Comparison between the effects of propranolol and chlordiazepoxide on timing behaviour in the rat

Ten rats were trained to lever press for food reward on a schedule of differential reinforcement of low rates of response with a 20-s criterion (DRL 20). Ten more were trained on a new schedule of punishment, designed to be comparable to DRL 20 — differential punishment of high rates of response (DPH 20). Under this schedule, responses with a latency of 20 s or more earned food rewards, while those of less than 20 s were followed by food reward and brief electric footshock. After 42 sessions, rats on each schedule showed temporal discrimination in the distribution of inter-response times. The effects on these baselines of the anxiolytic chlordiazepoxide (CDP; 1 mg/kg IP) and the beta-blocker propranolol (2, 5 and 10 mg/kg IP) were investigated. Both drugs reduced numbers of responses reaching criterion (criterion resonses) in DPH, CDP increasing total responses. CDP acted similarly under DRL, but propranolol only affected performance at the highest doese, which reduced criterion responses, probably because of changes in total responding. Each drug increased response bursts. It is concluded that propranolol can exert a disinhibitory action in these schedules, although with some differences from that of the benzodiazepine CDP.     

Opposing acute and chronic behavioural effects of a beta-blocker,propranolol, in the rat

Rats were trained over 40 days to lever-press for food reward under a schedule of differential reinforcement of low rates of response with a 20-s criterion (DRL 20), following seven sessions of continuous reinforcement. The effect of injecting a beta-adrenergic blocker, propranolol (5 mg/kg IP), before and at two different delays after each daily session of DRL were investigated. In Experiment I, rats drugged 5–8 min before every session earned fewer reinforcements compared to controls, and showed impaired temporal discrimination. In Experiment II, this result was not replicated, but similar effects were clear in animals drugged pre-session from the 15th day of acquisition. By contrast, an improved temporal discrimination, and increased number of reinforcements were seen in rats drugged 5–8 min after every session. In Experiment III, the postsession effects were replicated and found also in rats drugged 4–5.5 h after each session. These results suggest that propranolol has an acute effect on DRL responding which resembles that of anxiolytics, and a chronic effect which opposes the acute one.     

Interaction between the antidepressant-like behavioral effects of beta adrenergic agonists and the cyclic AMP PDE inhibitor rolipram in rats

Rationale Type 4 phosphodiesterase (PDE4) is critical for hydrolysis of cAMP formed by stimulation of beta adrenergic receptors. However, it is not known if PDE4 is associated with beta adrenergic receptors in the mediation of antidepressant-like effects. Objective The aim of the study is to determine the relationship between PDE4 and beta adrenergic receptor-mediated cAMP signaling in mediating antidepressant-like effects. Methods The effects of the PDE4 inhibitor rolipram, alone or combined with dobutamine or clenbuterol, selective beta-1 and beta-2 adrenergic agonists, respectively, on behavior were examined in rats under a differential reinforcement of low rate (DRL) schedule and rats trained to discriminate rolipram from vehicle. Their effects on cAMP in primary cultures of rat cerebral cortical neurons also were determined. Results Rolipram (0.01–0.3 mg/kg), dobutamine (1–30 mg/kg), and clenbuterol (0.03–0.3 mg/kg) dose-dependently produced antidepressant-like effects on DRL behavior, decreasing response rate and increasing reinforcement rate. The effects of beta adrenergic agonists were potentiated by rolipram. Isobolographic analysis revealed that rolipram enhanced the antidepressant-like effect of dobutamine additively and that of clenbuterol synergistically. Consistently, a combination of ineffective doses of rolipram (0.03 mg/kg) and dobutamine (3 mg/kg) or clenbuterol (0.03 mg/kg) completely substituted for the rolipram discrimination stimulus. Further, incubation with an ineffective concentration of clenbuterol, but not dobutamine, in the presence of a subeffective concentration of rolipram, significantly increased cAMP in cultured cortical neurons. Conclusions PDE4 plays an important role in regulating cAMP signaling by either beta-1 or beta-2 adrenergic receptors that mediate antidepressant-like actions; beta-2 adrenergic receptor-mediated cAMP signaling appears more responsive than beta-1 cAMP signaling to PDE4 inhibition.     

