Signaling mechanisms of growth hormone-releasing hormone receptor in LPS-induced acute ocular inflammation.

Ocular inflammation is a major cause of visual impairment attributed to dysregulation of the immune system. Previously, we have shown that the receptor for growth-hormone-releasing hormone (GHRH-R) affects multiple inflammatory processes. To clarify the pathological roles of GHRH-R in acute ocular inflammation, we investigated the inflammatory cascades mediated by this receptor. In human ciliary epithelial cells, the NF-κB subunit p65 was phosphorylated in response to stimulation with lipopolysaccharide (LPS), resulting in transcriptional up-regulation of GHRH-R. Bioinformatics analysis and coimmunoprecipitation showed that GHRH-R had a direct interaction with JAK2. JAK2, but not JAK1, JAK3, and TYK2, was elevated in ciliary body and iris after treatment with LPS in a rat model of endotoxin-induced uveitis. This elevation augmented the phosphorylation of STAT3 and production of proinflammatory factors, including IL-6, IL-17A, COX2, and iNOS. In explants of iris and ciliary body, the GHRH-R antagonist, MIA-602, suppressed phosphorylation of STAT3 and attenuated expression of downstream proinflammatory factors after LPS treatment. A similar suppression of STAT3 phosphorylation was observed in human ciliary epithelial cells. In vivo studies showed that blocking of the GHRH-R/JAK2/STAT3 axis with the JAK inhibitor Ruxolitinib alleviated partially the LPS-induced acute ocular inflammation by reducing inflammatory cells and protein leakage in the aqueous humor and by repressing expression of STAT3 target genes in rat ciliary body and iris and in human ciliary epithelial cells. Our findings indicate a functional role of the GHRH-R/JAK2/STAT3-signaling axis in acute anterior uveitis and suggest a therapeutic strategy based on treatment with antagonists targeting this signaling pathway.

Untargeted metabolomic analysis of aqueous humor in diabetic macular edema.

Background: The mechanism of diabetic macular edema (DME) was explored by comparing the intraocular metabolite profiles of the aqueous humor of patients with DME to those of diabetic patients without DME using untargeted metabolomic analysis. Methods: Aqueous samples from 18 type 2 diabetic patients with DME and 18 type 2 diabetic patients without DME used as controls were analyzed using liquid chromatography-mass spectrometry (LCMS). The two groups of patients were age and gender matched and had no systemic diseases other than diabetes mellitus (DM). The metabolites were analyzed using orthogonal partial least square discriminant analysis. Results: The metabolite profiles in DME patients differed from those in DM controls. This indicates the following metabolic derangements in DME: (a) a higher amount of oxidized fatty acids but a lower amount of endogenous antioxidants (oxidative stress); (b) higher levels of ß-glucose and homocysteine but a lower level of sorbitol (hyperglycemia); (c) a higher amount of prostaglandin metabolites (inflammation); (d) higher amounts of acylcarnitines, odd-numbered fatty acids, and 7,8-diaminononanoate (respiration deterioration); (e) a higher amount of neurotransmitter metabolites and homovanillic acid (neuronal damage); (f) a lower amount of extracellular matrix (ECM) constituents (ECM deterioration); and (g) a higher amount of di-amino peptides (microvascular damage). Conclusions: The change in the metabolic profiles in the aqueous humor of DME patients compared to DM controls without DME indicates that DME patients may have less capability to resist various stresses or damaging pathological conditions, such as oxidative stress, mitochondrial insufficiency, inflammation, and ECM deterioration.

Determination of exogenous epigallocatechin gallate peracetate in mouse plasma using liquid chromatography with quadrupole time-of-flight mass spectrometry.

A robust method for the quantitation of epigallocatechin gallate peracetate in plasma for pharmacokinetic studies is lacking. We have developed a validated method to quantify this compound using liquid chromatography with quadrupole time-of-flight mass spectrometry with isopropanol and tert-butyl methyl ether (3:10) extraction and thin-layer chromatography purification. The epigallocatechin gallate peracetate-1-(13) C8 isotope was used as an internal standard. The linear range (r(2) > 0.9950) was from 0.05 to 100.00 µg/mL. The lower limit of quantification of the method was 0.05 µg/mL. Reproducibility, coefficient of variation, was between 0.7 and 12.6% (n = 6), accuracy between 83.7 and 104.6% (n = 5), and recovery ranged from 82.4 to 109.0% (n = 4). Ion suppression was approximately 40%. No mass spectral peaks were found to interfere between the standard and internal standard or the blank plasma extracts. Epigallocatechin gallate peracetate in plasma was stably stored at -80°C over three months even after three freeze-thaw cycles. Extracts were stable in the sampler at 4°C for over 48 h. Plasma levels were maintained at 1.36 µg/mL for 360 min after intraorbital intravenous injection at 50 mg/kg in mice. This method can be used to reliably measure epigallocatechin gallate peracetate in plasma for pharmacokinetic studies.

