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Abstract This report summarizes the proceedings for day 2 sessions 1 and 3 of the 2‐day public workshop entitled “Regulatory Utility of Mechanistic Modeling to Support Alternative Bioequivalence Approaches,” a jointly sponsored workshop by the US Food and Drug Administration (FDA) and the Center for Research on Complex Generics (CRCG). The aims of this workshop were: (1) to discuss how mechanistic modeling, including physiologically‐based pharmacokinetic (PBPK) modeling and simulation, can support product development, and regulatory submissions; (2) to share the current state of mechanistic modeling for bioequivalence (BE) assessment through case studies; (3) to establish a consensus on best practices for using PBPK modeling for BE assessment to help drive further investment by the generic drug industry into mechanistic modeling and simulation; and (4) to introduce the concept of a Model Master File to improve model‐sharing. The theme of day 2 covered PBPK absorption model for oral products as an alternative BE approach and a tool for supporting risk assessment and biowaiver (session 1), oral PBPK for evaluating the impact of food on BE (session 2), successful cases, and challenges for oral PBPK (session 3). This report summarizes the topics of the presentations of day 2 sessions 1 and session 3 from FDA, academia, and pharmaceutical industry, including the current status of oral PBPK, case examples as well as the challenges and opportunities in this area. In addition, panel discussions on the utility of oral PBPK in both new drugs and generic drugs from regulatory and industry perspective are also summarized.

The full potential of single-cell RNA-sequencing applied to precision medicine has yet to be reached. Here, we propose a drug recommendation system ASGARD, which predicts drugs by considering cell clusters to address the intercellular heterogeneity within each patient.

Ninety percent of clinical drug development fails despite implementation of many successful strategies, which raised the question whether certain aspects in target validation and drug optimization are overlooked? Current drug optimization overly emphasizes potency/specificity using structure‒activity-relationship (SAR) but overlooks tissue exposure/selectivity in disease/normal tissues using structure‒tissue exposure/selectivity–relationship (STR), which may mislead the drug candidate selection and impact the balance of clinical dose/efficacy/toxicity. We propose structure‒tissue exposure/selectivity–activity relationship (STAR) to improve drug optimization, which classifies drug candidates based on drug's potency/selectivity, tissue exposure/selectivity, and required dose for balancing clinical efficacy/toxicity. Class I drugs have high specificity/potency and high tissue exposure/selectivity, which needs low dose to achieve superior clinical efficacy/safety with high success rate. Class II drugs have high specificity/potency and low tissue exposure/selectivity, which requires high dose to achieve clinical efficacy with high toxicity and needs to be cautiously evaluated. Class III drugs have relatively low (adequate) specificity/potency but high tissue exposure/selectivity, which requires low dose to achieve clinical efficacy with manageable toxicity but are often overlooked. Class IV drugs have low specificity/potency and low tissue exposure/selectivity, which achieves inadequate efficacy/safety, and should be terminated early. STAR may improve drug optimization and clinical studies for the success of clinical drug development.

Drug optimization, which improves drug potency/specificity by structure‒activity relationship (SAR) and drug-like properties, is rigorously performed to select drug candidates for clinical trials. However, the current drug optimization may overlook the structure‒tissue exposure/selectivity-relationship (STR) in disease-targeted tissues vs. normal tissues, which may mislead the drug candidate selection and impact the balance of clinical efficacy/toxicity. In this study, we investigated the STR in correlation with observed clinical efficacy/toxicity using seven selective estrogen receptor modulators (SERMs) that have similar structures, same molecular target, and similar/different pharmacokinetics. The results showed that drug's plasma exposure was not correlated with drug's exposures in the target tissues (tumor, fat pad, bone, uterus), while tissue exposure/selectivity of SERMs was correlated with clinical efficacy/safety. Slight structure modifications of four SERMs did not change drug's plasma exposure but altered drug's tissue exposure/selectivity. Seven SERMs with high protein binding showed higher accumulation in tumors compared to surrounding normal tissues, which is likely due to tumor EPR effect of protein-bound drugs. These suggest that STR alters drug's tissue exposure/selectivity in disease-targeted tissues vs. normal tissues impacting clinical efficacy/toxicity. Drug optimization needs to balance the SAR and STR in selecting drug candidate for clinical trial to improve success of clinical drug development.

