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We report herein the discovery of exceptionally potent and orally bioavailable PROTAC AR degraders with ARD-2585 being the most promising compound. ARD-2585 achieves DC(50) values of ≤0.1 nM in the VCaP cell line with AR gene amplification and in the LNCaP cell line carrying an AR mutation. It potently inhibits cell growth with IC(50) values of 1.5 and 16.2 nM in the VCaP and LNCaP cell lines, respectively, and achieves excellent pharmacokinetics and 51% of oral bioavailability in mice. It is more efficacious than enzalutamide in inhibition of VCaP tumor growth and does not cause any sign of toxicity in mice. ARD-2585 is a promising AR degrader for extensive investigations for the treatment of advanced prostate cancer.

Proteolysis targeting chimera (PROTAC) small-molecule degraders have emerged as a promising new type of therapeutic agents, but the design of PROTAC degraders with excellent oral pharmacokinetics is a major challenge. In this study, we present our strategies toward the discovery of highly potent PROTAC degraders of androgen receptor (AR) with excellent oral pharmacokinetics. Employing thalidomide to recruit cereblon/cullin 4A E3 ligase and through the rigidification of the linker, we discovered highly potent AR degraders with good oral pharmacokinetic properties in mice with ARD-2128 being the best compound. ARD-2128 achieves 67% oral bioavailability in mice, effectively reduces AR protein and suppresses AR-regulated genes in tumor tissues with oral administration, leading to the effective inhibition of tumor growth in mice without signs of toxicity. This study supports the development of an orally active PROTAC AR degrader for the treatment of prostate cancer and provides insights and guidance into the design of orally active PROTAC degraders.

[Image: see text] Signal transducer and activator of transcription 3 (STAT3) is an attractive cancer therapeutic target. We report herein our extensive in vitro and in vivo evaluations of SD-91, the product of the hydrolysis of our previously reported STAT3 degrader SD-36. SD-91 binds to STAT3 protein with a high affinity and displays >300-fold selectivity over other STAT family protein members. SD-91 potently and effectively induces degradation of STAT3 protein and displays a high selectivity over other STAT members and >7000 non-STAT proteins in cells. A single administration of SD-91 selectively depletes STAT3 protein in tumor tissues with a persistent effect. SD-91 achieves complete and long-lasting tumor regression in the MOLM-16 xenograft model in mice even with weekly administration. Hence, SD-91 is a potent, highly selective, and efficacious STAT3 degrader for extensive evaluations for the treatment of human cancers and other diseases for which STAT3 plays a key role.

Nanomedicine usually refers to nanoparticles that deliver the functional drugs and siRNAs to treat cancer. Recent research has suggested that cancer cells can also make nanoparticles that also deliver functional molecules in promoting cancer metastasis, which is the leading cause of various cancer mortalities. This nanoparticle is called tumor-derived vesicles, or better-known as tumor-derived exosomes (TEXs). TEXs are nanoscale membrane vesicles (30–140 nm) that are released continuously by various types of cancer cells and contain tumor-derived functional biomolecules, including lipids, proteins, and genetic molecules. These endogenous TEXs can interact with host immune cells and epithelial cells locally and systemically. More importantly, they can reprogram the recipient cells in favor of promoting metastasis through facilitating tumor cell local invasion, intravasation, immune evasion, extravasation, and survival and growth in distant organs. Growing evidence suggests that TEXs play a key role in cancer metastasis. Here, we will review the most recent findings of how cancer cells harness TEXs to promote cancer metastasis through modulating vascular permeability, suppressing systemic immune surveillance, and creating metastatic niches. We will also summarize recent research in targeting TEXs to treat cancer metastasis., Graphical abstract We reviewed recent progress on understanding how tumor-derived exosomes (TEXs) promote metastasis through facilitating tumor cell local invasion, intravasation, immune evasion, extravasation, and growth in distant organs.Image 1

Background: Triple-negative breast cancer (TNBC) is an aggressive cancer subtype lacking effective treatment options, and p53 is the most frequently mutated or deleted gene. Carboxypeptidase A4 (CPA4) is an extracellular metallocarboxypeptidase, which was closely associated with aggressiveness. Although a recent study indicated that CPA4 could induce epithelial‑mesenchymal transition in breast cancer cells, no studies investigated its stemness-related function and the correlation between CPA4 and p53 in TNBC. In this study, we aimed to investigate the CPA4 levels in breast cancer tissues and analyze its association with p53, and study its roles in cancer stemness maintenance. Methods: CPA4 mRNA level and its prognostic value were analyzed by using online database UALCAN (http://ualcan.path.uab.edu) and Kaplan-Meier plotter (www.kmplot.com), respectively. The expression of CPA4, p53 and ALDH1A1 in breast cancer and adjacent normal tissues were evaluated by IHC using the corresponding primary antibodies on a commercial tissue array (Shanghai Biochip Co., Ltd., Shanghai, China). siRNA knockdown was used to study the function of proliferation, colony formation assay and sphere formation in serum-free medium. Results: Analysis of the UALCAN datasets identified that CPA4 mRNA levels were elevated in TNBC, especially in the TP53-mutant subgroup. Furthermore, high levels of CPA4 mRNA were significantly associated with unfavourable overall survival OS in breast cancer patients. Immunohistochemistical analysis demonstrated that CPA4 levels were elevated in 32.1% of breast cancer samples (45/140), and the positive rates of ALDH1A1 and p53 in the breast cancer tissues were 25% (35/140) and 50% (70/140), respectively. Statistical analysis revealed high levels of CPA4 was significantly associated with TNBC phenotype. Correlation analysis indicated that CPA4 over-expression was positively associated with ALDH1A1 (P

Our systematic literature collection and annotation identified 106 chemical drugs and 31 antibodies effective against the infection of at least one human coronavirus (including SARS-CoV, SAR-CoV-2, and MERS-CoV) in vitro or in vivo in an experimental or clinical setting. A total of 163 drug protein targets were identified, and 125 biological processes involving the drug targets were significantly enriched based on a Gene Ontology (GO) enrichment analysis. The Coronavirus Infectious Disease Ontology (CIDO) was used as an ontological platform to represent the anti-coronaviral drugs, chemical compounds, drug targets, biological processes, viruses, and the relations among these entities. In addition to new term generation, CIDO also adopted various terms from existing ontologies and developed new relations and axioms to semantically represent our annotated knowledge. The CIDO knowledgebase was systematically analyzed for scientific insights. To support rational drug design, a “Host-coronavirus interaction (HCI) checkpoint cocktail” strategy was proposed to interrupt the important checkpoints in the dynamic HCI network, and ontologies would greatly support the design process with interoperable knowledge representation and reasoning.

We report herein the discovery of a class of potent small-molecule inhibitors of anaplastic lymphoma kinase (ALK) containing a fused indoloquinoline scaffold. The most promising compound CJ-2360 has an IC(50) value of 2.2 nM against wild-type ALK and low-nanomolar potency against several clinically reported ALK mutants. This compound is capable of achieving complete tumor regression in the ALK-positive KARPAS-299 xenograft model with oral administration in mice. CJ-2360 represents a promising ALK inhibitor for advanced preclinical development.

