Design and synthesis of pyrrolo[3,2-d]pyrimidine HER2/EGFR dual inhibitors: Improvement of the physicochemical and pharmacokinetic profiles for potent in vivo anti-tumor efficacy

During the course of our studies on a novel HER2/EGFR dual inhibitor (TAK-285), we found an alternative potent pyrrolo[3,2-d]pyrimidine compound (1a). To enhance the pharmacokinetic (PK) profile of this compound, we conducted chemical modifications into its N-5 side chain and conversion of the chemically modified compounds into their salts. Among them, 2cb, the tosylate salt of compound 2c, showed potent HER2/EGFR kinase inhibitory activity (IC₅₀: 11/11 nM) and cellular growth inhibitory activity (BT-474 cell GI₅₀: 56 nM) with a good drug metabolism and PK (DMPK) profile. Furthermore, 2cb exhibited significant in vivo antitumor efficacy in both mouse and rat xenograft models with transplanted 4-1ST gastric cancer cell lines (mouse, T/C = 0%, 2cb po bid at 100 mg/kg; rat, T/C: -1%, 2cb po bid at 25 mg/kg).     

Assessment of Still and Moving Images in the Diagnosis of Gastric Lesions Using Magnifying Narrow-Band Imaging in a Prospective Multicenter Trial

Objectives

Magnifying narrow-band imaging (M-NBI) is more accurate than white-light imaging for diagnosing small gastric cancers. However, it is uncertain whether moving M-NBI images have additional effects in the diagnosis of gastric cancers compared with still images.

Design

A prospective multicenter cohort study.

Methods

To identify the additional benefits of moving M-NBI images by comparing the diagnostic accuracy of still images only with that of both still and moving images. Still and moving M-NBI images of 40 gastric lesions were obtained by an expert endoscopist prior to this prospective multicenter cohort study. Thirty-four endoscopists from ten different Japanese institutions participated in the prospective multicenter cohort study. Each study participant was first tested using only still M-NBI images (still image test), then tested 1 month later using both still and moving M-NBI images (moving image test). The main outcome was a difference in the diagnostic accuracy of cancerous versus noncancerous lesions between the still image test and the moving image test.

Results

Thirty-four endoscopists were analysed. There were no significant difference of cancerous versus noncancerous lesions between still and moving image tests in the diagnostic accuracy (59.9% versus 61.5%), sensitivity (53.4% versus 55.9%), and specificity (67.0% versus 67.6%). And there were no significant difference in the diagnostic accuracy between still and moving image tests of demarcation line (65.4% versus 65.5%), microvascular pattern (56.7% versus 56.9%), and microsurface pattern (48.1% versus 50.9%). Diagnostic accuracy showed no significant difference between the still and moving image tests in the subgroups of endoscopic findings of the lesions.

Conclusions

The addition of moving M-NBI images to still M-NBI images does not improve the diagnostic accuracy for gastric lesions. It is reasonable to concentrate on taking sharp still M-NBI images during endoscopic observation and use them for diagnosis.

Trial registration

Umin.ac.jp UMIN-CTR000008048     

Attempt at cloning high-quality goldfish breed 'Ranchu' by fin-cultured cell nuclear transplantation

The viability of ornamental fish culture relies on the maintenance of high-quality breeds. To improve the profitability of culture operations we attempted to produce cloned fish from the somatic nucleus of the high-quality Japanese goldfish (Carassius auratus auratus) breed 'Ranchu'. We transplanted the nucleus of a cultured fin-cell from an adult Ranchu into the non-enucleated egg of the original goldfish breed 'Wakin'. Of the 2323 eggs we treated, 802 underwent cleavage, 321 reached the blastula stage, and 51 reached the gastrula stage. Two of the gastrulas developed until the hatching stage. A considerable number of nuclear transplants retained only the donor nucleus. Some of these had only a 2n nucleus derived from the same donor fish. Our results provide insights into the process of somatic cell nuclear transplantation in teleosts, and the cloning of Ranchu.     

Genetic analysis of the mode of interplay between an ATPase subunit and membrane subunits of the lipoprotein-releasing ATP-binding cassette transporter LolCDE

The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the inner membrane. Thus, functional interaction between LolD and LolC/E is critically important for coupling of ATP hydrolysis to the lipoprotein release reaction. LolD contains a characteristic sequence called the LolD motif, which is highly conserved among LolD homologs but not other ABC transporters of E. coli. The LolD motif is suggested to be a region in contact with LolC/E, judging from the crystal structures of other ABC transporters. To determine the functions of the LolD motif, we mutagenized each of the 32 residues of the LolD motif and isolated 26 dominant-negative mutants, whose overexpression arrested growth despite the chromosomal lolD(+) background. We then selected suppressor mutations of the lolC and lolE genes that correct the growth defect caused by the LolD mutations. Mutations of the lolC suppressors were mainly located in the periplasmic loop, whereas ones of lolE suppressors were mainly located in the cytoplasmic loop, suggesting that the mode of interaction with LolD differs between LolC and LolE. Moreover, the LolD motif was found to be critical for functional interplay with LolC/E, since some LolD mutations lowered the ATPase activity of LolCDE without affecting that of LolD.     

