HPLC法测定非诺贝特胶囊中非诺贝特的含量

摘 要 目的：建立非诺贝特胶囊中非诺贝特的含量测定方法。方法: 采用HPLC法,色谱柱：Dikma Diamonsil C₁₈柱（150 mm×4.6 mm,5 μm）,流动相：乙腈-水（70∶30,用磷酸调节pH值至2.5）,检测波长：286 nm,流速：1.0 ml·min^-1,柱温：30℃,进样量：20 μl。 结果: 非诺贝特浓度在10.07～60.42 μg·mL^-1范围内线性关系良好（r=0.999 6）,平均回收率为99.26%,RSD为0.5%（n=6）。结论:本法简便、准确、专属性强,可用于该制剂的含量测定。     

HPLC测定非诺贝特缓释胶囊的有关物质

目的 建立非诺贝特缓释胶囊有关物质的HPLC测定方法。方法 采用Kromasil-100-5 C₁₈柱,流动相为乙腈-水（用磷酸调节pH至3.0）（67：33）;流速1.0 mL·min^-1;检测波长为286 nm;柱温为40℃;进样量为10 μL。结果 非诺贝特与其他杂质的分离度较好,非诺贝特的线性范围为0.54~3.23 μg·mL^-1（r=0.999 9）,检测限为0.03 μg·mL^-1。结论 建立的方法简便、准确、专属性强,可作为非诺贝特缓释胶囊有关物质的检测方法。     

Ameliorating Effects of Sulfonylurea Drugs on Insulin Resistance in Otsuka Long-Evans Tokushima Fatty Rats

OLETF (Otsuka Long-Evans Tokushima Fatty) rats are characterized by obesity-related insulin resistance, which is a phenotype of type 2 diabetes. Sulfonylurea drugs or benzoic acid derivatives as inhibitors of the ATP-sensitive potassium (K_ATP) channel are commercially available to treat diabetes. The present study compared sulfonylurea drugs (glimepiride and gliclazide) with one of benzoic acid derivatives (repaglinide) in regard to their long-term effect on ameliorating insulin sensitivity in OLETF rats. Each drug was dissolved and fed with drinking water from 29 weeks of age. On high glucose loading at 45 weeks of age, response of blood glucose recovery was the greatest in the group treated with glimepiride. On immunohistochemistry analysis for the Kir6.2 subunit of K_ATP channels, insulin receptor β-subunits, and glucose transporters (GLUT) type 2 and 4 in liver, fat and skeletal muscle tissues, the sulfonylurea drugs (glimepiride and gliclazide) were more effective than repaglinide in recovery from their decreased expressions in OLETF rats. From these results, it seems to be plausible that K_ATP-channel inhibitors containing sulfonylurea moiety may be much more effective in reducing insulin resistance than those with benzoic acid moiety. In contrast to gliclazide, non-tissue selectivity of glimepiride on K_ATP channel inhibition may further strengthen an amelioration of insulin sensitivity unless considering other side effects.     

HPLC测定清洁验证残留物非诺贝特的含量

目的：建立清洁验证中残留物非诺贝特含量测定的高效液相色谱法。方法：色谱柱为 Agilent ZORBAX SB-C18（150 mm×4.6 mm,5μm）,流动相为水（磷酸调节pH至2.5）-乙腈（30∶70）,检测波长：286 nm,流速：1.0 mL/min,柱温：25℃,进样量：20μL。结果：非诺贝特在0.09～0.90μg/mL范围内线性关系良好, r2=0.9994;回收率：99.69%,RSD =0.16%（n=9）。结论：该法操作简便、结果准确,可以用于清洁验证残留物非诺贝特的定量分析。     

HPLC法测定非诺贝特缓释片中非诺贝特

目的采用HPLC法测定非诺贝特缓释片中非诺贝特。方法采用Dikma C18色谱柱(200 mm×4.6 mm,5μm);流动相:甲醇–水(90∶10);检测波长:288 nm;柱温:25℃;体积流量:1.0 mL/min;进样量10μL。结果非诺贝特在2.0~12.0μg/mL与其峰面积呈良好的线性关系(r=0.999 9);检测限和定量限分别为10、30 ng/mL;平均回收率为99.86%,RSD值为0.72%(n=9)。结论该方法准确、简便,可用于非诺贝特缓释片中非诺贝特的质量控制。     