Amelioration of Functional, Metabolic, and Morphological Deterioration in the Retina following Retinal Detachment by Green Tea Extract.

Retinal detachment (RD) can result in the loss of photoreceptors that cause vision impairment and potential blindness. This study explores the protective effects of the oral administration of green tea extract (GTE) in a rat model of RD. Various doses of GTE or epigallocatechin gallate (EGCG), the most active ingredient in green tea catechins, were administered to Sprague Dawley (SD) rats with experimentally induced retinal detachment. The rats received sub-retinal injections of hyaluronic acid (0.1%) to induce RD and were given different doses of GTE and EGCG twice daily for three days. Notably, a low dose of GTE (142.9 mg/kg) caused significantly higher signal amplitudes in electroretinograms (ERGs) compared to higher GTE doses and any doses of EGCG. After administration of a low dose of GTE, the outer nuclear layer thickness, following normalization, of the detached retina reduced to 82.4 ± 8.2% (Mean ± SEM, p < 0.05) of the thickness by RD treatment. This thickness was similar to non-RD conditions, at 83.5 ± 4.7% (Mean ± SEM) of the thickness following RD treatment. In addition, the number of TUNEL-positive cells decreased from 76.7 ± 7.4 to 4.7 ± 1.02 (Mean ± SEM, p < 0.0001). This reduction was associated with the inhibition of apoptosis through decreased sphingomyelin levels and mitigation of oxidative stress shown by a lowered protein carbonyl level, which may involve suppression of HIF-1α pathways. Furthermore, GTE showed anti-inflammatory effects by reducing inflammatory cytokines and increasing resolving cytokines. In conclusion, low-dose GTE, but not EGCG, significantly alleviated RD-induced apoptosis, oxidative stress, inflammation, and energy insufficiency within a short period and without affecting energy metabolism. These findings suggest the potential of low-dose GTE as a protective agent for the retina in RD.

Antiviral Effects and Mechanisms of Green Tea Catechins on Influenza: A Perception on Combating Symptoms from COVID-19.

Over the centuries, influenza and its associated epidemics have been a serious public health problem. Although vaccination and medications (such as neuraminidase inhibitors) are the mainstay of pharmacological approaches to prevent and treat influenza, however, frequent mutations in the influenza genome often result in treatment failure and resistance to standard medications which limit their effectiveness. In recent years, green tea catechins have been evaluated as potential anti-influenza agents. Herein, in this review, we highlighted the effects and mechanisms underlying the inhibitory effects of epigallocatechin 3-gallate (EGCG), the most abundant ingredient in green tea, against different influenza viral infections, and their clinical benefits toward prevention and treatment. In addition, as the severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2) causes the outbreak of COVID-19 pandemic, our review also delineates the current perspective on SARS-CoV-2 and future insights as to the potential application of EGCG on suppressing the flu-like symptoms caused by COVID-19.

Prodrug of green tea epigallocatechin-3-gallate (Pro-EGCG) as a potent anti-angiogenesis agent for endometriosis in mice.

