ABSTRACT
Pharmacological reactivation of the γ-globin gene for the production of fetal hemoglobin (HbF) is a promising approach for the management of ß-thalassemia and sickle cell disease (SCD). We conducted a phenotypic screen in human erythroid progenitor cells to identify molecules that could induce HbF, which resulted in identification of the hit compound 1. Exploration of structure-activity relationships and optimization of ADME properties led to 2-azaspiro[3.3]heptane derivative 18, which is more rigid and has a unique structure. In vivo using cynomolgus monkeys, compound 18 induced a significant dose-dependent increase in globin switching, with developable properties. Moreover, compound 18 showed no genotoxic effects and was much safer than hydroxyurea. These findings could facilitate the development of effective new therapies for the treatment of ß-hemoglobinopathies, including SCD.
Subject(s)
Azetidines/pharmacology , Erythroid Precursor Cells/drug effects , Fetal Hemoglobin/metabolism , Spiro Compounds/pharmacology , Animals , Azetidines/chemical synthesis , Azetidines/pharmacokinetics , Drug Design , Drug Stability , Gene Expression Regulation/drug effects , Humans , Isoxazoles/chemical synthesis , Isoxazoles/pharmacokinetics , Isoxazoles/pharmacology , Macaca fascicularis , Microsomes, Liver/metabolism , Molecular Structure , Spiro Compounds/chemical synthesis , Spiro Compounds/pharmacokinetics , Structure-Activity RelationshipABSTRACT
Heritable disorders associated with hemoglobin production are the most common monogenic disorders. These are mainly represented by disorders such as ß-thalassemia and sickle cell disease. Induction of fetal hemoglobin (HbF) has been known to ameliorate the clinical severity of these ß hemoglobinopathies. A high throughput phenotypic screening was used in this study to isolate novel compounds that may enhance the expression of γ-globin, the component of HbF, in human erythroid cell lines and primary erythroid progenitors derived from human CD34+ cells. The effect of lead compounds on epigenetic enzymes and key transcriptional factors was evaluated to identify their mode of action. One hit compound was further evaluated in vivo using monkey models. Among the ~18,000 compounds screened, 18 compounds were selected and tested to determine their ability to induce HbF in human erythroid cell lines and primary erythroid cells. One of these compounds, a 3-phenyl-isoxazole derivative, could potentially induce HbF in monkey bone marrow cells when administered orally. The compound downregulated negative transcriptional regulators of HbF, Bcl11a and LRF without inhibiting the known epigenetic enzymes. These studies demonstrated the advantages associated with phenotype-screening and identified novel fetal globin inducers that may be useful for treating hemoglobinopathies.
Subject(s)
Fetal Hemoglobin/genetics , Gene Expression Regulation/drug effects , Hemoglobinopathies/genetics , Repressor Proteins/genetics , Xenobiotics/pharmacology , Zinc Fingers , Animals , Antigens, CD34/metabolism , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Line , Down-Regulation/drug effects , Erythroblasts/cytology , Erythroblasts/drug effects , Erythroblasts/metabolism , Fetal Hemoglobin/metabolism , Hemoglobinopathies/metabolism , High-Throughput Screening Assays/methods , Humans , Macaca fascicularis , Phenotype , Repressor Proteins/metabolismABSTRACT
xCT, a well-known cystine transporter, is reported to be involved in the proliferation of various cells, such as cancer cells, immune cells, and fibroblasts. xCT inhibitor is expected to be a promising drug for cancer or immune diseases. However, there are little studies reporting that xCT inhibitors improve disease progression in vivo. To invent potent xCT inhibitors in vivo, we established a new in vivo model for assessing efficacy of xCT inhibition. dl-propargylglycine (PPG) was administered intraperitoneally to wild-type C57BL/6J mice. Concentration of cystathionine, another substrate of xCT, in the thymus and spleen was measured by LC-MS/MS. PPG increased cystathionine amounts in the thymus and spleen in a dose- and time-dependent manner. At 7 h after PPG administration, the efficacy of erastin, a representative xCT inhibitor, was clearly shown. We synthesized a new compound, Compound A, which had much higher inhibitory effect on xCT than erastin both in vitro and in vivo. We established a mouse model of PPG-induced cystathionine accumulation for assessing xCT inhibition in vivo. By using this model, we discovered that Compound A was approximately 15 times more effective in vivo than erastin.