GC测定非诺贝特制剂中十二烷基硫酸钠含量

目的 建立非诺贝特制剂中十二烷基硫酸钠的GC测定方法。方法 采用HP-5（30 m×0.53 mm,2.65 μm）毛细管柱,以氮气为载气,氢火焰离子化检测器,程序升温,直接进样,以十二烷醇为对照品外标法测定十二烷基硫酸钠的含量。结果 十二烷醇在0.165~1.32 mg·mL^-1内具有良好的线性关系,相关系数为0.999 5。国内企业产品的十二烷基硫酸钠用量远低于安全用量,但部分国内企业可能存在过量添加和非法添加的问题。结论 本方法准确,专属性强,可作为非诺贝特制剂中十二烷基硫酸钠的检测方法。     

HPLC法测定血浆中非诺贝特活性代谢物非诺贝酸的浓度

目的:建立高效液相色谱法测定血浆中非诺贝特活性代谢产物非诺贝特酸浓度。方法:以甲醇直接沉淀血浆蛋白,色谱柱为Waters sunfire C_(18)柱(150mm×4.6mm,5μm),流动相为0.05 mol·L~(-1)磷酸二氢钾溶液-甲醇(70:30),用磷酸调pH 2.5,检测波长286nm。结果:非诺贝酸的保留时间约为6.7 min,线性范围为0.2～20.0μg·ml~(-1)(r=0.999 9),最低定量限为0.2μg·ml~(-1),方法回收率99.28%～101.38%,提取回收率97.18%～107.28%,日内和日间RSD均小于10%。结论:本法简便、快捷、灵敏,适用于非诺贝特药物动力学研究。     

HPLC测定大鼠血浆中非诺贝特的活性代谢物及其药动学研究

目的采用RP-HPLC测定大鼠血浆中非诺贝特活性代谢物非诺贝特酸的浓度,并研究单次口服不同剂量非诺贝特在大鼠体内的药动学。方法将18只SD大鼠随机分为3组,分别按体重口服低、中、高剂量(10、50、100 mg·kg-1)非诺贝特混悬液后,采用经过验证的RP-HPLC法测定血浆中药物的浓度,绘制血药浓度-时间曲线,以DAS 2.1.1软件拟合药动学参数。结果高、中、低剂量非诺贝特酸的药-时曲线均符合口服吸收的二室模型,主要药动学参数:Cmax分别为18.15±3.02、8.68±1.69、1.68±0.42μg·mL-1;Tmax分别为6.80±0.75、6.33±1.75、6.50±1.38 h;t1/2分别为4.44±1.03、5.38±1.91、4.76±1.12 h;Cl/F分别为0.49±0.08、0.56±0.13、0.50±0.11 L.h-1;AUC0-t分别为209.07±36.25、92.20±23.47、20.17±6.54μg·mL-1·h-1;AUC0-∞分别为210.22±36.29、93.87±23.51、21.27±6.53μg·mL-1·h-1。结论所用RP-HPLC法能够准确地测定非诺贝特酸的血药浓度,满足药动学研究的要求。在给药剂量范围内,非诺贝特酸在大鼠体内的药动学符合线性药动学规律。     

大鼠在体肠循环结合颈动脉取血研究非诺贝特纳米混悬剂的体内吸收

目的考察非诺贝特纳米混悬剂在大鼠小肠内的吸收动力学及药动学过程。方法采用大鼠在体肠道灌流结合颈动脉取血实验考察非诺贝特纳米混悬剂的体内吸收情况。结果非诺贝特纳米混悬剂在大鼠体内的血药质量浓度y与肠腔中药物累计减少量x的关系为:y=7×10-5 x+0.521 9,r=0.993 6。结论非诺贝特纳米混悬剂在体肠吸收较好,动物实验体内药动学特性符合一室模型。血药质量浓度和肠腔内药物减少量的相关性良好。     

