大鼠肝纤维化精密切片技术的建立

目的：建立肝纤维化精密切片技术，摸索最佳体外培养条件，用于体外肝脏异物代谢和药物相互作用研究。方法：建立大鼠复合因素(高脂、酒精和CCl4)肝纤维化模型，制备肝纤维化状态下的肝切片，建立培养系统，以培养基中乳酸脱氢酶(LDH)漏出量、肝组织中谷胱甘肽S-转移酶(GST)活性、肝细胞噻唑蓝(MTT)还原能力为指标，观察切片厚度、培养液pH值和培养时间对肝切片活力的影响。结果：大鼠经复合因素处理后，3周即可造成肝纤维化早期病理改变。在切片厚度300μm、培养液pH7．0的条件下，肝切片在6h内能够维持最低的LDH漏出量和最高的GST活性、MTT还原量。结论：肝纤维化状态下的精密肝切片技术，其切片厚度300μm、培养液pH7．0、培养时间6h时为最佳培养条件。     

Utilization of ionotropic gelation technique for bioavailability enhancement of cinnarizine: in-vitro optimization and in-vivo performance in human

Gastro retentive drug delivery system techniques were adopted to deliver drugs having narrow absorption window from a particular site in the GIT. Therefore, gastro retentive dosage forms were retained in the stomach, thus improving absorption and bioavailability would be improved consequently. In this study, cinnarizine (CNZ) was employed as the model drug. CNZ is a poorly soluble basic drug, suffering from low and erratic bioavailability. This is attributed to its pH-dependant solubility (highly soluble at pH?<?4). CNZ is characterized by short half-life (3–6?h). Accordingly, floating CNZ emulsion gel calcium pectinate beads were developed. A mixture design was employed to study the effect of the percent of LM pectin (A), the percent of GMO (B) and the percent of Labrafac Lipophile (C) simultaneously on the percent of drug released and loaded. The optimized floating CNZ emulsion gel calcium pectinate beads and Stugeron® (the marketed reference product) were compared through a pharmacokinetic study carried on healthy human volunteers. Fortunately, simple floating CNZ emulsion gel calcium pectinate beads were prepared with zero-order release profile for 12?h. A promising in-vivo CNZ controlled release dosage form with higher bioavailability, when compared to once daily administration of Stugeron® tablets was achieved.     

The role of positron emission tomography in neuropharmacology in the living human brain and drug development]

Neuroimaging is a powerful and innovative tool for studying the pathology of psychiatric and neurological diseases and, more recently, for studying the drugs used in their treatment. Technological advances in imaging have made it possible to noninvasively extract information from the human brain regarding a drug's mechanism and site of action. Until now, our understanding of human brain pharmacology has depended primarily on indirect assessments or models derived from animal studies. However, the advent of multiple techniques for human brain imaging allows researchers to focus directly on human pharmacology and brain function. In this review article, our PET studies on the histaminergic neuron system were presented as an example. We have developed and used the PET techniques for 10 years in order to examine the H1 receptors in the living human brain. This review outlines available PET techniques and examines how these various methods have already been applied to the drug development process and neuropharmacology in the living human brain.     

Refinements in the Exposure Assessment Process

Abstract

Some of the most significant advances and refinements in human health risk assessments for environmental and occupational chemicals have come from investigations in the area of exposure assessment. This presentation summary describes three previously published examples of continuing advances in this process.     

Synthesis and neuropharmacology of cyclobutanecarbonylureas

Ten urea derivatives of cyclobutanecarboxylic acid were synthesized and examined for general CNS depressant properties, barbiturate potentiation, myorelaxant, antitremorine and anticonvulsant potencies. Water solubility seems to play an important part in the activity of these compounds. However, lipid solubility also plays a part as activity determinant. 1-Cyclo-butanecarbonyl-3-ethylthiourea appears to be the most active CNS depressant, whereas the parent compound, cyclobutanecarbonylurea, is the most active barbiturate potentiator. Cyclobutanecarbonylurea, 1-cyclobutanecarbonyl-3-n-butylurea and 1-cyclobutanecarbonyl-3-(2,4-xylyl)urea appear to be the most active myorelaxants, while 1-cyclobutanecarbonyl-3-n-butylurea and 1-cyclobutanecarbonyl-3-(1-adamantyl)urea are the most active against pentylenetetrazole-induced convulsions. Cyclobutanecarbonylurea, 1-cyclobutane-carbonyl-3-n-butylurea, 1-cyclobutancarbonyl-3-cyanoacetylurea, 1-cyclobuta-necarbonyl-3-(1-adamantyl)urea and 1-cyclobutanecarbonyl-3-(2,4-xylyl)urea are also slightly active oxotremorine antagonists. None of the compounds possess significant analgesic activity.     

