Use of Human Induced Pluripotent Stem Cells and Kidney Organoids To Develop a Cysteamine/mTOR Inhibition Combination Therapy for Cystinosis.

BACKGROUND: Mutations in CTNS-a gene encoding the cystine transporter cystinosin-cause the rare, autosomal, recessive, lysosomal-storage disease cystinosis. Research has also implicated cystinosin in modulating the mTORC1 pathway, which serves as a core regulator of cellular metabolism, proliferation, survival, and autophagy. In its severest form, cystinosis is characterized by cystine accumulation, renal proximal tubule dysfunction, and kidney failure. Because treatment with the cystine-depleting drug cysteamine only slows disease progression, there is an urgent need for better treatments. METHODS: To address a lack of good human-based cell culture models for studying cystinosis, we generated the first human induced pluripotent stem cell (iPSC) and kidney organoid models of the disorder. We used a variety of techniques to examine hallmarks of cystinosis-including cystine accumulation, lysosome size, the autophagy pathway, and apoptosis-and performed RNA sequencing on isogenic lines to identify differentially expressed genes in the cystinosis models compared with controls. RESULTS: Compared with controls, these cystinosis models exhibit elevated cystine levels, increased apoptosis, and defective basal autophagy. Cysteamine treatment ameliorates this phenotype, except for abnormalities in apoptosis and basal autophagy. We found that treatment with everolimus, an inhibitor of the mTOR pathway, reduces the number of large lysosomes, decreases apoptosis, and activates autophagy, but it does not rescue the defect in cystine loading. However, dual treatment of cystinotic iPSCs or kidney organoids with cysteamine and everolimus corrects all of the observed phenotypic abnormalities. CONCLUSIONS: These observations suggest that combination therapy with a cystine-depleting drug such as cysteamine and an mTOR pathway inhibitor such as everolimus has potential to improve treatment of cystinosis.

Optimisation of glutathione conjugation to liposomes quantified with a validated HPLC assay.

Glutathione (GSH) grafted onto nanoliposomes (GSH-liposomes) have the potential to enhance drug delivery into the brain. GSH is known to be an unstable tripeptide, however, despite widespread use to promote active transport its stability has been largely ignored to date. Therefore this study focuses on the optimisation of GSH conjugation with liposomes, supported with a validated HPLC assay for GSH. An isocratic stability-indicating HPLC assay of GSH was developed after derivatisation of GSH with 5,5'-dithio-bis-2-nitrobenzoic acid and applied for efficient conjugation of GSH to DSPE-PEG2000-maleimide lipid, either in solution or in preformed liposomes (4% molar ratio) at pH 7.4. The conjugation rate was monitored by the HPLC assay to optimise the conjugation conditions, including GSH concentration, GSH:lipid ratio, reaction time, temperature and medium. The physiochemical properties of the resulting GSH-liposomes and their GSH densities were characterised. The HPLC method was linear in the range of 0.05-50 µg/ml, highly sensitive (limit of quantification 50 ng/ml), and accurate (98-102% recoveries) with less than 4% intra-day and inter-day variability. Interestingly, enhanced GSH stability was observed at higher GSH concentrations ≥2 mg/ml and mass spectroscopy confirmed that GSH degradation occurred predominantly by oxidation. Both the proton nuclear magnetic resonance (1H NMR) spectra and HPLC analysis of GSH concentrations confirmed the formation of GSH-PEG-DSPE conjugate. Under the optimal conditions, complete conjugation was attained either by post-insertion or direct conjugation methods with the resulting GSH-liposomes attaining a GSH density of 4% with similar size (120 nm) and zeta potential (-26.7 ±â€¯0.9 mV or -29.8 ±â€¯1.5 mV). The study provides useful information on GSH stability for the optimisation of its conjugation in liposomal formulation.

A revised biosynthetic pathway for the cofactor F420 in prokaryotes.

Cofactor F420 plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence, antibiotic biosynthesis, pro-drug activation and methanogenesis. However, the biosynthetic pathway for F420 has not been fully elucidated: neither the enzyme that generates the putative intermediate 2-phospho-L-lactate, nor the function of the FMN-binding C-terminal domain of the γ-glutamyl ligase (FbiB) in bacteria are known. Here we present the structure of the guanylyltransferase FbiD and show that, along with its archaeal homolog CofC, it accepts phosphoenolpyruvate, rather than 2-phospho-L-lactate, as the substrate, leading to the formation of the previously uncharacterized intermediate dehydro-F420-0. The C-terminal domain of FbiB then utilizes FMNH2 to reduce dehydro-F420-0, which produces mature F420 species when combined with the γ-glutamyl ligase activity of the N-terminal domain. These new insights have allowed the heterologous production of F420 from a recombinant F420 biosynthetic pathway in Escherichia coli.

Bioavailable Blueberry-Derived Phenolic Acids at Physiological Concentrations Enhance Nrf2-Regulated Antioxidant Responses in Human Vascular Endothelial Cells.

