Microdialysis: An Alternative for in Vitro and in Vivo Protein Binding Studies

The aim of the present study was to compare the performance of conventional equilibrium dialysis method with a microdialysis method in studying drug protein binding. The two methods were assessed by comparing the measured mean unbound drug fraction in different plasma species in vitro in plasma of four different species and at two concentrations of the non-indolic melatonin analog S 20098. For the microdialysis study, the unbound drug fraction was calculated after correction for membrane recovery. Plasma protein binding of S 20098 ranged from 75 to 95%. In humans, rabbits and rats (10 ng/ml), equal unbound percentages were found between equilibrium dialysis and microdialysis. Microdialysis gave slightly but significantly higher values in rat (2000 ng/ml), and in monkey plasma independent of the drug concentration. Microdialysis was also performed in vivo in freely moving rats under steady-state conditions, yielding similar unbound fraction values (26.0 ± 0.9%) to those obtained using microdialysis probes in rat plasma in vitro (24.4 ± 1.6%). These results support the use of in vivo microdialysis in pharmacokinetic studies in freely moving animals.     

Synthesis and Receptor Binding Studies of (±)l-Iodo-MK-801

The glutamate analogue N-methyl-D-aspartate (NMDA) binds to a subset of glutamate receptors that are coupled to a voltage-sensitive cation channel. This NMDA-linked channel is the likely binding locus of the potent anticonvulsant MK-801. To develop single-photon emission computed tomography (SPECT) probes of this brain channel, we synthesized (±)l-iodo-MK-801 and (±)l-[¹²⁵I]iodo-MK-801. The effect of (±)l-iodo-MK-801 on ligand binding to the NMDA-linked glutamate receptor site was assessed using a rat brain homogenate assay. (±)l-Iodo-MK-801 displaced the dissociative anesthetic ligand [³H]N-[l-(2-thienyl)cyclohexyl]piperidine ([³H]TCP) binding with an IC₅₀ of 1 µM, which is a 10-fold lower binding affinity than that of (±)MK-801. In in vivo autoradiographic studies, (±)MK-801 failed to block selective uptake of (±)l-iodo-MK-801 in rat brain. These results suggest that (±)l-iodo-MK-801 may not be a suitable ligand for mapping NMDA-linked glutamate receptor channels.     

Sedative-Hypnotic and Receptor Binding Studies of Fermented Marine Organisms

This study was performed to investigate the sedative-hypnotic activity of γ-aminobutyric acid (GABA)-enriched fermented marine organisms (FMO), including sea tangle (FST) and oyster (FO) by Lactobacillus brevis BJ20 (L. brevis BJ20). FST and FO were tested for their binding activity of the GABA_A-benzodiazepine and 5-HT_2C receptors, which are well-known molecular targets for sleep aids. We also measured the sleep latency and sleep duration during pentobarbital-induced sleep in mice after oral administration of FST and FO. In GABA_A and 5-HT_2C receptor binding assays, FST displayed an effective concentration-dependent binding affinity to GABA_A receptor, similar to the binding affinity to 5-HT_2C receptor. FO exhibited higher affinity to 5-HT_2C receptor, compared with the GABA_A receptor. The oral administration of FST and FO produced a dose-dependent decrease in sleep latency and increase in sleep duration in pentobarbital-induced hypnosis. The data demonstrate that FST and FO possess sedative-hypnotic activity possibly by modulating GABA_A and 5-HT_2C receptors. We propose that FST and FO might be effective agents for treatment of insomnia.     

肺靶向海藻酸钙微球体内外溶蚀行为的探讨

采用乳化法制备肺靶向海藻酸钙微球,制品平均粒径(12.2±8.9)μm,在注射用水中的溶胀率约150%,在生理盐水中24h累积溶蚀率约60%.小鼠尾静脉注射微球后肺靶向效果显著,5min时肺组织中充斥微球,6h时可见微球溶蚀.     

