Comparison between human liver microsomes and the fungus Cunninghamella elegans for biotransformation of the synthetic cannabinoid JWH-424 having a bromo-naphthyl moiety analysed by high-resolution mass spectrometry

Purpose

JWH-424, (8-bromo-1-naphthyl)(1-pentyl-1H-indol-3-yl)methanone, is a synthetic cannabinoid, which is a brominated analogue of JWH-018, one of the best-known synthetic cannabinoids. Despite the structural similarity to JWH-018, little is known about JWH-424 including its metabolism. The aim of the study was to compare human liver microsomes (HLM) and the fungus Cunninghamella elegans as the metabolism catalysts for JWH-424 to better understand the characteristic actions of the fungus in the synthetic cannabinoid metabolism.

Methods

JWH-424 was incubated with HLM for 1 h and Cunninghamella elegans for up to 72 h. The HLM incubation mixtures were diluted with methanol and fungal incubation mixtures were extracted with dichloromethane and reconstituted in methanol before analyses by liquid chromatography–high-resolution mass spectrometry (LC-HRMS).

Results

HLM incubation resulted in production of ten metabolites through dihydrodiol formation, hydroxylation, and/or ipso substitution of the bromine with a hydroxy group. Fungal incubation led to production of 23 metabolites through carboxylation, dihydrodiol formation, hydroxylation, ketone formation, glucosidation and/or sulfation.

Conclusions

Generally, HLM models give good predictions of human metabolites and structural analogues are metabolised in a similar fashion. However, major hydroxy metabolites produced by HLM were those hydroxylated at naphthalene instead of pentyl moiety, the major site of hydroxylation for JWH-018. Fungal metabolites, on the other hand, had undergone hydroxylation mainly at pentyl moiety. The metabolic disagreement suggests the necessity to verify the human metabolites in authentic urine samples, while H9 and H10 (hydroxynaphthalene), H8 (ipso substitution), F22 (hydroxypentyl), and F17 (dihydroxypentyl) are recommended for monitoring of JWH-424 in urinalysis.

     

Contribution of Bcl-2, but not Bcl-xL and Bax, to antiapoptotic actions of hepatocyte growth factor in hypoxia-conditioned human endothelial cells

Angiogenic growth factors play important roles in angiogenic responses, such as vasculogenesis and angiogenesis in response to hypoxia. A novel angiogenic growth factor, hepatocyte growth factor (HGF), has been reported to inhibit endothelial cell death. However, its molecular mechanisms are largely unknown. Thus, we studied (1) the effects of HGF on hypoxia-induced endothelial apoptosis and (2) the molecular mechanisms of the antiapoptotic actions of HGF in endothelial cells. Severe hypoxia increased the cell death rate in human aortic endothelial cells, whereas HGF significantly attenuated cell death. In addition, hypoxic treatment resulted in a significant increase in apoptotic cells, whereas HGF could attenuate apoptosis, accompanied by attenuation of the increase in caspase-3-like activity (P<0.01). Of importance, HGF significantly increased Bcl-2, an inhibitor of apoptosis, in a dose-dependent manner under normoxic and hypoxic conditions (P<0.01), whereas hypoxic conditions resulted in a significant decrease in Bcl-2. In contrast, HGF failed to affect Bcl-xL, which is also well known as an inhibitor of apoptosis under both normoxic and hypoxic conditions, whereas Bcl-xL was significantly decreased in endothelial cells exposed to hypoxia (P<0.01). No significant change in Bax, a promoter of apoptosis, was also observed in endothelial cells under hypoxia, whereas HGF did not affect BAX: Overall, this study demonstrated that HGF prevented endothelial cell death induced by hypoxia through its antiapoptotic action. The antiapoptotic mechanisms of HGF in hypoxia-induced endothelial cell death largely depend on Bcl-2, but not Bcl-xL and BAX:     

Clinical significance of serum autoantibodies against Ras-like GTPases,RalA, in patients with esophageal squamous cell carcinoma

Background

The Ras-like GTPases, RalA and RalB are members of the Ras superfamily of small GTPases. Aberrant activation of Ral is a major cause of human tumorigenesis induced by oncogenic Ras. Serum anti-RalA antibodies are induced in esophageal carcinoma patients. However, detailed comparisons of their pathological characteristics are unavailable, and conventional serum markers have not been well evaluated.

Methods

Serum samples of 171 patients with esophageal squamous cell carcinoma and 73 healthy individuals were analyzed using specifically developed ELISA system for serum anti-RalA antibodies. A cut-off optical density value was fixed at 0.255 (the control mean + 2 SD). Clinicopathological characteristics and positive rates of conventional tumor markers were evaluated for seropositive patients.

