An orally active inhibitor of leukotriene synthesis accelerates healing in a rat model of colitis

1990 ◽  
Vol 258 (4) ◽  
pp. G527-G534 ◽  
Author(s):  
J. L. Wallace ◽  
C. M. Keenan
Keyword(s):  
Rat Model ◽  
Equilibrium Dialysis ◽  
Daily Treatment ◽  
Myeloperoxidase Activity ◽  
Trinitrobenzenesulfonic Acid ◽  
Tissue Samples ◽  
Active Inhibitor ◽  
Orally Active

Leukotrienes (LTs) have been implicated as mediators of the inflammation and ulceration associated with ulcerative colitis and Crohn's disease. In the present study, the effects of a novel, orally active inhibitor of LT synthesis (MK-886) were examined in a rat model of chronic colitis. Colitis was induced by intracolonic administration of trinitrobenzenesulfonic acid. Colonic LTB4 synthesis was measured after incubation of tissue samples in vitro and by in vivo equilibrium dialysis. A single dose of MK-886 (10 mg/kg) significantly inhibited colonic LTB4 synthesis for greater than 24 h. Daily treatment with this dose significantly reduced colonic damage, as assessed macroscopically and histologically, when the treatment was performed 2 h before induction of colitis and daily thereafter for 1 wk, but not when treatment was performed during the second week after induction of colitis. A less marked beneficial effect of MK-886 was observed when the pretreatment dose was excluded, suggesting a role for LTs in the early events of the inflammatory process. Inhibition of LT synthesis during the first 24 h after induction of colitis did not alter the extent of infiltration of neutrophils into the colon, as measured by tissue myeloperoxidase activity. Daily treatment with sulfasalazine (100 mg/kg po) either during the first or second week after induction of colitis did not significantly affect the rates of healing. At the dose used, sulfasalazine only produced a transient inhibition of colonic LTB4 synthesis. This study therefore demonstrates that a specific, orally active inhibitor of LT synthesis can significantly accelerate healing in this animal model of colitis when the treatment is performed during the early phase of the inflammatory response.

Potent Antitumor Activity of AP24534, An Orally Active Inhibitor of Bcr-Abl, Flt3 and Other Kinases, in Both in Vitro and in Vivo Models of Acute Myeloid Leukemia (AML)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2932-2932 ◽  
Author(s):  
Victor M. Rivera ◽  
Qihong Xu ◽  
Lori Berk ◽  
Jeffrey Keats ◽  
Scott Wardwell ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Tumor Regression ◽  
Hematologic Malignancies ◽  
Fgf Receptor ◽  
Active Inhibitor ◽  
Orally Active

