Structure-Activity Relationships of Flupirtine Analogues for Liver Esterase-Mediated Cleavage of the 4-Fluorobenzylamine Moiety and Its Possible Relevance to Liver Toxicity.

Flupirtine and retigabine were essential drugs to combat pain and epilepsy. However, the Kv 7 potassium channel openers are fraught with hepatotoxicity and tissue discoloration, respectively, limiting their therapeutic value. Both adverse events are likely due to reactive metabolites arising from oxidative metabolism. Designing safer analogues lacking the structural elements leading to described side effects is an active area of current research. One of the main metabolites of flupirtine is the biologically inactive 4-fluorohippuric acid. Hitherto unexplained, the proposed metabolic pathway leading to the formation of 4-fluorohippuric acid from flupirtine is verified here. Through the use of eighteen flupirtine analogues, mechanistic details of this pathway could be elucidated. A possible connection with the in vitro hepatotoxicity of the flupirtine analogues and the levels of 4-fluorobenzoic acid formed in enzyme incubations was examined by correlation analysis. These findings provide important information for the design of new flupirtine analogues as potential drug candidates.

Adult-onset CNS myelin sulfatide deficiency is sufficient to cause Alzheimer's disease-like neuroinflammation and cognitive impairment.

BACKGROUND: Human genetic association studies point to immune response and lipid metabolism, in addition to amyloid-beta (Aß) and tau, as major pathways in Alzheimer's disease (AD) etiology. Accumulating evidence suggests that chronic neuroinflammation, mainly mediated by microglia and astrocytes, plays a causative role in neurodegeneration in AD. Our group and others have reported early and dramatic losses of brain sulfatide in AD cases and animal models that are mediated by ApoE in an isoform-dependent manner and accelerated by Aß accumulation. To date, it remains unclear if changes in specific brain lipids are sufficient to drive AD-related pathology. METHODS: To study the consequences of CNS sulfatide deficiency and gain insights into the underlying mechanisms, we developed a novel mouse model of adult-onset myelin sulfatide deficiency, i.e., tamoxifen-inducible myelinating glia-specific cerebroside sulfotransferase (CST) conditional knockout mice (CSTfl/fl/Plp1-CreERT), took advantage of constitutive CST knockout mice (CST-/-), and generated CST/ApoE double knockout mice (CST-/-/ApoE-/-), and assessed these mice using a broad range of methodologies including lipidomics, RNA profiling, behavioral testing, PLX3397-mediated microglia depletion, mass spectrometry (MS) imaging, immunofluorescence, electron microscopy, and Western blot. RESULTS: We found that mild central nervous system (CNS) sulfatide losses within myelinating cells are sufficient to activate disease-associated microglia and astrocytes, and to increase the expression of AD risk genes (e.g., Apoe, Trem2, Cd33, and Mmp12), as well as previously established causal regulators of the immune/microglia network in late-onset AD (e.g., Tyrobp, Dock, and Fcerg1), leading to chronic AD-like neuroinflammation and mild cognitive impairment. Notably, neuroinflammation and mild cognitive impairment showed gender differences, being more pronounced in females than males. Subsequent mechanistic studies demonstrated that although CNS sulfatide losses led to ApoE upregulation, genetically-induced myelin sulfatide deficiency led to neuroinflammation independently of ApoE. These results, together with our previous studies (sulfatide deficiency in the context of AD is mediated by ApoE and accelerated by Aß accumulation) placed both Aß and ApoE upstream of sulfatide deficiency-induced neuroinflammation, and suggested a positive feedback loop where sulfatide losses may be amplified by increased ApoE expression. We also demonstrated that CNS sulfatide deficiency-induced astrogliosis and ApoE upregulation are not secondary to microgliosis, and that astrogliosis and microgliosis seem to be driven by activation of STAT3 and PU.1/Spi1 transcription factors, respectively. CONCLUSION: Our results strongly suggest that sulfatide deficiency is an important contributor and driver of neuroinflammation and mild cognitive impairment in AD pathology.

Discovery and Toxicological Profiling of Aminopyridines as Orally Bioavailable Selective Inhibitors of PI3-Kinase γ.

