Min In Mice Research Articles

7-Amino-3-phenyl-2-methyl-pyrazolopyrimidine derivatives inhibit human rhinovirus replication

Small molecules that exhibit broad-spectrum enteroviral inhibitory activity by targeting viral replication proteins are highly desired in antiviral drug discovery studies. To discover new human rhinovirus (hRV) inhibitors, we performed a high-throughput screening of 100,000 compounds from the Korea Chemical Bank library. This search led to identification of two phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) inhibitors having the pyrazolo-pyrimidine core structure, which display moderate anti-rhinoviral activity along with mild cytotoxicity. The results of a study aimed at optimizing the activity of the hit compounds showed that the pyrazolo-pyrimidine derivative 6f exhibits the highest activity (EC50 = 0.044, 0.066, and 0.083 μM for hRV-B14, hRV-A16, and hRV-A21, respectively) and moderate toxicity (CC50 = 31.38 μM). Furthermore, 6f has broad-spectrum activities against various hRVs, coxsackieviruses and other enteroviruses, such as EV-A71, EV-D68. An assessment of kinase inhibition potencies demonstrated that 6f possesses a high and selective kinase inhibition activity against PI4KIIIβ (IC50 value of 0.057 μM) and not against PI4KIIIα (>10 μM). Moreover, 6f exhibits modest hepatic stability (46.9 and 55.3 % remaining after 30 min in mouse and human liver microsomes, respectively). Finally, an in vivo study demonstrated that 6f possesses a desirable pharmacokinetic profile reflected in low systemic clearance (0.48 L∙h−1 kg−1) and modest oral bioavailability (52.4 %). Hence, 6f (KR-26549) appears to be an ideal lead for the development of new antiviral drugs.

Oral delivery of the amylin receptor agonist pramlintide

AbstractAmylin receptor agonism safely benefit diabetic patients, reducing the insulin requirements and glycemic excursions. Pramlintide is the triple proline human amylin analogue first used as injectable drug, but lacking physico‐chemical compatibility when co‐formulated with insulin. Here, we report the design and characterization of polymeric microparticles for oral delivery of pramlintide. Eudragit S100, a gastric‐resistant polymer, was used in preparation of pramlintide‐loaded spherical microcapsules by double emulsion and solvent evaporation technique, with approximately 66 μm ± 11 particle size, with 83.2% ± 2.7 efficiency for pramlintide entrapment and 67.6% ± 2.1 yield. Intra‐venous pramlintide free in solution showed a plasmatic half‐life of 6.8 min in mice. In contrast, oral delivery of acid‐resistant pramlintide‐loaded microparticles in mice showed a protracted release for 120 min compared to 30 min obtained for pramlintide in solution. Our data provide evidences for the potential use of the oral route in the therapeutic development of pramlintide formulations.

Novel CH25H+ and OASL+ microglia subclusters play distinct roles in cerebral ischemic stroke

BackgroundMicroglial polarization is one of the most promising therapeutic targets for multiple central nervous system (CNS) disorders, including ischemic stroke. However, detailed transcriptional alteration of microglia following cerebral ischemic stroke remains largely unclear.MethodsFocal cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) for 60 min in mice. Single-cell RNA sequencing (scRNA-seq) was performed using ischemic brain tissues from tMCAO and sham mice 3 days after surgery. Ch25h−/− mice were used to investigate the role of specific microglia subcluster on post-stroke infarct volume and neuroinflammation.ResultsWe identified a relatively homeostatic subcluster with enhanced antigen processing and three “ischemic stroke associated microglia” (ISAM): MKI67+, CH25H+ and OASL+ subclusters. We found the MKI67+ subcluster undergo proliferation and differentiation into CH25H+ and OASL+ subclusters. CH25H+ microglia was a critical subcluster of ISAM that exhibited increased phagocytosis and neuroprotective property after stroke. Ch25h−/− mice developed significantly increased infarct volume following ischemic stroke compared to Ch25h+/−. Meanwhile, the OASL+ subcluster accumulated in the ischemic brain and was associated with the evolving of neuroinflammation after stroke, which was further aggravated in the aged mice brain.ConclusionsOur data reveal previously unrecognized roles of the newly defined CH25H+ and OASL+ microglia subclusters following ischemic stroke, with novel insights for precise microglia modulation towards stroke therapy.

Synthesis and Evaluation of Fluorine-18-Labeled L-Rhamnose Derivatives.

