Stability and compatibility of an intramuscular fetal anesthetic cocktail for fetal intervention.

INTRODUCTION: To evaluate chemical stability and physical compatibility when combining fentanyl, rocuronium, and atropine in a fixed ratio to support intramuscular drug delivery during fetal intervention and surgery. METHODS: A highly concentrated combination of fentanyl, rocuronium, and atropine was created based on common prescribing practices at a maternal fetal care center. Chemical stability testing was completed using liquid chromatograph mass spectrometry-mass spectrometry (LC/MS-MS) to detect and quantitate atropine, rocuronium, and fentanyl, with fentanyl-d5 being an internal standard at 6-, 12, 24-, and 36-hours following sample preparation. Physical compatibility testing was completed using United State Pharmacopeia (USP)<788> recommended analytical technique of light obscuration (LO) in addition to novel backgrounded membrane imaging (BMI) at 6- and 24-hours following sample preparation. Physical compatibility was determined using USP<788> particle count limits for both techniques. RESULTS: Based on LC/MS-MS results, the samples retained expected medication concentrations at all time points tested. For physical compatibility testing, the particle counts met criteria to be considered compatible per USP<788> large volume particle count thresholds at 6 hours by both methods but exceeded tolerable thresholds at 24 hours. DISCUSSION/CONCLUSION: The combination of rocuronium, fentanyl, and atropine for intramuscular fetal administration are physically compatible and chemically stable for 6 hours.

LAT1 enables T cell activation under inflammatory conditions.

The aim of this study was to assess the L-type amino acid transporter-1 (LAT1) as a possible therapeutic target for rheumatoid arthritis (RA). Synovial LAT1 expression in RA was monitored by immunohistochemistry and transcriptomic datasets. The contribution of LAT1 to gene expression and immune synapse formation was assessed by RNA-sequencing and total internal reflection fluorescent (TIRF) microscopy, respectively. Mouse models of RA were used to assess the impact of therapeutic targeting of LAT1. LAT1 was strongly expressed by CD4+ T cells in the synovial membrane of people with active RA and the level of expression correlated with levels of ESR and CRP as well as DAS-28 scores. Deletion of LAT1 in murine CD4+ T cells inhibited the development of experimental arthritis and prevented the differentiation of CD4+ T cells expressing IFN-γ and TNF-α, without affecting regulatory T cells. LAT1 deficient CD4+ T cells demonstrated reduced transcription of genes associated with TCR/CD28 signalling, including Akt1, Akt2, Nfatc2, Nfkb1 and Nfkb2. Functional studies using TIRF microscopy revealed a significant impairment of immune synapse formation with reduced recruitment of CD3ζ and phospho-tyrosine signalling molecules in LAT1 deficient CD4+ T cells from the inflamed joints but not the draining lymph nodes of arthritic mice. Finally, it was shown that a small molecule LAT1 inhibitor, currently undergoing clinical trials in man, was highly effective in treating experimental arthritis in mice. It was concluded that LAT1 plays a critical role in activation of pathogenic T cell subsets under inflammatory conditions and represents a promising new therapeutic target for RA.

Retraction: Nuclear Morphometric Analysis of Leydig Cells of Male Pubertal Rats Exposed In Utero to Di(n-butyl) Phthalate.

[This retracts the article on p. 439 in vol. 26, PMID: 24526819.].

Extended Stability of Isoproterenol Hydrochloride Injection in Polyvinyl Chloride Bags Stored in Amber Ultraviolet Light Blocking Bags.

Purpose:Evaluate the stability of isoproterenol hydrochloride injection in 0.9% sodium chloride in polyvinyl chloride bags for up to 90 days. Methods: Dilutions of isoproterenol hydrochloride injection to a concentration of 4 µg/mL were performed under aseptic conditions. The bags were stored in amber ultraviolet light blocking bags at room temperature (23°C-25°C) or under refrigeration (3°C-5°C). Three samples of each preparation and storage environment were analyzed on days 0, 2, 14, 30, 45, 60, and 90. Physical stability was performed by visual examination. The pH was assessed at baseline, each analysis day, and upon final degradation evaluation. Sterility of the samples was not assessed. Chemical stability of isoproterenol hydrochloride was evaluated using liquid chromatography with tandem mass spectrometry. Samples were considered stable if there was <10% degradation of the initial concentration. Results: Isoproterenol hydrochloride diluted to 4 µg/mL with 0.9% sodium chloride injection was physically stable throughout the study. No precipitation was observed. At days 2, 14, 30, 45, 60, and 90 all bags diluted to 4 µg/mL had <10% degradation when stored under refrigeration (3°C-5°C) or stored at room temperature (23°C-25°C). Conclusion: Isoproterenol hydrochloride diluted to a concentration of 4 µg/mL with 0.9% sodium chloride for injection in ultraviolet light blocking bags was stable for 90 days at room temperature and under refrigeration.

