α-Latrotoxin-induced transmitter release in feline oesophageal smooth muscle: focus on nitric oxide and vasoactive intestinal peptide

1. The effects of α-latrotoxin (αLTX) on muscle tone, resting membrane potential, cyclic nucleotide content, and ultrastructure were examined in feline oesophageal smooth muscle, including the lower oesophageal sphincter (LOS).
2. In circular smooth muscle strips from LOS developing active tone, αLTX (1 nM) induced a 94±3% (n=16) relaxation. Intermittent treatment with αLTX for 4 h abolished the response. Pretreatment with N^G-nitro-L-arginine (L-NOARG; 0.1 mM) attenuated the relaxation.
3. In carbachol-contracted circular smooth muscle strips from the LOS and oesophageal body (OB), αLTX induced a 95±5% (n=6) and 73±9% (n=8) relaxation, respectively. The relaxations were attenuated by L-NOARG, and in LOS strips, the relaxation was abolished by the combination of L-NOARG and vasoactive intestinal peptide (VIP)-antiserum (1:25). At resting tension in circular smooth muscle strips from the OB, αLTX induced a scopolamine sensitive contraction in the presence of L-NOARG.
4. In circular LOS and OB preparations, αLTX changed the resting membrane potential from −49±2 mV to −59±3 mV (n=4), and −62±2 mV to −71±3 mV (n=4), respectively.
5. The αLTX-induced relaxation of LOS and OB muscle was associated with a 138% and 72% increase in cyclic GMP levels, respectively. No changes in cyclic AMP levels were observed.
6. Ultrastructural analysis of LOS and OB revealed a rich supply of nerve profiles containing small synaptic and large dense core vesicles. αLTX treatment resulted in a loss of both types of vesicle.
7. These results suggest that αLTX induces relaxation of oesophageal circular smooth muscle associated with NO-generation and transmitter release from synaptic vesicles. Beside NO, VIP seems to be involved in the relaxant effects of αLTX on the LOS. In addition, αLTX may have contractile effects by release of acetylcholine.

     

Dissecting the genetic association of C-reactive protein with PTSD,traumatic events,and social support

Inflammatory markers like C-reactive protein (CRP) have been associated with post-traumatic stress disorder (PTSD) and traumatic experiences, but the underlying mechanisms are unclear. We investigated the relationship among serum CRP, PTSD, and traits related to traumatic events and social support using genetic association data from the Psychiatric Genomics Consortium (23,185 PTSD cases and 151,309 controls), the UK Biobank (UKB; up to 117,900 individuals), and the CHARGE study (Cohorts for Heart and Aging Research in Genomic Epidemiology, 148,164 individual). Linkage disequilibrium score regression, polygenic risk scoring, and two-sample Mendelian randomization (MR) analyses were used to investigate genetic overlap and causal relationships. Genetic correlations of CRP were observed with PTSD (rg = 0.16, p = 0.026) and traits related to traumatic events, and the presence of social support (−0.28 < rg < 0.20; p < 0.008). We observed a bidirectional association between CRP and PTSD (CRP → PTSD: β = 0.065, p = 0.015; PTSD → CRP: β = 0.008, p = 0.009). CRP also showed a negative association with the “felt loved as a child” trait (UKB, β = −0.017, p = 0.008). Owing to the known association of socioeconomic status (SES) on PTSD, a multivariable MR was performed to investigate SES as potential mediator. We found that household income (univariate MR: β = −0.22, p = 1.57 × 10⁻⁷; multivariate MR: β = −0.17, p = 0.005) and deprivation index (univariate MR: β = 0.38, p = 1.63 × 10⁻⁹; multivariate MR: β = 0.27, p = 0.016) were driving the causal estimates of “felt loved as a child” and CRP on PTSD. The present findings highlight a bidirectional genetic association between PTSD and CRP, also suggesting a potential role of SES in the interplay between childhood support and inflammatory processes with respect to PTSD risk.Subject terms: Post-traumatic stress disorder, Trauma, Genetics     

Validity of (−)-[3H]-CGP 12177A as a radioligand for the ‘putative β4-adrenoceptor' in rat atrium

