ABSTRACT
The pathogenesis of atopic dermatitis (AD) results from complex interactions between environmental factors, barrier defects, and immune dysregulation resulting in systemic inflammation. Therefore, we sought to characterize circulating inflammatory profiles in pediatric AD patients and identify potential signaling nodes which drive disease heterogeneity and progression. We analyzed a sample set of 87 infants that were at high risk for atopic disease based on atopic dermatitis diagnoses. Clinical parameters, serum, and peripheral blood mononuclear cells (PBMCs) were collected upon entry, and at one and four years later. Within patient serum, 126 unique analytes were measured using a combination of multiplex platforms and ultrasensitive immunoassays. We assessed the correlation of inflammatory analytes with AD severity (SCORAD). Key biomarkers, such as IL-13 (rmcorr=0.47) and TARC/CCL17 (rmcorr=0.37), among other inflammatory signals, significantly correlated with SCORAD across all timepoints in the study. Flow cytometry and pathway analysis of these analytes implies that CD4 T cell involvement in type 2 immune responses were enhanced at the earliest time point (year 1) relative to the end of study collection (year 5). Importantly, forward selection modeling identified 18 analytes in infant serum at study entry which could be used to predict change in SCORAD four years later. We have identified a pediatric AD biomarker signature linked to disease severity which will have predictive value in determining AD persistence in youth and provide utility in defining core systemic inflammatory signals linked to pathogenesis of atopic disease.
ABSTRACT
Atopic dermatitis (AD) is a heterogeneous systemic inflammatory skin disease associated with dysregulated immune responses, barrier dysfunction and activated sensory nerves. To characterize circulating inflammatory profiles and underlying systemic disease heterogeneity within AD patients, blood samples from adult patients (N = 123) with moderate-to-severe AD in a phase 2 study of baricitinib (JAHG) were analysed. Baseline levels of 131 markers were evaluated using high-throughput and ultrasensitive proteomic platforms, patient clusters were generated based on these peripheral markers. We implemented a novel cluster reproducibility method to validate cluster outcomes within our study and used publicly available AD biomarker data set (73 markers, N = 58 patients) to validate our findings. Cluster reproducibility analysis demonstrated best consistency for 2 clusters by k-means, reproducibility of this clustering outcome was validated in an independent patient cohort. These unique JAHG patient subgroups either possessed elevated pro-inflammatory mediators, notably TNFß, MCP-3 and IL-13, among a variety of immune responses (high inflammatory) or lower levels of inflammatory biomarkers (low inflammatory). The high inflammatory subgroup was associated with greater baseline disease severity, demonstrated by greater EASI, SCORAD Index, Itch NRS and DLQI scores, compared with low inflammatory subgroup. African-American patients were predominantly associated with the high inflammatory subgroup and increased baseline disease severity. In patients with moderate-to-severe AD, heterogeneity was identified by the detection of 2 disease subgroups, differential clustering amongst ethnic groups and elevated pro-inflammatory mediators extending beyond traditional polarized immune responses. Therapeutic strategies targeting multiple pro-inflammatory cytokines may be needed to address this heterogeneity.