The use of radiolabeled glucose for PET imaging resulted in the most commonly used tracer in the clinic, 2-deoxy-2-[18F]fluoroglucose (FDG). More recently, other radiolabeled sugars have been reported for various applications, including imaging tumors and infections. Therefore, in this study, we developed a series of fluorine-18-labeled L-rhamnose derivatives as potential PET tracers of various fungal and bacterial strains. Acetyl-protected triflate precursors of rhamnose were prepared and radiolabeled with fluorine-18 followed by hydrolysis to produce L-deoxy [18F]fluororhamnose. The overall radiochemical yield was 7-27% in a 90 min synthesis time with a radiochemical purity of 95%. In vivo biodistribution of the ligands using PET imaging showed that 2-deoxy-2-[18F]fluoro-L-rhamnose is stable for at least up to 60 min in mice and eliminated via renal clearance. The tracer also exhibited minimal tissue or skeletal uptake in healthy mice resulting in a low background signal.

Assessment of the Pharmacokinetics, Disposition, and Duration of Action of the Tumour-Targeting Peptide CEND-1

CEND-1 (iRGD) is a bifunctional cyclic peptide that can modulate the solid tumour microenvironment, enhancing the delivery and therapeutic index of co-administered anti-cancer agents. This study explored CEND-1's pharmacokinetic (PK) properties pre-clinically and clinically, and assessed CEND-1 distribution, tumour selectivity and duration of action in pre-clinical tumour models. Its PK properties were assessed after intravenous infusion of CEND-1 at various doses in animals (mice, rats, dogs and monkeys) and patients with metastatic pancreatic cancer. To assess tissue disposition, [3H]-CEND-1 radioligand was administered intravenously to mice bearing orthotopic 4T1 mammary carcinoma, followed by tissue measurement using quantitative whole-body autoradiography or quantitative radioactivity analysis. The duration of the tumour-penetrating effect of CEND-1 was evaluated by assessing tumour accumulation of Evans blue and gadolinium-based contrast agents in hepatocellular carcinoma (HCC) mouse models. The plasma half-life was approximately 25 min in mice and 2 h in patients following intravenous administration of CEND-1. [3H]-CEND-1 localised to the tumour and several healthy tissues shortly after administration but was cleared from most healthy tissues by 3 h. Despite the rapid systemic clearance, tumours retained significant [3H]-CEND-1 several hours post-administration. In mice with HCC, the tumour penetration activity remained elevated for at least 24 h after the injection of a single dose of CEND-1. These results indicate a favourable in vivo PK profile of CEND-1 and a specific and sustained tumour homing and tumour penetrability. Taken together, these data suggest that even single injections of CEND-1 may elicit long-lasting tumour PK improvements for co-administered anti-cancer agents.

Harmonization of sensorimotor deficit assessment in a registered multicentre pre-clinical randomized controlled trial using two models of ischemic stroke

Multicentre preclinical randomized controlled trials (pRCTs) are a valuable tool to improve experimental stroke research, but are challenging and therefore underused. A common challenge regards the standardization of procedures across centres. We here present the harmonization phase for the quantification of sensorimotor deficits by composite neuroscore, which was the primary outcome of two multicentre pRCTs assessing remote ischemic conditioning in rodent models of ischemic stroke. Ischemic stroke was induced by middle cerebral artery occlusion for 30, 45 or 60 min in mice and 50, 75 or 100 min in rats, allowing sufficient variability. Eleven animals per species were video recorded during neurobehavioural tasks and evaluated with neuroscore by eight independent raters, remotely and blindly. We aimed at reaching an intraclass correlation coefficient (ICC) ≥0.60 as satisfactory interrater agreement. After a first remote training we obtained ICC = 0.50 for mice and ICC = 0.49 for rats. Errors were identified in animal handling and test execution. After a second remote training, we reached the target interrater agreement for mice (ICC = 0.64) and rats (ICC = 0.69). In conclusion, a multi-step, online harmonization phase proved to be feasible, easy to implement and highly effective to align each centre’s behavioral evaluations before project’s interventional phase.