Distribution of lacosamide in the rat brain assessed by in vitro slice technique

Lacosamide is a newer anticonvulsant and is the only one that enhances the slow inactivation of voltage gated sodium channels. It is also claimed to have disease-modifying potential, but its pharmacokinetic properties have been much less discussed in the literature. In rats, lacosamide shows restricted distribution to tissues, and the brain-to-plasma partition coefficient (K_p) is only 0.553. In this study, the brain disposition of lacosamide was evaluated in rat brains, and its neuropharmacokinetic parameters (i.e., protein binding and intracellular accumulation) were assessed using in vitro methods. Brain slice experiments and brain homogenate binding studies were performed for several drugs acting on the central nervous system, and drugs were assayed by using a liquid chromatography-mass spectrometry system. By applying a combined approach, it was found that (1) the unbound volume of distribution in the brain for lacosamide (V_u,brain = 1.37) was lower than that of other classical anticonvulsants; (2) the unbound fraction of lacosamide in the brain (0.899) was slightly lower than its unbound fraction in plasma (0.96); (3) the unbound intracellular-to-extracellular concentration ratio of lacosamide was 1.233, meaning that lacosamide was accumulated in the intracellular space because of its physicochemical properties and zwitterionic structure; and (4) the unbound brain-to-plasma concentration ratio of lacosamide was lower than the total brain-to-plasma concentration ratio (K_p,uu,brain = 0.42 vs. K_p = 0.553). In conclusion, the limited brain distribution of lacosamide is not related to its nonspecific protein-binding capacity; rather, an active transport mechanism across the blood–brain barrier may be involved, which reduces the anticonvulsant and/or antiepileptogenic actions of this drug.     

Proteomic approaches in brain research and neuropharmacology

Numerous applications of genomic technologies have enabled the assembly of unprecedented inventories of genes, expressed in cells under specific physiological and pathophysiological conditions. Complementing the valuable information generated through functional genomics with the integrative knowledge of protein expression and function should enable the development of more efficient diagnostic tools and therapeutic agents. Proteomic analyses are particularly suitable to elucidate posttranslational modifications, expression levels and protein–protein interactions of thousands of proteins at a time. In this review, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) investigations of brain tissues in neurodegenerative diseases such as Alzheimer's disease, Down syndrome and schizophrenia, and the construction of 2D-PAGE proteome maps of the brain are discussed. The role of the Human Proteome Organization (HUPO) as an international coordinating organization for proteomic efforts, as well as challenges for proteomic technologies and data analysis are also addressed. It is expected that the use of proteomic strategies will have significant impact in neuropharmacology over the coming decade.     

Functional genomics in neuropsychiatric disorders and in neuropharmacology

The rapidly accumulating amount of information concerning gene and protein expression patterns produced by functional genomics, proteomics and bioinformatics is presently providing new targets for drug development. Furthermore, the analysis of gene expression in cells and tissues affected by a disease may reveal the underlying metabolic pathways and cellular processes affected. Finally, changes in gene expression may be used in either diagnostics or the monitoring of drug responses. This review focuses on advances in the use of functional genomics in neurological and neuropsychiatric diseases and neuropsychopharmacology. Although the number of published studies in this field is still limited, it already appears that this strategy may become a fruitful means in the analysis of the aetiology of neuropsychiatric disorders and the search for novel neuropharmacological drugs.     