Effects of 2‐(1‐hydroxypentyl)‐benzoate on platelet aggregation and thrombus formation in rats

2‐(1‐hydroxypentyl)‐benzoate (dl‐PHPB), a derivate of 3‐n‐butylphthalide (NBP), is a novel therapeutic agent for treatment of cerebral ischemia. In the present study, the antiplatelet and antithrombotic activities of dl‐PHPB were evaluated in ex vivo platelet aggregation and in vivo arteriovenous (A‐V) shunt models. dl‐PHPB inhibited platelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid (AA), and collagen (COL) in a dose‐dependent manner when given orally (12.9–129.5 mg/kg). The inhibitory potency was similar to 3‐n‐butylphthalide (NBP) and aspirin (ASP). Inhibition on platelet aggregation was also observed after iv administration of dl‐PHPB (1.29–12.9 mg/kg). The time‐course of these effects showed the maximal inhibition on platelet aggregation at 1 h after oral administration and 30 min after iv injection. dl‐PHPB (12.9–129.5 mg/kg, p.o.) caused dose‐dependent inhibition of thrombus formation in the rat A‐V shunt thrombosis model. These results show that dl‐PHPB is an orally and iv antiplatelet and antithrombotic agent and may be useful for treatment of ischemia stroke. Drug Dev Res 63:174–180, (2004). © 2004 Wiley‐Liss, Inc.     

Rapid determination of memantine in human plasma by using nanoring carboxyl‐functionalized paramagnetic molecularly imprinted polymer d‐μ‐SPE and UFLC‐MS/MS

A novel, simple, and sensitive method based on the use of dispersive micro‐solid‐phase extraction (d‐μ‐SPE) procedure combined with ultra‐fast liquid chromatography‐tandem quadrupole mass spectrometry (UFLC‐MS/MS) for the determination of memantine (ME) was developed and validated over the linearity range 0.05–10.0 µg/L with 100 μL of human plasma using memantine‐D6 (ME‐D6) as the internal standard. The novel nanoring carboxyl‐functionalized paramagnetic molecularly imprinted polymer (NR‐CF‐Mag‐MIP) was synthesized by ultrasound‐assisted suspension polymerization, using ME as a template molecule, methacrylic acid as a functional monomer, and divinylbenzene as a cross‐linking agent. The NR‐CF‐Mag‐MIP was used as the d‐μ‐SPE sorbent to extract ME from human plasma samples. The obtained results demonstrated the higher extraction capacity of NR‐CF‐Mag‐MIP with recoveries between 97.6 and 101%. The limits of quantification (LOQs) for ME was 0.015 µg/L. Validation results on linearity, specificity, accuracy, precision, and stability, as well as on application to the analysis of samples taken up to 480 h after oral administration of 20 mg (two 10 mg capsules) of ME in healthy volunteers demonstrated the applicability to bioequivalence studies. Copyright © 2014 John Wiley & Sons, Ltd.     

Enantiomeric separation and quantification of R/S‐amphetamine in serum using semi‐automated liquid‐liquid extraction and ultra‐high performance supercritical fluid chromatography‐tandem mass spectrometry

