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Genetic variation in drug metabolizing enzymes is an important contributor to interindividual variation in drug disposition and response and is associated with significant clinical consequences. Many commonly used drugs are dependent on the cytochrome P450 monooxygenase enzymes (CYP450) for their metabolism and elimination. At present, more than 57 active human CYP450 genes are known, and the majority of these genes are polymorphic. Despite the large number of CYP450 genes, only the CYP1, CYP2, and CYP3 families of enzymes have a major role in drug metabolism. Approximately 10 CYP450s are responsible for the metabolism of a large number of pharmacologic agents in human beings. The polymorphic forms of the CYP450s are responsible for the development of a significant number of adverse drug reactions and may also contribute to drug response. Genetic polymorphisms have now been identified in the genes encoding all the main CYP450s that contribute to drug and other xenobiotic metabolism, and there are marked interethnic differences in the distribution and frequency of variant alleles. A review of the progress in the pharmacogenetics of P450s that are important for drug metabolism is presented with particular emphasis on the clinical relevance of this research. [ABSTRACT FROM AUTHOR]

Experiments have shown that many phenylpropanoid genes are highly expressed in light-grown Arabidopsis roots. Studies employing reporter gene constructs have indicated that the expression of these genes is localized not only to the lignifying root vasculature, but also to non-lignifying tissues, such as the root cortex, suggesting that the proteins encoded by these genes may be involved in aspects of phenylpropanoid metabolism other than lignification. Consistent with this hypothesis, roots of etiolated and soil-grown plants contain almost no soluble phenylpropanoids, but exposure to light leads to the accumulation of flavonoids, as well as high levels of coniferin and syringin (coniferyl and sinapyl-4-O-glycosides), compounds not previously reported to be accumulated in Arabidopsis. To elucidate the mechanism by which light induces root secondary metabolism, extracts of mutants defective in light perception and light responses were analyzed for phenylpropanoid content. The results of these assays showed that phytochrome (PHY)B and cryptochrome (CRY)2 are the primary photoreceptors involved in light-dependent phenylpropanoid accumulation, and that the hypocotyl elongated (HY5) transcription factor is also required for this response. The presence of phenylpropanoids in etiolated roots of cop (constitutively photomorphogenic) 1, cop9, and det (de-etiolated) 1 mutants indicate that the corresponding wild-type genes are required to repress root phenylpropanoid biosynthesis in the absence of light. Biochemical analysis of root cell walls and analysis of phenylpropanoid gene expression suggest that coniferin and syringin accumulation may be the result of both increased biosynthesis and decreased conversion of these compounds into other phenylpropanoid end products. Finally, our data suggest that the accumulation of coniferin, syringin, and flavonoids in Arabidopsis roots is a high-irradiance response (HIR), and suggest that comparative analysis of light- and dark-grown Arabidopsis roots may provide new insights into both phenylpropanoid biosynthesis and light signaling in plants. [ABSTRACT FROM AUTHOR]

Highlights: What are the main findings? The validated method was robust, reliable, and sensitive for quantifying ABZ, ABZ-OX, ABZ-ON, and PR simultaneously from the plasma and dried blood spots. The method is well suited for therapeutic drug monitoring due to its high sensitivity, minimal sample volume requirement, and the ability for remote collection. What is the implication of the main finding? Dried blood spot (DBS) analysis represents an alternative sampling technique over conventional blood-sampling methods, particularly for sample collection, enabling a non-clinical setup without requiring special equipment or trained healthcare personnel. Additionally, it minimizes the solvent volume and analysis time and reduces transportation and storage needs. Millions of individuals throughout the world suffer from lymphatic filariasis (LF), which is a morbid disease caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori. These infections belong to tissue-invading nematodes and are one of the major neglected tropical diseases that often result in permanent and enduring disability among individuals in endemic regions. Due to combination therapy, LF eradication has drastically decreased infections globally. The development of blood micro-sampling techniques allowing precise quantitation of drugs in blood would facilitate pharmacokinetic (PK) studies in remote populations. Therefore, an LC-MS/MS bioanalytical method was utilized to analyze albendazole (ABZ), albendazole sulfone (ABZ-ON), albendazole sulfoxide (ABZ-OX), and probenecid (PR) in plasma and dried blood spots. Solid-phase extraction was utilized to extract the analyte from both plasma and blood-spiked DBS. Analytes of interest were eluted with a gradient mobile system using 0.05% formic acid in water (A) and 0.05% formic acid in methanol (B) and separated using a reversed-phase Acquity ®BEH C18 UPLC column (100 × 2.1 mm, 1.7 µm). Precision and accuracy at each QC level were within the acceptable limit, i.e., ±15% for all analytes in both the matrices. Tests for stability under laboratory and storage conditions indicated that no notable changes were observed for plasma and DBS. The LC-MS/MS method demonstrated its capability to consistently identify all target analytes (ABZ, ABZ-ON, ABZ-OX, and PR) at low concentrations, even at the small specimen volumes obtained from DBS cards. This confirms the efficacy and durability of DBS cards as a micro-sampling technique. Moreover, it enhances collection efforts for therapeutic drug monitoring in remote locations for patients infected with lymphatic filariasis. [ABSTRACT FROM AUTHOR]

Background: The activity of MP1, a pyrrolomycin, was studied in MYCN amplified neuroblastoma (NB) alone and combined with temsirolimus (TEM).Methods: Activity of MP1 was tested in MYCN amplified (BE-2c, IMR) and non amplified (SKN-AS) NB cells. The effect of MP1 on MYCN, MCL-1, cleaved PARP, LC3II/LC3I, bcl-2, BAX, and BRD-4 were determined by western blot and RNAseq. The effect of MP1 on metabolism, mitochondrial morphology, and cell cycle was determined. Toxicology and efficacy of MP1 plus TEM were evaluated.Results: The IC50 of MP1 was 0.096 μM in BE-2c cells compared to 0.89 μM in IMR, and >50 μM in SKN-AS. The IC50 of MP1 plus TEM in BE-2c cells was 0.023 μM. MP1 inhibited metabolism leading to quiescence and produced a decline in cell cycle S-phase. Electron microscopy showed cristae loss and rounding up of mitochondria. Gene and protein expression for MYCN and MCL-1 declined while LCII and cleaved PARP increased. Protein expression of BAX, bcl-2, and BRD-4 were not significantly changed after MP1 treatment. The in-vivo concentrations of MP1 in blood and tumor were sufficient to produce the biologic effects seen in-vitro. MP1 plus TEM produced a complete response in 3 out of 5 tumor bearing mice. In a second mouse study, the combination of MP1 and TEM slowed tumor growth compared to control.Conclusions: MP1 has a potent inhibitory effect on the viability of MYCN amplified NB. Inhibition of metabolism by MP1 induced quiescence and autophagy with a favorable toxicology and drug distribution profile. When combined with TEM anti-tumor activity was potentiated in-vitro and in-vivo. [ABSTRACT FROM AUTHOR]

