Stability and in vitro activity of nystatin and its γ-cyclodextrin complex against Candida albicans

γ-Cyclodextrin (γ-CD) was found to form an inclusion complex with nystatin. In the presence of γ-CD, cells of Candida albicans absorbed less nystatin than in its absence. The γ-CD/nystatin complex had no or insignificant antimicrobial activity. Upon diffusion into a γ-CD-free medium the antibiotic was released from the complex and inhibited growth of the test organism. The stability of nystatin and the γ-CD complex was studied using both a microbiological and a spectrophotometric assay. The stability of nystatin in aqueous preparations was improved by γ-CD complexation. Degradation of pure nystatin did not follow first-order kinetics. The loss of antimycotic potency was more rapid than the decrease in concentration calculated from the spectrophotometric data for the pure antibiotic as well as its γ-CD inclusion complex.     

Beating the odds: efficacy and toxicity of dihydropyrimidine dehydrogenase‐driven adaptive dosing of 5‐FU in patients with digestive cancer

Aims

5‐FU is the backbone of most regimens in digestive oncology. Administration of standard 5‐FU leads to 15–30% of severe side effects, and lethal toxicities are regularly reported with fluoropyrimidine drugs. Dihydropyrimidine dehydrogenase (DPD) deficiency is a pharmacogenetic syndrome responsible for most cases of life‐threatening toxicities upon 5‐FU intake, and pre‐treatment checking for DPD status should help to reduce both incidence and severity of side effects through adaptive dosing strategies.

Methods

We have used a simple method for rapidly establishing the DPD phenotype of patients with cancer and used it prospectively in 59 routine patients treated with 5‐FU‐based therapy for digestive cancers. No patient with total DPD deficiency was found but 23% of patients exhibited poor metabolizer phenotype, and one patient was phenotyped as profoundly deficient. Consequently, 5‐FU doses in poor metabolizer patients were cut by an average 35% as compared with non deficient patients (2390 ± 1225 mg vs. 3653 ± 1371 mg, P < 0.003, t‐test).

Results

Despite this marked reduction in 5‐FU dosing, similar efficacy was achieved in the two subsets (clinical benefit: 40 vs. 43%, stable disease: 40 vs. 37%, progressive disease: 20% in both subsets, P = 0.893, Pearson''s chi‐square). No difference in toxicities was observed (P = 0.104, Fisher''s exact test). Overall, only 3% of early severe toxicities were recorded, a value markedly lower than the 15‐30% ones usually reported with 5‐FU.

Conclusions

This feasibility study shows how simplified DPD‐based adaptive dosing of 5‐FU can reduce sharply the incidence of treatment‐related severe toxicities while maintaining efficacy as part of routine clinical practice in digestive oncology.     

Opposite effects of sex steroids on 11β-hydroxysteroid dehydrogenase activity in the normal and adrenalectomized rat testis

• 1.1. Sex steroids have been shown to regulate the biosynthesis of 11β-hydroxysteroid dehydrogenase (11β-HSD).
• 2.2. In vitro studies showed that oestradiol (E2) or testosterone (T) can interfere with the bioassay of enzyme activity, but not progesterone (P4).
• 3.3. For in vivo studies, the activity of 11β-HSD in the testis of normal and adrenalectomized (ADX) adult male Wistar rats was determined following a daily IM injection of sex steriods for 7 days.
• 4.4. The 11β-HSD activity was significantly reduced (P<0.01) either by E2 or T in normal and ADX rats. The enzyme activity in normal rats given both T and E2 was even lower (P<0.001) than when E2 was given alone.
• 5.5. P4 given to normal and ADX rats increased the enzyme activity higher than normal (P<0.001).
• 6.6. The presence of corticosteroids influenced the effects of E2, but not of T and P4, on 11β-HSD activity.
• 7.7. E2 and T downregulate 11β-HSD activity, whereas P4 increased it. E2 did not act through lowering T level.

