Optimization and in vivo evaluation of an oral dual controlled-release tablet dosage form of insulin and duck ovomucoid

The objectives of the present study were to (1) optimize the release rate of insulin from compressed microparticulates and (2) compare the in vivo hypoglycemic effect of optimized insulin microparticulates with compressed enzyme inhibitor (duck ovomucoid) and without inhibitor. A 3-factor, 15-run Box Behnken design was used to construct polynomial models correlating the dependent and independent variables. Independent processing variables were rate of addition of the alcoholic Eudragit L100 dispersion, volume of the antisolvent, and compression pressure. Responses were cumulative percent of insulin released from 1-6 hours. Insulin and ovomucoid release was simultaneously analyzed by high-performance liquid chromatography. They demonstrated variable release rates, which were optimized to the Higuchi's square root of time model to release the insulin and the inhibitor over 6 hours. The relationship between dissolution profiles and process parameters were demonstrated by contour and response surface plots. In vivo hypoglycemic effect was evaluated in rabbits in a 3-way crossover design. Cocompressed microparticulates of insulin and duck ovomucoid displayed a 3.2-fold greater hypoglycemic effect when compared with a similar preparation without ovomucoid. This study demonstrated the potential benefits of dosage forms with dual controlled-release mechanisms for both the protein and enzyme inhibitor.     

Polymethyacrylate based microparticulates of insulin for oral delivery: preparation and in vitro dissolution stability in the presence of enzyme inhibitors

The purpose of this investigation was to (a) evaluate the coprecipitation technique for preparing microparticulates of insulin, (b) study the effect of variables such as addition of salts in the precipitating medium and ratio of polymeric solution to volume of precipitating medium on the dissolution and encapsulation efficiency of insulin microparticulates, and (c) evaluate the in-vitro enzymatic dissolution stability of insulin microparticulates in the presence of chicken ovomucoid (CkOVM) and duck ovomucoid (DkOVM) as inhibitors. Insulin dissolved in 0.01 N HCl was mixed with alcohol USP to get a final concentration of 32% v/v. Eudragit L100, a representative polymethyacrylate polymer, was then dissolved in this solution which was transferred to a beaker containing cold water with homogenization to obtain microparticulates. Dissolution studies were carried out in pH 6.8 phosphate buffer using a 100-ml conversion kit in a standard dissolution assembly. Dissolution stability of microparticulates was evaluated in the presence of 0.5 microM trypsin and 0.l microM chymotrypsin at various ratios of CkOVM and DkOVM. The results indicated that insulin microparticulates could be prepared using the coprecipitation technique with high encapsulation efficiency by proper selection of experimental conditions and amount of polymer. Presence of salts in the precipitating medium decreased the dissolution of insulin from the microparticulates. As the ratio of precipitating medium with respect to the polymeric solution was increased, the encapsulation efficiency increased. In dissolution stability experiments, insulin was not detected in the presence of enzymes alone. When CkOVM and DkOVM were incorporated, the stability of insulin increased significantly in a concentration dependent fashion.     

尼莫地平胃内滞留漂浮型缓释片的研究

将尼莫地平先制成速释型固体分散体,再压制成胃内滞留漂浮型缓释片(NM-FSRT)。均匀设计法优选处方,并考察处方因素对尼莫地平释放的影响,在人体内对NM-FSRT进行了初步评价。结果表明优选处方于体外漂浮达10h,0.15～6h释放符合零级动力学。HPMC量越大,药物释放越慢,PEG 6000量越大,释放越快。饮食后NM-FSRT于胃内滞留时间约5h,空腹时约3h;对照非漂浮片饭后服滞留时间为3h,空腹2h排空。体内相对生物利用度为391.46%,MRT较普通片延长一倍多。     

Transport studies of insulin across rat jejunum in the presence of chicken and duck ovomucoids