Extended Stability of Vasopressin Injection in Polyvinyl Chloride Bags and Polypropylene Syringes and Its Impact on Critically Ill Patient Care and Medication Waste.

Background. Vasopressin is frequently utilized for a variety of shock states in critically ill patients. Short stability (≤24 hours) after intravenous admixture with current manufacturer labeling requires just in time preparation and may lead to delays in therapy and increased medication waste. We aimed to evaluate vasopressin stability in 0.9% sodium chloride stored in polyvinyl chloride bags and polypropylene syringes for up to 90 days. Additionally, we evaluated the impact of extended stability on the time to administration and cost savings from reduced medical waste at an academic medical center. Methods. Dilutions of vasopressin to concentrations of 0.4 and 1.0 unit/mL were performed under aseptic conditions. The bags and syringes were stored at room temperature (23°C-25°C) or under refrigeration (3°C-5°C). Three samples of each preparation and storage environment were analyzed on days 0, 2, 14, 30, 45, 60, and 90. Physical stability was performed by visual examination. The pH was assessed at each point and upon final degradation evaluation. Sterility of the samples was not assessed. Chemical stability of vasopressin was evaluated using liquid chromatography with tandem mass spectrometry. Samples were considered stable if there was <10% degradation of the initial concentration. Results. Vasopressin diluted to 0.4 and 1.0 unit/mL with 0.9% sodium chloride injection was physically stable throughout the study. No precipitation was observed. At days 2, 14, 30, 45, 60, and 90 all bags and syringes diluted to 0.4 units/mL had <10% degradation. Vasopressin diluted to 1 unit/mL and stored under refrigeration had <10% degradation at all measured days, but when stored under room temperature was found to have >10% degradation at day 30. Implementation of a batching process resulted in reduced waste ($185 300) and improved time to administration (26 vs 4 minutes). Conclusion. Vasopressin diluted to a concentration of 0.4 units/mL with 0.9% sodium chloride injection is stable for 90 days at room temperature and under refrigeration. When diluted to 1.0 unit/mL with 0.9% sodium chloride injection it is stable for 90 days under refrigeration. Use of extended stability and sterility testing to batch prepare infusions may lead to improved time to administration and cost savings from reduced medication waste.

Circulating cyclic adenosine monophosphate concentrations in milrinone treated paediatric patients after congenital heart surgery.

BACKGROUND: Milrinone is a phosphodiesterase type 3 inhibitor that results in a positive inotropic effect in the heart through an increase in cyclic adenosine monophosphate. The purpose of this study was to evaluate circulating cyclic adenosine monophosphate and milrinone concentrations in milrinone treated paediatric patients undergoing congenital heart surgery. METHODS: Single-centre prospective observational pilot study from January 2015 to December 2017 including children aged birth to 18 years. Milrinone and circulating cyclic adenosine monophosphate concentrations were measured at four time points through the first post-operative day and compared between patients with and without low cardiac output syndrome, defined using clinical and laboratory criteria. RESULTS: Fifty patients were included. Nine (18%) developed low cardiac output syndrome. For all patients, 22% had single ventricle heart disease. The density and distribution of cyclic adenosine monophosphate concentrations varied between those with and without low cardiac output syndrome but were not significantly different. Milrinone concentrations increased in all patients. Paired t-tests demonstrated an increase in circulating cyclic adenosine monophosphate concentrations during the post-operative period among patients without low cardiac output syndrome. CONCLUSIONS: In this prospective observational study, circulating cyclic adenosine monophosphate concentrations increased in those without low cardiac output syndrome during the first 24 post-operative hours and milrinone concentrations increased in all patients. Further study of the utility of cyclic adenosine monophosphate concentrations in milrinone treated patients is necessary.