ABSTRACT Inhaled corticosteroids (ICS) are commonly prescribed first-line treatments for asthma and chronic obstructive pulmonary disease (COPD). Recent evidence has shown that ICS use is associated with changes in the airway microbiome, which may impact clinical outcomes such as potential increased risk for pneumonia in COPD. Although the immunomodulatory effects of corticosteroids are well appreciated, whether ICS could directly influence the behavior of respiratory tract bacteria has been unknown. In this pilot study we explored the effects of fluticasone proprionate, a commonly prescribed inhaled corticosteroid, on respiratory bacteria with an expanded focus on Klebsiella pneumoniae, a species previously implicated in fluticasone-associated pneumonia in COPD. We observed significant effects of fluticasone proprionate on growth responses of K. pneumoniae, as well as other bacterial species isolated from asthmatic patients. Fluticasone-exposed K. pneumoniae displayed altered expression of several bacterial genes and reduced the metabolic activity of bronchial epithelial cells and their expression of human β-defensin 2. Targeted assays identified a fluticasone metabolite from fluticasone-exposed K. pneumoniae cells, suggesting this species may be capable of metabolizing fluticasone proprionate. Collectively, these observations support the hypothesis that specific members of the airway microbiota possess the functional repertoire to respond to or potentially utilize corticosteroids in their microenvironment. These findings lay a foundation for novel research directions into the potential direct effects of ICS, often prescribed long term to patients, on the broader airway microbial community and on the behavior of specific microbial species implicated in asthma and COPD outcomes. IMPORTANCE Inhaled corticosteroids are widely prescribed for many respiratory diseases, including asthma and COPD. While they benefit many patients, corticosteroids can also have negative effects. Some patients do not improve with treatment and even experience adverse side effects. Recent studies have shown that inhaled corticosteroids can change the make-up of bacteria in the human respiratory tract. However, whether these medications can directly impact the behavior of such bacteria has been unknown. Here, we explored the effects of fluticasone propionate, a commonly prescribed inhaled corticosteroid, on Klebsiella pneumoniae and other airway bacteria of interest, including primary species isolated from adult asthma patients. We provide evidence of growth responses to direct fluticasone exposure in culture and further examined fluticasone’s effects on K. pneumoniae, including gene expression changes and effects of fluticasone-exposed bacteria on airway cells. These findings indicate that members of the human airway bacterial community possess the functional ability to respond to corticosteroids, which may have implications for the heterogeneity of treatment response observed clinically.

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.

The histone methyltransferase ASH1L plays a role in various diseases, including cancer, and has been validated as a therapeutic target; however, no inhibitors of ASH1L have been reported. Here the authors present small molecule inhibitors of ASH1L and demonstrate their on-target activity in leukemia cells and a mouse model of leukemia.

Lower circulating levels of glycine are consistently reported in association with cardiovascular disease (CVD), but the causative role and therapeutic potential of glycine in atherosclerosis, the underlying cause of most CVDs, remain to be established. Here, following the identification of reduced circulating glycine in patients with significant coronary artery disease (sCAD), we investigated a causative role of glycine in atherosclerosis by modulating glycine availability in atheroprone mice. We further evaluated the atheroprotective potential of DT-109, a recently identified glycine-based compound with dual lipid/glucose-lowering properties. Glycine deficiency enhanced, while glycine supplementation attenuated, atherosclerosis development in apolipoprotein E-deficient (Apoe−/−) mice. DT-109 treatment showed the most significant atheroprotective effects and lowered atherosclerosis in the whole aortic tree and aortic sinus concomitant with reduced superoxide. In Apoe−/− mice with established atherosclerosis, DT-109 treatment significantly reduced atherosclerosis and aortic superoxide independent of lipid-lowering effects. Targeted metabolomics and kinetics studies revealed that DT-109 induces glutathione formation in mononuclear cells. In bone marrow-derived macrophages (BMDMs), glycine and DT-109 attenuated superoxide formation induced by glycine deficiency. This was abolished in BMDMs from glutamate-cysteine ligase modifier subunit-deficient (Gclm−/-) mice in which glutathione biosynthesis is impaired. Metabolic flux and carbon tracing experiments revealed that glycine deficiency inhibits glutathione formation in BMDMs while glycine-based treatment induces de novo glutathione biosynthesis. Through a combination of studies in patients with CAD, in vivo studies using atherosclerotic mice and in vitro studies using macrophages, we demonstrated a causative role of glycine in atherosclerosis and identified glycine-based treatment as an approach to mitigate atherosclerosis through antioxidant effects mediated by induction of glutathione biosynthesis.