Green tea epigallocatechin-3-gallate (EGCG) can inhibit angiogenesis and development of an experimental endometriosis model in mice, but it suffers from poor bioavailability. A prodrug of EGCG (pro-EGCG, EGCG octaacetate) is utilized to enhance the stability and bioavailability of EGCG in vivo. In this study, the potential of pro-EGCG as a potent anti-angiogenesis agent for endometriosis in mice was investigated. Homologous endometrium was subcutaneously transplanted into mice to receive either saline, vitamin E, EGCG or pro-EGCG treatment for 4 weeks. The growth of the endometrial implants were monitored by IVIS(®) non-invasive in vivo imaging during the interventions. Angiogenesis of the endometriotic lesions was determined by Cellvizio(®) in vivo imaging and SCANCO(®) Microfil microtomography. The bioavailability, anti-oxidation and anti-angiogenesis capacities of the treatments were measured in plasma and lesions. The implants with adjacent outer subcutaneous and inner abdominal muscle layers were collected for histological, microvessel and apoptosis examinations. The result showed that EGCG and pro-EGCG significantly decreased the growth of endometrial implants from the 2nd week to the 4th week of intervention. EGCG and pro-EGCG significantly reduced the lesion size and weight, inhibited functional and structural microvessels in the lesions, and enhanced lesion apoptosis at the end of interventions. The inhibition by pro-EGCG in all the angiogenesis parameters was significantly greater than that by EGCG, and pro-EGCG also had better bioavailability and greater anti-oxidation and anti-angiogenesis capacities than EGCG. Ovarian follicles and uterine endometrial glands were not affected by either EGCG or pro-EGCG. Vitamin E had no effect on endometriosis. In conclusion, pro-EGCG significantly inhibited the development, growth and angiogenesis of experimental endometriosis in mice with high efficacy, bioavailability, anti-oxidation and anti-angiogenesis capacities. Pro-EGCG could be a potent anti-angiogenesis agent for endometriosis.

Green Tea Catechins as Therapeutic Antioxidants for Glaucoma Treatment.

Glaucoma is the leading cause of irreversible blindness and visual impairment, affecting more than 80 million individuals worldwide. Oxidative stress and inflammation-induced neurodegenerative insults to retinal ganglion cells are the main pathogenesis of glaucoma. Retinal ganglion cells, the retinal neurons transmitting the visual signals to the visual cortex in the brain, have very limited regeneration or recovery capacity after damages. Apart from intraocular pressure-lowering treatments, there is still no clinically effective treatment to rescue the degeneration of retinal ganglion cells in glaucoma. Dietary antioxidants are easily accessible and can be applied as supplements assisting in the clinical treatments. Catechins, a chemical family of flavonoids, are the phenolic compounds found in many plants, especially in green tea. The anti-oxidative and anti-inflammatory properties of green tea catechins in vitro and in vivo have been well proven. They could be a potential treatment ameliorating retinal ganglion cell degeneration in glaucoma. In this review, the chemistry, pharmacokinetics, and therapeutic properties of green tea catechins were summarized. Research updates on the biological effects of green tea catechins in cellular and animal experimental glaucoma models were reviewed. In addition, clinical potentials of green tea catechins for glaucoma treatment were also highlighted.

Quantification and structure elucidation of in vivo bevacizumab modification in rabbit vitreous humor after intravitreal injection.

Off-label and intravitreal use of bevacizumab, a recombinant immunoglobulin against VEGF, has been practiced widely for ophthalmic treatments. However, longitudinal data of its intravitreal status is unavailable due to a lack of reliable methods for bevacizumab determination. Thus its pharmacokinetics and pharmacodynamics are uncertain. We developed and validated a high performance liquid chromatographic method to determine bevacizumab in vitreous humor and utilized a novel strategy to assess in vivo temporal binding changes by affinity chromatography. Mass spectrometry and tandem mass spectrometry detection were used for structural evaluation. The coefficient of variation (CV) for intrabatch imprecision varied from 0.5 to 14.3% and for interbatch imprecision from 1.9 to 11.6%. The linearity was over 0.9982, lower limit of quantification 1.95 µg, recoveries over 95%, and accuracy between 90 and 112% over the range of 1.95-250 µg of bevacizumab in 100 µL of vitreous humor. Blank vitreous humor showed no interference peak. It was stable at room temperature for 5 h. Bevacizumab elimination in the vitreous followed first order kinetics with half-life as 5.7 days and elimination rate as 0.1221 day(-1). Peptide mapping and tandem mass spectrometry revealed structural modifications of the in vivo bevacizumab mainly on the heavy chain in both variable and constant regions 7 days after intravitreal injection. Minor changes were also discovered on the light chain. Affinity chromatography showed significant affinity changes in samples 21 days after intravitreal injection. The changes were consistent with structural modifications as found in endothelial cell migration assays results. In conclusion, we have established a robust chromatographic method for determination of bevacizumab and strategies with affinity chromatography and molecular mass detection that revealed bevacizumab structural and possible functional changes in vitreous.

Systemic and Ocular Anti-Inflammatory Mechanisms of Green Tea Extract on Endotoxin-Induced Ocular Inflammation.