Bitter melon juice intake with gemcitabine intervention circumvents resistance to gemcitabine in pancreatic patient-derived xenograft tumors.

Chemoresistance to gemcitabine (GEM)-a frontline chemotherapeutic, resulting from its dysfunctional uptake and metabolism in cancer cells, is a major contributing factor for failed therapy in pancreatic cancer (PanC) patients. Therefore, there is an urgent need for agents that could reverse GEM resistance and allow continued chemosensitivity to the drug. We employed natural nontoxic agent (with anti-PanC potential) bitter melon juice (BMJ) and GEM to examine their combinatorial benefits against tumorigenesis of PanC patient-derived xenograft (PDX)-pancreatic ductal adenocarcinomas explants PDX272 (wild-type KRAS), PDX271 (mutant KRAS and SMAD4), and PDX266 (mutant KRAS). Anti-PanC efficacy of single agents vs combination in the three tumor explants, both at the end of active dosing regimen and following a drug-washout phase were compared. In animal studies, GEM alone treatment significantly inhibited PDX tumor growth, but effects were not sustained, as GEM-treated tumors exhibited regrowth posttreatment termination. However, combination-regimen displayed enhanced and sustained efficacy. Mechanistic assessments revealed that overcoming GEM resistance by coadministration with BMJ was possibly due to modulation of GEM transport/metabolism pathway molecules (ribonucleotide reductase regulatory subunit M1, human equilibrative nucleoside transporter 1, and deoxycytidine kinase). Study outcomes, highlighting significantly higher and sustained efficacy of GEM in combination with BMJ, make a compelling case for a clinical trial in PanC patients, wherein BMJ could be combined with GEM to target and overcome GEM resistance. In addition, given their specific effectiveness against KRAS-mutant tumors, this combination could be potentially beneficial to a broader PanC patient population.

Discovery and characterization of small molecules that target the GTPase Ral.

The Ras-like GTPases RalA and RalB are important drivers of tumour growth and metastasis. Chemicals that block Ral function would be valuable as research tools and for cancer therapeutics. Here we used protein structure analysis and virtual screening to identify drug-like molecules that bind to a site on the GDP-bound form of Ral. The compounds RBC6, RBC8 and RBC10 inhibited the binding of Ral to its effector RALBP1, as well as inhibiting Ral-mediated cell spreading of murine embryonic fibroblasts and anchorage-independent growth of human cancer cell lines. The binding of the RBC8 derivative BQU57 to RalB was confirmed by isothermal titration calorimetry, surface plasmon resonance and (1)H-(15)N transverse relaxation-optimized spectroscopy (TROSY) NMR spectroscopy. RBC8 and BQU57 show selectivity for Ral relative to the GTPases Ras and RhoA and inhibit tumour xenograft growth to a similar extent to the depletion of Ral using RNA interference. Our results show the utility of structure-based discovery for the development of therapeutics for Ral-dependent cancers.

Evaluation of intravenous direct thrombin inhibitor monitoring tests: Correlation with plasma concentrations and clinical outcomes in hospitalized patients.

The parenterally administered direct thrombin inhibitors (DTIs) argatroban and bivalirudin are effective anticoagulants for acute heparin-induced thrombocytopenia (HIT) treatment. The activated partial thromboplastin time (aPTT) has classically been used as the monitoring test to assess degree of anticoagulation, however concerns exist with using aPTT to monitor DTI therapy. In this observational study plasma samples from DTI treated patients were analyzed by aPTT, dilute thrombin time (dTT) and ecarin chromogenic assay (ECA) to delineate results into concordant and discordant groups. Discordant samples were further analyzed via liquid chromatography with tandem mass spectrometry (LC MS/MS). In total 101 patients with 198 samples were evaluated. Discordance between tests were frequent (59% of DTI treated patients). Bivalirudin aPTT vs dTT discordance was observed in 45% (57/126) of samples. Amongst bivalirudin samples with test discordance dTT results were more likely to be concordant with LC MS/MS than the aPTT (77% vs 9%, p < 0.0001). Argatroban aPTT vs dTT discordance was observed in 43% (31/72) and aPTT vs ECA discordance was observed in 40% (29/72) of samples. Amongst argatroban samples with test discordance both the dTT and ECA tests were more likely to have concordant results with LC MS/MS than the aPTT (88% vs 9%, p < 0.0001 for both dTT and ECA tests). There were no differences between discordant and concordant patient groups in a composite outcome of bleeding/thrombosis rate (23% vs 27%, p = 0.689). Further investigation is warranted to elucidate the effect of suitable monitoring assays on patient outcomes in the setting of DTI therapy.