There remain no approved therapies for rare but devastating neuronopathic glyocosphingolipid storage diseases, such as Sandhoff, Tay-Sachs, and Gaucher disease type 3. We previously reported initial optimization of the scaffold of eliglustat, an approved therapy for the peripheral symptoms of Gaucher disease type 1, to afford 2, which effected modest reductions in brain glucosylceramide (GlcCer) in normal mice at 60 mg/kg. The relatively poor pharmacokinetic properties and high Pgp-mediated efflux of 2 prompted further optimization of the scaffold. With a general objective of reducing topological polar surface area, and guided by multiple metabolite identification studies, we were successful at identifying 17 (CCG-222628), which achieves remarkably greater brain exposure in mice than 2. After demonstrating an over 60-fold improvement in potency over 2 at reducing brain GlcCer in normal mice, we compared 17 with Sanofi clinical candidate venglustat (Genz-682452) in the CBE mouse model of Gaucher disease type 3. At doses of 10 mg/kg, 17 and venglustat effected comparable reductions in both brain GlcCer and glucosylsphingosine. Importantly, 17 achieved these equivalent pharmacodynamic effects at significantly lower brain exposure than venglustat.

Triple-negative breast cancer (TNBC) is an aggressive cancer subtype for which effective therapies are unavailable. TNBC has a high frequency of tumor protein p53 (Tp53/p53)- and phosphatase and tensin homolog (PTEN) deficiencies, and combined p53- and PTEN-deficiency is associated with poor prognosis and poor response to anticancer therapies. In this study, we discovered that combined p53- and PTEN-deficiency in TNBC activates expression of the transcription factor mesenchyme homeobox 1 (MEOX1). We found that MEOX1 is expressed only in TNBC cells with frequent deficiencies in p53 and PTEN, and that its expression is undetectable in luminal A, luminal B, and HER2+ subtypes, as well as in normal breast cells with wild-type (WT) p53 and PTEN. Notably, siRNA knockdown of both p53 and PTEN activated MEOX1 expression in breast cancer cells, whereas individual knockdowns of either p53 or PTEN had only minimal effects on MEOX1 expression. MEOX1 knockdown abolished cell proliferation of p53- and PTEN-deficient TNBC in vitro and inhibited tumor growth in vivo, but had no effect on the proliferation of luminal and HER2+ cancer cells and normal breast cells. RNA-Seq and immunoblotting analyses showed that MEOX1 knockdown decreased expression of tyrosine kinase 2 (TYK2), signal transducer and activator of transcription 5B (STAT5B), and STAT6 in p53- and PTEN-deficient TNBC cells. These results reveal the effects of combined p53- and PTEN-deficiency on MEOX1 expression and TNBC cell proliferation, suggesting that MEOX1 may serve as a potential therapeutic target for managing p53- and PTEN-deficient TNBC.

Yan Li,1 Maria Palmisano,1 Duxin Sun,2 Simon Zhou1 1Translational Development and Clinical Pharmacology, Celgene Corporation, Summit, NJ 07920, USA; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USACorrespondence: Simon ZhouTranslational Development and Clinical Pharmacology, Celgene Corporation, 556 Morris Avenue, Summit, NJ 07920, USATel +1 908-673-9284Fax +1 908-673-2842Email szhou@celgene.comBackground: Voclosporin, a more potent derivative of cyclosporine, has been studied extensively in patients with immunologic disorders such as psoriasis, organ transplantation, uvetitis and lupus nephritis. Although better tolerated and safer than cyclosporine, voclosporin is inferior to cyclosporine in treating psoriasis, non-inferior to tacrolimus in organ transplantation and efficacious in treating lupus nephritis.Methods: The pharmacokinetic dispositions of voclosporin and cyclosporine in central and peripheral compartments were analyzed and correlated with their distinct clinical efficacy and safety profiles.Results: Both drugs demonstrated non-linear pharmacokinetics with increasing doses, more prominently at lower doses of voclosporin than at 10-fold higher doses of cyclosporine. Repeated lower dosing of voclosporin produced preferential calcineurin inhibition in and near blood circulation, leading to relatively lower cardiovascular and renal adverse effects but inferior efficacy for psoriasis compared to cyclosporine. With 10-fold higher plasma levels and deeper tissue penetration, cyclosporine has more prevalent renal and cardiac toxicities but superior efficacy to treat psoriasis.Conclusion: Although the two drugs are similar in structure and mechanism of action, the high potency and low dose compounded by the non-linear disposition of voclosporin resulted in more systemic versus local calcineurin inhibition than with cyclosporine. The dispositional difference between voclosporin and cyclosporine accounted for the puzzling efficacy and safety observations in different patients and was the basis for their optimal and differential use in treating diverse immunologic disorders.Keywords: pharmacokinetic, disposition, cyclosporine and voclosporin

Inhibition of embryonic ectoderm development (EED) is a new cancer therapeutic strategy. Herein, we report our discovery of EEDi-5285 as an exceptionally potent, efficacious, and orally active EED inhibitor. EEDi-5285 binds to the EED protein with an IC(50) value of 0.2 nM and inhibits cell growth with IC(50) values of 20 pM and 0.5 nM in the Pfeiffer and KARPAS422 lymphoma cell lines, respectively, carrying an EZH2 mutation. EEDi-5285 is approximately 100 times more potent than EED226 in binding to EED and >300 times more potent than EED226 in inhibition of cell growth in the KARPAS422 cell line. EEDi-5285 has excellent pharmacokinetics and achieves complete and durable tumor regression in the KARPAS422 xenograft model in mice with oral administration. The cocrystal structure of EEDi-5285 in a complex with EED defines the precise structural basis for their high binding affinity. EEDi-5285 is the most potent and efficacious EED inhibitor reported to date.

TP53 mutation in acute myeloid leukemia (AML) is associated with poor prognosis. Since no targeted therapy is available to restore p53 function, it is of great interest to test whether other pathways activated by TP53 mutations can be therapeutically targeted. Here, we showed HIF-1α target genes are enriched in TP53-mutated versus TP53-wild-type AML. To determine the role of this activation, we tested efficacy of HIF-1α inhibitor echinomycin in TP53-mutated AML samples in vitro and in vivo. Echinomycin was broadly effective against a panel of primary AML blast cells, with low nanomolar IC50s and, based on colony-forming unit assay, was tenfold more effective in eliminating AML stem cells. Echinomycin selectively eliminated CD34+CD38− AML cells. To test the therapeutic efficacy of echinomycin, we established a xenograft model of TP53-mutated AML. Echinomycin was broadly effective against xenografts from multiple AML samples in vivo, and more effective than cytarabine + daunorubicin chemotherapy. Importantly, while cytarabine + daunorubicin enriched for AML stem cells, echinomycin nearly eliminated this population. Using TP53-mutated AML cell line THP1 and patient-derived AML cells, we tested a new echinomycin formulation with longer half-life and significantly improved therapeutic effect. Our data suggest a novel approach to treat AML with TP53 mutations.

With roughly 2 billion people infected, the neurotropic protozoan Toxoplasma gondii remains one of the most pervasive and infectious parasites. Toxoplasma infection is the second leading cause of d...

Aim: We aim to demonstrate that a local nanoparticle-mediated hyperthermia can effectively eliminate tumor-associated Tregs and thereby boost checkpoint blockade-based immunotherapy. Materials & methods: Photothermal therapy (PTT), mediated with systemically administered stealthy iron-oxide nanoparticles, was applied to treat BALB/c mice bearing 4T1 murine breast tumors. Flow cytometry was applied to evaluate both Treg and CD8+ T-cell population. Tumor growth following combination therapy of both PTT and anti-CTLA-4 was further evaluated. Results: Our data reveal that tumor-associated Tregs can be preferentially depleted via iron-oxide nanoparticles-mediated PTT. When combining PTT with anti-CTLA-4 immunotherapy, we demonstrate a significant inhibition of syngeneic 4T1 tumor growth. Conclusion: This study offers a novel strategy to overcome Treg-mediated immunosuppression and thereby to boost cancer immunotherapy.

