Considering the selectivity of pore size gradient size exclusion chromatography columns.

One of the most significant performance determining variables of a size exclusion column is the pore size of its packing material. This is most definitely the case for assigning the suitability of a given column for differently sized analytes. As technologies for particle and column manufacturing continue to advance, it is worth contemplating the value of more finely controlled manipulation of this parameter. The change in a packing material's pores across the length of a size exclusion column was thus explored. A change in average pore diameter and pore size distribution was studied by means of theoretical modeling. These parameters were investigated for independent and combinatorial effects. From our predictions, versus tandem column chromatography, a gradient column apparatus does not yield sizable increases in monomer to dimer selectivity of any given critical pair. Instead, our modeling suggests it can yield more universally effective separations of multiple pairs of species at once, as is sometimes necessary when analyzing the high molecular weight components of highly aggregated drug substances.

Site-specific detection of circulating tumor DNA methylation in biological samples utilizing phosphorothioated primer-based loop-mediated isothermal amplification.

DNA methylation, a kind of epigenetic alteration, plays a vital role in tumorigenesis and offers a new class of targets for cancer treatment. DNA hypermethylation at the E-Box site (CACGTG, -288 bp) in the SLC22A2 promoter was related to multidrug resistance of renal cell carcinoma (RCC), which can provide the target for both treatment and monitoring. Herein, we developed a novel phosphorothioated primer based loop-mediated isothermal amplification (PS-LAMP) assay to detect circulating tumor DNA (ctDNA) methylation levels in E-Box sites in tumor tissue, urine, and plasma samples from patients with RCC. Bisulfite treatment converted methylated/unmethylated discrepancy to a single base discrepancy (C/U). PS-LAMP amplified the templates to a tremendous amount. One-step strand displacement (OSD) probe provided single base resolution in amplified products and finally realized the specific site methylation detection. Our proposed method provided a linear range from 0% to 100% for methylation levels and was available in samples at low concentrations (102 copies/µL). Visually observable colorimetric detection can be achieved by incorporating the OSD probe with gold nanoparticles (AuNP). Our assay performed better than traditional methods in biological samples with low ctDNA concentration. Further, we found a potential consistency of methylation levels between tumor tissue and plasma sample from the same patient (Spearman's ρ = 0.886, P = 0.019, n = 6). In general, this work provides a PS-LAMP assay combining OSD probes for site-specific methylation detection in various biological samples, offering a method for noninvasive detection.

Clinical characteristics and management of hemodialysis patients with pre-dialysis hypertension: a multicenter observational study.

BACKGROUND: Hypertension is a leading preventable risk factor for cardiovascular disease in hemodialysis patients. Pre-dialysis systolic blood pressure (SBP) more than 160 mmHg was thought to be associated with increased risk of cardiovascular events and all-cause mortality. The present study was performed to explore the clinical characteristics and management of hemodialysis patients with pre-dialysis SBP ≥ 160 mmHg. METHODS: A total of 1233 patients undergoing hemodialysis from nine hemodialysis centers were enrolled. Pre-dialysis and home BP were measured and clinical data were collected. The characteristics of patients with pre-dialysis SBP ≥ 160 mmHg were explored. Clinical parameters between hypertensive and non-hypertensive patients were compared. The partial correlation analyses performed to identify the associations between BP and clinical parameters. RESULTS: There were 24.6% of the hemodialysis patients had pre-dialysis SBP ≥ 160 mmHg and the average SBP was 173.8 ± 10.9 mmHg. Only 21.4% of the patients achieved dry weight after dialysis and up to 30.2% of patients were not given combination therapies of antihypertensive drugs. Compared to patients with pre-hemodialysis SBP < 160 mmHg, patients with pre-dialysis SBP ≥ 160 mmHg had lower target-reaching rate of Kt/v and higher incidences of intradialytic hypotension and muscle spasm. Most patients (96%) with pre-dialysis SBP ≥ 160 mmHg had home SBP≥ 135 mmHg. Patients with home SBP ≥ 160 mmHg had higher left ventricular weight index and lower hemoglobin levels when compared to their counterparts with home SBP <160 mmHg. CONCLUSIONS: Pre-dialysis SBP ≥ 160 mmHg is common in clinical practice and most of the patients could diagnosed to be hypertensive according to their home SBP. Patients with pre-dialysis SBP ≥ 160 mmHg are more likely to be subjected to dialysis insufficiency and intradialytic complications. Achieving dry weight and sufficient pharmacologic interventions should be strengthened to improve BP control in the hemodialysis population.