Bitter melon juice-intake modulates glucose metabolism and lactate efflux in tumors in its efficacy against pancreatic cancer.

The established role of bitter melon juice (BMJ), a natural product, in activating master metabolic regulator adenosine monophosphate-activated protein kinase in pancreatic cancer (PanC) cells served as a basis for pursuing deeper investigation into the underlying metabolic alterations leading to BMJ efficacy in PanC. We investigated the comparative metabolic profiles of PanC cells with differential KRAS mutational status on BMJ exposure. Specifically, we employed nuclear magnetic resonance (NMR) metabolomics and in vivo imaging platforms to understand the relevance of altered metabolism in PanC management by BMJ. Multinuclear NMR metabolomics was performed, as a function of time, post-BMJ treatment followed by partial least square discriminant analysis assessments on the quantitative metabolic data sets to visualize the treatment group clustering; altered glucose uptake, lactate export and energy state were identified as the key components responsible for cell death induction. We next employed PANC1 xenograft model for assessing in vivo BMJ efficacy against PanC. Positron emission tomography ([18FDG]-PET) and magnetic resonance imaging on PANC1 tumor-bearing animals reiterated the in vitro results, with BMJ-associated significant changes in tumor volumes, tumor cellularity and glucose uptake. Additional studies in BMJ-treated PanC cells and xenografts displayed a strong decrease in the expression of glucose and lactate transporters GLUT1 and MCT4, respectively, supporting their role in metabolic changes by BMJ. Collectively, these results highlight BMJ-induced modification in PanC metabolomics phenotype and establish primarily lactate efflux and glucose metabolism, specifically GLUT1 and MCT4 transporters, as the potential metabolic targets underlying BMJ efficacy in PanC.

Inhibition of the amino-acid transporter LAT1 demonstrates anti-neoplastic activity in medulloblastoma.

Most cases of medulloblastoma (MB) occur in young children. While the overall survival rate can be relatively high, current treatments combining surgery, chemo- and radiotherapy are very destructive for patient development and quality of life. Moreover, aggressive forms and recurrences of MB cannot be controlled by classical therapies. Therefore, new therapeutic approaches yielding good efficacy and low toxicity for healthy tissues are required to improve patient outcome. Cancer cells sustain their proliferation by optimizing their nutrient uptake capacities. The L-type amino acid transporter 1 (LAT1) is an essential amino acid carrier overexpressed in aggressive human cancers that was described as a potential therapeutic target. In this study, we investigated the therapeutic potential of JPH203, a LAT1-specific pharmacological inhibitor, on two independent MB cell lines belonging to subgroups 3 (HD-MB03) and Shh (DAOY). We show that while displaying low toxicity towards normal cerebral cells, JPH203 disrupts AA homeostasis, mTORC1 activity, proliferation and survival in MB cells. Moreover, we demonstrate that a long-term treatment with JPH203 does not lead to resistance in MB cells. Therefore, this study suggests that targeting LAT1 with JPH203 is a promising therapeutic approach for MB treatment.

The glutamine transporter ASCT2 (SLC1A5) promotes tumor growth independently of the amino acid transporter LAT1 (SLC7A5).

The transporters for glutamine and essential amino acids, ASCT2 (solute carrier family 1 member 5, SLC1A5) and LAT1 (solute carrier family 7 member 5, SLC7A5), respectively, are overexpressed in aggressive cancers and have been identified as cancer-promoting targets. Moreover, previous work has suggested that glutamine influx via ASCT2 triggers essential amino acids entry via the LAT1 exchanger, thus activating mechanistic target of rapamycin complex 1 (mTORC1) and stimulating growth. Here, to further investigate whether these two transporters are functionally coupled, we compared the respective knockout (KO) of either LAT1 or ASCT2 in colon (LS174T) and lung (A549) adenocarcinoma cell lines. Although ASCT2KO significantly reduced glutamine import (>60% reduction), no impact on leucine uptake was observed in both cell lines. Although an in vitro growth-reduction phenotype was observed in A549-ASCT2KO cells only, we found that genetic disruption of ASCT2 strongly decreased tumor growth in both cell lines. However, in sharp contrast to LAT1KO cells, ASCT2KO cells displayed no amino acid (AA) stress response (GCN2/EIF2a/ATF4) or altered mTORC1 activity (S6K1/S6). We therefore conclude that ASCT2KO reduces tumor growth by limiting AA import, but that this effect is independent of LAT1 activity. These data were further supported by in vitro cell proliferation experiments performed in the absence of glutamine. Together these results confirm and extend ASCT2's pro-tumoral role and indicate that the proposed functional coupling model of ASCT2 and LAT1 is not universal across different cancer types.