Abstract 5437: Preclinical characterization of EB160, a novel, highly potent and brain-penetrant KRASG12C inhibitor

Abstract The KRASG12C mutation is present in nearly 13% of lung adenocarcinoma, 3% of colorectal cancer and 2% of other solid tumors. AMG510 developed by Amgen and MRTX849 developed by Mirati Therapeutics both target the KRASG12C mutation through covalent modification of the Cys 12 of the mutated protein. According to the disclosed clinical data, AMG510 and MRTX849 have an ORR of 36-45% in NSCLC and of 7-22% in CRC patients harboring a KRASG12C mutation. Incomplete target occupancy is considered to be a potential reason for only less than 50% of patients with KRASG12C mutation benefiting from those KRASG12C inhibitors. Brain metastases developed from lung cancer, accounting for 40-50% of all brain metastases, were described as the primary or contributing cause of death in a large proportion of patients with brain metastases from NSCLC. About 55% of NSCLC patients with KRAS mutations develop brain metastases. The KRAS mutant patients with untreated brain metastasis from NSCLC have a median OS of only 5 months. Hence, in order to achieve maximal clinical benefit in patients with KRASG12C mutation, a highly potent, brain-penetrant KRASG12C inhibitor with sufficient continuous exposure for the complete dose interval to maximize depth and duration of antitumor activity is required. Here, we report the discovery of EB160, a novel, highly potent and brain-penetrant KRASG12C covalent inhibitor. In a panel of KRASG12C mutant cancer cell lines, EB160 inhibited the proliferation of these cells with higher potency compared to AMG510 and MRTX849. EB160 also inhibited phosphor-ERK in H358 cells with an IC50 value of 17.54 nM at 1 h, while the IC50 values of AMG510 and MRTX849 were 58.98 nM and 41.89 nM, respectively. In vitro ADME, EB160 showed moderate or high stability in liver microsomes across the species tested, and the whole blood t1/2 was greater than 240 min in mouse, rat, dog and human, which was comparable to MRTX849. Following single PO dose (5 mg/kg) in mice, rats and dogs, the mean AUC(0-∞) were 2679 ng*h/mL, 2855 ng*h/mL and 1511 ng*h/mL, respectively, and the absolute bioavailabilities were 86%, 78% and 32%, respectively. A H358 cell line-derived lung cancer xenograft model was selected for in vivo efficacy assay. At 10 mg/kg QD of EB160 for 28 days, the tumor growth inhibition (TGI) was 108.2%, with 5 out of 6 mice showing tumor regression > 80% from initial baseline. And in the 30 mg/kg treatment group, whole 6 mice showed near complete tumor regression. In the H1373Luc KRASG12C brain metastases model, 30 mg/kg QD EB160 oral dose in mice for 28 days results in tumor regression 92%, while in the same dosage of MRTX849 treatment group, the tumor regression was 50%. Citation Format: Xuxing Chen, Jing Li, Yanhong Chen. Preclinical characterization of EB160, a novel, highly potent and brain-penetrant KRASG12C inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5437.

MicroRNA-140–5p exacerbates vascular cognitive impairment by inhibiting neurogenesis in the adult mouse hippocampus after global cerebral ischemia

Vascular cognitive impairment (VCI) is the most common type of dementia after Alzheimer’s disease (AD). Effective treatments for VCI are currently lacking. MicroRNA (miR)− 140–5p is associated with cerebral ischemia and poststroke depression, but its relationship with VCI remains unknown. A VCI model was established by bilateral common carotid artery occlusion (BCCAO) for 17 min in mice. Neurogenesis was evaluated by immunostaining for Nestin/bromodeoxyuridine (BrdU), NeuN/BrdU, and doublecortin (DCX)/BrdU. Neuroplasticity was assessed by quantifying synapsin-I and postsynaptic density protein 95 (PSD-95) protein levels. Predicted target genes were screened and verified using the dual luciferase reporter gene system. MiR-140–5p was upregulated in the hippocampus of the BCCAO mice 2 weeks following ischemia. Compared with control groups, the AAV-miR-140–5p group exhibited poorer cognitive performance alongside lower numbers of DCX/BrdU and NeuN/BrdU and less synapsin-I and PSD-95 in the dentate gyrus (P < 0.05). MiR-140–5p overexpression decreased the predicted target gene Prox1. Dual luciferase reporter system confirmed that Prox1 was a direct target site for miR-140–5p. In conclusion, our results suggest that miR-140–5p inhibits neurogenesis and neuroplasticity via downregulation of Prox1 and aggravates VCI. Our findings highlight that miR-140–5p is involved in the pathological process of VCI and provides information for the development of new treatments, which may need further inhibition tests to verify.