1. We have recently suggested the existence in the heart of a ‘putative β₄-adrenoceptor'' based on the cardiostimulant effects of non-conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block β₁- and β₂-adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (−)-[³H]-CGP 12177A ((−)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one) in rat atrium. We investigated (−)-[³H]-CGP 12177A for this purpose for two reasons, because it is a non-conventional partial agonist and also because it is a hydrophilic radioligand.
2. Increasing concentrations of (−)-[³H]-CGP 12177A, in the absence or presence of 20 μM (−)-CGP 12177A to define non-specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to β₁- and β₂-adrenoceptors (pK_D 9.4±0.1, B_max 26.9±3.1 fmol mg^-1 protein) and higher concentrations bound to the ‘putative β₄-adrenoceptor'' (pK_D 7.5±0.1, B_max 47.7±4.9 fmol mg⁻¹ protein). In other experiments designed to exclude β₁- and β₂-adrenoceptors, (−)-[³H]-CGP 12177A (1–200 nM) binding in the presence of 500 nM (−)-propranolol was also saturable (pK_D 7.6±0.1, B_max 50.8±7.4 fmol mg⁻¹ protein).
3. The non-conventional partial agonists (−)-CGP 12177A (pK_i 7.3±0.2), (±)-cyanopindolol (pK_i 7.6±0.2), (−)-pindolol (pK_i 6.6±0.1) and (±)-carazolol (pK_i 7.2±0.2) and the antagonist (−)-bupranolol (pK_i 6.6±0.2), all competed for (−)-[³H]-CGP 12177A binding in the presence of 500 nM (−)-propranolol at the ‘putative β₄-adrenoceptor'', with affinities closely similar to potencies and affinities determined in organ bath studies.
4. The catecholamines competed with (−)-[³H]-CGP 12177A at the ‘putative β₄-adrenoceptor'' in a stereoselective manner, (−)-noradrenaline (pK_iH 6.3±0.3, pK_iL 3.5±0.1), (−)-adrenaline (pK_iH 6.5±0.2, pK_iL 2.9±0.1), (−)-isoprenaline (pK_iH 6.2±0.5, pK_iL 3.4±0.1), (+)-isoprenaline (pK_i<1.7), (−)-RO363 ((−)-(1-(3,4-dimethoxyphenethylamino)-3-(3,4-dihydroxyphenoxy)-2-propranol)oxalate, pK_i 5.5±0.1).
5. The inclusion of guanosine 5-triphosphate (GTP 0.1 mM) had no effect on binding of (−)-CGP 12177A or (−)-isoprenaline to the ‘putative β₄-adrenoceptor''. In competition binding studies, (−)-CGP 12177A competed with (−)-[³H]-CGP 12177A for one receptor state in the absence (pK_i 7.3±0.2) or presence of GTP (pK_i 7.3±0.2). (−)-Isoprenaline competed with (−)-[³H]-CGP 12177A for two states in the absence (pK_iH 6.6±0.3, pK_iL 3.5±0.1; % H 25±7) or presence of GTP (pK_iH 6.2±0.5, pK_iL 3.4±0.1; % H 37±6). In contrast, at β₁-adrenoceptors, GTP stabilized the low affinity state of the receptor for (−)-isoprenaline.
6. The specificity of binding to the ‘putative β₄-adrenoceptor'' was tested with compounds active at other receptors. High concentrations of the β₃-adrenoceptor agonists, BRL 37344 ((RR+SS)[4-[2-[[2-(3-chlorophenyl)-2-hydroxy - ethyl]amino]propyl]phenoxy]acetic acid, 6 μM), SR 58611A (ethyl{(7S)-7-[(2R)-2 - (3 - chlorophenyl) - 2 - hydroxyethylamino] - 5,6,7,8 - tetrahydronaphtyl2 - yloxy} acetate hydrochloride, 6 μM), ZD 2079 ((±)-1-phenyl-2-(2-4-carboxymethylphenoxy)-ethylamino)-ethan-1-ol, 60 μM), CL 316243 (disodium (R,R)-5-[2-[2-(3-chlorophenyl)-2-hydroxyethyl-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate, 60 μM) and antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-2S-2-propanol oxalate, 6 μM) caused less than 22% inhibition of (−)-[³H]-CGP 12177A binding in the presence of 500 nM (−)-propranolol. Histamine (1 mM), atropine (1 μM), phentolamine (10 μM), 5-HT (100 μM) and the 5-HT₄ receptor antagonist SB 207710 ((1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1,4-benzodioxan-5-carboxylate, 10 nM) caused less than 26% inhibition of binding.
7. Non-conventional partial agonists, the antagonist (−)-bupranolol and catecholamines all competed for (−)-[³H]-CGP 12177A binding in the absence of (−)-propranolol at β₁-adrenoceptors, with affinities (pK_i) ranging from 1.6–3.6 log orders greater than at the ‘putative β₄-adrenoceptor''.
8. We have established and validated a radioligand binding assay in rat atrium for the ‘putative β₄-adrenoceptor'' which is distinct from β₁-, β₂- and β₃-adrenoceptors. The stereoselective interaction with the catecholamines provides further support for the classification of the receptor as ‘putative β₄-adrenoceptor''.

     

N-Acetylcysteine Effects on Transforming Growth Factor-β and Tumor Necrosis Factor-α Serum Levels as Pro-Fibrotic and Inflammatory Biomarkers in Patients Following ST-Segment Elevation Myocardial Infarction

Background and Aims

Ischemia following acute myocardial infarction (AMI) increases the level of pro-fibrotic and inflammatory cytokines, including transforming growth factor (TGF)-β and tumor necrosis factor (TNF)-α. N-acetylcysteine (NAC) has therapeutic benefits in the management of patients with AMI. To the best of our knowledge, this is the first study that has evaluated the effect of NAC on TNF-α and TGF-β levels in patients with AMI.

Methods

Following confirmation of AMI, 88 patients were randomly administered NAC 600 mg (Fluimucil^®, Zambon, Ticino, Switzerland) or placebo orally twice daily for 3 days. For quantification of TGF-β and TNF-α serum levels after 24 and 72 h of NAC or placebo administration, peripheral venous blood (10 mL) samples were collected at these time points.