Differential effect of grape seed extract and its active constituent procyanidin B2 3,3″-di-O-gallate against prostate cancer stem cells.

The present study aimed to determine whether grape seed extract (GSE) procyanidin mix, and its active constituent procyanidin B2 3,3″-di-O-gallate (B2G2) have the potential to target cancer stem cells (CSCs) in prostate cancer (PCa). The CSC populations were isolated and purified based on CD44+ -α2ß1high surface markers in PCa cell lines LNCaP, C4-2B, 22Rv1, PC3, and DU145, and then subjected to prostasphere formation assays in the absence or presence of GSE or B2G2. Results indicated that at lower doses (<15 µg) , the GSE procyanidin mix produced activity in unsorted prostate cancer antigen (PCA) cells, but not in sorted; however, multiple treatments with low dose GSE over a course of time inhibited sphere formation by sorted PCA CSCs. Importantly, B2G2 demonstrated significant potential to target both unsorted and sorted CSCs at lower doses. As formation of spheroids, under specific in vitro conditions, is a measure of stemness, these results indicated the potential of both GSE and B2G2 to target the self-renewal of CSC in PCa cell lines, though B2G2 was more potent in its efficacy. Subsequent mechanistic studies revealed that both GSE procyanidins and B2G2 strongly decreased the constitutive as well as Jagged1 (Notch1 ligand)-induced activated Notch1 pathway. In totality, these in vitro studies warrant extensive dose-profiling-based assessments in vivo settings to conclusively determine the impact on CSC pool kinetics on the efficacy of both GSE and B2G2 to target PCa growth as well as tumor relapse.

Carnosine protects cardiac myocytes against lipid peroxidation products.

Endogenous histidyl dipeptides such as carnosine (ß-alanine-L-histidine) form conjugates with lipid peroxidation products such as 4-hydroxy-trans-2-nonenal (HNE and acrolein), chelate metals, and protect against myocardial ischemic injury. Nevertheless, it is unclear whether these peptides protect against cardiac injury by directly reacting with lipid peroxidation products. Hence, to examine whether changes in the structure of carnosine could affect its aldehyde reactivity and metal chelating ability, we synthesized methylated analogs of carnosine, balenine (ß-alanine-Nτ-methylhistidine) and dimethyl balenine (DMB), and measured their aldehyde reactivity and metal chelating properties. We found that methylation of Nτ residue of imidazole ring (balenine) or trimethylation of carnosine backbone at Nτ residue of imidazole ring and terminal amine group dimethyl balenine (DMB) abolishes the ability of these peptides to react with HNE. Incubation of balenine with acrolein resulted in the formation of single product (m/z 297), whereas DMB did not react with acrolein. In comparison with carnosine, balenine exhibited moderate acrolein quenching capacity. The Fe2+ chelating ability of balenine was higher than that of carnosine, whereas DMB lacked chelating capacity. Pretreatment of cardiac myocytes with carnosine increased the mean lifetime of myocytes superfused with HNE or acrolein compared with balenine or DMB. Collectively, these results suggest that carnosine protects cardiac myocytes against HNE and acrolein toxicity by directly reacting with these aldehydes. This reaction involves both the amino group of ß-alanyl residue and the imidazole residue of L-histidine. Methylation of these sites prevents or abolishes the aldehyde reactivity of carnosine, alters its metal-chelating property, and diminishes its ability to prevent electrophilic injury.

Identification of a novel biomarker for pyridoxine-dependent epilepsy: Implications for newborn screening.