Subject(s)
Azetidines/therapeutic use , Dermatitis, Atopic/blood , Dermatitis, Atopic/drug therapy , Purines/therapeutic use , Pyrazoles/therapeutic use , Sulfonamides/therapeutic use , Adult , Biomarkers/blood , Dermatitis, Atopic/complications , Female , Humans , Male , Middle Aged , Severity of Illness Index , Young AdultABSTRACT
LY2775240 is a highly selective, potent and orally-administered inhibitor of phosphodiesterase 4 (PDE4), and is being investigated as a treatment option for inflammatory disorders, such as psoriasis. LY2775240 was investigated in rodent and rhesus monkey nonclinical models. Treatment with LY2775240 led to significant reductions in TNFα production, a marker of PDE4 engagement upon immune activation, in both nonclinical models. In the first part of a 2-part first-in-human randomized study, a wide dose range of LY2775240 was safely evaluated and found to be well-tolerated with common adverse events (AEs) of nausea, diarrhea, and headache. No serious AEs were reported. The pharmacokinetic profile of LY2775240 was well-characterized, with a half-life that can support once-a-day dosing. An ex vivo pharmacodynamic (PD) assay demonstrated dose-dependent PDE4 target engagement as assessed by reduction in TNFα production. A 20 mg dose of LY2775240 led to near-maximal TNFα inhibition in this PD assay in the first part of the study and was selected for comparison with the clinical dose of apremilast (30 mg) in the crossover, second part of this study. The 20 mg dose of LY2775240 demonstrated sustained maximal (50%-80%) inhibition of TNFα over all timepoints over the 24-h duration. The comparator apremilast achieved peak inhibition of ~ 50% at only 4 h postdose with a return to about 10% inhibition within 12 h of dosing. In summary, the nonclinical data and safety, tolerability, and PK/PD data in healthy subjects supports further investigation of LY2775240 in inflammatory indications. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Phosphodiesterase 4 (PDE4) inhibitors, such as apremilast, are currently approved to treat autoimmune disorders, such as psoriasis. LY2775240 is an oral PDE4 inhibitor being developed for treatment of a variety of inflammatory disorders. The degree of enzymatic inhibition achieved by PDE4 inhibitors clinically is poorly understood. WHAT QUESTION DID THIS STUDY ADDRESS? This study investigated single ascending doses of LY2775240, a highly selective oral PDE4 inhibitor, in healthy subjects. LY2775240 was well-tolerated over the dose range evaluated, and pharmacokinetic/pharmacodynamic (PD) profiles were well-characterized. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? This study evaluated different doses of LY2775240 and subsequently compared a selected LY2775240 dose with the clinical dose of apremilast with an ex vivo assay. This information builds a connection between target engagement and clinical efficacy. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? This is the first report of an ex vivo PD assay that has been systematically implemented in a PDE4 inhibitor Phase 1 study. Early investigation of exposure-response relationships versus a comparator can support evaluation of clinically meaningful doses of investigational agents.
Subject(s)
Drugs, Investigational/pharmacology , Phosphodiesterase 4 Inhibitors/pharmacology , Administration, Oral , Adult , Animals , Cross-Over Studies , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Drug Evaluation, Preclinical , Drugs, Investigational/therapeutic use , Enzyme Assays , Female , Healthy Volunteers , Humans , Macaca mulatta , Male , Mice , Middle Aged , Phosphodiesterase 4 Inhibitors/therapeutic use , Psoriasis/drug therapy , Thalidomide/analogs & derivatives , Thalidomide/pharmacology , Thalidomide/therapeutic useABSTRACT
Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.
Subject(s)
Eukaryotic Initiation Factor-4E/metabolism , Gene Expression Regulation, Neoplastic , Neoplasms/metabolism , Neoplasms/therapy , Protein Biosynthesis/genetics , Animals , Apoptosis , Base Sequence , Cells, Cultured , Endothelial Cells/metabolism , Eukaryotic Initiation Factor-4E/genetics , Humans , Mice , Neoplasms/blood supply , Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
