Mutated GM-CSF-based CAR-T cells targeting CD116/CD131 complexes exhibit enhanced anti-tumor effects against acute myeloid leukaemia.

OBJECTIVES: As the prognosis of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains poor, novel treatment strategies are urgently needed. Clinical trials have shown that chimeric antigen receptor (CAR)-T cells for AML are more challenging than those targeting CD19 in B-cell malignancies. We recently developed piggyBac-modified ligand-based CAR-T cells that target CD116/CD131 complexes, also known as the GM-CSF receptor (GMR), for the treatment of juvenile myelomonocytic leukaemia. This study therefore aimed to develop a novel therapeutic method for R/R AML using GMR CAR-T cells. METHODS: To further improve the efficacy of the original GMR CAR-T cells, we have developed novel GMR CAR vectors incorporating a mutated GM-CSF for the antigen-binding domain and G4S spacer. All GMR CAR-T cells were generated using a piggyBac-based gene transfer system. The anti-tumor effect of GMR CAR-T cells was tested in mouse AML xenograft models. RESULTS: Nearly 80% of the AML cells predominant in myelomonocytic leukaemia were found to express CD116. GMR CAR-T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. Furthermore, GMR CAR-T cells incorporating a G4S spacer significantly improved long-term in vitro and in vivo anti-tumor effects. By employing a mutated GM-CSF at residue 21 (E21K), the anti-tumor effects of GMR CAR-T cells were also improved especially in long-term in vitro settings. Although GMR CAR-T cells exerted cytotoxic effects on normal monocytes, their lethality on normal neutrophils, T cells, B cells and NK cells was minimal. CONCLUSIONS: GMR CAR-T cell therapy represents a promising strategy for CD116+ R/R AML.

Skeletal muscle cells derived from mouse skin cultures.

We have established a novel, simple, and highly reproducible method to generate skeletal muscle cells from mouse skin. Small pieces of skin from the back of mice were cultured in extracellular material-coated dishes in typical culture medium for about 3 weeks. Myotubes formed after about a week, grew into twitching myotubes, and became twitching myotube clumps after 3 weeks. Skeletal muscle cells are formed spontaneously with no induction. Myotubes were immunologically positive for myosin heavy chains, MyoD, and myogenin. Ultrastructural analysis revealed the presence of the sarcomere structure. Furthermore, PAX7+/MyoD- muscle stem cells proliferated around these myotubes, and MyoD+/myogenin+/MHC-- cells were also observed. Moreover, we investigated the formation of skeletal muscle cells from the sialidosis mouse skin, and showed that it is decreased compared to that of the wild type. Our method to generate skeletal muscle cells from skin is thought to be useful for the investigation of muscle cell development and muscle-related disorders.

Novel oestrogen receptor ß-selective ligand reduces obesity and depressive-like behaviour in ovariectomized mice.

Hormonal changes due to menopause can cause various health problems including weight gain and depressive symptoms. Multiple lines of evidence indicate that oestrogen receptors (ERs) play a major role in postmenopausal obesity and depression. However, little is known regarding the ER subtype-specific effects on obesity and depressive symptoms. To delineate potential effects of ERß activation in postmenopausal women, we investigated the effects of a novel oestrogen receptor ß-selective ligand (C-1) in ovariectomized mice. Uterine weight, depressive behaviour, and weight gain were examined in sham-operated control mice and ovariectomized mice administered placebo, C-1, or 17ß-oestradiol (E2). Administration of C-1 or E2 reduced body weight gain and depressive-like behaviour in ovariectomized mice, as assessed by the forced swim test. In addition, administration of E2 to ovariectomized mice increased uterine weight, but administration of C-1 did not result in a significant increase in uterine weight. These results suggest that the selective activation of ERß in ovariectomized mice may have protective effects against obesity and depressive-like behaviour without causing an increase in uterine weight. The present findings raise the possibility of the application of ERß-ligands such as C-1 as a novel treatment for obesity and depression in postmenopausal women.

Design, synthesis, and structure-activity relationship (SAR) of N-[7-(4-hydroxyphenoxy)-6-methylindan-4-yl]malonamic acids as thyroid hormone receptor ß (TRß) selective agonists.

Highly TRß selective thyromimetics have several potential therapeutic applications. Based on the novel indane derivative KTA-439 with high receptor (TRß) and organ (liver) selectivity, a series of thyroid hormone analogues were prepared, in which the isopropyl at the 3'-position was replaced with alkyl and aralkyl moieties of variable lengths and branches. Binding assays for these human TRs and reporter cell assays showed that 2-arylethyl derivatives had higher TRß selectivity than KTA-439. KTA-574, a representative 2-arylethyl derivative, had TRß specificity in a binding assay and exhibited full agonism in a reporter cell assay.