     

In Vitro Regioselective Stability of β‐1‐O‐ and 2‐O‐Acyl Glucuronides of Naproxen and Their Covalent Binding to Human Serum Albumin

β‐1‐O‐ (NAG) and 2‐O‐glucuronides (2‐isomer) of (S)‐naproxen (NA) were prepared to determine which positional isomer‐(s) of the acyl glucuronide of NA is responsible for forming covalent adducts with human serum albumin (HSA). Their comparative stability and covalent binding adduct formation with HSA were investigated at pH 7.4 and at 37 °C. NA and its acyl glucuronides were simultaneously determined by HPLC. Three positional isomers were formed successively after incubation of NAG in the buffer only. However, when NAG was incubated with HSA (30 mg/mL), isomers other than the 2‐isomer were formed in little or negligible quantities. In HSA solution, NAG (k_d = 2.08 ± 0.08 h⁻¹) was four times less stable than 2‐isomer (k_d = 0.51 ± 0.02 h⁻¹). NAG was degraded by hydrolysis (k_hyd = 1.01 ± 0.10 h⁻¹) and isomerization (k_iso = 1.07 ± 0.07 h⁻¹) to the same extent; however, hydrolysis was predominant for the 2‐isomer (k_d = 0.51 ± 0.02 h⁻¹). The incubation of both NAG and 2‐isomer with HSA led to the formation of a covalent adduct; however, the adduct formation from the 2‐isomer proceeded more slowly than that from NAG. The present results suggest that the covalent binding of NA to HSA via its acyl glucuronides proceeds through both transacylation (direct nucleophilic displacement) and glycation mechanisms; NAG rapidly forms an adduct that may be unstable, and the protein adduct from the 2‐O‐acyl glucuronide is as important for the covalent binding as those from the 1‐O‐acyl glucuronides.     

The acute and long‐term L‐DOPA effects are independent from changes in the activity of dorsal raphe serotonergic neurons in 6‐OHDA lesioned rats

Background and Purpose

L‐DOPA is still the most efficacious pharmacological treatment for Parkinson''s disease. However, in the majority of patients receiving long‐term therapy with L‐DOPA, its efficacy is compromised by motor complications, notably L‐DOPA‐induced dyskinesia. Evidence suggests that the serotonergic system is involved in the therapeutic and the side effects of L‐DOPA. Here, we investigate if long‐term L‐DOPA treatment alters the activity of the dorsal raphe nucleus (DRN) and its responses to serotonergic drugs.

Experimental Approach

We measured the responses of serotonergic neurons to acute and chronic L‐DOPA treatment using in vivo electrophysiological single unit‐extracellular recordings in the 6‐OHDA‐lesion rat model of Parkinson''s disease.

Key Results

The results showed that neither acute nor chronic L‐DOPA administration (6 mg·kg⁻¹ s.c.) altered the properties of serotonergic‐like neurons. Furthermore, no correlation was found between the activity of these neurons and the magnitude of L‐DOPA‐induced dyskinesia. In dyskinetic rats, the inhibitory response induced by the 5‐HT_1A receptor agonist 8‐OH‐DPAT (0.0625–16 μg·kg⁻¹, i.v.) was preserved. Nonetheless, L‐DOPA impaired the ability of the serotonin reuptake inhibitor fluoxetine (0.125–8 mg·kg⁻¹, i.v) to inhibit DRN neuron firing rate in dyskinetic animals.

Conclusions and Implications

Although serotonergic neurons are involved in the dopaminergic effects of L‐DOPA, we provide evidence that the effect of L‐DOPA is not related to changes of the activity of DRN neurons. Rather, L‐DOPA might reduce the efficacy of drugs that normally enhance the extracellular levels of serotonin.

Linked Articles

This article is part of a themed section on Updating Neuropathology and Neuropharmacology of Monoaminergic Systems. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.13/issuetoc

Abbreviations

8‐OH‐DPAT

8‐hydroxy‐2‐(dipropylamino)tetralin

AIM

abnormal involuntary movements

DRN

dorsal raphe nucleus

L‐DOPA

L‐3,4‐dihydroxyphenylalanine

LID

L‐DOPA‐induced dyskinesia

6‐OHDA

6‐hydroxydopamine

PD

Parkinson''s disease

SSRI

selective serotonin reuptake inhibitor

     

Pro‐cognitive activity in rats of 3‐furan‐2‐yl‐N‐p‐tolyl‐acrylamide,a positive allosteric modulator of the α7 nicotinic acetylcholine receptor

Background and Purpose

α7 nicotinic acetylcholine receptors (α7 nAChRs) may represent useful targets for cognitive improvement. The aim of this study is to compare the pro‐cognitive activity of selective α7‐nAChR ligands, including the partial agonists, DMXBA and A‐582941, as well as the positive allosteric modulator, 3‐furan‐2‐yl‐N‐p‐tolyl‐acrylamide (PAM‐2).

