Activation of CYP3A-mediated testosterone 6β-hydroxylation by tanshinone IIA and midazolam 1-hydroxylation by cryptotanshinone in human liver microsomes

1. This study evaluated the in vitro activation of CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation by tanshinone I, tanshinone IIA, and cryptotanshinone.

2. The abilities of tanshinones to activate CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation in human liver microsomes (HLMs) were tested. Substrate- and effector-dependent activation of CYP3A by tanshinones were both observed.

3. Cryptotanshinone was shown to activate CYP3A-mediated midazolam 1-hydroxylation in a concentration-dependent manner. In contrast, tanshinone IIA and tanshinone I did not activate this hydroxylation reaction. In addition, tanshinone IIA activated CYP3A-mediated testosterone 6β-hydroxylation, whereas cryptotanshinone and tanshinone I did not.

4. The results from our study enhance the understanding of CYP3A activation by tanshinone IIA and cryptotanshinone in HLMs. Additionally, these data allow for an accurate prediction of the magnitude and likelihood of Danshen-drug interactions.

     

α-Adrenoceptor activity of arylalkylimidazoles is improved byα-methylation and impaired byα-hydroxylation

Summary To investigate the effects of hydroxyl and methyl substitution of the alkyl bridge bond on the-adrenoceptor activity of arylalkylimidazole derivatives, the cardiovascular effects of the molecules were studied in anaesthetized and pithed rats. The compounds studied were 4(5)-substituted imidazole derivatives with a methano, ethano or etheno bridge between the imidazole and the 2-, 2,3- or 2,6-methyl substituted phenyl rings. The hypotensive and bradycardic activities of the molecules in the anaesthetized rat were always reduced by-hydroxylation and usually augmented by-methylation of the bridge between the imidazole and phenyl rings. Hydroxylation was associated with a consistent, marked decrease in vasopressor and sympatho-inhibitory activity in the cardiovascular system of the pithed rat, but a methyl moiety as a bulky substituent in the-position of the alkyl bridge did not decrease but even caused an increase in-adrenoceptor activity in this test system. The detrimental effect of-hydroxylation of the compounds at ₁- and ₂-adrenoceptors supports the notion that the interaction of the imidazoles at-adrenoceptor is different from that of the classical, noradrenaline-like phenethylamines. The results also suggest that the alkyl bridge between the phenyl and imidazole rings of the imidazoles may contribute directly to the binding process.     

Effect of buffer conditions on CYP2C8-mediated paclitaxel 6α-hydroxylation and CYP3A4-mediated triazolam α- and 4-hydroxylation by human liver microsomes

Abstract

1.?Buffer conditions in in vitro metabolism studies using human liver microsomes (HLM) have been reported to affect the metabolic activities of several cytochrome P450 (CYP) isozymes in different ways, although there are no reports about the dependence of CYP2C8 activity on buffer conditions.

2.?The present study investigated the effect of buffer components (phosphate or Tris-HCl) and their concentration (10–200?mM) on the CYP2C8 and CYP3A4 activities of HLM, using paclitaxel and triazolam, respectively, as marker substrates.

3.?The K_m (or S₅₀) and V_max values for both paclitaxel 6α-hydroxylation and triazolam α- and 4-hydroxylation, estimated by fitting analyses based on the Michaelis–Menten or Hill equation, greatly depended on the buffer components and their concentration.

4.?The CL_int values in phosphate buffer were 1.2–3.0-fold (paclitaxel) or 3.1–6.4-fold (triazolam) higher than in Tris-HCl buffer at 50–100?mM. These values also depended on the buffer concentration, with a maximum 2.3-fold difference observed between 50 and 100?mM which are both commonly used in drug metabolism studies.

5.?These findings suggest the necessity for optimization of the buffer conditions in the quantitative evaluation of metabolic clearances, such as in vitro–in vivo extrapolation and also estimating the contribution of a particular enzyme in drug metabolism.     