Introduction: Green tea extract (GTE) alleviated ocular inflammations in endotoxin-induced uveitis (EIU) rat model induced by lipopolysaccharide (LPS) but the underlying mechanism is unclear. Objectives: To investigate the systematic and local mechanisms of the alleviation by untargeted metabolomics using liquid chromatography-tandem mass spectrometry. Methods: Sprague-Dawley rats were divided into control group, LPS treatment group, and LPS treatment group treated with GTE two hours after LPS injection. The eyes were monitored by slip lamp and electroretinography examination after 24 hours. The plasma and retina were collected for metabolomics analysis. Results: In LPS treated rats, the iris showed hyperemia. Plasma prostaglandins, arachidonic acids, corticosteroid metabolites, and bile acid metabolites increased. In the retina, histamine antagonists, corticosteroids, membrane phospholipids, free antioxidants, and sugars also increased but fatty acid metabolites, N-acetylglucosamine-6-sulphate, pyrocatechol, and adipic acid decreased. After GTE treatment, the a- and b- waves of electroretinography increased by 13%. Plasma phosphorylcholine lipids increased but plasma prostaglandin E1, cholanic metabolites, and glutarylglycine decreased. In the retina, tetranor-PGAM, pantothenic derivatives, 2-ethylacylcarinitine, and kynuramine levels decreased but anti-oxidative seleno-peptide level increased. Only phospholipids, fatty acids, and arachidonic acid metabolites in plasma and in the retina had significant correlation (p < 0.05, r > 0.4 or r < -0.4). Conclusions: The results showed GTE indirectly induced systemic phosphorylcholine lipids to suppress inflammatory responses, hepatic damage, and respiratory mitochondrial stress in EIU rats induced by LPS. Phospholipids may be a therapeutic target of GTE for anterior chamber inflammation.

Green Tea Epigallocatechin-3-Gallate Regulates Autophagy in Male and Female Reproductive Cancer.

With a rich abundance of natural polyphenols, green tea has become one of the most popular and healthiest nonalcoholic beverages being consumed worldwide. Epigallocatechin-3-gallate (EGCG) is the predominant catechin found in green tea, which has been shown to promote numerous health benefits, including metabolic regulation, antioxidant, anti-inflammatory, and anticancer. Clinical studies have also shown the inhibitory effects of EGCG on cancers of the male and female reproductive system, including ovarian, cervical, endometrial, breast, testicular, and prostate cancers. Autophagy is a natural, self-degradation process that serves important functions in both tumor suppression and tumor cell survival. Naturally derived products have the potential to be an effective and safe alternative in balancing autophagy and maintaining homeostasis during tumor development. Although EGCG has been shown to play a critical role in the suppression of multiple cancers, its role as autophagy modulator in cancers of the male and female reproductive system remains to be fully discussed. Herein, we aim to provide an overview of the current knowledge of EGCG in targeting autophagy and its related signaling mechanism in reproductive cancers. Effects of EGCG on regulating autophagy toward reproductive cancers as a single therapy or cotreatment with other chemotherapies will be reviewed and compared. Additionally, the underlying mechanisms and crosstalk of EGCG between autophagy and other cellular processes, such as reactive oxidative stress, ER stress, angiogenesis, and apoptosis, will be summarized. The present review will help to shed light on the significance of green tea as a potential therapeutic treatment for reproductive cancers through regulating autophagy.

Determination of (-)-epigallocatechin-3-gallate octaacetate and its metabolites in plasma of rats for pharmacokinetic study by ultra-performance-liquid-chromatography coupled to quadrupole-time-of-flight-mass-spectrometry.

(-)-Epigallocatechin-gallate octaacetate (pro-EGCG), a prodrug of epigallocatechin-gallate (EGCG), has been used for pre-clinical study for the treatment of endometriosis. A validated analytical method has been developed for the determination of plasma pro-EGCG and its metabolites after oral administration using ultra-performance-liquid-chromatography coupled to quadrupole-time-of-flight-mass-spectrometry (UPLC-Qtof-MS). This method is more robust, rapid, sensitive, simpler, and able to detect pro-EGCG metabolites compared to our previous method. Pro-EGCG in the plasma was stabilized from rapid degradation by formic acid, extracted by isopropanol/methyl-tert-butyl ether mixture, separated by UPLC core column, and quantified by an exact mass method with Qtof-MS. The lower limit of quantification (LLOQ), intra-day and inter-day precision, and accuracy for the range of 0.01-2.5 µg/mL were within acceptable limits. The sensitivity was improved by 25 folds using pro-EGCG ammonium adduct [M + NH4]+. This is the first report on the pharmacokinetics of oral administration with maximum-concentration (Cmax) was 0.067 ± 0.04 µg/mL, time-of-maximum-concentration (Tmax) was 1.33 h, area-under-curve (AUC) was 0.20 ± 0.05 h × µg/mL, and elimination-rate was 0.20 ± 0.11 hr-1. The pharmacokinetic profiles of pro-EGCG metabolites, (-)-epigallocatechin-gallate (EGCG) diacetates and EGCG triacetates, were also presented. This method is robust, rapid, and sensitive for the pharmacokinetic study of pro-EGCG and metabolites.