依托泊苷白蛋白微球小鼠体内分布及药代动力学

目的 :制备依托泊苷白蛋白微球 ,并对其动物体内分布及药代动力学进行研究。方法 :用高效液相色谱法测定各组织中依托泊苷浓度。结果 :小鼠尾静脉注射微球后15min ,肺内依托泊苷量占注入总量的(47.88±2.56) %,微球小鼠肺组织内药代动力学方程为 :C=149.0897e -1.7780t +3.9627e -0.0398t—153.0524e -3.5054t。结论 :依托泊苷白蛋白微球具有明显的肺靶向效果 ,小鼠肺组织中药代动力学规律可以二室模型拟合。     

Receptor Occupancy in Myocardium,Adrenal Cortex,and Brain by TH-142177, a Novel AT1 Receptor Antagonist in Rats,in Relation to Its Plasma Concentration and Hypotensive Effect

Purpose. To study the relationship between angiotensin II (All) receptor occupancy ex vivo in tissues plasma concentration and hypotensive effect of a novel All receptor antagonist, TH-142177 and losartan in rats. Methods. At 2, 8 and 24 hr after oral administration of TH-142177 and losartan in rats, All receptors in myocardium, adrenal cortex and cerebral cortex were determined by radioligand binding assay using [¹²⁵I]Sar¹,Ile⁸-AII. Plasma concentrations of both drugs and metabolite in rats were also measured using validated HPLC assays. Further, systolic blood pressure (SBP) in conscious renal hypertensive rats treated orally with TH-142177 and losartan were measured by using a tail cuff plethysmographic method. Results. Oral administration of TH-142177 (1.8 and 5.5 mol/kg) and losartan (6.5 and 21.7 mol/kg) in rats brought about dose-dependent decreases in [¹²⁵I]Sar¹,Ile⁸-AII binding sites (Bmax) in myocardium and adrenal cortex. The extent of receptor occupancy by both drugs in adrenal cortex was maximal at 2 hr later but that in myocardium at 8 hr later. Further, the receptor occupancy was more sustained in myocardium than adrenal cortex. The ex vivo binding affinity of TH-142177 for All receptors in these tissues was roughly three times higher than that of losartan. Also, cerebral cortical [¹²⁵I]Sar¹,Ile⁸-AII binding was significantly reduced by oral administration of losartan but not by TH-142177. The time course of All receptor occupancy by both drugs in adrenal cortex appeared to be in parallel with that of their plasma concentrations, while the time course in myocardium correlated with that of their hypotensive effects rather than plasma concentrations. Conclusions. TH-142177 produced a relatively selective and sustained occupancy ex vivo of All receptors in myocardium and adrenal cortex of rats with approximately three times greater potency than losartan. Its time course of myocardial receptor occupancy was in parallel with that of hypotensive effect rather than plasma concentration.     

Synthesis and in Vitro Receptor Binding Studies of Fluorotamoxifen Analogues

We describe the synthesis of new fluorotamoxifen analogues with the fluorine atom positioned on the end of the aliphatic chain of tamoxifen. The binding of fluorotamoxifens to cytosol estrogen receptors of rat uteri was determined with [³H]estradiol (5 nM). The fluorotamoxifens had similar or superior binding affinities compared with tamoxifen. The IC₅₀ value was as follows: tamoxifen, 5 × 10^–7 M; fluorotamoxifen (VII), 5 × 10^–7 M; N,N-diethylfluorotamoxifen (IV)—cis, 1 × 10^–6 M, and trans, 2 × 10^–7 M; and (cis) fluoromethyl-N,N-diethyltamoxifen (VI) 1 × 10^–7 M. Therefore, the fluorinated tamoxifens have potential use in imaging estrogen receptors by PET.     

Biorecognition of HPMA Copolymer-Adriamycin Conjugates by Lymphocytes Mediated by Synthetic Receptor Binding Epitopes

Purpose. The EDPGFFNVE nonapeptide (NP) was recognized as the CD21 (CR2) binding epitope of the Epstein-Barr virus (EBV) gp350/ 220 envelope glycoprotein which mediates the virus attachment to human B lymphocytes (Nemerow et al., Cell 56:369-377, 1989). Here we evaluated the targeting potential of a synthetic receptor binding epitope (NP) covalently attached to a water-soluble polymeric drug carrier. In particular, the biorecognition of N-(2-hydroxypropyl) metha-crylamide (HPMA) copolymer-NP conjugates by B- and T-cells and the cytotoxicity of HPMA copolymer-NP-adriamycin (ADR) conjugates toward B-cells, T-cells, and peripheral blood lymphocytes (PBL) were evaluated. Methods. HPMA copolymer-NP and optionally ADR conjugates varying in the NP density and mode of NP attachment were incubated with Raji B-cells (human Burkitt's lymphoma), CCRF-CEM T-cells (acute human lymphoblastic leukemia), and CCRF-HSB-2 T-cells (human lymphoblastic leukemia). The kinetics of binding was studied, the Langmuir adsorption isotherms analyzed, binding constants calculated, and IC₅₀ doses determined. Results. Flow cytometry studies revealed that binding was homogeneous to both cell types. The apparent binding constants to T-cells were about two times higher when compared to B-cells. The binding and cytotoxicity increased with increased amount of epitopes per polymer chain. Attachment of the NP via a GFLG spacer resulted in increased biorecognition when compared with conjugates containing NP bound via a GG spacer. HPMA copolymer-NP-ADR conjugates possessed specific cytotoxicity to T- and B-malignant cells. Concentrations, which were lethal to the latter, were not toxic for PBL. Conclusions. The data obtained seem to indicate the potential of the HPMA copolymer-NP conjugates as polymer anticancer drug carriers targetable to immunocompetent cells.     