Drug dependence: neuropharmacology and management

This overview has attempted to highlight the brain regions associated with reward, and the pathways and neurotransmitters responsible for communication between these regions. Work conducted in this field has shown that stimulants and opioids, despite interactions with different receptor types and different neurotransmitter reuptake transporters, appear to share a common action on brain reward pathways. Their effects on these pathways (the distinct brain regions making up the mesocorticolimbic dopaminergic system) are predominantly mediated through changes in dopamine neurotransmission, and compounds aimed at selectively modulating these effects may form the basis of drugs to treat addiction. Other transmitters such as GABA, acetylcholine and serotonin inevitably have a role to play in reward, although at present the exact nature of their effects remains unclear. Diverging from manipulating the CNS directly as a management strategy for dependence, it might be possible to exploit the immune system to prevent administered psychostimulants penetrating the brain, but antibody saturation and specificity are problematic.     

Toxicity and neuropharmacology of cyclopiazonic acid

Cyclopiazonic acid (CPA) was found to have many pharmacological properties in common with the antipsychotic drugs chlorpromazine and reserpine. Thus, in mice CPA at ip doses of 5-14 mg/kg body weight produced hypokinesia, hypothermia, catalepsy, ptosis, sedation without loss of righting reflex, tremor, gait disturbance, dyspnoea, opisthotonus, atypical convulsion and prolonged barbiturate-induced sleep. The ip LD50 of CPA was found to be 13 +/- 0.05 mg/kg. The tremors induced by near-lethal doses of CPA were associated with voluntary or forced movements (action tremors); they worsened during the days following treatment, but they were weak compared with the exhausting and continuous tremors of the whole body caused by 20 mg tremorine/kg (used for comparison). When death occurred only 24-259 min after administration of CPA (11-14 mg/kg), it was preceded by dypsnoea, cyanosis, opisthotonus and clonic leg movements and tonic extension of hind legs (convulsions). When death was delayed (2-6 days after CPA administration), it was preceded by prostration, ptosis, hypothermia, tremor and cessation of food and water intake resulting in cachexia; convulsions were not seen in this group of mice. CPA did not affect the rate of convulsion or death caused by either maximal electroshock or metrazol administration but it did delay the onset of metrazol-induced seizures. In rabbits, 10 mg CPA/kg body weight initially produced tachycardia, tachypnoea and sedation with an activated electroencephalogram. Of three rabbits given 10 mg CPA/kg one died, and in this rabbit slow delta waves were seen just before and during a brief period with clonic leg movements. In this animal death was accompanied by tonic extension of the hind legs, respiratory arrest and cardiac fibrillation; and epileptiform EEG was not seen at any time. The unexpected EEG activation with sedation in rabbits treated with CPA was similar to the effect of reserpine on EEG.     

The neuropharmacology of serotonin and noradrenaline in depression

Several classes of antidepressant drug exist, divided into three broad families, the monoamine reuptake inhibitors, the monoamine oxidase inhibitors and the monoamine receptor antagonists. All these drugs have a common pharmacological effect, to raise the synaptic concentrations of noradrenaline and serotonin. Although different drugs have different relative selectivity for noradrenaline and serotonin systems, these two neurotransmitter pathways work in parallel and in a coherent manner to produce the same final antidepressant response. The lag-time in the onset of action of antidepressants can be explained by the activation of inhibitory autoreceptors on serotonergic and noradrenergic neurones which initially attenuate the effects of antidepressants on synaptic transmitter levels. Over time, these autoreceptors desensitize, allowing the emergence of an overt antidepressant response. This theory has led to the proposition that antagonists at these autoreceptors such as pindolol may be useful adjuncts to antidepressant treatment, in order to hasten the appearance of a clinical response. Evidence for the clinical validity of this idea remains equivocal, however. The use of central monoamine depletion studies has demonstrated that it is elevated synaptic monoamine levels themselves, rather than some downstream postsynaptic changes in, for example, receptor sensitivity, that are responsible for the therapeutic effect of antidepressant drugs. Taken together, the data collected over the last 40 years have allowed the emergence of a unified monoamine hypothesis of antidepressant drug action.     

Techniques: applications of the nerve-bouton preparation in neuropharmacology

Single mammalian neurons can be isolated with adherent functional synaptic terminals using an enzyme-free, mechanical dissociation procedure. This allows investigations of the effects of presynaptic modulators of synaptic transmission with unprecedented ease and accuracy. Furthermore, single presynaptic terminals and boutons can be visualized using fluorescent markers and can also be focally stimulated with electrical pulses. In this article, the isolated-nerve-adherent-synaptic-bouton preparation and some examples of its general properties and uses are discussed.     

