OBJECTIVES: To evaluate the effects on the formation of benzidine-hemoglobin, and benzidine metabolite-hemoglobin adducts, caused by pretreatment with the known xenobiotic metabolism effectors, ethanol and phenobarbital, in rats administered Direct Black 38 dye. METHODS: The experimental rats were divided into three groups: a control group, an ethanol group and a phenobarbital group. Rats were pretreated with ethanol (1g/kg) or phenobarbital (80mg/kg) 24 hours prior to the oral administration of Direct Black 38 (0.5mmol/kg), with the control group being administered the same amount of distilled water. Blood samples were obtained from the vena cava of 5 rats from each group prior to, and at 30 min, 3 h, 6 h, 9 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 144 h following the oral administration of Direct Black 38. Directly after sampling the blood was separated into hemoglobin and plasma, with the adducts being converted into aromatic amines by basic hydrolysis. Hydrolyzed benzidiene, monoacetylbenzidine and 4-aminobiphenyl were analyzed by reverse-phase liquid chromatography with an electrochemical detector. The quantitative amount of the metabolites was expressed by the hemoglobin binding index (HBI). RESULTS: In the ethanol group, benzidine-, monoacetylben-zidine-, and 4-aminobiphenyl-HBI were increased to a greater extent than those in the control group. These results were attributed to the ethanol inducing N-hydroxylation, which is related to the formation of the hemoglobin adduct. In the phenobarbital group, all the HBIs, with the exception of the benzidine-HBI, were increased to a greater extent than those of the control group. These results were attributed to the phenobarbital inducing N-hydroxylation related to the formation of the hemoglobin adduct. The N-acetylation ratio was only increased with the phenobarbital pretreatment due to the lower benzidine-HBI of the phenobarbital group compared to those of the control and ethanol groups. The N-acetylation ratios for all groups were higher than 1 for the duration of the experimental period. Although the azo reduction was unaffected by the ethanol, it was inhibited by the phenobarbital. The ratio of the benzidine-HBI in the phenobarbital group was lower than those of the ethanol the control groups for the entire experiment. CONCLUSION: Our results indicate that both ethanol and phenobarbital increase the formation of adducts by the induction of N-hydroxylation, but also induced N-acetylation. Phenobarbital decreased the formation of benzidine-HBI due to the decrease of the azo reduction. These results suggest that the effects of ethanol and phenobarbital need to be considered in the biochemical monitoring of Direct Black 38.
Administration, Oral ; Amines ; Animals ; Chromatography, Reverse-Phase ; Ethanol* ; Hydrolysis ; Metabolism ; Phenobarbital* ; Plasma ; Rats* ; Water

OBJECTIVES: The effects of ethanol and phenobarbital,which are known to affect metabolism of xenobiotics, on the formation of benzidine-and its metabolites-plasma protein adducts in rats administered benzidine were evaluated. METHODS: The experimental rats were divided into the control,ethanol and phenobar-bital groups. The experimental groups (ethanol and phenobarbital group)were pretreated with ethanol (1g/kg)or phenobarbital (80mg/kg)24 hours prior to the oral administration of benzidine (0.5mmol/kg). Blood samples were obtained from the vena cava from 5 rats in each group; and at 30 min,3 h,6 h,9 h,12 h,24 h,48 h,72 h,96 h,and 144 h after the administration of benzidine using heparin treated syringes.The plasma protein levels were separated immediately after taking blood samples. The adducts were underwent basic hydrolysis to convert them into aromatic amines. The hydrolyzed benzidine, monoacetylbenzidine, and 4-aminobiphenyl were analyzed by reverse-phased liquid chro-matography with an electrochemical detector. The quantitative amount of the metabolites was expressed by the plasma protein binding index(PBI). RESULTS: Similar to the hemoglobin adducts,the levels of the plasma protein adducts of the ethanol and phenobarbital groups (benzidine-, monoacetylbenzidine-, and 4-amino-biphenyl-PBI)were higher than those of the control group. These results are attributable to the fact that ethanol and phenobarbital induced to the plasma protein adduct formation. The N-acetylation ratio in the control group was highest at 72 h with 2.34.In the ethanol group,it was highest at 72 h with a ratio of 2.46 and was highest in the phenobarbital group at 72 h with a ratio of 2.43. The N-acetylation ratio of the plasma protein adducts was relatively lower than that of the hemoglobin adducts.The level of the plasma protein adduct increased more rapidly than the hemoglobin adducts in all experimental groups regardless of the pretreatment,and decreased rapidly after reaching the maximum level. CONCLUSION: The above results indicate that ethanol and phenobarbital increased the level of plasma protein adduct formation. The plasma protein adducts tended to decrease more rapidly than the hemoglobin adducts in the body after benzidine exposure. This results in this study result suggests that the effects of ethanol or phenobarbital need to be considered in the biochemical monitoring,and that the level of the plasma protein adducts be a more proper biomarker than the hemoglobin adducts for assessing the short term exposure to a benzidine and benzidine based dye.
Administration, Oral ; Amines ; Animals ; Environmental Monitoring* ; Ethanol* ; Heparin ; Hydrolysis ; Metabolism ; Phenobarbital* ; Plasma* ; Protein Binding ; Rats* ; Xenobiotics