Abstract 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.

Background: Atherosclerosis-related cardiovascular diseases (CVDs) are the leading cause of mortality worldwide. Cholesterol crystals (CCs) induce inflammation in atherosclerosis and are associated with unstable plaques and poor prognosis, but no drug can remove CCs in the clinic currently. Methods: We generated a phospholipid-based and high-density lipoprotein (HDL)-like nanoparticle, miNano, and determined CC-dissolving capacity, cholesterol efflux property, and anti-inflammation effects of miNano in vitro. Both normal C57BL/6J and Apoe-deficient mice were used to explore the accumulation of miNano in atherosclerotic plaques. The efficacy and safety of miNano administration to treat atherosclerosis were evaluated in the Ldlr-deficient atherosclerosis model. The CC-dissolving capacity of miNano was also detected using human atherosclerotic plaques ex vivo. Findings: We found that miNano bound to and dissolved CCs efficiently in vitro, and miNano accumulated in atherosclerotic plaques, co-localized with CCs and macrophages in vivo. Administration of miNano inhibited atherosclerosis and improved plaque stability by reducing CCs and macrophages in Ldlr-deficient mice with favorable safety profiles. In macrophages, miNano prevented foam cell formation by enhancing cholesterol efflux and suppressed inflammatory responses via inhibiting TLR4-NF-κB pathway. Finally, in an ex vivo experiment, miNano effectively dissolved CCs in human aortic atherosclerotic plaques. Interpretation: Together, our work finds that phospholipid-based and HDL-like nanoparticle, miNano, has the potential to treat atherosclerosis by targeting CCs and stabilizing plaques. Funding: This work was supported by the National Institutes of Health HL134569, HL109916, HL136231, and HL137214 to Y.E.C, HL138139 to J.Z., R21NS111191 to A.S., by the American Heart Association 15SDG24470155, Grant Awards (U068144 from Bio-interfaces and G024404 from M-BRISC) at the University of Michigan to Y.G., by the American Heart Association 19PRE34400017 and Rackham Helen Wu award to M.Y., NIH T32 GM07767 to K. H., Barbour Fellowship to D.L.

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.

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.

Purpose: Due to a lack of recognized molecular targets for therapy, patients with triple-negative breast cancer (TNBC), unlike other subtypes of breast cancers, generally have not benefited from the advances made with targeted agents. The CXCR4/SDF-1 axis is involved in tumor growth and metastasis of TNBC. Therefore, down-regulation of the expression of CXCR4 in cancer cells is a potential therapeutic strategy for inhibiting primary tumor growth and metastasis of TNBC. In order to identify bioactive compounds that inhibit the expression of CXCR4 in traditional Chinese medicines, we investigated the capacity of saikosaponin A (SSA), one of the active ingredients isolated from Radix bupleuri, to affect CXCR4 expression and function in TNBC cells.Methods: Analyses of cell growth, migration, invasion, and protein expression were performed. Knockdowns by small interfering RNA (siRNA) and non-invasive bioluminescence were also used.Results: SSA reduced proliferation and colony formation of SUM149 and MDA-MB-231 cells. SSA inhibited migration and invasion of TNBC cells. For mice, SSA inhibited primary tumor growth and reduced lung metastasis of highly metastatic, triple-negative 4T1-luc cells. SSA inhibited CXCR4 expression but did not regulate CXCR7 expression in vitro and in vivo. The inhibitory effects on the migration and invasion of TNBC cells were reversed by down-regulation of CXCR4 expression. In addition, SSA inactivated the Akt/mTOR signaling pathway and inhibited MMP-9 and MMP-2 expression.Conclusions: The results show that SSA exerts an anti-TNBC effect through the inhibition of CXCR4 expression and thus has the potential to be a candidate therapeutic agent for TNBC patients.