Results

Overall positive rate for serum anti-RalA antibodies was 18 %, which gradually increased with the tumor stages. Although the positive rate for serum anti-RalA antibodies was comparable with that of carcinoembryonic antigen (24 %) and CYFRA21-1 (21 %), it was lower than the rate for serum p53 antibodies (31 %) and squamous cell carcinoma antigen (37 %). Although serum anti-RalA antibodies were not associated with other serum markers, it was inversely associated with serum p53 antibodies. No clear association was observed between serum anti-RalA antibodies and RalA immunoreactivity.

Conclusions

Presence of serum anti-RalA antibodies is associated with tumor stages, but not with conventional tumor markers. Serum anti-RalA antibodies may be candidate serum markers in combination with other serum markers for esophageal squamous cell carcinoma.

     

Identification of protein Salpha gene mutations including four novel mutations in eight unrelated patients with protein S deficiency

Eight distinct and potentially causative mutations were identified in eight unrelated Japanese patients with protein S (PS) deficiency, by direct DNA sequencing of the protein Salpha (PSalpha) gene-specific polymerase chain reaction products of all 15 exons and exon/intron boundaries. There were five missense mutations, including two novel mutations (Cys80Tyr and Arg314His), and three showed a major impact on the expected gene products: novel mutations of a 5-bp deletion (delCTCTG887:Cys206Stop) and a nonsense mutation (Glu208Stop), as well as a previously reported splice site (exon 10 +5 A-->G) mutation. One of the patients showed compound heterozygosity for delCTCTG887 and 732A-->G. Investigation for the cosegregation state of these two mutations with PS deficiency in the patient's family suggested that the delCTCTG887 mutation was responsible for the abnormal phenotype and that the 732A-->G (Lys155Glu) mutation did not appear to play a key role. However, we also identified the same 732A-->G (Lys155Glu) mutation in an unrelated patient with apparent PS deficiency with severe pulmonary embolism, and found that this mutation seemed to cosegregate with a PS-deficient state in her family members. These data implied that unknown factor(s) other than the 732A-->G mutation itself might influence phenotypic expression of PS status in different individuals.     

Anti-arthritic effects of combined treatment with histone deacetylase inhibitor and low-intensity ultrasound in the presence of microbubbles in human rheumatoid synovial cells

Objective. The therapeutic effects of histone deacetylase (HDAC)inhibitor combined with ultrasound (US) (1 MHz, 10% duty factor,0.1 or 0.2 W/cm²) in RA synovial fibroblasts (RASFs) were examined. Methods. RASFs were isolated from rheumatoid synovial tissuesobtained from patients with RA during total knee arthroplasty.RASFs were treated with an HDAC inhibitor, trichostatin A (TSA),with or without US. Cell viability was estimated using the Trypanblue dye exclusion test and cell cycle was examined by flowcytometry using propidium iodide (PI) staining. Gene expressionof cell cycle-related genes cyclin D, cyclin A, cyclin B andp21^WAF1/Cip1 was analysed by semi-quantitative RT-PCR. Detectionof apoptosis was examined by flow cytometry using annexin V-FITCand PI staining. Microarray analysis was carried out to profilegene expression of inflammation-related genes. Results. Dose-dependent decreases in cell viability, cell cyclearrest and apoptosis in RASFs due to TSA were observed. US treatmentin the presence of microbubbles increased cellular uptake, butdid not induce cell cycle arrest or apoptosis. The combinationof TSA and US modulated cell cycle-related gene expression andsignificantly decreased S phase cells and increased G₂–Mphase cells. US also further enhanced TSA-induced RASF apoptosisand regulated expression of inflammation-related genes. Conclusions. HDAC inhibitor in combination with US effectivelyreduces cell viability and induces apoptosis in RASFs. The combinationtherapy could be useful to control synovial proliferation andinflammation, since US can be easily applied to targeted jointsas local physiotherapy. KEY WORDS: Rheumatoid, Synovial fibroblasts, Histone deacetylase inhibitor, Low-intensity ultrasound, Sonoporation Submitted 11 October 2007; revised version accepted 2 January 2008.     

Clinical evaluation of cepharanthin for chronic idiopathic thrombocytopenic purpura]

Twenty-two patients with steroid-unresponsive idiopathic thrombocytopenic purpura (ITP) were treated with cepharanthin, biscoclaurin alkaloid. Cepharanthin was administered orally at a daily dose of 40 mg. Continuous elevation of platelet counts of 6.6-15.0 x 10(4)/microliters and 3-5 x 10(4)/microliters was attained in five and two patients, respectively, 1 to 4 months after the administration. Treatment with cepharanthin might be worth attempting in refractory ITP.