Subject(s)
Alkynes/pharmacology , Amino Acid Transport System y+/antagonists & inhibitors , Glycine/analogs & derivatives , Animals , Cystathionine/metabolism , Female , Glycine/pharmacology , Mice , Mice, Inbred C57BL , Models, Animal , Piperazines/pharmacology , Spleen/drug effects , Spleen/metabolism , Tandem Mass Spectrometry/methods , Thymus Gland/drug effects , Thymus Gland/metabolismABSTRACT
Objectives: In Japan, sodium-glucose co-transporter type 2 (SGLT2) inhibitors have been reported to be associated with serious skin and subcutaneous tissue disorders. A post-marketing surveillance (PMS) study suggested that the association was specific for ipragliflozin and, to a lesser extent for dapagliflozin. These studies were performed to confirm the association of 6 SGLT2 inhibitors with serious skin disorders in a clinical setting, to elucidate the role of melanin in serious skin disorders and to understand the underlying mechanisms. Methods: The latest PMS records were retrieved from the Japanese Adverse Drug Event Report (JADER) database, and the associations were analyzed by data mining techniques. In silico 3-D docking simulation of SGLT2 inhibitors with melanin was performed using the MOE software. The skin tissue distribution of SGLT2 inhibitors was evaluated using albino rats after oral administration at clinical doses. Results: The adjusted reporting odds ratio (95% confidential limit) was 1.667 (1.415, 1.963) for ipragliflozin, 0.514 (0.317, 0.835) for dapagliflozin, 0.149 (0.048, 0.465) for tofogliflozin, 0.624 (0.331, 1.177) for luseogliflozin, 0.590 (0.277, 1.257) for canagliflozin and 0.293 (0.073, 1.187) for empagliflozin, when drugs other than the SGLT2 inhibitors were referred, and the association was detected only for ipragliflozin in clinical use. In silico 3-D docking simulation suggested the influence of melanin in ipragliflozin-specific serious skin disorders. The skin tissue-to-plasma concentration ratio of ipragliflozin was 0.45 ± 0.20 (±SD) at 1 hr after administration and increased in a time-dependent manner to 5.82 ± 3.66 at 24 hr (p<0.05), but not in case of other SGLT2 inhibitors. Conclusions: Serious skin disorders were suggested to be specific for ipragliflozin. Interaction with melanin might be implicated in ipragliflozin-specific serious skin disorders. Ipragliflozin was retained in the skin tissue, which suggested its interaction with the skin tissue in serious skin disorders.
Subject(s)
Glucosides/adverse effects , Skin Diseases/chemically induced , Sodium-Glucose Transporter 2 Inhibitors/adverse effects , Thiophenes/adverse effects , Animals , Glucose , Glucose Transporter Type 2 , Glucosides/pharmacokinetics , Glucosides/pharmacology , Humans , Hypoglycemic Agents , Japan , Rats , Sodium , Sodium-Glucose Transporter 2 , Sodium-Glucose Transporter 2 Inhibitors/pharmacokinetics , Sodium-Glucose Transporter 2 Inhibitors/pharmacology , Subcutaneous Tissue , Thiophenes/pharmacokinetics , Thiophenes/pharmacology , Tissue DistributionABSTRACT
The possibility of using dissolving microneedles (DMs) as a skin allergy test device was studied in rats. Poly-L-arginine was used as a model allergen. Dextran was used to prepare three kinds of DM array chips containing different doses of poly-L-arginine: 17.1±0.5 µg (low-dose DM), 42.2±0.8 µg (medium-dose DM), and 87.4±1.1 µg (high-dose DM); each 1.0 cm2 chip contained 300 DMs. The mean lengths of the low-, medium-, and high-dose DM were 489±3, 485±3, and 492±1 µm and mean diameters of the base were 301±2, 299±1, and 299±2 µm, respectively. Furthermore, for the low-, medium-, and high-dose DM, the administered doses of poly-L-arginine were estimated to be 9.3±1.9, 31.1±1.3, and 61.9±4.7 µg and the scratching behavior per 30 min was 9.8±3.4, 60.4±8.3, and 95.7±10.6 times, respectively. These results demonstrate the dose dependence of the immunoreactivity of the poly-L-arginine DMs, suggesting that DMs can be used an alternative skin allergy device.
