Metabolic study of new psychoactive substances

University of Technology Sydney. Faculty of Science.The rapid and continuous emergence of new psychoactive substances has been a trend of the recreational drug market over the last decade. The sheer number of the new drugs has posed a challenge for the forensic and clinical laboratories to detect the consumption of the drugs in urine drug testing, especially when the metabolism of the drugs is unknown. This thesis aims to demonstrate that the fungus Cunninghamella elegans is a suitable model for metabolism studies of synthetic cannabinoids, as well as to provide new knowledge on metabolism of several new psychoactive substances to aid the forensic and clinical laboratories in detection of these drugs in urine testing. Seven synthetic cannabinoids JWH-018, JWH-073, AM2201, 5F-PB-22, PB-22, XLR-11 and UR-144 were incubated with C. elegans for 72 h and the obtained metabolites were analysed by liquid chromatography–high resolution mass spectrometry (LC-HRMS). The cannabinoids underwent various biotransformations including, either alone or in combination, carboxylation, defluorination, dehydrogenation, demethylation, dihydrodiol formation, dihydroxylation, ester hydrolysis, hydroxylation, ketone formation, N-dealkylation, oxidative defluorination, oxidative defluorination to carboxylic acid, and trihydroxylation. Glucosidation and sulfation were the observed phase II biotransformations, although uncommon. The fungal metabolites were generally consistent with the human-relevant metabolites of these drugs reported in literature, except that glucuronidation is the common phase II human metabolic pathway instead of glucosidation and that the prevalence of the fungal metabolites was not always reflective of the human metabolite prevalence. Large amounts of fungal metabolites of UR-144 were obtained in an upscaled experiment and analysed by nuclear magnetic resonance (NMR) spectroscopy after isolating several metabolites by preparative high performance liquid chromatography (HPLC). Ten metabolites were characterised including dihydroxy metabolites, carboxy and hydroxy metabolites, a hydroxy and ketone metabolite, and a carboxy and ketone metabolite. Use of these metabolites as reference standards for the UR-144 metabolites after human liver microsomes (HLM) incubation indicated that a dihydroxy metabolite, carboxy and hydroxy metabolites, and a hydroxy and ketone metabolite generated by the fungus were also produced by HLM. The metabolism of the synthetic cannabinoid AM1220 was investigated after incubation with HLM and the fungus using liquid chromatography–tandem mass spectrometry (LC-MS/MS) and LC-HRMS. AM1220 was found to be a high clearance drug and hydroxylation, demethylation and dihydrodiol formation were the major biotransformations. The metabolism of acetylfentanyl, acrylfentanyl, 4-fluoro-isobutyrylfentanyl, and furanylfentanyl was studied in authentic human urine and human hepatocytes samples using LC-HRMS. N-dealkylation, hydroxylation, and hydroxylation and methoxylation were the major biotransformations for all but furanylfentanyl, which was mainly transformed by amide hydrolysis and dihydrodiol formation. It illustrates the need to examine the metabolism of individual drugs rather than predicting them based on the previous knowledge. In conclusion, C. elegans has the ability to mimic human metabolism and, by allowing large production of metabolites, enables more comprehensive characterisation of metabolites by NMR analysis. Therefore, the fungus can be a useful complementary model for metabolism of synthetic cannabinoids

Increased [¹⁸F]FMISO accumulation under hypoxia by multidrug-resistant protein 1 inhibitors