Anti-inflammatory Effects of GTE in Eye Diseases.

Ocular inflammation is a common complication of various eye diseases with wide consequences from irritations to potentially sight-threatening complications. Green tea is a popular beverage throughout the world. One of the proven health benefits of consuming green tea extract (GTE) is anti-inflammation. Catechins are the biologically active constituents of GTE. In in vitro and in vivo studies, GTE and catechins present inhibition of inflammatory responses in the development of ocular inflammation including infectious, non-infectious or autoimmune, and oxidative-induced complications. Research on the ocular inflammation in animal models has made significant progress in the past decades and several key disease mechanisms have been identified. Here we review the experimental investigations on the effects of GTE and catechins on various ocular inflammation related diseases including glaucoma, age-related macular degeneration, uveitis and ocular surface inflammation. We also review the pharmacokinetics of GTE constituents and safety of green tea consumption. We discuss the insights and perspectives of these experimental results, which would be useful for future development of novel therapeutics in human.

Green tea catechins alleviate autoimmune symptoms and visual impairment in a murine model for human chronic intraocular inflammation by inhibiting Th17-associated pro-inflammatory gene expression.

Autoimmune uveitis is a sight-threatening disease mainly caused by dysregulation of immunity. We investigated the therapeutic effects of green tea extract (GTE) and its major component, epigallocatechin-3-gallate (EGCG), on a murine model of experimental autoimmune uveoretinitis (EAU). Oral administration of GTE, EGCG, dexamethasone, or water, which started 5 days before the induction, was fed every two days to each group. On day 21 post induction, the eyes were examined by confocal scanning laser ophthalmoscopy, optical coherence tomography (OCT), fundus fluorescein angiography (FFA) and electroretinography (ERG) prior to sacrificing the animals for histological assessments and gene expression studies. Retinal-choroidal thicknesses (RCT) and major retinal vessel diameter were measured on OCT sections and FFA images, respectively. Comparing to water-treated EAU animals, GTE attenuated uveitis clinical manifestations, RCT increase (1.100 ± 0.013 times vs 1.005 ± 0.012 times, P < 0.001), retinal vessel dilation (308.9 ± 6.189 units vs 240.8 units, P < 0.001), ERG amplitudes attenuation, histopathological ocular damages, and splenomegaly in EAU mice. The therapeutic effects of GTE were dose dependent and were comparable to dexamethasone. EGCG, a major active constituent of GTE, partially alleviated uveitic phenotypes including recovering visual function. Th-17 associated pro-inflammatory gene [interleukin 1 beta (IL-1ß), IL-6, IL-17A, and tumor necrosis factor alpha (TNF-α)] expressions were down regulated by GTE and EGCG treatments, which showed no detectable morphological defects in liver and kidney in non-induced and EAU mice. Our findings suggest that GTE consumption can serve as a potent therapeutic agent as well as a food supplement for developing alternative treatments against autoimmune uveitis.

Metabolomics of Green-Tea Catechins on Vascular-Endothelial-Growth-Factor-Stimulated Human-Endothelial-Cell Survival.

