Reduced Cytochrome P-450 Research Articles

Discovery of VU6008677: A Structurally Distinct Tricyclic M4 Positive Allosteric Modulator with Improved CYP450 Profile.

This Letter details our efforts to develop novel tricyclic muscarinic acetylcholine receptor subtype 4 (M4) positive allosteric modulator (PAM) scaffolds with improved pharmacological properties. This endeavor involved a "tie-back" strategy to replace the 3-amino-5-chloro-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide core, which led to the discovery of two novel tricyclic cores: an 8-chloro-9-methylpyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amine core and 8-chloro-7,9-dimethylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-4-amine core. Both tricyclic cores displayed low nanomolar potency against human M4 and greatly reduced cytochrome P450 inhibition when compared with parent compound ML253.

Efficacy of prasugrel versus clopidogrel for early neurological deterioration in branch atheromatous disease: An exploratory study

AbstractBackgroundDual antiplatelet therapy (DAPT) with clopidogrel is one of the initial treatment regimens for early neurological deterioration (END) in branch atheromatous disease (BAD). However, its effectiveness is affected by cytochrome P450 2C19 polymorphism. Prasugrel, with reduced cytochrome P450 2C19 interactions, has been approved for cerebrovascular diseases in Japan.AimTo explore the efficacy of DAPT with prasugrel and with clopidogrel in preventing END.MethodsPatients with BAD, admitted within 48 h of symptom onset between June 2022 and September 2023, were enrolled. The primary endpoint was the proportion of patients with END within 7 days, which was defined as an increase of 1 point or more on the National Institutes of Health Stroke Scale (NIHSS). Patients meeting specific magnetic resonance imaging (MRI) criteria, including lesions of ≥15 mm in the lenticulostriate artery (LSA) region or infarcts extending to the ventral side of the paramedian pontine region, were included. The patients were randomly assigned to the clopidogrel with loading and prasugrel without loading groups and administered common medications.ResultsThe study included 9 and 10 patients in clopidogrel and prasugrel groups, of whom 56% and 50% had END, respectively. The prasugrel group had earlier exacerbations after admission (days 0–1) than that of the clopidogrel group (days 1–2). The bleeding complications of both groups were not significantly different.ConclusionThe two groups had similar proportions of patients with END, suggesting comparable efficacies of both drugs for patients with BAD.

Combination of Cysteine and Glutathione Prevents Ethanol-Induced Hangover and Liver Damage by Modulation of Nrf2 Signaling in HepG2 Cells and Mice.

Excessive alcohol consumption increases oxidative stress, leading to alcoholic liver disease. In this study, the protective effects of a mixture of cysteine and glutathione against ethanol-induced hangover and liver damage were evaluated in mice and HepG2 cells. Ethanol (2 mL/kg) was orally administered to the mice 30 min before receiving the test compounds (200 mg/kg), and the behavioral and oxidative stress-related biochemical parameters altered by ethanol were analyzed. Acute ethanol administration increased anxiety behavior and decreased balance coordination in mice (p < 0.001); however, a mixture of cysteine and glutathione (MIX) in a 3:1 ratio improved alcohol-induced behavior more effectively than the individual compounds (p < 0.001). The MIX group showed higher ethanol-metabolizing enzyme activity than the control group (p < 0.001) and significantly suppressed the elevation of serum alcohol (p < 0.01) and acetaldehyde (p < 0.001) levels after 1 h of ethanol administration. In HepG2 cells, 2.5 mM MIX accelerated ethanol metabolism and reduced cytochrome P450 2E1 mRNA expression (p < 0.001). MIX also increased the expression of antioxidant enzymes through the upregulation of nuclear erythroid 2-related factor 2 (Nrf2) signaling and consequently suppressed the overproduction of reactive oxygen species and malondialdehyde (p < 0.001). Collectively, MIX alleviates the hangover symptoms and attenuates the alcohol-induced oxidative stress by regulating the Nrf2 pathway.

Evaluation of effects of indoxyl sulfate and parathyroid hormone on CYP3A activity considering the influence of CYP3A5 gene polymorphisms.