BACKGROUND: [¹⁸F]Fluoromisonidazole ([¹⁸F]FMISO) is a PET imaging probe widely used for the detection of hypoxia. We previously reported that [¹⁸F]FMISO is metabolized to the glutathione conjugate of the reduced form in hypoxic cells. In addition, we found that the [¹⁸F]FMISO uptake level varied depending on the cellular glutathione conjugation and excretion ability such as enzyme activity of glutathione-S-transferase and expression levels of multidrug resistance-associated protein 1 (MRP1, an efflux transporter), in addition to the cellular hypoxic state. In this study, we evaluated whether MRP1 activity affected [¹⁸F]FMISO PET imaging. METHODS: FaDu human pharyngeal squamous cell carcinoma cells were pretreated with MRP1 inhibitors (cyclosporine A, lapatinib, or MK-571) for 1 h, incubated with [¹⁸F]FMISO for 4 h under hypoxia, and their radioactivity was then measured. FaDu tumor-bearing mice were intravenously injected with [¹⁸F]FMISO, and PET/CT images were acquired at 4 h post-injection (1st PET scan). Two days later, the same mice were pretreated with MRP1 inhibitors (cyclosporine A, lapatinib, or MK-571) for 1 h, and PET/CT images were acquired (2nd PET scan). RESULTS: FaDu cells pretreated with MRP1 inhibitors exhibited significantly higher radioactivity than those without inhibitor treatment (cyclosporine A: 6.91 ± 0.27, lapatinib: 10.03 ± 0.47, MK-571: 10.15 ± 0.44%dose/mg protein, p < 0.01). In the in vivo PET study, the SUVmean ratio in tumors [calculated as after treatment (2nd PET scan)/before treatment of MRP1 inhibitors (1st PET scan)] of the mice treated with MRP1 inhibitors was significantly higher than those of control mice (cyclosporine A: 2.6 ± 0.7, lapatinib: 2.2 ± 0.7, MK-571: 2.2 ± 0.7, control: 1.2 ± 0.2, p < 0.05). CONCLUSION: In this study, we revealed that MRP1 inhibitors increase [¹⁸F]FMISO accumulation in hypoxic cells. This suggests that [¹⁸F]FMISO-PET imaging is affected by MRP1 inhibitors independent of the hypoxic state

Behavioral factors to include in guidelines for lifelong oral healthiness: an observational study in Japanese adults

BACKGROUND: The aim of this study was to determine which behavioral factors to include in guidelines for the Japanese public to achieve an acceptable level of oral healthiness. The objective was to determine the relationship between oral health related behaviors and symptoms related to oral disease and tooth loss in a Japanese adult community. METHODS: Oral health status and lifestyle were investigated in 777 people aged 20 years and older (390 men and 387 women). Subjects were asked to complete a postal questionnaire concerning past diet and lifestyle. The completed questionnaires were collected when they had health examinations. The 15 questions included their preference for sweets, how many between-meal snacks they usually had per day, smoking and drinking habits, presence of oral symptoms, and attitudes towards dental visits. Participants were asked about their behaviors at different stages of their life. The oral health examinations included examination of the oral cavity and teeth performed by dentists using WHO criteria. Odds ratios were calculated for all subjects, all 10 year age groups, and for subjects 30 years or older, 40 years or older, 50 years or older, and 60 years or older. RESULTS: Frequency of tooth brushing (OR = 3.98), having your own toothbrush (OR = 2.11), smoking (OR = 2.71) and bleeding gums (OR = 2.03) were significantly associated with number of retained teeth in males. Frequency of between-meal snacks was strongly associated with number of retained teeth in females (OR = 4.67). Having some hobbies (OR = 2.97), having a family dentist (OR = 2.34) and consulting a dentist as soon as symptoms occurred (OR = 1.74) were significantly associated with number of retained teeth in females. Factors that were significantly associated with tooth loss in both males and females included alcohol consumption (OR = 11.96, males, OR = 3.83, females), swollen gums (OR = 1.93, males, OR = 3.04, females) and toothache (OR = 3.39, males, OR = 3.52, females). CONCLUSION: Behavioral factors that were associated with tooth retention were frequency of eating snacks between meals, tooth brushing frequency, having one's own toothbrush, smoking and drinking habits, having hobbies, having a family dentist and when they had dental treatment. Clinical factors included bleeding gums, swollen gums, and toothache

Not only Body Weight Perception but also Body Mass Index is Relevant to Suicidal Ideation and Self-Harming Behavior in Japanese Adolescents

Whether a low body mass index (BMI) is directly associated with a high risk of suicidal ideation or self-harming behavior in adolescents is still inconclusive. This study has, therefore, evaluated the relevance of BMI to suicidal ideation and self-harming behavior after controlling for body weight perception (BWP) and other potential confounding factors. BMI, BWP, suicidal ideation, and self-harming behavior were all assessed using a self-report questionnaire administered to 18,104 Japanese adolescents. Potential confounding factors were also evaluated. The data were then analyzed using bi-variate and multivariate logistic regression. Low BMI was associated with suicidal ideation and deliberate self-harm when controlling for sex, age, drug use, emotional distress, and BWP. Low BMI may be an independent risk factor for suicidal ideation and deliberate self-harming behavior in Japanese adolescents.ArticleJOURNAL OF NERVOUS AND MENTAL DISEASE. 200(4):305-309 (2012)journal articl