Experimental Approach

The attentional set‐shifting task (ASST) and the novel object recognition task (NORT) in rats, were used to evaluate the pro‐cognitive activity of each ligand [i.e., PAM‐2 (0.5, 1.0, and 2.0 mg·kg⁻¹), DMXBA and A‐582941 (0.3 and 1.0 mg·kg⁻¹)], in the absence and presence of methyllycaconitine (MLA), a selective competitive antagonist. To determine potential drug interactions, an inactive dose of PAM‐2 (0.5 mg·kg⁻¹) was co‐injected with inactive doses of either agonist ‐ DMXBA: 0.1 (NORT); 0.3 mg·kg⁻¹ (ASST) or A‐582941: 0.1 mg·kg⁻¹.

Key Results

PAM‐2, DMXBA, and A‐582941 improved cognition in a MLA‐dependent manner, indicating that the observed activities are mediated by α7 nAChRs. Interestingly, the co‐injection of inactive doses of PAM‐2 and DMXBA or A‐582941 also improved cognition, suggesting drug interactions. Moreover, PAM‐2 reversed the scopolamine‐induced NORT deficit. The electrophysiological results also support the view that PAM‐2 potentiates the α7 nAChR currents elicited by a fixed concentration (3 μM) of DMXBA with apparent EC₅₀ = 34 ± 3 μM and E_max = 225 ± 5 %.

Conclusions and Implications

Our results support the view that α7 nAChRs are involved in cognition processes and that PAM‐2 is a novel promising candidate for the treatment of cognitive disorders.

Abbreviations

α7 nAChR

nicotinic acetylcholine receptor with α7 subunit

AD

Alzheimer''s disease

apparent EC₅₀

enhancement potency

ASST

attentional set‐shifting task

CD

compound discrimination

DI

discrimination index

E

exploration time

ED

extra‐dimensional

E_max

ligand efficacy

ID

intra‐dimensional

ITI

inter‐trial interval

MLA

methyllycaconitine

NORT

novel object recognition task

n_H

Hill coefficient

PAM

positive allosteric modulator

PAM‐2

3‐furan‐2‐yl‐N‐p‐tolyl‐acrylamide

Rev

reversal of discrimination

SD

simple discrimination

T1

familiarisation trial

T2

retention trial

     

The aggregative stability of β-lactoglobulin in glassy mixtures with sucrose, trehalose and dextran

The aim of this study was to investigate the effect of the addition of different carbohydrates on the thermally induced aggregation of a model globular protein, β-lactoglobulin (BLG), in the glass state. Amorphous mixtures of BLG with trehalose, sucrose and dextran were prepared by freeze-drying, their glass behaviour was characterised using calorimetry and thermally induced aggregation was measured using size exclusion chromatography. Pure BLG shows increasing levels of aggregation when heated in the temperature range 70-100 °C for 48-144 h. The addition of the disaccharides sucrose and trehalose both resulted in a decrease in aggregation rate which approached negligible rates at 50 wt.% carbohydrate. The effect of dextran addition was similar to that of the disaccharides when preparations containing 9 wt.% carbohydrate were heated at 70 °C for 2 days. However, when the concentration exceeded 23 wt.%, the reaction temperature was 70 °C or above or the reaction time was longer than 48 h, the addition of the polysaccharide did not protect the protein from thermally induced aggregation, suggesting that protein-polymer phase separation could have occurred during freeze-drying. Overall the results support the proposal that one aspect of carbohydrate additive functionality is as a diluent with the added condition that the carbohydrate remains miscible with the protein during processing.     