组胺H4受体在肺癌中减弱表达研究

目的研究肺癌组织中组胺H4受体（HH4R）的mRNA表达。方法应用RT-PCR方法,测定人类36例肺正常组织、51例肺癌患者癌旁组织、5l例肺癌组织的组胺H4受体基因mRNA的表达情况。结果人类肺组织富含组胺H4受体表达,HH4R表达为肺癌组织〈癌旁组织〈正常组织。结论在肺癌发展过程中,人类肺组织中组胺I-14受体mRNA表达明显减弱。     

乙醇和丙二醇单用与联用对曲安奈德喷雾溶液透皮性能的影响

目的 对曲安奈德喷雾溶液进行体外透皮试验,考察乙醇和丙二醇单用与联用时对曲安奈德喷雾溶液体外透皮功能的影响。方法 选取新西兰白兔腹部皮肤,用Franz扩散池法对曲安奈德喷雾溶液进行体外透皮试验,用高效液相色谱法（HPLC）测定曲安奈德含量,用单因素方差分析法对各组间的透皮吸收速率进行对比分析。结果 乙醇和丙二醇联用时的透皮吸收速率均显著高于单用时的透皮吸收速率（P<0.05）,且乙醇和丙二醇联用时对曲安奈德喷雾溶液的促透作用顺序为10%乙醇+25%丙二醇>10%乙醇+20%丙二醇>15%乙醇+25%丙二醇>15%乙醇+20%丙二醇。结论 10%乙醇和25%丙二醇联用时可使曲安奈德喷雾溶液的透皮功能达到最佳化。     

Paranoid Schizophrenia is Characterized by Increased CB1 Receptor Binding in the Dorsolateral Prefrontal Cortex

A number of studies suggest a dysregulation of the endogenous cannabinoid system in schizophrenia (SCZ). In the present study, we examined cannabinoid CB₁ receptor (CB₁R) binding and mRNA expression in the dorsolateral prefrontal cortex (DLPFC) (Brodmann''s area 46) of SCZ patients and controls, post-mortem. Receptor density was investigated using autoradiography with the CB₁R ligand [³H] CP 55 940 and CB₁R mRNA expression was measured using quantitative RT-PCR in a cohort of 16 patients with paranoid SCZ, 21 patients with non-paranoid SCZ and 37 controls matched for age, post-mortem interval and pH. All cases were obtained from the University of Sydney Tissue Resource Centre. Results were analyzed using one-way analysis of variance (ANOVA) and post hoc Bonferroni tests and with analysis of covariance (ANCOVA) to control for demographic factors that would potentially influence CB₁R expression. There was a main effect of diagnosis on [³H] CP 55 940 binding quantified across all layers of the DLPFC (F(2,71)=3.740, p=0.029). Post hoc tests indicated that this main effect was due to patients with paranoid SCZ having 22% higher levels of CB₁R binding compared with the control group. When ANCOVA was employed, this effect was strengthened (F(2,67)=6.048, p=0.004) with paranoid SCZ patients differing significantly from the control (p=0.004) and from the non-paranoid group (p=0.016). In contrast, no significant differences were observed in mRNA expression between the different disease subtypes and the control group. Our findings confirm the existence of a CB₁R dysregulation in SCZ and underline the need for further investigation of the role of this receptor particularly in those diagnosed with paranoid SCZ.     

ODN2088减轻线粒体DNA致脓毒症大鼠急性肺损伤

目的:探讨血浆线粒体DNA(mtDNA)含量与脓毒症大鼠急性肺损伤(ALI)程度的关系及选择性TLR9受体抑制剂ODN2088对脓毒症大鼠ALI的保护作用.方法:按随机数表法将80只大鼠分为假手术(sham)组、脓毒症(sepsis)组、脓毒症大鼠腹腔注射mtDNA 1.4 mg/kg(sepsis+mtDNA)组、脓...     

