Combining Classic and Novel Neutrophil-Related Biomarkers to Identify Non-Small-Cell Lung Cancer.

BACKGROUND: Recent studies have revealed that neutrophils play a crucial role in cancer progression. This study aimed to explore the diagnostic value of neutrophil-related biomarkers for non-small-cell lung cancer (NSCLC). METHODS: We initially assessed the associations between classic neutrophil-related biomarkers (neutrophil-to-lymphocyte ratio (NLR), absolute neutrophil counts (NEU), absolute lymphocyte counts (LYM)) and NSCLC in 3942 cases and 6791 controls. Then, we measured 11 novel neutrophil-related biomarkers via Luminex Assays in 132 cases and 66 controls, individually matching on sex and age (±5 years), and evaluated their associations with NSCLC risk. We also developed the predictive models by sequentially adding variables of interest and assessed model improvement. RESULTS: Interleukin-6 (IL-6) (odds ratio (OR) = 10.687, 95% confidence interval (CI): 3.875, 29.473) and Interleukin 1 Receptor Antagonist (IL-1RA) (OR = 8.113, 95% CI: 3.182, 20.689) shows strong associations with NSCLC risk after adjusting for body mass index, smoking status, NLR, and carcinoembryonic antigen. Adding the two identified biomarkers to the predictive model significantly elevated the model performance from an area under the receiver operating characteristic curve of 0.716 to 0.851 with a net reclassification improvement of 97.73%. CONCLUSIONS: IL-6 and IL-1RA were recognized as independent risk factors for NSCLC, improving the predictive performance of the model in identifying disease.

Human radiolabeled mass balance studies supporting the FDA approval of new drugs.

Human radiolabeled mass balance studies are an important component of the clinical pharmacology programs supporting the development of new investigational drugs. These studies allow for understanding of the absorption, distribution, metabolism, and excretion of the parent drug and metabolite(s) in the human body. Understanding the drug's disposition as well as metabolite profiling and abundance via mass balance studies can help inform the overall drug development program. A survey of the US Food and Drug Administration (FDA)-approved new drug applications (NDAs) indicated that about 66% of the drugs had relied on findings from the mass balance studies to help understand the pharmacokinetic characteristics of the drug and to inform the overall drug development program. When such studies were not available in the original NDA, adequate justifications were routinely provided. Of the 104 mass balance studies included in this survey, most of the studies were conducted in healthy volunteers (90%) who were mostly men (>86%). The studies had at least six evaluable participants (66%) and were performed using the final route(s) of administration (98%). Eighty-five percent of the studies utilized a dose within the pharmacokinetic linearity range with 54% of the studies using a dose the same as the approved dose. Nearly all studies were performed as a single-dose (97%) study using a fit-for-purpose radiolabeled formulation. In this analysis, we summarized the current practices for conducting mass balance studies and highlighted the importance of conducting appropriately designed human radiolabeled mass balance studies and the challenges associated with inadequately designed or untimely studies.

Recommendations for Dose Selection for Adolescent Patients in Relevant Adult Oncology Clinical Trials.

Our research supported the dose selection recommendations for adolescents in the US Food and Drug Administration (FDA) Guidance on Inclusion of Adolescent Patients in Adult Oncology Clinical Trials. The FDA Guidance states that for drugs administered as a flat dose in adults and data showing no clinically meaningful effect of body size on drug exposure and toxicity in adults, a minimum body weight threshold may need to be defined to prevent adolescents who have a lower body weight from exceeding adult exposures. Our review of adult population pharmacokinetic analyses of new molecular entities approved for oncology between January 2015 and March 2021 suggested that 40 kg (the approximate median body weight of a 12-year-old) is generally the lower end of the body weight range that has no clinically relevant effect on drug pharmacokinetics or safety. The minimum body weight threshold and selection of an appropriate dose for adolescents in relevant adult oncology clinical trials should ultimately be determined based on available data on pharmacokinetics or pharmacodynamics of the investigational drug with consideration of body size effect on drug exposure, toxicity, and efficacy data (if available), the therapeutic index of the drug, and dose- and exposure-response relationships in adults.

Cadmium exposure impairs pancreatic ß-cell function and exaggerates diabetes by disrupting lipid metabolism.