Formulation and in vitro/in vivo correlation of a drug‐in‐adhesive transdermal patch containing azasetron

The aim of the present study was to develop a transdermal drug delivery system for azasetron and evaluate the correlation between in vitro and in vivo release. The effects of different adhesives, permeation enhancers, and loadings of azasetron used in patches on the penetration of azasetron through rabbit skin were investigated using two‐chamber diffusion cells in vitro. For in vivo studies, azasetron pharmacokinetic parameters in Bama miniature pigs were determined according to a noncompartment model method after topical application of transdermal patches and intravenous administration of azasetron injections. The best permeation profile was obtained with the formulation containing DURO‐TAK 87‐9301 as adhesive, 5% of isopropyl myristate as penetration enhancer, and 5% of azasetron. The optimal patch formulation exhibited sustained release profiles in vivo for 216 h. The in vivo absorption curve in Bama miniature pigs obtained by deconvolution approach using WinNonlin® program was correlated well with the in vitro permeation curve of the azasetron patch. These findings indicated that the developed patch for azasetron is promising for the treatment of delayed chemotherapy‐induced nausea and vomiting, and the in vitro skin permeation experiments could be useful to predict the in vivo performance of transdermal azasetron patches. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:4540–4548, 2012     

Asthma-inducing potential of 28 substances in spray cleaning products—Assessed by quantitative structure activity relationship (QSAR) testing and literature review

Exposure to spray cleaning products constitutes a potential risk for asthma induction. We set out to review whether substances in such products are potential inducers of asthma. We identified 101 spray cleaning products for professional use. Twenty-eight of their chemical substances were selected. We based the selection on (a) positive prediction for respiratory sensitisation in humans based on quantitative structure activity relationship (QSAR) in the Danish (Q)SAR Database, (b) positive QSAR prediction for severe skin irritation in rabbits and (c) knowledge on the substances' physico-chemical characteristics and toxicity. Combining the findings in the literature and QSAR predictions, we could group substances into four classes: (1) some indication in humans for asthma induction: chloramine, benzalkonium chloride; (2) some indication in animals for asthma induction: ethylenediaminetetraacetic acid (EDTA), citric acid; (3) equivocal data: hypochlorite; (4) few or lacking data: nitriloacetic acid, monoethanolamine, 2-(2-aminoethoxy)ethanol, 2-diethylaminoethanol, alkyldimethylamin oxide, 1-aminopropan-2-ol, methylisothiazolinone, benzisothiazolinone and chlormethylisothiazolinone; three specific sulphonates and sulfamic acid, salicylic acid and its analogue sodium benzoate, propane-1,2-diol, glycerol, propylidynetrimethanol, lactic acid, disodium malate, morpholine, bronopol and benzyl alcohol. In conclusion, we identified an asthma induction potential for some of the substances. In addition, we identified major knowledge gaps for most substances. Thus, more data are needed to feed into a strategy of safe-by-design, where substances with potential for induction of asthma are avoided in future (spray) cleaning products. Moreover, we suggest that QSAR predictions can serve to prioritise substances that need further testing in various areas of toxicology.     

Structure-activity relationships of phthalates in inhibition of human placental 3β-hydroxysteroid dehydrogenase 1 and aromatase

Phthalates are associated with preterm delivery. However, the mechanism is unclear. Progesterone formed by 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) and estradiol by aromatase (CYP19A1) in placenta are critical for maintaining pregnancy. In this study, we compared structure-activity relationships (SAR) of 14 phthalates varied in carbon atoms in alcohol moiety to inhibit human HSD3B1 in COS1 and CYP19A1 in JEG-3 cells. There were responses in that only diphthalates with 4–7 carbon atoms were competitive HSD3B1 inhibitors and diphthalates with 6 carbon atoms were CYP19A1 inhibitors. IC₅₀s of dipentyl (DPP), bis(2-butoxyethyl) (BBOP), dicyclohexyl (DCHP), dibutyl (DBP), and diheptyl phthalate (DHP) were 50.12, 32.41, 31.42, 9.69, and 4.87 μM for HSD3B1, respectively. DCHP and BBOP inhibited CYP19A1, with IC₅₀s of 64.70 and 56.47 μM. DPP, BBOP, DCHP, DBP, and DHP inhibited progesterone production in JEG-3 cells. In conclusion, our results indicate that there is clear SAR for phthalates in inhibition of HSD3B1 and CYP19A1.     