Discovery of novel indane derivatives as liver-selective thyroid hormone receptor ß (TRß) agonists for the treatment of dyslipidemia.

Thyromimetics that specifically target TRß have been shown to reduce plasma cholesterol levels and avoid atherosclerosis through the promotion of reverse cholesterol transport in an animal model. We designed novel thyromimetics with high receptor (TRß) and organ (liver) selectivity based on the structure of eprotirome (3) and molecular modeling. We found that indane derivatives are potent and dual-selective thyromimetics expected to avoid hypothyroidism in some tissues as well as heart toxicity. KTA-439 (29), a representative indane derivative, showed the same high human TRß selectivity in a binding assay as 3 and higher liver selectivity than 3 in a cholesterol-fed rat model.

Structure-activity relationship studies of 5-benzylaminoimidazo[1,2-c]pyrimidine-8-carboxamide derivatives as potent, highly selective ZAP-70 kinase inhibitors.

Zeta-associated protein, 70 kDa (ZAP-70), a spleen tyrosine kinase (Syk) family kinase, is normally expressed on T cells and natural killer cells and plays a crucial role in activation of the T cell immunoresponse. Thus, selective ZAP-70 inhibitors might be useful not only for treating autoimmune diseases, but also for suppressing organ transplant rejection. In our recent study on the synthesis of Syk family kinase inhibitors, we discovered that novel imidazo[1,2-c]pyrimidine-8-carboxamide derivatives possessed potent ZAP-70 inhibitory activity with good selectivity for ZAP-70 over other kinases. In particular, compound 26 showed excellent ZAP-70 kinase inhibition and high selectivity for ZAP-70 over structurally related Syk. The discovery of a potent, highly selective ZAP-70 inhibitor would contribute a new therapeutic tool for autoimmune diseases and organ transplant medication.

Structure-activity relationship studies of imidazo[1,2-c]pyrimidine derivatives as potent and orally effective Syk family kinases inhibitors.

Spleen tyrosine kinase (Syk) and zeta-associated protein kinase of 70k Da (ZAP-70) are members of the Syk family and non-receptor-type protein tyrosine kinases, which play crucial roles in B- and T-cell activation. Therefore, a Syk family tyrosine kinases inhibitor would be a useful therapeutic agent for the treatment of various allergic disorders and autoimmune diseases. Previously, we reported that 1,2,4-triazolo[4,3-c]pyrimidine derivative 1 and 1,2,4-triazolo[1,5-c]pyrimidine derivative 2 showed strong inhibitory activities against Syk family kinases. These compounds also exhibited high-level suppression of IL-2 in cellular assays. However, their oral efficacies were poor in a mouse model of IL-2 production. To improve oral effectiveness, we investigated a new series of Syk family kinases inhibitors. We found that imidazo[1,2-c]pyrimidine derivatives potently inhibited the Syk family kinases. Among these agents, compound 9f not only showed strong inhibitory activities against Syk and ZAP-70 kinases in vitro, but its oral administration resulted in the in vivo suppression of both the passive cutaneous anaphylaxis reaction and Concanavalin A-induced IL-2 production in a mouse model.

A novel Syk family kinase inhibitor: design, synthesis, and structure-activity relationship of 1,2,4-triazolo[4,3-c]pyrimidine and 1,2,4-triazolo[1,5-c]pyrimidine derivatives.

Splenic tyrosine kinase (Syk) family kinases, which are members of the protein tyrosine kinase family, play crucial roles in immune responses, with Syk participating in B-cell activation and the zeta-associated protein 70 kDa (ZAP-70) kinase being involved in T-cell activation. Therefore, Syk family kinase inhibitors are candidate therapeutic agents for the treatment of various allergic disorders and autoimmune diseases. We designed 1,2,4-triazolo[4,3-c]pyrimidine and 1,2,4-triazolo[1,5-c]pyrimidine derivatives as Syk family kinase inhibitors, based on literature reports and structure-based drug design. These derivatives showed significant Syk inhibitory activities, with ZAP-70 inhibition. Representative compounds 10d and 11 not only exhibited strong inhibition of both Syk and ZAP-70 kinase but also suppressed IL-2 production by peripheral blood mononuclear cells and whole blood.

Novel pyrazolo[1,5-a]pyrimidines as c-Src kinase inhibitors that reduce IKr channel blockade.

To improve the in vitro potency of the c-Src inhibitor 1a and to address its hERG liability, a structure-activity study was carried out, focusing on two regions of the lead compound. The blockade of the delayed cardiac current rectifier K(+) (I(Kr)) channel was overcome by replacing the ethylenediamino group with an amino alcohol group at the 7-position. In addition, modifying the substituents at the 5-position and the side chain groups on the amino alcohols at the 7-position enhanced the intracellular c-Src inhibitory activity and increased central nervous system (CNS) penetration. In the present study, 6l exhibited significant in vivo efficacy in a middle cerebral artery (MCA) occlusion model in rats.