Our aim was to evaluate the transport of insulin across rat jejunum in the presence of ovomucoids and to assess the effect of ovomucoids on intestinal tissue by studying the permeation of a lipophilic and a hydrophilic marker. Rat jejunal segments were mounted in a side-by-side diffusion chamber filled with Krebs bicarbonate buffer, bubbled with 95% O2/5% CO2 at a fixed flow rate and maintained at 37 degrees C. The permeation of insulin, a lipophilic marker ([7- 3H] testosterone) and a hydrophilic marker (D-[1- 14C] mannitol) was evaluated in the presence of 0.5-1.5 microM duck ovomucoid (DkOVM) or chicken ovomucoid (CkOVM). For stability and permeation of insulin in the presence of alpha-chymotrypsin, an enzyme-to-inhibitor ratio of 1:1 and 1:2 was used. In the absence of alpha-chymotrypsin, the permeability coefficient (Papp) of insulin at pH 7.4 was 0.922+/- 0.168 x 10(-7) cm s(-1), which decreased with increasing concentrations of DkOVM or CkOVM. Conversely, the permeation of the hydrophilic and lipophilic marker increased with increasing concentrations of CkOVM and DkOVM. In stability studies, the percentage of drug remaining was found to be 2-fold higher at the 1:2 ratio than with the 1:1 ratio of enzyme to inhibitor. This was in agreement with the 2-fold increase in flux values of insulin in the presence of alpha-chymotrypsin and DkOVM at the 1:2 ratio of enzyme to inhibitor. The decrease in permeation of insulin in ovomucoids was unexpected. Marker transport studies indicated that ovomucoids have the potential to modulate transcellular and paracellular permeability. The flux enhancement of insulin in the presence of alpha-chymotrypsin and DkOVM is encouraging. The use of ovomucoids offers potential to enhance oral delivery of insulin and warrants further investigation.     

HA-GMS-INS胰岛素口服纳米给药系统研究

目的:建立透明质酸-单硬脂酸甘油酿-胰岛素(HA-GMS-INS)口服纳米给药系统,并进行成药性研究.方法:利用酿化反应合成载体HA-GMS,单因素法筛选制备工艺,通过红外光谱和核磁共振氢谱对结构进行表征;采用低能乳化法制备HA-GMS-INS,用多分散激光粒度仪和透射电镜测定纳米乳的粒径及形态,并对胰岛素的体外释放性...     

羧甲基壳聚糖-聚乙二醇作为胰岛素载体的研究

目的：制备胰岛素-羧甲基壳聚糖-聚乙二醇纳米粒。方法：利用红外光谱（FTIR）和核磁共振氢谱（¹H-NMR）对羧甲基壳聚糖-聚乙二醇的结构进行表征,用粒度分析仪测定纳米粒的粒径分布及电位,采用动态透析法考察纳米粒的释药性能,用CCK-8试剂盒检测纳米粒细胞毒性,以糖尿病小鼠为模型,研究纳米粒的降血糖作用。结果：聚乙二醇成功接枝到羧甲基壳聚糖上,包埋胰岛素的纳米粒的平均粒径为（257.5±12.1）nm,Zeta电位为（-15.2±0.3）mV,负载胰岛素的羧甲基壳聚糖-聚乙二醇纳米粒在中性释放介质中,5 h内胰岛素的释放速度较快,之后8 h趋于平稳,胰岛素的累计释放量可达到80%,CCK-8试剂盒显示纳米粒对L929细胞基本无细胞毒性,50 U·kg^-1的纳米粒溶液经灌胃给药后,血糖浓度明显降低。结论：胰岛素-羧甲基壳聚糖-聚乙二醇纳米粒基本无毒性,具有良好的生物相容性,对糖尿病小鼠有效发挥降血糖作用。     

Hypoglycemic effect of gelatin microspheres with entrapped insulin and protease inhibitor in normal and diabetic rats

Insulin containing gelatin microspheres (IGM) with and without soyabean trypsin inhibitor (TI) were pre-pared and coated with enteric polymers to protect them from degradation in stomach and to release the insulin upon reaching the intestine. Four types of coated IGM were prepared: (i) IGM coated with natural polymers (chitosan inner coat-alginate outer coat), (ii) IGM-TI coated with cellulose acetate phthalate; (iii) IGM-TI coated with cellulose acetate butyrate, and (iv) IGM-TI coated with natural polymers (chitosan inner coat-alginate outer coat). The protective efficiency of uncoated and four types of coated microspheres to-ward digestive enzymes such as pepsin and trypsin was evaluated under simulated physiological conditions. The microspheres were characterized for their insulin content and particle size. The morphology of the micro-spheres was studied using scanning electron micros-copy. The in vitro release studies of insulin from uncoated and coated microspheres indicated that the release followed a zero-order pattern, prolonging for 6 days from 2 days in the case of uncoated spheres. The uncoated and coated microspheres containing insulin (20 IU/kg) were orally administered to albino Wistar rats by stomach tube, and insulin absorption was evaluated by assessing the hypoglycemic effect in normal and diabetic rats. A significant and continuous hypoglycemic effect was observed in diabetic rats following oral administration of coated IGM containing TI when compared to the effect following administration of coated IGM without TI.     