CYP2C19 genotype and S-mephenytoin 4′-hydroxylation phenotype in a Chinese Dai population

AIMS: To investigate the incidence of the CYP2C19 polymorphism in the Chinese Dai population. METHODS: One hundred and ninety-three healthy Chinese Dai volunteers were identified with respect to CYP2C19 by genotype and phenotype analyses. A polymerase chain reaction-restriction fragment length polymorphism method was performed for genotyping procedures. The 4'-hydroxymephenytoin (4'-OH-MP) and S/R-mephenytoin ( S/R-MP) excreted in the urine were determined by high-performance liquid chromatography and gas chromatography, respectively. RESULTS: Eighteen subjects were identified as poor metabolisers (PMs). The frequency of PMs in the Chinese Dai subjects was 9.3% (95% confidence interval 5.2, 13.4), which is lower than that in the Chinese Han population ( P<0.05). Chinese Dai subjects had a higher frequency of the mutant CYP2C19*2 allele (0.303) and a lower frequency of the mutant CYP2C19*3 allele (0.034). These two mutant alleles could explain all deficiencies of CYP2C19 activity in the Chinese Dai subjects. The frequency of the CYP2C19*3 allele is significantly lower than that in the Chinese Han population ( P<0.05). The mean S/R ratio was lower in the homozygous extensive metabolisers (EMs) compared with that in heterozygous EMs ( P<0.01), and the latter was lower than that in the PMs ( P<0.01). Furthermore, the mean S/R ratio in CYP2C19*3/ CYP2C19*2 heterozygous PMs was possibly lower than that in the CYP2C19*2/ CYP2C19*2 homozygous PMs ( P<0.05). CONCLUSION: The frequencies of PMs and CYP2C19*3 allele in the Chinese Dai population are significantly lower than those in the Han population. The CYP2C19 genotype analysis is largely consistent with the mephenytoin phenotype analysis. The variability of S/R ratios in EMs and PMs shows a gene-dosage effect.     

Strain differences in age-associated change in testosterone 6β-hydroxylation in Wistar and Dark Agouti rats

This study examines strain differences in testosterone (T)-hydroxylations between Wistar and Dark Agouti (DA) rats of both genders. The DA rat, an animal model, is a poor metabolizer of such drugs as debrisoquine, which are metabolized by cytochrome P450 (CYP) 2D. T-16α-, 2α-hydroxylations, which are linked to CYP2C11, were catalyzed at similar rates by the microsomes of both strains. In contrast, the liver microsomes from mature male DA rats catalyzed T-6β-hydroxylation, the CYP3A mediated activity, at higher rates (～ 2-fold) than Wistar rat liver microsomes did. There was no difference between immature male DA and Wistar rats for T-6β-hydroxylation, indicating that the activity in male DA rat increases with maturation. Polyclonal antibodies raised against rat liver microsomal CYP3A2 and a CYP3A inhibitor, troleandomycin (TAO), effectively inhibited T-6β-hydroxylation by liver microsomes from both strains of rats. The level of T-6β- hydroxylation activity correlated well with the amount of CYP3A protein in the microsomes in mature as well as in immature male and female Wistar and DA rats. Northern blot analysis repeatedly indicated that the cellular contents of CYP3A2 mRNA are slightly (～ 20%) higher in the liver of mature DA rats than in that of mature Wistar rats. These results indicate that the increased levels of CYP3A are responsible for the increased T-6β-hydroxylation activity and protein in DA rat.     

Relative roles of CYP2C19 and CYP3A4/5 in midazolam 1′-hydroxylation

1. During the characterization of recombinant CYP2C19, it was observed that this enzyme metabolized midazolam, which is generally regarded as CYP3A4/5 substrate, and we therefore decided to pursue this observation further.

2. CYP2C19 showed a Michaelis-Menten pattern for midazolam 1′-hydroxylation and was inhibited by (+)-N-3-benzylnirvanol and S-mephenytoin, which are a standard potent inhibitor and a substrate of CYP2C19, respectively.

3. The inhibitory potency by CYP3A4/5 inhibitor on the midazolam 1′-hydroxylation in human liver microsomes (HLM) was correlated with the CYP3A4/5 specific catalytic activity, but such correlation was not observed in CYP2C19 enzyme. The in vitro intrinsic clearance value for midazolam 1′-hydroxylation was not changed by the addition of (+)-N-3-benzylnirvanol in four individual HLM preparations.