Results

Comparisons between levels of TGF-β and TNF-α after 24 and 72 h within the NAC or placebo groups revealed that there was not any significant difference except for TGF-β levels in the placebo group, which increased significantly over time (p = 0.042). Significant relationships existed between patients’ ejection fraction (p = 0.005) and TGF-β levels.

Conclusions

Receiving NAC could prevent TGF-β levels from increasing after 72 h as compared with not receiving NAC. As TGF-β had strong correlations with the ejection fraction, its antagonism seems to be important in the prevention of remodeling.     

Telomere attrition and inflammatory load in severe psychiatric disorders and in response to psychotropic medications

Individuals with severe psychiatric disorders have a reduced life expectancy compared to the general population. At the biological level, patients with these disorders present features that suggest the involvement of accelerated aging, such as increased circulating inflammatory markers and shorter telomere length (TL). To date, the role of the interplay between inflammation and telomere dynamics in the pathophysiology of severe psychiatric disorders has been scarcely investigated. In this study we measured T-lymphocytes TL with quantitative fluorescent in situ hybridization (Q-FISH) and plasma levels of inflammatory markers in a cohort comprised of 40 patients with bipolar disorder (BD), 41 with schizophrenia (SZ), 37 with major depressive disorder (MDD), and 36 non-psychiatric controls (NPC). TL was shorter in SZ and in MDD compared to NPC, while it was longer in BD (model F_{6, 137} = 20.128, p = 8.73 × 10⁻¹⁷, effect of diagnosis, F₃ = 31.870; p = 1.08 × 10⁻¹⁵). There was no effect of the different classes of psychotropic medications, while duration of treatment with mood stabilizers was associated with longer TL (Partial correlation controlled for age and BMI: correlation coefficient = 0.451; p = 0.001). Levels of high-sensitivity C-Reactive Protein (hsCRP) were higher in SZ compared to NPC (adjusted p = 0.027), and inversely correlated with TL in the whole sample (r = −0.180; p = 0.042). Compared to NPC, patients with treatment resistant (TR) SZ had shorter TL (p = 0.001), while patients with TR MDD had higher levels of tumor necrosis factor-α (TNFα) compared to NPC (p = 0.028) and to non-TR (p = 0.039). Comorbidity with cardio-metabolic disorders did not influence the observed differences in TL, hsCRP, and TNFα among the diagnostic groups. Our study suggests that patients with severe psychiatric disorders present reduced TL and increased inflammation.Subject terms: Schizophrenia, Diagnostic markers, Bipolar disorder, Depression     

Pharmacokinetics of PEGylated recombinant human endostatin (M2ES) in rats

Aim:

M₂ES is PEGylated recombinant human endostatin. In this study we investigated the pharmacokinetics, tissue distribution, and excretion of M₂ES in rats.

Methods:

¹²⁵I-radiolabeled M₂ES was administered to rats by intravenous bolus injection at 3 mg/kg. The pharmacokinetics, tissue distribution and excretion of M₂ES were investigated using the trichloroacetic acid (TCA) precipitation method.

Results:

The serum M₂ES concentration-time curve after a single intravenous dose of 3 mg/kg in rats was fitted with a non-compartment model. The pharmacokinetic parameters were evaluated as follows: C_max=28.3 μg·equ/mL, t_1/2=71.5 h, AUC_(0–∞)=174.6 μg·equ·h/mL, Cl=17.2 mL·h⁻¹·kg⁻¹, MRT=57.6 h, and V_ss=989.8 mL/kg for the total radioactivity; C_max=30.3 μg·equ/mL, t_1/2=60.1 h, AUC_(0–∞)=146.2 μg·equ·h/mL, Cl=20.6 mL·h⁻¹·kg⁻¹, MRT=47.4 h, and V_ss=974.6 mL/kg for the TCA precipitate radioactivity. M₂ES was rapidly and widely distributed in various tissues and showed substantial deposition in kidney, adrenal gland, lung, spleen, bladder and liver. The radioactivity recovered in the urine and feces by 432 h post-dose was 71.3% and 8.3%, respectively. Only 0.98% of radioactivity was excreted in the bile by 24 h post-dose.

Conclusion:

PEG modification substantially prolongs the circulation time of recombinant human endostatin and effectively improves its pharmacokinetic behavior. M₂ES is extensively distributed in most tissues of rats, including kidney, adrenal gland, lung, spleen, bladder and liver. Urinary excretion was the major elimination route for M₂ES.     

Comparing the cost,glycaemic control and medication adherence of utilizing patients’ own medicines (POMs) versus usual dispensing among diabetic patients in an outpatient setting

