Synthesis and Anticholinesterase Activity Evaluation of Asymmetric Azines

Pharmaceutical Chemistry Journal - Azines or di-imines are compounds containing R1R2C=N-N=CR3R4 fragment in its structure. These compounds have received special attention due to their importance as...     

Synthesis and Anticholinesterase Activity of N-Oxyalkyl-substituted Pyridinium and Hydroquinolinium Salts and Their Isologs

N-oxyalkylpiperidines (perhydroquinolines), N-oxyalkylpyridinium (hydroquinolinium) salts, and oxazolo(oxazino)hydroquinolines obtained from 1,5-diketones and the products of their O-heterocyclization exhibit moderate anticholinesterase activity in comparison to that of galanthamine. The maximum inhibiting effect was observed for N-oxyalkylpyridinium salts. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 39, No. 9, pp. 23 – 25, September, 2005.     

Structure Activity Relationship of Human Microsomal Epoxide Hydrolase Inhibition by Amide and Acid Analogues of Valproic Acid

Purpose. The purpose of this study was to evaluate the in vitro inhibitory potency of various amide analogues and derivatives of valproicacid toward human microsomal epoxide hydrolase (mEH). Methods. mEH inhibition was evaluated in human liver microsomeswith 25 (S)-(+)-styrene oxide as the substrate. Inhibitory potencyexpressed as the median inhibitory concentration (IC₅₀) was calculatedfrom the formation rate of the enzymatic product,(S)-(+)-1-phenyl-1,2-ethanediol. Results. Inhibitory potency was directly correlated with lipophilicityand became significant for amides with a minimum of eight carbonatoms. Branched eight-carbon amides were more potent inhibitors thantheir straight chain isomer, octanamide. N-substituted valproylamideanalogues had reduced or abolished inhibition potency with theexception of valproyl hydroxamic acid being a potent inhibitor. Inhibitionpotency was not stereoselective in two cases of chiral valpromideisomers. Valproyl glycinamide, a new antiepileptic drug currentlyundergoing phase II clinical trials and its major metabolite valproylglycine were weak mEH inhibitors. Acid isomers of valproic acid werenot potent mEH inhibitors. Conclusions. The structural requirements for valproylamide analoguesfor potent in vitro mEH inhibition are: an unsubstituted amide moiety;two saturated alkyl side chains; a minimum of eight carbons in themolecule.     

Purification of Human Serum Paraoxonase and Effect of Acetylsalicylic Acid on Paraoxonase Activity

Abstract

Paraoxonase was purified from human serum using sepharose-4B-l-tyrosine-1-napthylamine affinity chromatography. This enzyme was purified 1797-fold. SDS-polyacrylamide electrophoresis of the enzyme indicates a single protein staining band with an apparent Mr of 43 kDa. The kinetic properties of the purified enzyme were determined. The V._max and K_m are 231.5 μ/mg protein (EU) and 3.96 mM using substrate paraoxon respectively. The effect of acetylsalicylic acid on purified human serum paraoxonase has been investigated in vitro.. The inhibition and activation effects of acetylsalicylic acid on serum paraoxonase activity were measured spectrophotometrically using paraoxon as the substrate. The phenotypic distribution of paraoxonase was determined using the dual substrate paraoxon and phenyl acetate as substrates. It was observed that acetylsalicylic acid inhibited human serum paraoxonase activity. In addition, an in vivo. study was performed for acetylsalicylic acid in Sprague-Dawley rats. It was demonstrated that paraoxonase in the serum of Sprague-Dawley rat was activated, but activity in liver and heart was significantly inhibited.     