ObjectivesDespite safe handling guidelines published by several groups, health care worker exposure to hazardous drugs continues to occur due to suboptimal engineering controls and low use of protective equipment. Simple, multi-target and specific analytical methods are needed so that acute exposures to these drugs in the workplace can be assessed rapidly. Our aim was to develop an analytical method for simultaneous detection and quantification of widely used cancer drugs to rule out accidental acute chemotherapy exposures in health care workers.MethodsWe examined the feasibility of alternate high-performance liquid chromatographic-tandem mass spectrometry methods to simultaneously detect eighteen chemotherapy analytes in plasma and urine. The linear concentration ranges tested during assay development were 0.1–50 ng/mL. After development of a multi-analyte assay protocol, plasma samples (n = 743) from a multi-center cluster-randomized clinical trial (n = 12 sites) of an hazardous drug educational intervention were assayed. Confirmatory assays were performed based on the individual acute-spill case-histories.ResultsAn innovative HPLC-multiple reaction monitoring-information dependent acquisition-enhanced production ion (MRM-IDA-EPI) analytical method was developed to simultaneously detect: cytarabine, gemcitabine, dacarbazine, methotrexate, topotecan, mitomycin, pemetrexed, irinotecan, doxorubicin, vincristine, vinblastine, ifosamide, cyclophosphamide, vinorelbine, bendamustine, etoposide, docetaxel, and paclitaxel. The retention times ranged from 4 min to 13 min for the analytical run. The limit of detection (MRM-IDA-EPI) and limit of quantitation (MRM) was 0.25 ng/mL and 0.1 ng/mL, respectively for most analytes. No detectable plasma concentrations were measured at baseline, post-intervention and in cases of documented acute spills. Use of a secondary tandem mass spectrometry approach was able to successfully rule out false positive results.ConclusionsDevelopment of a sensitive high-throughput multi-analyte cancer chemotherapy assay is feasible using an MRM-IDA-EPI method. This method can be used to rapidly rule out systemic exposure to accidental acute chemotherapy spills in health care workers.

nab-Paclitaxel ( nab-P), an albumin-bound formulation of paclitaxel, was developed to improve the tolerability and antitumor activity of taxanes. The neonatal Fc receptor (FcRn) is a transport protein that can bind to albumin and regulate the homeostasis of circulating albumin. Therefore, the pharmacokinetics and pharmacodynamics of nab-P may be impacted by FcRn expression. This study aimed to investigate the effects of FcRn on nab-P elimination and distribution to targeted tissues. Wild-type and FcRn-knockout (FcRn-KO) mice were treated with nab-P, mouse-specific nab-P (distribution experiments only), and solvent-based paclitaxel (pac-T). Blood and tissue samples were collected for distribution analyses. Organ, urine, and fecal samples were collected for elimination analyses. The nab-P tissue penetration in the pancreas, fat pad, and kidney of wild-type mice, as reflected by the ratio of tissue/plasma concentration, was significantly higher (ranging from 5 to 80 fold) than that of FcRn-KO mice. In contrast, the tissue penetration of pac-T in these organs of FcRn-KO mice was similar to that of wild-type mice. More importantly, the excretion of nab-P in feces of FcRn-KO mice (45-68%) was significantly higher than that of wild-type mice (26-46%) from 8 to 48 h post treatment. In comparison, the difference of excretion of pac-T in feces between FcRn-KO mice and wild-type mice was smaller than that of nab-P. Furthermore, greater tissue penetration and fecal excretion were observed with nab-P than pac-T in both FcRn-KO and wild-type mice. These findings suggest that FcRn enhances the tissue distribution and penetration of nab-P in the targeted organs, while FcRn prevents excretion of nab-P to feces in the intestinal lumen. The findings support the notion that albumin nanoparticle delivery alters drug distribution and elimination through an FcRn-mediated process to impact drug efficacy and toxicity.

Through a phenotypic high throughput screen (HTS) using a serum response element luciferase (SRE.L) promoter, we identified a novel 5-aryl-1,3,4-oxadiazol-2-ylthiopropionic acid lead inhibitor of Rho/MRTF/SRF-mediated gene transcription with good potency (IC(50) = 180 nM). We were able to rapidly improve the cellular potency by 5 orders of magnitude guided by sharply defined and synergistic SAR. The remarkable potency and depth of the SAR, as well as the relatively low molecular weight of the series, suggests, but does not prove, that binding to the unknown molecular target may be occurring through a covalent mechanism. The series nevertheless has no observable cytotoxicity up to 100 μM. Ensuing pharmacokinetic optimization resulted in the development of two potent and orally bioavailable anti-fibrotic agents that were capable of dose-dependently reducing connective tissue growth factor (CTGF) gene expression in vitro as well as significantly reducing the development of bleomycin-induced dermal fibrosis in mice in vivo.

As part of a program toward making analogues of amlexanox (1), currently under clinical investigation for the treatment of type 2 diabetes and obesity, we have synthesized derivative 5 in which deuterium has been introduced into two sites of metabolism on the C-7 isopropyl function of amlexanox. The synthesis of 5 was completed in an efficient three-step process utilizing reduction of key olefin 7b to 8 by Wilkinson’s catalyst to provide specific incorporation of di-deuterium across the double bond. Compound 5 displayed nearly equivalent potency to amlexanox (IC(50) 1.1 μM vs 0.6 μM, respectively) against recombinant human TBK1. When incubated with human, rat, and mouse liver microsomes, amlexanox (1) and d(2)-amlexanox (5) were stable (t(1/2) > 60 min) with 1 showing marginally greater stability relative to 5 except for rat liver microsomes. These data show that incorporating deuterium into two sites of metabolism does not majorly suppress Cyp-mediated metabolism relative to amlexanox.

Prior reports have suggested that piperine enhances curcumin’s anti-carcinogenesis. We tested the hypothesis that piperine increases the intracellular concentrations of curcumin by improving intracellular uptake or reducing curcumin efflux or metabolism in breast cells. We incubated SUM149, MCF10A, primary normal human breast cells, ALDH+, and ALDH-CD44+24- SUM149 cells with curcumin ± piperine at concentrations 1 μM to 15 μM for time periods of 15 minutes to 24 hours. We assayed cell viability by MTT assay and proliferation by primary mammosphere assay. Curcumin and its metabolites were assayed using liquid chromatography mass spectroscopy. Curcumin, but not piperine, showed significantly higher effects on the viability of breast cancer SUM149 cells than in non-tumorigenic MCF10A cells. Curcumin + piperine synergistically reduced viability of SUM149 cells but had a concentration dependent effect upon MCF10A cell viability. Cellular uptake of curcumin in SUM149 is significantly higher, while the efflux in SUM149 is significantly lower than in MCF10A, which correlated with cell viability. Piperine did not alter curcumin cellular uptake, efflux, or metabolism in any of the cell models. The observed synergism of piperine+curcumin in reducing breast stem cell self renewal is likely due to independent anti-carcinogenesis effects rather than any effects upon intracellular curcumin concentrations.