Root-to-shoot Cd translocation via the xylem is the major process determining shoot and grain cadmium accumulation in rice

Physiological properties involved in divergent cadmium (Cd) accumulation among rice genotypes were characterized using the indica cultivar ‘Habataki’ (high Cd in grains) and the japonica cultivar ‘Sasanishiki’ (low Cd in grains). Time-dependence and concentration-dependence of symplastic Cd absorption in roots were revealed not to be responsible for the different Cd accumulation between the two cultivars because root Cd uptake was not greater in the Cd-accumulating cultivar ‘Habataki’ compared with ‘Sasanishiki’. On the other hand, rapid and greater root-to-shoot Cd translocation was observed in ‘Habataki’, which could be mediated by higher abilities in xylem loading of Cd and transpiration rate as a driving force. To verify whether different abilities in xylem-mediated shoot-to-root translocation generally account for the genotypic variation in shoot Cd accumulation in rice, the world rice core collection, consisting of 69 accessions which covers the genetic diversity of almost 32 000 accessions of cultivated rice, was used. The results showed strong correlation between Cd levels in xylem sap and shoots and grains among the 69 rice accessions. Overall, the results presented in this study revealed that the root-to-shoot Cd translocation via the xylem is the major and common physiological process determining the Cd accumulation level in shoots and grains of rice plants

Low-dose betamethasone-acetate for fetal lung maturation in preterm sheep

BackgroundAntenatal steroids are standard of care for women who are at risk of preterm delivery; however, antenatal steroid dosing and formulation have not been evaluated adequately. The standard clinical 2-dose treatment with betamethasone-acetate+betamethasone-phosphate is more effective than 2 doses of betamethasone-phosphate for the induction of lung maturation in preterm fetal sheep. We hypothesized that the slowly released betamethasone-acetate component induces similar lung maturation to betamethasone-phosphate+betamethasone-acetate with decreased dose and fetal exposure.ObjectiveThe purpose of this study was to investigate pharmacokinetics and fetal lung maturation of antenatal betamethasone-acetate in preterm fetal sheep.Study designGroups of 10 singleton-pregnant ewes received 1 or 2 intramuscular doses 24 hours apart of 0.25 mg/kg/dose of betamethasone-phosphate+betamethasone-acetate (the standard of care dose) or 1 intramuscular dose of 0.5 mg/kg, 0.25 mg/kg, or 0.125 mg/kg of betamethasone-acetate. Fetuses were delivered 48 hours after the first injection at 122 days of gestation (80% of term) and ventilated for 30 minutes, with ventilator settings, compliance, vital signs, and blood gas measurements recorded every 10 minutes. After ventilation, we measured static lung pressure-volume curves and sampled the lungs for messenger RNA measurements. Other groups of pregnant ewes and fetuses were catheterized and treated with intramuscular injections of betamethasone-phosphate 0.125 mg/kg, betamethasone-acetate 0.125 mg/kg, or betamethasone-acetate 0.5 mg/kg. Maternal and fetal betamethasone concentrations in plasma were measured for 24 hours.ResultsAll betamethasone-treated groups had increased messenger RNA expression of surfactant proteins A, B, and C, ATP-binding cassette subfamily A member 3, and aquaporin-5 compared with control animals. Treatment with 1 dose of intramuscular betamethasone-acetate 0.125mg/kg improved dynamic and static lung compliance, gas exchange, and ventilation efficiency similarly to the standard treatment of 2 doses of 0.25 m/kg of betamethasone-acetate+betamethasone-phosphate. Betamethasone-acetate 0.125 mg/kg resulted in lower maternal and fetal peak plasma concentrations and decreased fetal exposure to betamethasone compared with betamethasone-phosphate 0.125 mg/kg.ConclusionA single dose of betamethasone-acetate results in similar fetal lung maturation as the 2-dose clinical formulation of betamethasone-phosphate+betamethasone-acetate with decreased fetal exposure to betamethasone. A lower dose of betamethasone-acetate may be an effective alternative to induce fetal lung maturation with less risk to the fetus