Interlaboratory analysis of teicoplanin plasma concentration assays among Chinese laboratories.

WHAT IS KNOWN AND OBJECTIVE: Teicoplanin is widely used for the treatment of infections caused by drug-resistant Gram-positive bacteria. Since there is a good correlation between trough levels and clinical outcome, therapeutic drug monitoring (TDM) is recommended to achieve better clinical curative effects. However, TDM of teicoplanin is not routine in China. So, a programme was initiated in 2017, including both HPLC method establishment and interlaboratory quality assessment, for the measurement of teicoplanin. METHODS: A main centre and a quality control centre were set up in the study. An HPLC-based method of teicoplanin determination in plasma was developed by the main centre. Analysis was performed using a Waters Symmetry C18 column (250 mm × 4.6 mm, 5 µm). The mobile phase was NaH2 PO4 (0.01 mol/L) and acetonitrile (75:25 v/v; pH 3.3), with a flow rate of 1.0 mL/min and a detection wavelength of 215 nm. Piperacillin sodium was selected as an internal standard (IS). Twenty-six additional TDM centres were then recruited to adopt this method. Then, all the centres were asked to take part in a quality control assessment evaluated by the quality control centre. RESULTS: For all TDM centres, linearity of teicoplanin concentration ranges was between 3.125 and 100 µg/mL. Intraday and interday accuracies ranged from 87.1% to 118.4%. Intraday and interday precision ranged from 0.3% to 13.8%. Therapeutic drug monitoring centres all passed inter-room quality assessment. All samples tested met the acceptance criteria. Then, 542 samples were collected. Patients with sub-optimal (≤10 mg/L) plasma teicoplanin concentrations constituted 42% of the total study population. WHAT IS NEW AND CONCLUSIONS: For the first time, a simple, rapid and accurate HPLC method for determining teicoplanin levels was successfully applied to therapeutic drug monitoring in clinical practice for twenty-seven TDM centres in China. The results demonstrated excellent interlaboratory agreement for teicoplanin testing and provide support for clinical laboratory quality management and results inter-accreditation.

Changes in intestinal microbiota affect metabolism of ginsenoside Re.

Ginsenoside Re, an active ingredient in Panax ginseng, is widely used as a therapeutic and nutriment. The intestinal microbiota plays crucial roles in modulating the pharmacokinetics and pharmacological actions of ginsenoside Re. The aim of this study was to explore the relationship between bacterial community variety and the metabolic profiles of ginsenoside Re. We developed two models with intestinal dysbacteriosis: a pseudo-germ-free model induced by a nonabsorbable antimicrobial mixture (ATM), and Qi-deficiency model established via over-fatigue and acute cold stress (OACS). First, the bacterial community structures in control, ATM and OACS rats were compared via 16S ribosomal RNA amplicon sequencing. Then, the gut microbial metabolism of ginsenoside Re was assessed qualitatively and quantitatively in the three groups by UPLC-Q-TOF/MS and HPLC-TQ-MS, respectively. Ten metabolites of ginsenoside Re were detected and tentatively identified, three of which were novel. Moreover, owing to significant differences in bacterial communities, deglycosylated products, as the main metabolites of ginsenoside Re, were produced at lower levels in ATM and OACS models. Importantly, the levels of these deglycosylated metabolites correlated with alterations in Prevotella, Lactobacillus and Bacteroides populations, as well as glycosidase activities. Collectively, biotransformation of ginsenoside Re is potentially influenced by regulation of the composition of intestinal microbiota and glycosidase activities.

Chaperone heat shock protein 70 in nucleus accumbens core: a novel biological target of behavioural sensitization to morphine in rats.

Drug addiction is a major public health issue, yet the underlying adaptation of neural networks by drugs of abuse is not fully understood. We have previously linked chaperone heat shock protein 70 (Hsp70) to drug-induced adaptations. Focusing on the NAc core and shell, the present study aims to provide further findings for our understanding of the relation between behavioural sensitization to morphine and Hsp70 at transcriptional and functional levels in rats. Firstly, we delineated the characteristics of behavioural sensitization induced by a single morphine exposure (1-10 mg/kg, s.c.). Secondly, Hsp70 protein expression in the NAc core was time- and dose-relatedly induced during the development of behavioural sensitization to a single morphine exposure in rats, and Pearson analysis indicated a positive correlation between behavioural sensitization and Hsp70 expression in NAc core. Thirdly, at the transcriptional level, intra-NAc core injection of the specific heat shock factor-I (HSF-I) inhibitor N-Formyl-3,4-methylenedioxy-benzylidine-γ-butyrolactam (KNK437) suppressed Hsp70 expression and the development of behavioural sensitization, while the HSF-I specific inducer geranylgeranylacetone (GGA) promoted both of them. Interestingly, intra-NAc shell injection of KNK437 or GGA did not affect the development of behavioural sensitization. Finally, both the functional inhibition of Hsp70 ATPase activity by methylene blue (MB), and the antagonism of Hsp70 substrate binding site (SBD) activity by pifithrin-µ (PES) impaired the development of behavioural sensitization when they were microinjected into the NAc core. Taken together, the critical involvement of chaperone Hsp70 in behavioural sensitization to morphine identifies a biological target for long-lasting adaptations with relevance to addiction.