SCOPE: Blueberry consumption is believed to confer a cardiovascular health advantage, but the active compounds and effects require characterization. This study aims to identify the polyphenol metabolites in plasma after blueberry juice intake and determine their bioactivity on endothelial cells. METHODS AND RESULTS: Three healthy individuals are recruited to obtain profiles of bioavailable plasma polyphenol metabolites following intake of blueberry juice. Of 33 phenolic compounds screened, 12 aglycone phenolic acids are detected and their maximum plasma concentrations and circulation time determined. Using this information, the effect of three physiologically relevant mixtures of blueberry-derived phenolic acids is investigated for their ability to induce nuclear factor erythroid 2-related factor 2 (Nrf2)-nuclear translocation and downstream gene expression in human endothelial cells. Pretreatment with the phenolic acids for 18 h results in a significant upregulation of the Nrf2-regulated antioxidant response proteins heme oxygenase 1 (HO-1) and glutamate-cysteine ligase modifier subunit (GCLM), following 6 h exposure to 2.5 µm H2 O2 . CONCLUSION: Physiologically relevant concentrations of blueberry-derived aglycone phenolic acids can induce Nrf2-regulated antioxidant response proteins in vascular endothelial cells in response to low µm concentrations of H2 O2 . Our results represent an advance over previous studies that have used single compounds or high concentrations in cell-based investigations.

Pharmacokinetic Properties of Adenosine Amine Congener in Cochlear Perilymph after Systemic Administration.

Noise-induced hearing loss (NIHL) is a global health problem affecting over 5% of the population worldwide. We have shown previously that acute noise-induced cochlear injury can be ameliorated by administration of drugs acting on adenosine receptors in the inner ear, and a selective A1 adenosine receptor agonist adenosine amine congener (ADAC) has emerged as a potentially effective treatment for cochlear injury and resulting hearing loss. This study investigated pharmacokinetic properties of ADAC in rat perilymph after systemic (intravenous) administration using a newly developed liquid chromatography-tandem mass spectrometry detection method. The method was developed and validated in accordance with the USA FDA guidelines including accuracy, precision, specificity, and linearity. Perilymph was sampled from the apical turn of the cochlea to prevent contamination with the cerebrospinal fluid. ADAC was detected in cochlear perilymph within two minutes following intravenous administration and remained in perilymph above its minimal effective concentration for at least two hours. The pharmacokinetic pattern of ADAC was significantly altered by exposure to noise, suggesting transient changes in permeability of the blood-labyrinth barrier and/or cochlear blood flow. This study supports ADAC development as a potential clinical otological treatment for acute sensorineural hearing loss caused by exposure to traumatic noise.

Development of a novel stability indicating RP-HPLC method for quantification of Connexin43 mimetic peptide and determination of its degradation kinetics in biological fluids.

Connexin43 mimetic peptide (Cx43MP) has been intensively investigated for its therapeutic effect in the management of inflammatory eye conditions, spinal cord injury, wound healing and ischemia-induced brain damage. Here, we report on a validated stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method for the quantification of Cx43MP under stress conditions. These included exposure to acid/base, light, oxidation and high temperature. In addition, the degradation kinetics of the peptide were evaluated in bovine vitreous and drug-free human plasma at 37 °C. Detection of Cx43MP was carried out at 214 nm with a retention time of 7.5 min. The method showed excellent linearity over the concentration range of 0.9-250 µg/mL (R2 ≥ 0.998), and the limits of detection (LOD) and quantification (LOQ) were found to be 0.90 and 2.98 µg/mL, respectively. The accuracy of the method determined by the mean percentage recovery at 7.8, 62.5 and 250 µg/mL was 96.79%, 98.25% and 99.06% with a RSD of < 2.2%. Accelerated stability studies revealed that Cx43MP was more sensitive to basic conditions and completely degraded within 24 h at 37 °C (0% recovery) and within 12 h at 80 °C (0.34% recovery). Cx43MP was found to be more stable in bovine vitreous (t1/2slow= 171.8 min) compared to human plasma (t1/2slow = 39.3 min) at 37 °C according to the two phase degradation kinetic model. These findings are important for further pre-clinical development of Cx43MP.

Development of a novel stability indicating RP-HPLC method for quantification of Connexin43 mimetic peptide and determination of its degradation kinetics in biological fluids / 药物分析学报