Using a novel physiologically relevant in vitro human whole blood neutrophil shape change assay, an aminopyrazine series of selective PI3Kγ inhibitors was identified and prioritized for further optimization. Severe solubility limitations associated with the series leading to low oral bioavailability and poor exposures, especially at higher doses, were overcome by moving to an aminopyridine core. Compound 33, with the optimal balance of on-target activity, selectivity, and pharmacokinetic parameters, progressed into in vivo studies and demonstrated good efficacy (10 mg/kg) in a rat model of airway inflammation. Sufficient exposures were achieved at high doses to support toxicological studies, where unexpected inflammatory cell infiltrates in cardiovascular tissue prevented further compound development.

Microglia Depletion-Induced Remodeling of Extracellular Matrix and Excitatory Synapses in the Hippocampus of Adult Mice.

The extracellular matrix (ECM) plays a key role in synaptogenesis and the regulation of synaptic functions in the central nervous system. Recent studies revealed that in addition to dopaminergic and serotoninergic neuromodulatory systems, microglia also contribute to the regulation of ECM remodeling. In the present work, we investigated the physiological role of microglia in the remodeling of perineuronal nets (PNNs), predominantly associated with parvalbumin-immunopositive (PV+) interneurons, and the perisynaptic ECM around pyramidal neurons in the hippocampus. Adult mice were treated with PLX3397 (pexidartinib), as the inhibitor of colony-stimulating factor 1 receptor (CSF1-R), to deplete microglia. Then, confocal analysis of the ECM and synapses was performed. Although the elimination of microglia did not alter the overall number or intensity of PNNs in the CA1 region of the hippocampus, it decreased the size of PNN holes and elevated the expression of the surrounding ECM. In the neuropil area in the CA1 str. radiatum, the depletion of microglia increased the expression of perisynaptic ECM proteoglycan brevican, which was accompanied by the elevated expression of presynaptic marker vGluT1 and the increased density of dendritic spines. Thus, microglia regulate the homeostasis of pre- and postsynaptic excitatory terminals and the surrounding perisynaptic ECM as well as the fine structure of PNNs enveloping perisomatic-predominantly GABAergic-synapses.

Mitochondria of teleost radial glia: A novel target of neuroendocrine disruption by environmental chemicals?

In teleost fish, radial glial cells (RGCs) are progenitor cells for neurons and the major cell type synthesizing neuroestrogens. We hypothesized that chemical exposure impairs mitochondrial bioenergetics of RGCs, which then may lead to downstream consequences for neuroestrogen production. Here we provide proof of concept that mitochondria of RGCs can be perturbed by fungicides. We isolated RGCs from a mixed sex population of goldfish (Carassius auratus) and measured metabolic capacity of primary cells to a model mitotoxin fluazinam, a broad-spectrum fungicide that inhibits mitochondria electron transport chain (or ETC) Complex I. Using immunocytochemistry and real-time PCR, we demonstrate that the goldfish primary cell cultures are highly enriched for glia after multiple passages. Cytotoxicity assays revealed that glia treated with >25 µM fluazinam for 24 and 48-h showed reduced viability. As such, metabolic assays were conducted with non-cytotoxic concentrations (0.25-12.5 µM). Fluazinam did not affect oxygen consumption rates of RGCs at 24 h, but after 48 h, oligomycin induced ATP-linked respiration was decreased by both 6.25 and 12.5 µM fluazinam. Moreover, concentrations as low as 0.25 µM disrupted the mitochondrial membrane potential of RGCs, reflecting strong uncoupling effects of the fungicide on mitochondria. Here we provide proof of concept that mitochondrial bioenergetics of teleostean RGCs can be responsive to agrochemicals. Additional studies are required to address low-dose exposures in vivo and to determine if metabolic disruption impairs neuroendocrine functions of RGCs. We propose this mechanism constitutes a novel aspect of neuroendocrine disruption, significant because dysregulation of neuron-glia communication is expected to contribute to neuroendocrine disruption.

JTP-117968, a novel selective glucocorticoid receptor modulator, exhibits significant anti-inflammatory effect while maintaining bone mineral density in mice.