Molecular chaperone heat shock protein 70 participates in the labile phase of the development of behavioural sensitization induced by a single morphine exposure in mice.

De-novo protein synthesis is required in the development of behavioural sensitization. A prior screening test from our laboratory has implicated heat shock protein 70 (Hsp70) as one of the proteins required in this behavioural plasticity. Thus, this study was designed to extend our understanding of the role of Hsp70 in the development of behavioural sensitization induced by a single morphine exposure in mice. First, by employing transcription inhibitor actinomycin D (AD) and protein synthesis inhibitor cycloheximide (CHX), we identified a protein synthesis-dependent labile phase (within 4 h after the first morphine injection) in the development of behavioural sensitization to a single morphine exposure. Second, Hsp70 protein expression in the nucleus accumbens correlated positively with locomotor responses of sensitized mice and, more importantly, the expression of Hsp70 increased within 1 h after the first morphine injection. Third, AD and CHX both prevented expression of Hsp70 and disrupted the development of the single morphine induced behavioural sensitization, which further implied Hsp70 was highly associated with behavioural sensitization. Finally, the selective Hsp70 inhibitor pifithrin-µ (PES) i.c.v. injected in mice prevented the development of behavioural sensitization and, critically, this inhibitory effect occurred only when PES was given within 1 h after the first morphine injection, which was within the labile phase of the development period. Taken together, we draw the conclusion that Hsp70 is crucially involved in the labile phase of the development of behavioural sensitization induced by a single morphine exposure, probably functioning as a molecular chaperone.

The phosphodiesterase-4 (PDE4) inhibitor rolipram decreases ethanol seeking and consumption in alcohol-preferring Fawn-Hooded rats.

BACKGROUND: Alcohol dependence is a complex psychiatric disorder demanding development of novel pharmacotherapies. Because the cyclic adenosine monophosphate (cAMP) signaling cascade has been implicated in mediating behavioral responses to alcohol, key components in this cascade may serve as potential treatment targets. Phosphodiesterase-4 (PDE4), an enzyme that specifically catalyzes the hydrolysis of cAMP, represents a key point in regulating intracellular cAMP levels. Thus, it was of interest to determine whether PDE4 was involved in the regulation of alcohol use and abuse. METHODS: Male Fawn-Hooded (FH/Wjd) rats were tested for 5% (v/v) ethanol (EtOH) and 10% (w/v) sucrose operant oral self-administration following treatment with the selective PDE4 inhibitor rolipram (0.0125, 0.025, or 0.05 mg/kg, subcutaneous [s.c.]); rolipram at higher doses (0.05, 0.1, and 0.2 mg/kg, s.c.) was tested to determine its impact on the intake of EtOH, sucrose, or water using the 2-bottle choice drinking paradigm. Subsequent open-field testing was performed to evaluate the influence of higher doses of rolipram on locomotor activity. RESULTS: Acute administration of rolipram dose-dependently reduced operant self-administration of 5% EtOH, but had no effect on 10% sucrose responding. Time-course assessment revealed significant decreases in EtOH consumption after rolipram (0.1, 0.2 mg/kg) treatment in continuous- and intermittent access to EtOH at 5% or 10%, respectively. Moreover, chronic rolipram treatment time-dependently decreased 5% EtOH consumption and preference during treatment days and after the termination of rolipram administration. Rolipram at the highest doses (0.1 and 0.2 mg/kg) did decrease locomotor activity, but the effect lasted only 10 and 20 minutes, respectively, which did not likely alter long-term EtOH drinking. CONCLUSIONS: These results suggest that PDE4 plays a role in alcohol seeking and consumption behavior. Drugs interfering with PDE4 may be a potential pharmacotherapy for alcohol dependence.

Transcription and protein synthesis inhibitors reduce the induction of behavioural sensitization to a single morphine exposure and regulate Hsp70 expression in the mouse nucleus accumbens.

New protein synthesis has been implicated as necessary for long-lasting changes in neuronal function. Behavioural sensitization to a single exposure to addictive drugs is a form of neuroplasticity, but little is known about the importance of new protein synthesis in the underlying mechanism. This study was designed to investigate the effects of the transcription inhibitor actinomycin D (AD) and the protein synthesis inhibitor cycloheximide (CHX) on induction of behavioural sensitization to a single morphine exposure in mice. In combination with behavioural experiments, changes in gene and protein expression in the mouse nucleus accumbens (NAc) were analysed by RT-PCR array and Western blot respectively. Behavioural sensitization was evident in mice pretreated only once with morphine at the doses of 20 and 40 mg/kg, but not 5 and 10 mg/kg. Mice pretreated with morphine (20 mg/kg) and challenged with a lower dose (5 mg/kg) after a period of 4-21 d washout showed sensitized locomotion. At the doses that did not affect locomotion in mice, AD or CHX significantly suppressed hyperactivity induced by acute treatment, but not challenge with morphine, and blocked induction of behavioural sensitization to a single morphine exposure in a dose-related manner. The results from RT-PCR array and Western blot indicated that the changes of Hsp70 expression in the NAc of mice were associated with behavioural sensitization induced by a single morphine exposure. Together, these findings suggest that induction of behavioural sensitization to a single morphine exposure requires new protein synthesis, potentially involving Hsp70 expression in the NAc of mice.