IL-21 is a pleiotropic cytokine that plays a key role in modulating inflammatory responses, including the promotion of autoimmune diseases. Several groups have quantitated circulating levels of IL-21 in plasma and serum samples using various commercial ELISAs. We determined, however, that the most commonly used commercial assays in published literature were not specific or sensitive enough to detect levels of IL-21 in heparin plasma or serum from healthy human individuals. This finding prompted an effort to develop more specific and sensitive methods to quantitate IL-21 in complex biological matrices using proprietary anti-IL-21 antibodies with the Quanterix SiMoA platform and the Meso Scale Discovery (MSD) S-PLEX® format. Assays developed on both technology platforms were characterized in heparin plasma and serum using spike recoveries across a range of concentrations. Each method was able to detect sub-pg/mL levels of IL-21 (predicted Limit of Detection [LOD] of approximately 1.0â¯fg/mL for both the Quanterix SiMoA and MSD S-PLEX® platforms) which is 200-500 times lower than current commercial assays. Additionally we demonstrated that rheumatoid factor did not interfere with measuring IL-21 in the Quanterix SiMoA assay. Results obtained with the two new ultrasensitive assays showed a strong correlation (râ¯=â¯0.9428; pâ¯<â¯.0001). Additionally, IL-21 levels were significantly increased in samples from patients with Systemic Lupus Erythematosus (mean+/- SD: nâ¯=â¯14, 202.64 +/- 111.47â¯fg/mL, pâ¯=â¯.0001 for Quanterix SiMoA and 275.4 +/- 174.66â¯fg/mL pâ¯=â¯.0001 for MSD S-PLEX®) as well as in samples from patients with Sjögren's Syndrome (mean+/- SD: nâ¯=â¯11, 122.18 +/- 84.50â¯fg/mL, pâ¯=â¯.0029 for Quanterix SiMoA and 183.64 +/- 153.00â¯fg/mL, pâ¯=â¯.0082 for MSD S-PLEX®) when compared to healthy donors (mean+/- SD: nâ¯=â¯11, 38.1 +/- 27.8â¯fg/mL for Quanterix SiMoA and 58.1 +/- 30.7â¯fg/mL for MSD S-PLEX®). These ultrasensitive assays, for the first time, allow for the accurate quantitation of human IL-21 in heparin plasma and serum. In addition, these experiments also provide a direct comparison of the MSD S-PLEX® format and Quanterix SiMoA platform technologies, which may have broader implications to future application of these methods to evaluate low abundance proteins in complex biological matrices.
Subject(s)
Enzyme-Linked Immunosorbent Assay , Interleukins/blood , Healthy Volunteers , Humans , Interleukins/immunologyABSTRACT
BACKGROUND: Biomarkers that predict response to anabolic therapies could expedite the development of function-promoting anabolic drugs. This study aimed to identify serum biomarkers that are responsive to testosterone administration and associated with increases in fat-free mass (FFM). METHODS: Serum samples were obtained from the 5α-Reductase Trial, a randomized trial that compared the effects of graded doses of testosterone enanthate for 20 weeks in healthy men randomized with placebo or dutasteride (dual SRD5A inhibitor). Testosterone's effects on FFM or strength measures did not differ between placebo vs dutasteride groups. Accordingly, 54 subjects treated with testosterone plus placebo were included in the discovery cohort, and 48 subjects randomized to dutasteride were included in the validation cohort. A total of 1162 biomarkers were evaluated using prespecified criteria. RESULTS: In the discovery cohort, testosterone administration increased propeptide of type III collagen (PRO-C3) and propeptide of type VI collagen (PRO-C6) levels in a dose- and concentration-dependent manner; increases in these biomarkers from baseline to week 12 were associated with changes in FFM from baseline to week 20 (PRO-C3: r2 = 0.437, P < 0.001; PRO-C6: r2 = 0.434, P < 0.001). Changes in PRO-C3 and PRO-C6 levels were significantly associated with changes in chest press strength (PRO-C3: r2 = 0.394, P < 0.001; PRO-C6: r2 = 0.530, P < 0.001). In the SOMAscan, changes in IGF binding protein-6 (IGFBP6) and glypican 3 (GPC3) were associated with changes in total and free testosterone levels and FFM. These findings were replicated in the Validation cohort. CONCLUSION: PRO-C3, PRO-C6, IGFBP6, and GPC3 fulfilled the prespecified criteria for biomarkers of testosterone-induced muscle anabolism. Changes in these biomarkers were associated with changes in total and free testosterone concentrations and with testosterone-induced gains in FFM.
