ABSTRACT
Early kinetics of lymphocyte subsets involved in tolerance and rejection following heart transplantation (HTx) are barely defined. Here, we aimed to delineate the early alloimmune response immediately after HTx. Therefore, blood samples from 23 heart-transplanted patients were collected before (pre-), immediately (T0), 24 hours (T24), and 3 weeks (3 wks) after HTx. Immunophenotyping was performed using flow cytometry. A significant increase was detected for terminally differentiated (TEMRA) CD4+ or CD8+ T cells and CD56dim CD16+ NK cells immediately after HTx linked to a decrease in naïve CD8+ and CM CD4+ T as well as CD56bright CD16- NK cells, returning to baseline levels at T24. More detailed analyses revealed increased CD69+ CD25- and diminished CD69- CD25- CD4+ or CD8+ T-cell proportions at T0 associated with decreasing S1PR1 expression. Passenger T and NK cells were found at low frequencies only in several patients at T0 and did not correlate with lymphocyte alterations. Collectively, these results suggest an immediate, transient shift toward memory T and NK cells following HTx. Opposite migratory properties of naïve versus memory T and NK cells occurring in the early phase after HTx could underlie these observations and may impinge on the development of allo-specific immune responses.
Subject(s)
CD8-Positive T-Lymphocytes , Heart Transplantation , Humans , Killer Cells, Natural , Lymphocyte Subsets , Immunophenotyping , CD56 Antigen/metabolismABSTRACT
Structure-based design was utilized to optimize 6,6-diaryl substituted dihydropyrone and hydroxylactam to obtain inhibitors of lactate dehydrogenase (LDH) with low nanomolar biochemical and single-digit micromolar cellular potencies. Surprisingly the replacement of a phenyl with a pyridyl moiety in the chemical structure revealed a new binding mode for the inhibitors with subtle conformational change of the LDHA active site. This led to the identification of a potent, cell-active hydroxylactam inhibitor exhibiting an in vivo pharmacokinetic profile suitable for mouse tumor xenograft study.
Subject(s)
Enzyme Inhibitors/pharmacology , L-Lactate Dehydrogenase/antagonists & inhibitors , Lactams/pharmacology , Animals , Cell Line , Dogs , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Humans , L-Lactate Dehydrogenase/metabolism , Lactams/chemistry , Mice , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Molecular Structure , Structure-Activity RelationshipABSTRACT
Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.
Subject(s)
Cell Plasticity/drug effects , Enzyme Inhibitors/pharmacology , L-Lactate Dehydrogenase/antagonists & inhibitors , Pyridones/pharmacology , Thiophenes/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Humans , L-Lactate Dehydrogenase/metabolism , Models, Molecular , Molecular Structure , Pyridones/chemistry , Structure-Activity Relationship , Thiophenes/chemistryABSTRACT
Introduction: For end-stage lung diseases, double lung transplantation (DLTx) is the ultimate curative treatment option. However, acute and chronic rejection and chronic dysfunction are major limitations in thoracic transplantation medicine. Thus, a better understanding of the contribution of immune responses early after DLTx is urgently needed. Passenger cells, derived from donor lungs and migrating into the recipient periphery, are comprised primarily by NK and T cells. Here, we aimed at characterizing the expression of killer cell immunoglobulin-like receptors (KIR) on donor and recipient NK and T cells in recipient blood after DLTx. Furthermore, we investigated the functional status and capacity of donor vs. recipient NK cells. Methods: Peripheral blood samples of 51 DLTx recipients were analyzed pre Tx and at T0, T24 and 3wk post Tx for the presence of HLA-mismatched donor NK and T cells, their KIR repertoire as well as activation status using flow cytometry. Results: Within the first 3 weeks after DLTx, donor NK and T cells were detected in all patients with a peak at T0. An increase of the KIR2DL/S1-positive subset was found within the donor NK cell repertoire. Moreover, donor NK cells showed significantly higher frequencies of KIR2DL/S1-positive cells (p<0.01) 3wk post DLTx compared to recipient NK cells. This effect was also observed in donor KIR+ T cells 3wk after DLTx with higher proportions of KIR2DL/S1 (p<0.05) and KIR3DL/S1 (p<0.01) positive T cells. Higher activation levels of donor NK and T cells (p<0.001) were detected compared to recipient cells via CD25 expression as well as a higher degranulation capacity upon activation by K562 target cells. Conclusion: Higher frequencies of donor NK and T cells expressing KIR compared to recipient NK and T cells argue for their origin in the lung as a part of a highly specialized immunocompetent compartment. Despite KIR expression, higher activation levels of donor NK and T cells in the periphery of recipients suggest their pre-activation during the ex situ phase. Taken together, donor NK and T cells are likely to have a regulatory effect in the balance between tolerance and rejection and, hence, graft survival after DLTx.