Moxidectin (MOX) has recently been approved by the US Food and Drug Administration for the treatment of river blindness in select populations. It is also being evaluated as an alternative for the use of ivermectin, widespread resistance to which is becoming a global health issue. Moreover, MOX is becoming increasingly used as a prophylactic antiparasitic in the cattle industry. In this study, we developed and validated an LC–MS/MS method of MOX in human, monkey and mouse plasma. The separation was achieved on an ACE C18 (50 × 3.0 mm, 3 μm) column with isocratic elution using 0.1% acetic acid and methanol–acetonitrile (1:1, v/v) as mobile phase. MOX was quantitated using MS/MS with an electrospray ionization source operating in negative multiple reaction monitoring mode. The multiple reaction monitoring precursor ion → product ion transitions for MOX and abamectin (IS) were m/z 638.40 → 236.30 and m/z 871.50 → 565.35 respectively. The MS/MS response was linear over the concentration range 0.1–1000 ng/mL in plasma with a correlation coefficient (r2) of 0.997 or better. The within‐ and between‐day precision (relative standard deviation, RSD) and accuracy were within the acceptable limits per US Food and Drug Administration guidelines. The method was successfully applied to an in vitro metabolic stability study of MOX. [ABSTRACT FROM AUTHOR]

Isoprenoids (IsoP) are an important class of molecules involved in many different cellular processes including cholesterol synthesis. We have developed a sensitive and specific LC-MS/MS method for the quantitation of three key IsoPs in bio-matrices, geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP). LC-MS/MS analysis was performed using a Nexera UPLC System connected to a LCMS-8060 (Shimadzu Scientific Instruments, Columbia, MD) with a dual ion source. The electrospray ionization source was operated in the negative MRM mode. The chromatographic separation and detection of analytes was achieved on a reversed phase ACCQ-TAG Ultra C18 (1.7 µm, 100 mm × 2.1 mm I.D.) column. The mobile phase consisted of (1) a 10 mM ammonium carbonate with 0.1% ammonium hydroxide in water, and (2) a 0.1% ammonium hydroxide in acetonitrile/methanol (75/25). The flow rate was set to 0.25 mL/min in a gradient condition. The limit of quantification was 0.04 ng/mL for all analytes with a correlation coefficient (r2) of 0.998 or better and a total run time of 12 min. The inter- and intra-day accuracy (85–115%) precision (<15%), and recovery (40–90%) values met the acceptance criteria. The validated method was successfully applied to quantitate basal concentrations of GPP, FPP and GGPP in human plasma and in cultured cancer cell lines. Our LC-MS/MS method may be used for IsoP quantification in different bio-fluids and to further investigate the role of these compounds in various physiological processes. [ABSTRACT FROM AUTHOR]

Chemotherapy together with surgery and/or radiotherapy are the most common therapeutic methods for treating cancer. However, the off-target effects of chemotherapy are known to produce side effects and dose-limiting toxicities. Novel delivery platforms based on natural and synthetic polymers with enhanced pharmacokinetic and therapeutic potential for the treatment of cancer have grown tremendously over the past 10 years. Polymers can facilitate selective targeting, enhance and prolong circulation, improve delivery, and provide the controlled release of cargos through various mechanisms, including physical adsorption, chemical conjugation, and/or internal loading. Notably, polymers that are biodegradable, biocompatible, and physicochemically stable are considered to be ideal delivery carriers. This biomimetic and bio-inspired system offers a bright future for effective drug delivery with the potential to overcome the obstacles encountered. This review focuses on the barriers that impact the success of chemotherapy drug delivery as well as the recent developments based on natural and synthetic polymers as platforms for improving drug delivery for treating cancer. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OS) is a primary malignant bone tumor characterized by frequent metastasis, rapid disease progression, and a high rate of mortality. Treatment options for OS have remained largely unchanged for decades, consisting primarily of cytotoxic chemotherapy and surgery, thus necessitating the urgent need for novel therapies. Tropolones are naturally occurring seven‐membered non‐benzenoid aromatic compounds that possess antiproliferative effects in a wide array of cancer cell types. MO‐OH‐Nap is an α‐substituted tropolone that has activity as an iron chelator. Here, we demonstrate that MO‐OH‐Nap activates all three arms of the unfolded protein response (UPR) pathway and induces apoptosis in a panel of human OS cell lines. Co‐incubation with ferric chloride or ammonium ferrous sulfate completely prevents the induction of apoptotic and UPR markers in MO‐OH‐Nap‐treated OS cells. MO‐OH‐Nap upregulates transferrin receptor 1 (TFR1) protein levels, as well as TFR1, divalent metal transporter 1 (DMT1), iron‐regulatory proteins (IRP1, IRP2), ferroportin (FPN), and zinc transporter 14 (ZIP14) transcript levels, demonstrating the impact of MO‐OH‐Nap on iron‐homeostasis pathways in OS cells. Furthermore, MO‐OH‐Nap treatment restricts the migration and invasion of OS cells in vitro. Lastly, metabolomic profiling of MO‐OH‐Nap‐treated OS cells revealed distinct changes in purine and pyrimidine metabolism. Collectively, we demonstrate that MO‐OH‐Nap‐induced cytotoxic effects in OS cells are dependent on the tropolone's ability to alter cellular iron availability and that this agent exploits key metabolic pathways. These studies support further evaluation of MO‐OH‐Nap as a novel treatment for OS. [ABSTRACT FROM AUTHOR]

Background: Medulloblastoma (MB) patients with MYC oncogene amplification or overexpression exhibit extremely poor prognoses and therapy resistance. However, MYC itself has been one of the most challenging targets for cancer treatment. Here, we identify a novel marinopyrrole natural derivative, MP1, that shows desirable anti-MYC and anti-cancer activities in MB. Methods: In this study, using MYC-amplified (Group 3) and non-MYC amplified MB cell lines in vitro and in vivo, we evaluated anti-cancer efficacies and molecular mechanism(s) of MP1. Results: MP1 significantly suppressed MB cell growth and sphere counts and induced G2 cell cycle arrest and apoptosis in a MYC-dependent manner. Mechanistically, MP1 strongly downregulated the expression of MYC protein. Our results with RNA-seq revealed that MP1 significantly modulated global gene expression and inhibited MYC-associated transcriptional targets including translation/mTOR targets. In addition, MP1 inhibited MYC-target metabolism, leading to declined energy levels. The combination of MP1 with an FDA-approved mTOR inhibitor temsirolimus synergistically inhibited MB cell growth/survival by downregulating the expression of MYC and mTOR signaling components. Our results further showed that as single agents, both MP1 and temsirolimus, were able to significantly inhibit tumor growth and MYC expression in subcutaneously or orthotopically MYC-amplified MB bearing mice. In combination, there were further anti-MB effects on the tumor growth and MYC expression in mice. Conclusion: These preclinical findings highlight the promise of marinopyrrole MP1 as a novel MYC inhibition approach for MYC-amplified MB. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to a global pandemic of coronavirus disease 2019 (COVID-19). Early in the pandemic, efforts were made to test the SARS-CoV-2 antiviral efficacy of repurposed medications that were already approved and available for other indications, including hydroxychloroquine (HCQ) and azithromycin (AZI). To reduce the risk of SARS-CoV-2 exposure for clinical-trial study participants and to conform with lockdowns and social distancing guidelines, biospecimen collection for HCQ and AZI included at-home dried blood spot (DBS) collection rather than standard venipuncture by trained clinicians. In this study, we developed and validated the first sensitive and selective simultaneous LC–MS/MS method to accurately quantitate the concentration of HCQ, HCQ metabolites (Desethylchloroquine [DCQ], Bisdesethylchloroquine [BDCQ], Monodesethylhydroxychloroquine [DHCQ]) and AZI extracted from DBS. The validated method was successfully applied for the quantification of over 2000 DBS specimens to evaluate the pharmacokinetic profile of AZI, HQC, and its metabolites. This new method has a small sample volume requirement (~ 10 µL), results in high sensitivity (1 ng/mL), and would facilitate remotely conducted therapeutic drug monitoring. [ABSTRACT FROM AUTHOR]