Summary: Dysregulated glycine metabolism is emerging as a common denominator in cardiometabolic diseases, but its contribution to atherosclerosis remains unclear. In this study, we demonstrate impaired glycine-oxalate metabolism through alanine-glyoxylate aminotransferase (AGXT) in atherosclerosis. As found in patients with atherosclerosis, the glycine/oxalate ratio is decreased in atherosclerotic mice concomitant with suppression of AGXT. Agxt deletion in apolipoprotein E-deficient (Apoe−/−) mice decreases the glycine/oxalate ratio and increases atherosclerosis with induction of hepatic pro-atherogenic pathways, predominantly cytokine/chemokine signaling and dysregulated redox homeostasis. Consistently, circulating and aortic C-C motif chemokine ligand 5 (CCL5) and superoxide in lesional macrophages are increased. Similar findings are observed following dietary oxalate overload in Apoe−/− mice. In macrophages, oxalate induces mitochondrial dysfunction and superoxide accumulation, leading to increased CCL5. Conversely, AGXT overexpression in Apoe−/− mice increases the glycine/oxalate ratio and decreases aortic superoxide, CCL5, and atherosclerosis. Our findings uncover dysregulated oxalate metabolism via suppressed AGXT as a driver and therapeutic target in atherosclerosis.

Alterations in the gut microbiome have been associated with various human diseases. Most existing gut microbiome studies stopped at the stage of identifying microbial alterations between diseased or healthy conditions. As inspired by reverse vaccinology (RV), we developed a new strategy called Reverse Microbiomics (RM) that turns this process around: based on the identified microbial alternations, reverse-predicting the molecular mechanisms underlying the disease and microbial alternations. Our RM methodology starts by identifying significantly altered microbiota profiles, performing bioinformatics analysis on the proteomes of the microbiota identified, and finally predicting potential virulence or protective factors relevant to a microbiome-associated disease. As a use case study, this reverse methodology was applied to study the molecular pathogenesis of rheumatoid arthritis (RA), a common autoimmune and inflammatory disease. Those bacteria differentially associated with RA were first identified and annotated from published data and then modeled and classified using the Ontology of Host-Microbiome Interactions (OHMI). Our study identified 14 species increased and 9 species depleted in the gut microbiota of RA patients. Vaxign was used to comparatively analyze 15 genome sequences of the two pairs of species: Gram-negative Prevotella copri (increased) and Prevotella histicola (depleted), as well as Gram-positive Bifidobacterium dentium (increased) and Bifidobacterium bifidum (depleted). In total, 21 auto-antigens were predicted to be related to RA, and five of them were previously reported to be associated with RA with experimental evidence. Furthermore, we identified 94 potential adhesive virulence factors including 24 microbial ABC transporters. While eukaryotic ABC transporters are key RA diagnosis markers and drug targets, we identified, for the first-time, RA-associated microbial ABC transporters and provided a novel hypothesis of RA pathogenesis. Our study showed that RM, by broadening the scope of RV, is a novel and effective strategy to study from bacterial level to molecular level factors and gain further insight into how these factors possibly contribute to the development of microbial alterations under specific diseases.

Nonalcoholic fatty liver disease (NAFLD) including nonalcoholic steatohepatitis (NASH) has reached epidemic proportions with no pharmacological therapy approved. Lower circulating glycine is consistently reported in patients with NAFLD, but the causes for reduced glycine, its role as a causative factor, and its therapeutic potential remain unclear. We performed transcriptomics in livers from humans and mice with NAFLD and found suppression of glycine biosynthetic genes, primarily alanine-glyoxylate aminotransferase 1 (AGXT1). Genetic (Agxt1(−/−) mice) and dietary approaches to limit glycine availability resulted in exacerbated diet-induced hyperlipidemia and steatohepatitis, with suppressed mitochondrial/peroxisomal fatty acid β-oxidation (FAO) and enhanced inflammation as the underlying pathways. We explored glycine-based compounds with dual lipid/glucose-lowering properties as potential therapies for NAFLD, and identified a tripeptide (Gly-Gly-L-Leu, DT-109) that improved body composition and lowered circulating glucose, lipids, transaminases, pro-inflammatory cytokines and steatohepatitis in mice with established NASH induced by a high-fat, cholesterol, and fructose diet. We applied metagenomics, transcriptomics and metabolomics to explore the underlying mechanisms. The bacterial genus Clostridium sensu stricto was markedly increased in mice with NASH and decreased following DT-109 treatment. DT-109 induced hepatic FAO pathways, lowered lipotoxicity, and stimulated de novo glutathione synthesis. In turn, inflammatory infiltration and hepatic fibrosis were attenuated via suppression of nuclear factor-kappa B (NFκB) target genes and transforming growth factor-beta (TGFβ)/SMAD signaling. Unlike its effects on the gut microbiome, DT-109 stimulated FAO and glutathione synthesis independent of NASH. In conclusion, impaired glycine metabolism may play a causative role in NAFLD. Glycine-based treatment attenuates experimental NAFLD by stimulating hepatic FAO and glutathione synthesis, thus warranting clinical evaluation.

The three design criteria of anticancer nanomedicines to improve anticancer efficacy and to reduce toxicity have been debated for decades: (1) Nanomedicines increase drug accumulation through enhanced permeability and retention (EPR) in tumors to improve anticancer efficacy. (2) Long systemic circulation of nanomedicines with high plasma concentration reduces reticuloendothelial system (RES) clearance and decreases drug accumulation in the normal organs to reduce toxicity, and to enhance the EPR effect. (3) A universal nanodelivery platform based on EPR and long systemic circulation can be developed to deliver different anticancer drugs. Although these criteria have repeatedly been confirmed in preclinical xenograft cancers, the majority of anticancer nanomedicines have failed to improve clinical efficacy, while the clinical efficacies/safety of successful nanomedicines are inconsistent with these design criteria. First, the debate over tumor EPR may have mixed two different questions and missed more clinically relevant comparisons for nanomedicines versus free drugs. When tumors are compared with normal tissues, tumor EPR has been confirmed in both mouse xenograft tumors and human cancers. However, nanomedicines may not enhance drug accumulation in human tumors compared with free drugs, despite outstanding improvement in preclinical cancers. Heterogeneity of enhanced permeability and retention in human cancers occurs for small/large molecules and nanomedicines, which cannot fully explain the poor translation of nanomedicines' efficacy from preclinical cancer models to cancer patients. Second, long-circulation nanomedicine should not be used as a universal design criterion because it does not further improve tumor accumulation by tumor EPR in human patients nor universally reduce distribution in normal organs. In contrast, nanomedicines change the drug tissue distribution to alter anticancer efficacy/safety. Third, a universal nanodelivery platform that uses the same design criteria for different drugs is not feasible. Rather, drug-specific nanodelivery systems are required to overcome the intrinsic shortcomings of delivered drugs, which are determined by the physicochemical, pharmacokinetic, and pharmacodynamic properties of the delivered drugs and nanocarriers to improve their efficacy/safety.