Cadmium is known as an environmental pollutant that contributes to pancreatic damage and the pathogenesis of diabetes. However, less attention has been devoted to elucidating the mechanisms underlying Cd-induced pancreatic ß-cell dysfunction and the role of Cd toxicity in the development of diabetes. In this study, we demonstrated that exposure to Cd caused remarkable pancreatic ß-cell dysfunction and death, both in vitro and in vivo. Lipidomic analysis of Cd-exposed pancreatic ß-cells using high-resolution mass spectrometry revealed that Cd exposure altered the profile and abundance of lipids. Cd exposure induced intracellular lipid accumulation, promoted lipid biogenesis, elevated pro-inflammatory lipid contents and inhibited lipid degradation. Furthermore, Cd exposure upregulated the expression levels of TNF-α, IL-1ß and IL-6 in pancreatic ß-cells and elevated the TNF-α, IL1-ß and IL-6 levels in the serum and pancreas. Taken together, the results of our study demonstrated that environmental relevant Cd exposure causes pro-inflammatory lipids elevation and insulin secretion dysfunction in ß-cells and hence exaggerates diabetes development. Combined exposure to environmental hazardous chemicals might markedly increase the probability of developing diabetes in humans. This study provides new metabolic and pharmacological targets for antagonizing Cd toxicity.

Immunomodulatory Therapeutic Proteins in COVID-19: Current Clinical Development and Clinical Pharmacology Considerations.

The coronavirus disease 2019 (COVID-19) pandemic caused by infection with SARS-CoV-2 has led to more than 600 000 deaths worldwide. Patients with severe disease often experience acute respiratory distress characterized by upregulation of multiple cytokines. Immunomodulatory biological therapies are being evaluated in clinical trials for the management of the systemic inflammatory response and pulmonary complications in patients with advanced stages of COVID-19. In this review, we summarize the clinical pharmacology considerations in the development of immunomodulatory therapeutic proteins for mitigating the heightened inflammatory response identified in COVID-19.

A Comprehensive Updated Review on SARS-CoV-2 and COVID-19.

This literature review aims to provide a comprehensive current summary of the pathogenesis, clinical features, disease course, host immune responses, and current investigational antiviral and immunomodulatory pharmacotherapies to facilitate the development of future therapies and measures for prevention and control.

A Novel Pharmacokinetic Bridging Strategy to Support a Change in the Route of Administration for Biologics.

Determination of appropriate pharmacokinetic end point to bridge different dosing regimens is often a challenge when developing a new route of administration. Trough concentrations (Ctrough) are often considered the most relevant PK end point to predict efficacy (ACR20/DAS28) in the treatment of rheumatoid arthritis for biologics. However, no systematic research has been conducted to evaluate this approach. We developed a novel strategy to predict the most relevant PK variables that may be used to support a change in the route of administration for biological products. Our analysis indicated that matching only Ctrough when switching from intravenous dosing to subcutaneous dosing with decreasing dosing interval may result in a lower treatment response. If only average concentration (Cave) is considered as the relevant variable, our analysis showed that treatment response may be worsened when switching from subcutaneous dosing to intravenous dosing with increasing dosing interval. The study results demonstrated that matching a single pharmacokinetic end point (Ctrough or Cave) may not be sufficient to ensure efficacy when switching between intravenous dosing and subcutaneous dosing. A practical novel pharmacokinetic bridging approach is provided to support a change in the route of administration for biological products.

Outdoor air pollution pregnancy exposures are associated with behavioral problems in China's preschoolers.

There are mounting evidences indicated that maternal exposure to outdoor air pollutants in pregnancy affects children's neural development, but the researches on children's behavioral difficulties are seldom. We explored the association between maternal exposure to outdoor air pollution during different trimesters of pregnancy and the prevalence of behavioral difficulties among 657 preschool children aged 3-4 from three kindergartens in Wuhan, China. This is a cross-sectional study. Children's behavioral difficulties were assessed by the Strengths and Difficulties Questionnaire (SDQ) (reported by parents). Maternal exposure to outdoor air pollutants during pregnancy were estimated based on the daily average measured concentration levels from ground monitoring stations. Potential confounding factors including children-related, maternal, and socio-economic status (SES) were adjusted in the study. We calculated the prevalence of each type of behavioral difficulties and used binary logistic regression method to estimate the crude odds ratio (cOR), adjusted odds ratio (aOR), and corresponding 95% confidence intervals (95% CIs) for 1 µg/m3 increase in each air pollutant during every exposure window in single- and two-pollutant models. The prevalence of participants' total behavioral difficulties was 9.6%. In single-pollutant models, during full gestation, positive associations were observed between exposure to NO2 (aOR = 1.204, 95% CI 1.042, 1.392), particle matter (PM)10 (aOR = 1.070, 95% CI 1.018, 1.125), PM2.5 (aOR = 1.095, 95% CI 1.021, 1.176) and total difficulties, exposure to PM10 (aOR = 1.040, 95% CI 1.001, 1.081), PM2.5 (aOR = 1.053, 95% CI 1.000, 1.109) and prosocial behavior, respectively. In the first trimester, exposure to SO2 (aOR = 1.047, 95% CI 1.009, 1.086), NO2 (aOR = 1.039, 95% CI 1.013, 1.066), PM10 (aOR = 1.013, 95% CI 1.004, 1.023), and PM2.5 (aOR = 1.016, 95% CI 1.004, 1.028) were all positively associated with total difficulties. The associations between second and third trimesters' exposure to all pollutants and outcomes were not statistically significant. However, in the two-pollutant models, second trimester exposure to PM2.5 (aOR = 1.078, 95%CI 1.023, 1.137) was positively associated with total behavioral difficulties after adjusting for PM10. Exposure to outdoor air pollutants SO2, NO2, PM10, and PM2.5 during pregnancy may be associated with behavioral difficulties, especially in the first trimester.

