Interaction of Cucurbit[7]uril with Oxime K027, Atropine, and Paraoxon: Risky or Advantageous Delivery System?

Antidotes against organophosphates often possess physicochemical properties that mitigate their passage across the blood-brain barrier. Cucurbit[7]urils may be successfully used as a drug delivery system for bisquaternary oximes and improve central nervous system targeting. The main aim of these studies was to elucidate the relationship between cucurbit[7]uril, oxime K027, atropine, and paraoxon to define potential risks or advantages of this delivery system in a complex in vivo system. For this reason, in silico (molecular docking combined with umbrella sampling simulation) and in vivo (UHPLC-pharmacokinetics, toxicokinetics; acetylcholinesterase reactivation and functional observatory battery) methods were used. Based on our results, cucurbit[7]urils affect multiple factors in organophosphates poisoning and its therapy by (i) scavenging paraoxon and preventing free fraction of this toxin from entering the brain, (ii) enhancing the availability of atropine in the central nervous system and by (iii) increasing oxime passage into the brain. In conclusion, using cucurbit[7]urils with oximes might positively impact the overall treatment effectiveness and the benefits can outweigh the potential risks.

Aberrant methylation of tumour suppressor genes WT1, GATA5 and PAX5 in hepatocellular carcinoma.

BACKGROUND: Aberrant hypermethylation of tumour suppressor genes (TSGs) occurring in hepatocellular carcinoma (HCC) could provide a mean of molecular characterisation of this cancer. The aim of this study was to investigate promoter methylation and gene expression of selected TSGs in HCC to identify candidate genes for further validation as potential biomarkers. METHODS: Methylation-specific multiplex ligation-dependent probe amplification method was used to measure the methylation status of 25 TSGs in 49 HCC samples and 36 corresponding non-cancerous liver tissue samples. Relative expression of the differentially methylated genes was assessed at the mRNA level using quantitative PCR. RESULTS: We observed a significantly higher methylation in genes WT1, PAX5, PAX6, PYCARD and GATA5 in HCC compared with control samples. The expression of PAX5 was significantly decreased by methylation; conversely methylation of WT1 was associated with higher mRNA levels. Methylation of GATA5 was significantly associated with overall survival and methylation of WT1 and PAX5 significantly varied between patients with ALBI score 1 vs. 2+3. Moreover, PAX5 was significantly more methylated in patients with tumour grade 2+3 vs. grade 1, and methylation of the PAX5 correlated with the patient's age at the time of diagnosis. CONCLUSIONS: HCC evince aberrant promoter methylation of WT1, PAX5, PAX6, PYCARD and GATA5 genes. Correlation between GATA5, WT1 and PAX5 methylation and clinical/histological parameters is suggestive of applicability of these markers in non-invasive (epi)genetic testing in HCC.

Effect of mirtazapine on rat bone tissue after orchidectomy.

OBJECTIVE: Our study aimed to investigate the effect of mirtazapine on bone metabolism in the orchidectomized rat model. METHODS: Rats were divided into three groups. A sham-operated control group (SHAM group) and a control group after orchidectomy (ORX group) received the standard laboratory diet (SLD). An experimental group after orchidectomy (ORX MIRTA group) received SLD enriched with mirtazapine for 12 weeks. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry. Bone marker concentrations of osteoprotegerin (OPG), amino-terminal propeptide of procollagen type I, bone alkaline phosphatase (BALP), sclerostin and bone morphogenetic protein 2 were examined in bone homogenate. The femurs were used for biomechanical testing. RESULTS: Compared with the control ORX group, we found a lower BMD in the ORX MIRTA group. The differences were statistically significant, although not in the lumbar vertebrae. BMD was lower in the MIRTA group, suggesting a preferential effect on cortical bone. However, although the thickness of the diaphyseal cortical bone was not different, the fragility in the femoral neck area was statistically significantly different between the groups in biomechanical testing. Regarding the bone metabolism markers, there was a significant decrease in OPG and BALP levels, suggesting a reduction in osteoid synthesis. CONCLUSIONS: The results suggest that prolonged use of mirtazapine may have a negative effect on the synthesis of bone and on its mechanical strength, especially in the femoral neck. Further studies are warranted to establish whether mirtazapine may have a clinically significant adverse effect on bone exclusively in the model of gonadectomized rats, or whether the effect occurs also in the model of gonadally intact animals and in respective human models.

Serum Citrulline and Ornithine: Potential Markers of Coeliac Disease Activity.