Classic glucocorticoids have been prescribed for various inflammatory diseases, such as rheumatoid arthritis, due to their outstanding anti-inflammatory effects. However, glucocorticoids cause numerous unwanted side effects, including osteoporosis and diabetes. Hence, selective glucocorticoid receptor modulators (SGRMs), which retain anti-inflammatory effects with minimized side effects, are among the most anticipated drugs in the clinical field. The assumption is that there are two major mechanisms of action via glucocorticoid receptors, transrepression (TR) and transactivation (TA). In general, anti-inflammatory effects of glucocorticoids are largely due to TR, while the side effects associated with glucocorticoids are mostly mediated through TA. We previously reported that JTP-117968, a novel SGRM, maintained partial TR activity while remarkably reducing the TA activity. In this study, we investigated the anti-inflammatory effect of JTP-117968 on a lipopolysaccharide (LPS) challenge model and collagen-induced arthritis (CIA) model in mice. Meanwhile, we tested the effect of JTP-117968 on the bone mineral density (BMD) in mouse femur to evaluate the side effect. Based on the evaluation, JTP-117968 reduced the plasma levels of tumor necrosis factor α induced by LPS challenge in mice significantly. Remarkably, CIA development was suppressed by JTP-117968 comparably with prednisolone and PF-802, an active form of fosdagrocorat that has been developed clinically as an orally available SGRM. Strikingly, the side effect of JTP-117968 on mouse femoral BMD was much lower than those of PF-802 and prednisolone. Therefore, JTP-117968 has attractive potential as a new therapeutic option against inflammatory diseases with minimized side effects compared to classic glucocorticoids.

Safety Assessment of 2-Amino-3-Hydroxypyridine as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the safety of 2-Amino-3-Hydroxypyridine, which is reported to function as an oxidative hair dye ingredient. The Panel reviewed relevant animal and human data provided in this safety assessment, and concluded that 2-Amino-3-Hydroxypyridine is safe in the present practices of use and concentration for use in oxidative hair dye formulations.

Drug efficacy and toxicity prediction: an innovative application of transcriptomic data.

Drug toxicity and efficacy are difficult to predict partly because they are both poorly defined, which I aim to remedy here from a transcriptomic perspective. There are two major categories of drugs: (1) restorative drugs aiming to restore an abnormal cell, tissue, or organ to normal function (e.g., restoring normal membrane function of epithelial cells in cystic fibrosis), and (2) disruptive drugs aiming to kill pathogens or malignant cells. These two types of drugs require different definition of efficacy and toxicity. I outlined rationales for defining transcriptomic efficacy and toxicity and illustrated numerically their application with two sets of transcriptomic data, one for restorative drugs (treating cystic fibrosis with lumacaftor/ivacaftor aiming to restore the cellular function of epithelial cells) and the other for disruptive drugs (treating acute myeloid leukemia with prexasertib). The conceptual framework presented will help and sensitize researchers to collect data required for determining drug toxicity.

Time of flight mass spectrometry based in vitro and in vivo metabolite profiling of ribociclib and their toxicity prediction.

The metabolic investigation in the drug discovery process is an imperative aspect for selection of drug candidates with excellent therapeutic efficacy and safety profile. Ribociclib (RIBO), an orally active Cyclin dependent kinases inhibitor recently approved by USFDA for its clinical efficacy against human epithelial growth factor receptor negative and hormonal receptor positive advanced breast cancer. Although an in vitro metabolite identification study of RIBO is available in literature, no systematic metabolic investigation including detailed structural characterization and toxicity prediction of the metabolites generated in in vivo system is reported till date. Therefore, in this study, we focused on the characterization of its entire metabolites generated in in vitro as well as in vivo matrices. In vitro study includes incubation of RIBO in rat and human liver microsomes and human S9 fraction, while in vivo study was carried out using plasma, urine and faeces samples of male Sprague Dawley rats. A total of 22 metabolites were successfully separated on Agilent SB C18 (100 × 4.6 mm, 2.7µ) column using ammonium formate (pH 3.5) and acetonitrile as mobile phase. Metabolites were identified with the help of UHPLC-ESI-Q-TOF-MS/MS by accurate mass measurement. RIBO was found to be metabolised by N- dealkylation, sulphation, acetylation, oxidation, hydroxylation, carbonylation, dehydrogenation and by a combination of these reactions. The in silico toxicity profiling of all the metabolites was carried out with the help of ProTox-II software. Ten out of twenty two newly identified metabolites showed to have potential for possessing immunotoxicity. Novelty of this investigation can be justified by the unavailability of any previously published literature on complete in vitro and in vivo metabolite profiling of RIBO. Moreover, in silico toxicity of the metabolites were also not known till date.