Indoxyl sulfate and parathyroid hormone (PTH), which accumulate in chronic kidney disease (CKD), have been reported to reduce cytochrome P450(CYP)3A activity. Homozygotes of the CYP3A5*3 allele have reduced CYP3A5 activity compared to carriers of at least one CYP3A5*1 allele. 4β-Hydroxycholesterol (4β-OHC) has been established as an endogenous substrate reflecting CYP3A activity. 4β-OHC is produced through hydroxylation by CYP3A4 and CYP3A5 and by autoxidation of cholesterol, whereas 4α-hydroxycholesterol (4α-OHC) is produced solely by autoxidation of cholesterol. This study focused on CKD patients and evaluated the effects of plasma indoxyl sulfate and intact-PTH concentrations on plasma 4β-OHC concentration, 4β-OHC/total cholesterol ratio and 4β-OHC-4α-OHC, with consideration of the influence of CYP3A5 polymorphism. Sixty-three CKD patients were analysed and divided into CYP3A5 carrier group (n = 26) and non-carrier group (n = 37). Plasma indoxyl sulfate significantly correlated inversely with 4β-OHC concentration and with 4β-OHC-4α-OHC in both the CYP3A5*1 carrier group (r = -0.42, P = .034; r = -0.39, P = .050, respectively) and the non-carrier group (r = -0.45, P = .0054; r = -0.39, P = .019, respectively). However, multiple regression analysis did not identify plasma indoxyl sulfate concentration as a significant independent factor associated with any of the CYP3A activity indices. There was no significant correlation between plasma intact-PTH concentration and any of the CYP3A activity indices. The present results suggest that plasma indoxyl sulfate and intact-PTH concentrations do not have clinically significant effects on CYP3A activity in patients with CKD.

Design, synthesis, and pharmacological evaluation of N-(3-carbamoyl-1H-pyrazol-4-yl)-1,3-oxazole-4-carboxamide derivatives as interleukin-1 receptor-associated kinase 4 inhibitors with reduced potential for cytochrome P450 1A2 induction

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a critical molecule in Toll-like receptor/interleukin-1 receptor signaling and an attractive therapeutic target for a wide range of inflammatory and autoimmune diseases as well as cancers. In our search for novel IRAK4 inhibitors, we conducted structural modification of a thiazolecarboxamide derivative 1, a lead compound derived from high-throughput screening hits, to elucidate structure–activity relationship and improve drug metabolism and pharmacokinetic (DMPK) properties. First, conversion of the thiazole ring of 1 to an oxazole ring along with introduction of a methyl group at the 2-position of the pyridine ring aimed at reducing cytochrome P450 (CYP) inhibition were conducted to afford 16. Next, modification of the alkyl substituent at the 1-position of the pyrazole ring of 16 aimed at improving CYP1A2 induction properties revealed that branched alkyl and analogous substituents such as isobutyl (18) and (oxolan-3-yl)methyl (21), as well as six-membered saturated heterocyclic groups such as oxan-4-yl (2), piperidin-4-yl (24, 25), and dioxothian-4-y (26), are effective for reducing induction potential. Representative compound AS2444697 (2) exhibited potent IRAK4 inhibitory activity with an IC50 value of 20 nM and favorable DMPK properties such as low risk of drug–drug interactions mediated by CYPs as well as excellent metabolic stability and oral bioavailability.

Spontaneous Remission of Primary Aldosteronism with Mineralocorticoid Receptor Antagonist Therapy: A Review.