INTRODUCTION: To date, there is not generally accepted and universal indicator of activity, and functional integrity of the small intestine in patients with coeliac disease. The aim of our study was to investigate whether serum concentrations of the non-essential amino acids citrulline and ornithine might have this function. METHODS: We examined serum citrulline and ornithine concentrations in a subgroup of patients with proven coeliac disease and healthy controls (blood donors). RESULTS: A total of 94 patients with coeliac disease (29 men, mean age 53 ± 18 years; 65 women, mean age 44 ± 14 years) and 35 healthy controls (blood donors) in whom coeliac disease was serologically excluded (10 men, mean age 51 ± 14 years; 25 women, mean age 46 ± 12 years) were included in the study. Significantly lower concentrations of serum ornithine were found in patients with coeliac disease (mean 65 ± 3 µmol/L; median 63 µmol/L, IQR 34 µmol/L, p < 0.001). No statistically nor clinically significant differences were found in the citrulline concentrations between the study and control group. CONCLUSIONS: Serum ornithine (but not citrulline) may be useful for assessing the functional status of the small intestine in uncomplicated coeliac disease. Further studies involving more detailed analysis of dietary and metabolic changes in patients will be needed to reach definitive conclusions.

Differential adsorption of an analyte and its D4, D5 and 13C6 labeled analogues combined with instrument-specific carry-over issues: The Achilles' heel of ibrutinib TDM.

At present, therapeutic drug monitoring is the standard in pharmacotherapy using medications with a narrow therapeutic index or showing serious adverse effects, such as in the case of ibrutinib. A technique commonly used for this purpose is liquid chromatography-tandem mass spectrometry combined with isotope dilution in sample processing. Although this method provides a high degree of reliability, its use can be complicated with some specific factors and does not guarantee trouble-free analysis. This paper is focused on investigating issues related to the differential adsorption of ibrutinib and its D4, D5 and 13C6 isotopically labeled analogues combined with instrument-specific carry-over. The results of the research point out the significantly different adsorption behavior of ibrutinib in fluidics of LC-MS compared with that of its D4, D5 and 13C6 stable isotope labeled analogues, showing preferential adsorption of non-labeled compound. The investigation also pointed to a strong affinity of ibrutinib to polymeric surfaces under specific conditions, which has to be taken into consideration during sample preparation and analysis. Our work opens a new field for the discussion of scarcely reported problem related to the use of stable isotope labeled internal standards in LC-MS/MS analysis.

In vitro and in vivo metabolism of 3-quinuclidinyl benzilate by high-resolution mass spectrometry.

3-Quinuclidinyl benzilate (QNB) is an anticholinergic compound that affects the nervous system. Its hallucinogenic action has led to its potential utility as an incapacitating warfare agent, and it is listed in Schedule 2 by the Organization for the Prohibition of Chemical Weapons. Although this compound has been known for a long time, limited information is available regarding its metabolism and mass spectrometric data of the metabolites, the information that could facilitate the identification of QNB in case of suspected intoxication. To the best of our knowledge, the analytical methods previously described in the literature are based on outdated procedures, which may result in a significantly lower number of observable metabolites. The aim of this work was to obtain deeper insight into QNB biotransformation using a combination of in vitro and in vivo approach. The development of a suitable method for the separation and detection of metabolites using mass spectrometry together with the identification of reliable diagnostic fragments for the unambiguous identification of QNB metabolites in the different biological matrices are also presented in this work. A screening of rat plasma, urine and tissue homogenates revealed 26 new metabolites related to the cytochrome P450 biotransformation pathway, which involves N-oxidation and hydroxylation(s) followed by O-methylation and O-glucuronosylation within phase II of the metabolism. A study showed that the brain is not metabolically active in the case of QNB and that the metabolites do not cross the blood-brain barrier; thus, the toxicodynamic effects are due to QNB itself. In addition, in vitro experiments performed using isolated human liver microsomes revealed N-oxidation as the principal metabolic pathway in human tissue. In light of current global events, the abuse of QNB by terrorists or para-military groups is a real possibility, and our findings may improve the detection systems used in laboratories involved in postexposure investigations.

Novel tacrine-tryptophan hybrids: Multi-target directed ligands as potential treatment for Alzheimer's disease.

A combination of tacrine and tryptophan led to the development of a new family of heterodimers as multi-target agents with potential to treat Alzheimer's disease. Based on the in vitro biological profile, compound S-K1035 was found to be the most potent inhibitor of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE), demonstrating balanced IC50 values of 6.3 and 9.1 nM, respectively. For all the tacrine-tryptophan heterodimers, favorable inhibitory effect on hAChE as well as on hBChE was coined to the optimal spacer length ranging from five to eight carbon atoms between these two pharmacophores. S-K1035 also showed good ability to inhibit Aß42 self-aggregation (58.6 ±â€¯5.1% at 50 µM) as well as hAChE-induced Aß40 aggregation (48.3 ±â€¯6.3% at 100 µM). The X-ray crystallographic analysis of TcAChE in complex with S-K1035 pinpointed the utility of the hybridization strategy applied and the structures determined with the two K1035 enantiomers in complex with hBChE could explain the higher inhibition potency of S-K1035. Other in vitro evaluations predicted the ability of S-K1035 to cross blood-brain barrier and to exert a moderate inhibition potency against neuronal nitric oxide synthase. Based on the initial promising biochemical data and a safer in vivo toxicity compared to tacrine, S-K1035 was administered to scopolamine-treated rats being able to dose-dependently revert amnesia.