Preparation and release of salbutamol from chitosan and chitosan co-spray dried compacts and multiparticulates.

Chitosan microparticulates were prepared by spray drying from aqueous media containing hydrochloric acid or acetic acid. The medium affected the morphology and degree of acetylation of chitosan, the presence of acetic acid resulting in increased acetylation of the polymer during processing. Co-spray drying salbutamol sulphate/chitosan systems with the crosslinking agent formaldehyde had no detectable effect on particle morphology. However, with increasing salbutamol loading particles became less spherical, taking on a collapsed appearance. Spray dried chitosan-salbutamol sulphate microparticulates were X-ray amorphous. Chitosan-salbutamol sulphate composites were compressed into discs to quantify drug release and showed delayed release of salbutamol sulphate. The general power law equation fitted the data better than the t0.5, mono- or bi-exponential models and gave n indices greater than 0.5, i.e. in the range 0.53-0.71. Crosslinking did not dramatically alter the drug release behaviour. Both crosslinked and non-crosslinked composites swelled during release, the former to the greater extent. The release data for crosslinked composites gave slightly higher n values than the corresponding non-crosslinked composites, consistent with the increased swelling of these systems. Release studies were also conducted on the microparticulates. Because of the small particle size and large surface area present, the release of the highly soluble drug salt was extremely rapid (> 90% release in 5 min). Twin impinger analysis indicated good in vitro deposition of the microparticulates and potential for pulmonary delivery.     

Spray-freeze-dried dry powder inhalation of insulin-loaded liposomes for enhanced pulmonary delivery

Nowadays, growing attention has been paid to the pulmonary region as a target for the delivery of peptide and protein drugs, especially macromolecules with systemic effect like insulin, since the pulmonary route exhibits numerous benefits to be an alternative for repeated injection. Furthermore, encapsulation of insulin into liposomal carriers is an attractive way to increase drug retention time and control the drug release in the lung; however, its long-term stability during storage in the reservoir and the process of aerosolization might be suspected when practically applied. Thus, the aim of this study was to design and characterize dry powder inhalation of insulin-loaded liposomes prepared by novel spray-freeze-drying method for enhanced pulmonary delivery. Process variables such as compressed air pressure, pump speed, and concentration were optimized for parameters such as mean particle diameter, moisture content, and fine particle fraction of the produced powders. Influence of different kinds and amounts of lyoprotectants was also evaluated for the best preservation of the drug entrapped in the liposome bilayers after the dehydration–rehydration cycle. The in vivo study of intratracheal instillation of insulin-loaded liposomes to diabetic rats showed successful hypoglycemic effect with low blood glucose level and long-lasting period and a relative pharmacological bioavailability as high as 38.38% in the group of 8 IU/kg dosage.     

Absorption of insulin from pluronic F-127 gels following subcutaneous administration in rats.

The main objective of this work was to evaluate the use of Pluronic (PF127) gels, polylactic-co-glycolic acid (PLGA) nanoparticles and their combination for parenteral delivery of peptides and proteins having short half-lives using insulin as a model drug. The in vitro insulin release profiles of various PF127 formulations were evaluated at 37 degrees C using a membraneless in vitro model. In vivo evaluation of the serum glucose and insulin levels was performed following subcutaneous administration of various insulin formulations in normal rats. The in vitro results demonstrated that the higher the concentration of PF127 in the gel, the slower the release of insulin from the matrices, independent of the vehicle used. The acute hypoglycemic peak resulting from administration of an insulin solution between 0.5 and 2.0 h after administration (peak at 1 h) is replaced after administration of insulin-PLGA nanoparticles by an almost constant hypoglycemic effect with a slower recovery of the serum glucose levels at about 2 h after administration. By loading insulin into PF127 gels, a slower and more prolonged hypoglycemic effect of insulin was obtained in inverse proportion to the polymer concentration. PF127 gel formulations containing insulin-PLGA nanoparticles had the most long-lasting hypoglycemic effects of all formulations. From the current in vitro and in vivo study, we concluded that PF127 gel formulations containing either drug or drug-nanoparticles could be useful for the preparation of a controlled delivery system for peptides and proteins having short half-lives. Copyright     