Neovascularization causes serious oculopathy related to upregulation of vascular-endothelial-growth factor (VEGF) causing new capillary growth via endothelial cells. Green-tea-extract (GTE) constituents possess antiangiogenesis properties. We used VEGF to induce human umbilical-vein endothelial cells (HUVECs) and applied GTE, epigallocatechin gallate (EGCG), and mixtures of different compositions of purified catechins (M1 and M2) to evaluate their efficacies of inhibition and their underlying mechanisms using cell-cycle analysis and untargeted metabolomics techniques. GTE, EGCG, M1, and M2 induced HUVEC apoptosis by 22.1 ± 2, 20.0 ± 0.7, 50.7 ± 8.5, and 69.8 ± 4.1%, respectively. GTE exerted a broad, balanced metabolomics spectrum, involving suppression of the biosynthesis of cellular building blocks and oxidative-phosphorylation metabolites as well as promotion of the biosynthesis of membrane lipids and growth factors. M2 mainly induced mechanisms associated with energy and biosynthesis suppression. Therefore, GTE exerted mechanisms involving both promotion and suppression activities, whereas purified catechins induced extensive apoptosis. GTE could be a more promising antineovascularization remedy for ocular treatment.

Tea epigallocatechin-3-gallate increases 8-isoprostane level and induces caudal regression in developing rat embryos.

Tea is the most common beverage after water. Concerns have been raised about the safety of tea during pregnancy, especially for embryo development. We aimed at studying the effects of active tea components on developing embryos by in vitro rat embryo culture. Rat embryos during early organogenesis were cultivated in serum supplemented with one of the tea catechins. Developmental hallmarks and malformations (Mal) in the developing embryos were compared and evaluated by a standard morphological scoring system. The embryotoxicity of each tea catechin was classified according to the European Center for the Validation of Alternative Methods. Cell viability was assessed by supervital dye staining, apoptosis by TUNEL assay, and peroxidation by the 8-isoprostane EIA method. We found that (+)-catechin had the least effect on developing embryos (Mal(50)=715.1 mg/L; IC50(Mal)=435 mg/L), whereas (-)-epigallocatechin gallate had the most adverse effect (Mal(50)=54.2 mg/L; IC50(Mal)=45.8 mg/L). The major malformation in affected embryos included caudal retardation with abnormal axial flexion and delayed hind-limb formation. All catechins were classified as nonembryotoxic except (-)-epigallocatechin gallate, which was classified as weakly embryotoxic. With (-)-epigallocatechin gallate, increased numbers of nonviable and apoptotic cells in the malformed embryos were associated with increased embryo 8-isoprostane.

Differential aqueous and vitreous concentrations of moxifloxacin and ofloxacin after topical administration one hour before vitrectomy.

PURPOSE: To investigate the penetration of ofloxacin and moxifloxacin into the aqueous and vitreous after topical administration one hour before vitrectomy surgery. DESIGN: Prospective, randomized, double-blind case series study. METHODS: Twenty-seven patients undergoing vitrectomy were randomized to receive either topical ofloxacin 0.3% or moxifloxacin 0.5% every 10 minutes for one hour before surgery. Aqueous and vitreous samples were obtained and analyzed using high-performance liquidation chromatography. RESULTS: The moxifloxacin aqueous (1.576 +/- 0.745 microg/ml) and vitreous (0.225 +/- 0.013 microg/ml) levels were significantly higher than the ofloxacin aqueous (0.816 +/- 0.504 microg/ml) (P = .0009) and vitreous (0.225 +/- 0.013 microg/ml) [P = .0054] levels, respectively. The mean moxifloxacin aqueous and vitreous levels exceeded the minimum inhibitory concentration for 90% of isolates (MIC(90)) for a wide variety of bacteria implicated in endophthalmitis. In contrast, the aqueous level of ofloxacin exceeded the MIC(90) of only a few organisms. CONCLUSIONS: Moxifloxacin applied every 10 minutes during the hour before vitrectomy penetrated the eye significantly better than ofloxacin.

Method to determine stability and recovery of carboprost and misoprostol in infusion preparations.

The two synthetic prostaglandin analogues, carboprost and misoprostol, are used extensively in obstetric and gynaecological practice. Our recent research of these compounds' use for intra-umbilical injection to treat adherent placenta necessitated their storage in solution for 3-4 days. This raised concerns over the stability and applied dosage in the in-house infusion preparations. It requires various pharmacological preparations before administration in clinical practice. We used LCMS to develop a simultaneous, valid, fast and simple method to assess the stability and recovery of their in-house preparations in different conditions. The linearity between 0-40 microg/ml was above 0.995. The reproducibility (CV) was within 5.2%. The limit of quantitation of the method for both compounds is about 2 microg/ml. The accuracy of both compounds from 0.4-40 microg/ml is 96.4-104.3% while the precision is 0.4-7.4%. The recoveries of carboprost in the infusion were from 100.3+/-4.0 to 102.4+/-1.6% and that of misoprostol in Cytotec tablet was from 44.9+/-3.5 to 50.0+/-5.0% in water and saline at 4 degrees C and room temperature. No interference was found from the matrix and between the tested compounds. The compounds were basically stable for 6 days in water and in saline, whether they were stored at 4 degrees C or at room temperature. However, only half of the dosage of misoprostol was recovered in the solution. Therefore, misoprostol dosage should be adjusted before clinical application.