In this review, we describe previous basic and clinical studies on autonomous aldosterone production. Over the past decades, mineralocorticoid receptor antagonists (MRAs) have been found to concentration-dependently inhibit steroidogenesis in different degrees. However, many studies have proven the suppressive effects of MRAs on the activities of hormone synthase. The probable factors of cytochrome P-450 reduction, both in microsomes and mitochondria, have also been considered: (1) one of the spironolactone metabolite forms had destructive function, except canrenone, (2) 7α-thio-spironolactone was an obligatory intermediate in the spironolactone-induced CYP450 decrease, and (3) the contributing steroids should have 7α-methylthio or 7α-methylsulfone groups. In previous clinical research, spironolactone-body-containing cells showed a type II pattern of enzyme activity (i.e., enhanced 3β-hydroxysteroid dehydrogenase, glucose-6-phosphate, and NADP-isocitrate dehydrogenase activities and weaken succinate dehydrogenase activity), and the subcapsular micronodules composed of spironolactone-body-containing cells also exhibited a type II pattern and excess aldosterone secretion, indicating that the subcapsular micronodules might be the root of aldosterone-producing adenoma. Moreover, combined with the potential impeditive function to aldosterone secretion, a few cases of spontaneous remission of primary aldosteronism, with normal ranges of blood pressure, plasma potassium, plasma renin activity, and aldosterone renin ratio, have been reported after long-term treatment with MRAs.

WITHDRAWN: Anti-apoptotic and Anti-oxidative Effects of Aqueous Ex-tract Prepared from Steamed Daylily Flower on Normal Cardiac and Hepatic Cells against Ethanol

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In vivo evaluation of pharmacokinetic drug–drug interactions between fluorinated pyrimidine anticancer drugs, 5-fluorouracil and capecitabin, and an anticoagulant, warfarin

Warfarin is a common anticoagulant and has demonstrated interactions with several drugs. Among them, as a serious adverse event, a case of death due to the enhanced warfarin action owing to its combined use with a fluoropyrimidine anticancer drug has been reported, but the detailed mechanism has not been elucidated. Some reports have advocated that fluorinated pyrimidine anticancer drugs reduce cytochrome P450 2C9 expression, leading to the enhanced pharmacological effects of warfarin. The purpose of this study was to clarify the mechanisms of drug–drug interactions between warfarin and 5-fluorouracil (5-FU) and capecitabine in vivo using rats. Rats were administered warfarin in combination with 5-FU (15 mg/kg/d) or capecitabine (15 mg/kg/d) for 7 d. Prothrombin time (PT) and activated partial thromboplastin time were significantly prolonged in the warfarin plus 5-FU or capecitabine groups compared with those in the warfarin alone group. No significant difference was observed in the area under the plasma concentration-time curve of the warfarin alone group compared with the warfarin with 5-FU or capecitabine groups. These data suggest that the enhancement of warfarin efficacy caused by the combination of 5-FU or capecitabine was due to a pharmacological interaction rather than a pharmacokinetic interaction.

Physical Evidence of Oil Uptake and Toxicity Assessment of Amphiphilic Grafted Nanoparticles Used as Oil Dispersants

Herein, we report the toxicity evaluation of a new prototype dispersant system, silicon dioxide nanoparticles (NPs) functionalized with (3-glycidoxypropyl)triethoxysilane (GPS) and grafted poly(ε-caprolactone)-block-poly[oligo(ethylene glycol)methyl methacrylate mono-methyl ether] (NP-PCL-POEGMA). This serves as a follow up of our previous study where grafted silicon dioxide NPs functionalized with GPS and grafted hyperbranched poly(glycidol) (NP-HPG) were evaluated for reducing the toxicity in embryo, juvenile, and adult fish populations. In this study, the NP-HPG sample is used as a baseline to compare against the new NP-PCL-POEGMA samples. The relative size was established for three NP-PCL-POEGMA samples via cryogenic transmission electron microscopy. A quantitative mortality study determined that these NPs are non-toxic to embryo populations. An ethoxyresorufin-O-deethylase assay was performed on these NP-PCL-POEGMA samples to test for reduced cytochrome P450 1A after the embryos were exposed to the water-accommodated fraction of crude oil. Overall, these NP-PCL-POEGMA NPs better protected the embryo populations than the previous NP-HPG sample (using a protein activity end point), showing a trend in the right direction for prototype dispersants to replace the commercially utilized Corexit.