Abstract AP24534 is a potent, orally active inhibitor of Bcr-Abl and its mutants, including T315I, inhibiting kinase activity with IC50s of 0.3–2 nM. Potent cellular and in vivo activity of the compound has been demonstrated in models of chronic myeloid leukemia (CML). AP24534 also potently inhibits a discrete subset of other kinases, including Flt3 (IC50 13 nM), c-Kit (13 nM) and members of the FGF receptor family (2–18 nM), suggesting the potential for activity against other hematologic disorders characterized by activation of these proteins, such as acute myeloid leukemia (AML). Methods: In this study, we examined the effects of AP24534 on AML cell lines characterized by expression of various activated kinase targets, including the internal tandem duplication (ITD) variant of Flt3, FGFR1 and c-Kit. Effects on cell viability in vitro were determined using an MTS assay, and correlated with biochemical assessment of target inhibition by Western blot analysis. In vivo activity was determined by daily oral administration of AP24534 for 4 weeks in a subcutaneous tumor model using Flt3-ITD-expressing cell line MV4-11. Results: AP24534 potently inhibited the viability of AML cell lines expressing Flt3-ITD (MV4-11 cells), an activated FGFR1 fusion (KG-1 cells) or an activating c-Kit mutant N822K (Kasumi-1 cells) with IC50s of 0.7, 2.5 and 2.4 nM, respectively. In MV4-11, KG-1 and Kasumi-1 cells western blot analysis demonstrated that AP24534 inhibited the phosphorylation of the putative targets with IC50s of approximately 1, 10 and 18 nM, respectively. Furthermore, potent cellular activity (<10 nM) against all 3 activated kinases was a unique characteristic of AP24534 compared with other multi-targeted kinase inhibitors tested, including sunitinib and sorafenib. In vivo activity of AP24534 was examined in an MV4-11 mouse xenograft model. Statistically significant inhibition of tumor growth was demonstrated with once-daily oral doses as low as 1 mg/kg, and partial tumor regression with doses of 2.5 mg/kg. Doses of 5 or 10 mg/kg led to complete and durable tumor regression with no palpable tumors detected during a 4 week follow-up period. A single 10 mg/kg dose of AP24534 was sufficient to block phosphorylation of STAT5, a major downstream target of Flt-3. These potencies and responses observed in AML cell lines are comparable to the observed effects of AP24534 in analogous in vitro and xenograft studies using the Bcr-Abl-driven CML cell line K562. Conclusions: These results indicate that AP24534 has the potential to be an effective treatment for AML, including the approximately one-third of AML cases characterized by the Flt3- ITD mutation that is correlated with a poor prognosis. The compound was particularly potent on the Flt3-driven cell line MV4-11. Inhibition by AP24534 of non-Flt3-dependent AML cell lines, such as those driven by c-Kit or FGF receptors, indicates the potential for activity across diverse AML subtypes and other c-Kit or FGF receptor-driven malignancies, such as multiple myeloma. Together with previous data showing potent activity in CML models, these results suggest a broad potential for AP24534 in hematologic malignancies. Based on these observations, a phase 1 clinical trial is now underway to evaluate AP24534 in patients with a range of hematologic malignancies, including AML and CML.

Pharmacokinetics of Darolutamide, its Diastereomers and Active Metabolite in the Mouse: Response to Saini NK et al. (2020)

2020 ◽  
Vol 14 ◽  
Author(s):  
Pirjo Nykänen ◽  
Timo Korjamo ◽  
Hille Gieschen ◽  
Christian Zurth ◽  
Mikko Koskinen
Keyword(s):  
Protein Binding ◽  
Equilibrium Dialysis ◽  
In Vivo Studies ◽  
Pharmacokinetic Parameters ◽  
Tissue Samples ◽  
High Protein Binding ◽  
Mouse Hepatocytes ◽  
Mouse Response

Background: Saini et al. recently investigated the pharmacokinetics of darolutamide and its diastereomers in vitro and in vivo in Balb/c mice, reporting higher levels of (S,S)-darolutamide than (S,R)-darolutamide following intravenous or oral dosing, and interconversion of (S,R)-darolutamide to (S,S)-darolutamide. Objective: To present our in vitro and in vivo studies of darolutamide pharmacokinetics in mice, which contrast with the findings of Saini et al. Methods: Nude male Balb/c mice were orally dosed for 7 days with 25, 50, or 100 mg/kg of darolutamide twice daily. Pharmacokinetic parameters in plasma and tissue samples were assessed by liquid chromatography-tandem mass spectrometry. Metabolism and interconversion of darolutamide and diastereomers was investigated in cryopreserved Balb/c mouse hepatocytes. Protein binding was determined in plasma samples by equilibrium dialysis. Results and Discussion: At day 7, Cmax was reached 30 min after last dose. Rapid formation and greater exposure of ketodarolutamide versus darolutamide were observed. Plasma exposure of (S,R)-darolutamide was 3–5-fold higher than that of (S,S)-darolutamide. The fraction of unbound keto-darolutamide was almost 6-fold lower than for darolutamide. In mouse hepatocytes, conversion of (S,S)- to (S,R)-darolutamide was observed but conversion of (S,R)- to (S,S)- darolutamide was not detectable. Back-formation of keto-darolutamide to both diastereomers occurred at low levels. Conclusion: The darolutamide diastereomer ratio changes upon administration in mice and other species, due to interconversion through keto-darolutamide. This is not considered clinically relevant since both diastereomers and ketodarolutamide are pharmacologically similar in vitro. Based on the high protein binding of keto-darolutamide, its contribution in vivo in humans is considered low.