Efficient biosynthesis of 3-hydroxypropionic acid from ethanol in metabolically engineered Escherichia coli.

Engineering microbial cell factories to convert CO2-based feedstock into chemicals and fuels provide a feasible carbon-neutral route for the third-generation biorefineries. Ethanol became one of the major products of syngas fermentation by engineered acetogens. The key building block chemical 3-hydroxypropionic acid (3-HP) can be synthesized from ethanol by the malonyl-CoA pathway with CO2 fixation. In this study, the effect of two ethanol consumption pathways on 3-HP synthesis were studied as well as the effect of TCA cycle, gluconeogenesis pathway, and transhydrogenase. And the 3-HP synthesis pathway was also optimized. The engineered strain synthesized 1.66 g/L of 3-HP with a yield of 0.24 g/g. Furthermore, the titer and the yield of 3-HP increased to 13.17 g/L and 0.57 g/g in the whole-cell biocatalysis system. This study indicated that ethanol as feedstock had the potential to synthesize 3-HP, which provided an alternative route for future biorefinery.

Pharmacokinetics and brain distribution studies of 6-hydroxykynurenic acid and its structural modified compounds.

OBJECTIVES: 6-Hydroxykynurenic acid (6-HKA) is an organic acid component in extracts of Ginkgo biloba leaves and acts as a major contributor to neurorestorative effects, while its oral bioavailability was low. Therefore, using prodrug method to improve the bioavailability and brain content of 6-HKA is significant. METHODS: Three structural modified compounds of 6-HKA were synthesized, and ultra performance liquid chromatography-tandem mass spectrometry methods for quantification of these structural modified compounds in rat plasma and rat brain homogenate were established and comprehensively validated. The methods were effectively applied to investigate the effects of structural modification on apparent permeability coefficients in cells, the pharmacokinetics and the brain distribution in rats. KEY FINDINGS: The results illustrated that esterification can greatly improve the apparent permeability coefficient and bioavailability of 6-HKA. Comparing with direct oral administration of 6-HKA, the bioavailability of isopropyl ester was greatly improved (from 3.96 ± 1.45% to 41.8 ± 15.3%), and the contents of 6-HKA in rat brains (49.7 ± 9.2 ng/g brain) were significantly higher after oral administration. CONCLUSIONS: The bioavailability and the brain content of 6-HKA can be improved by the prodrug method. Among three structural modified compounds, isopropyl-esterified 6-HKA was the most promising treatment.

The characterisation of the in vitro metabolism and transport of 6-hydroxykynurenic acid, an important constituent of Ginkgo biloba extracts.

6-Hydroxykynurenic acid (6-HKA) is a nitrogen-containing phenolic acid compound in Ginkgo biloba leaves. The pharmacological activities of 6-HKA have been reported and shown that 6-HKA has the potential to become a therapeutic drug and may play an important role in the treatment of nervous system diseases. However, there are few studies on the drug metabolism and transport of 6-HKA. The aim of this study is to investigate the in vitro metabolism of 6-HKA and its interaction with multiple important drug transporters.The in vitro metabolism experiments in the present study demonstrate that 6-HKA might not undergo phase-I or phase-II metabolism in hepatic microsomes/S9 of rats. In addition, some drug transporters, including OAT1/3, OCT2, MDR1, OATP1B1, MATE1/2K and OCTN2, were investigated. The cellular uptake assays indicate that 6-HKA exhibits inhibition to the transport of classical substrates mediated by OAT3, OCT2, MATE2K and OCTN2 but has no significant effect on the transport of substrates mediated by MDR1, OAT1, OATP1B1 or MATE1. Further investigation of cellular accumulation assays shows that 6-HKA might be the substrate of OAT3, but not OCT2 or OCTN2. The bidirectional transport study suggests that 6-HKA is not a substrate of MDR1.The information about the in vitro metabolism of 6-HKA and the interaction between 6-HKA and some transporters will help us to better understand the pharmacokinetic properties of 6-HKA and provide reference for its pharmacodynamics, DDIs and drug-food interactions studies.

