LC-MS/MS法分析人血浆中西格列汀浓度及其应用研究

摘要：目的 建立一种测定人血浆中西格列汀浓度的液相色谱-串联质谱（LC-MS/MS）分析方法,并将该方法应 用于西格列汀在人体内的药代动力学研究。方法 以西格列汀-d4为内标,血浆样品经CleanertPPT沉淀板沉淀后, 通过Diamonsil C18色谱柱（100 mm×4.6 mm,5 μm）进行分离,使用甲醇-10 mmol/L甲酸铵水溶液（含10%甲醇,0.1% 甲酸）作为流动相,进行梯度洗脱,流速为0.5 mL/min。通过电喷雾电离源（ESI）,以多反应监测（MRM）模式进行正离 子检测。从选择性、残留、线性范围与定量下限、精密度与准确度、基质效应和回收率、稳定性方面进行方法学验证。 同时考察健康人口服西格列汀片100 mg后的主要药代动力学参数。结果 西格列汀、西格列汀-d4的MRM离子对 分别为 m/z 408.0→235.2、m/z 412.1→239.0。人血浆中西格列汀在 0.5～1 000 μg/L 浓度范围内线性关系良好（R2> 0.99）,定量下限为0.5 μg/L;定量下限和质控样品的批内、批间精密度（RSD）在0.83%~12.80%之间,准确度（RE）在± 10.0%以内。健康人口服西格列汀片100 mg后主要药代动力学参数：达峰时间（Tmax）、达峰浓度（Cmax）、、生物利用度 （AUC）、半衰期（T1/2）分别为（2.44±1.29）h、（375±138）μg/L、（2 915±585）h·μg/L、（11.10±2.41）h。结论 本LC-MS/ MS分析方法敏感度高且样品处理方法简单快速,满足生物分析的法规要求,可应用于人体内西格列汀的药代动力学 研究。     

Mechanism-based pharmacokinetic/pharmacodynamic modeling of the effects of sitagliptin on DPP-4 activity,insulin and glucose in diabetic rats

Dipeptidyl peptidase-4 (DPP-4) inhibitors exert their antihyperglycemic effects through repressing inactivation of certain incretin hormones and thus increasing insulin secretion and controlling glucose level. In this study, the plasma concentrations of sitagliptin, a potent DPP-4 inhibitor, after a single oral dose of 300 mg/kg in streptozotocin-induced type 2 diabetic rats were determined by HPLC. A one-compartment pharmacokinetic (PK) model with first order absorption was developed to describe the PK profile of sitagliptin, and the drug concentrations at the doses given in the pharmacodynamic (PD) study were simulated accordingly. The dynamic changes in DPP-4 activity, insulin concentration and blood glucose level in diabetic rats at doses of 1, 5 and 10 mg/kg were measured, and a mechanism-based PK/PD model was established subsequently. In this model, the inhibitory effect of sitagliptin on DPP-4 activity was demonstrated using the Hill’s function with direct link, and the downstream increase in insulin secretion and inhibition of glucose production were characterized using indirect response (IDR) models. This model interpreted the mechanism of antihyperglycemic action of sitagliptin, and may be modified and applied to other species or other agents in this class.     

磷酸西格列汀片处方工艺研究

目的 筛选磷酸西格列汀片的最佳处方工艺。方法 设计不同处方工艺,通过检测溶出曲线及粉体学数据,确定最佳处方工艺。结果 自制片的4个处方中,粉体学数据及压片流畅性均较好,其中湿法制粒工艺两个处方溶出比参比制剂慢,粉末直压工艺中当交联羧甲基纤维素钠为16 mg时溶出曲线与参比制剂拟合较好。结论 通过对磷酸西格列汀片的处方工艺研究,最终确定了磷酸西格列汀片的工艺和崩解剂用量。     