Effect of PLGA hydrophilia on the drug release and the hypoglucemic activity of different insulin-loaded PLGA microspheres

The effects of viscosity and hydrophilic characteristics of different PLGA polymers on the microencapsulation of insulin have been studied in?vitro and in?vivo after subcutaneous administration to hyperglycemic rats. Hydrophilic PLGA polymers produced a higher burst effect than the hydrophobic ones. Moreover, an incomplete insulin release was observed with the hydrophilic PLGA polymers in comparison with the hydrophobic ones. An explanation for that incomplete release can be the development of polymer-insulin interactions associated to the polymer hydrophilic/hydrophobic character, as detected by DSC analysis. Differences in the release rate of microsphere formulations lead to differences in the hypoglycemic action and the weight of animals. Hydrophobic PLGA was able to prolong the hypoglycemic action up to 4 weeks which is at least double than that obtained with hydrophilic PLGA of a similar viscosity. Comparing insulin microspheres with an immediate release formulation, microspheres can increase insulin relative bioavailability up to four times.     

A simple HPLC method for the simultaneous analysis of insulin and ovomucoid

An analytical HPLC method is reported for the simultaneous determination of insulin and its enzyme inhibitor, chicken ovomucoid. Verapamil was used as an internal standard. The elution was achieved using a gradient technique (10-15% B for 4 min, 15-35% B from 5th to 11th min and 35-10% B from 12th to 22nd min). The mobile phase used was 0.05% v/v trifluoroacetic acid (TFA) in water and 0.05% v/v TFA in acetonitrile with a flow rate of 1.2 ml/min. The analytes were detected at 210 nm after resolution using a reversed phase C-18 column. Insulin, ovomucoid and verapamil (IS) were eluted at 11.9, 14.2, and 18 min, respectively, free from any interfering endogenous peaks during a run time of 22 min. Linear relationships were observed between the detector response and the concentrations of the analytes (0.05-1 I.U/ml for insulin (r2 = 0.9975) and 5-100 microg/ml for the chicken ovomucoid (r2 = 0.9993)). The assay was found to be highly selective and sensitive due to the absence of any interfering peaks. The lower C.V and % error values of the assay indicates that the assay could accurately and precisely quantitate both insulin and ovomucoid in the examined concentration range. This method can be used for the simultaneous quantitation of insulin and chicken ovomucoid.     

肠溶包衣胰岛素-壳聚糖复合物纳米粒的制备及体内外性质

目的制备肠溶包衣的胰岛素壳聚糖复合物纳米粒,并对其理化性质、体外释药以及在糖尿病模型大鼠体内的降血糖效果进行研究。方法采用离子交联法制备胰岛素壳聚糖复合物纳米粒,使用羟丙基甲基纤维素酞酸酯(HP55)对其进行肠溶包衣;通过扫描电子显微镜观察其表观形态,用激光粒度测定仪测定其粒径大小,用Zeta电势测定仪测定其Zeta电势,使用HPLC法测定离心上清夜中胰岛素浓度,计算包封率。结果制备得到的纳米粒均匀、圆整,包衣前后粒径分别为(281±10)nm和(328±13)nm,Zeta电势分别为(30.4±6.97)mV和(33.7±6.69)mV,包封率分别为78.5%和74.3%;肠溶包衣纳米粒在人工胃液和肠液中的释药速率均明显低于未包衣纳米粒,突释效应显著减小;未包衣复合物纳米粒能够显著降低糖尿病模型大鼠的血糖浓度,其降糖效果能持续20 h以上,肠溶包衣后,降糖效果明显增强;肠溶包衣前后在模型大鼠体内24 h相对生物利用度分别为11.12%和16.29%。结论肠溶包衣胰岛素壳聚糖复合物纳米粒可以有效抑制胰岛素的突释,促进其吸收,显著降低模型大鼠的血糖浓度,能够作为胰岛素口服给药的有效载体。     