Effects of losartan in combination with or without exercise on insulin resistance in Otsuka Long–Evans Tokushima Fatty rats

Hypertension often complicates type 2 diabetes mellitus, and angiotensin converting enzyme inhibitor treatment has been shown to improve insulin resistance in such cases. However, the effect of angiotensin II type-1 (AT₁) receptor antagonists on insulin resistance is still controversial. To gain further information on this effect, we examined the effect of losartan on insulin resistance in Otsuka Long–Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes mellitus. Losartan administration alone lowered systolic blood pressure, but did not improve oral glucose tolerance test or insulin resistance in OLETF rats. However, the administration of losartan with exercise significantly improved both systolic blood pressure and insulin resistance relative to control OLETF rats. On the other hand, losartan treatment, regardless of exercise, increased glucose uptake in excised soleus muscle and fat cells. To explore the beneficial effect of losartan on skeletal muscle glucose uptake, we examined intracellular signaling of soleus muscle. Although Akt activity and glucose transporter type 4 (GLUT4) expressions were not affected by losartan with or without exercise, extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein (MAP) kinase activities were increased by both interventions. These results indicate that angiotensin AT₁ receptor antagonist improved local insulin resistance, but not systemic insulin resistance. These findings may explain the controversy over the effect of angiotensin AT₁ receptor antagonists on insulin resistance in clinical use. The enhancing effect of angiotensin AT₁ receptor antagonist on skeletal muscle glucose uptake may be attributable to MAP kinase activation or other mechanisms rather than phosphatidylinositol 3-kinase activation.     

非诺贝特PEG2000-DSPE胶束的制备及在大鼠体内的口服药动学研究

目的:为提高非诺贝特溶解度,将非诺贝特包载于PEG₂₀₀₀-DSPE胶束中,研究其在SD大鼠体内的口服药动学情况。方法:对非诺贝特PEG₂₀₀₀-DSPE胶束进行表征,大鼠单剂量灌胃给予非诺贝特PEG₂₀₀₀-DSPE胶束和非诺贝特混悬液,眼底静脉丛取血,HPLC法测定血浆中非诺贝特酸含量,并用药物与统计（Drug and Statistics,DAS）软件分析处理药动学数据。结果:成功制备了非诺贝特PEG₂₀₀₀-DSPE胶束,平均粒径为（23.40±3.62）nm,包封率和载药量分别为（97.65±3.32）%和（1.33±0.32）%。大鼠体内口服药动学结果表明非诺贝特PEG₂₀₀₀-DSPE胶束和非诺贝特混悬液的药动学行为均符合二室模型,非诺贝特PEG20 00-DSPE胶束和非诺贝特混悬液的AUC_（0-24）分别为（61.41±5.71）μg·h·ml^-1和（8.49±0.66）μg·h·ml^-1,C_max分别为（9.67±1.65）μg·ml^-1和（0.71±0.09）μg·ml^-1。非诺贝特PEG₂₀₀₀-DSPE胶束的AUC_（0-24）和C_max相比于非诺贝特混悬液组分别提高了7倍和14倍。非诺贝特PEG₂₀₀₀-DSPE胶束相对于原料药生物利用度为723.3%。结论:非诺贝特PEG₂₀₀₀-DSPE胶束显著提高了非诺贝特在大鼠体内的口服吸收速度和生物利用度。PEG₂₀₀₀-DSPE胶束作为口服药物载体具有优良的应用前景。     

The anorectic action of naloxone is attenuated by adaptation to a food-deprivation schedule

Recent studies have shown that naloxone and other opiate antagonists can reduce the amounts of food and water consumed by laboratory animals, a finding consistent with a role for endogenous opioids in the control of appetite. Because there have also been some failures to observe an anorectic action of naloxone, a study was carried out in which the effects of the drug on food intake were investigated using two different experimental procedures. In naive rats deprived of food for 24 h, both naloxone (0.1, 1.0 and 10.0 mg/kg) and fenfluramine (1.0, 3.0 and 10.0 mg/kg) produced dose-related decreases in food and water intake. In rats which has been adapted to receiving food for only 6 h each day, fenfluramine produced a similar effect whereas naloxone had no effect on food intake and reduced water consumption only at the highest dose. A second experiment showed that the different actions of a 1.0 mg/kg dose of naloxone in the two procedures were not due to differences in the duration of the immediately preceding period of food deprivation or in the time during which the rats were handled. These results show that the anorectic action of naloxone can be attenuated by adaptation to a schedule of repeated food deprivation.     