Temporal patterns of increased growth hormone secretion in mice after oral administration of L-ornithine: possible involvement of ghrelin receptors.

l-Ornithine is known to stimulate growth hormone (GH) release in mammals. Here, we demonstrated that increases in plasma GH levels after oral administration of l-ornithine were first observed 150 min after administration, and the elevated levels were sustained for more than 90 min in mice. The increase was significantly delayed compared with the reported timing of plasma and tissue levels of l-ornithine after administration. The l-ornithine-induced increase in GH release was completely blocked by [D-Lys3]-GHRP-6, a ghrelin receptor antagonist, but not by cyclosomatostatin or JV-1-38, antagonists of somatostatin and GH-releasing hormone, respectively. These results suggest the involvement of ghrelin receptor-mediated pathways in l-ornithine-induced increases in GH release.

Recommended doses of medetomidine-midazolam-butorphanol with atipamezole for preventing hypothermia in mice.

A non-narcotic anesthetic combination (Me/Mi/Bu) of medetomidine (Me), midazolam (Mi), and butorphanol (Bu) has been recommended as the injectable anesthesia in mice. An original dose of Me/Mi/Bu (0.3/4.0/5.0 mg/kg) has provided sufficient anesthetic duration of 40–50 min in mice. In addition, atipamezole is available for reversal of Me/Mi/Bu anesthesia. As an adverse effect of Me/Mi/Bu anesthesia, however, severe hypothermia has been also observed in mice. In the present study, we investigated 1) the main agent in Me/Mi/Bu to cause of hypothermia, 2) the effects of the differential doses of atipamezole on hypothermia induced by Me/Mi/Bu anesthesia and on the plasma levels of creatinine phosphokinase and transaminases, and 3) those recommended doses for preventing hypothermia induced by Me/Mi/Bu anesthesia in mice. The results suggested that 1) the α2-agonist medetomidine is most likely to induce hypothermia in mice under Me/Mi/Bu anesthesia, 2) the antagonism of atipamezole within proper dose range is effective in promoting the recovery from Me/Mi/Bu-induced hypothermia, and 3) Me/Mi/Bu at the recommended dose of 0.2/6.0/10.0 mg/kg enable to provide anesthetic effects for 40 min and is more considerable to prevent the hypothermia than that at the original dose of 0.3/4.0/5.0 mg/kg.

Participation of Monocarboxylate Transporter 8, But Not P-Glycoprotein, in Carrier-Mediated Cerebral Elimination of Phenytoin across the Blood-Brain Barrier.

In this study, we investigated in detail the transport of phenytoin across the blood-brain barrier (BBB) to identify the transporter(s) involved in BBB-mediated phenytoin efflux from the brain. We evaluated the brain-to-blood efflux transport of phenytoin in vivo by determining the brain efflux index (BEI) and uptake in brain slices. We additionally conducted brain perfusion experiments and BEI studies in P-glycoprotein (P-gp)-deficient mice. In addition, we determined the mRNA expression of monocarboxylate transporter (MCT) in isolated brain capillaries and performed phenytoin uptake studies in MCT-expressing Xenopus oocytes. [14C]Phenytoin brain efflux was time-dependent with a half-life of 17min in rats and 31min in mice. Intracerebral pre-administration of unlabeled phenytoin attenuated BBB-mediated phenytoin efflux transport, suggesting carrier-mediated phenytoin efflux transport across the BBB. Pre-administration of P-gp substrates in rats and genetic P-gp deficiency in mice did not affect BBB-mediated phenytoin efflux transport. In contrast, pre-administration of MCT8 inhibitors attenuated phenytoin efflux. Moreover, rat MCT8-expressing Xenopus oocytes exhibited [14C]phenytoin uptake, which was inhibited by unlabeled phenytoin. Our data suggest that MCT8 at the BBB participates in phenytoin efflux transport from the brain to the blood.