Subject(s)
Allergens/administration & dosage , Hypersensitivity/metabolism , Microinjections/methods , Skin Absorption/drug effects , Administration, Cutaneous , Allergens/adverse effects , Animals , Dose-Response Relationship, Drug , Hypersensitivity/etiology , Male , Needles , Rats , Rats, Wistar , Skin Absorption/physiology , Skin Tests/methodsABSTRACT
OBJECTIVES: Cadherin-11 (CDH11) is an adhesion molecule that anchors ß-catenin and is involved with various functions of synovial fibroblast cells (SFCs) during the development of rheumatoid arthritis (RA). However, the mechanism of CDH11 during RA-SFC proliferation is unclear. The aim of our study was to clarify the involvement of CDH11 and ß-catenin signalling during proliferation. METHODS: IL-1ß-induced and tumour necrosis factor-α (TNF-α)-induced cell proliferation, with CDH11 siRNAs, ß-catenin-specific siRNAs and a CDH11-neutralizing antibody, were assessed by 5-Bromo-2'-deoxy-uridine ELISA. KEY FINDINGS: Using CDH11 siRNAs, there were a 42% reduction in IL-1ß-induced proliferation and a 64% reduction in ß-catenin protein. When ß-catenin siRNAs were applied, there was a 63% reduction in IL-1ß-induced proliferation. The median effective concentration (EC50 ) values for IL-1ß-induced proliferation via CDH11-mediated ß-catenin-dependent, total ß-catenin-dependent and ß-catenin-independent signalling were 0.0015, 0.016 and 0.18 ng/ml, respectively. Blocking CDH11 ligation with a CDH11-neutralizing antibody did not decrease IL-1ß-induced proliferation. CONCLUSIONS: CDH11-mediated ß-catenin signalling was 42% involved in IL-1ß-induced proliferation and had the highest susceptibility to IL-1ß among the proliferative signallings analysed in this study. The mode of action for CDH11 during the cell proliferation was likely associated with a pool of ß-catenin protein. In contrast, CDH11 and ß-catenin were not involved in TNF-α-induced RA-SFC proliferation.
Subject(s)
Arthritis, Rheumatoid/physiopathology , Cadherins/pharmacology , Fibroblasts/metabolism , Interleukin-1beta/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , beta Catenin/pharmacology , Cell Proliferation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , RNA, Small Interfering/metabolism , Signal Transduction , Synovial Membrane/physiopathologyABSTRACT
The melanin granules are synthesized in melanocytes in the epidermal basal layer and the hair matrix. For the effective passage of melanin granules to the adjacent keratinocytes, melanocytes utilize unique cytoplasmic delivery system in which cytoskeletal network is prominently involved. Here, we show that the t-SNARE protein syntaxin3, a member of a family of key mediators of the cytoplasmic vesicle fusion and potent modulators of cytoskeletal dynamics, dramatically affects melanocyte cell behavior. Although plasmalemmal syntaxin3 has been detected also on the melanosomes of normal human melanocytes, we noticed that mouse melanoma B16 cells had completely lost endogenous syntaxin3. In response to the forcible expression of syntaxin3, B16 cells formed well-developed dendritic filopodia and accumulated melanin granules in the cytoplasm. We found that exogenous syntaxin3 was not expressed at the plasma membrane, but rather, localized with non-fibrous F-actin and melanin-packed melanosomes in the cytoplasm, by which the assembly/polymerization of actin was dramatically impacted and the melanosome secretion was severely suppressed. The syntaxin3-triggered phenotypic changes were also induced by a syntaxin3 mutant lacking SNARE and transmembrane domains, and they were completely reverted by the subsequent knockdown of exogenous syntaxin3. This t-SNARE protein may act as a regulator of the actin dynamics, rather than a direct vesicle fusion mediator, to determine the fundamental properties of melanocytes.
Subject(s)
Cell Movement/physiology , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Qa-SNARE Proteins/metabolism , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Actins/metabolism , Animals , Cell Adhesion/physiology , Cell Line, Tumor , Cells, Cultured , Disease Models, Animal , Humans , Melanins/metabolism , Melanocytes/metabolism , Melanocytes/pathology , Melanosomes/metabolism , Mice , Phenotype , Pseudopodia/physiologyABSTRACT
PEGL-DOX is an excellent treatment for recurrent ovarian cancer that rarely causes side-effects like cardiotoxicity or hair loss, but frequently results in Hand-Foot Syndrome (HFS). In severe cases, it can become necessary to reduce the PEGL-DOX concentration or the duration of the drug therapy, sometimes making it difficult to continue treatment. In this study, we prepared an animal model to compare the effects of DOX versus PEGL-DOX, and we noticed that only treatment with PEGL-DOX resulted in HFS, which led us to conclude that extravasation due to long-term circulation was one of the causes of HFS. In addition, we were able to show that the primary factor leading to the skin-specific outbreaks in the extremities was the appearance of reactive oxygen species (ROS) due to interactions between DOX and the metallic Cu(II) ions abundant in skin tissue. ROS directly disturb the surrounding tissue and simultaneously induce keratinocyte-specific apoptosis. Keratinocytes express the thermoreceptor TRPM2, which is thought to be able to detect ROS and stimulate the release of chemokines (IL-8, GRO, Fractalkine), which induce directed chemotaxis in neutrophils and other blood cells. Those cells and the keratinocytes then undergo apoptosis and simultaneously release IL-1ß, IL-1α, and IL-6, which brings about an inflammatory state. In the future, we plan to develop preventative as well as therapeutic treatments by trapping the ROS.