Texture Optimization of Water‐in‐Oil Emulsions

The aim of this research is to demonstrate the effect of variations in certain parameters of the oily phase (OP) in water‐in‐oil (W/O) emulsions on rheological and texture properties of finished products. The formulated emulsions were selected according to an optimal experimental procedure. The applied variations were nature of the OP, its volume fraction, the hydrophilic‐lipophilic balance (HLB) value, and the surfactant proportion. Results are presented for the followed tests carried out on the emulsions: texture analysis, rheology, and particle size analysis. The oils used in the study were sweet almond oil, liquid paraffin, maize oil, cyclomethicone, dimethicone, and wheat germ oil. The resulting data demonstrate a notable influence of the volume fraction oil on hardness, viscosity, adhesiveness, and cohesiveness of W/O emulsions. Emulsion hardness and viscosity increased as the OP percentage increased; this effect being even more pronounced for the vegetable oils. In contrast, emulsion adhesiveness and cohesiveness decreased as the volume fraction oil increased. The HLB value of the surfactant mixture of the emulsion also influenced hardness, adhesiveness, and elasticity, increasing or decreasing as HLB value did.     

Antioxidative activity of 4-oxy- and 4-hydroxy-nitroxides in tisues and erythrocytes from rats

研究氮氧自由基的抗脂质过氧化作用．方法：用·ＯＨ生成系统Ｆｅ２＋＋抗坏血酸刺激大鼠心、肝、肾组织匀浆，用ＴＢＡ比色法测ＭＤＡ生成；用分光光度法测Ｈ２Ｏ２诱导的大鼠ＲＢＣ溶血度；用ＮＢＴ还原法测酵母多糖诱导大鼠中性白细胞生成的Ｏ－２．结果：氮氧自由基ＯＴＭＰＯ和ＨＴＭＰＯ抑制ＭＤＡ生成，对抗溶血反应，但不影响Ｏ－２生成．１羟基化合物ＯＴＭＰＯＨ和ＨＴＭＰＯＨ也有类似作用．但非自由基ＯＴＭＰ和ＨＴＭＰ对组织脂质过氧化无任何影响．结论：氮氧自由基通过清除·ＯＨ而对抗脂质过氧化，其捕捉·ＯＨ不是通过ＮＯ·的还原作用，ＮＯ·基和ＮＯＨ基都是必需活性基团．     

Concomitant use of tramadol and venlafaxine – evaluation of antidepressant-like activity and other behavioral effects in rats

BackgroundThe aim of this study was to evaluate antidepressant-like effect (Porsolt test), locomotor activity and motor coordination of joint administration of tramadol (TRM) and venlafaxine (VEN) in rats.MethodsThe tests were performed on male Wistar rats after single and chronic treatment (7 and 14 days) with TRM intraperitoneally (ip) and VEN orally (po) administered once a day. The controls were given 0.5% carboxymethylcellulose (CMC) solution (0.5 ml per rat, ip and po).ResultsIt was found that combination of TRM (5 mg/kg ip) with VEN (20 mg/kg po) caused an increased antidepressant effect compared to TRM and VEN administered alone, with no effect on locomotor activity or motor coordination in rats, which may be of clinical significance.It was also observed that reduced time of active swimming of animals in Porsolt test with an increased dose (10 and 20 mg/kg) and time of administration (7 and 14 days) of TRM were correlated with a decreased locomotor activity in rats. It may indicate the development of tolerance to TRM’s antidepressant effect in rats during chronic treatment with doses higher than 5 mg/kg.ConclusionIt can be expected that combination of low doses of TRM and VEN could potentially be feasible and relatively safe in cases with acute pain with co-existing depression, however, further investigations are needed.     

Pharmacokinetics of paracetamol and its metabolites in women at delivery and post‐partum

Aim

A recent report on intravenous (i.v.) paracetamol pharmacokinetics (PK) showed a higher total clearance in women at delivery compared with non‐pregnant women. To describe the paracetamol metabolic and elimination routes involved in this increase in clearance, we performed a population PK analysis in women at delivery and post‐partum in which the different pathways were considered.