4. These results indicated that although CYP2C19 is capable of catalyzing midazolam 1′-hydroxylation, CYP3A4/5 play a more important role.     

Inhibition by viozan of extravasation induced in rat trachea by capsaicin is mediated exclusively by β2-adrenoceptors

The mechanism by which 2(3H)-benzothiazolone, 4-hydroxy-7-[2-[[2-[[3-(2-phenylethoxy)propyl]-sulphonyl]ethyl]amino]ethyl]-monohydrochloride (AR-C68397AA; viozan), a dual dopamine D2/beta2-adrenoceptor agonist which has shown promise in the treatment of chronic obstructive pulmonary disease (COPD), inhibits the extravasation of plasma protein induced by capsaicin in the tracheas of Brown Norway rats has been re-evaluated. Viozan (10-30 microg/kg given intratracheally; i.t.) inhibited dose-dependently the extravasation of plasma protein tagged with Evans Blue into rat trachea induced by capsaicin (10 microg/kg i.t.). Similar effects were seen with the selective beta2-adrenoceptor agonist, salbutamol (3-10 microg/kg i.t.), but the selective dopamine D2 receptor agonist, quinagolide (10-30 microg/kg i.t.), was inactive. The effects of viozan and salbutamol were abolished by propranolol (3 mg/kg) given intraperitoneally (i.p.) but unaffected by sulpiride (3 mg/kg i.p.). Thus, in c,ontrast to claims in the literature, a functional response to dopamine D2 receptor activation in a preclinical model of oedema arising from sensory nerve fibre activation in the rat lung could not be demonstrated. Moreover, no qualitative difference could be demonstrated between the response to a dual D2/beta2-adrenoceptor agonist and a selective beta2-adrenoceptor agonist. The observations call into question whether a dual D2/beta2-adrenoceptor agonist such as viozan would bring added benefit over established selective beta2-adrenoceptor agonists in the therapy     

Pro-IL-1β accumulation in macrophages by alendronate and its prevention by clodronate

Nitrogen-containing bisphosphonates (NBPs), anti-bone-resorptive drugs, exhibit inflammatory side effects (fever, jaw osteomyelitis or osteonecrosis, etc.). We previously reported that in mice: (i) a single intraperitoneal injection of alendronate (an NBP, 40 μmol/kg or less) induces various inflammatory reactions, (ii) these effects, which are minimal in IL-1-deficient mice, can be prevented by co-administration of clodronate (a non-NBP, 40 μmol/kg or less), and (iii) alendronate increases IL-1β in tissues (liver, spleen, and lung), but strangely not in blood. Here, we found the following in mice. (a) The IL-1β in tissues is pro-IL-1β. (b) Unlike LPS, alendronate induces minimal activation of caspase-1 (pro-IL-1β-processing enzyme). (c) The tissue pro-IL-1β elevations are largely absent in macrophage-depleted mice. (d) In vitro, 100 μM alendronate directly stimulates RAW 264 cells (murine macrophage-like cells) to produce pro-IL-1β, and 1 μM clodronate inhibits this effect. These results suggest that in mice: (i) the major pro-IL-1β-producing cells in response to alendronate are macrophages, (ii) alendronate directly stimulates them to produce pro-IL-1β, but the release of mature IL-1β is below detectable levels due to insufficient activation of caspase-1, and (iii) clodronate inhibits the pro-IL-1β production by acting directly on macrophages, although the in vivo mechanism may differ from the in vitro one.     

Increased psychotropic drug consumption by children in the Netherlands during 1995–2001 is caused by increased use of methylphenidate by boys

Objective The aim of the study was to determine the changes in consumption of psychotropic drugs by children aged less than 18 years during the years 1995 to 2001 in the Netherlands.Methods The year prevalence of antipsychotics, benzodiazepines, antidepressants and psychostimulants for boys and girls under 18 years was determined using electronic pharmacy dispensing records obtained from the PHARMO database.Results The overall prevalence of psychotropic drugs increased from 11.1 per 1000 in 1995 to 22.9 per 1000 in 2001. This increase could almost completely be attributed to the increase in the use of psychostimulants, i.e. methylphenidate, which increased from 1.7 per 1000 children in 1995 to 10.0 per 1000 in 2001. For the other psychotropic drugs, no or only a small increase was seen. For both boys and girls, the use of psychostimulants was highest in the age group of 5–14 years.Conclusion During the years 1995–2001, the consumption of psychotropic drugs by children in the Netherlands has more than doubled. This increase could largely be attributed to an increased use of the psychostimulant methylphenidate by boys of the age 5–14 years.     