Pyridoxine-dependent epilepsy (PDE) is often characterized as an early onset epileptic encephalopathy with dramatic clinical improvement following pyridoxine supplementation. Unfortunately, not all patients present with classic neonatal seizures or respond to an initial pyridoxine trial, which can result in the under diagnosis of this treatable disorder. Restriction of lysine intake and transport is associated with improved neurologic outcomes, although treatment should be started in the first year of life to be effective. Because of the documented diagnostic delay and benefit of early treatment, we aimed to develop a newborn screening method for PDE. Previous studies have demonstrated the accumulation of Δ1 -piperideine-6-carboxylate and α-aminoadipic semialdehyde in individuals with PDE, although these metabolites are unstable at room temperature (RT) limiting their utility for newborn screening. As a result, we sought to identify a biomarker that could be applied to current newborn screening paradigms. We identified a novel metabolite, 6-oxo-pipecolate (6-oxo-PIP), which accumulates in substantial amounts in blood, plasma, urine, and cerebral spinal fluid of individuals with PDE. Using a stable isotope-labeled internal standard, we developed a nonderivatized liquid chromatography tandem mass spectrometry-based method to quantify 6-oxo-PIP. This method replicates the analytical techniques used in many laboratories and could be used with few modifications in newborn screening programs. Furthermore, 6-oxo-PIP was measurable in urine for 4 months even when stored at RT. Herein, we report a novel biomarker for PDE that is stable at RT and can be quantified using current newborn screening techniques.

The genotypic spectrum of ALDH7A1 mutations resulting in pyridoxine dependent epilepsy: A common epileptic encephalopathy.

Pyridoxine dependent epilepsy (PDE) is a treatable epileptic encephalopathy characterized by a positive response to pharmacologic doses of pyridoxine. Despite seizure control, at least 75% of individuals have intellectual disability and developmental delay. Current treatment paradigms have resulted in improved cognitive outcomes emphasizing the importance of an early diagnosis. As genetic testing is increasingly accepted as first tier testing for epileptic encephalopathies, we aimed to provide a comprehensive overview of ALDH7A1 mutations that cause PDE. The genotypes, ethnic origin and reported gender was collected from 185 subjects with a diagnosis of PDE. The population frequency for the variants in this report and the existing literature were reviewed in the Genome Aggregation Database (gnomAD). Novel variants identified in population databases were also evaluated through in silico prediction software and select variants were over-expressed in an E.coli-based expression system to measure α-aminoadipic semialdehyde dehydrogenase activity and production of α-aminoadipic acid. This study adds 47 novel variants to the literature resulting in a total of 165 reported pathogenic variants. Based on this report, in silico predictions, and general population data, we estimate an incidence of approximately 1:64,352 live births. This report provides a comprehensive overview of known ALDH7A1 mutations that cause PDE, and suggests that PDE may be more common than initially estimated. Due to the relative high frequency of the disease, the likelihood of under-diagnosis given the wide clinical spectrum and limited awareness among clinicians as well as the cognitive improvement noted with early treatment, newborn screening for PDE may be warranted.

Biomarkers of oxidative stress, inflammation, and vascular dysfunction in inherited cystathionine ß-synthase deficient homocystinuria and the impact of taurine treatment in a phase 1/2 human clinical trial.

STUDY OBJECTIVE: A phase 1/2 clinical trial was performed in individuals with cystathionine ß synthase (CBS) deficient homocystinuria with aims to: (a) assess pharmacokinetics and safety of taurine therapy, (b) evaluate oxidative stress, inflammation, and vascular function in CBS deficiency, and (c) evaluate the impact of short-term taurine treatment. METHODS: Individuals with pyridoxine-nonresponsive CBS deficiency with homocysteine >50 µM, without inflammatory disorder or on antioxidant therapy were enrolled. Biomarkers of oxidative stress and inflammation, endothelial function (brachial artery flow-mediated dilation [FMD]), and disease-related metabolites obtained at baseline were compared to normal values. While maintaining current treatment, patients were treated with 75 mg/kg taurine twice daily, and treatment response assessed after 4 hours and 4 days. RESULTS: Fourteen patients (8-35 years; 8 males, 6 females) were enrolled with baseline homocysteine levels 161 ± 67 µM. The study found high-dose taurine to be safe when excluding preexisting hypertriglyceridemia. Taurine pharmacokinetics showed a rapid peak level returning to near normal levels at 12 hours, but had slow accumulation and elevated predosing levels after 4 days of treatment. Only a single parameter of oxidative stress, 2,3-dinor-8-isoprostaglandin-F2α, was elevated at baseline, with no elevated inflammatory parameters, and no change in FMD values overall. Taurine had no effect on any of these parameters. However, the effect of taurine was strongly related to pretreatment FMD values; and taurine significantly improved FMD in the subset of individuals with pretreatment FMD values <10% and in individuals with homocysteine levels >125 µM, pertinent to endothelial function. CONCLUSION: Taurine improves endothelial function in CBS-deficient homocystinuria in patients with preexisting reduced function.