Roxadustat, a Hypoxia-Inducible Factor 1α Activator, Attenuates Both Long- and Short-Term Alcohol-Induced Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is a worldwide healthcare problem featured by inflammation, reactive oxygen species (ROS), and lipid dysregulation. Roxadustat is used for chronic kidney disease anemia treatment. As a specific inhibitor of prolyl hydroxylase, it can maintain high levels of hypoxia-inducible factor 1α (HIF-1α), through which it can further influence many important pathways, including the three featured in ALD. However, its effects on ALD remain to be elucidated. In this study, we used chronic and acute ALD mouse models to investigate the protective effects of roxadustat in vivo. Our results showed that long- and short-term alcohol exposure caused rising activities of serum transaminases, liver lipid accumulation, and morphology changes, which were reversed by roxadustat. Roxadustat-reduced fatty liver was mainly contributed by the reducing sterol-responsive element-binding protein 1c (SREBP1c) pathway, and enhancing β-oxidation through inducing peroxisome proliferator-activated receptor α (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) expression. Long-term alcohol treatment induced the infiltration of monocytes/macrophages to hepatocytes, as well as inflammatory cytokine expression, which were also blocked by roxadustat. Moreover, roxadustat attenuated alcohol caused ROS generation in the liver of those two mouse models mainly by reducing cytochrome P450 2E1 (CYP2E1) and enhancing superoxidase dismutase 1 (SOD1) expression. In vitro, we found roxadustat reduced inflammation and lipid accumulation mainly via HIF-1α regulation. Taken together, our study demonstrates that activation of HIF-1α can ameliorate ALD, which is contributed by reduced hepatic lipid synthesis, inflammation, and oxidative stress. This study suggested that roxadustat could be a potential drug for ALD treatment.

Oyster broth concentrate and its major component taurine alleviate acute alcohol-induced liver damage.

Our previous study showed that oyster hydrolysate (OH) protected against the liver damage caused by a single instance of ethanol (EtOH) binge drinking. Oyster broth concentrate (OBC) was discovered in the process of searching for a different substance derived from oysters (Crassostrea gigas) with economic value. OBC is a by‐product of boiling oysters at 95°C for 3 min. In this study, we investigated the effects of OBC and its major component taurine on blood and liver tissues obtained from a single‐EtOH‐binge‐drinking mouse model. The preadministration of OBC enhanced EtOH metabolism by increasing the activities of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and catalase. In addition, the preadministration of OBC reduced cytochrome P450 2E1 (CYP2E1) activity, reactive oxygen species (ROS) generation, Ca2+ concentrations, apoptotic signals, and inflammatory mediators in liver tissues. The reduction of apoptotic and inflammatory signals by OBC resulted from the downregulation of endoplasmic reticulum (ER) stress molecules and NF‐κB activity. Taurine administration showed similar effects to OBC. These results show that OBC protected against acute EtOH‐induced liver damage through the action of taurine. Our findings suggest that OBC could be an economically valuable substance and a functional food with hepatoprotective effects.

Diallyl disulfide prevents 1,3-dichloro-2-propanol-induced hepatotoxicity through mitogen-activated protein kinases signaling

We investigated whether diallyl disulfide (DADS) has protective effects against 1,3-dichloro-2-propanol (1,3-DCP)-induced hepatotoxicity and oxidative damage in rats and HepG2 cells. DADS was administered to rats once daily for 7 days at doses of 30 and 60 mg/kg/day. One hour after the final DADS treatment, the rats were administered 90 mg/kg 1,3-DCP to induce acute hepatotoxicity. DADS treatment significantly suppressed the increase in serum aminotransferase levels induced by 1,3-DCP administration, and reduced histopathological alterations in the liver. DADS treatment reduced 1-3-DCP-induced apoptotic changes in the liver, as revealed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining and immunohistochemistry for caspase-3. DADS treatment competitively inhibited or reduced cytochrome p450 2E1 (CYP2E1) expression, which is involved in the metabolic activation of 1,3-DCP, and enhanced antioxidant properties. Furthermore, DADS treatment inhibited phosphorylation of mitogen-activated protein kinases (MAPKs) and apoptotic signaling. In in vitro experiments, MAPKs inhibitors reduced the expression of Bax/Bcl-2/Caspase 3 signaling, which effects were more significant in co-treated cells with DADS and MAPKs inhibitors. In conclusion, the protective effect of DADS against 1,3-DCP-induced hepatotoxicity may be related to blocking the metabolic activation of 1,3-DCP by suppressing CYP2E1 expression, inducing antioxidant enzyme activity, and reducing apoptotic activity by inhibiting phosphorylation of MAPKs.