Full-length-transcriptomic analysis in mice and human heart using Single-Molecule Real-time Sequencing (SMRT) identified 15 novel isoforms and a novel promoter region of PGC1-alpha

Abstract Background Alternative splicing is a process by which exons within a pre-mRNA are joined or skipped, resulting in isoforms being encoded by a single gene. Alternative Splicing affecting transcription factors may have substantial impact on cellular dynamics. The PPARG Coactivator 1 Alpha (PGC1-α), is a major modulator in energy metabolism. Data from murine skeletal muscle revealed distinctive isoform patterns giving rise to different phenotypes, i.e. mitogenesis and hypertrophy. Here, we aimed to establish a complete dataset of isoforms in murine and human heart applying single-molecule real-time (SMRT)-sequencing as novel approach to identify transcripts without need for assembly, resulting in true full-length sequences. Moreover, we aimed to unravel functional relevance of the various isoforms during experimental ischemia reperfusion (I/R). Methods RNA-Isolation was performed in murine (C57Bl/6J) or human heart tissue (obtained during LVAD-surgery), followed by library preparation and SMRT-Sequencing. Bioinformatic analysis was done using a modified IsoSeq3-Pipeline and OS-tools. Identification of PGC1-α isoforms was fulfilled by similarity search against exonic sequences within the full-length, non-concatemere (FLNC) reads. Isoforms with Open-Reading-Frame (ORF) were manually curated and validated by PCR and Sanger-Sequencing. I/R was induced by ligature of the LAD for 45 min in mice on standard chow as well as on high-fat-high-sucrose diet. Area At Risk (AAR) and remote tissue were collected three and 16 days after I/R or sham-surgery (n=4 per time point). Promotor patterns were analyzed by qPCR. Results Deciphering the full-length transcriptome of murine and human heart resulted in ∼60000 Isoforms with 99% accuracy on mRNA-sequence. Focusing on murine PGC1-α-isoforms we discovered and verified 15 novel transcripts generated by hitherto unknown splicing events. Additionally, we identified a novel Exon 1 originating between the known promoters followed by a valid ORF, suggesting the discovery of a novel promoter. Remarkably, we found a homologous novel Exon1 in human heart, suggesting conservation of the postulated promoter. In I/R the AAR exhibited a significant lower expression of established and novel promoters compared to remote under standard chow 3d post I/R. 16d post I/R, the difference between AAR &amp; Remote equalized in standard chow while remaining under High-Fat-Diet. Conclusion Applying SMRT-technique, we generated the first time a complete full-length-transcriptome of the murine and human heart, identifying 15 novel potentially coding transcripts of PGC1-α and a novel exon 1. These transcripts are differentially regulated in experimental I/R in AAR and remote myocardium, suggesting transcriptional regulation and alternative splicing modulating PGC1-α function in heart. Differences between standard chow and high fat diet suggest impact of impaired glucose metabolism on regulatory processes after myocardial infarction. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Collaborative Research Centre 1116 (German Research Foundation)

Metabolic profiling of senkyunolide A and identification of its metabolites in hepatocytes by ultra-high-performance liquid chromatography combined with diode-array detector and high-resolution mass spectrometry.

Senkyunolide A is one of the bioactive constituents originally isolated from Ligusticum chuanxiong Hort. To better understand the action of this constituent, it is necessary to study the metabolic profiles in different species. For the metabolic stability study, senkyunolide A at a concentration of 0.5 μM was individually incubated with hepatocytes of mouse, rat, dog, monkey and human at 37°C for 2 h. For metabolite profiling and identification, senkyunolide A (10 μM) was incubated with hepatocytes and the incubation samples were analyzed by ultra-high-performance liquid chromatography combined with diode-array detector and high-resolution mass spectrometry (UHPLC/DAD-HRMS). The identities of the metabolites were characterized by accurate masses, product ions and retention times. Senkyunolide A was metabolically unstable in hepatocytes. The in vitro half-lives were 136.2, 60.6, 33.65, 55.96 and 138 min in mouse, rat, dog, monkey and human hepatocytes, respectively. Furthermore, a total of 14 metabolites were detected. M1 and M9 were the most abundant metabolites in all species. The metabolic pathways of senkyunolide A involved the following pathways: (1) hydroxylation to form 10- and 11-hydroxysenkyunolide A, which further underwent epoxidation followed by GSH conjugation; (2) epoxidation followed by epoxide hydrolysis or GSH conjugation; and (3) aromatization to form 3-butylphthalide followed by hydroxylation. Hydroxylation, epoxidation, aromatization and GSH conjugation were the main metabolic pathways of senkyunolide A. This study provides an overview of the metabolic profiles of senkyunolide A, which is helpful for a better understanding of the action of this compound.

Pharmacokinetics of Panaxynol in Mice.