There is strong evidence that inhibition of one or more Aldehyde Dehydrogenase 1A (ALDH1A) isoforms may be beneficial in chemotherapy-resistant ovarian cancer and other tumor types. While many previous efforts have focused on development of ALDH1A1 selective inhibitors, the most deadly ovarian cancer subtype, high-grade serous (HGSOC), exhibits elevated expression of ALDH1A3. Herein, we report continued development of pan-ALDH1A inhibitors to assess whether broad spectrum ALDH1A inhibition is an effective adjunct to chemotherapy in this critical tumor subtype. Optimization of the CM39 scaffold, aided by metabolite ID and several new ALDH1A1 crystal structures, led to improved biochemical potencies, improved cellular ALDH inhibition in HGSOC cell lines, and substantial improvements in microsomal stability culminating in orally bioavailable compounds. We demonstrate that two compounds 68 and 69 are able to synergize with chemotherapy in a resistant cell line and patient-derived HGSOC tumor spheroids, indicating their suitability for future in vivo proof of concept experiments.

Redox modulators have been developed as an attractive approach to treat cancer. Herein, we report the synthesis, identification, and biological evaluation of a quinazolinedione reactive oxygen species (ROS) inducer, QD394, with significant cytotoxicity in pancreatic cancer cells. QD394 shows a transcriptomic profile remarkably similar to napabucasin, a cancer stemness inhibitor. Both small molecules inhibit STAT3 phosphorylation, increase cellular ROS, and decrease the GSH/GSSG ratio. Moreover, QD394 causes an iron- and ROS-dependent, GPX4 mediated cell death, suggesting ferroptosis as a major mechanism. Importantly, QD394 decreases the expression of LRPPRC and PNPT1, two proteins involved in mitochondrial RNA catabolic processes and both negatively correlated with the overall survival of pancreatic cancer patients. Pharmacokinetics-guided lead optimization resulted in the derivative QD394-Me, which showed improved plasma stability and reduced toxicity in mice compared to QD394. Overall, QD394 and QD394-Me represent novel ROS-inducing drug-like compounds warranting further development for the treatment of pancreatic cancer.

Remdesivir is one of the most promising drugs to treat COVID-19 based on the following facts: remdesivir has a broad-spectrum antiviral mechanism of action; it demonstrated in vitro activity against SARS-CoV-2 and in vivo efficacy in animal models against the similar coronavirus MERS-CoV; its safety profile has been tested in Ebola patients and in compassionate use in COVID-19 patients. Currently, remdesivir is being investigated in ten randomized controlled trials against COVID-19. The dose regimen of remdesivir is an IV loading dose of 200 mg on day 1 followed by daily IV maintenance doses of 100 mg for 5–9 days. Based on our data analysis, however, remdesivir with IV administration alone is unlikely to achieve excellent clinical efficacy. This analysis is based on the following observations: plasma exposures of remdesivir and its active metabolite are unlikely to be correlated with its clinical efficacy; remdesivir and its active metabolites are unlikely to be adequate in the lung to kill the SARS-CoV-2 virus. Even if remdesivir demonstrates benefits in the current randomized controlled trials, its efficacy may be limited. We suggest that a combination of an IV and pulmonary delivery dose regimen should be studied immediately to realize a potentially more effective antiviral therapy against COVID-19. Graphical abstract

Triple-negative breast cancer (TNBC) has a high rate of metastasis, which is associated with breast cancer stem-like cells (CSCs). Although Taxol (micelle formulation of paclitaxel) is the first line chemotherapy to treat TNBC, it increases CSCs in residual tumors. Abraxane, albumin nanoparticle of paclitaxel, showed lower plasma concentration compared to Taxol in both human and animal models, but it is not clear why Abraxane showed superior efficacy to Taxol in treatment of metastatic breast cancer in humans. In this study, we intend to investigate if Abraxane eliminates CSCs for its better efficacy. The results showed that Abraxane showed similar cytotoxicity in SUM149 cells in comparison with Taxol. Although Abraxane showed 3- to 5-fold lower blood drug concentration compared to Taxol, it achieved similar tumor drug concentration and 10-fold higher tumor/plasma ratio in SUM149 xenograft NOD/SCID mouse model. In addition, Abraxane and Taxol showed similar efficacy to shrink the tumor size in orthotopic breast cancer NOD/SCID mouse model. However, Abraxane decreased breast CSCs frequency by 3- to 9-fold, while Taxol increased breast CSCs frequency in an orthotopic breast cancer NOD/SCID mouse model. Furthermore, Abraxane increased 3- to 15-fold intracellular uptake in both ALDH+ CSCs and differentiated ALDH- cells in comparison with Taxol, which provides a mechanism for Abraxane's superior efficacy to eliminate CSCs in comparison with Taxol. Our data suggest albumin nanoparticle Abraxane may have a broad implication to enhance drug's efficacy by eliminating breast cancer stem cells for treatment of metastatic diseases.

Remdesivir is one of the most promising drugs to treat COVID-19 based on the following facts: remdesivir has a broad-spectrum antiviral mechanism of action; it demonstrated in vitro activity against SARS-CoV-2 and in vivo efficacy in animal models against the similar coronavirus MERS-CoV; its safety profile has been tested in Ebola patients and in compassionate use in COVID-19 patients. Currently, remdesivir is being investigated in ten randomized controlled trials against COVID-19. The dose regimen of remdesivir is an IV loading dose of 200 mg on day 1 followed by daily IV maintenance doses of 100 mg for 5-9 days. Based on our data analysis, however, remdesivir with IV administration alone is unlikely to achieve excellent clinical efficacy. This analysis is based on the following observations: plasma exposures of remdesivir and its active metabolite are unlikely to be correlated with its clinical efficacy; remdesivir and its active metabolites are unlikely to be adequate in the lung to kill the SARS-CoV-2 virus. Even if remdesivir demonstrates benefits in the current randomized controlled trials, its efficacy may be limited. We suggest that a combination of an IV and pulmonary delivery dose regimen should be studied immediately to realize a potentially more effective antiviral therapy against COVID-19. Graphical abstract.

Background/aims High-resolution methods have advanced esophageal and anorectal manometry interpretation but are incompletely established for intestinal manometry. We characterized normal fasting duodeno-jejunal manometry parameters not measurable by standard techniques using clustered closely-spaced recordings. Methods Ten fasting recordings were performed in 8 healthy controls using catheters with 3-4 gastrointestinal manometry clusters with 1-2 cm channel spacing. Migrating motor complex phase III characteristics were quantified. Spatial-temporal contour plots measured propagation direction and velocity of individual contractions. Coupling was defined by pressure peak continuity within clusters. Results Twenty-three phase III complexes (11 antral, 12 intestinal origin) with 157 (95% CI, 104-211) minute periodicities, 6.99 (6.25-7.74) minute durations, 10.92 (10.68-11.16) cycle/minute frequencies, 73.6 (67.7-79.5) mmHg maximal amplitudes, and 4.20 (3.18-5.22) cm/minute propagation velocities were recorded. Coupling of individual contractions was 39.1% (32.1-46.1); 63.0% (54.4-71.6) of contractions were antegrade and 32.8% (24.1-41.5) were retrograde. Individual phase III contractions propagated > 35 fold faster (2.48 cm/sec; 95% CI, 2.25-2.71) than complexes themselves. Phase III complexes beyond the proximal jejunum were longer in duration (P = 0.025) and had poorer contractile coupling (P = 0.025) than proximal complexes. Coupling was greater with 1 cm channel spacing vs 2 cm (P ＜ 0.001). Conclusions Intestinal manometry using clustered closely-spaced pressure ports characterizes novel antegrade and retrograde propagation and coupling properties which degrade in more distal jejunal segments. Coupling is greater with more closely-spaced recordings. Applying similar methods to dysmotility syndromes will define the relevance of these methods.

