12. Central veins, but not arteries, were significantly enlarged on P8. These abnormalities were similar to those observed in KRN633-treated pups and probably represent a common phenotype induced by short-term treatment with VEGF receptor inhibitors in newborn mice. Therefore, mice treated postnatally with VEGFR inhibitors could serve as an animal model for studying the mechanisms of retinal vascular formation and patterning.
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http://dx.doi.org/10.1248/bpb.b14-00540DOI Listing
August 2015

Treatment of mid-pregnant mice with KRN633, an inhibitor of vascular endothelial growth factor receptor tyrosine kinase, induces abnormal retinal vascular patterning in their newborn pups.

Birth Defects Res B Dev Reprod Toxicol 2014 Aug 15;101(4):293-9. Epub 2014 May 15.

Department of Molecular Pharmacology, Shirokane, Minato-ku, Tokyo, Japan.

We previously reported that treatment of mid-pregnant mice with KRN633, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, caused fetal growth restriction resulting from diminished vascularization in the placenta and fetal organs. In this study, we examined how the treatment of mid-pregnant mice with KRN633 affects the development and morphology of vascular components (endothelial cells, pericytes, and basement membrane) in the retinas of their newborn pups. Pregnant mice were treated with KRN633 (5 mg/kg) once daily from embryonic day 13.5 until the day of delivery. Vascular components were examined using immunohistochemistry with specific markers for each component. Radial vascular growth in the retina was slightly delayed until postnatal day 4 (P4) in the newborn pups of KRN633-treated mothers. On P8, compared with the pups of control mothers, the pups of KRN633-treated mothers exhibited decreased numbers of central arteries and veins and abnormal branching of the central arteries. No apparent differences in pericytes or basement membrane were observed between the pups of control and KRN633-treated mothers. These results suggest that a critical period for determining retinal vascular patterning is present at the earliest stages of retinal vascular development, and that the impaired vascular endothelial growth factor signaling during this period induces abnormal architecture in the retinal vascular network.
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http://dx.doi.org/10.1002/bdrb.21112DOI Listing
August 2014

Effects of pre- and post-natal treatment with KRN633, an inhibitor of vascular endothelial growth factor receptor tyrosine kinase, on retinal vascular development and patterning in mice.

Exp Eye Res 2014 Mar 23;120:127-37. Epub 2014 Jan 23.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.

The impaired function of angiogenic factors, including vascular endothelial growth factor (VEGF), during pregnancy is associated with preeclampsia and intrauterine growth restriction. To determine how the attenuation of VEGF signals during retinal vascular development affects retinal vascular growth and patterns, we examined the effects of pre- and post-natal treatment of mice with KRN633, a VEGF receptor tyrosine kinase inhibitor, on retinal vascular development and structure. Delays in retinal vascular development were observed in the pups of mother mice that were treated daily with KRN633 (5 mg/kg/day) from embryonic day 13.5 until the day of delivery. A more marked delay was seen in pups treated with the inhibitor (5 mg/kg/day) on the day of birth and on the following day. Pups treated postnatally with KRN633 showed abnormal retinal vascular patterns, such as highly dense capillary networks and decreased numbers of central arteries and veins. The high-density vascular networks in KRN633-treated pups showed a greater sensitivity to VEGF signaling inhibition than the normal vascular networks in vehicle-treated pups. Compared to vehicle-treated pups, more severe hypoxia and stronger VEGF mRNA expression were observed in avascular areas in KRN633-treated pups. These results suggest that a short-term loss of VEGF function at the earliest stages of vascular development suppresses vascular growth, leading to abnormal vascular patterning, at least in part via mechanisms involving VEGF in the mouse retina.
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http://dx.doi.org/10.1016/j.exer.2014.01.009DOI Listing
March 2014

KRN633, an inhibitor of vascular endothelial growth factor receptor tyrosine kinase, induces intrauterine growth restriction in mice.

Birth Defects Res B Dev Reprod Toxicol 2013 Aug 18;98(4):297-303. Epub 2013 Jun 18.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan.