In Vitro and In Vivo Studies of AT-1362, a Newly Synthesized and Orally Active Inhibitor of Thrombin

2000 ◽  
Vol 100 (1) ◽  
pp. 97-107 ◽  
Author(s):  
Jae-hyung Cho ◽  
Hyoung-sik Seo ◽  
Chi-ho Yun ◽  
Bon-am Koo ◽  
Shoshin Yoshida ◽  
...  
Keyword(s):  
In Vivo Studies ◽  
Active Inhibitor ◽  
Orally Active

Potent Antitumor Activity of AP24534, an Orally Active Inhibitor of Bcr-Abl Variants Including T315I, in In Vitro and In Vivo Models of Chronic Myeloid Leukemia (CML).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1032-1032 ◽  
Author(s):  
Victor M. Rivera ◽  
Qihong Xu ◽  
Frank Wang ◽  
Joseph Snodgrass ◽  
Thomas O’Hare ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Oral Administration ◽  
Complete Regression ◽  
Dependent Manner ◽  
In Vivo Models ◽  
Active Inhibitor ◽  
Short Term Exposure ◽  
Orally Active

Abstract Resistance to current Bcr-Abl kinase inhibitors in CML patients is associated with the emergence of Bcr-Abl point mutations, including the T315I variant which represents 15–20% of clinically observed mutants and is resistant to all approved agents (e.g. imatinib and dasatinib). AP24534 is a novel, orally active Bcr-Abl inhibitor that also potently inhibits T315I and other clinically relevant mutants. We previously showed that daily oral administration of AP24534 can induce complete regression of T315I-expressing tumors in mice. In this study, we set out to characterize the antitumor activity of AP24534 through correlative in vitro and in vivo efficacy and pharmacodynamic studies. AP24534 inhibited the kinase activity of unmutated and T315I Abl enzymes with IC50s of 12 and 58 nM respectively, and inhibited the proliferation of their respective Bcr-Abl Ba/F3-derived cell lines with IC50s of 1 and 8 nM in a 3-day assay. Exposure to AP24534 for three hours led to the inhibition of Bcr-Abl phosphorylation with IC50s of 25 and 78 nM, respectively, with substantial apoptosis observed within 24 hours, suggesting that short term exposure to AP24534 is sufficient for efficacy. In mononuclear cells isolated from 3 patients with the T315I mutation (2 CML patients in blast crisis and a Ph+ ALL patient), treatment with 50 nM AP24534 resulted in a >50% decrease in phospho-CrkL levels. To further investigate the relationship between dose, exposure and response, we tested the activity of AP24534 on the human CML cell line K-562. In vitro, exposure of K-562 cells to 60 nM AP24534 for only 3 hr was sufficient to reduce cell proliferation by >90% when measured 3 days later. Oral administration of AP24534 to mice bearing K-562 xenografts inhibited tumor growth in a dose-dependent manner; a 2.5 mg/kg daily dose induced complete tumor regression. Bcr-Abl phosphorylation was only transiently inhibited at this dose, confirming that sustained target inhibition is not required for antitumor activity. Indeed, complete regression could also be achieved by intermittent (twice-weekly) dosing. Compared to AP24534, dasatinib required significantly higher transient exposure levels to achieve >90% K-562 cell killing in vitro (300–600 nM), and showed reduced efficacy in the xenograft model when dosed daily or intermittently. Pharmacokinetic analyses showed that the longer half-life of AP24534 versus dasatinib results in higher levels of exposure in mice. The in vitro kinase specificity profile of AP24534 suggests that Bcr-Abl inhibition is the dominant mechanism for its activity in CML models. Specifically, it is inactive against Aurora kinases (IC50 > 1000 nM). AP24534 potently inhibits a discrete subset of other kinases, including Flt3 and members of the Src, VEGFR and FGFR families (IC50s = 0.4–58 nM), activities which may contribute to its potency against CML and suggest the potential for efficacy in other tumor types. Together these data indicate that AP24534 has the potential to be an effective treatment for CML, including in patients refractory to current targeted agents. A phase 1 clinical trial is being planned.