左旋咪唑与咪唑啉2受体的放射性受体配基实验观察

目的:观察左旋咪唑（LMS）在体外实验中能否与正常大鼠脑组织咪唑啉2受体（I₂R）结合;LMS在体内长期处理能否引起大鼠脑内I₂R的变化。方法:通过正常大鼠脑组织I₂R与放射性配基的竞争结合实验,测定其平衡解离常数（K_i）及50%的放射性配基结合被取代所需的竞争剂浓度(IC₅₀),观察LMS与I₂R在体外的结合能力。通过LMS长期体内处理大鼠脑组织I₂R饱和结合实验,测定LMS长期处理大鼠和对照组大鼠脑组织I₂R密度和亲和力的变化。结果:体外实验发现,LMS能与[³H]-2BFI竞争结合大鼠脑组织的I₂R,且具有浓度依赖性。IC₅₀值为1.292×10^-5mol·L^-1,K_i值为9.796×10^-6mol·L^-1。大鼠体内长期LMS处理,其脑组织I₂R密度明显上调（P<0.05）,而亲和力两组相比差异无统计学意义。结论:左旋咪唑在体外能与脑组织I₂R直接结合,可能是I₂R的配基。左旋咪唑长期作用于体内能引起脑组织I₂R密度的上调。     

Uptake and Stereoselective Binding of the Enantiomers of MK-927, a Potent Carbonic Anhydrase Inhibitor,by Human Erythrocytes in Vitro

MK-927 [5,6-dihydro-4H-4(isobutylamino)thieno(2,3-B)thiopyran-2-sulfonamide-7.7 dioxide], a potent carbonic anhydrase inhibitor, contains a chiral center and exists as a racemate. In order to understand the kinetic behavior of the enantiomers of MK-927 in the body, the uptake and binding of these compounds were studied in human erythrocytes in vitro. Since no degradation or metabolism of the enantiomers occurred during incubation in blood, one can describe the equilibration of the drugs between plasma and erythrocytes by a closed two-compartment system. Erythrocytes were considered as a compartment composed of two parts: one in which free drug is exchangeable to plasma and the other in which drug is tightly bound to carbonic anhydrase in a Michaelis–Menten type binding. After the addition of the enantiomers individually to fresh blood, they were taken up by erythrocytes rapidly in a concentration-dependent manner. The time to achieve equilibrium decreased as the concentration increased, suggesting saturation of binding sites. With the assumption of simple diffusion, the binding and transfer kinetics were determined simultaneously by computer fitting. There were no Stereoselective differences in the transfer process of the enantiomers across the erythrocyte membrane, while binding of the enantiomers exhibited stereoselectivity. The penetration of the unbound enantiomer across the erythrocyte cell membrane was rapid, with a mean transit time of about 3 sec. The S-( + )-enantiomer was bound to the high-affinity carbonic anhydrase isoenzyme more strongly than the R-( – )-enantiomer by approximately 10-fold. For the low-affinity isoenzyme, the R-( – )-enantiomer was bound more strongly than the S-( + )-enantiomer.     

Thermodynamic In Vitro Studies as a Method to Investigate the Pharmacodynamic Behavior of Adenosine A1 Receptor Ligands

Purpose. A thermodynamic analysis of the binding to rat cortex adenosine A₁, receptor of N⁶-substituted (full agonists) and N⁶-substituted-deoxyribose (partial agonists) adenosine derivatives was performed. The intrinsic activity of the compounds was evaluated by measurements of the inhibition of forskolin stimulated 3, 5-cyclic adenosine mono-phosphate (c-AMP) levels in isolated epididymal rat adipocytes. Methods. The thermodynamic parameters G° (standard free energy), H °(standard enthalpy), and S° (standard entropy) of the binding equilibrium were determined by means of affinity measurements carried out at different temperatures (0, 10, 20, 25, 30° C). Levels of c-AMP were evaluated performing competitive protein binding assays. Results. The binding of the ligands increases with temperature enhancement and, as a consequence, is totally entropy driven. Standard entropy values correlate significantly with intrinsic activity ones. Conclusions. It is proposed the data obtained by these in vitro experiments can be used to investigate the in vivo pharmacodynamic of A₁, full and partial agonists.     

Contribution of Monoamine Oxidase(MAO) to the Binding of Tertiary Basic Drugs in Isolated Perfused Rat Lung

The effect of tertiary basic drugs on mitochondrial MAO activity and the effect of MAO inhibitors (MAOIs) on basic drug accumulation in the isolated perfused rat lung were studied to clarify the role of MAO in drug binding to lung tissue. In the perfused lung preparation, the inhibition of MAO by basic drugs correlated well with their lipid solubilities and followed competitive kinetics. The inhibitory rank order (imipramine diphenhydramine > quinine > metoclopramide > procainamide) also correlated with their accumulation in the perfused lung. Moreover, MAOI treatment decreased the accumulation of basic drugs in the lung, and the potency of MAOIs to inhibit drug accumulation in the lung correlated with their MAO inhibitory activity. These results indicate that lung MAO has specific binding sites for basic drugs and may function as a drug reservoir.     