We previously reported that treatment with KRN633, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, during mid-pregnancy caused intrauterine growth restriction resulting from impairment of blood vessel growth in the labyrinthine zone of the placenta and fetal organs. However, the relative sensitivities of blood vessels in the placenta and fetal organs to vascular endothelial growth factor (VEGF) inhibitors have not been determined. In this study, we aimed to examine the effects of KRN633 on the vasculatures of organs in mother mice and their newborn pups by immunohistochemical analysis. Pregnant mice were treated daily with KRN633 (5 mg/kg) either from embryonic day 13.5 (E13.5) to E17.5 or from E13.5 to the day of delivery. The weights of the pups of KRN633-treated mice were lower than those of the pups of vehicle-treated mothers. However, no significant difference in body weight was observed between the vehicle- and KRN633-treated mice. The vascular development in the organs (the pancreas, kidney, and intestine) and intestinal lymphatic formation of the pups of KRN633-treated mothers was markedly impaired. In contrast, the KRN633 treatment showed no significant effect on the vascular beds in the organs, including the labyrinthine zone of the placenta, of the mother mice. These results suggest that blood vessels in fetal organs are likely to be more sensitive to reduced VEGF signaling than those in the mother. A partial loss of VEGF function during pregnancy could suppress vascular growth in the fetus without affecting the vasculature in the mother mouse, thereby increasing the risk of intrauterine growth restriction.
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http://dx.doi.org/10.1002/bdrb.21064DOI Listing
August 2013

Successful treatment with noninvasive positive-pressure ventilation based on the prediction of disease onset using CT and respiratory function tests in an elderly patient with relapsing polychondritis.

Intern Med 2013 15;52(10):1085-9. Epub 2013 May 15.

Department of Respiratory Medicine, Yokohama-City Seibu Hospital of St. Marianna University School of Medicine, Japan.

An 83-year-old man who had been receiving treatment for bronchial asthma since 62 years of age experienced difficulty breathing on exertion and was admitted to the hospital. On admission, computed tomography revealed tracheal wall thickening, while test results for antinuclear antibodies and anti-type II collagen antibodies were positive. Since a saddle nose deformity, malacia of the auricles and sensorineural deafness were also observed, relapsing polychondritis was diagnosed. Measuring the peak expiratory flow rate was useful in the early airway assessment. During the follow-up period, the patient's dyspnea worsened and noninvasive positive-pressure ventilation was introduced. As a result, the subjective symptoms improved.
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http://dx.doi.org/10.2169/internalmedicine.52.9035DOI Listing
January 2014

Superiority of nebulized corticosteroids over dry powder inhalers in certain patients with cough variant asthma or cough-predominant asthma.

Allergol Int 2012 Sep 25;61(3):411-7. Epub 2012 May 25.

Department of Pulmonology, National Hospital Organization Disaster Medical Center, 3256Midori-machi, Tachikawa-shi, Tokyo, Japan.

Background: The particle distribution might differ between nebulizer therapy and metered-dose inhaler (MDI) or dry powder inhaler (DPI) therapy because the particles repeatedly enter/re-enter the airways with the nebulizer. Inhaled corticosteroids (ICS) were administered with a nebulizer to assess the benefit of changes in the distribution of particles in patients with cough variant asthma (CVA) and cough-predominant asthma (CPA).

Methods: Patients whose symptoms were not controlled by their current therapy were enrolled. In patients receiving high-dose ICS by MDI or DPI (ICS-MDI/DPI), steroid therapy was switched to 1,320μg/day of nebulized dexamethasone (1,600μg as dexamethasone sodium phosphate) (chronic steroid-independent group). In patients receiving systemic steroids regardless of their ICS-MDI/DPI therapy, nebulized dexamethasone was added and any concurrent ICS-MDI/DPI therapy was halted to detect a steroid-sparing effect (chronic steroid-dependent group). In patients with acute exacerbation of CVA or CPA and persistent symptoms despite systemic corticosteroids, nebulized dexamethasone was added to assess its effect (acute group).

Results: Superior symptom control was achieved in 10 out of 12 steroid-independent patients, 3 out of 6 steroid-dependent patients, and all 7 acute patients.

Conclusions: Delivery of ICS via a nebulizer has advantages over ICS-MDI/DPI in some patients with CVA or CPA.
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http://dx.doi.org/10.2332/allergolint.11-OA-0357DOI Listing
September 2012

[A case of severe influenza (H1N1) 2009 virus pneumonia identified by virus culture instead of PCR].

Kansenshogaku Zasshi 2011 Nov;85(6):670-3

Department of Respiratory Internal Medicine, St. Marianna University, School of Medicine, Yokohama-City Seibu Hospital.

A 70-year-old man was being treated for asthma and chronic obstructive pulmonary disease, which had been well controlled. He was seen at our emergency outpatient department on October 24, 2009, for respiratory distress and mild fever. Point-of-care testing for influenza, general bacteria, and acid-fast bacilli in the sputum, were negative. With antibiotics ineffective, his respiratory status worsened, requiring him to be intubated and ventilated mechanically. Steroid pulse therapy temporarily improved his condition, as confirmed by imaging studies, but he died on hospital day 38. Polymerase chain reaction (PCR) analysis of tracheal secretion and bronchial washings collected on hospital day 14 and 21 were negative for influenza (H1N1) 2009 virus, which was identified in a subsequent culture. Negative results for reverse transcriptase-PCR analysis leave (H1N1) 2009 virus unable to be diagnosed clinically. Culture tests and repeated PCR analysis have been done in cases of strongly suspected clinical infection to confirm results. Our case, in which the virus was identified by culture, suggests that the viral load may have been too low or the time of culture inappropriate.
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http://dx.doi.org/10.11150/kansenshogakuzasshi.85.670DOI Listing
November 2011

A case of yellow nail syndrome with dramatically improved nail discoloration by oral clarithromycin.