Measuring triamcinolone acetonide in aqueous humor by gas chromatography-electron-capture negative-ion mass spectrometry.

Intravitreal triamcinolone acetonide (IVTA) injection has been used in the treatment of various posterior segment diseases. One of the side effects of IVTA is raised intraocular pressure, which may be secondary to triamcinolone acetonide (TAA)'s effects on the trabecular meshwork that affects aqueous outflow. In order to study the biological effects of TAA on the trabecular meshwork, we firstly need to reliably and accurately detect the concentration of TAA in tissue cells or fluids. In this study we have described a technique of using gas chromatography-electron-capture-negative-ion mass spectrometry (GC-NCI-MS) to develop a simple, sensitive, selective and validated method to detect TAA in aqueous humor (AH) of rabbits following IVTA and subconjunctival TAA injections. We derivatized TAA from extracted aqueous sample by acetic anhydride and BSTFA, respectively, and analyzed by GC-NCI-MS. The detection limit was 0.3ng/ml, linearity over 0.995 from 0 to 300ng/ml. The reproducibility ranged from 10.4 to 3.9 for concentrations from 3 to 300ng/ml, and recovery was over 95% for the concentrations 10, 60, and 200ng/ml. No interference was found from 159 aqueous samples. There was no TAA residue carried to the next injection from previously high concentration injection, 10,000ng/ml. We have provided an alternative, rapid, and robust method other than LC-MS-MS for TAA detection in AH.

Quantitative Characterization of Autoimmune Uveoretinitis in an Experimental Mouse Model.

Purpose: To accurately evaluate the autoimmune inflammation, we aim to develop three quantitative measurements to monitor the inflammatory changes in the retina: retinal-choroidal thickness, major retinal vessel diameter, and electroretinography amplitudes. Methods: During a 21-day experimental period, eyes were examined by confocal scanning laser ophthalmoscopy, optical coherence tomography, fundus fluorescein angiography, and electroretinography in living mice, which were then subsequently killed for histologic assessments. Results: On day 21 postimmunization, inflammation was observed both in vivo and in vitro. Fold change of retinal-choroidal thickness and major retinal vessel diameter in experimental autoimmune uveoretinitis mice were significantly greater than controls (P < 0.001). Both scotopic and photopic electroretinography amplitudes were significantly attenuated when compared with control mice (P < 0.01). Our results showed that these three quantifiable indicators provided an objective and accurate evaluation of autoimmune inflammation, which are in good correlations with the reported clinical and histopathologic scoring systems (P < 0.05). Conclusions: These three indicators will be useful to detect the small but significant differences in the severity of experimental autoimmune uveoretinitis for future longitudinally therapeutic studies.

Pharmacokinetic studies of green tea catechins in maternal plasma and fetuses in rats.

We carried out a pharmacokinetic study to determine the levels and profiles of catechins in pregnant rats and their fetuses after ingestion of green tea extract (GTE). We measured total catechin levels after enzyme digestions. Dams, at 15.5 days of gestation, were fed with GTE and catechins were measured in the maternal plasma, placenta, and fetus 0, 0.5, 1, 2, 3, 5, 8, 10, 12, 16, and 20 h after maternal GTE intake. The pharmacokinetic changes were analyzed by non compartmental models. We found that maternal plasma concentrations of catechins were about 10 times higher than in placenta and 50-100 times higher than in the fetus. AUC and Cmax levels of (-)-epicatechin (EC) were the highest in plasma while the levels of (-)-epigallocatechin gallate (EGCG) were the highest in the placenta and the fetus. The exposure level of catechin derivatives was higher than the gallate derivatives in maternal plasma after normalization but reversed in the placenta and fetus. The absorption of epi-isomers in plasma was found to be more favorable than their non epi-isomer counterparts. EGCG had the highest level of exposure (AUC) and the highest Cmax in the fetus, implying it may have potential for in utero antioxidant protection.