scholarly journals Novel Identified Circular Transcript of RCAN2, circ-RCAN2, Shows Deviated Expression Pattern in Pig Reperfused Infarcted Myocardium and Hypoxic Porcine Cardiac Progenitor Cells In Vitro

2021 ◽  
Vol 22 (3) ◽  
pp. 1390
Author(s):  
Julia Mester-Tonczar ◽  
Patrick Einzinger ◽  
Johannes Winkler ◽  
Nina Kastner ◽  
Andreas Spannbauer ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Progenitor Cells ◽  
Regulatory Networks ◽  
Circular Rnas ◽  
Cardiac Progenitor Cells ◽  
Tissue Samples ◽  
Healthy Myocardium ◽  
Acute Mi

Circular RNAs (circRNAs) are crucial in gene regulatory networks and disease development, yet circRNA expression in myocardial infarction (MI) is poorly understood. Here, we harvested myocardium samples from domestic pigs 3 days after closed-chest reperfused MI or sham surgery. Cardiac circRNAs were identified by RNA-sequencing of rRNA-depleted RNA from infarcted and healthy myocardium tissue samples. Bioinformatics analysis was performed using the CIRIfull and KNIFE algorithms, and circRNAs identified with both algorithms were subjected to differential expression (DE) analysis and validation by qPCR. Circ-RCAN2 and circ-C12orf29 expressions were significantly downregulated in infarcted tissue compared to healthy pig heart. Sanger sequencing was performed to identify the backsplice junctions of circular transcripts. Finally, we compared the expressions of circ-C12orf29 and circ-RCAN2 between porcine cardiac progenitor cells (pCPCs) that were incubated in a hypoxia chamber for different time periods versus normoxic pCPCs. Circ-C12orf29 did not show significant DE in vitro, whereas circ-RCAN2 exhibited significant ischemia-time-dependent upregulation in hypoxic pCPCs. Overall, our results revealed novel cardiac circRNAs with DE patterns in pCPCs, and in infarcted and healthy myocardium. Circ-RCAN2 exhibited differential regulation by myocardial infarction in vivo and by hypoxia in vitro. These results will improve our understanding of circRNA regulation during acute MI.

scholarly journals Splicing factor SRSF1 promotes breast cancer progression via oncogenic splice switching of PTPMT1

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jun-Xian Du ◽  
Yi-Hong Luo ◽  
Si-Jia Zhang ◽  
Biao Wang ◽  
Cong Chen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Progression ◽  
High Risk Group ◽  
Clinical Samples ◽  
Binding Motif ◽  
Ki 67 ◽  
Tissue Samples