Moxidectin (MOX) is a milbemycin endectocide recently approved by the U.S. FDA for the treatment of onchocerciasis in persons at least 12 years of age. MOX has been shown to have a good safety profile in recent clinical trials. The efficacy of MOX for the treatment of lymphatic filariasis (LF) and its potential use in mass drug administration protocols for the elimination of LF is currently under evaluation. In the context of a clinical trial, we investigated the pharmacokinetics and drug interactions of a combination of MOX plus albendazole (ALB) with or without diethylcarbamazine (DEC) compared to ivermectin (IVM) plus ALB with or without DEC in the following four different treatment arms: (I) IVM (0.2mg/kg) plus DEC (6 mg/kg) and ALB (400mg); (II) IVM plus ALB; (III) MOX (8 mg) plus DEC and ALB; and (IV) MOX plus ALB. Drug concentrations were determined using validated liquid chromatography-mass spectrometric methods. Pharmacokinetic parameters were determined using standard non-compartmental analysis methods. Statistical analysis was performed using JMP software. Fifty-eight of 164 study participants (53 men and five women) were included with ages ranging from 18 to 63 yrs (mean = 37). MOX apparent oral clearance (Cl/F) ranged from 0.7 to 10.8 L/hr with Cmax values ranging from 20.8 to 314.5 ng/mL. The mean (range) area under the curve (AUC)0-∞ for MOX, 3405 ng*hr/mL (742–11376), and IVM 1906 ng*hr/mL (692–5900), varied over a ~15.3 and ~8.5-fold range, respectively. The geometric mean ratio for Cmax, AUC0–t, and AUC0–∞ were within the no-drug interaction range of 80–125% for all drugs. This indicates that the addition of MOX to ALB alone or ALB plus DEC for LF therapy did not alter the drug exposure of co-administered drugs compared to IVM combinations. Clinical Trial Registration: NCT04410406, https://clinicaltrials.gov/. Author summary: Mass Drug administration (MDA) with multiple drugs is common for the treatment and control of lymphatic filariasis (LF). This study aimed to determine whether the incorporation of moxidectin (MOX) into MDA regimens for LF will alter the pharmacokinetics (PK) of co-administered drugs. In the context of a clinical trial, we investigated the PK and drug interactions of a combination of moxidectin (MOX) plus albendazole (ALB) with or without diethylcarbamazine (DEC) compared to ivermectin (IVM) plus ALB with or without DEC. The study was conducted in adult patients located in Côte d'Ivoire. The addition of MOX did not alter the exposure (AUC0-t) or maximum concentration (Cmax) of co-administered drugs. These results suggest the incorporation of MOX into current MDA LF treatment programs will not result in clinically significant alterations in drug exposure. [ABSTRACT FROM AUTHOR]

Hydroxychloroquine (HCQ) is Food and Drug Administration (FDA)‐approved for malaria, systemic and chronic discoid lupus erythematosus, and rheumatoid arthritis. Because HCQ has a proposed multimodal mechanism of action and a well‐established safety profile, it is often investigated as a repurposed therapeutic for a range of indications. There is a large degree of uncertainty in HCQ pharmacokinetic (PK) parameters which complicates dose selection when investigating its use in new disease states. Complications with HCQ dose selection emerged as multiple clinical trials investigated HCQ as a potential therapeutic in the early stages of the COVID‐19 pandemic. In addition to uncertainty in baseline HCQ PK parameters, it was not clear if disease‐related consequences of SARS‐CoV‐2 infection/COVID‐19 would be expected to impact the PK of HCQ and its primary metabolite desethylhydroxychloroquine (DHCQ). To address the question whether SARS‐CoV‐2 infection/COVID‐19 impacted HCQ and DHCQ PK, dried blood spot samples were collected from SARS‐CoV‐2(−)/(+) participants administered HCQ. When a previously published physiologically based pharmacokinetic (PBPK) model was used to fit the data, the variability in exposure of HCQ and DHCQ was not adequately captured and DHCQ concentrations were overestimated. Improvements to the previous PBPK model were made by incorporating the known range of blood to plasma concentration ratios (B/P) for each compound, adjusting HCQ and DHCQ distribution settings, and optimizing DHCQ clearance. The final PBPK model adequately captured the HCQ and DHCQ concentrations observed in SARS‐CoV‐2(−)/(+)participants, and incorporating COVID‐19‐associated changes in cytochrome P450 activity did not further improve model performance for the SARS‐CoV‐2(+) population. [ABSTRACT FROM AUTHOR]

Background: Ivermectin (IVM) is a broad–spectrum anthelmintic drug used to treat diseases caused by filarial worms, such as onchocerciasis and lymphatic filariasis (LF). IVM is part of a triple–drug therapy used by the Mass Drug Administration (MDA) as a preventive strategy to eradicate LF in sub–Saharan Africa. The drug shows high variability in drug exposure in previous pharmacokinetic studies. This study aims to build a population pharmacokinetic (PopPK) model to identify and quantify the possible sources of the variability of IVM exposure after a single–oral dose in LF–infected subjects and healthy individuals. Methodology / Principal findings: In this analysis, 724 samples were collected from treatment–naïve Wuchereria bancrofti–infected (n = 32) and uninfected (n = 24) adults living in Côte d'Ivoire who had received one dose of IVM as a part of triple–drug therapy. PopPK analysis was conducted using Phoenix NLME 8.3 software. The Monte Carlo simulation based on the final model was performed to simulate drug exposure among different dosing groups (200 μg/kg, 18 mg, and 36 mg). A two–compartment model with zero–order dose input into the absorption compartment with a lag time function followed by first–order absorption and linear elimination best described the IVM's pharmacokinetic (PK) parameters. The final model identifies that the PK parameters of IVM are not affected by LF infection. Sex was a significant covariate on the peripheral volume of distribution (Vp/F, 53% lower in men than in women). IVM drug exposure shows linear pharmacokinetic behavior among the simulated dosing groups with similar drug exposure based on sex. Conclusion/Significance: We have developed a PopPk model to describe and identify possible sources of the variability of IVM exposure. To our knowledge, this is the first PopPK study of IVM in patients with LF. Trial registration: NCT02845713; NCT03664063 Author summary: Ivermectin has been shown to be a valuable tool in mass drug administration campaigns against lymphatic filariasis. The drug shows high variability in drug exposure in previous pharmacokinetic studies. Variability in drug exposure can lead to either a higher exposure with the potential for toxic effects or a lower exposure that may result in treatment failure. Identifying and quantifying the source of high variability using non–linear mixed effects modeling is crucial for future dosing recommendations that seek to maximize therapeutic benefit while minimizing toxicity risks. In this study, the variability of IVM exposure after a single oral dose was investigated with a population pharmacokinetic model using data from Lymphatic filariasis infected subjects and healthy individuals. [ABSTRACT FROM AUTHOR]