Methods

Population PK parameters using non‐linear mixed effect modelling were estimated in a two‐period PK study in women to whom i.v. paracetamol (2 g loading dose followed by 1 g every 6 h up to 24 h) was administered immediately following Caesarean delivery and in a subgroup of the same women to whom single 2 g i.v.loading dose was administered 10–15 weeks post‐partum.

Results

Population PK analysis was performed based on 255 plasma and 71 urine samples collected in 39 women at delivery and in eight of these 39 women 12 weeks post‐partum. Total clearance was higher in women at delivery compared with 12th post‐partum week (21.1 vs. 11.7 l h⁻¹) due to higher clearances to paracetamol glucuronide (11.6 vs. 4.76 l h⁻¹), to oxidative metabolites (4.95 vs. 2.77 l h⁻¹) and of unchanged paracetamol (1.15 vs. 0.75 l h⁻¹). In contrast, there was no difference in clearance to paracetamol sulphate.

Conclusion

The increased total paracetamol clearance at delivery is caused by a disproportional increase in glucuronidation clearance and a proportional increase in clearance of unchanged paracetamol and in oxidation clearance, of which the latter may potentially limit further dose increase in this patient group.     

Analgesic, antioedematous and antioxidant activity of γ-butyrolactone derivatives in rodents

In this paper, the analgesic, antioedematous, motor-impairing and antioxidant properties of four γ-butyrolactone derivatives (BM113, BM113A, BM138 and BM138A) are described. Pain was induced by thermal (hot-plate test), chemical (writhing test) or mechanical (Randall-Selitto model) stimulation. All in-vivo assays were carried out in mice pretreated intraperitoneally with the test compounds, except for the evaluation of anti-inflammatory and analgesic activities in the carrageenan-induced paw oedema model, in which rats were pretreated orally with these compounds. In the hot-plate assay, BM113A and BM138A dose dependently prolonged the latency of the nociceptive reaction. Their analgesic activity, measured as a median effective dose (ED(50)=4.7 mg/kg), was similar to that of morphine (2.4 mg/kg). In the writhing test, all four compounds, in particular BM113A and BM138A, showed higher potency than the reference drug acetylsalicylic acid (the ED(50) values were 3.7, 2.3 and 46.1 mg/kg, respectively). BM138 caused a dose-dependent diminution of paw oedema (up to 49%) in the carrageenan model and BM138A at 200 mg/kg reduced mechanical hyperalgesia in the Randall-Selitto test (～30% when compared with the control). None of the γ-butyrolactone derivatives tested at the ED(50) obtained in the hot-plate test influenced the locomotor activity of mice, although in the rotarod test at 24 rpm, BM113A and BM138 at 100 mg/kg showed some motor-impairing properties. In vitro, a concentration-dependent ABTS radical cation-scavenging activity of BM138 and BM138A (up to 80% inhibition of the radical absorbance) was observed. The results of the present study suggest that BM138 and BM138A could be of interest for future investigations as antinociceptive and antioedematous agents with potential free radical-scavenging properties.     

Benzofuran derivatives inhibit 11β-hydroxysteroid dehydrogenase type 1 activity in rat adipose tissue

Excess glucocorticoids promote visceral obesity and insulin resistance. The main regulator of intracellular glucocorticoid levels are 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts inactive glucocorticoid into bioactive glucocorticoid such as cortisol in humans and corticosterone in rodents; therefore, the inhibition of 11β-HSD1 has considerable therapeutic potential for metabolic diseases including obesity and diabetes. Benzofuran is a key structure in many biologically active compounds such as benzbromarone, malibatol A and (+)-liphagal. The aim of this study was to investigate the inhibitory effect of benzofuran derivatives on 11β-HSD1 in mesenteric adipose tissue from rodents. 11β-HSD1 activity was determined by incubation of rat mesenteric adipose tissue microsomes in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) with and without benzofuran derivatives (Compounds 1-14). The corticosterone produced was measured by HPLC. More than 40% of 11β-HSD1 inhibition was observed in Compounds 1, 5, 7 and 8. Moreover, Compounds 7 and 8 inhibited the 11β-HSD1 activity in adipose microsomes dose- and time-dependently, as well as in 3T3-L1 adipocytes. Compounds 7 and 8 did not inhibit 11β-HSD type 2 (11β-HSD2), whereas Compounds 1 and 5 inhibited 11β-HSD2 by 18.7% and 56.3%, respectively. Further, a kinetic study revealed that Compounds 7 and 8 acted as non-competitive inhibitors of 11β-HSD1. Ki (nmol/h/mg protein) values of Compounds 7 and 8 were 17.5 and 24.0, respectively, with IC50 (μM) of 10.2 and 25.6, respectively. These data indicate that Compounds 7 and 8 are convincing candidates for seed compounds as specific inhibitors of 11β-HSD1 and have the potential to be developed as anti-obesity drugs.     