Subject(s)
Antibiotics, Antineoplastic/adverse effects , Doxorubicin/adverse effects , Hand-Foot Syndrome/etiology , Keratinocytes/drug effects , Animals , Cell Survival/drug effects , Cells, Cultured , Chemokines/metabolism , Cytokines/metabolism , Female , Hand-Foot Syndrome/metabolism , Humans , Keratinocytes/metabolism , Liposomes , Polyethylene Glycols/adverse effects , Rats , Rats, Hairless , Rats, Sprague-Dawley , Superoxide Dismutase/pharmacologyABSTRACT
BACKGROUND: We recently reported that over-expression of skin fibroblast-derived elastase (SFE) plays a pivotal role in the mechanism of UVB-induced skin wrinkling. Since UVB penetrates only modestly to the dermis, we hypothesized that factors secreted by UVB-exposed keratinocytes in the epidermis trigger fibroblasts in the dermis to increase their expression of SFE which then degrades the elastic fibers. OBJECTIVE: In this study, we characterized the paracrine interaction between human keratinocytes (HK) and human fibroblasts (HF) which leads to increased expression of SFE. METHODS AND RESULTS: Medium conditioned by UVB-exposed HK contained increased levels of IL-1α, GM-CSF, IL-6, TNFα and IL-8. While HF cultured with those conditioned medium slightly down-regulated the gene expression of collagen and elastin, they significantly increased their expression of SFE at the transcriptional, translational and enzymatic levels. Neutralizing antibodies to IL-1α or GM-CSF significantly abolished the increased expression of SFE at the translational and/or enzymatic levels in HF cultured with those conditioned medium, while neutralizing antibodies to IL-6, IL-8 or TNFα had no such effect. The addition of IL-1α or GM-CSF, but not TNFα, IL-6 or IL-8, at concentrations ranging from 1 to 10nm, significantly stimulated the enzymatic levels of SFE in HF. CONCLUSIONS: The sum of these findings suggests that IL-1α and GM-CSF are intrinsic cytokines secreted by UVB-exposed HK that stimulate expression of SFE by HF, leading to UVB-induced wrinkle formation.
Subject(s)
Cytokines/metabolism , Fibroblasts/enzymology , Fibroblasts/radiation effects , Keratinocytes/radiation effects , Neprilysin/metabolism , Paracrine Communication/radiation effects , Ultraviolet Rays , Adult , Antibodies, Neutralizing , Cell Line , Cells, Cultured , Collagenases/genetics , Collagenases/metabolism , Culture Media, Conditioned/pharmacology , Dermis/cytology , Epidermal Cells , Female , Fibroblasts/drug effects , Gene Expression Regulation/drug effects , Gene Expression Regulation/radiation effects , Humans , Keratinocytes/drug effects , Keratinocytes/metabolism , Male , Neprilysin/genetics , Paracrine Communication/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
We recently reported that overexpression of the elastase NEP (neutral endopeptidase) by fibroblasts plays a pivotal role in the mechanism of UVB-induced skin wrinkling by degrading dermal elastic fibres. Since UVB does not penetrate to the dermis, we hypothesized that factors secreted by UVB-exposed keratinocytes in the epidermis trigger fibroblasts in the dermis to increase their expression of NEP which then degrades the elastic fibres. In the present study, we characterized the epithelial-mesenchymal interaction between keratinocytes and fibroblasts which leads to increased expression of NEP. Human fibroblasts co-cultured with UVB-exposed human keratinocytes in cell inserts significantly increased their expression of NEP at the transcriptional, translational and enzymatic levels. Neutralizing antibodies to IL (interleukin)-1α or GM-CSF (granulocyte/macrophage colony-stimulating factor) significantly abolished the increased expression of NEP at the enzymatic levels in human fibroblasts co-cultured with UVB-exposed human keratinocytes, whereas neutralizing antibodies to IL-6, IL-8 or TNFα (tumour necrosis factor α) had no such effect. The addition of IL-1α or GM-CSF, but not TNFα, IL-6 or IL-8, at concentrations ranging from 1 to 10 nM, significantly stimulated the expression of NEP in human fibroblasts at the transcriptional and translational levels. These findings suggest that IL-1α and GM-CSF are intrinsic cytokines secreted by UVB-exposed keratinocytes that stimulate expression of NEP by fibroblasts.