BackgroundMillions worth of unused drugs particularly those indicated for chronic diseases such as diabetes were returned and disposed leading to substantial wastage. Use of patients’ own medications (POMs) in the inpatient setting has reduced wastage and saved cost. The impact of utilizing POMs in the outpatient setting has hitherto not been determined.PurposeThis study aims to compare the cost, medication adherence and glycaemic control of utilizing POMs versus usual dispensing.MethodsProspective randomized controlled study was conducted among diabetic patients that required monthly medication refill in the Outpatient Pharmacy in 2017. Patients who consented were equally divided into POMs and control groups. Both groups brought excess medications from home at week-0 and week-12. Patients in the POMs group brought excess medications monthly and sufficient amount of drugs were added until the next refill date. Drugs were dispensed as usual in the control group. Total cost consisting of the cost of drugs, staff and building was calculated. Glycosylated haemoglobin (HbA1c) was measured at baseline and week-12. Adherence was measured based on pill counting.ResultsThirty patients aged 56.77 ± 14.67 years with 13.37 ± 7.36 years of diabetes participated. Baseline characteristics were similar between the groups. POMs minimized the total cost by 38.96% which translated to a cost saving of USD 42.76 ± 6.98, significantly different versus USD 0.02 ± 0.52 in the control group, p = 0.025. Mean HbA1c reduced significantly (−0.79%, p = 0.016) in the POMs group but not significant in the control group (−0.11%, p = 0.740). Medication adherence improved significantly in both groups at week-12 (p < 0.010). Nevertheless, patients in the POMs group were more adherent, 87.20% vs. 66.32%, p = 0.034.ConclusionUtilizing POMs resulted in cost saving, improved adherence and better glycaemic control. Use of POMs should be practiced in the outpatient pharmacy to reduce wastage and cost.Graphical abstract      

Synthesis,in vivo and in silico analgesic and anti-inflammatory studies of α-D-ribofuranose derivatives

Five α-D-ribofuranose analogues (2, 3, 4, 5 and 6) were synthesized in good yields from 3-O-benzyl-4-C-(hydroxymethyl)-1, 2-O-isopropylidene-α-D-ribofuranose (1). The synthesized compounds were then subjected to analgesic, anti-inflammatory, antimicrobial and antioxidant assays. Compound 3 demonstrated 79.74% (P < 0.001) writhing inhibition and highest reaction time of 2.55 ± 0.13 min (P < 0.001) after 30 min of oral administration in peripheral and central analgesic assay, respectively, at 50 mg/kg dose. Compound 2 and 6 exhibited significant anti-inflammatory activity at 100 mg/kg dose with paw edema inhibition of 91.15% (P < 0.001) and 95.13% (P < 0.001), respectively, in 4th hour. The synthesized analogues did not show notable antioxidant and antibacterial properties. Molecular docking study revealed higher binding affinity of −8.1 kcal/mol and −8.9 kcal/mol of compound 3 towards cyclooxygenase-1 and phospholipase A_2, respectively, compared to −7.7 and −7.6 kcal/mol respectively for corresponding native ligands. Compound 2 demonstrated binding affinity of −9.1 kcal/mol towards interleukin-1 receptor-associated kinase-4 compared to −8.7 kcal/mol of the native ligand. The molecular properties related to drug likeness of compounds were found to be within acceptable range. Synthesized D-ribofuranose analogues demonstrated promising analgesic and anti-inflammatory activities and further development may lead to new potent analgesic and anti-inflammatory agents.     

Association between polygenic risk for Alzheimer’s disease,brain structure and cognitive abilities in UK Biobank

Previous studies testing associations between polygenic risk for late-onset Alzheimer’s disease (LOAD-PGR) and brain magnetic resonance imaging (MRI) measures have been limited by small samples and inconsistent consideration of potential confounders. This study investigates whether higher LOAD-PGR is associated with differences in structural brain imaging and cognitive values in a relatively large sample of non-demented, generally healthy adults (UK Biobank). Summary statistics were used to create PGR scores for n = 32,790 participants using LDpred. Outcomes included 12 structural MRI volumes and 6 concurrent cognitive measures. Models were adjusted for age, sex, body mass index, genotyping chip, 8 genetic principal components, lifetime smoking, apolipoprotein (APOE) e4 genotype and socioeconomic deprivation. We tested for statistical interactions between APOE e4 allele dose and LOAD-PGR vs. all outcomes. In fully adjusted models, LOAD-PGR was associated with worse fluid intelligence (standardised beta [β] = −0.080 per LOAD-PGR standard deviation, p = 0.002), matrix completion (β = −0.102, p = 0.003), smaller left hippocampal total (β = −0.118, p = 0.002) and body (β = −0.069, p = 0.002) volumes, but not other hippocampal subdivisions. There were no significant APOE x LOAD-PGR score interactions for any outcomes in fully adjusted models. This is the largest study to date investigating LOAD-PGR and non-demented structural brain MRI and cognition phenotypes. LOAD-PGR was associated with smaller hippocampal volumes and aspects of cognitive ability in healthy adults and could supplement APOE status in risk stratification of cognitive impairment/LOAD.Subject terms: Risk factors, Predictive markers, Genetic markers     

Pharmacological characterization of CGRP receptors mediating relaxation of the rat pulmonary artery and inhibition of twitch responses of the rat vas deferens