Species-specific differences in the inhibition of human and zebrafish 11β-hydroxysteroid dehydrogenase 2 by thiram and organotins

Dithiocarbamates and organotins can inhibit enzymes by interacting with functionally essential sulfhydryl groups. Both classes of chemicals were shown to inhibit human 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), which converts active cortisol into inactive cortisone and has a role in renal and intestinal electrolyte regulation and in the feto-placental barrier to maternal glucocorticoids. In fish, 11β-HSD2 has a dual role by inactivating glucocorticoids and generating the major androgen 11-ketotestosterone. Inhibition of this enzyme may enhance glucocorticoid and diminish androgen effects in fish. Here, we characterized 11β-HSD2 activity of the model species zebrafish. A comparison with human and mouse 11β-HSD2 revealed species-specific substrate preference. Unexpectedly, assessment of the effects of thiram and several organotins on the activity of zebrafish 11β-HSD2 showed weak inhibition by thiram and no inhibition by any of the organotins tested. Sequence comparison revealed the presence of an alanine at position 253 on zebrafish 11β-HSD2, corresponding to cysteine-264 in the substrate-binding pocket of the human enzyme. Substitution of alanine-253 by cysteine resulted in a more than 10-fold increased sensitivity of zebrafish 11β-HSD2 to thiram. Mutating cysteine-264 on human 11β-HSD2 to serine resulted in 100-fold lower inhibitory activity. Our results demonstrate significant species differences in the sensitivity of human and zebrafish 11β-HSD2 to inhibition by thiram and organotins. Site-directed mutagenesis revealed a key role of cysteine-264 in the substrate-binding pocket of human 11β-HSD2 for sensitivity to sulfhydryl modifying agents.     

Correlation between increased dopamine-β- hydroxylase activity and catecholamine concentration in plasma: Determination of acute changes in sympathetic activity in man

Summary In 11 healthy untrained volunteers the increase in plasma dopamine--hydroxylase (DBH) activity during graded physical exercise has been examined as a true measure of increased activity of the sympathetic nervous system. The correlation between DBH activity, catecholamine concentration (CA) in plasma and heart rate was studied. When work on an electrically braked bicycle ergometer was gradually increased from 12.5 to 100, 200 and 300 watts there was a linear increase in DBH activity and heart rate; the increase in CA concentrations followed an exponential function. The peak values for DBH activity and CA concentration in plasma after the 300 watt work load (as percentages of the resting levels) were 130±3% and 820±71%, respectively; the adrenaline concentration in plasma increased only to 150±19% (p>0.05). There were significant correlations between heart rate and work load, DBH and work load and log CA and work load. The data imply direct correlations between heart rate and DBH, heart rate and log CA and DBH and log CA. The exponential increase in noradrenaline concentration in plasma might be due either to a greater net overflow from sympathetic nerve endings, and/or to increased secretion by the adrenal medulla. In the latter case, the release of noradrenaline would not be accompanied by secretion either of adrenaline or DBH. After work ceased there were sharp falls in heart rate and CA concentration, which indicate an immediate drop in sympathetic activity. DBH activity in plasma returned to normal very slowly; it reached half maximum values after 20 – 22 min. It is concluded that increased sympathetic activity in man can be estimated in vivo as changes in DBH and/or CA concentration in plasma. In contrast, a rapid decrease in sympathetic activity is directly reflected only by a rapid fall in the plasma concentrations of CA.Part of this work was presented at the Symposion on Hypertension. Mainz, November 1973; Wietholdet al., 1973.Supported by a grant from the Bundesinstitut für Sportwissenschaft, Köln.