ABSTRACT
Matrix metalloproteinases (MMPs) have been implicated in the invasion, metastasis, and angiogenesis associated with human cancer by mediating the degradation of extracellular matrix components. In this paper, we report data that show that BAY 12-9566, a novel inhibitor of MMPs, inhibits angiogenesis, tumor regrowth, and the growth of lung metastases. BAY 12-9566, at 15-100 microM, inhibited tubule formation by human endothelial cells in an in vitro model, but did not prevent the proliferation of endothelial and human breast cancer cells. In the MDA-MB-435 human mammary carcinoma xenograft model, in which the primary tumor is transplanted into the murine mammary fat pad, BAY 12-9566, administered daily at a dose of 100 mg/kg/day p.o. after resection of the primary tumor, inhibited local tumor regrowth by 58% without causing any toxic effect. In addition, BAY 12-9566 treatment inhibited the number and volume of lung metastases by 57 and 88%, respectively. These effects were highly correlated with the serum concentration of BAY 12-9566 at the end of treatment. The serum of the treated animals, harvested 24 h after the last treatment, and the tumor regrown at the site of tumor transplant in the treated animals, contained less protein with MMP-9 activity (as measured in a gelatin zymography assay) than the corresponding controls. However, no difference in the activity of MMP-2 was observed. Although all clinical trials in cancer involving BAY 12-9566 have been halted, this MMP inhibitor has never been used in clinical trials in breast cancer. These results suggest that the novel MMP inhibitor BAY 12-9566 maybe a useful and safe oral treatment for breast cancer, adjunctive to surgery.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/enzymology , Enzyme Inhibitors/pharmacology , Organic Chemicals , Animals , Biphenyl Compounds , Breast Neoplasms/drug therapy , Cell Division , Cells, Cultured , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Humans , Matrix Metalloproteinase 2/biosynthesis , Matrix Metalloproteinase 9/biosynthesis , Mice , Mice, Nude , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Transplantation , Phenylbutyrates , Time Factors , Tumor Cells, Cultured , Umbilical Veins/cytologyABSTRACT
DNA-dependent RNA polymerase II (RNAP II) largest subunit RPB1 C-terminal domain (CTD) kinases, including CDK9, are serine/threonine kinases known to regulate transcriptional initiation and elongation by phosphorylating Ser 2, 5, and 7 residues on CTD. Given the reported dysregulation of these kinases in some cancers, we asked whether inhibiting CDK9 may induce stress response and preferentially kill tumor cells. Herein, we describe a potent CDK9 inhibitor, LY2857785, that significantly reduces RNAP II CTD phosphorylation and dramatically decreases MCL1 protein levels to result in apoptosis in a variety of leukemia and solid tumor cell lines. This molecule inhibits the growth of a broad panel of cancer cell lines, and is particularly efficacious in leukemia cells, including orthotopic leukemia preclinical models as well as in ex vivo acute myeloid leukemia and chronic lymphocytic leukemia patient tumor samples. Thus, inhibition of CDK9 may represent an interesting approach as a cancer therapeutic target, especially in hematologic malignancies.
Subject(s)
Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Cyclin-Dependent Kinase 9/genetics , Cyclohexylamines/administration & dosage , Indazoles/administration & dosage , Leukemia/drug therapy , Cell Line, Tumor , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Humans , Leukemia/pathology , Myeloid Cell Leukemia Sequence 1 Protein/biosynthesis , Phosphorylation/drug effects , Serine/metabolismABSTRACT
Protein kinases play important roles in many disease processes and are primary targets for drug development. Because cellular phosphorylation cascades are complex multidirectional pathways, the behavior of a drug in a biochemical enzyme assay may not accurately reflect its performance in the context of a whole cell. We have developed a near-infrared cytoblot assay that can be used to investigate both kinase signaling and effects of kinase inhibitors. Adherent cells were grown in either 96- or 384-well plates. Following stimulation, protein phosphorylation was detected immunohistochemically by simultaneous staining with two primary antibodies: a phospho-specific primary and normalization antibody that recognized either the target protein regardless of phosphorylation status (pan protein) or a housekeeping protein. Secondary antibodies labeled with two spectrally distinct near-infrared dyes were used for visualization. Nuclear staining with TO-PRO-3 was also used in place of the normalization antibody. Normalization for well-to-well variability was accomplished by ratiometric analysis of the two wavelengths. The near-infrared cytoblot was used to analyze phosphorylation of EGFR, Akt, Stat3, MEK 1, and ERK1/2. This assay format was also able to simultaneously assess the phosphorylation of multiple signaling proteins in response to known kinase inhibitors. We observed that the IC50 for the EGFR inhibitor PD168393 was similar for EGFR and Stat3 but was significantly higher for ERK1/2, a downstream modulator of EGFR function. The observation that the receptor and its effectors show different IC50 values for the same inhibitory drug could be important for target selection in drug development.