Case Rep Dermatol 2011 Sep 30;3(3):251-8. Epub 2011 Nov 30.

Department of Respiratory Medicine, National Center for Global Health and Medicine, Infection, Oncology, Nippon Medical School, Tokyo.

An 80-year-old woman was admitted to our hospital with pneumonia and exacerbation of sinobronchial syndrome (SBS). She presented with yellow discoloration of the nail beds of all fingers and toes, and her nails were recognized as growing slowly. Chest X-ray revealed bronchiectasis in the bilateral lower lobe and bilateral pleural effusion. We diagnosed her as having yellow nail syndrome (YNS), based on the triad of yellow nails, lymphedema, and lung disease. After treatment with antibiotics [ampicillin/sulbactam and clarithromycin (CAM)] for pneumonia and SBS, her general condition improved, and the yellow nails disappeared in some fingers. When she was previously treated with 200 mg CAM for SBS, her yellow nails had not shown improvement. This time, her yellow nails improved after treatment with 400 mg CAM. The literature reports vitamin E, zinc, and topical corticosteroid plus active vitamin D3 to be effective in the treatment of yellow nails. Two studies have reported treatment for YNS using CAM, though they found a lack of efficacy. Thus, the present case is the first to report improved yellow nails using CAM alone. We conclude that not only SBS and lung disease but also YNS were improved by treatment with 400 mg CAM.
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http://dx.doi.org/10.1159/000334734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3250669PMC
September 2011

Paragonimiasis in a person whose symptoms were shown 22 years after emigrating to Japan from Laos.

J Infect Chemother 2010 Feb;16(1):49-52

Department of Respiratory Medicine, Yokohama-City Seibu Hospital, St. Marianna University School of Medicine, Asahi-ku, Yokohama, Japan.

We report a patient, a 52-year-old man from Laos, who had come to Japan at 30 years of age, but had maintained a habit of eating raw freshwater crabs. The patient visited a physician for left chest pain in January 2007. Infiltration and mass-like shadows were noted in the left superior and inferior lobes on chest X-ray. Diagnosis could not be made by bronchial brushing, but eggs were present in sputum cytology 3 days after bronchoscopy. Therefore, paragonimiasis was diagnosed. The peripheral eosinophil count had increased to 2550/μl and the serum IgE level was elevated, at 71000 IU/ml. Multiple-dot enzyme-linked immunosorbent assay (ELISA) for specific IgG antibodies in serum was positive for Paragonimus westermani and P. miyazakii. Paragonimiasis may have been caused by the style of Laotian cooking without heating. Because the habit of eating raw freshwater crabs is common in Laos, Laos is one of the countries where paragonimiasis is prevalent. For patients from Laos with lung diseases, differentiation including paragonimiasis is required.
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http://dx.doi.org/10.1007/s10156-009-0005-4DOI Listing
February 2010

[A case of lung adenocarcinoma successfully treated with dose-reescalated gefitinib after resistance was acquired].

Gan To Kagaku Ryoho 2009 Aug;36(8):1333-6

Dept. of Respiratory Medicine, International Medical Center of Japan.

Gefitinib is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, and it shows favorable antitumor activity against chemorefractory non-small cell lung cancer (NSCLC), especially with EGFR gene mutations. On the other hand, patients with NSCLC have few treatment options available if they acquire resistance to gefitinib or severe adverse events occur. We report a 73-year-old small woman diagnosed with NSCLC who was treated with gefitinib (250 mg/day) every other day or by 7-days-on followed by 7-days-off therapy dose schedule after severe paronychia appeared. The best response was stable disease (SD), which lasted 9 months. Dose reescalation of gefitinib to 250 mg/day was chosen after progression of disease was demonstrated. Most lesions decreased in size again and this lasted for more than 5 months. EGFR gene analysis showed point mutation of codon 861 in exon21 (L861Q). On the other hand, T790M was not detected. These observations suggest the possibility that treatment with dose-escalated gefitinib might be useful even after resistance to initial dose of gefitinib is acquired if initial treatment shows a favorable clinical response.
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August 2009
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