Background: MEOX1 is a homeobox transcriptional factor, and plays essential roles in regulating somite development. Our previous study indicated that MEOX1 is a critical molecular target in mesenchymal-like cancer cells in PTEN-deficient Trastuzumab resistant breast cancer. Despite the potential implication of MEOX1 for the cancer progression, no previous studies examined its level and clinical significance in lung cancer tissues. In this study, we aimed to detect the MEOX1 expression and correlate its level with clinical outcome in non-small-cell lung cancer patients (NSCLC). Methods: MEOX1 gene expression in lung cancer was examined by using the Oncomine database. MEOX1 protein levels were evaluated by IHC using the corresponding primary antibody on two different commercial lung cancer tissue arrays. siRNA knockdown was used to elucidate the function of MEOX1. Results: Analysis of the Oncomine datasets identified that an elevation of MEOX1 in gene amplification in lung cancer tissues in comparison to normal lung tissues. Immunohistochemistical analysis demonstrated that MEOX1 was localized predominantly in the nucleus, and positive rate was 67.3% (111/165) in NSCLC samples. Statistical analysis revealed high levels of MEOX1 significantly correlated with Lymph Node Metastasis and Stage. Kaplan-Meier survival analysis showed that high levels of MEOX1 were significantly associated with unfavorable survival in NSCLC patients, and MEOX1 nucleus staining had worse survival, than did patients with overall expression in lung squamous cell carcinoma patients. Multivariate Cox's regression analysis found that MEOX1 was an independent poor prognostic predictor for patients with NSCLC. Silencing of MEOX1 by specific SiRNA significantly inhibited H460 and H1299 cell proliferation and sphere formation in serum-free medium. Conclusions: Our results firstly indentified that high levels of MEOX1 especially nuclear staining was an independent prognostic factor for NSCLC, and it served a essential roles in the regulation of cell proliferation and colony formation in vitro. It may represent a potential target for the NSCLC treatment.

Ginger is known to have antiinflammatory and antioxidative effects and has traditionally been used as an herbal supplement in the treatment of various chronic diseases. Here, we report antineutrophil properties of 6-gingerol, the most abundant bioactive compound of ginger root, in models of lupus and antiphospholipid syndrome (APS). Specifically, we demonstrate that 6-gingerol attenuates neutrophil extracellular trap (NET) release in response to lupus- and APS-relevant stimuli through a mechanism that is at least partially dependent on inhibition of phosphodiesterases. At the same time, administration of 6-gingerol to mice reduces NET release in various models of lupus and APS, while also improving other disease-relevant endpoints, such as autoantibody formation and large-vein thrombosis. In summary, this study is the first to our knowledge to demonstrate a protective role for ginger-derived compounds in the context of lupus. Importantly, it provides a potential mechanism for these effects via phosphodiesterase inhibition and attenuation of neutrophil hyperactivity.

Overlapping substrate specificities within the family of matrix metalloproteinases (MMPs), usually caused by their highly conserved structural topology, increase the potential for a substrate to be cleaved by multiple enzymes within this family, which leads to the decrease in the selectivity of MMP substrate-based probes. To resolve this issue, MT1-MMP activatable fluorogenic probes for tumor detection with enhanced specificity were developed by combining a fluorescence resonance energy transfer (FRET) peptide substrate and its specific binding peptide with different lengths of linkers. The specificity of the probes increased profiting from the high affinity of the MT1-MMP specific binding peptide while keeping the ability to amplify the output imaging signals in response to MMP activity with the FRET substrate. Enzyme kinetics analysis clearly demonstrated that the conjugation of P-1 and MT1-AF7p enhanced both the specificity and selectivity of the fluorogenic probes for MT1-MMP, and introducing a linker composed of 12 PEG subunits into these two fragments led to optimized specificity and selectivity of the fluorogenic probe for MT1-MMP. Both in vitro and in vivo results revealed that the imaging probe with the linker composed of 12 PEG subunits based on our designed strategy could be effectively applied for MT1-MMP positive tumor imaging. Since this strategy for enhancing the specificity of protease sensing probes can be applied to other proteases and is not just limited to MT1-MMP, it is an appealing platform to achieve selective tumor imaging.

Exploring the intraluminal behavior of an oral drug product in the human gastrointestinal (GI) tract remains challenging. Many in vivo techniques are available to investigate the impact of GI physiology on oral drug behavior in fasting state conditions. However, little is known about the intraluminal behavior of a drug in postprandial conditions. In a previous report, we described the mean solution and total concentrations of ibuprofen after oral administration of an immediate-release (IR) tablet in fed state conditions. In parallel, blood samples were taken to assess systemic concentrations. The purpose of this work was to statistically evaluate the impact of GI physiology (e.g., pH, contractile events) within and between individuals (intra and intersubject variability) for a total of 17 healthy subjects. In addition, a pharmacokinetic (PK) analysis was performed by noncompartmental analysis, and PK parameters were correlated with underlying physiological factors (pH, time to phase III contractions postdose) and study parameters (e.g., ingested amount of calories, coadministered water). Moreover, individual plasma profiles were deconvoluted to assess the fraction absorbed as a function of time, demonstrating the link between intraluminal and systemic behavior of the drug. The results demonstrated that the in vivo dissolution of ibuprofen depends on the present gastric pH and motility events at the time of administration. Both intraluminal factors were responsible for explaining 63% of plasma Cmax variability among all individuals. For the first time, an in-depth analysis was performed on a large data set derived from an aspiration/motility study, quantifying the impact of physiology on systemic behavior of an orally administered drug product in fed state conditions. The data obtained from this study will help us to develop an in vitro biorelevant dissolution approach and optimize in silico tools in order to predict the in vivo performance of orally administered drug products, especially in fed state conditions. ispartof: MOLECULAR PHARMACEUTICS vol:15 issue:12 pages:5468-5478 ispartof: location:United States status: published

Purpose: The response rates of Head and Neck Squamous Cell Carcinoma (HNSCC) to checkpoint blockade are below 20%. We aim to develop a mechanism-based vaccine to prevent HNSCC immune escape. Experimental Design: We performed RNA-Seq of sensitive and resistant HNSCC cells to discover central pathways promoting resistance to immune killing. Using biochemistry, animal models, HNSCC microarray, and immune cell deconvolution, we assessed the role of SOX2 in inhibiting STING-type I interferon (IFN-I) signaling-mediated antitumor immunity. To bypass SOX2-potentiated STING suppression, we engineered a novel tumor antigen–targeted nanosatellite vehicle to enhance the efficacy of STING agonist and sensitize SOX2-expressing HNSCC to checkpoint blockade. Results: The DNA-sensing defense response is the most suppressed pathway in immune-resistant HNSCC cells. We identified SOX2 as a novel inhibitor of STING. SOX2 facilitates autophagy-dependent degradation of STING and inhibits IFN-I signaling. SOX2 potentiates an immunosuppressive microenvironment and promotes HNSCC growth in vivo in an IFN-I-dependent fashion. Our unique nanosatellite vehicle significantly enhances the efficacy of STING agonist. We show that the E6/E7–targeted nanosatellite vaccine expands the tumor-specific CD8+ T cells by over 12-fold in the tumor microenvironment and reduces tumor burden. A combination of nanosatellite vaccine with anti-PD-L1 significantly expands tumor-specific CTLs and limits the populations expressing markers for exhaustion, resulting in more effective tumor control and improved survival. Conclusions: SOX2 dampens the immunogenicity of HNSCC by targeting the STING pathway for degradation. The nanosatellite vaccine offers a novel and effective approach to enhance the adjuvant potential of STING agonist and break cancer tolerance to immunotherapy. Clin Cancer Res; 24(17); 4242–55. ©2018 AACR.