Background: Prior work supports delayed gastric emptying in anorexia nervosa and bulimia nervosa (BN) but not binge-eating disorder, suggesting that neither low body weight nor binge eating fully accounts for slowed gastric motility. Specifying a link between delayed gastric emptying and self-induced vomiting could offer new insights into the pathophysiology of purging disorder (PD). Methods: Women (N = 95) recruited from the community meeting criteria for DSM-5 BN who purged (n = 26), BN with nonpurging compensatory behaviors (n = 18), PD (n = 25), or healthy control women (n = 26) completed assessments of gastric emptying, gut peptides, and subjective responses over the course of a standardized test meal under two conditions administered in a double-blind, crossover sequence: placebo and 10 mg of metoclopramide. Results: Delayed gastric emptying was associated with purging with no main or moderating effects of binge eating in the placebo condition. Medication eliminated group differences in gastric emptying but did not alter group differences in reported gastrointestinal distress. Exploratory analyses revealed that medication caused increased postprandial PYY release, which predicted elevated gastrointestinal distress. Conclusions: Delayed gastric emptying demonstrates a specific association with purging behaviors. However, correcting disruptions in gastric emptying may exacerbate disruptions in gut peptide responses specifically linked to the presence of purging after normal amounts of food. [ABSTRACT FROM AUTHOR]

Vitamin D plays a critical role in bone development and maintenance, and in other physiological functions. The quantitation of endogenous levels of individual vitamin D and its metabolites is crucial for assessing several disease state conditions. With cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to the coronavirus disease 2019 (COVID-19) pandemic, there are several studies that have associated lower levels of serum vitamin D with severity of infection in COVID-19 patients. In this context, we have developed and validated a robust LC–MS/MS method for simultaneous quantitation of vitamin D and its metabolites in human dried blood spot (DBS) obtained from participants tested for COVID-19. The chromatographic separation for vitamin D and metabolites was performed using an ACE Excel C18 PFP column protected with a C18 guard column (Phenomenex, Torrance, CA, USA). The mobile phase consisted of formic acid in water (0.1% v/v) as mobile phase A and formic acid in methanol (0.1% v/v) as mobile phase B, operated at a flow rate of 0.5 mL/min. Analysis was performed utilizing the LC–MS/MS technique. The method was sensitive with a limit of quantification of 0.78 ng/mL for all analytes, and had a large dynamic range (200 ng/mL) with a total run time of 11 min. The inter- and intraday accuracy and precision values met the acceptance criteria per the US Food and Drug Administration guidelines. Blood concentrations of 25(OH)D3, vitamin D3, 25(OH)D2, and vitamin D2 over a range of 2–195.6, 0.5–121.5, 0.6–54.9, and 0.5–23.9 ng/mL, respectively, were quantified in 909 DBS samples. In summary, our developed LC−MS/MS method may be used for quantification of vitamin D and its metabolites in DBS, and may be applied to investigations of the emerging role of these compounds in various physiological processes. [ABSTRACT FROM AUTHOR]

A selective, sensitive, and rapid liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of ICG in rat plasma. The chromatographic separation was achieved using an ACE excel C18 (3 µm, 50 × 3.0 mm) column, with a mobile phase composition of 0.1% formic acid and 0.1% formic acid in acetonitrile, using a gradient flow at a rate of 0.3 mL/min. The MS was operated at a unit resolution in the multiple reaction monitoring mode, using the precursor ion → product ion combinations of 753.3 → 330.2 m/z (ICG) and 747.45 → 717.50 (Cy7.5 amine) with a run time of 5 min. The assay was linear over a concentration range of 1–1000 ng/mL with a regression coefficient (r2) of 0.998 or better. The inter and intra-batch precision (% relative standard deviation, %RSD) was lower than 13.5%, with accuracy (%Bias) between −10.03% and 11.56%. The ICG was stable under laboratory storage and handling conditions. The validated method was successfully applied to preclinical pharmacokinetic (PK) studies of ICG at a dose of 0.39 mg/kg in rats. PK parameters suggested the highest plasma concentration within 2 min of intravenous dosing with restricted systemic distribution and rapid clearance. [ABSTRACT FROM AUTHOR]

Rab GTPases are critical regulators of protein trafficking in the cell. To ensure proper cellular localization and function, Rab proteins must undergo a posttranslational modification, termed geranylgeranylation. In the isoprenoid biosynthesis pathway, the enzyme geranylgeranyl diphosphate synthase (GGDPS) generates the 20‐carbon isoprenoid donor (geranylgeranyl pyrophosphate [GGPP]), which is utilized in the prenylation of Rab proteins. We have pursued the development of GGDPS inhibitors (GGSI) as a novel means to target Rab activity in cancer cells. Osteosarcoma (OS) and Ewing sarcoma (ES) are aggressive childhood bone cancers with stagnant survival statistics and limited treatment options. Here we show that GGSI treatment induces markers of the unfolded protein response (UPR) and triggers apoptotic cell death in a variety of OS and ES cell lines. Confirmation that these effects were secondary to cellular depletion of GGPP and disruption of Rab geranylgeranylation was confirmed via experiments using exogenous GGPP or specific geranylgeranyl transferase inhibitors. Furthermore, GGSI treatment disrupts cellular migration and invasion in vitro. Metabolomic profiles of OS and ES cell lines identify distinct changes in purine metabolism in GGSI‐treated cells. Lastly, we demonstrate that GGSI treatment slows tumor growth in a mouse model of ES. Collectively, these studies support further development of GGSIs as a novel treatment for OS and ES. [ABSTRACT FROM AUTHOR]

Nucleic acid (NA) therapy has gained importance over the past decade due to its high degree of selectivity and minimal toxic effects over conventional drugs. Currently, intravenous (IV) or intramuscular (IM) formulations constitute majority of the marketed formulations containing nucleic acids. However, oral administration is traditionally preferred due to ease of administration as well as higher patient compliance. To leverage the benefits of oral delivery for NA therapy, the NA of interest must be delivered to the target site avoiding all degrading and inhibiting factors during its transition through the gastrointestinal tract. The oral route presents myriad of challenges to NA delivery, making formulation development challenging. Researchers in the last few decades have formulated various delivery systems to overcome such challenges and several reviews summarize and discuss these strategies in detail. However, there is a need to differentiate between the approaches based on target so that in future, delivery strategies can be developed according to the goal of the study and for efficient delivery to the desired site. The goal of this review is to summarize the mechanisms for target specific delivery, list and discuss the formulation strategies used for oral delivery of NA therapies and delineate the similarities and differences between local and systemic targeting oral delivery systems and current challenges. [ABSTRACT FROM AUTHOR]

Fatty acids are widespread naturally occurring compounds, and essential constituents for living organisms. Short chain fatty acids (SCFAs) appeared as physiologically relevant metabolites for their involvement with gut microbiota, immunology, obesity, and other pathophysiological functions. This has raised the demand for reliable analytical detection methods in a variety of biological matrices. Here, we describe an updated overview of sample pretreatment techniques and liquid chromatography-mass spectrometry (LC-MS)-based methods for quantitative analysis of SCFAs in blood, plasma, serum, urine, feces and bacterial cultures. The present review incorporates various procedures and their applications to help researchers in choosing crucial parameters, such as pretreatment for complex biological matrices, and variables for chromatographic separation and detection, to establish a simple, sensitive, and robust quantitative method to advance our understanding of the role of SCFAs in human health and disease as potential biomarkers. [ABSTRACT FROM AUTHOR]

Baclofen, an antispasmodic agent that acts as a GABA agonist, resembles phenylalanine in structure and has been reported to be a substrate of the large amino acid transporter 1 (LAT-1). The objective of this study was to investigate the absorption of baclofen across the nasal mucosa both in vitro and in vivo. Baclofen transport was measured across excised bovine olfactory and respiratory mucosae to investigate site-specific uptake of baclofen, and the intranasal bioavailability of R- and S-baclofen was determined in rats. Increasing flux with increasing baclofen donor concentration and the absence of polarized transport was observed in vitro, and similar distribution profiles were observed for both enantiomers following intranasal administration in rats. The absence of stereospecificity in nasal absorption indicates limited involvement of the amino acid or other transporters in the nasal absorption of baclofen. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:2717-2723, 2011 [ABSTRACT FROM AUTHOR]