Effects of a Brain-Enhanced Chemical Delivery System for Estradiol on Body Weight and Food Intake in Intact and Ovariectomized Rats

Studies were undertaken to determine the effects on body weight of a brain-enhanced chemical delivery system for estradiol. This estradiol-chemical delivery system (E₂-CDS) has a long half-life in the brain, where it slowly releases estradiol but is quickly cleared from peripheral tissues. We administered, by a single iv injection, E₂-CDS (0.2, 1.0, or 5.0 mg/kg), equimolar doses of another 17-hydroxy-substituted estrogen, estradiol valerate (E₂-VAL), or the dimethyl sulfoxide (DMSO) vehicle to female rats. Daily food intake and body weight was determined for 24 days thereafter. E₂-CDS caused an initial dose-dependent suppression in body weight for up to 8 days and a suppression in food intake for up to 4 days. In response to E₂-VAL, the initial declines in body weight and food intake were lower in magnitude, were shorter in duration, and showed no dose dependency. Following this period of weight loss, E₂-CDS-treated rats gained weight at a rate greater than that of the DMSO controls, and at the 0.2- and 1.0-mg/kg doses, body weights achieved were greater than control levels. To determine the role of the ovaries on this biphasic response to E₂-CDS, long-term ovariectomized rats were treated with E₂-CDS (1.0 mg/kg) or the vehicle and parameters of body weight regulation were determined for 25 days. Ovariectomized rats responded to E₂-CDS with a prompt and sustained decrease in body weight which did not recover over the 25-day course of the study. The body-weight loss in ovariectomized rats was associated with a marked reduction in food intake for 8 days. Finally, when intact female rats were administered the E₂-CDS on the day of diestrus I, rats exhibited cornified vaginal epithelial lavages for 3.5 days, during which weight loss was observed, followed by a 7.8-day period of pseudopregnancy during which animals rapidly gained weight. Collectively, these data indicate that delivery of E₂ to the brain with E₂-CDS causes a marked decline in body weight and food intake in female rats. The phase of increased body weight which follows this drug-induced weight loss appears to be ovarian dependent, since in ovariectomized rats this phase of response to the drug is not observed.     

Reduction of heroin intake in baboons by an economic constraint

Baboons earned their total food ration in a situation where they were periodically given an opportunity to choose between food and an intravenous infusion of heroin. As the number of daily choices was restricted, food intake remained relatively constant, while heroin intake decreased dramatically.     