Monooxygenase System and NO Metabolism in Liver Microsomes of Rats with Toxic Hepatitis and the Effect of Sesquiterpene Lactones.

We analyzed changes in activities of enzymes of phases I and II of xenobiotic biotransformation and parameters of NO metabolism in liver microsomes of rats with toxic CCl4-induced hepatitis after a 14-day course of sesquiterpene lactones from Artemisia leucodes (10 mg/kg). It was found that toxic hepatitis was associated with significant inhibition of NADPH-cytochrome c-reductase, benzo(a)pyrene hydroxylase, and NADPH-diaphorase, reduced cytochrome P-450 content, and enhanced induction of nitrate/nitrite reductase with accumulation of NO metabolites in the liver. Administration of sesquiterpene lactones stimulated activity of the studied components of the cytochrome P-450 system and promoted recovery of the NOergic system components; the effects were most pronounced in 7 and 14 days after treatment.

Progesterone receptor membrane component 1 (PGRMC1) binds and stabilizes cytochromes P450 through a heme-independent mechanism

Progesterone receptor membrane component 1 (PGRMC1) is a heme-binding protein implicated in a wide range of cellular functions. We previously showed that PGRMC1 binds to cytochromes P450 in yeast and mammalian cells and supports their activity. Recently, the paralog PGRMC2 was shown to function as a heme chaperone. The extent of PGRMC1 function in cytochrome P450 biology and whether PGRMC1 is also a heme chaperone are unknown. Here, we examined the function of Pgrmc1 in mouse liver using a knockout model and found that Pgrmc1 binds and stabilizes a broad range of cytochromes P450 in a heme-independent manner. Proteomic and transcriptomic studies demonstrated that Pgrmc1 binds more than 13 cytochromes P450 and supports maintenance of cytochrome P450 protein levels posttranscriptionally. In vitro assays confirmed that Pgrmc1 KO livers exhibit reduced cytochrome P450 activity consistent with reduced enzyme levels. Mechanistic studies in cultured cells demonstrated that PGRMC1 stabilizes cytochromes P450 and that binding and stabilization do not require PGRMC1 binding to heme. Importantly, Pgrmc1-dependent stabilization of cytochromes P450 is physiologically relevant, as Pgrmc1 deletion protected mice from acetaminophen-induced liver injury. Finally, evaluation of Y113F mutant Pgrmc1, which lacks the axial heme iron-coordinating hydroxyl group, revealed that proper iron coordination is not required for heme binding, but is required for binding to ferrochelatase, the final enzyme in heme biosynthesis. PGRMC1 was recently identified as the causative mutation in X-linked isolated pediatric cataract formation. Together, these results demonstrate a heme-independent function for PGRMC1 in cytochrome P450 stability that may underlie clinical phenotypes.

Effect of 5-fluorouracil on mRNA expression of drug metabolizing enzyme and transporter genes in human hepatoma cell lines.

Fluoropyrimidines such as 5-fluorouracil (5-FU) are well known to have drug-drug interactions with anticoagulant medications such as warfarin. This study investigated the mRNA expression of pharmacokinetic (PK)-related genes in response to 5-FU using the hepatocarcinoma cell lines after examining relevant gene expression via RNA sequencing. We used HepaRG cells for 5-FU treatment analysis because these cells displayed PK-related gene expression. 5-FU exposure significantly reduced cytochrome P450 3A4 (CYP3A4) mRNA expression. Additionally, the mRNA expression of nuclear receptor subfamily 1 group I member 2 (also known as pregnane X receptor), a nuclear receptor transcription factor that promotes the expression of many CYP genes, was also decreased in HepaRG cells following 5-FU treatment. The mRNA expressions of the CYP2B6 and ATP-binding cassette transporter genes were decreased after 5-FU treatment. This study revealed that 5-FU treatment reduced PK-related gene expression in HepaRG cells. These findings should be useful for further drug-drug interaction research.