Mutant prevention concentration of ozenoxacin for quinolone-susceptible or -resistant Staphylococcus aureus and Staphylococcus epidermidis.

Ozenoxacin (OZN) belongs to a new generation of non-fluorinated quinolones for the topical treatment of skin infections which has shown to be effective in the treatment of susceptible and resistant Gram-positive cocci. The mutant prevention concentration (MPC) of ozenoxacin, levofloxacin and ciprofloxacin was determined in quinolone-susceptible and -resistant strains including methicillin-susceptible S. aureus, methicillin-resistant S. aureus, methicillin-susceptible S. epidermidis and methicillin-resistant S. epidermidis with different profile of mutation in the quinolone resistance determining regions (QRDR). The MPC value of OZN for the methicillin-susceptible S. aureus strain susceptible to quinolones, without mutations in QRDR, was 0.05 mg/L, being 280-fold lower than that observed with ciprofloxacin and levofloxacin. In methicillin-susceptible and-resistant S. aureus strains with mutations in the gyrA or/and grlA genes the MPC of OZN went from 0.1 to 6 mg/L, whereas the MPC of levofloxacin and ciprofloxacin was > 50 mg/L for the same strains. For methicillin-susceptible and-resistant S. epidermidis the results were similar to those abovementioned for S. aureus. According to our results, the MPC of OZN was far below the quantity of ozenoxacin achieved in the epidermal layer, suggesting that the in vivo selection of mutants, if it occurs, will take place at low frequency. Ozenoxacin is an excellent candidate for the treatment of bacterial infections caused by susceptible and quinolone-resistant staphylococci isolated usually from skin infections.

PI3K/AKT inhibitors aggravate death receptor-mediated hepatocyte apoptosis and liver injury.

The PI3K/AKT signaling pathway is one of the most frequently activated signaling networks in human cancers and has become a valuable target in anticancer therapy. However, accumulating reports suggest that adverse effects such as severe liver injury and inflammation may accompany treatment with pan-PI3K and pan-AKT inhibitors. Our prior work has demonstrated that activation of the PI3K/AKT pathway has a protective role in Fas- or TNFα-induced hepatocytic cell death and liver injury. We postulated that PI3K or AKT inhibitors may exacerbate liver damage via the death factor-mediated hepatocyte apoptosis. In this study we found that several drugs targeting PI3K/AKT either clinically used or in clinical trials sensitized hepatocytes to agonistic anti-Fas antibody- or TNFα-induced apoptosis and significantly shortened the survival of mice in in vivo liver damage models. The PI3K or AKT inhibitors promoted Fas aggregation, inhibited the expression of cellular FLICE-inhibitory protein S and L (FLIPL/S), and enhanced procaspase-8 activation. Conversely, cotreatment with the AKT specific activator SC79 reversed these effects. Taken together, these findings suggest that PI3K or AKT inhibitors may render hepatocytes hypersensitive to Fas- or TNFα-induced apoptosis and liver injury.

Concurrent radiotherapy with palbociclib or ribociclib for metastatic breast cancer patients: Preliminary assessment of toxicity.