Pelleted bioadhesive polymeric nanoparticles for buccal delivery of insulin: preparation and characterization

The study was an attempt to develop an alternative buccal delivery system for insulin. Insulin bearing nanoparticles were prepared by the emulsion internal phase evaporation method. The effect of some formulation variables viz., polymer/drug ratio and emulsifier concentration was studied on particle size and entrapment efficiency. Nanoparticles were pelleted to impart three-dimensional structural conformity and coherence thereby facilitating buccal application. Solid lateral and horizontal sedimentaton in the pellet can be avoided by nanoparticulation and ensuring uniform drug distribution throughout the pellet. The in vitro studies of the pellets included bioadhesion and drug release profile. In vivo studies were performed on diabetic rats. A significant hypoglycemic response was observed after 7 h, without any detectable fluctuation in blood glucose profile and risk of hypoglycemia.     

壳聚糖及其EDTA轭合物双层包覆胰岛素脂质体的处方工艺优化

目的:研究制剂因素对壳聚糖(CH)及其EDTA轭合物(CEC)双层包覆胰岛素脂质体降血糖作用的影响.方法:采用逆相蒸发法制得胰岛素脂质体,并用CH和CEC进行双层包覆.以小鼠口服降血糖效果作为实验指标,分别应用L16(215)和L8(27)正交实验设计优化CH-CEC双层包覆胰岛素脂质体的处方与工艺.结果:最佳处方组成为胰岛素100IU,pH 7.4磷酸盐缓冲液,磷脂150mg,胆固醇25mg,维生素E 15mg,0.2% CH 1.5mL和1% CEC 1.5mL.最佳制备工艺为乙醚10mL,旋转蒸发温度20℃,探针式超声时间0.5min,加CH和CEC后的孵化时间30min,孵化温度10℃,CH要先于CEC加入.经优化得到的双层包覆胰岛素脂质体经糖尿病模型大鼠口服后,具有平稳持久的降血糖作用,以皮下注射胰岛素为对照,其相对药理生物利用度(PA)达14.78%.结论:CH-CEC双层包覆胰岛素脂质体的组成和制备因素的变化会影响降血糖效果,用CH-CEC双层包覆的胰岛素脂质体具有较好降血糖作用.     

口服降糖药临床应用分析

目的：分析常熟医院口服降糖药应用现状和趋势。方法：对2007—2009年我院口服降糖药应用品种、销售金额、用药频度（DDDs）和日均费用（DDC）进行回顾性分析。结果：3年口服降糖药销售金额占抗糖尿病药总金额48.12%;销售金额排序前3位的依次是：磺酰脲类、非磺酰脲类胰岛素促泌剂和α-糖苷酶抑制剂;DDDs排序前3位的依次是：磺酰脲类、双胍类和非磺酰脲类胰岛素促泌剂;3年中,阿卡波糖、瑞格列奈和格列吡嗪缓释片位列单品种销售金额前3位;格列吡嗪缓释片、二甲双胍和格列吡嗪普通片位列单品种DDDs的前3位;噻唑烷二酮类的DDC最高,双胍类最低。结论：我院口服降糖药用药合理且占主导地位.     

Effective insulin delivery using starch nanoparticles as a potential trans-nasal mucoadhesive carrier

Mucoadhesive nanoparticles (NPs) could be an exciting prospect for trans-nasal insulin delivery as they have higher surface area to cover highly vascularised nasal absorptive area providing a greater concentration gradient; hence the present study makes an attempt in this regard. Starch NPs were prepared by different crosslinkers using various methodologies and were loaded with insulin. Emulsion crosslinked particles were smaller in size compared to gel method (351 vs 997 nm), and size is further reduced when epichlorohydrin is used as crosslinking agent compared to POCl3 (194 vs 810 nm). NPs of epichlorohydrin emulsion were further optimized with variable crosslinking to evaluate the effect of degree of crosslinking on in vivo performance. In vitro, a size dependent first order diffusion controlled insulin release with an initial burst effect was found, which is higher with NPs of small size and least crosslinking. Formulation of EE-NPs with Na glycocholate showed a superior hypoglycemic action compared to other NPs formulations containing the former and lysophosphatidylcholine as permeation enhancers. The hypoglycemic effects were more pronounced with medium crosslinked NPs (EE-L2-NPs), which showed a nadir of 70% reduction of plasma glucose and significant effects until 6h. The peak plasma insulin level (Cmax) of medium crosslinked EE-L2-NPs (258 muIU/ml at 1h) vindicates the pharmacodynamic effect, which was found to be superior compared to all other formulations. The release rate and higher associated surface area might work in tandem, and could be greatly amplified when combined with permeation enhancers to make starch NPs an efficient trans-nasal mucoadhesive carrier of insulin.     