Hypersensitivities for acetaldehyde and other agents among cancer cells null for clinically relevant Fanconi anemia genes.

Large-magnitude numerical distinctions (>10-fold) among drug responses of genetically contrasting cancers were crucial for guiding the development of some targeted therapies. Similar strategies brought epidemiological clues and prevention goals for genetic diseases. Such numerical guides, however, were incomplete or low magnitude for Fanconi anemia pathway (FANC) gene mutations relevant to cancer in FANC-mutation carriers (heterozygotes). We generated a four-gene FANC-null cancer panel, including the engineering of new PALB2/FANCN-null cancer cells by homologous recombination. A characteristic matching of FANCC-null, FANCG-null, BRCA2/FANCD1-null, and PALB2/FANCN-null phenotypes was confirmed by uniform tumor regression on single-dose cross-linker therapy in mice and by shared chemical hypersensitivities to various inter-strand cross-linking agents and γ-radiation in vitro. Some compounds, however, had contrasting magnitudes of sensitivity; a strikingly high (19- to 22-fold) hypersensitivity was seen among PALB2-null and BRCA2-null cells for the ethanol metabolite, acetaldehyde, associated with widespread chromosomal breakage at a concentration not producing breaks in parental cells. Because FANC-defective cancer cells can share or differ in their chemical sensitivities, patterns of selective hypersensitivity hold implications for the evolutionary understanding of this pathway. Clinical decisions for cancer-relevant prevention and management of FANC-mutation carriers could be modified by expanded studies of high-magnitude sensitivities.

FAM190A deficiency creates a cell division defect.

Like the p16, SMAD4, and RB1 genes, FAM190A (alias CCSER1) lies at a consensus site of homogeneous genomic deletions in human cancer. FAM190A transcripts in 40% of cancers also contain in-frame deletions of evolutionarily conserved exons. Its gene function was unknown. We found an internal deletion of the FAM190A gene in a pancreatic cancer having prominent focal multinuclearity. The experimental knockdown of FAM190A expression by shRNA caused focal cytokinesis defects, multipolar mitosis, and multinuclearity as observed in time-lapse microscopy. FAM190A was localized to the γ-tubulin ring complex of early mitosis and to the midbody in late cytokinesis by immunofluorescence assay and was present in the nuclear fraction of unsynchronized cells by immunoblot. FAM190A interacted with EXOC1 and Ndel1, which function in cytoskeletal organization and the cell division cycle. Levels of FAM190A protein peaked 12 hours after release from thymidine block, corresponding to M-phase. Slower-migrating phosphorylated forms accumulated toward M-phase and disappeared after release from a mitotic block and before cytokinesis. Studies of FAM190A alterations may provide mechanistic insights into mitotic dysregulation and multinuclearity in cancer. We propose that FAM190A is a regulator or structural component required for normal mitosis and that both the rare truncating mutations and common in-frame deletion alteration of FAM190A may contribute to the chromosomal instability of cancer.

Unbiased discovery of interactions at a control locus driving expression of the cancer-specific therapeutic and diagnostic target, mesothelin.