Trace amines are endogenous molecules distributed in the central nervous system and peripheral tissues that resemble common biogenic amines in terms of subcellular localization, chemical structure, and metabolism. Trace amine-associated receptor (TAAR) is a kind of evolutionarily conserved G-protein-coupled receptors in vertebrates, in which TAAR1 is a functional regulator of monoamine transmitters such as dopamine and serotonin. TAAR1 is widely considered as a potential therapeutic target for schizophrenia, depression and drug addiction. Moreover, TAAR1 is also expressed in peripheral tissues. The homeostasis imbalance of trace aminergic system can induce over-activation of peripheral immune system and central immune inflammatory response. TAAR1 modulators are becoming potential emerging drugs for the treatment of immune-related illnesses, because they may play a major role in the activation or modulation of immune response.
Animals ; Humans ; Receptors, G-Protein-Coupled/metabolism* ; Biogenic Amines ; Dopamine ; Substance-Related Disorders

ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t_1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T¹⁰₅₀) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.
Transaminases/chemistry* ; Molecular Docking Simulation ; Amines/chemistry* ; Pyruvic Acid/metabolism* ; Enzyme Stability

AIMTo investigate factors affecting the properties of antisense oligodeoxy nucleotides (ASON)-liposomes complex and their cellular uptake.

METHODSThree types of blank liposomes were prepared by reverse-phase evaporation vesicles, and the complex were obtained through physical absorption. The light microscope was used to observe morphology characteristics of the complex. Drug loading capacity was analyzed by agarose gel electrophoresis. The transfected cell percentage and means fluorescence intensity were determined by flow cytometric analysis using M3 myeloma cell as a model.

RESULTSThe neutral liposome showed no aggregation while the cationic liposomes appeared some different extent aggregation in different medium when associated antisense oligodeoxynucleotides. The drug loading capacity depended on the ratio of +/- and the cationic charge density on the lipid membrane. The two kinds of cationic liposomes appeared different principles of loading ASON. As far as cellular uptake, The neutral liposomes showed no improvement of cellular uptake of ASON. However, the cationic liposomes were shown to enhance the cellular uptake of ASON if the appropriate +/- charge ratio was used. The optimal cellular uptake was achieved when +/- charge ratio was at 0.5:1 and 1:1 for SA-I liposome and SA-II liposomes, respectively.

CONCLUSIONThe cationic liposomes improved the loading capacity and cell uptake of antisense oligodeoxynucleotides, which was determined by +/- charge ratio and charge density.

Amines ; metabolism ; Biological Transport ; Drug Carriers ; Drug Delivery Systems ; Humans ; Liposomes ; pharmacokinetics ; Multiple Myeloma ; Oligodeoxyribonucleotides, Antisense ; administration & dosage ; pharmacokinetics ; Random Allocation ; Tumor Cells, Cultured ; metabolism

N-Acyl-4-phenylthiazole-2-amines were designed and synthesized, moreover their effects on acetylcholinesterase activities were tested. N-Acyl-4-phenylthiazole-2-amines were prepared from substituted 2-bromo-1-acetophenones by three steps reaction, and their AChE inhibitory activities were measured by Ellman method in vitro. The results showed that the target compounds had a certain inhibitory activity on AChE in vitro. Among them, 8c was the best, and IC50 of 8c was 0.51 micromol x L(-1), better than that of rivastigmine and Huperzine-A. The inhibitory activities of N-acyl-4-phenylthiazole-2-amines on acetylcholinesterase are worth while to be further studied.
Acetylcholinesterase ; metabolism ; Alkaloids ; pharmacology ; Amines ; chemical synthesis ; pharmacology ; Cholinesterase Inhibitors ; chemical synthesis ; pharmacology ; Drug Design ; Rivastigmine ; pharmacology ; Sesquiterpenes ; pharmacology ; Structure-Activity Relationship ; Thiazoles ; pharmacology

A series of novel N-acyl-thiochromenothiazol-2-amine derivatives were designed and synthesized, furthermore, their inhibition effect on acetylcholinesterase was investigated. N-Acyl-thiochromenothiazol-2-amines were prepared from thiophenol by Hantzsch reaction, acylation reaction and substitution reaction. Moreover, their bioactivities as AChE inhibitors in vitro were measured with Ellman spectrophotometry. The results showed that most of them had a certain inhibition activity on AChE, and the compound 10a was the best in them. The IC50 of 10a to AChE is 7.92 μmol x L(-1), and the value is better than that of rivastigmine. N-Acyl-thiochromenothiazol-2-amine derivatives showed a certain bioactivity in vitro, which were worth further investigation.
Acetylcholinesterase ; metabolism ; Amines ; chemical synthesis ; pharmacology ; Benzopyrans ; chemical synthesis ; pharmacology ; Cholinesterase Inhibitors ; chemical synthesis ; pharmacology ; Rivastigmine ; Structure-Activity Relationship ; Thiazoles ; chemical synthesis ; pharmacology

BACKGROUNDGabapentin has been widely and successfully used in the clinic for many neuropathic pain syndromes since last decade, however its analgesic mechanisms are still elusive. Our study was to investigate whether Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) contributes to the analgesic effect of gabapentin on a chronic constriction injury (CCI) model.