The amphetamine molecule contains a chiral center and its enantiomers exhibit differences in pharmacological effects, with the S‐enantiomer mediating most of the central nervous system stimulating activity. The majority of prescribed amphetamine consists of the pure S‐enantiomer, but therapeutic formulations containing the R‐enantiomer in various proportions are also available. Illegal amphetamine remains available mainly as a racemic mixture of the R‐ and S‐enantiomers. To distinguish between legal and illegal consumption of amphetamine a method for enantiomeric separation and quantification of R/S‐amphetamine in serum was developed and validated using ultra‐high performance supercritical fluid chromatography‐tandem mass spectrometry (UHPSFC‐MS/MS). Sample preparation prior to UHPSFC‐MS/MS analysis was performed by a semi‐automated liquid–liquid extraction method. The UHPSFC‐MS/MS method used a Chiralpak AD‐3 column with a mobile phase consisting of CO₂ and 0.1% ammonium hydroxide in 2‐propanol/methanol (50/50, v/v). The injection volume was 2 μL and run time was 4 minutes. MS/MS detection was performed with positive electrospray ionization and two multiple reaction monitoring transitions (m/z 136.1 > 119.0 and m/z 136.1 > 91.0). The calibration range was 12.5–1,000 nM for each analyte. The between‐assay relative standard deviations were in the range of 1.3–3.0%. Recovery was 73% and matrix effects ranged from 95 to 100% when corrected with internal standard. After development and validation, the method has been successfully implemented in our laboratory for both separation and quantification of R/S‐amphetamine and has proved to be a reliable and useful tool for distinguishing intake of R‐ and S‐amphetamine in authentic patient samples.     

Species differences of organic anion transporters involved in the renal uptake of 4‐amino‐3‐chlorophenyl hydrogen sulfate,a metabolite of resatorvid,between rats and dogs

Previous studies on the metabolic fate of resatorvid (TAK‐242) have shown that species differences in the pharmacokinetics of 4‐amino‐3‐chlorophenyl hydrogen sulfate (M‐III), a metabolite of TAK‐242, between rats and dogs are mainly attributable to the urinary excretion process. In the present study, the renal uptake mechanism of M‐III was investigated using kidney slices and Xenopus laevis oocytes expressing rat organic anion transporter 1 (rOat1; Slc22a6) and rOat3 (Slc22a8). The uptake of p‐aminohippuric acid (PAH), a substrate for Oats, by kidney slices from rats and dogs increased at 37 °C and M‐III inhibited the uptake. The initial uptake clearance of M‐III by rat kidney slices was 0.295 and 0.0114 ml/min/g at 37 °C and 4 °C, respectively. The Eadie‐Hofstee plot of M‐III uptake at 37 °C revealed two‐component transport processes with K_m values being 6.48 and 724 µmol/l. The uptake was inhibited by probenecid (PBC), PAH and benzylpenicillin (PCG). In contrast, in dog kidney slices, the initial uptake clearance of M‐III was 8.70 × 10^‐3 and 9.00 × 10^‐3 ml/min/g at 37 °C and 4 °C, respectively, and the uptake was not inhibited by PBC. Furthermore, rOat1‐ and rOat3‐expressing oocytes mediated M‐III uptake and the uptake was inhibited by PAH and PCG, respectively. These results suggest that rOat1 and rOat3 are responsible for the renal uptake of M‐III in rats. Moreover, it is speculated that Oat(s) is unable to transport M‐III in dogs and that the difference in the substrate recognition of Oat(s) contributes to the species difference in the pharmacokinetics of M‐III between rats and dogs. Copyright © 2013 John Wiley & Sons, Ltd.     

The development of decision limits for the GH‐2000 detection methodology using additional insulin‐like growth factor‐I and amino‐terminal pro‐peptide of type III collagen assays