Context- and time-dependent neurobiological and behavioral sensitization induced by a single morphine exposure in mice.

RATIONALE: Drug-induced sensitization in the mesocorticolimbic systems is thought to play an important role in certain aspects of drug addiction, including the involvement of drug-associated cues and environments in mediating drug-seeking behaviors. Our previous studies have identified the significance of heat shock protein 70 (Hsp70) in the development of a single morphine exposure-induced behavioral sensitization. OBJECTIVES: The present study expands upon these findings by investigating the effect of environment on the expression of behavioral sensitization induced by a single morphine exposure, and the potential involvement of Hsp70 protein levels in these effects. METHODS: Mice were pretreated with a single morphine injection in test chambers (morphine-paired) or home cages (morphine-unpaired) on day 1 and challenged on day 2 or 8, in test chambers. Hsp70 expression in the nucleus accumbens (NAc) was analyzed after the challenge. RESULTS: The expression of single morphine exposure-induced behavioral sensitization was accompanied by a significant increase in Hsp70 expression in NAc. In contrast, the unpaired morphine-treated group failed to exhibit behavioral sensitization or higher Hsp70 expression. Additionally, by adding a habituation process prior to the challenge, we demonstrated that conditioned hyperactivity, which was not accompanied by an increased expression of Hsp70, is not essential for behavioral sensitization. CONCLUSIONS: Behavioral sensitization induced by a single morphine exposure in mice exhibits context and time dependency, with environmental context likely functioning via an inhibitory conditioning mechanism. Furthermore, alterations in Hsp70 expression in the NAc may represent a neurobiological sensitization mechanism mediating context- and time-dependent behavioral sensitization.

Septal nuclei critically mediate the development of behavioral sensitization to a single morphine injection in rats.

Behavioral sensitization to a single morphine injection is a unique model to study the neuroanatomical substrates of long-lasting behavioral plasticity associated with opioid reward and abuse. Earlier observations have demonstrated that septal nuclei are critically involved in the processes of reward, learning and memory. In the present study, we investigated the effects of septal nuclei lesions on behavioral sensitization to a single morphine injection, morphine induced conditioned place preference and antinociception in rats. Behavioral sensitization was established by a single injection of 3-30 mg/kg morphine in rats. Bilateral electrical lesions of septal nuclei were carried out 7 days before morphine pretreatment. Acute morphine injection induced hyperactivity in the non-surgery control, sham surgery and septal nuclei-lesioned rats. Seven days later, the challenge injection with 3mg/kg morphine induced significant behavioral sensitization in rats with no surgery and sham surgery, but failed to induce behavioral sensitization in septal nuclei-lesioned rats. When the septal nuclei ablation was carried out after acute morphine pretreatment, the expression of behavioral sensitization was unaffected and not different among rats. In addition, septal nuclei lesions did not impact the rewarding and antinociceptive effects of 10 mg/kg morphine when the rats were tested in a conditioned place preference test and tail-flick test, respectively. Collectively, these results suggest that septal nuclei may be selectively involved in the initiation of behavioral sensitization to morphine, which is separable from the effects of morphine for exerting its rewarding and antinociceptive effects.

Effect of the histone deacetylase inhibitors on behavioural sensitization to a single morphine exposure in mice.

Behavioural sensitization to a single morphine exposure has been considered to be a long-term form of behavioural plasticity associated with opioid addiction. Accumulated evidence has shown that histone modification plays a key role in behavioural plasticity. Therefore, this study was designed to investigate whether the histone deacetylase inhibitors sodium butyrate (SB) and valproic acid (VPA) could disrupt behavioural sensitization to a single morphine exposure. Mice were pretreated with a single injection of morphine and elicited subsequent behavioural sensitization by a challenge-dosage of morphine after a 7-day drug-free period. At doses that did not affect the locomotor activity, both SB and VPA inhibited the acute morphine induced hyperactivity and significantly attenuated the development of behavioural sensitization to a single morphine exposure. Furthermore, the combination of SB and VPA at the sub-effective doses could additionally reduce the development of morphine sensitization. Western blot analysis revealed that multiple administration with the effective dose of SB (160 mg/kg, i.p.) or VPA (150 mg/kg, i.p.) in the behavioural experiments induced hyperacetylation of histone H3 in the NAc of mice. Taken together, these findings suggest that histone acetylation may be involved in morphine sensitization.