Although significant effort is expended on identifying transcripts/proteins that are up-regulated in cancer, there are few reports on systematic elucidation of transcriptional mechanisms underlying such druggable cancer-specific targets. The mesothelin (MSLN) gene offers a promising subject, being expressed in a restricted pattern normally, yet highly overexpressed in almost one-third of human malignancies and a target of cancer immunotherapeutic trials. CanScript, a cis promoter element, appears to control MSLN cancer-specific expression; its related genomic sequences may up-regulate other cancer markers. CanScript is a 20-nt bipartite element consisting of an SP1-like motif and a consensus MCAT sequence. The latter recruits TEAD (TEA domain) family members, which are universally expressed. Exploration of the active CanScript element, especially the proteins binding to the SP1-like motif, thus could reveal cancer-specific features having diagnostic or therapeutic interest. The efficient identification of sequence-specific DNA-binding proteins at a given locus, however, has lagged in biomarker explorations. We used two orthogonal proteomics approaches--unbiased SILAC (stable isotope labeling by amino acids in cell culture)/DNA affinity-capture/mass spectrometry survey (SD-MS) and a large transcription factor protein microarray (TFM)--and functional validation to explore systematically the CanScript interactome. SD-MS produced nine candidates, and TFM, 18. The screens agreed in confirming binding by TEAD proteins and by newly identified NAB1 and NFATc. Among other identified candidates, we found functional roles for ZNF24, NAB1 and RFX1 in MSLN expression by cancer cells. Combined interactome screens yield an efficient, reproducible, sensitive, and unbiased approach to identify sequence-specific DNA-binding proteins and other participants in disease-specific DNA elements.

Structural analysis of the cancer-specific promoter in mesothelin and in other genes overexpressed in cancers.

Mesothelin (MSLN) may be the most "dramatic" of the tumor markers, being strongly overexpressed in nearly one-third of human malignancies. The biochemical cause is unclear. We previously ascribed this cancer-specific overexpression to an element, Canscript, residing around 50 bp 5' of the transcription start site in cancer (Hucl, T., Brody, J. R., Gallmeier, E., Iacobuzio-Donahue, C. A., Farrance, I. K., and Kern, S. E. (2007) Cancer Res. 67, 9055-9065). Herein, we found a Canscript promoter activity elevated over 100-fold in cancer cells. In addition to a highly conserved TEAD1 (TEA domain family member 1)-binding MCAT motif, nucleotide substitution revealed the consensus core sequence (WCYCCACCC) of an SP1-like motif in Canscript. The unknown transcription factor binding to the SP1-like motif may hold the key for the cancer specificity of Canscript. SP1, GLI1, and RUNX1, -2, and -3 appeared unlikely to be the direct transcription factors acting at the SP1-like motif, but KLF6 had some features of such a candidate. YAP1, a TEAD1-binding protein, appeared necessary, but not sufficient, for Canscript activity; knockdown of YAP1 by small interfering RNAs greatly reduced MSLN levels in MSLN-overexpressing cells, but overexpressing YAP1 in MSLN-negative cells did not induce MSLN expression. Cansript-like sequences were found in other genes up-regulated in pancreatic cancer; reporters driven by the sequences from FXYD3, MUC1, and TIMP1 had activities more than 2 times that of the control. This suggested that the cause of MSLN overexpression might also contribute mechanistically to the overexpression of other tumor markers.

Improved DNA electrophoresis in conditions favoring polyborates and lewis acid complexation.

Spatial compression among the longer DNA fragments occurs during DNA electrophoresis in agarose and non-agarose gels when using certain ions in the conductive buffer, impairing the range of fragment sizes resolved well in a single gel. Substitutions using various polyhydroxyl anions supported the underlying phenomenon as the complexation of Lewis acids to DNA. We saw significant improvements using conditions (lithium borate 10 mM cations, pH 6.5) favoring the formation of borate polyanions and having lower conductance and Joule heating, delayed electrolyte exhaustion, faster electrophoretic run-speed, and sharper separation of DNA bands from 100 bp to 12 kb in a single run.

Cholesterol trafficking is required for mTOR activation in endothelial cells.

Mammalian target of rapamycin (mTOR) constitutes a nodal point of a signaling network that regulates cell growth and proliferation in response to various environmental cues ranging from growth factor stimulation to nutrients to stress. Whether mTOR is also affected by cholesterol homeostasis, however, has remained unknown. We report that blockade of cholesterol trafficking through lysosome by a newly identified inhibitor of angiogenesis, itraconazole, leads to inhibition of mTOR activity in endothelial cells. Inhibition of mTOR by itraconazole but not rapamycin can be partially restored by extracellular cholesterol delivered by cyclodextrin. Moreover, other known inhibitors of endosomal/lysosomal cholesterol trafficking as well as siRNA knockdown of Niemann-Pick disease type C (NPC) 1 and NPC2 also cause inhibition of mTOR in endothelial cells. In addition, both the accumulation of cholesterol in the lysosome and inhibition of mTOR caused by itraconazole can be reversed by thapsigarin. These observations suggest that mTOR is likely to be involved in sensing membrane sterol concentrations in endothelial cells, and the cholesterol trafficking pathway is a promising target for the discovery of inhibitors of angiogenesis.