Abstract Background Intensive evidence has highlighted the effect of aberrant alternative splicing (AS) events on cancer progression when triggered by dysregulation of the SR protein family. Nonetheless, the underlying mechanism in breast cancer (BRCA) remains elusive. Here we sought to explore the molecular function of SRSF1 and identify the key AS events regulated by SRSF1 in BRCA. Methods We conducted a comprehensive analysis of the expression and clinical correlation of SRSF1 in BRCA based on the TCGA dataset, Metabric database and clinical tissue samples. Functional analysis of SRSF1 in BRCA was conducted in vitro and in vivo. SRSF1-mediated AS events and their binding motifs were identified by RNA-seq, RNA immunoprecipitation-PCR (RIP-PCR) and in vivo crosslinking followed by immunoprecipitation (CLIP), which was further validated by the minigene reporter assay. PTPMT1 exon 3 (E3) AS was identified to partially mediate the oncogenic role of SRSF1 by the P-AKT/C-MYC axis. Finally, the expression and clinical significance of these AS events were validated in clinical samples and using the TCGA database. Results SRSF1 expression was consistently upregulated in BRCA samples, positively associated with tumor grade and the Ki-67 index, and correlated with poor prognosis in a hormone receptor-positive (HR+) cohort, which facilitated proliferation, cell migration and inhibited apoptosis in vitro and in vivo. We identified SRSF1-mediated AS events and discovered the SRSF1 binding motif in the regulation of splice switching of PTPMT1. Furthermore, PTPMT1 splice switching was regulated by SRSF1 by binding directly to its motif in E3 which partially mediated the oncogenic role of SRSF1 by the AKT/C-MYC axis. Additionally, PTPMT1 splice switching was validated in tissue samples of BRCA patients and using the TCGA database. The high-risk group, identified by AS of PTPMT1 and expression of SRSF1, possessed poorer prognosis in the stage I/II TCGA BRCA cohort. Conclusions SRSF1 exerts oncogenic roles in BRCA partially by regulating the AS of PTPMT1, which could be a therapeutic target candidate in BRCA and a prognostic factor in HR+ BRCA patient.

scholarly journals Circular RNA hsa_circ_0006401 promotes proliferation and metastasis in colorectal carcinoma

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Chenjing Zhang ◽  
Xiaolu Zhou ◽  
Xiaoge Geng ◽  
Yu Zhang ◽  
Jingya Wang ◽  
...  
Keyword(s):  
Splice Junction ◽  
Circular Rna ◽  
Host Gene ◽  
Cell Counting ◽  
Tissue Samples ◽  
Proliferation And Metastasis ◽  
And Migration ◽  
Proliferation And Migration

AbstractDysregulation of circular RNA (circRNA) expression is involved in the progression of cancer. Here, we aimed to study the potential function of hsa_circ_0006401 in colorectal cancer (CRC). CircRNA hsa_circ_0006401 expression levels in CRC and adjacent nontumor tissues were analyzed by real-time quantitative PCR (qRT-PCR) and circRNA in situ hybridization (RNA-ISH). Then, CRC cell proliferation was assessed by cell counting. Wound-healing and transwell assays were utilized to detect the effect of hsa_circ_0006401 on CRC migration. A circRNA-ORF construct was created, and a specific antibody against the splice junction of hsa_circ_0006401 was prepared. Finally, the proteins directly binding to hsa_circ_0006401 peptides were identified by immunoprecipitation combined with mass spectrometry. In our study, we found hsa_circ_0006401 was closely related to CRC metastasis and exhibited upregulated expression in metastatic CRC tissue samples. Proliferation and migration were inhibited in vitro when hsa_circ_0006401 expression was silenced. Downregulation of hsa_circ_0006401 expression decreased CRC proliferation and liver metastasis in vivo. A 198-aa peptide was encoded by sequences of the splice junction absent from col6a3. Hsa_circ_0006401 promoted CRC proliferation and migration by encoding the hsa_circ_0006401 peptide. Hsa_circ_0006401 peptides decreased the mRNA and protein level of the host gene col6a3 by promoting col6a3 mRNA stabilation. In conclusion, our study revealed that circRNAs generated from col6a3 that contain an open-reading frame (ORF) encode a novel 198-aa functional peptide and hsa_circ_0006401 peptides promote stability of the host gene col6a3 mRNA to promote CRC proliferation and metastasis.

scholarly journals A Collagen-Based Scaffold for Promoting Neural Plasticity in a Rat Model of Spinal Cord Injury

Polymers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2245
Author(s):  
Jue-Zong Yeh ◽  
Ding-Han Wang ◽  
Juin-Hong Cherng ◽  
Yi-Wen Wang ◽  
Gang-Yi Fan ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Rat Model ◽  
Neural Plasticity ◽  
Collagen Scaffold ◽  
Glial Scar ◽  
Beneficial Effects ◽  
Cord Injury