RETRACTED ARTICLE: Ornithine-A urea cycle metabolite enhances autophagy and controls Mycobacterium tuberculosis infection

Macrophages are professional phagocytes known to play a vital role in controlling Mycobacterium tuberculosis (Mtb) infection and disease progression. Here we compare Mtb growth in mouse alveolar (AMs), peritoneal (PMs), and liver (Kupffer cells; KCs) macrophages and in bone marrow-derived monocytes (BDMs). KCs restrict Mtb growth more efficiently than all other macrophages and monocytes despite equivalent infections through enhanced autophagy. A metabolomics comparison of Mtb-infected macrophages indicates that ornithine and imidazole are two top-scoring metabolites in Mtb-infected KCs and that acetylcholine is the top-scoring in Mtb-infected AMs. Ornithine, imidazole and atropine (acetylcholine inhibitor) inhibit Mtb growth in AMs. Ornithine enhances AMPK mediated autophagy whereas imidazole directly kills Mtb by reducing cytochrome P450 activity. Intranasal delivery of ornithine or imidazole or the two together restricts Mtb growth. Our study demonstrates that the metabolic differences between Mtb-infected AMs and KCs lead to differences in the restriction of Mtb growth.

Crocin attenuates CCl4-induced liver fibrosis via PPAR-γ mediated modulation of inflammation and fibrogenesis in rats.

Liver fibrosis is a chronic pathological condition with a leading cause of liver-related mortality worldwide. In the present study, we have evaluated the antifibrotic effect of crocin, a carotenoid present in the stigma of Crocus sativus, and also explored its putative mechanism of action. Liver fibrosis was induced by intraperitoneal administration of 30% carbon tetrachloride (CCl4). The crocin was administered orally at 20, 40 and 80 mg/kg body weight along with CCl4 up to 8 weeks. Chronic exposure to CCl4 resulted in elevated levels of liver enzymes and reduced cytochrome P450 2E1 (CYP2E1) activity in the liver. The liver tissue showed cellular swelling, vacuolization, necrosis, infiltration of inflammatory cells and fibrotic changes. The crocin treatment significantly lowered the levels of liver enzymes in serum and improved the liver CYP2E1 mRNA levels. The pathological changes in the liver were also lowered by crocin treatment. The level of pro-inflammatory cytokines, nuclear factor-kappa B, interleukin-6 and tumor necrosis factor α and fibrogenic factor, transforming growth factor β, and α-smooth muscle actin were elevated by the CCl4 in the liver tissue. However, crocin treatment at different doses significantly reduced the expression of these factors. The increased caspase 3/7 activity was also lowered by crocin. CCl4 administration decreased the expression of peroxisome proliferator-activated receptor γ (PPAR-γ) in liver tissue. The improved PPAR-γ expression in the liver by crocin treatment indicates its role in the therapeutic effect of crocin. Crocin attenuated the various events in the progression of liver fibrosis via PPAR-γ mediated modulation of inflammatory and fibrogenic pathways.

Ornithine-A urea cycle metabolite enhances autophagy and controls Mycobacterium tuberculosis infection