Suppression of oxygen toxicity by melatonin

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Is Drug Use by the Elderly with Cognitive Impairment Influenced by Type of Dementia?

Patterns of drug use among the elderly vary greatly depending on level of cognitive function, yet no systematic evaluation of drug use by type of dementia has been performed. We compared patterns of drug use among patients with Alzheimer's disease (AD) and vascular dementia (VaD) to examine their relation to cognitive impairment. We used a population-based data set with over 350,000 residents admitted between 1992 and 1995 to all Medicare-Medicaid-certified nursing homes in five states. After excluding patients with a history of mental disorders or retardation, we identified 23,073 patients age 65 years and over with a diagnosis of AD and 76,087 with VaD. We examined over 350 resident data items (demographic, diagnostic, clinical, treatment) collected with the federally mandated Minimum Data Set, drug data (brand name, dosage, route and frequency of administration for all drugs), and Medicare hospital claims. Cognitive status was measured with a 7-point cognitive performance scale. Estimates of drug use were adjusted for age, gender, race, and prevalence of respective disease. Patients with AD were younger and had more severe cognitive impairment than those with VaD. The latter had more comorbid clinical conditions (3.1 +/- 1.9 vs 2.3 +/- 1.7 for patients with AD) and received a greater number of total drugs (6.1 +/- 4.6 vs 5.3 +/- 4.3). Overall use of cardiovascular, anti-Parkinson, pulmonary, antineoplastic, and nutritional agents was less frequent among patients with AD than those with VaD. Results were consistent across different levels of cognitive impairment. Thus, patients with AD have fewer associated diseases and appear to be less intensively medically treated.     

Attenuation of Aβ toxicity by promotion of mitochondrial fusion in neuroblastoma cells by liquiritigenin

Mitochondrial dynamics control mitochondrial morphology and function, and aberrations in these are associated with various neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease. To identify novel regulators of mitochondrial dynamics, we screened a phytochemical library and identified liquiritigenin as a potent inducer of mitochondrial fusion. Treatment with liquiritigenin induced an elongated mitochondrial morphology in SK-N-MC cells. In addition, liquiritigenin rescued mitochondrial fragmentation induced by knockout of mitochondrial fusion mediators such as Mfn1, Mfn2, and Opa1. Furthermore, we found that treatment with liquiritigenin notably inhibited mitochondrial fragmentation and cytotoxicity induced by Aβ in SK-N-MC cells.     

Anti-nociception mediated by a κ opioid receptor agonist is blocked by a δ receptor agonist

BACKGROUND AND PURPOSE

The opioid receptor family comprises four structurally homologous but functionally distinct sub-groups, the μ (MOP), δ (DOP), κ (KOP) and nociceptin (NOP) receptors. As most opioid agonists are selective but not specific, a broad spectrum of behaviours due to activation of different opioid receptors is expected. In this study, we examine whether other opioid receptor systems influenced KOP-mediated antinociception.

EXPERIMENTAL APPROACH

We used a tail withdrawal assay in C57Bl/6 mice to assay the antinociceptive effect of systemically administered opioid agonists with varying selectivity at KOP receptors. Pharmacological and genetic approaches were used to analyse the interactions of the other opioid receptors in modulating KOP-mediated antinociception.

KEY RESULTS

Etorphine, a potent agonist at all four opioid receptors, was not anti-nociceptive in MOP knockout (KO) mice, although etorphine is an efficacious KOP receptor agonist and specific KOP receptor agonists remain analgesic in MOP KO mice. As KOP receptor agonists are aversive, we considered KOP-mediated antinociception might be a form of stress-induced analgesia that is blocked by the anxiolytic effects of DOP receptor agonists. In support of this hypothesis, pretreatment with the DOP antagonist, naltrindole (10 mg·kg⁻¹), unmasked etorphine (3 mg·kg⁻¹) antinociception in MOP KO mice. Further, in wild-type mice, KOP-mediated antinociception by systemic U50,488H (10 mg·kg⁻¹) was blocked by pretreatment with the DOP agonist SNC80 (5 mg·kg⁻¹) and diazepam (1 mg·kg⁻¹).