ABSTRACT Although the microbiota in the proximal gastrointestinal (GI) tract have been implicated in health and disease, much about these microbes remains understudied compared to those in the distal GI tract. This study characterized the microbiota across multiple proximal GI sites over time in healthy individuals. As part of a study of the pharmacokinetics of oral mesalamine administration, healthy, fasted volunteers (n = 8; 10 observation periods total) were orally intubated with a four-lumen catheter with multiple aspiration ports. Samples were taken from stomach, duodenal, and multiple jejunal sites, sampling hourly (≤7 h) to measure mesalamine (administered at t = 0), pH, and 16S rRNA gene-based composition. We observed a predominance of Firmicutes across proximal GI sites, with significant variation compared to stool. The microbiota was more similar within individuals over time than between subjects, with the fecal microbiota being unique from that of the small intestine. The stomach and duodenal microbiota displayed highest intraindividual variability compared to jejunal sites, which were more stable across time. We observed significant correlations in the duodenal microbial composition with changes in pH; linear mixed models identified positive correlations with multiple Streptococcus operational taxonomic units (OTUs) and negative correlations with multiple Prevotella and Pasteurellaceae OTUs. Few OTUs correlated with mesalamine concentration. The stomach and duodenal microbiota exhibited greater compositional dynamics than the jejunum. Short-term fluctuations in the duodenal microbiota were correlated with pH. Given the unique characteristics and dynamics of the proximal GI tract microbiota, it is important to consider these local environments in health and disease states. IMPORTANCE The gut microbiota are linked to a variety of gastrointestinal diseases, including inflammatory bowel disease. Despite this importance, microbiota dynamics in the upper gastrointestinal tract are understudied. Our article seeks to understand what factors impact microbiota dynamics in the healthy human upper gut. We found that the upper gastrointestinal tract contains consistently prevalent bacterial OTUs that dominate the overall community. Microbiota variability is highest in the stomach and duodenum and correlates with pH.

Interleukin-6 (IL-6) and Notch signaling are important regulators of breast cancer stem cells (CSCs), which drive the malignant phenotype through self-renewal, differentiation, and development of therapeutic resistance. We investigated the role of integrin-linked kinase (ILK) in regulating IL-6–driven Notch1 activation and the ability to target breast CSCs through ILK inhibition. Ectopic expression/short hairpin RNA-mediated knockdown of ILK, pharmacological inhibition of ILK with the small molecule T315, Western blot analysis, immunofluorescence, and luciferase reporter assays were used to evaluate the regulation of IL-6–driven Notch1 activation by ILK in IL-6–producing triple-negative breast cancer cell lines (MDA-MB-231, SUM-159) and in MCF-7 and MCF-7IL-6 cells. The effects of ILK on γ-secretase complex assembly and cellular localization were determined by immunofluorescence, Western blots of membrane fractions, and immunoprecipitation. In vivo effects of T315-induced ILK inhibition on CSCs in SUM-159 xenograft models were assessed by mammosphere assays, flow cytometry, and tumorigenicity assays. Results show that the genetic knockdown or pharmacological inhibition of ILK suppressed Notch1 activation and the abundance of the γ-secretase components presenilin-1, nicastrin, and presenilin enhancer 2 at the posttranscriptional level via inhibition of caveolin-1-dependent membrane assembly of the γ-secretase complex. Accordingly, knockdown of ILK inhibited breast CSC-like properties in vitro and the breast CSC subpopulation in vivo in xenograft tumor models. Based on these findings, we propose a novel function of ILK in regulating γ-secretase–mediated Notch1 activation, which suggests the targeting of ILK as a therapeutic approach to suppress IL-6–induced breast CSCs.