Antimicrobial peptides are important candidates for developing new classes of antibiotics because of their potency against antibioticresistant pathogens. Current research focuses on topical applications and it is unclear how to design peptides with systemic efficacy. To address this problem, we designed two potent peptides by combining database-guided discovery with structure-based design. When bound to membranes, these two short peptides with an identical amino acid composition can adopt two distinct amphipathic structures: A classic horizontal helix (horine) and a novel vertical spiral structure (verine). Their horizontal and vertical orientations on membranes were determined by solid-state 15N NMR data. While horine was potent primarily against gram-positive pathogens, verine showed broad-spectrum antimicrobial activity. Both peptides protected greater than 80% mice from infection-caused deaths. Moreover, horine and verine also displayed significant systemic efficacy in different murine models comparable to conventional antibiotics. In addition, they could eliminate resistant pathogens and preformed biofilms. Significantly, the peptides showed no nephrotoxicity to mice after intraperitoneal or intravenous administration for 1 wk. Our study underscores the significance of horine and verine in fighting drug-resistant pathogens. [ABSTRACT FROM AUTHOR]

The persistence of drug resistance poses a significant obstacle to the advancement of efficacious malaria treatments. The remarkable efficacy displayed by 1,2,3-triazole-based compounds against Plasmodium falciparum highlights the potential of triazole conjugates, with diverse pharmacologically active structures, as potential antimalarial agents. We aimed to synthesize 7-dichloroquinoline-triazole conjugates and their structure-activity relationship (SAR) derivatives to investigate their anti-plasmodial activity. Among them, QP11, featuring a m -NO 2 substitution, demonstrated efficacy against both chloroquine-sensitive and -resistant parasite strains. QP11 selectively inhibited FP2, a cysteine protease involved in hemoglobin degradation, and showed synergistic effects when combined with chloroquine. Additionally, QP11 hindered hemoglobin degradation and hemozoin formation within the parasite. Metabolic stability studies indicated high stability of QP11, making it a promising antimalarial candidate. In vivo evaluation using a murine malaria model demonstrated QP11's efficacy in eradicating parasite growth without neurotoxicity, presenting it as a promising compound for novel antimalarial development. [Display omitted] • Quinoline-triazole conjugates with various linkers & their SAR studies. • IC 50 of QP11 found to be 0.97 (3D7) & 1.8 μM (RKL9) strains of P. falciparum. • QP11 showed synergistic effect in combination with CQ on the parasite growth. • In vivo studies proved the efficacy, safety profile & non-toxicity of QP11. [ABSTRACT FROM AUTHOR]

Highlights • Summary of vitamin K analysis in various biological matrices. • This review collectively describes an updated overview of the sample pretreatment methodologies and methods for quantitative determination of vitamin K that have been used in last two decades. • LC–MS/MS detection of vitamin K will further help to evaluate the role of vitamin K in health and disease. Abstract Vitamin K is an essential nutrient in the body and involved in numerous physiological and pathophysiological functions. Both the lack and surplus of vitamin K can put human health at risk. Therefore, it becomes necessary to monitor vitamin K concentrations in different biomatrices through establishing sensitive and specific analytical methods. This review collectively describes an updated overview of the sample pretreatment methodologies and methods for quantitative determination of vitamin K that have been used in last two decades. High Performance Liquid Chromatography (HPLC) is commonly utilized as a standard for separation of vitamin K in combination with different detection including spectroscopic, spectrometric, fluorometric and mass spectroscopy. Recent progress in sample pretreatment technologies and quantitation methodologies have enhanced the ability to identify and quantitate vitamin K in biomatrices to further advance our understanding of the role of this vitamin in human health and disease. [ABSTRACT FROM AUTHOR]

Highlights • Iron prevents induction of myeloma cell death by the novel tropolone MO-OH-Nap. • Iron prevents MO-OH-Nap induction of the unfolded protein response (UPR) pathway. • The iron chelator deferoxamine induces upregulation of the UPR in myeloma cells. • MO-OH-Nap directly chelates iron and upregulates transferrin receptor expression. Abstract Tropolones are naturally occurring seven-membered non-benzenoid aromatic compounds that are of interest due to their cytotoxic properties. MO-OH-Nap is a novel α-substituted tropolone that induces caspase cleavage and upregulates markers associated with the unfolded protein response (UPR) in multiple myeloma (MM) cells. Given previous reports that tropolones may function as iron chelators, we investigated the effects of MO-OH-Nap, as well as the known iron chelator deferoxamine (DFO), in MM cells in the presence or absence of supplemental iron. The ability of MO-OH-Nap to induce apoptosis and upregulate markers of the UPR could be completely prevented by co-incubation with either ferric chloride or ammonium ferrous sulfate. Iron also completely prevented the decrease in BrdU incorporation induced by either DFO or MO-OH-Nap. Ferrozine assays demonstrated that MO-OH-Nap directly chelates iron. Furthermore, MO-OH-Nap upregulates cell surface expression and mRNA levels of transferrin receptor. In vivo studies demonstrate increased Prussian blue staining in hepatosplenic macrophages in MO-OH-Nap-treated mice. These studies demonstrate that MO-OH-Nap-induced cytotoxic effects in MM cells are dependent on the tropolone's ability to alter cellular iron availability and establish new connections between iron homeostasis and the UPR in MM. [ABSTRACT FROM AUTHOR]

Drug-resistant pathogens are a major cause of hospital- and community-associated bacterial infections in the United States and around the world. These infections are increasingly difficult to treat due to the development of antibiotic resistance and the formation of bacterial biofilms. In the paper, a series of phenazines were synthesized and evaluated for their in vitro antimicrobial activity against Gram positive (methicillin resistant staphylococcus aureus , MRSA) and Gram negative ( Escherichia coli , E. coli ) bacteria. The compound 6,9-dichloro- N -(methylsulfonyl)phenazine-1-carboxamide ( 18c) proved to be the most active molecule (MIC = 16 μg/mL) against MRSA whereas 9-methyl- N -(methylsulfonyl)phenazine-1-carboxamide ( 30e) showed good activity against both MRSA (MIC = 32 μg/mL) and E. coli (MIC = 32 μg/mL). Molecule 18c also demonstrated significant biofilm dispersion and inhibition against S. aureus . Preliminary studies indicate the molecules do not disturb bacterial membranes and there activity is not directly linked to the generation of reactive oxygen species. Compound 18c displayed minor toxicity against mammalian cells. Metabolic stability studies of the most promising compounds indicate stability towards phase I and phase II metabolizing enzymes. [ABSTRACT FROM AUTHOR]