OBJECTIVE: To evaluate the early toxicity of concurrent use of radiotherapy in association with CDK4/6 inhibitors (palbociclib or ribociclib) in patients with hormone-receptors positive metastatic breast cancer. MATERIAL AND METHODS: Records of patients with histologically proven metastatic or locally advanced breast cancer treated in our institution were reviewed. Patients who received radiotherapy and concurrent palbociclib or ribociclib were selected. Toxicity was assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0 (NCI-CTCAE V4.0). RESULTS: Sixteen consecutive metastatic breast cancer patients with 24 radiotherapy treatments were studied. Thirteen patients (81.3%) received palbociclib, 3 (18.7%) patients received ribociclib concurrently with RT (18 and 5 radiotherapy courses respectively). The majority of patients (68.7%) received palliative radiotherapy to the bones (median dose 30 Gy, range 8-36 Gy). Five patients (31.2%) were treated in oligo-metastatic or oligo-progressive sites of disease with higher doses (median dose = 50 Gy, range 39.6-60 Gy). The most common toxicity observed was hematological toxicity. Neutropenia was common (grade 2 = 12.5%; grade 3 = 25%, grade 4 = 6.3%); 60% of patients experiencing grade ≥ 3 neutropenia had already experienced neutropenia during previous cycles of palbociclib. One patient (6.3%) completed the RT course earlier (48 Gy of 50 Gy prescribed) and another patient (6.3%) suspended RT for 2 days. CONCLUSION: concomitant treatment of CDK4/6 and radiotherapy seems well tolerated; high grade hematological toxicity is common, but did not change treatment course in the majority of patients. Previous toxicity should be carefully evaluated as it usually reoccurs.

In vitro approach to elucidate the relevance of carboxylesterase 2 and N-acetyltransferase 2 to flupirtine-induced liver injury.

The use of flupirtine, an analgesic, has been restricted in European countries because it causes liver injury in rare cases. Flupirtine is primarily metabolized to D-13223, an acetylamino form. In the process of D-13223 formation, it has been hypothesized that a reactive metabolite is formed which may be involved in flupirtine hepatotoxicity. The purpose of this study was to identify the potential reactive metabolite and the responsible enzymes in the human liver to get a clue to the mechanism of hepatotoxicity. Using recombinant enzymes, we found that D-13223 was formed from flupirtine via hydrolysis by carboxylesterase 2 (CES2) and subsequent acetylation by N-acetyltransferase (NAT) 2. A conjugate of N-acetyl-l-cysteine (NAC), a nucleophile, was detected by incubation of flupirtine with CES2, and the conjugate formation in human liver microsomes was inhibited by CES2 inhibitors, indicating that a reactive metabolite, which may be a quinone diimine, was produced in the process of CES2-mediated hydrolysis of flupirtine. The formation of the NAC conjugate in liver S9 samples from NAT2 slow acetylators was significantly higher than that from NAT2 rapid/intermediate acetylators, indicating that NAT2 could function as a detoxification enzyme for flupirtine. CES2-overexpressing HepG2 cells showed remarkable lactate dehydrogenase leakage under flupirtine treatment, while no cytotoxicity was observed in control cells, suggesting that the reactive metabolite formed by CES2-mediated hydrolysis of flupirtine would be a trigger of hepatotoxicity. NAT2 slow acetylators with high CES2 activity could be highly susceptible to flupirtine-induced liver injury.

Studies on articular and general toxicity of orally administered ozenoxacin in juvenile rats and dogs.

AIM: Ozenoxacin is a nonfluorinated quinolone antibacterial approved for topical treatment of impetigo. Because quinolones have known chondrotoxic effects in juvenile animals, the potential toxicity of ozenoxacin was assessed in preclinical studies. MATERIALS & METHODS: Ozenoxacin or ofloxacin (300 mg/kg/day for 5 days, for each compound) was orally administered to juvenile rats, and oral ozenoxacin (10-100 mg/kg/day for 14 days) was administered to juvenile dogs. RESULTS: In juvenile rats, ozenoxacin showed no chondrotoxicity, whereas ofloxacin produced typical quinolone-induced lesions in articular cartilage in three of ten rats. Oral ozenoxacin administration to juvenile dogs showed no chondrotoxicity or toxicologically relevant findings in selected target organs. CONCLUSION: Ozenoxacin was generally well-tolerated in juvenile rats and dogs, with no evidence of quinolone-induced arthropathy.

Flupirtine drug-induced liver injury in a patient developing acute liver failure.