1. CGRP receptors mediating vasorelaxation of the rat isolated pulmonary artery and inhibition of contractions of the rat isolated prostatic vas deferens were investigated using CGRP agonists, homologues and the antagonist CGRP_8-37.
2. In the pulmonary artery, human (h)α-CGRP-induced relaxation of phenylephrine-evoked tone was abolished either by removal of the endothelium or by N^G-nitro-L-arginine (10⁻⁵ M). The inhibitory effect of N^G-nitro-L-arginine was stereoselectively reversed by L- but not by D-arginine (10⁻⁴ M). Thus, CGRP acts via nitric oxide released from the endothelium.
3. In the endothelium-intact artery, hα-CGRP, hβ-CGRP and human adrenomedullin (10⁻¹⁰–3×10⁻⁷ M), dose-dependently relaxed the phenylephrine-induced tone with similar potency. Compared with hα-CGRP, rat amylin was around 50 fold less potent, while [Cys(ACM^2,7)] hα-CGRP (10⁻⁷–10⁻⁴ M) was at least 3000 fold less potent. Salmon calcitonin was inactive (up to 10⁻⁴ M).
4. Human α-CGRP_8-37 (3×10⁻⁷–3×10⁻⁶ M) antagonized hα-CGRP (pA₂ 6.9, Schild plot slope 1.2±0.1) and hβ-CGRP (apparent pK_B of 7.1±0.1 for hα-CGRP_8-37 10⁻⁶ M) in the pulmonary artery. Human β-CGRP_8-37 (10⁻⁶ M) antagonized hα-CGRP responses with a similar affinity (apparent pK_B 7.1±0.1). Human adrenomedullin responses were not inhibited by hα-CGRP_8-37 (10⁻⁶ M).
5. In the prostatic vas deferens, hα-CGRP, hβ-CGRP and rat β-CGRP (10⁻¹⁰–3×10⁻⁷ M) concentration-dependently inhibited twitch responses with about equal potency, while rat amylin (10⁻⁸–10⁻⁵ M) was around 10 fold less potent and the linear analogue [Cys(ACM^2,7)] hα-CGRP was at least 3000 fold weaker. Salmon calcitonin was inactive (up to 10⁻⁴ M).
6. The antagonist effect of hα-CGRP_8-37 (10⁻⁵–3×10⁻⁵) in the vas deferens was independent of the agonist, with pA₂ values against hα-CGRP of 6.0 (slope 0.9±0.1), against hβ-CGRP of 5.8 (slope 1.1±0.1), and an apparent pK_B value of 5.8±0.1 against both rat β-CGRP and rat amylin. Human β-CGRP_8-37 (3×10⁻⁵–10⁻⁴ M) competitively antagonized hα-CGRP responses (pA₂ 5.6, slope 1.1±0.2). The inhibitory effect of hα-CGRP on noradrenaline-induced contractions in both the prostatic and epididymal vas deferens was antagonized by hα-CGRP_8-37 (pA₂ 5.8 and 5.8, slope 1.0±0.2 and 1.0±0.3, respectively).
7. The effects of hα-CGRP and hα-CGRP_8-37 in both rat pulmonary artery and vas deferens were not significantly altered by pretreatment with peptidase inhibitors (amastatin, bestatin, captopril, phosphoramidon and thiorphan, all at 10⁻⁶ M). The weak agonist activity of [Cys(ACM^2,7)] hα-CGRP in the vas deferens was not increased by peptidase inhibitors.
8. These data demonstrate that two different CGRP receptors may exist in the rat pulmonary artery and vas deferens, a CGRP₁ receptor subtype in the rat pulmonary artery (CGRP_8-37 pA₂ 6.9), while the lower affinity for CGRP_8-37 (pA₂ 6.0) in the vas deferens is consistent with a CGRP₂ receptor.

     

Pharmacokinetics of trapidil,an antagonist of platelet derived growth factor,in healthy subjects and in patients with liver cirrhosis

1Pharmacokinetic parameters of trapidil (an antagonist of platelet derived growth factor) were evaluated in 12 healthy male subjects (study I) and in a group of 10 patients with liver cirrhosis (Child B) and five control subjects, respectively (study II). 2Investigations were carried out after a single dose trapidil (200 mg) and at steady state after application of 200 mg trapidil three times daily for 5 days (study 1) or 4 days (study II). 3Study I: The concentration-time curves of the terminal elimination phase of trapidil exhibited a slight convexity which might reflect nonlinear kinetics. The AUC of trapidil obtained after the first dose (20.5 [±7.0 s.d.] μg ml⁻¹ h) was markedly higher than the AUC determined at steady state (13.2 [±3.8 s.d.] μg ml⁻¹ h), the non-parametric 90% confidence intervals of the ratio day 5/day 1 was 0.58–0.73 (point estimator 0.64). 4Study II: AUC averaged (21.4 [±9.1 s.d.] μg ml⁻¹ h) in controls and (34.4 [±14.9 s.d.] μg ml⁻¹ h) in cirrhotic patients. The 90% confidence intervals for the difference group 1 vs group 2 was 0.95–2.97 (point estimator 1.48, P=0.066). At steady state, AUC averaged (13.7 [±5.7 s.d.] μg ml⁻¹ h) in controls and (20.8 [±6.8 s.d.] μg ml⁻¹ h) in cirrhotic patients (90% confidence intervals group 1 vs group 2: 0.88–2.20 [point estimator 1.45, P=0.05]). As seen in study I, the AUC of trapidil obtained after the first dose was markedly higher than the AUC determined at steady state , the non-parametric 90% confidence intervals of the ratio day 5/day 1 was 0.48–0.84 (point estimator 0.66) in control subjects and 0.54–0.72 (point estimator 0.64) in cirrhotic patients, respectively. 5An inverse correlation was seen between the results of the monoethylglycin-xilidid (MEGX)-test and the AUC of trapidil (single dose: r=−0.516, P=0.048; steady state: r=−0.548, P=0.042). 6Results of study I and study II indicate an autoinduction of trapidil metabolism after repeated oral doses. Although trapidil elimination is decreased in patients with liver cirrhosis (study II), the elimination half-life at steady state is relatively short (2.4 [±1.1 s.d.] h) and therefore should prevent cumulation of trapidil even in cirrhotic patients.     