Some aspects of the neuropharmacology of phenylurea

The neuropharmacology of phenylurea was studied. The compound is a potent sedative hypnotic agent with a fairly good margin of safety. It produced significant motor incoordination and behavioral changes in sub-sedative doses, thus suggesting that it acts on the areas of the brain which control motor coordination (neurological deficit) and spontaneous motor movements (awareness of environment). The activity pattern of phenylurea as measured on the rotarod treadmill was identical with that of an ataractic drug, meprobamate, and different from that of the sedative drug, 2-ethylcrotonylurea (ectylurea). It exhibited significant meprobamate-like antistrychnine (myorelaxant) activity, and at high doses only, anticonvulsant activity (pentetrazole antagonism). Phenylurea also protected mice against tremorine-induced tremors. A sub-sedative dose of phenylurea significantly prolonged pentobarbital sleeping time.     

Resistance to glutathione depletion in diabetic and non-diabetic human erythrocytes in-vitro

We have investigated the resistance of erythrocytes from diabetics and non-diabetics to glutathione depletion caused by p-benzoquinone, 1-chloro-2,4-dinitrobenzene (CDNB), diethyl maleate and 4-aminophenol. Incubation of erythrocytes with 4-aminophenol (2 mM) caused a precipitous reduction (>80%) in cellular glutathione levels although there was no significant difference between 4-aminophenol-mediated glutathione depletion in the diabetic and non-diabetic cells. p-Benzoquinone and CDNB were both associated with a less severe initial reduction in glutathione levels (>50% at 30 min) although p-benzoquinone caused greater depletion (P < 0.001) at 4.5 h (21.1 +/- 3.1%, non-diabetic; 20.0 +/- 1.0%, diabetic) compared with CDNB (49.2 +/- 2.2%, non-diabetic; 51.3 +/- 1.1% diabetic). Although there was no significant difference between the two types of cell in terms of level of depletion, administration of diethyl maleate caused a significant reduction in glutathione levels at 30 min (P < 0.0005), 3.5 h (P < 0.05) and 4.5 h (P < 0.05) in erythrocytes from diabetic man compared with those from non-diabetic man. Co-administration of buthionine sulphoximine (20 mM) and 4-aminophenol (1 mM) also led to a significant reduction in glutathione levels in diabetic cells at 30 min (P < 0.05), 3.5 h (P < 0.02) and 4.5 h (P < 0.007) compared with those in non-diabetic cells. The observations that diabetic red cells' resistance to depletion was similar to that of nondiabetic cells for three of the four depletors, and that the combination of 4-aminophenol and buthionine sulphoximine-mediated inhibition of glutathione synthesis was required to illustrate differences suggests that diabetic complications might be a result of the long-term effect of small deficiencies in oxidative self-defence mechanisms such as glutathione.     

On the neuropharmacology of Harmane and other β-carbolines

β-Carbolines have been recently proposed as candidates for the unknown endogenous ligand of the benzodiazepine receptor. Out of the β-carboline derivatives already found in the mammalian CNS, harmane is clearly the most potent inhibitor of benzodiazepine receptor binding. Therefore, it has been considered as possible endogenous ligand for this new receptor system. However, a certain degree of specificity might be a basic condition to accept the hypothesis of harmane as the endogenous ligand. Thus, the effects of harmane as well as other β-carbolines on several neurotransmitter receptor binding systems in vitro and on some neuropharmacological tests in vivo were investigated. Harmane developed the highest affinity towards the benzodiazepine binding site among all systems investigated. Its IC₅₀-values for inhibiting opiate and muscarinic cholinergic receptor binding were about four times lower than those for dopamine and serotonin receptor binding but were about four times higher than that found for the benzodiazepine receptor binding. Norharmane exerted a remarkable displacing activity only at the benzodiazepine binding site. Harmine affected mainly the opiate and cholinergic muscarinic system, whereas tetrahydronorharmane turned out to be a potent inhibitor of serotonin and dopamine receptor binding. Doses of harmane needed to produce convulsions as indication of its possible benzodiazepine receptor agonistic properties are also sufficient to diminish nociception and decrease body temperature whereas the apomorphine-induced licking rate was affected at higher doses. The data demonstrate that harmane affects not only the benzodiazepine binding site but also other neuronal mechanisms. Furthermore, only minor changes of the β-carboline structure lead to substantially different effects. Therefore, the search for other β-carbolines with higher affinity for the benzodiazepine binding site as harmane seems to be promising.     