Genetic Differentiation of Populations of a Hydrothermal Vent-Endemic Gastropod, Ifremeria nautilei, between the North Fiji Basin and the Manus Basin revealed by Nucleotide Sequences of Mitochondrial DNA

The genetic differentiation of populations of a hydrothermal vent-endemic gastropod, Ifremeria nautilei, between two back-arc basins in the south Western Pacific, namely the Manus Basin and the North Fiji Basin, was analyzed on the basis of nucleotide sequences of the mitochondrial gene for cytochrome oxidase I. The two populations of I. nautilei had no common haplotypes and appeared, therefore, to be isolated from one another. All haplotypes obtained from the North Fiji Basin formed a monophyletic group supported by a high bootstrap probability and the genetic diversity of the population in the North Fiji Basin was much smaller than that of the population in the Manus Basin. The population in the North Fiji Basin might have been founded by relatively recent migrants from the Manus Basin. The present results suggest that the larval dispersal ability of I. nautilei might be lower than that of an undescribed species in the closely related genus Alviniconchay.     

Interleukin-1 derived from human monocytic leukemia cell line JOSK-I acts as an autocrine growth factor

Interleukin-1 (IL-1) enhances the growth of human monocytic leukemia cell line JOSK-I cells, which were recently established in our laboratory and which were demonstrated to produce a high level of IL-1 constitutively, in liquid as well as semisolid culture systems. Concomitantly, IL-1 stimulated the prostaglandin E2 synthesis and nitroblue tetrazolium dye-reducing capacity of JOSK-I cells. This indicates that IL-1 may act as autocrine growth factor for monocytes, and also suggests the possibility that this autocrine stimulation may play an important role in the pathophysiology of monocytic leukemia in vivo.     

Changes in spatial and temporal localization of Dictyostelium homologues of TRAP1 and GRP94 revealed by immunoelectron microscopy

TRAP1 (tumor necrosis factor receptor-associated protein 1) is a member of the molecular chaperone HSP90 (90-kDa heat shock protein) family. In this study, we mainly examined the behavior of Dictyostelium TRAP1 homologue, Dd-TRAP1, during Dictyostelium development by immunoelectron microscopy. In vegetatively growing D. discoideum Ax-2 cells, Dd-TRAP1 locates in nucleolus and vesicles in addition to the cell cortex including cell membrane. Many of Dd-TRAP1 molecules moved to the mitochondrial matrix in response to differentiation, although Dd-TRAP1 on the cell membrane seems to be retained. Some Dd-TRAP1 was also found to be secreted to locate outside the cell membrane in Ax-2 cells starved for 6 h. At the multicellular slug stage, Dd-TRAP1 was primarily located in mitochondria and cell membrane in both prestalk and prespore cells. More importantly, in differentiating prespore cells, a significant number of Dd-TRAP1 locates in the PSV (prespore-specific vacuole) that is a sole cell type-specific organelle and essential for spore wall formation, whereas some Dd-TRAP1 in the cell cortical region of prestalk cells. These findings strongly suggest the importance of Dd-TRAP1 regulated temporally and spatially during Dictyostelium development. Incidentally, we also have certified that the glucose-regulated protein 94 (Dd-GRP94) is predominantly located in Golgi vesicles and cisternae, followed by its colocalization with Dd-TRAP1 in the PSV.     

Effect of volume of oocyte cytoplasm on embryo development after parthenogenetic activation, intracytoplasmic sperm injection, or somatic cell nuclear transfer

Animal cloning methods are now well described and are becoming routine. Yet, the frequency at which live cloned offspring are produced remains below 5%, irrespective of the nuclear donor species or cell type. One possible explanation is that the reprogramming factor(s) of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we have increased the oocyte volume by oocyte fusion and examined its subsequent development. We constructed oocytes with volumes two to nine times greater than the normal volume by the electrofusion or mechanical fusion of intact and enucleated oocytes. We examined their in vitro and in vivo developmental potential after parthenogenetic activation, intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). When the fused oocytes were activated parthenogenetically, most developed to morulae or blastocysts, regardless of their original size. Diploid fused oocytes were fertilized by ICSI and developed normally and after embryo transfer, we obtained 12 (4-15%) healthy and fertile offspring. However, enucleated fused oocytes could not support the development of mice cloned by SCNT. These results suggest that double fused oocytes have normal potential for development after fertilization, but oocytes with extra cytoplasm do not have enhanced reprogramming potential.     

IL-15-dependent activation-induced cell death-resistant Th1 type CD8 alpha beta+NK1.1+ T cells for the development of small intestinal inflammation

To clarify the role of IL-15 at local sites, we engineered a transgenic (Tg) mouse (T3(b)-IL-15 Tg) to overexpress human IL-15 preferentially in intestinal epithelial cells by the use of T3(b)-promoter. Although IL-15 was expressed in the entire small intestine (SI) and large intestines of the Tg mice, localized inflammation developed in the proximal SI only. Histopathologic study revealed reduced villus length, marked infiltration of lymphocytes, and vacuolar degeneration of the villus epithelium, beginning at approximately 3-4 mo of age. The numbers of CD8(+) T cells, especially CD8alphabeta(+) T cells expressing NK1.1, were dramatically increased in the lamina propria of the involved SI. The severity of inflammation corresponded to increased numbers of CD8alphabeta(+)NK1.1(+) T cells and levels of production of the Th1-type cytokines IFN-gamma and TNF-alpha. Locally overexpressed IL-15 was accompanied by increased resistance of CD8alphabeta(+) NK1.1(+) T cells to activation-induced cell death. Our results suggest that chronic inflammation in the SI in this murine model is mediated by dysregulation of epithelial cell-derived IL-15. The model may contribute to understanding the role of CD8(+) T cells in human Crohn's disease involving the SI.