Childhood maltreatment and cognitive functioning: the role of depression,parental education,and polygenic predisposition

Childhood maltreatment is associated with cognitive deficits that in turn have been predictive for therapeutic outcome in psychiatric patients. However, previous studies have either investigated maltreatment associations with single cognitive domains or failed to adequately control for confounders such as depression, socioeconomic environment, and genetic predisposition. We aimed to isolate the relationship between childhood maltreatment and dysfunction in diverse cognitive domains, while estimating the contribution of potential confounders to this relationship, and to investigate gene–environment interactions. We included 547 depressive disorder and 670 healthy control participants (mean age: 34.7 years, SD = 13.2). Cognitive functioning was assessed for the domains of working memory, executive functioning, processing speed, attention, memory, and verbal intelligence using neuropsychological tests. Childhood maltreatment and parental education were assessed using self-reports, and psychiatric diagnosis was based on DSM-IV criteria. Polygenic scores for depression and for educational attainment were calculated. Multivariate analysis of cognitive domains yielded significant associations with childhood maltreatment (η²_p = 0.083, P < 0.001), depression (η²_p = 0.097, P < 0.001), parental education (η²_p = 0.085, P < 0.001), and polygenic scores for depression (η²_p = 0.021, P = 0.005) and educational attainment (η²_p = 0.031, P < 0.001). Each of these associations remained significant when including all of the predictors in one model. Univariate tests revealed that maltreatment was associated with poorer performance in all cognitive domains. Thus, environmental, psychopathological, and genetic risk factors each independently affect cognition. The insights of the current study may aid in estimating the potential impact of different loci of interventions for cognitive dysfunction. Future research should investigate if customized interventions, informed by individual risk profiles and related cognitive preconditions, might enhance response to therapeutic treatments.Subject terms: Risk factors, Genetic interaction, Depression     

Study of Sustained Blood Pressure-Lowering Effect of Azelnidipine Guided by Self-Measured Morning and Evening Home Blood Pressure: Subgroup Analysis of the At-HOME Study

Background

Morning hypertension is a risk factor for cardiovascular and cerebrovascular events, and consequently diagnosis and control of morning hypertension are considered very important. We previously reported the results of the Azelnidipine Treatment for Hypertension Open-label Monitoring in the Early morning (At-HOME) Study, which indicated that azelnidipine effectively controlled morning hypertension.

Objectives

The objective of this At-HOME subgroup analysis was to evaluate the sustained blood pressure (BP)-lowering effect of azelnidipine, using mean morning and evening systolic BP [ME average] and morning systolic BP minus evening systolic BP (ME difference).

Methods

We analyzed the self-measured home BP data (measured in the morning and at bedtime) from this 16-week prospective observational study to clarify the effect of morning dosing of azelnidipine (mean [± standard deviation] maximum dose 14.3 ± 3.6 mg/day). A subgroup of patients from the At-HOME Study who had an evening home BP measurement within 28 days prior to the baseline date were used for efficacy analysis (n = 2,546; mean age, 65.1 years; female, 53.6 %).

Results

Home systolic BP/diastolic BP levels in the morning and evening were significantly lowered (p < 0.0001) by −19.4 ± 17.1/−10.3 ± 10.6 and −16.9 ± 17.0/−9.4 ± 10.6 mmHg, respectively. Home pulse rates in the morning and evening were also significantly lowered (p < 0.0001) by −3.5 ± 7.8 and −3.5 ± 7.3 beats/min, respectively. At baseline, patients whose ME average was ≥135 mmHg and whose ME difference was ≥15 mmHg (defined as morning-predominant hypertension) accounted for 20.4 % of the study population. However, at the end of the study, the number of such patients was significantly reduced to 7.9 % (p < 0.0001). Patients whose ME average was ≥135 mmHg and whose ME difference was <15 mmHg (defined as sustained hypertension) accounted for 71.1 % of the study population at baseline. This was reduced significantly to 42.8 % at the end of the study (p < 0.0001). ME average decreased significantly from 153.8 ± 15.5 mmHg to 135.6 ± 11.9 mmHg, and ME difference also decreased significantly from 6.7 ± 13.1 mmHg to 4.7 ± 10.8 mmHg (both p < 0.0001).

Conclusion

These results suggest that azelnidipine improved morning hypertension with its sustained BP-lowering effect.

Electronic supplementary material

The online version of this article (doi:10.1007/s40268-013-0007-7) contains supplementary material, which is available to authorized users.     