利拉鲁肽对糖尿病前期大鼠胰岛海马胆碱能神经刺激肽前体蛋白的影响

目的明确利拉鲁肽对糖尿病前期OLETF大鼠的阻抑作用,观察对胰腺海马胆碱能神经刺激肽前体蛋白(hippocampal cholinergicneurostimulating peptide precursor protein,HCNP-pp)的影响。方法检测空腹及餐后2小时血糖,将处于糖耐量减低阶段12～14周龄OLETF大鼠随机分为3组,安慰剂组(PBO组)、100μg/kg利拉鲁肽处理组(L-100组)、200μg/kg利拉鲁肽处理组(L-200组),LETO大鼠为阴性对照组(LETO组)。12周后检测大鼠体重、空腹血糖、餐后2 h血糖和胰岛素,以免疫组织化学染色分析胰岛细胞形态学变化,应用蛋白印迹技术检测HCNP-pp及胰高血糖素样多肽-1受体(GLP-1R)蛋白含量;应用实时定量PCR检测HCNP-pp和LGP-1R、胆碱乙酰转移酶(ChAT)、3型毒蕈碱样受体(M3R)基因表达水平。结果PBO组OLETF大鼠空腹、餐后2 h血糖符合糖尿病诊断标准,而L-100组、L-200组及LETO组大鼠均未进展为糖尿病状态。PBO组OLETF大鼠体重及空腹胰岛素均明显高于L-100组、L-200组和LETO组(P<0.01)。与PBO组相比,L-200组、LETO组大鼠胰腺胰岛素阳性细胞表达密度明显增多(P<0.01),而与L-100组差别无统计学意义(P>0.05);L-100组、L-200组及LEITO组大鼠胰腺HCNP-pp蛋白相对含量明显减少(P<0.05或P<0.01);L-100组、L-200组及LEITO组大鼠胰腺GLP-1R蛋白相对含量明显增多(P<0.01);L-100组、L-200组和LETO组大鼠胰腺HCNP-pp mRNA水平明显下降(P<0.01);L-200组和LETO组大鼠胰GLP-1R,ChAT、M3R mRNA水平均明显增多(P<0.01);L-100组大鼠GLP-1R和ChAT mRNA水平均明显增多(P<0.01),而M3R mRNA水平则无变化(P>0.05)。结论利拉鲁肽可阻抑糖尿病前期进展,可能与胰腺HCNP-pp表达下降、GLP-1R表达增高及胰岛β细胞密度增加相关。利拉鲁肽改善胰岛素分泌状态,可能与胰腺HCNP增多、ChAT和M3R表达增高相关。     

Effects of ceruletide and cholecystokinin octapeptide on eating in mice

Summary Subcutaneous (SC) injections of ceruletide (caerulein diethylammonium hydrate, CER) and the octapeptide of cholecystokinin (CCK-8) reduced the intake of liquid food in male NMRI mice starved for 18 h. The corresponding ED₅₀ values were 2 g/kg for CER and 24 g/kg for CCK-8; hence, on a molar basis, CER was 14 times more potent than CCK-8. Naloxone (0.2 and 1 mg/kg, SC) inhibited eating. (d-Ala)² (MePhe)⁴-(Met(O)-ol)⁵-enkephalin (FK 33-824; 0.3 and 1 mg/kg) was only stimulatory. Naloxone enhanced the effect of CER, whereas FK 33-824 antagonized it. It is concluded that concerning the inhibition of food intake, opioid peptides can be antagonists of CCK-like peptides. This is consistent with the current view of the regulation of appetitive behaviour.     

The cannabinoid CB1 receptor antagonist SR141716A (Rimonabant) enhances the metabolic benefits of long-term treatment with oleoylethanolamide in Zucker rats

Anandamide and oleoylethanolamide (OEA) are lipid mediators that regulate feeding and lipid metabolism. While anandamide, a cannabinoid CB1 receptor agonist, promotes feeding and lipogenesis, oleoylethanolamide, an endogenous agonist of peroxisome proliferator activated receptor alpha (PPAR-alpha), decreases food intake and activates lipid mobilization and oxidation. The treatment with a cannabinoid CB1 receptor antagonist results in reduction of body weight gain and cholesterol in obese humans and rodents. In the present study, we show the benefits of the treatment of obese Zucker rats with a combination of a cannabinoid CB1 receptor antagonist (Rimonabant) and oleoylethanolamide. This combinational therapy improved the separate effects of Rimonabant and OEA, and resulted in marked decreases on feeding, body weight gain, and plasma cholesterol levels. Additionally, the treatment with both drugs reduced the hepatic steatosis observed in Zucker rats, decreasing liver fat deposits and damage, as revealed by the levels of alanine aminotransferase activity in serum. The combined treatment inhibits the expression of stearoyl coenzyme-A desaturase-1 (SCD-1), a pivotal enzyme in lipid biosynthesis and triglyceride mobilization that is linked to obesity phenotypes. These results support the use of combined therapies with cannabinoid CB1 receptor antagonists and PPAR-alpha agonists for the treatment of obesity associated with dyslipemia.     