Recombinant Human Serum Albumin Dimer has High Blood Circulation Activity and Low Vascular Permeability in Comparison with Native Human Serum Albumin

Purpose Human serum albumin (HSA) is used clinically as an important plasma expander. Albumin infusion is not recommended for critically ill patients with hypovolemia, burns, or hypoalbuminemia because of the increased leakage of albumin into the extravascular spaces, thereby worsening edema. In the present study, we attempted to overcome this problem by producing a recombinant HSA (rHSA) dimer with decreased vascular permeability and an increased half-life. Methods Two molecules of rHSA were genetically fused to produce a recombinant albumin dimer molecule. The pharmacokinetics and biodistribution of the recombinant proteins were evaluated in normal rats and carrageenin-induced paw edema mouse model. Results The conformational properties of this rHSA dimer were similar to those for the native HSA (the HSA monomer), as evidenced by the Western blot and spectroscopic studies. The biological half-life and area under the plasma concentration–time curve of the rHSA dimer were approximately 1.5 times greater than those of the monomer. Dimerization has also caused a significant decrease in the total body clearance and distribution volume at the steady state of the native HSA. rHSA dimer accumulated to a lesser extent in the liver, skin, muscle, and fat, as compared with the native HSA. Up to 96 h, the vascular permeability of the rHSA dimer was less than that of the native HSA in paw edema mouse models. A prolonged plasma half-life of the rHSA dimer was also observed in the edema model rats. Conclusions rHSA dimer has a high retention rate in circulating blood and a lower vascular permeability than that of the native HSA.     

Modulation of lnterleukin-2-Driven Proliferation of Human Large Granular Lymphocytes by Carbaryl, an Anticholinesterase Insecticide

Modulation of Interieukin-2-Driven Proliferation of Human LargeGranular Lymphocytes by Carbaryl, an Anticholinesterase Insecticide.Bavari, S., Casale, G. P., Gold, R. E., and Vitzthum, E. F.(1991). Fundam. Appl. Toxicol. 17,61-74. Studies in other laboratorieshave provided evidence that the interleukin-2 (IL2) signalingpathway in lymphocytes includes essential, serine proteases.Since the anticholinesterase (antiCHE) insecticides are potentserine hydrolase (esterase and protease) inhibitors, we assessedthe ability of carbaryl (CA, a widely used antiCHE insecticide)and a-naphthol (NA, a major metabolite of carbaryl) to modulateIL2-driven proliferation of large granular lymphocytes (LGL)purified from human peripheral blood. These cells express nearlyall of the natural killer (NK.) activity of human peripheralWood. NK cells are normal lymphocytes that respond to IL2 byproliferating and increasing their tumoricidal activity on aper cell basis. Cultures of purified LGL, initiated in the presenceof human recombinant IL2, were harvested on culture Day 4, thenproliferation was measured as [³H]thymidine incorporation. Whenadded only at the time cultures were initiated, CA inhibitedincorporation 10-32%, 35-53%, and 54-57% at 0.5, 5.0, and 50mm, respectively. In contrast, NA had no effect at 0.5 and 5.0mm, but was inhibitory (16-17%) at 50 mm. Reexposure to CA orNA, during the incorporation assay, had little effect on theobserved inhibition profiles. Chemically induced changes incell number during an 8-day culture period reflected the chemicallyinduced changes in [³H]thymidine incorporation. Neither CA norNA produced cell death. Quantitation of both CA and NA by HPLCindicated a rapid loss of CA (ca. 95% in 24 hr) and a much slowerloss of NA from the culture medium. CA inhibited human LGL proliferationat concentrations producing little or no inhibition of serumCHE, an indicator of exposure to antiCHE insecticides.     