Identification of inhibitors of ABCG2 by a bioluminescence imaging-based high-throughput assay.

ABCG2 is a member of the ATP-binding cassette (ABC) family of transporters, the overexpression of which is associated with tumor resistance to a variety of chemotherapeutic agents. Accordingly, combining ABCG2 inhibitor(s) with chemotherapy has the potential to improve treatment outcome. To search for clinically useful ABCG2 inhibitors, a bioluminescence imaging (BLI)-based assay was developed to allow high-throughput compound screening. This assay exploits our finding that d-luciferin, the substrate of firefly luciferase (fLuc), is a specific substrate of ABCG2, and ABCG2 inhibitors block the export of d-luciferin and enhance bioluminescence signal by increasing intracellular d-luciferin concentrations. HEK293 cells, engineered to express ABCG2 and fLuc, were used to screen the Hopkins Drug Library that includes drugs approved by the Food and Drug Administration (FDA) as well as drug candidates that have entered phase II clinical trials. Forty-seven compounds showed BLI enhancement, a measure of anti-ABCG2 activity, of > or =5-fold, the majority of which were not previously known as ABCG2 inhibitors. The assay was validated by its identification of known ABCG2 inhibitors and by confirming previously unknown ABCG2 inhibitors using established in vitro assays (e.g., mitoxantrone resensitization and BODIPY-prazosin assays). Glafenine, a potent new inhibitor, also inhibited ABCG2 activity in vivo. The BLI-based assay is an efficient method to identify new inhibitors of ABCG2. As they were derived from a FDA-approved compound library, many of the inhibitors uncovered in this study are ready for clinical testing.

Clofazimine inhibits human Kv1.3 potassium channel by perturbing calcium oscillation in T lymphocytes.

The Kv1.3 potassium channel plays an essential role in effector memory T cells and has been implicated in several important autoimmune diseases including multiple sclerosis, psoriasis and type 1 diabetes. A number of potent small molecule inhibitors of Kv1.3 channel have been reported, some of which were found to be effective in various animal models of autoimmune diseases. We report herein the identification of clofazimine, a known anti-mycobacterial drug, as a novel inhibitor of human Kv1.3. Clofazimine was initially identified as an inhibitor of intracellular T cell receptor-mediated signaling leading to the transcriptional activation of human interleukin-2 gene in T cells from a screen of the Johns Hopkins Drug Library. A systematic mechanistic deconvolution revealed that clofazimine selectively blocked the Kv1.3 channel activity, perturbing the oscillation frequency of the calcium-release activated calcium channel, which in turn led to the inhibition of the calcineurin-NFAT signaling pathway. These effects of clofazimine provide the first line of experimental evidence in support of a causal relationship between Kv1.3 and calcium oscillation in human T cells. Furthermore, clofazimine was found to be effective in blocking human T cell-mediated skin graft rejection in an animal model in vivo. Together, these results suggest that clofazimine is a promising immunomodulatory drug candidate for treating a variety of autoimmune disorders.

Digoxin and other cardiac glycosides inhibit HIF-1alpha synthesis and block tumor growth.

A library of drugs that are in clinical trials or use was screened for inhibitors of hypoxia-inducible factor 1 (HIF-1). Twenty drugs inhibited HIF-1-dependent gene transcription by >88% at a concentration of 0.4 microM. Eleven of these drugs were cardiac glycosides, including digoxin, ouabain, and proscillaridin A, which inhibited HIF-1alpha protein synthesis and expression of HIF-1 target genes in cancer cells. Digoxin administration increased latency and decreased growth of tumor xenografts, whereas treatment of established tumors resulted in growth arrest within one week. Enforced expression of HIF-1alpha by transfection was not inhibited by digoxin, and xenografts derived from these cells were resistant to the anti-tumor effects of digoxin, demonstrating that HIF-1 is a critical target of digoxin for cancer therapy.

Reading dynamic kinase activity in living cells for high-throughput screening.

Protein kinases, as crucial signaling molecules, represent an emerging class of drug targets, and the ability to assay their activities in living cells with high-throughput screening should provide exciting opportunities for drug discovery and chemical and functional genomics. Here, we describe a general method for high-throughput reading of dynamic kinase activities using ratiometric fluorescent sensors, and showcase an example of reading intracellular activities of protein kinase A (PKA) and the cyclic adenosine monophosphate (cAMP)/PKA pathway downstream of many G-protein coupled receptors (GPCRs). We further demonstrate the first compound screen based on the ability of compounds to modulate dynamic kinase activities in living cells and show that such screening of a collection of clinical compounds has successfully identified modulators of the GPCR/cAMP/PKA pathway.