The purpose of our study is to explore the pharmacokinetic parameters of panaxynol (PA) and understand its potential and dosage used in pre-clinical animal models. For in vitro analysis,5 μM of PA was added to liver microsomes of mouse and human species. Nicotinamide adenine dinucleotide phosphate was added to initiate enzyme reaction except for the negative control. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis was used to measure concentrations. For in vivo studies, CD-1 mice were treated with PA by intravenous (IV) injection or oral administration (PO). Concentrations of PA were measured in plasma and tissue using LC-MS/MS. Pharmacokinetic parameters were obtained using non-compartmental analysis. Area under the curve concentration versus time was calculated using a linear trapezoidal model.In vitro, PA’s half-life is 21.4 min and 48.1 min in mouse and human liver microsomes, respectively. In vivo, PA has a half-life of 1.5 hr when IV-injected, and 5.9 hr when administered via PO, with a moderate bioavailability of 50.4%. Mice show no signs of toxicity up to 300 mg/kg PO. PA concentrations were highest in colon tissue 2 hr post-treatment at 486 ng/g of colon tissue.PA’s pharmacokinetic properties and low toxicity point to the safety and compatibility of PA with mice.

Abstract 3893: Melatonin-tamoxifen drug conjugates: Mechanism against breast cancer cells, and pharmacokinetic assessment in female mice

Abstract Background: Melatonin-tamoxifen drug conjugate linked with a 5-carbon chain (C5) exhibited uterus protection in female mice, which did not occur when the same doses of unlinked melatonin and tamoxifen were co-administered. Anti-cancer actions of C5 and 4 others, with varying carbon lengths (C2, C4, C9, C15), were further tested in phenotypically diverse breast cancer (BC) cell lines—MCF-7(ER+/PR+), MMC(HER2+) and triple negatives(MDA-MB-231 and BT-549) in terms of cell viability and migration. In all BC lines, C4 and C5 exhibited superior potency and efficacy compared to C2, C9, C15 and unlinked melatonin and tamoxifen or 4-OH-tamoxifen controls. In this study, C4 and C5 were further tested for their anti-cancer effects in tamoxifen-resistant MCF-7 cells(TamR-MCF-7); their pharmacokinetic parameters were assessed in vitro and in vivo; and the mechanisms underlying C4- and C5-mediated anti-cancer actions were also determined by use of inhibitors against MEK1/2, MEK5 or PI3 kinase and western blot analysis. Results: C4 and C5 exhibited similar potency (TamR-MCF-7 IC50 = 4.22µM and 7.21µM; MCF-7 IC50 = 3.62nM and 440mM) and efficacy (TamR-MCF-7 = 83% and 81% inhibition; MCF-7= 71% and 90% inhibition) to inhibit TamR-MCF-7 cell viability as they did in MCF-7 cells. Regarding metabolism, C4 and C5 displayed similar CYP-mediated pharmacokinetic profiles as tamoxifen in both mouse and human liver microsomes where 40% loss of tamoxifen, C4 or C5 occurred by 10 min in mouse vs. 20 min in human. In vivo, the Tmax for C5 (oral) was 24h compared to 0.5h for C4 (oral) resulting in lower Cmax (8.9 ng/mL) and AUC0-24 (1.6 hr.ng/mL) for C5 compared to C4 (Cmax =102.6 ng/mL and AUC0-24 =143.7 hr.ng/mL) and lower oral bioavailability (0.11%) for C5 compared to C4 (4.52%). Regarding mechanisms, the effect of C4- or C5-mediated protein modulation was “context-specific” dependent upon BC phenotype (i.e., ER+, HER2+ or ER-/PR-/HER2-); the number of carbons linking melatonin to tamoxifen (i.e., 4 carbons for C4 or 5 carbons for C5); and the concentration (i.e., 1nM or 10µM) of C4 or C5 tested. For MCF-7, MMC and MDA- MB-231 cells, inhibition of MEK1/2, MEK5 and PI3K enhanced but did not block C4’s or C5’s anti-cancer actions; while for BT-549 cells, Bix02189 blocked C5’s effects. Also, in MCF-7 cells, pERK1/2, NF-κB and Runx2 were significantly modulated by C4 or C5; for MMCs, C4 and C5 modulated NF-κB and β1-integrin expression; for MDA-MB-231, MEK1/2 and MEK5 played a more central role possibly through co-modulation of NF-κB; and for BT-549, C5-mediated effects on pERK5 or PI3K-dependent regulation of pERK1/2 may underlie its anti-cancer actions. Conclusions: Melatonin-tamoxifen drug conjugates (C4 and C5) may be superior to tamoxifen therapy alone in a variety of cancers, including TamR BC. Citation Format: Mahmud Hasan, Thomas D. Wright, Saugat Adhikari, Mohamed A. Marzouk, Alaina Pericoloso, Kelsey Murgas, Miranda Burgman, Benton P. Miller, Ulrike Holzgrabe, Darius P. Zlotos, Robert E. Stratford, Paula A. Witt-Enderby. Melatonin-tamoxifen drug conjugates: Mechanism against breast cancer cells, and pharmacokinetic assessment in female mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3893.