Measurement of Microcystin Activity in Human Plasma Using Immunocapture and Protein Phosphatase Inhibition Assay

Microcystins are toxic chemicals generated by certain freshwater cyanobacteria. These chemicals can accumulate to dangerous levels during harmful algal blooms. When exposed to microcystins, humans are at risk of hepatic injury, including liver failure. Here, we describe a method to detect microcystins in human plasma by using immunocapture followed by a protein phosphatase inhibition assay. At least 279 microcystins have been identified, and most of these compounds share a common amino acid, the Adda side chain. We targeted this Adda side chain using a commercial antibody and extracted microcystins from human samples for screening and analysis. To quantitate the extracted microcystins, we fortified plasma with microcystin-LR, one of the most well-studied, commonly detected, and toxic microcystin congeners. The quantitation range for the detection of microcystin in human plasma using this method is 0.030–0.50 ng/mL microcystin-LR equivalents. This method detects unconjugated and conjugated forms (cysteine and glutathione) of microcystins. Quality control sample accuracies varied between 98.9% and 114%, with a precision of 7.18–15.8%. Finally, we evaluated plasma samples from a community health surveillance project of Florida residents living or working near harmful algae blooms.     

Activity of Doxorubicin Covalently Bound to a Novel Human Serum Albumin Microcapsule

Doxorubicin is widely used in the treatment of human malignancies, however is associated with significant cardiac, bone marrow and gastro-intestinal toxicity. Delivery systems may ameliorate this toxicity and increase treatment specificity by increasing the proportion of drug delivered to sites of disease. We have developed a novel preparation of doxorubicin (Dox) covalently linked to a heat stabilised human serum albumin microparticle (HSAM) carrier (median particle diameter of 4 m) and assessed its activity in 4 malignant cell lines.Materials and methods. Doxorubicin microcapsules were compared with free doxorubicin in the rat carcinoma cell line, WRC256, and the human lines, OVCAR3, MCF7 and the Dox resistant MCF7/Dox, using a cell counting technique. IC50 were calculated from regression analysis of the resulting survival curves. Endocytosis of the microcapsules by cells in culture was observed. The rate of microcapsule uptake was assessed using dual wavelength filtered fluorescence microscopy and flow cytometry.Results. The mean IC50 following incubation with the Dox microcapsules was around 5 times greater than Dox for WRC256 (p < 0.001), MCF7 (p < 0.01) and for OVCAR3 (p < 0.01). MCF7/Dox was significantly more sensitive to Dox microcapsules than free Dox (p = 0.034). A negative correlation between the rate of microcapsule uptake and the IC50 values for each cell line in culture exists (r = –0.96, p = 0.04).Conclusions. We conclude that: 1) Doxorubicin microcapsules retain activity in vitro and appear to overcome p-glycoprotein mediated Dox resistance. 2) The observed activity of Dox microcapsules correlates with the rate of particle uptake. Further studies in animal tumour models are in progress.     

Effect of Human Serum on Killing Activity of Vancomycin and Teicoplanin against Staphylococcus aureus

Study Objective . To investigate the effects of pooled human serum (PHS) on the killing activity of vancomycin and teicoplanin against two isolates of Staphylococcus aureus from patients treated for endocarditis. Design . An in vitro assessment of antibiotic susceptibility and killing rates. Setting . An urban university teaching hospital. Patients . Pooled human serum from patients treated for endocarditis. Interventions . Two clinical isolates of Staphylococcus aureus were obtained from patients treated for endocarditis. Media consisted of cation-supplemented Mueller-Hinton broth alone and in 1:1 dilutions with PHS, 2-hour heat-inactivated PHS (HI-PHS), ultrafiltrate (UF), and 2-hour heat-inactivated ultrafiltrate (HI-UF). Heat inactivation of PHS and UF was accomplished by treatment at 56°C for 2 hours. Measurements and Main Results . Killing curves with vancomycin and teicoplanin were performed using drug concentrations of 45 μg/ml and a starting inoculum of ∼1 × 10⁶ colony-forming units (cfu)/ml. Bactericidal rates (-log cfu/ml/hr) were calculated from the slope of the killing curves over 0–12 hours (mean 3–8 replicates). Conclusions . The killing activity of vancomycin in PHS and HI-PHS against both isolates was significantly greater than all other media tested (p<0.0001). Ultrafiltrate tended to reverse this enhancement effect. Addition of PHS or UF did not enhance teicoplanin's killing activity against either isolate. Further investigations in our laboratory will determine if the factor is antibiotic class or organism specific.     