Motivators for and Barriers to Exercise Rehabilitation in Hemodialysis Centers: A Multicenter Cross-Sectional Survey.

OBJECTIVES: The aim of the study was to explore motivators for and barriers to exercise rehabilitation in hemodialysis patients and the barriers perceived by the hemodialysis center staff. DESIGN: A cross-sectional study was performed in five hemodialysis centers using patient questionnaires designed for this study to evaluate the motivators for and barriers to exercise rehabilitation. Questionnaires were not yet validated. RESULTS: Of the 471 recruited patients, 63.3% were willing to participant in exercise rehabilitation. The greatest motivators included improving quality of life (98.0%) and wanting to be healthier (98.0%). Perceived barriers included discomfort (59.0%), concerns regarding safety (36.7%), and disinterest (27.0%). Among these, unwillingness, disinterest, and having peripheral arterial disease were independent risk factors of lack of participation in exercise rehabilitation. The most common perceived barriers among the 90 employees that participated were lack of professional guidance and advice from rehabilitation therapists (93.1%), lack of exercise rehabilitation knowledge (86.2%), and lack of special exercise equipment (86.2%). CONCLUSIONS: Most patients were willing to exercise to improve their health and quality of life. Barriers to exercise rehabilitation included patient and staff factors. It is essential to establish a rehabilitation team within dialysis centers, including general staff and rehabilitation therapists. These centers require improved rehabilitation policies and access to specialized rehabilitation equipment.

Effect of Intradialytic Exercise on Physical Performance and Cardiovascular Risk Factors in Patients Receiving Maintenance Hemodialysis: A Pilot and Feasibility Study.

INTRODUCTION: Although intradialytic exercise is considered a form of "nonpharmacological medicine" for patients receiving maintenance hemodialysis (MHD), this practice has not been widely implemented in most dialysis centers because of clinical limitations. We, therefore, aimed to design an intradialytic exercise training program to improve the implementation of this practice and determine its impact on physical performance and cardiovascular risk factors in patients receiving MHD. METHODS: A total of 132 MHD patients at 4 outpatient dialysis units were enrolled and assigned randomly into exercise (n = 67) and control groups (n = 65). During a 2-year period, patients in the exercise group participated in 20-min exercise training sessions within dialysis sessions on 3 days per week. All patients underwent assessments of physical function (6-min walk test) and cardiovascular risk factors (blood pressure [BP], total cholesterol [TC], low-density lipoprotein [LDL], high-sensitivity C-reactive protein [hsCRP], albumin [Alb], hemoglobin [Hb], and erythropoietin [EPO] dose) at the baseline and annually thereafter. RESULTS: Of the participants, 50.8% had completed the study after 2 years. No statistically significant intragroup or intergroup differences were observed in the measures of 6MD, BP, TC, hsCRP, Alb, Hb, and EPO dose. CONCLUSION: The results suggest that although this low-intensity, nonprogressive intradialytic exercise program may be practical, it was not sufficient to improve physiological function and reduce cardiovascular disease risk factors in patients receiving MHD.

Research advances in the detection of miRNA.

MicroRNAs (miRNAs) are a family of endogenous, small (approximately 22 nucleotides in length), noncoding, functional RNAs. With the development of molecular biology, the research of miRNA biological function has attracted significant interest, as abnormal miRNA expression is identified to contribute to serious human diseases such as cancers. Traditional methods for miRNA detection do not meet current demands. In particular, nanomaterial-based methods, nucleic acid amplification-based methods such as rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), strand-displacement amplification (SDA) and some enzyme-free amplifications have been employed widely for the highly sensitive detection of miRNA. MiRNA functional research and clinical diagnostics have been accelerated by these new techniques. Herein, we summarize and discuss the recent progress in the development of miRNA detection methods and new applications. This review will provide guidelines for the development of follow-up miRNA detection methods with high sensitivity and specificity, and applicability to disease diagnosis and therapy.