Simultaneous Effect of Temperature and Irradiance on Growth and Okadaic Acid Production from the Marine Dinoflagellate Prorocentrum belizeanum

Benthic marine dioflagellate microalgae belonging to the genus Prorocentrum are a major source of okadaic acid (OA), OA analogues and polyketides. However, dinoflagellates produce these valuable toxins and bioactives in tiny quantities, and they grow slowly compared to other commercially used microalgae. This hinders evaluation in possible large-scale applications. The careful selection of producer species is therefore crucial for success in a hypothetical scale-up of culture, as are appropriate environmental conditions for optimal growth. A clone of the marine toxic dinoflagellate P. belizeanum was studied in vitro to evaluate its capacities to grow and produce OA as an indicator of general polyketide toxin production under the simultaneous influence of temperature (T) and irradiance (I₀). Three temperatures and four irradiance levels were tested (18, 25 and 28 °C; 20, 40, 80 and 120 µE·m⁻²·s⁻¹), and the response variables measured were concentration of cells, maximum photochemical yield of photosystem II (PSII), pigments and OA. Experiments were conducted in T-flasks, since their parallelepipedal geometry proved ideal to ensure optically thin cultures, which are essential for reliable modeling of growth-irradiance curves. The net maximum specific growth rate (µ_m) was 0.204 day⁻¹ at 25 °C and 40 µE·m⁻²·s⁻¹. Photo-inhibition was observed at I₀ > 40 μEm⁻²s⁻¹, leading to culture death at 120 µE·m⁻²·s⁻¹ and 28 °C. Cells at I₀ ≥ 80 µE·m⁻²·s⁻¹ were photoinhibited irrespective of the temperature assayed. A mechanistic model for µ_m-I₀ curves and another empirical model for relating µ_m-T satisfactorily interpreted the growth kinetics obtained. ANOVA for responses of PSII maximum photochemical yield and pigment profile has demonstrated that P. belizeanum is extremely light sensitive. The pool of photoprotective pigments (diadinoxanthin and dinoxanthin) and peridinin was not able to regulate the excessive light-absorption at high I₀-T. OA synthesis in cells was decoupled from optimal growth conditions, as OA overproduction was observed at high temperatures and when both temperature and irradiance were low. T-flask culture observations were consistent with preliminary assays outdoors.     

Impact of Common KIBRA Allele on Human Cognitive Functions

The rs17070145 polymorphism (C → T substitution, intron 9) of the KIBRA gene has recently been associated with episodic memory and cognitive flexibility. These findings were inconsistent across reports though, and largely lacked gene–gene or gene–environment interactions. The aim of the present study was to determine the impact of the rs17070145 polymorphism on clinically relevant cognitive domains and its interaction with the modifiers ‘lifestyle'' and ‘cardiovascular risk factors''. Five-hundred forty-five elderly volunteers (mean age 64 years, ±7 years, 56% women) accomplished a comprehensive cognitive testing. Principal component analysis was used to reveal the internal structure of the data, rendering four composite scores: verbal memory, word fluency, executive function/psychomotor speed, and working memory. Lifestyle was assessed with a detailed questionnaire, age-associated risk factors by clinical interview and examination. There was no main effect of the rs17070145 genotype on any cognitive composite scores. However, we found worse performance in executive functions for T-allele carriers in the presence of arterial hypertension (β=−0.365, p=0.0077 and 0.031 after Bonferroni correction). This association was further modified by gender, showing the strongest association in hypertensive females (β=−0.500, p=0.0072 and 0.029 after Bonferroni correction). The effect of KIBRA on cognitive function seems to be complex and modified by gender and arterial hypertension.     

A validated HPTLC method for determination of terbutaline sulfate in biological samples: Application to pharmacokinetic study

Terbutaline sulfate (TBS) was assayed in biological samples by validated HPTLC method. Densitometric analysis of TBS was carried out at 366 nm on precoated TLC aluminum plates with silica gel 60F₂₅₄ as a stationary phase and chloroform–methanol (9.0:1.0, v/v) as a mobile phase. TBS was well resolved at R_F 0.34 ± 0.02. In all matrices, the calibration curve appeared linear (r² ⩾ 0.9943) in the tested range of 100–1000 ng spot⁻¹ with a limit of quantification of 18.35 ng spot⁻¹. Drug recovery from biological fluids averaged ⩾95.92%. In both matrices, rapid degradation of drug favored and the T_0.5 of drug ranged from 9.92 to 12.41 h at 4 °C and from 6.31 to 9.13 h at 20 °C. Frozen at −20 °C, this drug was stable for at least 2 months (without losses >10%). The maximum plasma concentration (Cp_max) was found to be 5875.03 ± 114 ng mL⁻¹, which is significantly higher than the maximum saliva concentration (Cs_max, 1501.69 ± 96 ng mL⁻¹). Therefore, the validated method could be used to carry out pharmacokinetic studies of the TBS from novel drug delivery systems.     