Summary of the In Vivo Predictive Dissolution (iPD) - Oral Drug Delivery (ODD) Conference 2018

Valproic acid (VPA) is an older first-line antiepileptic drug with a complex pharmacokinetic (PK) profile, currently under investigation for several novel neurologic and non-neurologic indications. Our study objective was to design and validate a mechanistic model of VPA disposition in adults and children; and evaluate its predictive performance of drug-drug interactions (DDIs). This study expands upon existing physiologically based pharmacokinetic (PBPK) models for VPA by incorporating UGT enzyme kinetics and an advanced dissolution, absorption, and metabolism (ADAM) model for extended-release (ER) formulation. PBPK models for VPA IR and ER formulations were constructed using Simcyp Simulator (Version 15). First-order absorption was used for the immediate-release (IR) formulation and the ADAM model, including a controlled-release profile, for ER. Data from twenty-one published clinical studies were used to assess model performance. The model accurately predicted the concentration-time profiles of IR formulation for single-dose and steady-state doses ranging from 200 mg to 1000 mg. Similarly profiles were also simulated for ER formulation after a single-dose and steady-state doses of 500 mg and 1000 mg, respectively. In addition, simulated PK profiles agreed well with the observed data from studies in which VPA ER formulation was given to pediatric patients and VPA IR formulation to adult patients with cirrhosis. The model was further validated with individual adult data from a Phase I clinical trial consisting of eight cohorts after IV infusion of VPA with doses ranging from 15 to 150 mg/kg. Co-administrations of VPA as an enzyme-inhibitor with victim drug phenytoin or lorazepam, as well as a substrate with enzyme inducer carbamazepine or phenobarbital, were simulated with the model to evaluate drug-drug interaction. The simulated serum concentration-time profiles were within the 5th and 95th percentiles, and the majority of the predicted area-under-the-curve (AUC) and peak plasma concentration (C(max)) values were within 25% of the reported average values. The comprehensive VPA PBPK model defined by this study may be used to support dosage regimen optimization to improve the safety and efficacy profile of this agent under different scenarios.

Anticancer nanomedicines are designed to improve anticancer efficacy by increasing drug accumulation in tumors through enhanced permeability retention (EPR) effect, and to reduce toxicity by decreasing drug accumulation in normal organs through long systemic circulation. However, the inconsistent efficacy/safety of nanomedicines in cancer patients versus preclinical cancer models have provoked debate for nanomedicine design criteria. In this study, we investigate nanomedicine design criteria in three types of preclinical cancer models using five clinically used nanomedicines, which identifies the factors for better clinical translations of their observed clinical efficacy/safety compared to free drug or clinical micelle formulation. When those nanomedicines were compared with drug solution or clinical micelle formulation in breast tumors, long and short-circulating nanomedicines did not enhance tumor accumulation by EPR effect in transgenic spontaneous breast cancer model regardless of their size or composition, although they improved tumor accumulations in subcutaneous and orthotopic breast cancer models. However, when tumors were compared to normal breast tissue, nanomedicines, drug solution and clinical micelle formulation showed enhanced tumor accumulation regardless of the breast cancer models. In addition, long-circulating nanomedicines did not further increase tumor accumulation in transgenic mouse spontaneous breast cancer nor universally decrease drug accumulations in normal organs; they decreased or increased accumulation in different organs, potentially changing the clinical efficacy/safety. In contrast, short-circulating nanomedicines decreased blood concentration and altered drug distribution in normal organs, which are correlated with their clinical efficacy/safety. A reappraisal of current nanomedicine design criteria is needed to ensure consistent clinical translation for improvement of their clinical efficacy/safety in cancer patients.

Inefficient cellular uptake and intracellular drug release at the tumor site are two major obstacles limiting the antitumor efficacy of nanoparticle delivery systems. To overcome both problems, we designed a smart nanoparticle that undergoes phase transition in a tumor microenvironment (TME). The smart nanoparticle is generated using a lipid-polypetide hybrid nanoparticle, which comprises a PEGylated lipid monolayer shell and a pH-sensitive hydrophobic poly-l-histidine core and is loaded with the antitumor drug doxorubicin (DOX). The smart nanoparticle undergoes a two-step phase transition at two different pH values in the TME: (i) At the TME (pHe: 7.0-6.5), the smart nanoparticle swells, and its surface potential turns from negative to neutral, facilitating the cellular uptake; (ii) After internalization, at the acid endolysosome (pHendo: 6.5-4.5), the smart nanoparticle dissociates and induces endolysosome escape to release DOX into the cytoplasm. In addition, a tumor-penetrating peptide iNRG was modified on the surface of the smart nanoparticle as a tumor target moiety. The in vitro studies demonstrated that the iNGR-modified smart nanoparticles promoted cellular uptake in the acidic environment (pH 6.8). The in vivo studies showed that the iNGR-modified smart nanoparticles exerted more potent antitumor efficacy against late-stage aggressive breast carcinoma than free DOX. These data suggest that the smart nanoparticles may serve as a promising delivery system for sequential uptake and intracellular drug release of antitumor agents. The easy preparation of these smart nanoparticles may also have advantages in the future manufacture for clinical trials and clinical use.

Background: Alpha-enolase is an important glycolytic enzyme, and its aberrant expression has been associated with multiple tumor progression. However, few studies investigated the expression of alpha-enolase and its clinical significance in pancreatic cancer (PC). Objectives: To evaluate alpha-enolase level in PC tissues by immunohistochemical (IHC) analysis, and investigate the association of alpha-enolase expression with clinicopathologic features. Methods: The alpha-enolase levels in pancreatic cancer tissues were analyzed by using the Oncomine database. The expression of alpha-enolase, Ki67 and p53 in pancreatic cancer and adjacent normal tissues were evaluated by IHC using the corresponding primary antibodies on the commercial tissue arrays. We also examined their association with clinicopathologic parameters, and explored their prognostic value in PC. Results: We identified an elevation of alpha-enolase mRNA level in pancreatic cancer independent datasets from Oncomine. IHC analysis showed that alpha-enolase protein levels were elevated in 47% (n=100) PC tissue samples, but there was weak or no staining in the normal tissues. Statistical analysis revealed that high levels of alpha-enolase were significantly associated with Stage and Lymph node metastasis. Correlation analysis indicated that over-expression of alpha-enolase was positively associated with Ki67 expression and inversely correlated with p53 expression. Furthermore, membranous expression of alpha-enolase was also observed in 29.8% (14/47) total alpha-enolase positive samples, and was significantly associated with Lymph node metastasis. Kaplan-Meier survival analysis demonstrated that high total alpha-enolase expression was significantly associated with unfavorable survival, while membranous alpha-enolase expression was significantly associated with better survival of PC patients. Multivariate Cox analysis demonstrated that total alpha-enolase expression was an independent prognostic factor for PC patients. Conclusions: Our results suggested that alpha-enolase level was significantly elevated in pancreatic cancer tissues, which was closely associated with PC progression. It might be a candidate target for targeted pancreatic cancer treatments.