Subject(s)
Fibroblasts/enzymology , Keratinocytes/radiation effects , Neprilysin/genetics , Ultraviolet Rays , Up-Regulation/radiation effects , Cells, Cultured , Coculture Techniques , Cytokines/pharmacology , Cytokines/physiology , Epidermal Cells , Foreskin/cytology , Gene Expression , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/physiology , Humans , Interleukin-1alpha/metabolism , Interleukin-1alpha/pharmacology , Interleukin-1alpha/physiology , Keratinocytes/metabolism , Male , Neprilysin/metabolism , Paracrine CommunicationABSTRACT
Excessive proliferation of epidermal keratinocytes is a typical aspect of chronic skin diseases such as psoriasis. In the present study, the effect of phosphodiesterase 7A (PDE7A) inhibitor ASB16165 on proliferation of keratinocytes was investigated to examine the role of PDE7A in keratinocyte proliferation and the possible therapeutic relevance of PDE7A inhibition in psoriasis. Topical application of ASB16165 inhibited the increase of thickness of skin as well as epidermis in a skin inflammation model induced by repeated painting of 12-O-tetradecanoylphorbol-13-acetate (TPA) in a concentration-dependent manner. The ASB16165 treatment also suppressed the increase in the number of Ki67-positive keratinocytes in the model, showing the disturbance of keratinocyte proliferation by the treatment. In addition, both ASB16165 and dibutyryl cAMP significantly decreased the proliferation of human keratinocytes in vitro, suggesting that PDE7A participates in keratinocyte proliferation probably by controlling intracellular cAMP, while the contribution of other mechanism(s) is not completely denied. The findings in the present study indicate that the effect of ASB16165 on skin and epidermal hyperplasia in the TPA-induced skin inflammation is mediated, at least in part, by the inhibition of keratinocyte proliferation. The inhibitors for PDE7A including ASB16165 might be useful for the treatment of psoriasis.
Subject(s)
Cell Proliferation/drug effects , Cyclic Nucleotide Phosphodiesterases, Type 7/antagonists & inhibitors , Epidermis/drug effects , Keratinocytes/drug effects , Phosphodiesterase Inhibitors/pharmacology , Psoriasis/drug therapy , Pyrazoles/pharmacology , Skin/drug effects , Thiophenes/pharmacology , Administration, Cutaneous , Animals , Bucladesine/pharmacology , Cells, Cultured , Disease Models, Animal , Epidermis/pathology , Female , Humans , Keratinocytes/pathology , Mice , Mice, Inbred BALB C , Phosphodiesterase Inhibitors/administration & dosage , Psoriasis/chemically induced , Pyrazoles/administration & dosage , Skin/pathology , Tetradecanoylphorbol Acetate , Thiophenes/administration & dosageABSTRACT
Possible role of phosphodiesterase 7A (PDE7A) in skin inflammation was examined using ASB16165, a specific inhibitor for PDE7A. Epicutaneous application of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse ear resulted in induction of skin edema, and topical treatment with ASB16165 inhibited the induction of skin edema in a dose-dependent manner. The TPA challenge also increased the level of TNF-alpha at the application site, and the ASB16165 treatment reduced the TNF-alpha level in the skin. In addition, ASB16165 suppressed the production of TNF-alpha by human keratinocytes stimulated in vitro with TPA and calcium ionophore. Forskolin, an activator of adenylyl cyclase, as well as dibutyryl cAMP also showed inhibitory effect on the TNF-alpha production in the cells, suggesting involvement of cAMP in TNF-alpha generation. These results demonstrate that PDE7A might regulate TNF-alpha production in keratinocytes in a cAMP-dependent fashion. As immunostaining analysis revealed that PDE7A is expressed in the epidermis and TNF-alpha is known to contribute to the TPA-induced edema, it is possible that the inhibitory effect of ASB16165 on skin edema in mouse TPA-induced dermatitis model is mediated by suppression of TNF-alpha production. This is the first report suggesting the association of PDE7A with the function of keratinocytes. ASB16165 will be useful as an agent for skin inflammation in which TNF-alpha plays a pathogenic role (e.g. psoriasis).