Connexin43 mimetic peptide (Cx43MP) has been intensively investigated for its therapeutic effect in the management of inflammatory eye conditions, spinal cord injury, wound healing and ischemia-induced brain damage. Here, we report on a validated stability–indicating reversed-phase high performance liquid chromatography(RP-HPLC)method for the quantification of Cx43MP under stress conditions.These included exposure to acid/base, light, oxidation and high temperature. In addition, the degradation kinetics of the peptide were evaluated in bovine vitreous and drug-free human plasma at 37 ℃. Detection of Cx43MP was carried out at 214 nm with a retention time of 7.5 min. The method showed excellent linearity over the concentration range of 0.9–250μg/mL(R2≥0.998),and the limits of detection(LOD)and quantification(LOQ) were found to be 0.90 and 2.98 μg/mL, respectively. The accuracy of the method determined by the mean percentage recovery at 7.8, 62.5 and 250μg/mL was 96.79%, 98.25% and 99.06% with a RSD of<2.2%. Accelerated stability studies revealed that Cx43MP was more sensitive to basic conditions and completely degraded within 24 h at 37 ℃(0% recovery)and within 12 h at 80 ℃(0.34% recovery).Cx43MP was found to be more stable in bovine vitreous(t1/2slow=171.8 min)compared to human plasma(t1/2slow=39.3 min)at 37 ℃ according to the two phase degradation kinetic model. These findings are important for further pre-clinical development of Cx43MP.

Elongation of the Poly-γ-glutamate Tail of F420 Requires Both Domains of the F420:γ-Glutamyl Ligase (FbiB) of Mycobacterium tuberculosis.

Cofactor F420is an electron carrier with a major role in the oxidoreductive reactions ofMycobacterium tuberculosis, the causative agent of tuberculosis. A γ-glutamyl ligase catalyzes the final steps of the F420biosynthesis pathway by successive additions ofl-glutamate residues to F420-0, producing a poly-γ-glutamate tail. The enzyme responsible for this reaction in archaea (CofE) comprises a single domain and produces F420-2 as the major species. The homologousM. tuberculosisenzyme, FbiB, is a two-domain protein and produces F420with predominantly 5-7l-glutamate residues in the poly-γ-glutamate tail. The N-terminal domain of FbiB is homologous to CofE with an annotated γ-glutamyl ligase activity, whereas the C-terminal domain has sequence similarity to an FMN-dependent family of nitroreductase enzymes. Here we demonstrate that full-length FbiB adds multiplel-glutamate residues to F420-0in vitroto produce F420-5 after 24 h; communication between the two domains is critical for full γ-glutamyl ligase activity. We also present crystal structures of the C-terminal domain of FbiB in apo-, F420-0-, and FMN-bound states, displaying distinct sites for F420-0 and FMN ligands that partially overlap. Finally, we discuss the features of a full-length structural model produced by small angle x-ray scattering and its implications for the role of N- and C-terminal domains in catalysis.

Identification of P450 Oxidoreductase as a Major Determinant of Sensitivity to Hypoxia-Activated Prodrugs.

Hypoxia is a prevalent feature of many tumors contributing to disease progression and treatment resistance, and therefore constitutes an attractive therapeutic target. Several hypoxia-activated prodrugs (HAP) have been developed, including the phase III candidate TH-302 (evofosfamide) and the preclinical agent SN30000, which is an optimized analogue of the well-studied HAP tirapazamine. Experience with this therapeutic class highlights an urgent need to identify biomarkers of HAP sensitivity, including enzymes responsible for prodrug activation during hypoxia. Using genome-scale shRNA screens and a high-representation library enriched for oxidoreductases, we identified the flavoprotein P450 (cytochrome) oxidoreductase (POR) as the predominant determinant of sensitivity to SN30000 in three different genetic backgrounds. No other genes consistently modified SN30000 sensitivity, even within a POR-negative background. Knockdown or genetic knockout of POR reduced SN30000 reductive metabolism and clonogenic cell death and similarly reduced sensitivity to TH-302 under hypoxia. A retrospective evaluation of head and neck squamous cell carcinomas showed heterogeneous POR expression and suggested a possible relationship between human papillomavirus status and HAP sensitivity. Taken together, our study identifies POR as a potential predictive biomarker of HAP sensitivity that should be explored during the clinical development of SN30000, TH-302, and other hypoxia-directed agents.

Adenosine amine congener as a cochlear rescue agent.

UNLABELLED: We have previously shown that adenosine amine congener (ADAC), a selective A1 adenosine receptor agonist, can ameliorate noise- and cisplatin-induced cochlear injury. Here we demonstrate the dose-dependent rescue effects of ADAC on noise-induced cochlear injury in a rat model and establish the time window for treatment. METHODS: ADAC (25-300 µg/kg) was administered intraperitoneally to Wistar rats (8-10 weeks old) at intervals (6-72 hours) after exposure to traumatic noise (8-16 kHz, 110 dB sound pressure level, 2 hours). Hearing sensitivity was assessed using auditory brainstem responses (ABR) before and 12 days after noise exposure. Pharmacokinetic studies investigated ADAC concentrations in plasma after systemic (intravenous) administration. RESULTS: ADAC was most effective in the first 24 hours after noise exposure at doses >50 µg/kg, providing up to 21 dB protection (averaged across 8-28 kHz). Pharmacokinetic studies demonstrated a short (5 min) half-life of ADAC in plasma after intravenous administration without detection of degradation products. CONCLUSION: Our data show that ADAC mitigates noise-induced hearing loss in a dose- and time-dependent manner, but further studies are required to establish its translation as a clinical otological treatment.