Evaluation of Drug Absorption After Intrapulmonary Administration Using Xenopus Pulmonary Membranes: Correlation with In Vivo Pulmonary Absorption Studies in Rats

Pharmaceutical Research -     

Contribution of Monoamine Oxidase (MAO) to the Binding of Tertiary Basic Drugs in Lung Mitochondria

The effects of tertiary amine-containing basic drugs on the enzymes located in the mitochondria and the effect of monoamine oxidase inhibitors (MAOIs) on drug accumulation in lung mitochondria have been studied. Various basic drugs inhibited MAO activity but not other mitochondrial marker enzymes. The potency of MAO inhibition correlated well with their lipid solubility, and the basic drugs inhibited MAO activity dose dependency and competitively. Further, MAO inhibition correlated well with binding affinity to lung mitochondria, and the binding of tertiary amine drugs to lung mitochondria was decreased by treatment with MAOIs. A good correlation was observed between the potency of MAOIs to inhibit the binding of the basic drug to the high-affinity site in mitochondria and the MAO inhibitory activity in mitochondria. These results indicate that mitochondrial MAO is one of the binding sites for tertiary basic drugs in the lung. We think that the action and/or adverse reaction of some drugs may result from inhibition of mitochondrial MAO to metabolize various biogenic amines and that mitochondrial MAO may function as a reservoir for basic drugs.     

前列腺素E1脂微球制剂对大鼠急性肺损伤的影响

目的:采用大鼠腹腔注射脂多糖(LPS)复制大鼠急性肺损伤(ALI)模型,评价前列腺素E1脂微球制剂(凯时)对急性肺损伤大鼠的保护作用。方法:雄性健康Sprague Dawley大鼠60只随机分为3组,A组:生理盐水组;B组:LPS模型组;C组:凯时治疗组。动物以硫喷妥纳腹腔注射麻醉,于实验3h分别测量每只大鼠的肺系数,肺通透指数,肺湿重/肺干重,肺组织中髓过氧化物酶(MPO)含量,丙二荃(MDA)和过氧化物歧化酶(SOD)的含量,血清细胞因子TNF-a、IL-12、IL10的浓度,肺组织中NF-KB蛋白的表达,并进行统计学分析。结果:①B组与A组比较,肺系数,肺通透指数,肺湿重/肺干重显著增加(P<0.05);血浆SOD含量,血清细胞因子IL10显著减少(P<0.05);肺组织中髓过氧化物(MPO)活性和丙二荃(MDA)含量,血清细胞因子TNF-a,IL-12显著增加(P<0.05)。②C组与B组比较,肺系数,肺通透指数,肺湿重/肺干重显著减少(P<0.05);肺组织SOD含量,血清细胞因子IL10显著升高(P<0.05);肺组织中髓过氧化物(MPO)活性和丙二荃(MDA)含量,血清细胞因子TNF-a,IL-12显著减少(P<0.05)。③C组与B组比较,NF-kB P65的表达明显低于A组(P<0.05)。结论:前列腺素E1脂微球制剂通过抑制NF-kB的表达,减少中性粒细胞的渗出,对LPS诱导的急性肺损伤大鼠有明显保护作用。     

血清血管内皮生长因子和表皮生长因子受体在非小细胞肺癌诊断中的价值

目的:探讨血清血管内皮生长因子(VEGF)和表皮生长因子受体(EGFR)测定在非小细胞肺癌诊断中的价值。方法:采用双抗体夹心ABC-ELISA法测定74例非小细胞肺癌患者及40例健康对照组血清VEGF和EGFR的含量。结果:非小细胞肺癌患者血清VEGF和EGFR测定值均高于健康对照组(P<0.01)。Ⅲ期和Ⅳ期非小细胞肺癌患者血清VEGF、EGFR高于Ⅱ期患者(P<0.01)。血清VEGF和EGFR测定值与非小细胞肺癌病理分型无关(P>0.05)。非小细胞肺癌患者血清VEGF与EGFR测定值之间呈正相关(rs=0.47,t=4.3,P<0.01)。结论:检测血清VEGF和EGFR水平对非小细胞肺癌的诊断和预后判定具有一定临床意义。