In spinal cord injury (SCI) therapy, glial scarring formed by activated astrocytes is a primary problem that needs to be solved to enhance axonal regeneration. In this study, we developed and used a collagen scaffold for glial scar replacement to create an appropriate environment in an SCI rat model and determined whether neural plasticity can be manipulated using this approach. We used four experimental groups, as follows: SCI-collagen scaffold, SCI control, normal spinal cord-collagen scaffold, and normal control. The collagen scaffold showed excellent in vitro and in vivo biocompatibility. Immunofluorescence staining revealed increased expression of neurofilament and fibronectin and reduced expression of glial fibrillary acidic protein and anti-chondroitin sulfate in the collagen scaffold-treated SCI rats at 1 and 4 weeks post-implantation compared with that in untreated SCI control. This indicates that the collagen scaffold implantation promoted neuronal survival and axonal growth within the injured site and prevented glial scar formation by controlling astrocyte production for their normal functioning. Our study highlights the feasibility of using the collagen scaffold in SCI repair. The collagen scaffold was found to exert beneficial effects on neuronal activity and may help in manipulating synaptic plasticity, implying its great potential for clinical application in SCI.

scholarly journals The glycosyltransferase ST3GAL2 is regulated by miR-615-3p in the intestinal tract of Campylobacter jejuni infected mice

Gut Pathogens ◽  
2021 ◽  
Vol 13 (1) ◽  
Author(s):  
De Xi ◽  
Lukas Hofmann ◽  
Thomas Alter ◽  
Ralf Einspanier ◽  
Stefan Bereswill ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Regulatory Networks ◽  
In Silico Analysis ◽  
Luciferase Reporter ◽  
Colonic Tissue ◽  
Tissue Samples ◽  
Vitro Model ◽  
Pathway Analyses

Abstract Background Campylobacter jejuni (C. jejuni) infections are of increasing importance worldwide. As a typical mucosal pathogen, the interaction of C. jejuni with mucins is a prominent step in the colonisation of mucosal surfaces. Despite recent advances in understanding the interaction between bacterial pathogens and host mucins, the mechanisms of mucin glycosylation during intestinal C. jejuni infection remain largely unclear. This prompted us to identify relevant regulatory networks that are concerted by miRNAs and could play a role in the mucin modification and interaction. Results We firstly used a human intestinal in vitro model, in which we observed altered transcription of MUC2 and TFF3 upon C. jejuni NCTC 11168 infection. Using a combined approach consisting of in silico analysis together with in vitro expression analysis, we identified the conserved miRNAs miR-125a-5p and miR-615-3p associated with MUC2 and TFF3. Further pathway analyses showed that both miRNAs appear to regulate glycosyltransferases, which are related to the KEGG pathway ‘Mucin type O-glycan biosynthesis’. To validate the proposed interactions, we applied an in vivo approach utilising a well-established secondary abiotic IL-10−/− mouse model for infection with C. jejuni 81-176. In colonic tissue samples, we confirmed infection-dependent aberrant transcription of MUC2 and TFF3. Moreover, two predicted glycosyltransferases, the sialyltransferases ST3GAL1 and ST3GAL2, exhibited inversely correlated transcriptional levels compared to the expression of the identified miRNAs miR-125a-5p and miR-615-3p, respectively. In this study, we mainly focused on the interaction between miR-615-3p and ST3GAL2 and were able to demonstrate their molecular interaction using luciferase reporter assays and RNAi. Detection of ST3GAL2 in murine colonic tissue by immunofluorescence demonstrated reduced intensity after C. jejuni 81-176 infection and was thus consistent with the observations made above. Conclusions We report here for the first time the regulation of glycosyltransferases by miRNAs during murine infection with C. jejuni 81-176. Our data suggest that mucin type O-glycan biosynthesis is concerted by the interplay of miRNAs and glycosyltransferases, which could determine the shape of intestinal glycosylated proteins during infection.

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