Ultrasensitive detection of miRNA via one-step rolling circle-quantitative PCR (RC-qPCR).

A novel microRNA (miRNA) quantification method has been developed using one-step rolling circle-quantitative PCR (RC-qPCR) analysis. Vent (exo-) DNA polymerase is firstly utilized to combine a rolling circle amplification (RCA) and qPCR in one step with high sensitivity and specificity in our RC-qPCR assay. Before performing the RC-qPCR, a padlock probe is ligated only when it is perfectly hybridized with miRNA. This ligation-based miRNA assay is highly specific for mature miRNAs, discriminating among related miRNAs that differ by as little as one nucleotide. It exhibits a dynamic range of seven orders of magnitude with a detection limit of 500 aM, and could be also used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs).

Influence of organic anion transporter 1/3 on the pharmacokinetics and renal excretion of ginkgolides and bilobalide.

ETHNOPHARMACOLOGICAL RELEVANCE: The major terpene lactones of ginkgo biloba extract (GBE) include ginkgolide A, B, C and bilobalide are used for the protection of cardiovascular, cerebrovascular and neurodegenerative diseases. Terpene lactones are orally bioavailable and predominantly eliminated via the renal pathway. However, information on the transporters involved in the pharmacokinetics (PK) and renal excretion of terpene lactones is limited. AIM OF THE STUDY: The objective of this study is to assess the role of OAT1/3 which are important transporters in the human kidney in the PK and renal excretion ginkgolide A, B, C and bilobalide. MATERIALS AND METHODS: Uptake of ginkgolide A, B, C and bilobalide in Madin-Darby Canine Kidney (MDCK) and human embryonic kidney 293 (HEK293) cells overexpressing OAT1 or OAT3, respectively were studied. To verify the result from in vitro cell models, the studies on PK, kidney accumulation and urinary excretion of ginkgolide A, B, C and bilobalide were carried out in rats. RESULTS: The result showed that ginkgolide A, B, C and bilobalide are low-affinity substrates of OAT1/3. Following co-administration with probenecid, a typical inhibitor of OAT1/3, the rat plasma concentrations of ginkgolide A, B, C and bilobalide increased significantly. AUC showed a significant increase in the probenecid-treated rats compared to control rats (893.48 vs. 1123.85, 314.91 vs. 505.74, and 2724.97 vs. 3096.40 µg/L*h for ginkgolide A, B and bilobalide, respectively), while the clearance of these compounds significantly decreased. The accumulation of ginkgolide A, B and bilobalide in the kidney of the probenecid-treated rats was reduced by 1.8, 2.4, and 1.5-fold, respectively; further reducing the cumulative urinary recovery of these compounds. CONCLUSION: The findings indicated that ginkgolide A, B and bilobalide are excreted via OAT1/3-mediated transport in the kidney and OAT1/3 inhibitor significantly influence the PK ginkgolides and bilobalide.

Development and validation of a sensitive LC-MS/MS method for the determination of 6-hydroxykynurenic acid in rat plasma and its application to pharmacokinetics study.