Hydroxychloroquine (HCQ) has been shown to disrupt autophagy and sensitize cancer cells to radiation and chemotherapeutic agents. However, the optimal delivery method, dose, and tumor concentrations required for these effects are not known. This is in part due to a lack of sensitive and reproducible analytical methods for HCQ quantitation in small animals. As such, we developed and validated a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method for simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues. The chromatographic separation and detection of analytes were achieved on a reversed phase Thermo Aquasil C 18 (50 × 4.6 mm, 3 μ) column, with gradient elution using 0.2% formic acid and 0.1% formic acid in methanol as mobile phase at a flow rate of 0.5 mL/min. Simple protein precipitation was utilized for extraction of analytes from the desired matrix. Analytes were separated and quantitated using MS/MS with an electrospray ionization source in positive multiple reaction monitoring (MRM) mode. The MS/MS response was linear over the concentration range from 1 to 2000 ng/mL for all analytes with a correlation coefficient (R 2 ) of 0.998 or better. The within- and between-day precision (relative standard deviation, % RSD) and accuracy were within the acceptable limits per FDA guidelines. The validated method was successfully applied to a preclinical pharmacokinetic mouse study involving low volume blood and tissue samples for hydroxychloroquine and metabolites. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is a leading cause of the cancer-related deaths worldwide. Thus, developing novel and targeted therapies for inhibiting CRC progression and metastasis is urgent. Several studies, including ours, have reported a causal role for an upregulated claudin-1 expression in promoting CRC metastasis through the activation of the Src and β-catenin-signaling. In murine models of colon tumorigenesis, claudin-1 overexpression promotes oncogenic properties such as transformation and invasiveness. Conversely, the downregulation of claudin-1 inhibits colon tumorigenesis. Despite being a desirable target for cancer treatment, there are currently no known claudin-1 inhibitors with antitumor efficacy. Using a rigorous analytical design and implementing in- vitro and in-vivo testing and a brief medicinal chemistry campaign, we identified a claudin-1-specific inhibitor and named it I-6. Despite its high potency, I-6 was rapidly cleared in human liver microsomes. We, therefore, synthesized I-6 analogs and discovered a novel small molecule, PDS-0330. We determined that PDS0330 inhibits claudin-1-dependent CRC progression without exhibiting toxicity in in-vitro and in-vivo models of CRC and that it binds directly and specifically to claudin-1 with micromolar affinity. Further analyses revealed that PDS-0330 exhibits antitumor and chemosensitizer activities with favorable pharmacokinetic properties by inhibiting the association with metastatic oncogene Src. Overall, our data propose that PDS-0330 interferes with claudin-1/Src association to inhibit CRC progression and metastasis. Our findings are of direct clinical relevance and may open new therapeutic opportunities in colon cancer treatment and/or management by targeting claudin-1. • This study focuses on preclinical testing for a new claudin-1 inhibitor PDS0330 in Colorectal Cancer. • PDS0330 inhibits claudin-1-dependent CRC progression. • PDS-0330 selectively binds to claudin-1 and inhibits the Src association. • PDS-0330 suppresses anoikis resistance, reduces invasion and enhances apoptosis. [ABSTRACT FROM AUTHOR]

The sigma 1 receptor is a multifunctional receptor with wide distribution in the nervous system and its function has been implicated with a number of neurological disorders including dementia and Alzheimer's disease (AD) and other neurodegenerative disorders. In addition, modulators of σ1 have been advanced into clinical trials for the treatment of pain. Starting from our previously disclosed piperidine scaffold, we have identified a class of potent sigma 1 modulators. This work highlights the key SAR components that lead to the divergence in D 4 and σ1 activity. In addition, we further profile lead compounds in a panel of off-target receptors, in vitro and in vivo pharmacokinetic studies. This has culminated in the discovery of multiple σ1 receptor modulators with properties that will allow for study in animal models. [Display omitted] • An SAR campaign from D 4 antagonists to sigma 1 modulators is presented. • Key SAR points are highlighted in the manuscript which identify key areas where the D 4 and σ1 binding diverge. • In vitro and in vivo PK is also presented showing these compounds to be highly brain penetrant. • Selectivity data is presented showing these compounds are highly selective for the σ1 receptor. [ABSTRACT FROM AUTHOR]

AIMS Vatalanib is an oral anti-angiogenesis agent that inhibits vascular endothelial growth factor receptor tyrosine kinases, which in patients showed auto induction of metabolism and variability in pharmacokinetic (PK) disposition. The objective was to characterize the population PK and time-dependent change in vatalanib clearance and assess exposure-toxicity relationship in patients with myelodysplastic syndrome (MDS). METHODS This was an open-label phase II study of vatalanib in MDS patients receiving 750-1250 mg once daily in 28-day cycles. Serial blood samples were obtained and plasma vatalanib concentrations measured by HPLC. Population PK analysis was performed using NONMEM 7.2 with FO estimation since FOCE failed. The final model was evaluated using goodness-of-fit plots, bootstrap analysis, and visual predictive check. METHODS This was an open-label phase II study of vatalanib in MDS patients receiving 750-1250 mg once daily in 28-day cycles. Serial blood samples were obtained and plasma vatalanib concentrations measured by HPLC. Population PK analysis was performed using NONMEM 7.2 with FO estimation since FOCE failed. The final model was evaluated using goodness-of-fit plots, bootstrap analysis, and visual predictive check. RESULTS Pharmacokinetic data were complete for 137 patients (86 M, 51 F), of median age 70 years (range 20-91). A one-compartment model with lagged first-order absorption and time-dependent change in oral clearance was fitted to the vatalanib plasma concentration versus time data. The population means for pre-induction and post-induction oral clearance were 24.1 l h-1 (range: 9.6-45.5) and 54.9 l h-1 (range: 39.8-75.6), respectively. The apparent oral clearance increased 2.3-fold, (range: 1.7-4.1-fold) from first dose to steady state. Our data did not identify a significant relationship of the predefined covariates with vatalanib pharmacokinetics, although power to detect such a relationship was limited. CONCLUSIONS Vatalanib pharmacokinetics were highly variable and the extent of auto induction was not determined to correlate with any of the pre-defined covariates. [ABSTRACT FROM AUTHOR]

Acute kidney injury (AKI) is characterized by a sudden decrease in renal function and impacts growing number of people worldwide. RNA interference (RNAi) showed potential to treat diseases with no or limited conventional therapies, including AKI. Suitable carriers are needed to protect and selectively deliver RNAi to target cells to fully explore this therapeutic modality. Here, we report on the synthesis of chitosan modified with α-cyclam- p -toluic acid (C-CS) as a novel siRNA carrier for targeted delivery to injured kidneys. We demonstrate that conjugation of the α-cyclam- p -toluic acid to chitosan imparts the C-CS polymer with targeting and antagonistic properties to cells overexpressing chemokine receptor CXCR4. In contrast, the parent α-cyclam- p -toluic acid showed no such properties. Self-assembled C-CS/siRNA nanoparticles rapidly accumulate in the injured kidneys and show long retention in renal tubules. Apoptosis and metabolic and inflammatory pathways induced by p53 are important pathological mechanisms in the development of AKI. Nanoparticles with siRNA against p53 (sip53) were formulated and intravenously injected for attenuation of IRI-AKI. Due to the favorable accumulation in injured kidneys, the treatment with C-CS/sip53 decreased renal injury, extent of renal apoptosis, macrophage and neutrophil infiltration, and improved renal function. Overall, our study suggests that C-CS/siRNA nanoparticles have the potential to effectively accumulate and deliver therapeutic siRNAs to injured kidneys through CXCR4 binding, providing a novel way for AKI therapy. [ABSTRACT FROM AUTHOR]