ATM/G6PD-driven redox metabolism promotes FLT3 inhibitor resistance in acute myeloid leukemia.

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML, they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism, resulting in impaired production of the antioxidant factor glutathione, which was further impaired by ATM or G6PD inactivation. Moreover, FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo, revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML.

Procyanidin B2 3,3″-di-O-gallate induces oxidative stress-mediated cell death in prostate cancer cells via inhibiting MAP kinase phosphatase activity and activating ERK1/2 and AMPK.

Neoplastic cells exhibit higher oxidative stress compared to normal cells; however, antioxidants based clinical trials have mostly failed. Another attractive therapeutic approach is to further increase the oxidative stress in cancer cells leading to cell death. Herein, we show that Procyanidin B2 3,3″-di-O-gallate (B2G2), the most active constituent of grape seed extract, treatment causes cell death in human prostate cancer (PCa) cells (LNCaP and 22Rv1) via increasing the reactive oxygen species (ROS) generation. Mechanistically, B2G2 treatment decreased the mitochondrial electron transport chain complex III activity leading to enhanced mitochondrial superoxide generation and decreased ATP production in LNCaP cells. Additional molecular studies revealed that B2G2-induced cell death was mediated mainly through ROS-induced sustained activation of ERK1/2, which was due to inhibition of MAP kinase phosphatase (MKP) activity as over-expression of MKP3 in LNCaP cells conferred significant protection against B2G2-induced cell death. Along with ERK1/2, AMP-activated protein kinase α (AMPKα) was also activated by B2G2 treatment, and pre-treatment with AMPKα inhibitor compound C significantly reversed the cytotoxic effects of B2G2 in LNCaP cells. Furthermore, pre-treatment of MKP3 over-expressing LNCaP cells with compound C further reduced the B2G2-induced cell death, suggesting the involvement of AMPKα along with MKP3 and ERK1/2 in the biological effects of B2G2. Together, these results for the first time identified that oxidative stress and MKP3 inhibition play a critical role in B2G2-induced cell death in PCa cells through sustained activation of both ERK1/2 and AMPKα. These results offer a unique opportunity to control this deadly malignancy through B2G2 use.

Bitter melon juice exerts its efficacy against pancreatic cancer via targeting both bulk and cancer stem cells.

Pancreatic cancer (PanC) is one of the deadliest malignancies worldwide and frontline treatment with gemcitabine becomes eventually ineffective due to increasing PanC resistance, suggesting additional approaches are needed to manage PanC. Recently, we have shown the efficacy of bitter melon juice (BMJ) against PanC cells, including those resistant to gemcitabine. As cancer stem cells (CSCs) are actively involved in PanC initiation, progression, relapse and drug-resistance, here we assessed BMJ ability in targeting pancreatic cancer-associated cancer stem cells (PanC-CSCs). We found BMJ efficacy against CD44+ /CD24+ /EpCAMhigh enriched PanC-CSCs in spheroid assays; BMJ also increased the sensitivity of gemcitabine-resistant PanC-CSCs. Exogenous addition of BMJ to PanC-CSC generated spheroids (not pre-exposed to BMJ) also significantly reduced spheroid number and size. Mechanistically, BMJ effects were associated with a decrease in the expression of genes and proteins involved in PanC-CSC renewal and proliferation. Specifically, immunofluorescence staining showed that BMJ decreases protein expression/nuclear localization of CSC-associated transcription factors SOX2, OCT4 and NANOG, and CSC marker CD44. Immunohistochemical analysis of MiaPaCa2 xenografts from BMJ treated animals also showed a significant decrease in the levels of CSC-associated transcription factors. Together, these results show BMJ potential in targeting PanC-CSC pool and associated regulatory pathways, suggesting the need for further investigation of its efficacy against PanC growth and progression including gemcitabine-resistant PanC.