西格列汀对糖尿病合并动脉粥样硬化患者的治疗作用

目的：观察西格列汀对糖尿病及合并动脉粥样硬化患者CRP水平及单核细胞趋化蛋白-1（MCP-1）及血管细胞黏附分子-1 （VCAM-1）mRNA表达的影响及优势,探讨西格列汀在糖尿病患者抗动脉粥样硬化的作用及机制.方法：选取2011-03～2013-06门诊就诊及住院的90例初次确诊2型糖尿病患者（经口服葡萄糖耐量试验及胰岛素、C肽激发试验）,年龄（50.77±12.54）岁,首次就诊内分泌科,未经降血糖、降脂、抗血小板等治疗,其中39例彩超提示颈动脉粥样硬化板块形成（硬斑或/和软斑）.分为单纯糖尿病组（51例）和已合并颈动脉粥样硬化组（39例）,90例患者经糖尿病宣教后,均给予糖尿病饮食及适当运动锻炼,同时给予西格列汀100mg qd降血糖治疗;测定治疗前及治疗6月后空腹胰岛素及CRP、糖基化血红蛋白,并采用逆转录多聚酶链反应（RT-PCR）方法检测静脉血中MCP-1、VCAM-1 mRNA表达水平的变化.结果：（1）两组治疗后,糖基化血红蛋白水平均显著降低（P＜0.01）,空腹血清胰岛素水平显著升高（P＜0.05）;（2）同期相比,糖尿病合并颈动脉粥样硬化组CRP水平显著高于于单纯糖尿病组（P＜0.05）;单纯糖尿病组治疗前后CRP水平无明显变化（P＞0.05）,糖尿病合并颈动脉粥样硬化组治疗3月后CRP水平显著降低（P＜0.01）;（3）同期相比,糖尿病合并颈动脉粥样硬化组MCP-1、VCAM-1mRNA表达水平显著高于于单纯糖尿病组（P＜0.05）;单纯糖尿病组治疗前后MCP-1、VCAM-1 mRNA表达水平无明显变化（P＞0.05）,糖尿病合并颈动脉粥样硬化组治疗3月后MCP-1、VCAM-1 mRNA表达水平水平显著降低（P＜0.01）.结论：CRP可导致动脉粥样硬化的发生;西格列汀具有降糖之外的直接抗动脉粥样硬化作用;西格列汀抗动脉粥样硬化作用部分是通过降低MCP-1、VCAM-1的表达实现的。     

Clinical Effects of Exposure to DPP-4 Inhibitors as Reported to the National Poison Data System

DPP-4 inhibitors (sitagliptin, saxagliptin, and linagliptin) are approved for the treatment of diabetes. They are considered safe due to their hyperglycemia dependent mechanism of action. We examined all isolated exposures to DPP-4 inhibitors reported to the National Poison Database System since 2006 to determine if significant toxicity occurs after exposure with attention to pediatric and intentional overdoses. NPDS data regarding DPP-4 ingestions in all age groups between January 2006 and March 2013 was collected. Cases were reviewed, and the following inclusion criteria applied: (1) reported ingestion of a DPP-4 inhibitor and (2) known clinical outcome. Exclusion criteria included the following: (1) exposure to more than a single substance, (2) no known outcome, and (3) clinical outcome judged to be unrelated to the exposure. One thousand four hundred seventy-six cases were reviewed while 826 were excluded. Of 650 included cases, 562 developed no clinical effects. Mild effects were noted in 77. There were no deaths. Moderate/major effect cases were investigated: two medication-naive nondiabetic individuals with accidental exposures developed clinically significant hypoglycemia requiring treatment. One diabetic patient on a DPP-4 inhibitor developed prolonged hypoglycemia requiring admission and continuous exogenous dextrose. Of 650 included exposures to DPP-4 inhibitors, 639 (98.3%) had either no or minor clinical effects. Three resulted in clinically significant hypoglycemia requiring intervention. None of the moderate or major clinical outcomes were the result of intentional overdoses for the purpose of self-injury. No exploratory ingestions resulted in moderate or major effects. Based on this data, exposure to DPP-4 inhibitors may rarely result in clinically significant hypoglycemia.     