A patient is admitted with complaints of recent onset nausea, discomfort, jaundice and blood tests that reveal severe hepatitis. At the time, she had been taking medication with Hypericum perforatum (St John's wort) for 6 months, and 6 weeks before this event, she took flupirtine maleate. A few days after being admitted, she developed encephalopathy progressing to acute liver failure (ALF) requiring unsuccessful liver transplantation. The patient was ultimately diagnosed with drug-induced liver injury (DILI). In this context, while H. perforatum could interfere with other medication or trigger DILI itself, flupirtine appears to have triggered the DILI, given its liver toxicity capacity. DILI is one of the major ALF causes and can jeopardise patient's life. Accordingly, all efforts to reduce medication potentially hazardous to the liver are recommended.

A Phase I Trial of the PI3K Inhibitor Buparlisib Combined With Capecitabine in Patients With Metastatic Breast Cancer.

BACKGROUND: Buparlisib is an oral pan-class I phosphotidyinositol-3-kinase (PI3K) inhibitor. The present phase I study evaluated the safety, pharmacokinetics, and efficacy of buparlisib with capecitabine in patients with metastatic breast cancer. PATIENTS AND METHODS: Patients received buparlisib once daily (range, 50 to 100 mg) for 3 weeks with capecitabine twice daily (range, 1000 to 1250 mg/m2) for 2 weeks with a 1-week break. Dose escalation used a traditional "3 + 3" design with standard definitions of dose-limiting toxicity (DLT) and maximum tolerated dose. RESULTS: Of the 25 patients enrolled, 23 were evaluable for DLT and 17 were evaluable for response. The maximum tolerated dose of the combination was buparlisib 100 mg daily and capecitabine 1000 mg/m2 twice daily. DLTs included grade 3 hyperglycemia and grade 3 confusion. The most common grade 3 toxicities were diarrhea and elevation of aspartate aminotransferase and alanine transaminase. One patient exhibited a complete response to treatment and four had a confirmed partial response. In cohorts 3 and 4, in which the buparlisib dose remained constant but the capecitabine dose was increased, significant increases in the buparlisib plasma concentration were noted. CONCLUSION: The combination of buparlisib with capecitabine in patients with metastatic breast cancer was generally well-tolerated, with several patients demonstrating prolonged responses. Unexpectedly low rates of PIK3CA mutations (3 of 17) were seen, and only 2 of 7 tumors with subtyping were luminal, making exploration of these putative predictive markers impossible. Further study of the combination is not unreasonable, with expanded pharmacokinetics and sequencing analysis to better elucidate potential drug-drug interactions and more accurate predictive biomarkers of response.

Potentialization of N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) cytotoxic activity by 2-(1-adamantylamino)-6-methylpyridine (AdAMP) in human ovarian cancer cells.

OBJECTIVES: TNF is one of the key cytokines involved in cancer development. TNF signaling can result in both stimulating and inhibitory signals that can result in opposite biological effects in cancerogenesis. 2-(1-adamantylamino)-6-methylpyridine (AdAMP) enhances TNF secretion whereas N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) is a NF-κB inhibitor potentially stimulating proapoptotic TNF signals. The aim of the study was to assess the effect of TPCK in combination with AdAMP on human ovarian cells. MATERIAL AND METHODS: CAOV-1 human ovarian cell line was incubated with TPCK and AdAMP for 24 hours. The cytotoxic effect was evaluated in a crystal violet assay. A monoclonal antibody against TNF, Infliximab, was added to examine the possible mechanism of interactions. RESULTS: Depending on concentration, AdAMP potentialized cytotoxic activity of TPCK or had a synergistic effect with TPCK. Infliximab did not reverse cytotoxicity of AdAMP and TPCK and in some cytotoxic and non-cytotoxic concentrations even enhanced their cytotoxicity. CONCLUSIONS: AdAMP and TPCK cytotoxicity seems to be dependent on TNF signaling, however, the exact mechanism of interactions remains unclear.

Discovery of wt RET and V804M RET Inhibitors: From Hit to Lead.