Adiponectin serum levels correlate with insulin resistance in type 2 diabetic patients

The adipose tissue is not only an inert storage depot for lipids, but also it secretes a variety of bioactive molecules, known as adipokines, which affect whole-body homeostasis. Adiponectin is the most abundant of these adipocytokines and is known to have a regulatory effect on the metabolism of glucose and lipid. The main objectives of this study were to evaluate the serum levels of adiponectin and to establish a correlation between adiponectin serum levels and the degree of insulin resistance in type 2 diabetic patients. Eighty participants were enrolled in this study; 61 type 2 diabetic patients and 19 apparently healthy subjects. Serum level of adiponectin was measured by enzyme-linked immunosorbent assay (ELISA) for each participant. Data collection sheet was filled with all required information for each participant. Adiponectin level in the diabetic patients (5.05 ± 2.61 μg/ml) was lower than in non-diabetic healthy controls (5.71 ± 2.35 μg/ml). When the results were compared according to gender, diabetic females showed significantly higher adiponectin levels (5.76 ± 2.64 μg/ml) than diabetic males (4.366 ± 2.43 μg/ml, P = 0.035). In addition, female diabetic patients with abdominal obesity (waist circumference (WC) ⩾ 88 cm) had lower adiponectin levels (5.58 ± 2.58 μg/ml) than diabetic females without abdominal obesity (6.96 ± 3.12 μg/ml). The correlation analysis indicated that adiponectin had a significant positive correlation with age (r = −0.450, P < 0.001). In conclusion, female diabetic patients had a statistically significant higher adiponectin level than male diabetic patients which could indicate a gender effect. Adiponectin levels were inversely related to insulin resistance; as patients with abdominal obesity had lower serum levels of adiponectin.     

Investigation of Cortical Glutamate–Glutamine and γ-Aminobutyric Acid in Obsessive–Compulsive Disorder by Proton Magnetic Resonance Spectroscopy

Glutamatergic abnormalities in corticostriatal brain circuits are thought to underlie obsessive–compulsive disorder (OCD). Whether these abnormalities exist in adults with OCD is not clear. We used proton magnetic resonance spectroscopy (¹H MRS) to test our hypothesis that unmedicated adults with OCD have reduced glutamate plus glutamine (Glx) levels in the medial prefrontal cortex (MPFC) compared with healthy controls. Levels of γ-aminobutyric acid (GABA) were also explored. Twenty-four unmedicated adults with OCD and 22 matched healthy control subjects underwent ¹H MRS scans at 3.0 T. Resonances of both Glx and GABA were obtained using the standard J-editing technique and assessed as ratios relative to voxel tissue water (W) in the MPFC (the region of interest) and the dorsolateral prefrontal cortex (DLPFC) to explore the regional specificity of any finding. In the MPFC, Glx/W did not differ by diagnostic group (p=0.98) or sex (p=0.57). However, GABA/W was decreased in OCD (2.16±0.46 × 10⁻³) compared with healthy controls (2.43±0.45 × 10⁻³, p=0.045); moreover, age of OCD onset was inversely correlated with MPFC GABA/W (r=−0.50, p=0.015). MPFC GABA/W was higher in females than in males. In the DLPFC, there were no main effects of diagnosis or gender on Glx/W or GABA/W. These data indicate that unmedicated adults with OCD do not have Glx abnormalities in a MPFC voxel that includes the pregenual anterior cingulate cortex. However, they may have decreased MPFC GABA levels. How GABA abnormalities might contribute to corticostriatal dysfunction in OCD deserves further study.     

Catecholamine synthesis and metabolism in the central nervous system of mice lacking α2-adrenoceptor subtypes

Background and purpose:

This study investigates the role of α₂-adrenoceptor subtypes, α_2A, α_2B and α_2C, on catecholamine synthesis and catabolism in the central nervous system of mice.

Experimental approach:

Activities of the main catecholamine synthetic and catabolic enzymes were determined in whole brains obtained from α_2A-, α_2B- and α_2C-adrenoceptor knockout (KO) and C56Bl\7 wild-type (WT) mice.

Key results:

Although no significant differences were found in tyrosine hydroxylase activity and expression, brain tissue levels of 3,4-dihydroxyphenylalanine were threefold higher in α_2A- and α_2C-adrenoceptor KO mice. Brain tissue levels of dopamine and noradrenaline were significantly higher in α_2A and α_2CKOs compared with WT [WT: 2.8 ± 0.5, 1.1 ± 0.1; α_2AKO: 6.9 ± 0.7, 1.9 ± 0.1; α_2BKO: 2.3 ± 0.2, 1.0 ± 0.1; α_2CKO: 4.6 ± 0.8, 1.5 ± 0.2 nmol·(g tissue)⁻¹, for dopamine and noradrenaline respectively]. Aromatic L-amino acid decarboxylase activity was significantly higher in α_2A and α_2CKO [WT: 40 ± 1; α_2A: 77 ± 2; α_2B: 40 ± 1; α_2C: 50 ± 1, maximum velocity (V_max) in nmol·(mg protein)⁻¹·h⁻¹], but no significant differences were found in dopamine β-hydroxylase. Of the catabolic enzymes, catechol-O-methyltransferase enzyme activity was significantly higher in all three α₂KO mice [WT: 2.0 ± 0.0; α_2A: 2.4 ± 0.1; α_2B: 2.2 ± 0.0; α_2C: 2.2 ± 0.0 nmol·(mg protein)⁻¹·h⁻¹], but no significant differences were found in monoamine oxidase activity between all α₂KOs and WT mice.

Conclusions and implications:

In mouse brain, deletion of α_2A- or α_2C-adrenoceptors increased cerebral aromatic L-amino acid decarboxylase activity and catecholamine tissue levels. Deletion of any α₂-adrenoceptor subtypes resulted in increased activity of catechol-O-methyltransferase. Higher 3,4-dihydroxyphenylalanine tissue levels in α_2A and α_2CKO mice could be explained by increased 3,4-dihydroxyphenylalanine transport.