西格列汀联合二甲双胍治疗初诊2型糖尿病的疗效观察

目的 探讨西格列汀联合二甲双胍治疗初发、肥胖2型糖尿病的临床疗效.方法 选取2012年1月至2013年12月期间在我院治疗的180例初发、肥胖2型糖尿病患者为研究对象,将其随机分为治疗组和对照组,其中治疗组92例,对照组88例.对照组患者给予格列美脲＋二甲双胍治疗,治疗组给予二甲双胍＋西格列汀治疗.入组前常规进行肝肾功能、血糖血脂、糖化血红蛋白、胰岛细胞分泌功能、胰岛素抵抗等评估,治疗期24周.治疗结束后分析两组患者的在体重、空腹血糖、餐后2小时血糖、糖化血红蛋白、胰岛细胞分泌功能、胰岛素抵抗指数等方面的差异.结果 治疗组患者各项监测指标（空腹及餐后血糖、糖化血红蛋白、胰岛细胞分泌功能、胰岛素抵抗指数）明显好于对照组患者（P＜0.05）,且治疗组的总有效率92.40％ （85/92）和对照组患者72.73％ （64/88）相比占优势,两组患者比较差异具有统计学意义（P＜0.05）.两组患者在体重、低血糖发作方面无明显差异（P＞0.05）.结论 二甲双胍联合西格列汀治疗初发、肥胖2型糖尿病患者安全有效,值得借鉴.     

西格列汀联合二甲双胍治疗2型糖尿病的临床观察

目的探讨西格列汀联合二甲双胍治疗2型糖尿病的疗效与安全性。方法选取100例2型糖尿病患者,按随机数字表法分为两组,每组50例,研究组采用磷酸西格列汀（100mg日1次口服）联合二甲双胍（500mg日3次口服）治疗,对照组采用瑞格列奈（1mg日3次口服）联合二甲双胍（500mg日3次口服）治疗,两组均治疗12周,治疗前后分别观察两组患者空腹血糖（FPG）、餐后2h血糖（2hPG）、糖化血红蛋白（HbA1c）、空腹胰岛素（FINS）、体质量指数（BMI）及低血糖事件发生率等变化。结果两组治疗后FPG、2hPG、HbA1C均较治疗前显著下降,差异有统计学意义（P〈0．01）。研究组治疗后BMI显著减低[（24．45±2．13）kg／m2比（25．46±2．62）kg／m2],FINS显著升高[（13．36．4±1.89）mU／L比（12．36±1．80）mU/L],与治疗前比较差异有统计学意义（P〈0．05或〈0．01）。对照组治疗后BMI、FINS与治疗前比较差异无统计学意义（P〉0．05）。研究组和对照组低血糖事件发生率比较差异有统计学意义[0比12％（6／50）]（P〈0．05）。结论西格列汀联合二甲双胍治疗2型糖尿病的方案是安全有效的,不仅能显著减少低血糖事件的风险,而且具有能控制并减轻体质量等优越性,是治疗2型糖尿病的优选方案。     

Short-term inhibition of DPP-4 enhances endothelial regeneration after acute arterial injury via enhanced recruitment of circulating progenitor cells

Background

Endothelial injuries regularly occur in atherosclerosis and during interventional therapies of the arterial occlusive disease. Disturbances in the endothelial integrity can lead to insufficient blood supply and bear the risk of thrombus formation and acute vascular occlusion. At present, effective therapeutics to restore endothelial integrity are barely available.We analyzed the effect of pharmacological DPP-4-inhibition by Sitagliptin on endogenous progenitor cell-based endothelial regeneration via the SDF-1α/CXCR4-axis after acute endothelial damage in a mouse model of carotid injury.