Pneumococcal pneumonia is a leading cause of morbidity and mortality worldwide. An increased susceptibility is due, in part, to compromised immune function. Zinc is required for proper immune function, and an insufficient dietary intake increases the risk of pneumonia. Our group was the first to reveal that the Zn transporter, ZIP8, is required for host defense. Furthermore, the gut microbiota that is essential for lung immunity is adversely impacted by a commonly occurring defective ZIP8 allele in humans. Taken together, we hypothesized that loss of the ZIP8 function would lead to intestinal dysbiosis and impaired host defense against pneumonia. To test this, we utilized a novel myeloid-specific Zip8KO mouse model in our studies. The comparison of the cecal microbial composition of wild-type and Zip8KO mice revealed significant differences in microbial community structure. Most strikingly, upon a S. pneumoniae lung infection, mice recolonized with Zip8KO-derived microbiota exhibited an increase in weight loss, bacterial dissemination, and lung inflammation compared to mice recolonized with WT microbiota. For the first time, we reveal the critical role of myeloid-specific ZIP8 on the maintenance of the gut microbiome structure, and that loss of ZIP8 leads to intestinal dysbiosis and impaired host defense in the lung. Given the high incidence of dietary Zn deficiency and the ZIP8 variant allele in the human population, additional investigation is warranted to improve surveillance and treatment strategies. [ABSTRACT FROM AUTHOR]

Retrospective studies revealed that cigarette smoking enhances risk of incidence and worsens prognosis in pancreatic cancer (PC) patients. Poor prognosis in smoker cohort of PC patients indicates prevalence of cigarette smoke stimulated survival mechanisms yet to be explored in PC. In this study, cigarette smoke induced metabolic pathways were explored and targeted in PC. Human pancreatic ductal adenocarcinoma cell (PDAC) lines, genetically engineered mice models (GEMMs), mass spectrometry based heavy isotope-based metabolite analysis, cytotoxicity assays and Nuclear factor kappa-B (NF-kB) targeting were utilized in this study. Cigarette smoke extract (CSE) was prepared fresh each day by bubbling cell culture media with the smoke emitted from 85 mm, filtered, Code 1R6F reference cigarettes and used for in vitro procedures. High dose cigarette smoke exposure of GEMMs was achieved by daily exposure of animals to similar cigarettes, 6 h/day for a total period of 180 days. We observed that PDAC cells upregulate glutathione anabolism through cysteine uptake and glutamate cysteine ligase (GCLM), supporting survival, upon CSE exposure. In vivo, cigarette smoke exposure leads to concomitant upregulation of GCLM and activated NF-kB in the PDAC consistent with in vitro , in CSE-exposed PDAC. Finally, either inhibition of NF-kB or depletion of cysteine impaired PDAC cell survival in cigarette smoke exposed conditions through suppression of glutathione and ROS enhancement, reverted by glutathione supplementation. Our findings demonstrate scope for targeting smoke induced, NF-kB mediated, cysteine and glutathione metabolism for improving the survival of smoke addicted PDAC. [Display omitted] • Pancreatic ductal adenocarcinoma (PDAC) tolerates high dose cigarette smoke. • Cysteine uptake and de novo glutathione synthesis enhance in cigarette smoke exposed PDAC. • NF-κB regulates glutathione synthesis in cigarette smoke exposed PDAC. • Targeting NF-κB impairs ROS balance in cigarette smoke exposed PDAC. • PDAC succumbs to cigarette smoke upon cysteine depletion. [ABSTRACT FROM AUTHOR]

• Optimized sensitive and rapid LC–MS/MS method of phylloquinone and menaquinone-4 quantification in human plasma. • The assay was linear over the concentration range from 0.01 to 50 ng/mL for both analytes. • The validated method was successfully applied to a clinical study in patients with neurodegenerative diseases. A rapid and sensitive liquid chromatographic-tandem mass spectrometric method was developed and validated to simultaneously quantitate vitamin K 1 (PK) and vitamin K 2 (MK-4) concentrations in human plasma to evaluate nutritional interventions. Charcoal-stripped human plasma was used for standard and calibration preparation with vitamin E-d6 as the internal standard. Patient plasma samples were extracted using n-hexane and analytes separated using an ACE –PFP C18 column (50 × 2.1 mm, 3 μ) equipped with standard C 18 guard. The mobile phase consisted of 0.1 % formic acid in water and 0.1 % formic acid in methanol at a flow rate of 0.5 mL/min. Analyte quantification was achieved by using MS/MS with a positive atmospheric pressure chemical ionization in multiple reaction monitoring (MRM) mode. The lower limit of quantification was 0.01 ng/mL for both PK and MK-4. The assay was linear over the concentration range from 0.01–50 ng/mL for all analytes with a determination coefficient (r2) of 0.998 or better. The intra-and inter-day accuracy and precision were within the acceptable limits per FDA guidance. The validated method was successfully applied to a clinical study for quantification of PK and MK-4 in human plasma to assess nutritional status and dietary interventions in patients with neurodegenerative diseases. Baseline plasma concentrations of PK and MK-4 ranged from 0.23 to 1.81 ng/mL and 0.33–0.57 ng/mL, respectively. [ABSTRACT FROM AUTHOR]

A rapid, selective, and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of MO-OH-Nap tropolone (MO-OH-Nap) in mouse plasma. MO-OH-Nap is an α-substituted tropolone with anti-proliferative properties in various cancer cell lines. Detection and separation of analytes was achieved on an ACE Excel C18 (1.7 µm, 100 × 2.1 mm, MAC-MOD Analytical, Chadds Ford, PA, USA) column with mobile phase consisting of 0.05% trifluoroacetic acid in water (mobile phase A) and 0.05% trifluoroacetic acid in acetonitrile (mobile phase B), with an isocratic elution of 15:85% (A:B) at a total flow rate of 0.25 mL/min. The LC-MS/MS system was operated at unit resolution in multiple reaction monitoring (MRM) mode, using precursor ion > product ion combination of 249.10 > 202.15 m / z for MO-OH-Nap and 305.10 > 215.05 m / z for the internal standard (IS), BA-SM-OM. The MS/MS response was linear over a concentration range of 1 to 500 ng/mL with a correlation coefficient (r 2 ) of ≥0.987. The within- and between-batch precision (%RSD) and accuracy (%Bias) were within acceptable limits. The validated method was successfully applied to determine MO-OH-Nap metabolic stability, plasma protein binding, and bio-distribution studies of MO-OH-Nap in CD-1 mice.

Congenital heart disease (CHD) is the most common birth defect, occurring in roughly 40,000 U.S. births annually. Malnutrition and feeding intolerance (FI) in CHD range from 30% to 42% and are associated with longer hospitalization and increased mortality. Cardiopulmonary bypass (CPB) required for surgical repair of CHD induces a systemic inflammatory response worsening intestinal dysbiosis and leading to intestinal epithelial barrier dysfunction (EBD), possibly contributing to postoperative FI. The objective of this study was to determine the relationship of postoperative FI with intestinal microbiome, short-chain fatty acids (SCFAs), and EBD in pediatric CHD after cardiac surgery. This was a prospective study of patients aged 0-15 years undergoing cardiac surgery with CPB. Samples were collected preoperatively and postoperatively to evaluate the gut microbiome, plasma EBD markers, short-chain fatty acids (SCFAs), and plasma cytokines. Clinical data were collected to calculate a FI score and evaluate patient status postoperatively. We enrolled 26 CPB patients and identified FI ( n = 13). Patients with FI had unique microbial shifts with the reduced SCFA-producing organisms Rothia , Clostridium innocuum , and Intestinimonas . Patients who developed FI had associated elevations in the plasma EBD markers claudin-2 ( P < 0.05), claudin-3 ( P < 0.01), and fatty acid binding protein ( P < 0.01). Patients with FI had reduced plasma and stool SCFAs. Mediation analysis showed the microbiome functional shift was associated with reductions in stool butyric and propionic acid in patients with FI. In conclusion, we provide novel evidence that intestinal dysbiosis, markers of EBD, and SCFA depletion are associated with FI. These data will help identify mechanisms and therapeutics to improve clinical outcomes following pediatric cardiac surgery. NEW & NOTEWORTHY Feeding intolerance contributes to postoperative morbidity following pediatric cardiac surgery. The intestinal microbiome and milieu play a vital role in gut function. Short-chain fatty acids are gut and cardioprotective metabolites produced by commensal bacteria and help maintain appropriate barrier function. Depletion of these metabolites and barrier dysfunction contribute to postoperative feeding intolerance following cardiac surgery. Identifying mechanistic targets to improve the intestinal milieu with the goal of improved nutrition and clinical outcomes is critical.