Inhibition of Human Plasma and Serum Butyrylcholinesterase (EC 3.1.1.8) by {alpha}-Chaconine and {alpha}-Solanine

The purpose of these experiments was to determine the reversibilityof -chaconine and -solanine inhibition of human plasma butyrylcholinesterase(BuChE). For the substrate -naphthylacetate, optimal assay conditionswere 0.50 M sodium phosphate buffer and a substrate concentrationof 3–5 ' 10^–4 M. Dibucaine (1 ' 10^–5 M) indicatedthe usual phenotype for all subjects; -chaconine and -solanineat 2.88 ' 10^–6 M inhibited BuChE about 70 and 50%, respectively.One-and 24-hr incubations at 1 ' 10^–1 M with -chaconine,-solanine, paraoxon, eserine, and ethanol yielded reversibleinhibition with dilution except for paraoxon. Twenty-four-hourdialyses of incubations showed no inhibition except for paraoxon.PAGE enzyme activity gels of 1-and 24-hr incubations also showedno inhibition except for paraoxon. -Chaconine and -solanineare reversible inhibitors of human butyrylcholinesterase. Atestimated tissue levels, -chaconine, -solanine, and solanidineinhibited BuChE 10–86%. In assays which combined -chaconine,-solanine, and solanidine, inhibition of BuChE was less thanadditive. No inhibition of albumin -naphthylacetate esterase(an arylesterase) was noted with any inhibitor. The importanceof these data to adverse toxicological effects of potato alkaloidsis discussed.     

Inhibition of Acetylcholinesterase Activity by some Isoquinoline Alkaloids*,**.

The effects of quaternary protoberberine and benzophenanthridine alkaloids on acetylcholinesterase (ACHE, EC 3.1.1.7) activity IN VITRO and on isolated rat duodenum and guinea pig ileum tissue preparations have been studied. The ACHE inhibitory activity of quaternary alkaloids depends on the cationpseudobase equilibrium of the studied alkaloids in solution. The relationship between pK (R)+ values and the biological activity is described. The influence of surfactants on pseudobase formation of benzo-phenanthridine alkaloids has been discussed.     

鸡卵清蛋白对蛋白酶抑制作用的研究

以新鲜鸡蛋清为材料,采用三氯醋酸-丙酮提取,冷丙酮沉淀,碳酸盐透析和DEAE-Cel-lulose离子交换层析分离出鸡卵清蛋白。研究了该蛋白质对蛋白酶活性的影响,结果表明鸡卵清蛋白对胰蛋白酶有强烈抑制作用并且抑制程度与温度、pH值等密切相关,能部分仰制枯草杆菌蛋白酶活性,不抑制胰凝乳蛋白酶。     

HPLC法测定人血清中奈达铂浓度

目的:建立高效液相色谱法测定奈达铂的血清浓度。方法:采用色谱柱为Spherisorb NH2柱(4.0 mm×300 mm,10μm);流动相:40 mmol·L-1磷酸二氢钾-乙腈(8∶2);流速:0.8 mL·min-1;检测波长:210 nm。结果:奈达铂浓度在5～50μg·mL-1范围内呈良好的线性关系(r=0.9995),奈达铂浓度为5、20、40μg·mL-1时日内和日间平均回收率结果分别为:96.7%、97.2%、97.8%和98.4%、98.2%、99.6%,日内、日间RSD分别为2.09%、1.98%、3.19%和2.27%、2.13%、1.92%(n=5)。结论:该方法具有样品处理简单、稳定性高等特点,适用于奈达铂的药动学研究和治疗药物监测。     

Influence of Intoxication by Anticholinesterase Agents on Core Temperature in Rats: Relationships between Hypothermia and Acetylcholinesterase Inhibition in Different Brain Areas