Subject(s)
Killer Cells, Natural/immunology , Lung Transplantation , Lung/immunology , Receptors, KIR/blood , T-Lymphocytes/immunology , Adult , Cell Degranulation , Coculture Techniques , Cytotoxicity, Immunologic , Female , Flow Cytometry , Humans , Immunophenotyping , Interleukin-2 Receptor alpha Subunit/blood , K562 Cells , Killer Cells, Natural/metabolism , Lung/metabolism , Lung Transplantation/adverse effects , Male , Middle Aged , Phenotype , Receptors, KIR2DL3/blood , Receptors, KIR3DL1/blood , T-Lymphocytes/metabolism , Time Factors , Treatment OutcomeABSTRACT
The effect of anti-angiogenic agents on tumor oxygenation has been in question for a number of years, where both increases and decreases in tumor pO2 have been observed. This dichotomy in results may be explained by the role of vessel normalization in the response of tumors to anti-angiogenic therapy, where anti-angiogenic therapies may initially improve both the structure and the function of tumor vessels, but more sustained or potent anti-angiogenic treatments will produce an anti-vascular response, producing a more hypoxic environment. The first goal of this study was to employ multispectral (MS) 19F-MRI to noninvasively quantify viable tumor pO2 and evaluate the ability of a high dose of an antibody to vascular endothelial growth factor (VEGF) to produce a strong and prolonged anti-vascular response that results in significant tumor hypoxia. The second goal of this study was to target the anti-VEGF induced hypoxic tumor micro-environment with an agent, tirapazamine (TPZ), which has been designed to target hypoxic regions of tumors. These goals have been successfully met, where an antibody that blocks both murine and human VEGF-A (B20.4.1.1) was found by MS 19F-MRI to produce a strong anti-vascular response and reduce viable tumor pO2 in an HM-7 xenograft model. TPZ was then employed to target the anti-VEGF-induced hypoxic region. The combination of anti-VEGF and TPZ strongly suppressed HM-7 tumor growth and was superior to control and both monotherapies. This study provides evidence that clinical trials combining anti-vascular agents with hypoxia-activated prodrugs should be considered to improved efficacy in cancer patients.
Subject(s)
Fluorine-19 Magnetic Resonance Imaging/methods , Tumor Hypoxia/physiology , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Survival/drug effects , Cell Survival/physiology , Female , HT29 Cells , Humans , Mice , Mice, Nude , Tirapazamine , Triazines/pharmacology , Triazines/therapeutic use , Tumor Hypoxia/drug effects , Vascular Endothelial Growth Factor A/metabolism , Xenograft Model Antitumor Assays/methodsABSTRACT
A series of trisubstituted hydroxylactams was identified as potent enzymatic and cellular inhibitors of human lactate dehydrogenase A. Utilizing structure-based design and physical property optimization, multiple inhibitors were discovered with <10 µM lactate IC50 in a MiaPaca2 cell line. Optimization of the series led to 29, a potent cell active molecule (MiaPaca2 IC50 = 0.67 µM) that also possessed good exposure when dosed orally to mice.
ABSTRACT
OBJECTIVE: Experiments on human erythropoietin (EPO) demonstrated that there is a direct relationship between the sialic acid-containing carbohydrate content of EPO, its circulating half-life, and in vivo bioactivity. This led to the hypothesis that an EPO analogue engineered to contain additional oligosaccharide chains would have enhanced biological activity. Darbepoetin alfa, a hyperglycosylated recombinant human EPO (rHuEPO) analogue with two extra carbohydrate chains, was designed and developed to test this hypothesis. MATERIALS AND METHODS: Comparative pharmacokinetic and pharmacodynamic studies and biochemical analyses of darbepoetin alfa and rHuEPO were performed to define the consequences of the increased carbohydrate content. RESULTS: Due to its increased sialic acid-containing carbohydrate content, darbepoetin alfa has a higher molecular weight, a greater negative charge, and a approximately fourfold lower EPO receptor binding activity than rHuEPO. It also has a threefold longer circulating half-life than rHuEPO in rats and dogs. In spite of its lower receptor binding, and perhaps counterintuitively, darbepoetin alfa is significantly more potent in vivo than rHuEPO. Due to the pharmacokinetic differences, the relative potency of the two molecules varies as a function of the dosing frequency. Darbepoetin alfa is 3.6-fold more potent than rHuEPO in increasing the hematocrit of normal mice when each is administered thrice weekly, but when the administration frequency is reduced to once weekly, darbepoetin alfa is approximately 13-fold to 14-fold more potent than rHuEPO. CONCLUSIONS: Increasing the sialic acid-containing carbohydrate content beyond the maximum found in EPO leads to a molecule with a longer circulating half-life and thereby an increased in vivo potency that can be administered less frequently.
Subject(s)
Erythropoietin/analogs & derivatives , Erythropoietin/pharmacokinetics , Animals , Carbohydrates/analysis , Darbepoetin alfa , Dogs , Dose-Response Relationship, Drug , Electrochemistry , Electrophoresis, Polyacrylamide Gel , Erythropoietin/metabolism , Erythropoietin/pharmacology , Half-Life , Hematocrit , Humans , Kinetics , Male , Mice , Molecular Weight , N-Acetylneuraminic Acid/analysis , Rats , Rats, Sprague-Dawley , Receptors, Erythropoietin/metabolism , Recombinant ProteinsABSTRACT
This communication highlights the development of a nicotinamide series of histone deacetylase inhibitors within the benzamide structural class. Extensive exploration around the nicotinamide core led to the discovery of a class I selective HDAC inhibitor that possesses excellent intrinsic and cell-based potency, acceptable ancillary pharmacology, favorable pharmacokinetics, sustained pharmacodynamics in vitro, and achieves in vivo efficacy in an HCT116 xenograft model.