Establishment of rat precision-cut fibrotic liver slice technique and its application in verapamil metabolism

1. Liver fibrosis is the compensatory state of cirrhosis. In the long asymptomatic period, it is imperative to select a proper dosing regimen for drugs that are applicable to hepatic fibrosis. Otherwise, progressive deterioration to uncompensated cirrhosis may occur. The present study explored the characteristics of drug metabolism in fibrotic liver. 2. A rat precision-cut fibrotic liver slice (PCFLS) technique was established and the metabolism of verapamil was studied employing this technique. A rat hepatic fibrosis model was successfully induced integrating complex factors that included a high-fat diet, alcohol and CCl4. The PCFLS were incubated under different conditions and lactate dehydrogenase leakage, glutathione S-transferase activity and 3[4,5-dimethythiazole-2-yl]-2,5-diphenyltetrazolium bromide reduction were used as indices to assess PCFLS viability. Activities of phase I and phase II metabolizing enzymes were monitored following treatment with cytochrome P450 (CYP) inducers. Normal and fibrotic liver slices were incubated individually with 10 micromol/L verapamil. The concentration of verapamil in the medium was determined by high-performance liquid chromatography and intrinsic clearance (Cl(int)) was calculated on the basis of the concentration-time curve. 3. The results showed that the PCFLS viability remained steady throughout the 6 h of culture when the thickness of slices was 300 microm and pH of the medium was 7.0; CYP inducers (phenobarbital and ethanol) enhanced CYP2E1, CYP3A1/2 and uridine diphosphate-glucuronate transferase (UDPGT) activities, respectively, in a time-dependent manner. The Cl(int) (microL/min per mg) values differed significantly between normal (9.7 +/- 1.8) and fibrotic (5.6 +/- 1.4) liver slices (P < 0.01). 4. These results suggested that the PCFLS could remain viable for 2-6 h under appropriate conditions. The stability and inducibility of drug-metabolizing enzymes of PCFLS were also demonstrated. Furthermore, the metabolic rate of verapamil in PCFLS was decreased. These findings add further support to the use of PCFLS as a tool to study drug metabolism and to guide clinical medication.     

Venous Catheter Implantation in the Minipig Surgical Approaches and Refinements

Abstract: In spite of requiring invasive implantation procedures, chronic venous catheters offer a number of benefits in biomedical research involving pig:

• – avoidance of long term or repeated stress due to restraint for venepuncture
• – assured delivery of compounds into venous blood
• – sampling is always from the same site
• – use of large central veins ensures representative samples, in large quantities
• – central venous sites allow rapid dilution or bufferng of compounds whcich may potentially irritant
• – facilitate long term and infusion studies

Common sites of access to the venous system are jugular vein or its tributaries and the femoral vein. Experience has shown the jugular approach to the cranial vena cava to give superior performance. The presentation will focus on this approach by surgical exposure of the vessel with reference to the potential use of percutaneous introducer sheaths and Seldinger technique which offer reduced invasiveness of catheter insertion. Catheters may be either exteriorised through the skin or attached to a totally implanted vascular access port (VAP). The management of the skin-implant interface with materials such a Dacron velour which favour adherence to the animal's own tissue, obliterating space in which infection may arise and providing anchorage is advocated. Fully implantable pumps for infusion studies may also be used. Catheter related sepsis (CRS) is the chief and most significant complication of chronic vascular access in any species, in the pig, CRS is particularly resistant to therapy in comparison with other species. Methods for prevention of catheter related will be outlined.     

无损光声层析成像技术及其在神经药理学中的应用前景

光声层析成像(photoacoustic tomography,PAT)是一种新兴的生物医学成像技术,它整合了组织的光学特性和超声探测技术,提供生物组织的光学吸收信息,具有灵敏度高、对比性强、成像深度较深的特点。该文综述了PAT技术及其在神经药理学研究中的应用及发展前景。PAT技术为无创研究神经系统疾病,尤其是脑血管病形态学改变,提供了强有力的神经影像学研究工具,并为其他脑疾病的研究和神经系统新药研发提供新的思路。