Abstract: The early and late effects of three anticholinesterase agents (physostigmine, paraoxon and soman) on core temperature and brain acetylcholinesterase (AcChE) inhibition are compared. The study was performed in adult male rats using sublethal doses of all drugs. AcChE activity was determined in hypothalamus, striatum, hippocampus, medulla oblongata-pons and rest of the brain. The co-existence of intoxication symptoms, hypothermia and AcChE inhibition was clearly shown by the early effects. However, no relationship was found between the degree of hypothermia and that of inhibition whatever brain area. In contrast, late AcChE inhibition was not accompanied by symptoms and lowering of core temperature. Several hypotheses have been suggested to explain this phenomenon: de novo synthesis of enzyme, decreased sensitivity of neurotransmitter or return to normal of brain acetylcholine level through a negative feed back at the presynaptic level. The present data suggest that this latter assumption is the most likely.     

HPLC法测定人血清中甲氨蝶呤的浓度

目的:建立以高效液相色谱法测定人血清中甲氨蝶呤浓度的方法。方法:色谱柱为WatersC18,流动相为乙腈-pH7.2的磷酸盐缓冲液(12∶88),柱温为30℃,流速为1.0mL.min-1,检测波长为303nm。结果:甲氨蝶呤血药浓度在0.5～80μg.mL-1范围内与峰面积线性关系良好(r=0.9994);方法回收率均>90%,日内、日间RSD分别为3.03%～3.52%、2.57%～4.05%。结论:本方法简便、快速、准确、灵敏,适用于甲氨蝶呤体内血药浓度测定和药动学研究。     

HPLC测定人血清中胺碘酮浓度

采用高效液相色谱法测定人血清中胺碘酮的浓度 ,色谱柱为ShimpackC1 8柱 (1 5 0mm× 6mm ,5 μm)。流动相为甲醇 -水 -二乙胺 (75 0∶2 5 0∶5 ) (醋酸调pH值 5 5 )。流速为 1ml·min- 1 ,检测波长为 2 42nm。测得胺碘酮的平均回收率为 1 0 2 3 % ,RSD为 6 7% ,方法快速 ,简便     

HPLC法测定人血清中百草枯含量

摘 要 目的：建立HPLC法测定人血清中百草枯含量。方法: 色谱柱：Kromasil C18柱（200 mm×4.6 mm,5 μm）,流动相：乙腈 水（含0.03 mol·L-1庚烷磺酸钠,0.24 mol·L-1磷酸）=3〖KG*9〗∶〖KG-*2〗97（用三乙胺调pH至2.0）;检测波长258 nm;柱温25℃;进样体积20 μl;流速0.8 ml·min-1。结果: 百草枯在0.106~10.6 mg·L-1浓度范围内线性关系良好( r=0.999 3),最低检出浓度为0.065 mg·L-1。高、中、低3种浓度样品绝对回收率＞89.4%,方法回收率＞94.4%,日内精密度RSD在0.12%～1.74%之间,日间精密度RSD在0.44%～2.89%之间。结论: 该方法操作简便易行、灵敏度高、专属性强,适用于百草枯的人体血清浓度测定。     

HPLC测定人血清中多索茶碱的浓度

目的建立高效液相色谱测定多索茶碱血药浓度的方法。方法用乙醚提取血样,以卡马西平为内标进行检测。色谱柱采用Waters C18（3．9mm×150mm,5μm）,流动相为甲醇-水（35：65）,流速为0．6mL·min^-1,检测波长为273nm,柱温为30℃。结果多索茶碱血清浓度在0．7～28btg·mL^-1内线性关系良好,线性方程为Y=0．3549X＋0,0039（r=0．9997）方法回收率为100．4％,日内、日间RSD分别为1．10％～3．87％和2．11％～4．86％（n＝5）。结论该方法快速、简便、准确、安全,可用于临床多索荼碱血药浓度的测定。