蒲公英多糖的提取及降糖作用的研究

目的：探讨提取蒲公英多糖的提取最佳工艺和蒲公英多糖对四氧嘧啶（Alloxan）糖尿病小鼠的降糖作用。方法：采用酶提取法、超声波提取法和超声波协同酶提取法,利用正交试验,优化各种提取方法的试验条件;用四氧嘧啶小鼠进行糖尿病模型的建立,用降糖仪测定小鼠的血糖,考察其降糖效果。结果：酶提取法的提取率为：9.203%;超声提取的提取率为：13.98%;超声协同酶法的提取率为：15.83%。结论：提取的蒲公英多糖均有降低血糖的作用（P〈0.01）。     

基于研究组均值提取与统计推断法研究西格列汀降糖效果的影响因素

目的：探索药物疗效应答差异的影响因素研究模式,寻找西格列汀降糖效果的关键影响因素,为该类药物的个体化选择提供依据。方法：系统检索二肽基肽酶-Ⅳ抑制剂（DPP-Ⅳi）相关的临床研究,采用MINORS表进行文献质量评价。以研究组为单位,提取西格列汀组各变量的平均值。采用Pearson相关或Spearman秩相关分析连续型原因变量与结果变量的相关性;参数检验与非参数检验法研究定性原因变量的不同组间结果是否具有统计学差异;对可能影响西格列汀降糖效果的原因变量进行两两相关性分析,以确定原因变量对结果的影响是否独立存在;对联用药物、降糖药物数量及降糖疗效间的关系进行多水平分析,以确定降糖药物数量对西格列汀疗效的影响。结果：共纳入西格列汀相关文献43篇,研究组47个。结果显示,高糖化血红蛋白（HbA1c）和胰岛素敏感性指标（HOMAIR）,或未联用二甲双胍（Met）的患者能降低更多的HbA1c;基线期体质量越低的患者人群用药后HbA1c<7%的可能性增加;高C-肽、空腹血糖（FBG）和HbA1c,或未联用Met的患者降低FBG更甚;低体质量、BMI、HOMAIR和胰岛素水平,或未联用Met或磺酰脲类药物（SU）的人群能获得更多的餐后血糖（PBG）降低。BMI和体质量,HbA1c和FBG间存在相关性,提示可能存在共同作用。基础降糖药物数量越少的患者,用药后降低PBG的效果越好。结论：本法用于药物疗效影响因素的分析,具有更高的灵敏度。患者对西格列汀疗效的应答可能与其基线期HbA1c、血糖、BMI、胰岛素分泌和敏感性及联用降糖药物等有关。     

Controlled Release of Insulin from Injectable Biodegradable Triblock Copolymer Depot in ZDF Rats

PURPOSE: The purpose of this study was to design sustained release system which provides basal insulin release over a week by one injection in diabetic animals. For an effective injectable formulation and controlled release of insulin, a water soluble biodegradable triblock copolymer of PLGA-PEG-PLGA was used. METHODS: For in vitro release, samples were analyzed by reversed-phase high performance liquid chromatography. Animal studies using ZDF rats have been conducted to demonstrate the bioactivity of the released insulin. Insulin formulation was injected subcutaneously. At designated times, the blood glucose levels and insulin levels of the ZDF rats were measured. RESULTS: The in vitro release of zinc-complexed insulin showed no initial burst and demonstrated constant release rate with the duration of 14 days. Constant steady state plasma levels of exogenous insulin were detected for nearly two weeks indicating constant rate of insulin release in vivo upon single subcutaneous injection. CONCLUSIONS: We conclude that it is feasible to achieve basal insulin levels over a week by a single injection of ReGel formulation. This will provide various advantages, including depot formation without surgery, easy sterilization, straightforward drug loading, simple dose adjustment, system biocompatibility with no inflammatory reaction, and no requirement of using organic solvents.