A sensitive and selective liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of dual PI3K/BRD4 inhibitor SF2523 in mouse plasma. The analysis was performed on a UPLC system connected to a Shimadzu 8060 mass spectrometer by electrospray ionization in positive multiple reaction monitoring mode. Chromatographic separation was carried out on an ACE Excel C 18 column with a gradient elution containing 0.1% formic acid and methanol as the mobile phase. The linearity was conducted in the concentration range 0.1-500 ng/ml for SF2523 in 100 μl of plasma. The inter- and intra-batch precision (RSD) were both lower than 13.5%, with the accuracy (percentage bias) ranging from -10.03 to 11.56%. The validated method was successfully applied to plasma protein binding and in vitro metabolism studies. SF2523 was highly bound to mouse plasma proteins (>95% bound). Utilizing mouse S9 fractions, a total of seven phase I and II metabolites were identified with hydroxylation found to be the major metabolic pathway. Metabolite identification included analysis of retention behaviors, molecular weight changes and MS/MS fragment patterns of SF2523 and the metabolites. This newly developed and validated method allows the rapid and easy determination of the SF2523 concentration with high sensitivity in a low sample volume and can be applied to future pre-clinical studies., (© 2023 The Authors. Biomedical Chromatography published by John Wiley & Sons Ltd.)

Aldo-keto reductase 1C3 (AKR1C3) is overexpressed in castration-resistant prostate cancer where it acts to drive proliferation and aggressiveness by producing androgens. The reductive action of the enzyme leads to chemoresistance development against various clinical antineoplastics across a range of cancers. Herein, we report the continued optimization of selective AKR1C3 inhibitors and the identification of 5r , a potent AKR1C3 inhibitor (IC 50 = 51 nM) with >1216-fold selectivity for AKR1C3 over closely related isoforms. Due to the cognizance of the poor pharmacokinetics associated with free carboxylic acids, a methyl ester prodrug strategy was pursued. The prodrug 4r was converted to free acid 5r in vitro in mouse plasma and in vivo. The in vivo pharmacokinetic evaluation revealed an increase in systemic exposure and increased the maximum 5r concentration compared to direct administration of the free acid. The prodrug 4r demonstrated a dose-dependent effect to reduce the tumor volume of 22Rv1 prostate cancer xenografts without observed toxicity.

The intestinal microbiome is essential to human health and homeostasis, and is implicated in the pathophysiology of disease, including congenital heart disease and cardiac surgery. Improving the microbiome and reducing inflammatory metabolites may reduce systemic inflammation following cardiac surgery with cardiopulmonary bypass (CPB) to expedite recovery post-operatively. Limited research exists in this area and identifying animal models that can replicate changes in the human intestinal microbiome after CPB is necessary. We used a piglet model of CPB with two groups, CPB (n=5) and a control group with mechanical ventilation (n=7), to evaluate changes to the microbiome, intestinal barrier dysfunction and intestinal metabolites with inflammation after CPB. We identified significant changes to the microbiome, barrier dysfunction, intestinal short-chain fatty acids and eicosanoids, and elevated cytokines in the CPB/deep hypothermic circulatory arrest group compared to the control group at just 4 h after intervention. This piglet model of CPB replicates known human changes to intestinal flora and metabolite profiles, and can be used to evaluate gut interventions aimed at reducing downstream inflammation after cardiac surgery with CPB., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Acne vulgaris is a complex skin disease involving infection by Cutibacterium acnes, inflammation, and hyperkeratinization. We evaluated the activity of the retinoid 6-[3-(adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) and 16 other retinoid analogs as potential anti- C. acnes compounds and found that CD437 displayed the highest antimicrobial activity with an MIC against C. acnes (ATCC 6919 and HM-513) of 1 μg/mL. CD437 demonstrated an MBC of 2 μg/mL compared to up to 64 μg/mL for the retinoid adapalene and up to 16 μg/mL for tetracycline, which are commonly used clinically to treat acne. Membrane permeability assays demonstrated that exposure of C. acnes ATCC 6919 to CD437 damaged the integrity of C. acnes ATCC 6919 bacterial membranes, and this finding was confirmed with scanning electron microscopy. Additionally, CD437 downregulated the expression of C. acnes ATCC 6919 virulence factors, including the genes encoding Christie-Atkins-Munch-Petersen factor 1 (CAMP1), CAMP2, glycerol-ester hydrolase B (GehB), sialidase B, and neuraminidase. In a mouse skin infection model of C. acnes ATCC 6919, topical treatment with CD437 ameliorated skin lesions and reduced the bacterial burden in situ ( P < 0.001). In human NHEK primary cells, CD437 reduced the transcriptional levels of the coding genes for inflammatory cytokines (interleukin-1α, ~10-fold; interleukin-6, ~20-fold; interleukin-8, ~30-fold; and tumor necrosis factor-alpha, ~6-fold) and downregulated the transcriptional levels of KRT10 (~10-fold), FLG (~4-fold), and TGM1 (~2-fold), indicating that CD437 can diminish inflammation and hyperkeratinization. In summary, CD437 deserves further attention for its dual function as a potential acne therapeutic that potentially acts on both the pathogen and the host.

Background: Posttraumatic stress disorder (PTSD) is a mental health condition triggered by exposure to traumatic events in an individual's life. Patients with PTSD are also at a higher risk for comorbidities. However, it is not well understood how PTSD affects human health and/or promotes the risk for comorbidities. Nevertheless, patients with PTSD harbor a proinflammatory milieu and dysbiotic gut microbiota. Gut barrier integrity helps to maintain normal gut homeostasis and its dysregulation promotes gut dysbiosis and inflammation., Methods: We used a mouse model of repeated social defeat stress (RSDS), a preclinical model of PTSD. Behavioral studies, metagenomics analysis of the microbiome, gut permeability assay (on mouse colon, using an Ussing chamber), immunoblotting, and immunohistochemical analyses were performed. Polarized intestinal epithelial cells and 3-dimensional crypt cultures were used for mechanistic analysis., Results: The RSDS mice harbor a heightened proinflammatory gut environment and microbiota dysbiosis. The RSDS mice further showed significant dysregulation of gut barrier functions, including transepithelial electrical resistance, mucin homeostasis, and antimicrobial responses. RSDS mice also showed a specific increase in intestinal expression of claudin-2, a tight junction protein, and epinephrine, a stress-induced neurotransmitter. Treating intestinal epithelial cells or 3-dimensional cultured crypts with norepinephrine or intestinal luminal contents (fecal contents) upregulated claudin-2 expression and inhibited transepithelial electrical resistance., Conclusions: Traumatic stress induces dysregulation of gut barrier functions, which may underlie the observed gut microbiota changes and proinflammatory gut milieu, all of which may have an interdependent effect on the health and increased risk of comorbidities in patients with PTSD., (© 2023 The Authors.)