Djulis (&lt;i&gt;Chenopodium formosanum&lt;/i&gt;) and Its Bioactive Compounds for Management of Hyperlipidemia and Hyperglycemia in High-Fat Diet-Fed Mice

The aim of this study was to investigate the effect of Djulis (Chenopodium formosaneum) and its bioactive compounds on hyperlipidemia and hyperglycemia in high-fat diet (HFD)-fed mice. The study indicated that, total cholesterol (TC), and triglyceride (TG) levels were significantly higher in HFD-fed mice than in those fed with ethanolic extract of Djulis (EECF) at different doses (10, 25, 50 mg/kg bw), rutin (5 mg/kg bw), betanin (5 mg/kg bw) and quercetin (5 mg/kg bw) throughout the 12-week period, indicating alleviation of hyperlipidemia by EECF and bioactive compounds groups. Moreover, plasma insulin was significantly higher in HFD-fed mice than those fed with EECF at different doses, rutin, betanin and quercetin. In addition, results of oral glucose tolerance test (OGTT) revealed improved glucose tolerance and ameliorated insulin response at 60 and 90 min in mice fed with EECF, rutin, betanin and quercetin. The findings of this study suggest EECF has beneficial effects on hyperlipidemia and hyperglycemia of obese mice induced with HFD, which can be partly attributed to the bioactive compounds present in Djulis.

Development of an anti-hepatitis B virus (HBV) agent through the structure-activity relationship of the interferon-like small compound CDM-3008.

Hepatitis B, a viral infectious disease caused by hepatitis B virus (HBV), is a life-threatening disease that leads liver cirrhosis and liver cancer. Because the current treatments for HBV, such as an interferon (IFN) formulation or nucleoside/nucleotide analogues, are not sufficient, the development of a more effective agent for HBV is urgent required. CDM-3008 (1, 2-(2,4-bis(trifluoromethyl)imidazo[1,2-a][1,8]naphthyridin-8-yl)-1,3,4-oxadiazole) (RO8191)) is a small molecule with an imidazo[1,2-a][1,8]naphthyridine scaffold that shows anti-HCV activity with an IFN-like effect. Here, we report that 1 was also effective for HBV, although the solubility and metabolic stability were insufficient for clinical use. Through the structure-activity relationship (SAR), we discovered that CDM-3032 (11, N-(piperidine-4-yl)-2,4-bis(trifluoromethyl)imidazo[1,2-a][1,8]naphthyridine-8-carboxamide hydrochloride) was more soluble than 1 (>30 mg/mL for 11 versus 0.92 mg/mL for 1). In addition, the half-life period of 11 was dramatically improved in both mouse and human hepatic microsomes (T1/2, >120 min versus 58.2 min in mouse, and >120 min versus 34.1 min in human, for 11 and 1, respectively).

Apelin-13 analogues show potent in vitro and in vivo insulinotropic and glucose lowering actions

Nine structurally modified apelin-13 analogues were assessed for their in vitro and acute in vivo antidiabetic potential. Stability was assessed in mouse plasma and insulinotropic efficacy tested in cultured pancreatic BRIN-BD11 cells and isolated mouse pancreatic islets. Intracellular Ca2+ and cAMP production in BRIN-BD11 cells was determined, as was glucose uptake in 3T3-L1 adipocytes. Acute antihyperglycemic effects of apelin analogues were assessed following i.p. glucose tolerance tests (ipGGT, 18 mmol/kg) in normal and diet-induced-obese (DIO) mice and on food intake in normal mice. Apelin analogues all showed enhanced in vitro stability (up to 5.8-fold, t½ = 12.8 h) in mouse plasma compared to native apelin-13 (t½ = 2.1 h). Compared to glucose controls, stable analogues exhibited enhanced insulinotropic responses from BRIN-BD11 cells (up to 4.7-fold, p < 0.001) and isolated mouse islets (up to 5.3-fold) for 10−7 M apelin-13 amide (versus 7.6-fold for 10−7 M GLP-1). Activation of APJ receptors on BRIN-BD11 cells increased intracellular Ca2+ (up to 3.0-fold, p < 0.001) and cAMP (up to 1.7-fold, p < 0.01). Acute ipGTT showed improved insulinotropic and glucose disposal responses in normal and DIO mice (p < 0.05 and p < 0.01, respectively). Apelin-13 amide and (pGlu)apelin-13 amide were the most effective analogues exhibiting acute, dose-dependent and persistent biological actions. Both analogues stimulated insulin-independent glucose uptake by differentiated adipocytes (2.9–3.3-fold, p < 0.05) and inhibited food intake (26–33%, p < 0.001), up to 180 min in mice, versus saline. In contrast, (Ala13)apelin-13 and (Val13)apelin-13 inhibited insulin secretion, suppressed beta-cell signal transduction and stimulated food intake in mice. Thus, stable analogues of apelin-13 have potential for diabetes/obesity therapy.