Suppression of deprivation-induced food and water intake in rats and mice by naloxone

Naloxone, an opiate antagonist, was administered to male and female rats and male mice after periods of food or water deprivation ranging from 12 to 48 hr. Naloxone (0.01-10 mg/kg) reduced postdeprivational water intake in most groups of rats and mice in a dose-related manner. Naloxone suppression of water consumption appeared to be independent of sexual differences in rats, and phase of the diurnal cycle, and length of the deprivation interval in both rats and mice. Postdeprivational food intake in male rats and mice was also reduced by naloxone in a dose-dependent fashion. This naloxone effect was less pronounced than actions observed with water intake, and tended to diminish with lengthening food deprivation periods. In general, mice appeared to be less sensitive than rats to naloxone suppression of food and water intake. Naloxone appears to markedly reduce appetitive behavior, particularly water intake, following deprivation in both rats and mice. The fact that low doses of naloxone can elicit these effects suggests that the drug is acting at specific tissue sites, possibly endorphine recpetors.     

福辛普利联合非诺贝特对糖尿病小鼠视网膜病变干预作用的研究

目的 研究福辛普利（Fosinopril）联合非诺贝特（Fenofibrate）对糖尿病小鼠视网膜细胞凋亡的影响及相关基因的表达和视网膜组织中血管内皮生长因子（vascular endothelial growth factor,VEGF）、氧化相关物质的影响,以明确其抑制糖尿病视网膜病变（diabetic retinopathy,DR）的作用机制。方法 选取清洁级性成熟ICR小鼠150只,随机分为5组（每组30只）：A组（假造模组,普通饲料喂养并给予相同体积的生理盐水）、B组（模型组,高脂饲料喂养4 w后给予腹腔注射链脲佐菌素（streptozotocin,STZ）,给予相同体积生理盐水灌胃8 w）、C组（福辛普利对抗组,高脂饲料喂养4 w后给予腹腔注射链脲佐菌素,给予福辛普利干预8 w）、D组（非诺贝特对抗组,高脂饲料喂养4 w后给予腹腔注射链脲佐菌素,给予非诺贝特干预8 w）、E组（福辛普利+非诺贝特联合对抗组,高脂饲料喂养4 w后给予腹腔注射链脲佐菌素,给予福辛普利+非诺贝特干预8 w）,0w和8w时测血糖BG,处死小鼠取眼球制备视网膜组织匀浆取上清检测谷光甘肽过氧化物酶（Glutathione peroxidase,GSH-PX）、超氧化物歧化酶（Superioxide dismutase,SOD）、活性氧类物质（Reactive oxygen species,ROS）、丙二醛（Malondialdehyde,MDA）、VEGF浓度,用RT-PCR法检测视网膜Bax与Bcl-2基因mRNA水平的表达,并用Tunel染色法检测视网膜细胞凋亡情况。结果B、C、D、E组小鼠用药前后血糖无差别(p>0.05); 均较A组明显升高(p<0.05);A组小鼠视网膜组织GSH-PX、SOD活性值及Bcl-2基因mRNA水平表达均高于其他四组（P＜0.05）,而ROS、MDA、VEGF、Bax基因mRNA水平表达与Tunel指数均低于B、C、D组（P＜0.05）;B组小鼠GSH-PX、SOD活性值及Bcl-2基因mRNA水平表达均低于其他四组（P＜0.05）,而ROS、MDA、VEGF、Bax基因mRNA水平表达与Tunel指数均高于其他四组（P＜0.05）;E组小鼠GSH-PX、SOD活性值及Bcl-2基因mRNA水平表达均高于C、D组（P＜0.05）,而ROS、MDA、VEGF、Bax基因mRNA水平表达与Tunel指数均低于C、D组（P＜0.05）;D组小鼠GSH-PX、SOD活性及Bcl-2基因mRNA水平表达均高于C组（P＜0.05）,而ROS、MDA、Bax基因mRNA水平表达与Tunel指数均低于C组（P＜0.05）;C组小鼠VEGF浓度值低于D组（P＜0.05）。结论 福辛普利及非诺贝特均能改善DR,通过抑制凋亡与抗氧化对视网膜起到一定的保护作用,但两药联合应用效果更佳。