A sensitive and specific bioanalytical method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of 6-hydroxykynurenic acid (6-HKA) in a small quantity of rat plasma has been developed and comprehensively validated. Tolbutamide (Tol) was used as the internal standard (IS). An aliquot of 50 µL rat plasma sample was deproteinized by 150 µL methanol, and then 100 µL of its supernatant was mixed with 100 µL water after centrifugation. Chromatographic separation was performed on a ZORBAX Eclipse Plus C18 Rapid Resolution HD column (2.1 mm × 100 mm, 1.8 µm) with a gradient mobile phase consisting of water containing 2 mM ammonium formate and methanol at a flow rate of 0.2 mL/min over a total run time of 5.0 min. The mass spectrometer was operated under multiple reactions monitoring (MRM) mode with the transitions m/z 206.1 → 160.0 for 6-HKA and m/z 269.0 → 170.0 for Tol. The linear range was 2.5-250 ng/mL with the square regression coefficient (r2) of 0.997. The lower limit of quantification (LLOQ) was 2.5 ng/mL (Relative Error, RE +1.6% and RSD 4.8%, n = 5). The intra- and inter-day precision and accuracy was <13.3%. The mean recovery of 6-HKA in rat plasma was between the range of 99.3-103.4%. This method was successfully applied to study the pharmacokinetics of 6-HKA in rats after oral administration at a single dose of 20.0 mg/kg and after tail intravenous injection at a single dose of 2.0 mg/kg. Pharmacokinetic parameters bioavailability, Cmax, oral, Tmax, oral, AUC0-24h, oral, AUC0-∞, oral, AUC0-24h, iv and AUC0-∞, iv were 3.96%, 152.0 ±â€¯85.5 ng/mL, 0.4 ±â€¯0.1 h, 340.0 ±â€¯136.3 ng/mL ∗ h, 369.5 ±â€¯135.0 ng/mL ∗ h, 906.6 ±â€¯324.1 ng/mL*h and 932.9 ±â€¯336.5 ng/mL ∗ h, respectively.

A novel helper qPCR system for platinum detection via Pt-DNA coordination.

A novel real-time polymerase chain reaction (qPCR) platform for the simple and robust detection of platinum is described for the first time. Compared with conventional qPCR, a helper template, which is related to the active template for performing qPCR, was introduced in our helper qPCR system. Several guanine (G) bases were introduced in the helper template to obtain a platinum-responsive on/off switch based on G-Pt-G coordination. Because of the helper template, a slight change in platinum concentration would significantly change the signal in the qPCR. This novel helper qPCR technique easily detects platinum with high sensitivity (1 ng/mL) and selectivity over other metal ions. Therefore, it will be a promising technique for the detection of platinum in biomedical and environmental samples.

The metabolism and hepatotoxicity of ginkgolic acid (17 : 1) in vitro.

Pharmacological activities and adverse side effects of ginkgolic acids (GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA (17 : 1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA (17 : 1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA (17 : 1) metabolism were human CYP1A2, CYP3A4, UGT1A6, UGT1A9, and UGT2B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA (17 : 1) in HepG2 cells occurred in a time- and dose-dependent manner. Further investigation showed that GA (17 : 1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1A- and CYP3A-mediated metabolism.

Mechanism for ginkgolic acid (15 : 1)-induced MDCK cell necrosis: Mitochondria and lysosomes damages and cell cycle arrest.

Ginkgolic acids (GAs), primarily found in the leaves, nuts, and testa of ginkgo biloba, have been identified with suspected allergenic, genotoxic and cytotoxic properties. However, little information is available about GAs toxicity in kidneys and the underlying mechanism has not been thoroughly elucidated so far. Instead of GAs extract, the renal cytotoxicity of GA (15 : 1), which was isolated from the testa of Ginkgo biloba, was assessed in vitro by using MDCK cells. The action of GA (15 : 1) on cell viability was evaluated by the MTT and neutral red uptake assays. Compared with the control, the cytotoxicity of GA (15 : 1) on MDCK cells displayed a time- and dose-dependent manner, suggesting the cells mitochondria and lysosomes were damaged. It was confirmed that GA (15 : 1) resulted in the loss of cells mitochondrial trans-membrane potential (ΔΨm). In propidium iodide (PI) staining analysis, GA (15 : 1) induced cell cycle arrest at the G0/G1 and G2/M phases, influencing on the DNA synthesis and cell mitosis. Characteristics of necrotic cell death were observed in MDCK cells at the experimental conditions, as a result of DNA agarose gel electrophoresis and morphological observation of MDCK cells. In conclusion, these findings might provide useful information for a better understanding of the GA (15 : 1) induced renal toxicity.

The regioselective glucuronidation of morphine by dimerized human UGT2B7, 1A1, 1A9 and their allelic variants.