The GH‐2000 and GH‐2004 projects have developed a method for detecting GH misuse based on measuring insulin‐like growth factor‐I (IGF‐I) and the amino‐terminal pro‐peptide of type III collagen (P‐III‐NP). The objectives were to analyze more samples from elite athletes to improve the reliability of the decision limit estimates, to evaluate whether the existing decision limits needed revision, and to validate further non‐radioisotopic assays for these markers. The study included 998 male and 931 female elite athletes. Blood samples were collected according to World Anti‐Doping Agency (WADA) guidelines at various sporting events including the 2011 International Association of Athletics Federations (IAAF) World Athletics Championships in Daegu, South Korea. IGF‐I was measured by the Immunotech A15729 IGF‐I IRMA, the Immunodiagnostic Systems iSYS IGF‐I assay and a recently developed mass spectrometry (LC‐MS/MS) method. P‐III‐NP was measured by the Cisbio RIA‐gnost P‐III‐P, Orion UniQ? PIIINP RIA and Siemens ADVIA Centaur P‐III‐NP assays. The GH‐2000 score decision limits were developed using existing statistical techniques. Decision limits were determined using a specificity of 99.99% and an allowance for uncertainty because of the finite sample size. The revised Immunotech IGF‐I – Orion P‐III‐NP assay combination decision limit did not change significantly following the addition of the new samples. The new decision limits are applied to currently available non‐radioisotopic assays to measure IGF‐I and P‐III‐NP in elite athletes, which should allow wider flexibility to implement the GH‐2000 marker test for GH misuse while providing some resilience against manufacturer withdrawal or change of assays. Copyright © 2015 John Wiley & Sons, Ltd.     

Metabolic profiling of isomeric aglycones central‐icaritin (c‐IT) and icaritin (IT) in osteoporotic rats by UPLC‐QTOF‐MS

The isomers, although of similarly chemical structures, have different pharmacological activities due to their metabolic processes in vivo. Central‐icaritin (c‐IT) and icaritin (IT) are isomers and major bioactive aglycones of the Herba Epimedii. In this study, we found that the anti‐osteoporotic effect of c‐IT was stronger than IT on bone structural changes in osteoporotic rats evaluated by Micro‐μCT with the parameters of bone mineral density (BMD), bone mineral content (BMC), tissue mineral content (TMC), and tissue mineral density (TMD). c‐IT treatment significantly increased the bone microarchitecture, compared with IT (p < 0.05). In order to explain their differences in anti‐osteoporosis, the metabolic profiling and pathways of c‐IT and IT in the plasma, bile, urine, and faeces of ovariectomized (OVX) rats were investigated by ultra‐performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC‐QTOF‐MS) after oral administration of c‐IT or IT (80 mg/kg). Finally, 59 metabolites of c‐IT and 43 metabolites of IT were identified by elucidating their corresponding quasimolecular ions and fragment ions. IT could be quickly absorbed into blood and reached a maximum plasma concentration, and then be rapidly conversed to its glucuronidation metabolites, most of which were excreted out by urine. Interestingly, the absorbed and conjugated speeds of c‐IT were slower than IT. The metabolic processes of c‐IT existed enterohepatic circulation, which decreased the metabolism and excretion rate of c‐IT, and prolonged the anti‐osteoporosis effect. Our findings provided evidence on the difference on metabolic profiles of c‐IT and IT in osteoporotic rats, which might shed new lights on improving anti‐osteoporotic effects of IT and c‐IT. Copyright © 2014 John Wiley & Sons, Ltd.     

Generic sample preparation and dual polarity liquid chromatography—time‐of‐flight mass spectrometry for high‐throughput screening in doping analysis

The requirements on initial testing in doping control are getting tighter regarding efficiency and speed while the scope of analytes is getting more diverse and, consequently, the need for high‐throughput methods is apparent. In this study, a comprehensive screening method for doping agents in human urine is presented, based on solid phase extraction (SPE) and liquid chromatography–time‐of‐flight mass spectrometry (LCTOFMS). The method covers most of the compound groups in the list of prohibited substances by World Anti‐Doping Agency (WADA). Mixed‐mode SPE on two types of sorbent and the use of negative ionization mode besides the commonly used positive mode in electrospray ionization (ESI) allowed detection of acidic compounds, such as sulpho‐conjugated metabolites. A run time of 8 minutes for each of the two ESI polarities was achieved. The method was validated regarding relative ionization efficiency, selectivity and signal to noise at the WADA's minimum required performance limit (MRPL) level, resulting in the acceptance of 197 compounds. A selection of 20 compounds was submitted for a more thorough validation, including extraction recovery, repeatability and linearity. Recovery and linearity (R²) varied mainly between 83–115% and 0.78–0.99, respectively. Median values for repeatability at the MRPL and 10 × MRPL levels were below 20%. A mean and median mass accuracy of 1.2 and 0.80 mDa, respectively, was achieved. The present method represents at the moment the widest coverage of low molecular weight prohibited substances for the screening in sports, providing an approach for further rationalisation of the analytical work‐flow in the doping control laboratories. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of d-amphetamine on temporal and spatial discrimination in rats