Abstract Alveolar macrophages (AMs) are the first cells encountered by TB pathogen and serve as the primary defense against Mycobacterium tuberculosis (Mtb) in the lungs. Studies have shown that liver macrophages (Kupffer cells; KCs) can control Mtb growth but, it is not clear how KCs completely eliminate mycobacterial infections. Thus, identification of these mechanisms will facilitate the development of immunomodulatory strategies to boost AM-mediated immunity to Mtb. In the current study, we compared Mtb growth in murine AMs, peritoneal (PMs), liver macrophages (Kupffer cells; KCs) and bone marrow-derived monocytes (BDMs). KCs restricted Mtb growth more efficiently than all other macrophages and monocytes despite equivalent infections. Differences in Mtb growth restriction were not due to differences in cytokine production, expression of Toll-like receptors 2 and 4, M1/M2 paradigm or apoptosis. We provide evidence that the enhanced autophagy efficiently restricts Mtb growth in KCs using flow cytometry, western blot, RT-PCR and confocal microscopy. A metabolomic comparison of Mtb-infected macrophages by liquid chromatography mass spectrometry indicated that ornithine (VIP=1.8) and imidazole (VIP=1.6) were two top-scoring metabolites found in Mtb-infected KCs and that acetylcholine was top-scoring in Mtb-infected AMs. Ornithine and imidazole inhibited Mtb growth in AMs by enhancing AMPK mediated autophagy whereas imidazole directly killed Mtb by reducing cytochrome P450 activity. Intranasal delivery of ornithine or imidazole or together restricted Mtb growth in Mtb-infected mouse lungs. Our study demonstrates that the metabolic differences in Mtb-infected AMs and KCs leads to differences in the restriction of Mtb growth.

846 Resistance to apoptosis and PD-L1 induction in melanoma is associated with suppressed aryl hydrocarbon receptor signaling

Metastatic melanoma is an aggressive form of skin cancer with a 5-year survival rate of 15% to 20%. Unlike primary melanoma, metastatic disease is difficult to treat due to acquisition of chemoresistance and immune evasion through PD-L1 expression. Highlighting the intrinsic differences in signaling networks that are overactive or suppressed between primary and metastatic melanoma will provide essential cues to understanding mechanisms of therapeutic resistance and immune evasion. We detected reduced cytochrome p450 enzymes, Cyp1a1 and Cyp1b1, in vertical growth phase melanoma cell line, WM793, and its metastatic variant, 1205Lu, compared to primary melanoma cells WM1552c. Since their expression is directly controlled by aryl hydrocarbon receptor (AhR), whose expression was also decreased in 1205Lu and WM793 cells, we tested whether reduced AhR signaling in melanoma cells confers chemoresistance and immune evasion properties. We used paclitaxel (ptx)-mediated apoptosis and interferon gamma (IFNg)-mediated PD-L1 induction as end points to evaluate chemotherapeutic resistance and immune evasion, respectively. 1205Lu and WM793 cells were resistant to ptx-mediated apoptosis (<10%) and PD-L1 expression increased by 3 to 6-fold following IFNg treatment. WM1552c cells, however, were sensitive to ptx-mediated apoptosis (>25%) with minimal induction of PD-L1 (1.5-fold) following IFNg treatment. Treatment with AhR antagonist, CH223191, prior to ptx decreased the percentage of apoptotic cells to <10% in WM1552 cells. IFNg-induced PD-L1 expression also increased by 3-folds in WM1552c cells following AhR inhibition although the mean fluorescence intensity was still lower than 1205Lu cells. Our data indicates that a suppressed AhR signaling in vertical phase and metastatic melanoma cells may have been evolved to resist apoptosis to chemotherapy and promote immune evasion via enhanced PD-L1 induction. Elucidating components of AhR pathway that are affected can be of high therapeutic value for metastatic melanoma.

Novel Tetrazole-Containing Analogues of Itraconazole as Potent Antiangiogenic Agents with Reduced Cytochrome P450 3A4 Inhibition.

Itraconazole has been found to possess potent antiangiogenic activity, exhibiting promising antitumor activity in several human clinical studies. The wider use of itraconazole in the treatment of cancer, however, has been limited by its potent inhibition of the drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4). In an effort to eliminate the CYP3A4 inhibition while retaining its antiangiogenic activity, we designed and synthesized a series of derivatives in which the 1,2,4-triazole ring is replaced with various azoles and nonazoles. Among these analogues, 15n with tetrazole in place of 1,2,4-triazole exhibited optimal inhibition of human umbilical vein endothelial cell proliferation with an IC50 of 73 nM without a significant effect on CYP3A4 (EC50 > 20 μM). Similar to itraconazole, 15n induced Niemann-Pick C phenotype (NPC phenotype) and blocked AMPK/mechanistic target of rapamycin signaling. These results suggest that 15n is a promising angiogenesis inhibitor that can be used in combination with most other known anticancer drugs.