Uridine diphosphate-glucuronosyltransferase (UGT) 2B7 is expressed mostly in the human liver, lung and kidney and can transfer endogenous glucuronide group into its substrate and impact the pharmacological effects of several drugs such as estriol, AZT and morphine. UGT2B7 and its allelic variants can dimerize with the homologous enzymes UGT1A1 and UGT1A9, as well as their allelic variants, and then change their enzymatic activities in the process of substrate catalysis. The current study was designed to identify this mechanism using morphine as the substrate of UGT2B7. Single-recombinant allozymes, including UGT2B7*1 (wild type), UGT2B7*71S (A71S, 211G>T), UGT2B7*2 (H268Y, 802C>T), UGT2B7*5 (D398N, 1192G>A), and double-recombinant allozymes formed by the dimerization of UGT1A9*1 (wild type), UGT1A9*2 (C3Y, 8G>A), UGT1A9*3 (M33T, 98T>C), UGT1A9*5 (D256N, 766G>A), UGT1A1 (wild type) with its splice variant UGT1A1b were established and incubated with morphine in vitro. Each sample was analyzed with HPLC-MS/MS. All enzyme kinetic parameters were then measured and analyzed. From the results, the production ratio of its aberrant metabolism and subsequent metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), changes regioselectively. Double-recombinant allozymes exhibit stronger enzymatic activity catalyzing morphine than the single-recombinant alloyzymes. Compared to UGT2B7*1, UGT2B7*2 singles or doubles have lower Km values for M3G and M6G, whereas UGT2B7*5 allozymes perform opposite effects. The double allozymes of UGT1A9*2 or UGT1A9*5 with UGT2B7 tend to produce M6G. Interestingly, the majority of single or double allozymes significantly reduce the ratio of M3G to M6G. The UGT1A9*2-UGT2B7*1 double enzyme has the lowest M3G:M6G ratio, reflecting that more M6G would form in morphine glucuronide metabolism. This study demonstrates that UGT2B7 common SNPs and their dimers with UGT1A1 and UGT1A9 and their allelic variants can regioselectively affect the generation of two metabolites of morphine via altering the CLint ratios of M3G to M6G. These results may predict the effectiveness of morphine antinociception in individualized opioid treatment.

Brain-derived neurotrophic factor involved epigenetic repression of UGT2B7 in colorectal carcinoma: A mechanism to alter morphine glucuronidation in tumor.

Uridine diphosphate-glucuronosyltransferase (UGT) 2B7, as one of significant drug enzymes, is responsible on the glucuronidation of abundant endobiotics or xenobiotics. We here report that it is markedly repressed in the tumor tissues of colorectal carcinoma (CRC) patients. Accordingly, morphine in CRC cells will stimulate the expression of its main metabolic enzyme, UGT2B7 during tolerance generation by activating the positive signals in histone 3, especially for trimethylated lysine 27 (H3K4Me3) and acetylated lysine 4 (H3K27Ac). Further study reveals that brain-derived neutrophilic factor (BDNF), a secretory neurotrophin, enriched in CRC can interact and inhibit UGT2B7 by primarily blocking the positive signals of H3K4Me3 as well as activating H3K27Ac on the promoter region of UGT2B7. Meanwhile, BDNF repression attributes to the sensitizations of main core factors in poly-comb repressive complex (PRC) 1 rather than PRC2 as the reason of the depression of SUZ12 in the later complex. Besides that, the productions of two main morphine glucuronides are both increased in the BDNF deficient or TSA and BIX-01294 treated morphine tolerance-like HCT-116 cells. On the same condition, active metabolite, morphine-6-glucuronide (M6G) was accumulated more than inactive M3G. Our findings imply that enzymatic activity enhancement and substrate regioselective catalysis alteration of UGT2B7 may release morphine tolerance under the cure of tumor-induced pain.

Reverse of Acute and Chronic Morphine Tolerance by Lithocholic Acid via Down-Regulating UGT2B7.