Methods and Results

Induction of a defined endothelial injury was performed in the carotid artery of C57Bl/6 mice which led to a local upregulation of SDF-1α expression. Animals were treated with placebo, Sitagliptin or Sitagliptin + AMD3100. Using mass spectrometry we could prove that Sitagliptin prevented cleavage of the chemokine SDF-1α. Accordingly, increased SDF-1α concentrations enhanced recruitment of systemically applied and endogenous circulating CXCR4 + progenitor cells to the site of vascular injury followed by a significantly accelerated reendothelialization as compared to placebo-treated animals. Improved endothelial recovery, as well as recruitment of circulating CXCR4 + progenitor cells (CD133 +, Flk1 +), was reversed by CXCR4-antagonization through AMD3100. In addition, short-term Sitagliptin treatment did not significantly promote neointimal or medial hyperplasia.

Conclusion

Sitagliptin can accelerate endothelial regeneration after acute endothelial injury. DPP-4 inhibitors prevent degradation of the chemokine SDF-1α and thus improve the recruitment of regenerative circulating CXCR4 + progenitor cells which mediate local endothelial cell proliferation without adversely affecting vessel wall architecture.     

A comparative study of the role of crocin and sitagliptin in attenuation of STZ-induced diabetes mellitus and the associated inflammatory and apoptotic changes in pancreatic β-islets

Type 1 diabetes mellitus (T1DM) describes a complex group of metabolic disorders associated with elevated blood glucose levels and increased risks of complications development. Exploring new drug therapies would reduce the increased diabetes-associated morbidity and mortality and will reduce the excessive health care costs. Crocin is the major active ingredient of saffron. In the current study, DM was induced by single intraperitoneal injection of streptozocin (50 mg/kg).DM progression was associated with a significant increase in blood glucose level with reduced insulin and increased glucagon secretion. Pancreatic malondialdehyde (MDA) content significantly escalated, while superoxide dismutase (SOD) activity, reduced glutathione (GSH) concentration, catalase activity, thioredoxin level and serum total antioxidant capacity significantly declined. This was associated with a significant increase in pancreatic caspase-3 contents and pancreatic infiltration with inflammatory cells in β-islets. Both sitagliptin and crocin significantly reduced blood glucose levels, enhanced pancreatic insulin expression and secretion and suppressed glucagon secretion with enhancement of anti-oxidant defenses and reduction of oxidative burden, with evident anti-inflammatory impacts. Interestingly, the effect of crocin on DM indices, inflammatory and apoptotic changes was comparable to that of sitagliptin; the standard oral hypoglycemic agent. Nevertheless, crocin had a superior effect compared to sitagliptin on blood sugar level, β-islets diameter and insulin immune-reactivity. In conclusion, crocin reduced blood glucose level mainly via reduction of oxidative burden, modulation of apoptotic pathway and attenuation of pancreatic inflammation.     

Sitagliptin: Anti-platelet effect in diabetes and healthy volunteers

Sitagliptin, a selective dipeptidyl peptidase-4 inhibitor drug is used to treat type-2 diabetes (T2DM). We investigated the anti-platelet activity of sitagliptin in patients with T2DM and in in vitro samples obtained from healthy humans. Patients with T2DM (27 male + 23 female) were selected and followed up before (control) and after treatment with sitagliptin for up to 3 months. Platelets were isolated from the blood of sitagliptin treated patients and controls. Patients with T2DM treated with sitagliptin for 1and 3 months, showed 10?±?2% and 30?±?5% inhibition of platelet aggregation, respectively. For the in vitro study, platelets from 10 normal humans (n?=?10) were isolated. Platelet aggregation, intracellular free calcium and tyrosine phosphorylation of multiple proteins were measured by aggregometer, spectrofluorometer and western blotting, respectively. Platelets pre-treated with 5 and 10?µg/ml of sitagliptin, showed 25?±?4% and 40?±?6% inhibition of thrombin-induced platelet aggregation, respectively. Sitagliptin decreased intracellular free calcium (2.5-fold) and tyrosine phosphorylation of multiple proteins in thrombin-induced platelet activation. Sitagliptin inhibited platelet aggregation in T2DM as well as in healthy humans. Sitagliptin has significant concentration-dependent anti-platelet activity. This activity was due to its inhibitory effect on intracellular free calcium and tyrosine phosphorylation.