Oncogenic activation of RET kinase has been found in several neoplastic diseases, like medullary thyroid carcinoma, multiple endocrine neoplasia, papillary thyroid carcinoma, and non-small-cell lung cancer. Currently approved RET inhibitors were not originally designed to be RET inhibitors, and their potency against RET kinase has not been optimized. Hence, novel compounds able to inhibit both wild-type RET (wt RET) and its mutants (e.g., V804M RET) are needed. Herein we present the development and the preliminary evaluation of a new sub-micromolar wt RET/V804M RET inhibitor, N-(2-fluoro-5-trifluoromethylphenyl)-N'-{4'-[(2''-benzamido)pyridin-4''-ylamino]phenyl}urea (69), endowed with a 4-anilinopyridine structure, starting from our previously identified 4-anilinopyrimidine hit compound. Profiling against a panel of kinases indicated 69 as a multi cKIT/wt RET/V804M RET inhibitor.

Comparison of cell-based assays for the identification and evaluation of competitive CXCR4 inhibitors.

The chemokine receptor CXCR4 is activated by its unique chemokine ligand CXCL12 and regulates many physiological and developmental processes such as hematopoietic cell trafficking. CXCR4 is also one of the main co-receptors for human immunodeficiency virus (HIV) entry. Dysfunction of the CXCL12/CXCR4 axis contributes to several human pathologies, including cancer and inflammatory diseases. Consequently, inhibition of CXCR4 activation is recognized as an attractive target for therapeutic intervention. In this regard, numerous agents modifying CXCR4 activity have been evaluated in in vitro experimental studies and pre-clinical models. Here, we evaluated a CXCL12 competition binding assay for its potential as a valuable initial screen for functional and competitive CXCR4 inhibitors. In total, 11 structurally diverse compounds were included in a side-by-side comparison of in vitro CXCR4 cell-based assays, such as CXCL12 competition binding, CXCL12-induced calcium signaling, CXCR4 internalization, CXCL12-guided cell migration and CXCR4-specific HIV-1 replication experiments. Our data indicated that agents that inhibit CXCL12 binding, i.e. the anti-CXCR4 peptide analogs T22, T140 and TC14012 and the small molecule antagonists AMD3100, AMD3465, AMD11070 and IT1t showed inhibitory activity with consistent relative potencies in all further applied CXCR4-related assays. Accordingly, agents exerting no or very weak receptor binding (i.e., CTCE-9908, WZ811, Me6TREN and gambogic acid) showed no or very poor anti-CXCR4 inhibitory activity. Thus, CXCL12 competition binding studies were proven to be highly valuable as an initial screening assay and indicative for the pharmacological and functional profile of competitive CXCR4 antagonists, which will help the design of new potent CXCR4 inhibitors.

Use of in silico models for prioritization of heat-induced food contaminants in mutagenicity and carcinogenicity testing.

Numerous Maillard reaction and lipid oxidation products are present in processed foods such as heated cereals, roasted meat, refined oils, coffee, and juices. Due to the lack of experimental toxicological data, risk assessment is hardly possible for most of these compounds. In the present study, an in silico approach was employed for the prediction of the toxicological endpoints mutagenicity and carcinogenicity on the basis of the structure of the respective compound, to examine (quantitative) structure-activity relationships for more than 800 compounds. Five software tools for mutagenicity prediction (T.E.S.T., SARpy, CAESAR, Benigni-Bossa, and LAZAR) and three carcinogenicity prediction tools (CAESAR, Benigni-Bossa, and LAZAR) were combined to yield so-called mutagenic or carcinogenic scores for every single substance. Alcohols, ketones, acids, lactones, and esters were predicted to be mutagenic and carcinogenic with low probability, whereas the software tools tended to predict a considerable mutagenic and carcinogenic potential for thiazoles. To verify the in silico predictions for the endpoint mutagenicity experimentally, twelve selected compounds were examined for their mutagenic potential using two different validated in vitro test systems, the bacterial reverse mutation assay (Ames test) and the in vitro micronucleus assay. There was a good correlation between the results of the Ames test and the in silico predictions. However, in the case of the micronucleus assay, at least three substances, 2-amino-6-methylpyridine, 6-heptenoic acid, and 2-methylphenol, were clearly positive although they were predicted to be non-mutagenic. Thus, software tools for mutagenicity prediction are suitable for prioritization among large numbers of substances, but these predictions still need experimental verification.