Lithocholic acid (LCA) deposited in human livers always induces drastic pains which need analgesic drug, like morphine to release. Our research showed that LCA can effectively inhibit uridine 5'-diphospho-glucuronosyltransferase 2B7 (UGT2B7) in morphine tolerance-like human normal liver cells, HL-7702, then increase µ-opioid receptor (MOR) and calcium-calmodulin dependent protein kinase IIα (CaMKIIα) expression. In vivo assay, UGT2B7 was significantly repressed in the livers of acute or chronic morphine tolerance mice pretreated with LCA (10, 50, and 100 mg/kg, p.o.). To investigate the connections between LCA function performance and change of UGT2B7 enzymatic activity in mice livers, two morphine metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) were quantified by solid phase extraction (SPE)-HPLC-MS/MS. The result indicated no matter in acute or chronic morphine tolerance, the concentrations of M3G and M6G were all decreased, the later one fell even more. Besides that, 50 mg/kg of LCA administration can prevent auto-phosphorylation of CaMKIIα at Thr286 in acute or chronic morphine tolerance mice prefrontal cortexes (mPFCs) due to synthesis increase of cyclic adenosine monophosphate. As a consequence, UGT2B7 depression mediated by LCA can affect its selective catalysis ability to morphine, that may be responsible to acute or chronic morphine tolerance alleviation. These findings might assist to modify antinociception of morphine in clinic.

siRNA capsulated brain-targeted nanoparticles specifically knock down OATP2B1 in mice: a mechanism for acute morphine tolerance suppression.

Regulating main brain-uptake transporter of morphine may restrict its tolerance generation, then modify its antinociception. In this study, more than 2 fold higher intracellular uptake concentrations for morphine and morphine-6-glucuronide (M6G) were observed in stable expression cells, HEK293-hOATP2B1 than HEK293-MOCK. Specifically, the Km value of morphine to OATP2B1 (57.58 ± 8.90 µM) is 1.4-time more than that of M6G (80.31 ± 21.75 µM); Cyclosporine A (CsA), an inhibitor of OATP2B1, can inhibit their intracellular accumulations with IC50 = 3.90 ± 0.50 µM for morphine and IC50 = 6.04 ± 0.86 µM for M6G, respectively. To further investigate the role of OATP2B1 in morphine brain transport and tolerance, the novel nanoparticles of DGL-PEG/dermorphin capsulated siRNA (OATP2B1) were applied to deliver siRNA into mouse brain. Along with OATP2B1 depressed, a main reduction was found for each of morphine or M6G in cerebrums or epencephalons of acute morphine tolerance mice. Furthermore, calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) in mouse prefrontal cortex (mPFC) underwent dephosphorylation at Thr286. In conclusion, OATP2B1 downregulation in mouse brain can suppress tolerance via blocking morphine and M6G brain transport. These findings might help to improve the pharmacological effects of morphine.

Simultaneous analysis of gemfibrozil, morphine, and its two active metabolites in different mouse brain structures using solid-phase extraction with ultra-high performance liquid chromatography and tandem mass spectrometry with a deuterated internal standard.

A rapid and sensitive bioassay was established and validated to simultaneously determine gemfibrozil, morphine, morphine-3ß-glucuronide, and morphine-6ß-glucuronide in mouse cerebrum, epencephalon, and hippocampus based on ultra-high performance liquid chromatography and tandem mass spectrometry. The deuterated internal standard, M6G-d3, was mixed with the prepared samples at 10 ng/mL as the final concentration. The samples were transferred into the C18 solid-phase extraction columns with gradient elution for solid-phase extraction. The mobile phase consisted of methanol and 0.05% formic acid (pH 3.2). Multiple reaction monitoring has been applied to analyze gemfibrozil (m/z 249.0 → 121.0) in anion mode, and M6G-d3 (m/z 465.1 → 289.1), morphine (m/z 286.0 → 200.9), and M3G and M6G (m/z 462.1 → 286.1) in the positive ion mode. The method has a linear calibration range from 0.05 to 10 ng for gemfibrozil, morphine, and M3G and M6G with correlation coefficients >0.993. The lower limit of quantitation for all four analytes was 0.05 ng/mL, relative standard deviation of intra- and interday precision was less than 10.5%, and the relative error of accuracy was from -8.2 to 8.3% at low, medium, and high concentrations for all the analytes. In conclusion, gemfibrozil can influence the morphine antinociception after coronary heart disease induced chronic angina by the change in one of morphine metabolites', M3G, distribution in mouse brain.