METHODSGabapentin (2%, 100 mg/kg) or saline (0.5 ml/100 g) was injected intraperitoneally 15 minutes prior to surgery and then every 12 hours from postoperative day 0 - 4 to all rats in control, sham and CCI groups. The analgesic effect of gabapentin was assessed by measuring mechanical allodynia and thermal hyperalgesia of rats. Expression and activation of CaMKII were quantified by reverse-transcriptional polymerase chain reaction and Western blotting.

RESULTSThe analgesic effect of gabapentin on mechanical allodynia and thermal hyperalgesia was significant in the CCI model, with maximal reduction reached on postoperative day 8. Gabapentin decreased the expression of the total CaMKII and phosphorylated CaMKII in CCI rats.

CONCLUSIONThe analgesic effect of gabapentin on CCI rats may be related to the decreased expression and phosphorylation of CaMKII in the spinal cord.

Amines ; therapeutic use ; Analgesics ; therapeutic use ; Animals ; Blotting, Western ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Cyclohexanecarboxylic Acids ; therapeutic use ; Male ; Neuralgia ; drug therapy ; metabolism ; Rats ; Rats, Sprague-Dawley ; gamma-Aminobutyric Acid ; therapeutic use

We examined the possible anti-inflammatory mechanisms of gabapentin in the attenuation of neuropathic pain and the interaction between the anti-allodynic effects of gabapentin and interleukin-10 (IL-10) expression in a rat model of neuropathic pain. The anti-allodynic effect of intrathecal gabapentin was examined over a 7-day period. The anti-allodynic effects of IL-10 was measured, and the effects of anti-IL-10 antibody on the gabapentin were assessed. On day 7, the concentrations of pro-inflammatory cytokines and IL-10 were measured. Gabapentin produced an anti-allodynic effect over the 7-day period, reducing the expression of pro-inflammatory cytokines but increasing the expression of IL-10 (TNF-alpha, 316.0 +/- 69.7 pg/mL vs 88.8 +/- 24.4 pg/mL; IL-1beta, 1,212.9 +/- 104.5 vs 577.4 +/- 97.1 pg/mL; IL-6, 254.0 +/- 64.8 pg/mL vs 125.5 +/- 44.1 pg/mL; IL-10, 532.1 +/- 78.7 pg/mL vs 918.9 +/- 63.1 pg/mL). The suppressive effect of gabapentin on pro-inflammatory cytokine expression was partially blocked by the anti-IL-10 antibody. Expression of pro-inflammatory cytokines was significantly attenuated by daily injections of IL-10. The anti-allodynic effects of gabapentin may be caused by upregulation of IL-10 expression in the spinal cord, which leads to inhibition of the expression of pro-inflammatory cytokines in the spinal cords.
Amines/pharmacology/*therapeutic use ; Analgesics/pharmacology/*therapeutic use ; Animals ; Antibodies/immunology/pharmacology ; Behavior, Animal/drug effects ; Cyclohexanecarboxylic Acids/pharmacology/*therapeutic use ; Cytokines/*metabolism ; Disease Models, Animal ; Injections, Spinal ; Interleukin-10/genetics/immunology/*metabolism ; Male ; Neuralgia/*drug therapy/metabolism/pathology ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins/biosynthesis/genetics/pharmacology ; Spinal Cord/metabolism ; Up-Regulation ; gamma-Aminobutyric Acid/pharmacology/*therapeutic use

Spinal gabapentin and adenosine have been known to display an antinociceptive effect. We evaluated the nature of the interaction between gabapentin and adenosine in formalin-induced nociception at the spinal level. Male Sprague-Dawley rats were prepared for intrathecal catheterization. Pain was evoked by injection of formalin solution (5%, 50 microliter) into the hindpaw. After examination of the effects of gabapentin and adenosine, the resulting interaction was investigated with isobolographic and fractional analyses. Neither gabapentin nor adenosine affected motor function. Gabapentin or adenosine decreased the sum of the number of flinches during phase 2, but not during phase 1 in the formalin test. Isobolographic analysis, in phase 2, revealed an additive interaction between gabapentin and adenosine. Taken together, intrathecal gabapentin and adenosine attenuated the facilitated state and interacted additively with each other.
*Adenosine/administration & dosage/metabolism/therapeutic use ; *Amines/administration & dosage/metabolism/therapeutic use ; *Analgesics/administration & dosage/metabolism/therapeutic use ; Animals ; *Cyclohexanecarboxylic Acids/administration & Dose-Response Relationship, Drug ; Formaldehyde/*toxicity ; Injections, Spinal ; Male ; Motor Activity/physiology ; Pain Measurement ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; *gamma-Aminobutyric Acid/administration & dosage/metabolism/therapeutic