Discovery of novel 2-anilinopyrazolo[1,5-a]pyrimidine derivatives as c-Src kinase inhibitors for the treatment of acute ischemic stroke.

We synthesized a series of novel 2-anilinopyrazolo[1,5-a]pyrimidine derivatives and evaluated their ability to inhibit c-Src kinase; 7-(2-amino-2-methylpropylamino)-5-cyclopropyl-2-(3,5-dimethoxyphenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide 7o and 7-(2-amino-2-methylpropylamino)-2-(3,5-dimethoxyphenylamino)-5-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide 7f showed potent inhibitory activity. Compound 7f inhibited c-Src selectively and exhibited satisfactory central nervous system (CNS) penetration. Furthermore, 7f.HCl reduced the infarct volume in vivo in a rat middle cerebral artery (MCA) occlusion model when administrated intraperitoneally.

Different inhibitory effects in the early and late phase of treatment with KAT-681, a liver-selective thyromimetic, on rat hepatocarcinogenesis induced by 2-acetylaminofluorene and partial hepatectomy after diethylnitrosamine initiation.

We recently reported that short-term treatment with KAT-681 (KAT), a liver-selective thyromimetic, inhibits the development of preneoplastic lesions in rat livers and may be a candidate chemopreventive agent for hepatocarcinogenesis. In this study, time-course observations of hepatocellular proliferative lesions were carried out during short-term and long-term treatment with KAT to investigate its anti-hepatocarcinogenic effects. The hepatocellular proliferative lesions in male F344 rats were induced by the initiation treatment of diethylnitrosamine (DEN), followed by treatment with 2-acetylaminofluorene (2-AAF) and partial hepatectomy (PH). The rats were administered KAT orally at a dose of 0.25 mg/kg/day for 3 weeks (experiment 1) or 0.1 mg/kg/day for 20 weeks (experiment 2). In experiment 1, a serial reduction in the number of altered hepatocellular foci (AHF) with positive expression of glutathione S-transferase placental form (GST-P) was observed until day 14 of the treatment period. The proliferative index (PI) of hepatocytes in the AHF significantly increased in the KAT group throughout the treatment period, with a peak on day 2. KAT treatment showed no obvious effects on GST-P-positive hepatocellular adenomas (HCAs) at any time point. In contrast, long-term KAT treatment in experiment 2 revealed a reduction in the mean size of HCAs in addition to reductions in the number and mean size of AHF. The PIs within the lesions in KAT-treated rats were significantly lower than those in controls. The present study indicates that KAT has different inhibitory effects on hepatocarcinogenesis in the early and late phases of KAT treatment; there is a reduction in AHF with enhanced cell proliferation in the early phase and the inhibition of development of AHF and HCAs with suppression of cell proliferation in the late phase. These results may suggest further potential of KAT as a promising chemopreventive agent for hepatocarcinogenesis.

Inhibitory effects of KAT-681, a liver-selective thyromimetic, on development of hepatocellular proliferative lesions in rats induced by 2-acetylaminofluorene and partial hepatectomy after diethylnitrosamine initiation.

To examine the potential inhibitory effects of a novel liver-selective thyromimetic, KAT-681 (KAT), on the development of hepatocellular proliferative lesions, male F344 rats were given a single intraperitoneal injection of 150 mg/kg diethylnitrosamine (DEN), followed by gavage administration of 7.5 mg/kg per day of 2-acetylaminofluorene (2-AAF) twice daily from weeks 2 to 4 with partial hepatectomy (PH) at week 3. From 5 weeks after the completion of 2-AAF administration, the rats were orally dosed with 0.04, 0.1, or 0.25 mg/kg per day KAT for 3 weeks, and subjected to morphometric analysis of the induced glutathione S-transferase placental form (GST-P)-positive lesions and hepatocellular adenomas (HCAs). Administration of KAT significantly and dose-dependently reduced the total area of GST-P-positive lesions (by 34-48%) and also their numbers (by 20-44%), their mean size not being significantly changed. No effects on the number of HCAs were apparent, although a reduction in their mean size was detected at a dose of 0.25 mg/kg per day KAT (by 34%). On biochemical analysis, serum activity of gamma-glutamyl transpeptidase, an enzyme related to hepatocarcinogenesis, was markedly reduced in rats given 0.25 mg/kg per day KAT (by 64%). The results of the present study thus suggest that KAT inhibits the development of altered hepatocellular foci and might be a promising chemopreventive agent for hepatocarcinogenesis.