The New Acetylcholinesterase Inhibitors PC-37 and PC-48 (7-Methoxytacrine-Donepezil-Like Compounds): Characterization of Their Metabolites in Human Liver Microsomes, Pharmacokinetics and In Vivo Formation of the Major Metabolites in Rats.

The objective of this study was to elucidate the pharmacokinetics and metabolite formation of newly developed non-selective AChE/BChE 7-MEOTA-donepezil-like inhibitors for potential therapeutic use in Alzheimer's disease (AD) patients. The chemical structures of metabolites were defined during incubation with human liver microsomes, and subsequently, the metabolization was verified in in vivo study. In vitro metabolic profiling revealed the formation of nine major metabolites in the case of PC-37 and eight metabolites of PC-48. Hydroxylation and the enzymatic hydrolysis of bonds close to the piperazine ring appeared to be the principal metabolic pathways in vitro. Of these metabolites, M1-M7 of PC-37 and M1-M6 of PC-48 were confirmed under in vivo conditions. Pilot pharmacokinetic experiments in rats were focused on the absorption, distribution and elimination of these compounds. Absorption after i.m. application was relatively fast; the bioavailability expressed as AUCtotal was 28179 ± 4691 min.ng/mL for PC-37 and 23374 ± 4045 min.ng/mL for PC-48. Both compounds showed ability to target the central nervous system, with brain concentrations exceeding those in plasma. The maximal brain concentrations are approximately two times higher than the plasma concentrations. The relatively high brain concentrations persisted throughout the experiment until 24 hr after application. Elimination via the kidneys (urine) significantly exceeded elimination via the liver (bile). All these characteristics are crucial for new candidates intended for AD treatment. The principle metabolic pathways that were verified in the in vivo study do not show any evidence for formation of extremely toxic metabolites, but this needs to be confirmed by further studies.

An HPLC-MS method for the quantification of new acetylcholinesterase inhibitor PC 48 (7-MEOTA-donepezil like compound) in rat plasma: Application to a pharmacokinetic study.

A simple, rapid and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated for the quantitative determination in rat plasma of a new candidate for AD treatment, namely PC 48 (a 7-MEOTA-donepezil like compound) in rat plasma. Sample preparation involved pH adjustment with sodium hydroxide followed by solvent extraction with ethyl acetate:dichloromethane (80:20, v/v). The chromatographic separation was achieved on an Ascentis Express RP-Amide column (75 mm × 2.1mm, 2.7 µm) with a gradient mobile phase consisting of 0.05 M aqueous formic acid and acetonitrile. Detection was carried out using positive-ion electrospray tandem mass spectrometry on an LTQ XL system using the MS/MS CID (collision-induced dissociation) mode. The method was linear in the range 0.1-1000 ng/ml (r(2)=0.999) with a lower limit of quantitation of 0.1 ng/mL. Extraction recovery was in the range 63.5-72.1% for PC 48 and 70.5% for reserpine (internal standard, IS). Intra- and inter-day precisions measured as relative standard deviation were below 10.8% and accuracy was from -7.2% to 7.4%. The method was successfully applied to a pharmacokinetic study involving intramuscular application of 3.86 mg/kg PC 48 to rats for the first time. Pharmacokinetic parameters for PC 48 include Cmax 39.09 ± 4.45 ng/mL,Tmax 5.00 ± 3.08 min, AUC0-t 23374 ± 4045 min ng/mL and t1/2 1065 ± 246 min.

Negative effect of serotonin-norepinephrine reuptake inhibitor therapy on rat bone tissue after orchidectomy.

Our goal was to determine if venlafaxine has a negative effect on bone metabolism. Rats were divided into three groups. The sham-operated control group (SHAM), the control group after orchidectomy (ORX), and the experimental group after orchidectomy received venlafaxine (VEN ORX) in standard laboratory diet (SLD) for 12 weeks. Bone mineral content (BMC) was measured by dual energy X-ray absorptiometry (DXA). Bone marker concentrations of carboxy-terminal cross-linking telopeptide of type I collagen (CTX-I), osteoprotegerin (OPG), amino-terminal propeptide of procollagen type I (P1NP), bone alkaline phosphatase (BALP), sclerostin and bone morphogenetic protein 2 (BMP-2) were examined in bone homogenate. The femurs were used for biomechanical testing. Compared to the ORX group we found lower BMD in the diaphysis area of the femur in the VEN ORX group, suggesting a preferential effect on cortical bone. Of the bone metabolism markers, there was significant decrease (ORX control group versus VEN ORX experimental group) in BALP levels and increase in sclerostin and CTX-I levels, suggesting a decrease in osteoid synthesis and increased bone resorption. The results suggest that the prolonged use of venlafaxine may have a negative effect on bone metabolism. Further studies are warranted to establish whether venlafaxine may have a clinically significant adverse effect on bone.