Esophageal cancer is an aggressive disease with poor prognosis because of early metastasis when diagnosed and recurrence after surgery. This study is aimed at investigating the expression of carboxypeptidaseA4 (CPA4) and aldehyde dehydrogenase 1A1 (ALDH1A1) in esophageal squamous cell carcinoma (ESCC) tumor tissues and analyzed their association and clinical significance. The expression of CPA4 and ALDH1A1 was determined by immunohistochemistry using the corresponding primary antibodies on two commercial tissue arrays. High level of CPA4 was observed in 87/150 (58%) ESCC samples and was significantly associated with histologic grade, lymph node metastasis, and TNM Classification of Esophageal cancer stage. The expression level of ALDH1A1 was much higher in ESCC than their corresponding normal epithelial tissues, with 66% positive rate. And, high levels of ALDH1A1 were significantly associated with lymph nodes metastasis (P < 0.05) and TNM stage (P < 0.05). Correlation analysis showed the expression level of CPA4 positively correlated with that of ALDH1A1 (r = 0.416, P < 0.01). In Kaplan-Meier survival analysis, either CPA4 or ALDH1A1 was significantly correlated with poor overall survival of ESCC patients. Multivariate Cox regression model showed that high expression of CPA4 was an independent prognostic factor for ESCC patients. In conclusion, our present study demonstrated for the first time that CPA4 might be used as an independent poor prognostic factor in ESCC. This study demonstrated for the first time that CPA4 was aberrantly expressed in ESCC tissues. Overexpression of CPA4 was closely associated with the putative cancer stem cell marker ALDH1A1 and might be used as an independent prognostic factor in ESCC.

The development of formulations and the assessment of oral drug absorption for Biopharmaceutical Classification System (BCS) class IIb drugs is often a difficult issue due to the potential for supersaturation and precipitation in the gastrointestinal (GI) tract. The physiological environment in the GI tract largely influences in vivo drug dissolution rates of those drugs. Thus, those physiological factors should be incorporated into the in vitro system to better assess in vivo performance of BCS class IIb drugs. In order to predict oral bioperformance, an in vitro dissolution system with multiple compartments incorporating physiologically relevant factors would be expected to more accurately predict in vivo phenomena than a one-compartment dissolution system like USP Apparatus 2 because, for example, the pH change occurring in the human GI tract can be better replicated in a multi-compartmental platform. The Gastrointestinal Simulator (GIS) consists of three compartments, the gastric, duodenal and jejunal chambers, and is a practical in vitro dissolution apparatus to predict in vivo dissolution for oral dosage forms. This system can demonstrate supersaturation and precipitation and, therefore, has the potential to predict in vivo bioperformance of oral dosage forms where this phenomenon may occur. In this report, in vitro studies were performed with dipyridamole and ketoconazole to evaluate the precipitation rates and the relationship between the supersaturation levels and oral absorption of BCS class II weak base drugs. To evaluate the impact of observed supersaturation levels on oral absorption, a study utilizing the GIS in combination with mouse intestinal infusion was conducted. Supersaturation levels observed in the GIS enhanced dipyridamole and ketoconazole absorption in mouse, and a good correlation between their supersaturation levels and their concentration in plasma was observed. The GIS, therefore, appears to represent in vivo dissolution phenomena and demonstrate supersaturation and precipitation of dipyridamole and ketoconazole. We therefore conclude that the GIS has been shown to be a good biopredictive tool to predict in vivo bioperformance of BCS class IIb drugs that can be used to optimize oral formulations.

Triple-negative breast cancers (TNBC) remain clinically challenging with a lack of options for targeted therapy. In this study, we report the development of a second-generation BET protein degrader, BETd-246, which exhibits superior selectivity, potency, and antitumor activity. In human TNBC cells, BETd-246 induced degradation of BET proteins at low nanomolar concentrations within 1 hour of exposure, resulting in robust growth inhibition and apoptosis. BETd-246 was more potent and effective in TNBC cells than its parental BET inhibitor compound BETi-211. RNA-seq analysis revealed predominant downregulation of a large number of genes involved in proliferation and apoptosis in cells treated with BETd-246, as compared with BETi-211 treatment that upregulated and downregulated a similar number of genes. Functional investigations identified the MCL1 gene as a critical downstream effector for BET degraders, which synergized with small-molecule inhibitors of BCL-xL in triggering apoptosis. In multiple murine xenograft models of human breast cancer, BETd-246 and a further optimized analogue BETd-260 effectively depleted BET proteins in tumors and exhibited strong antitumor activities at well-tolerated dosing schedules. Overall, our findings show that targeting BET proteins for degradation represents an effective therapeutic strategy for TNBC treatment. Cancer Res; 77(9); 2476–87. ©2017 AACR.

The bromodomain and extra-terminal (BET) family proteins, consisting of BRD2, BRD3, BRD4, and testis-specific BRDT members, are epigenetic “readers” and play a key role in the regulation of gene transcription. BET proteins are considered to be attractive therapeutic targets for cancer and other human diseases. Recently, heterobifunctional small-molecule BET degraders have been designed based upon the proteolysis targeting chimera (PROTAC) concept to induce BET protein degradation. Herein, we present our design, synthesis, and evaluation of a new class of PROTAC BET degraders. One of the most promising compounds, 23, effectively degrades BRD4 protein at concentrations as low as 30 pM in the RS4;11 leukemia cell line, achieves an IC50 value of 51 pM in inhibition of RS4;11 cell growth and induces rapid tumor regression in vivo against RS4;11 xenograft tumors. These data establish that compound 23 (BETd-260/ZBC260) is a highly potent and efficacious BET degrader.

Hypoxia-inducible factor 1α (HIF-1α) is recognized as a prime molecular target for metastatic cancer. However, no specific HIF-1α inhibitor has been approved for clinical use. Here, we demonstrated that in vivo efficacy of echinomycin in solid tumors with HIF-1α overexpression is formulation-dependent. Compared to previously-used Cremophor-formulated echinomycin, which was toxic and ineffective in clinical trials, liposomal-echinomycin provides significantly more inhibition of primary tumor growth and only liposome-formulated echinomycin can eliminate established triple-negative breast cancer (TNBC) metastases, which are the leading cause of death from breast cancer, as available therapies remain minimally effective at this stage. Pharmacodynamic analyses reveal liposomal-echinomycin more potently inhibits HIF-1α transcriptional activity in primary and metastasized TNBC cells in vivo, the latter of which are HIF-1α enriched. The data suggest that nanoliposomal-echinomycin can provide safe and effective therapeutic HIF-1α inhibition and could represent the most potent HIF-1α inhibitor in prospective trials for metastatic cancer.

Summary Signal transducer and activator of transcription 3 (STAT3) is an attractive cancer therapeutic target. Here we report the discovery of SD-36, a small-molecule degrader of STAT3. SD-36 potently induces the degradation of STAT3 protein in vitro and in vivo and demonstrates high selectivity over other STAT members. Induced degradation of STAT3 results in a strong suppression of its transcription network in leukemia and lymphoma cells. SD-36 inhibits the growth of a subset of acute myeloid leukemia and anaplastic large-cell lymphoma cell lines by inducing cell-cycle arrest and/or apoptosis. SD-36 achieves complete and long-lasting tumor regression in multiple xenograft mouse models at well-tolerated dose schedules. Degradation of STAT3 protein, therefore, is a promising cancer therapeutic strategy.