OBJECTIVES/SPECIFIC AIMS: Peripheral neuropathy is the dose limiting toxicity of paclitaxel treatment. Paclitaxel pharmacokinetics (PK), specifically the Cmax and amount of time the concentration remains above 0.05 µM (Tc>0.05), have been associated with occurrence of severe, clinician-documented neuropathy. The objective of this study was to confirm that paclitaxel PK predicts progression of patient-reported neuropathy. METHODS/STUDY POPULATION: This observational trial enrolled breast cancer patients receiving weekly 1-hour paclitaxel infusions (80 mg/m2×12 cycles) at the University of Michigan Comprehensive Cancer Center. Paclitaxel concentration was measured via LC/MS in plasma samples collected at the end of (Cmax) and 16–24 hours after (Tc>0.05) first infusion. Patient-reported neuropathy was collected (EORTC CIPN20) at baseline and each cycle. The rate of neuropathy severity increase per treatment cycle is being modeled for each patient. Cmax and Tc>0.05 values will be introduced into the model to confirm that PK independently contributes to neuropathy progression. RESULTS/ANTICIPATED RESULTS: PK and neuropathy data have been collected from 60 patients for ongoing analysis. Our initial model will characterize the expected severity of neuropathy after each cycle of paclitaxel treatment. The PK-neuropathy model will include either PK parameter to validate their contribution to the progression of neuropathy severity during treatment. We anticipate, based on our preliminary analysis of the first 16 patients, that both PK parameters will significantly contribute to the model but Tc>0.05 will be more strongly associated with neuropathy progression. DISCUSSION/SIGNIFICANCE OF IMPACT: This project will generate a model that can be used to predict a patient’s neuropathy severity throughout treatment using a single, conveniently collected and easily measured PK sample during their first cycle. The next steps of this project include identifying genetic and metabolomic biomarkers that predict which patients experienced more severe neuropathy than would be anticipated based on their paclitaxel PK, and a planned interventional trial of personalized paclitaxel dosing to enhance efficacy and/or prevent neuropathy.

STAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH(+)), or cell surface molecule CD44-positive (CD44(+)) but CD24-negative (CD24(-)) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells is unknown.We examined STAT3 activation in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH(+)) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH(-)) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH(+) subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH(+) subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH(+) subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH(+) breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH(+) cells were further selected for the stem cell markers CD44(+) and CD24(-).These studies demonstrate an important role for STAT3 signaling in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells which may have cancer stem cell properties and suggest that pharmacologic inhibition of STAT3 represents an effective strategy to selectively target the cancer stem cell-like subpopulation.

Purpose. The two purposes of this study were evaluating preclinical pharmacokinetics of MI-219 and predicting clearance (CL) and volume of distribution at steady-state (Vdss) of MI-219 in humans. Methods. Pharmacokinetic studies were conducted on mice, rats, dogs, and monkeys. Human CL of MI-219 was predicted using allometric scaling (SA), multi-exponential allometric scaling (ME), rule of exponents (RoE), single species scaling, two-term power equation (TTPE), physiologically based in vitro-in vivo extrapolation (IVIVE), and fu corrected intercept method (FCIM). In vitro assays were conducted to determine in vitro intrinsic CL, protein binding, and blood-plasma partition coefficients. To estimate half-life of MI-219, plasma concentration–time profile in humans was predicted using kallynochron and apolysichron time transformation (Dedrick plots) and normalization with MRT and Vdss (Wajima’s method). In addition, simultaneous interspecies scaling of CL, Vdss and concentration–time profile were performed by using Nonlinear Mixed Effects Modeling (NONMEM). Results. Preclinical studies showed that the elimination of MI-219 was mainly through metabolism. The validation using observed monkey CL and Vdss showed that MA, IVIVE and Oie-Tozer methods were accurately than the other methods. Human CL of MI-219 predicted by ME and IVIVE was between 0.237-0.342 L*h-1*kg-1. Human Vdss predicted by Oie-Tozer method and allometric scaling of unbound volume of distribution of tissues (VT/fuT) method was between 0.93-1.40 L*kg-1. Superimposition of rat, monkey and dog data was observed in Dedrick plots and Wajima’s transformations. Conclusions. The predicted human pharmacokinetics is useful for the design of first-in-human study. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.