Pharmacodynamic and Pharmacokinetic Properties of AFPep, a Novel Peptide for the Treatment of Breast Cancer

AFPep is a small synthetic cyclized peptide derived from alpha-fetoprotein that has been shown to have anti-estrogenic and anti-breast cancer activity. The purpose of this study was to establish blood levels of AFPep that are associated with biological activity. Blood levels of AFPep were measured by LC/MS/MS. Once daily treatment of mice with 4 mg/kg i.p. AFPep yielded a Cmax of 7 µg/ml and was sufficient to inhibit estrogen-stimulated growth of mouse uterus and of human breast cancer xenografts even though the half-life of this drug was only 11 min in mice, suggesting that its biological effects last much longer than its chemical half-life. In dose de-escalation studies, blood levels of 100 ng/ml of AFPep were still found to be biologically active. AFPep was effective by parenteral routes and with dose escalation also by the oral route. Blood levels of AFPep that were biologically effective in mice were readily achieved in dogs through parenteral as well as oral routes with no apparent evidence of host toxicity. In conclusion, AFPep blood levels can be measured by LC/MS/MS with accuracy into the ng/ml range. Blood levels in the 100 ng/ml range are associated with efficacious biological activity. This drug shows great promise for the treatment as well as prevention of breast cancer.

Conjugation to 10 kDa Linear PEG Extends Serum Half-Life and Preserves the Receptor-Binding Ability of mmTRAIL with Minimal Stimulation of PEG-Specific Antibodies.

The poor in vivo potencies of most therapeutic proteins might be attributed to their short serum half-lives. PEGylation is a well-established method and has been clinically proven to improve pharmacokinetics. mmTRAIL exhibited supercytotoxicity in a variety of tumor cells, but its serum half-life was less than 10 min in mice. Here, mmTRAIL-5K, mmTRAIL-10K, and mmTRAIL-20K were produced by N-terminus-specific PEGylation of mmTRAIL with 5, 10, or 20 kDa mPEG, respectively. The particle sizes of mmTRAIL-5K, mmTRAIL-10K, and mmTRAIL-20K were 9.09 ± 2.76, 12.62 ± 4.05, and 15.68 ± 4.95 nm, which were higher than the threshold (∼7 nm) of renal clearance. Accordingly, mmTRAIL-5K exhibited a serum half-life of 30 min only 3 times longer than that of mmTRAIL. However, both mmTRAIL-10K and mmTRAIL-20K exhibited similar serum half-lives ranging from 350 to 400 min, indicating that PEGylation with 10 or 20 kDa mPEG significantly improved the pharmacokinetics of mmTRAIL. However, death receptor binding of mmTRAIL-20K was reduced 5- to 8-fold, resulting in a 3-fold reduction of cytotoxicity. Additionally, repeated administration of mmTRAIL-20K elicited both mPEG-specific IgG and IgM antibody responses in rats. In contrast, the receptor binding and cytotoxicity of mmTRAIL-10K were similar to those of mmTRAIL. Repeated administration of mmTRAIL-10K did not obviously stimulate mPEG-specific antibody responses in rats and rhesus monkeys. Of the three PEGylated mmTRAIL analogues, mmTRAIL-10K exerted the greatest tumor suppression in mice bearing human tumor xenografts. These results demonstrated that conjugation of mmTRAIL to 10 kDa mPEG was better than that to 5 or 20 kDa mPEG for enhancing antitumor effects.