Rats were trained to lever-press for food reinforcers on a multiple schedule that had a fixed-interval (FI) and a differential reinforcement of low rate (DRL) component. Illumination of a stimulus light above the right-hand lever indicated that responses on this lever would be reinforced according to a FI 60-s schedule while responses on the left-hand lever were without programmed consequences. However, when the light above the left-hand lever was illuminated only responses on this lever were reinforced according to a DRL 15-s schedule. When the behaviour of the subjects had been brought under schedule control so that characteristic patterns of FI and DRL responding were emitted and there were relatively few responses on the incorrect levers, the effects of several doses of d-amphetamine (0.25, 0.5, 1.0, and 2.0 mg/kg) were assessed. The drug increased preference for responding on the right-hand lever. Thus, as dosage increased performance tended towards a constant high rate of responding on the right-hand lever throughout a session, with a much lower response rate on the left-hand lever. This result emphasises that the behavioural effects of drugs depend not only on patterns of ongoing behaviour but also on the context in which this behaviour occurs.     

Spatial distribution of theobromine – a low MW drug – in tissues via matrix‐free NALDI‐MS imaging

The ability of nano‐assisted laser desorption‐ionization mass spectrometry imaging (NALDI‐IMS) to provide selective chemical monitoring with appropriate spatial distribution of a low molecular drug in a biological tissue was investigated. NALDI‐IMS is a matrix‐free laser desorption ionization (LDI) protocol based on imprinting of tissue constituents on a nanostructured surface. Using the accumulation of theobromine in rat kidney as a model, NALDI‐IMS was found to provide well‐resolved images of the special distribution of this low molecular weight (MW) drug in tissue. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparison of anti‐inflammatory activities of ruscogenin,a major steroidal sapogenin from Radix Ophiopogon japonicus,and Its succinylated derivative,RUS‐2HS

Ruscogenin (RUS), first isolated from Ruscus aculeatus, is also a major steroidal sapogenin of the traditional Chinese herb Radix Ophiopogon japonicus. It has robust anti‐inflammatory activities. In previous studies, a ruscogenin affinity column, derived from succinylated ruscogenin (RUS‐2HS), was used to purify an antibody of ruscogenin. A ruscogenin affinity column can also be used to explore its protein targets. However, until now there have been no related pharmacological reports about ruscogenin derivatives. Whether the activity groups of ruscogenin have been blocked during the derivation process remains unknown. The present study was performed to compare the anti‐inflammatory activities in vitro of RUS‐2HS and ruscogenin. Both compounds reduced tumor necrosis factor‐α (TNF‐α)‐induced adhesion of human pro‐myelocytic leukemia cells (HL‐60) to endothelial ECV304 cells with IC₅₀ values of 6.90 nM and 7.45 nM, respectively. They were also inhibited overexpression of ICAM‐1 in ECV304 cells at the mRNA level as evaluated by real‐time PCR and at the protein level evaluated by flow cytometry with similar potency. Such data demonstrate that the functional groups of ruscogenin were not blocked by derivation, suggesting further use of the ruscogenin affinity column for target investigation. Meanwhile, RUS‐2HS was found to have remarkable anti‐inflammatory activity for the first time, indicating it would be a new lead compound with improved bioavailability. Drug Dev Res 69: 196–202, 2008. © 2008 Wiley‐Liss, Inc.