CONCLUSIONS AND IMPLICATIONS

Systemic DOP receptor agonists blocked systemic KOP antinociception, and these results identify DOP receptor agonists as potential agents for reversing stress-driven addictive and depressive behaviours mediated through KOP receptor activation.

LINKED ARTICLES

This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2     

Guillain-Barré syndrome complicated by myocarditis

Guillain-Barré syndrome (GBS) is an autoimmune disease affecting the peripheral nerves, and is frequently associated with triggering events several weeks prior to the onset of symptoms. We report the case of a 68-year-old female who was diagnosed with GBS and subsequently developed myocarditis. She was treated with inotropic support and intravenous immunoglobulin (IVIG), and her condition improved. This presentation of GBS complicated by myocarditis is very rare. We examined the literature regarding this association.     

Ochratoxin-induced toxicity,oxidative stress and apoptosis ameliorated by quercetin – Modulation by Nrf2

Ochratoxin (OTA) is one of the most abundant food contaminating mycotoxins and is commonly present in the food chain. Many of the effects associated with OTA, appear to be mediated through oxidative stress. Although the toxicity of OTA is fairly well characterized, antidotes for alleviating the toxicity are sparsely reported. Dietary antioxidants have gained much importance in the recent years for their antioxidative and therapeutic properties. In the present study the therapeutic strategy was directed towards use of quercetin, a dietary antioxidant to combat OTA-induced toxicity in Vero cell line. Our results demonstrate that quercetin pre-treatment suppressed OTA-induced cytotoxicity and oxidative stress. It modulated OTA-induced alteration on the antioxidant defence through activation of Nrf2 pathway. Morphological studies by scanning electron microscopy (SEM) and cell cycle analysis indicated that quercetin prevented OTA-induced apoptosis. It also inhibited the activation of caspase cascade that leads to DNA fragmentation. Quercetin also exhibited antigenotoxic potential by attenuating OTA-induced DNA damage and micronucleus (MN) formation. The results of the study demonstrate for the first time that quercetin pre-treatment prevents OTA-induced oxidative stress and apoptosis in Vero cell line.     

Increase by α-adrenolytic drugs of acetylcholine release evoked by field stimulation of the guinea-pig ileum

Summary The release of acetylcholine evoked by field stimulation of the guinea-pig ileum (3 Hz) is increased by yohimbine and tolazoline but not affected by phentolamine. It is proposed that yohimbine and tolazoline by blocking -adrenoceptors of the cholinergic nerves abolish the inhibition caused by endogenous noradrenaline, and thus facilitate the output of acetylcholine.     

Enhanced gene transfection efficiency by low-dose 25 kDa polyethylenimine by the assistance of 1.8 kDa polyethylenimine

Gene therapy is a promising strategy for treatments of various diseases. Efficient and safe introduction of therapeutic genes into targeted cells is essential to realize functions of the genes. High-molecular-weight polyethylenimines (HMW PEIs) including 25?kDa branched PEI and 22?kDa linear PEI are widely used for in vitro gene transfection. However, high-gene transfection efficiency is usually accompanied with high cytotoxicity, which hampers their further clinical study. On the contrary, low-molecular-weight polyethylenimines (LMW PEIs) such as 1.8?kDa PEI and 800?Da PEI show good biocompatibility but their applications are limited by the poor DNA condensation capability. In this study, we find that 1.8?kDa PEI, but not 800?Da PEI combined with low-dose 25?kDa PEI could significantly promote gene transfection with low cytotoxicity. Plasmids encoding enhanced green fluorescence protein (EGFP) were delivered by the combined PEI and gene transfection efficiency was evaluated by microscopic observation and flow cytometry. Parameters including concentrations of 25?kDa PEI and 1.8?kDa PEI and preparation ways were further optimized. This study presents an efficient and safe combined PEI-based non-viral gene delivery strategy with potential for in vivo applications.