Local and systemic delivery of high-molecular weight drugs by powder inhalation

The pulmonary route has recently attracted attention as a noninvasive administration route for peptide and protein drugs, and an insulin powder for inhalation was approved by authorities in Europe and the USA. The present study examined usefulness of insulin and gene powders for systemic and local inhalation therapy. We prepared several dry insulin powders by spray drying to examine the effect of additives on insulin absorption. Citric acid appears to be a safe and potent absorption enhancer for insulin in dry powder. However, in the powder with citric acid (MIC0.2 SD) insulin was unstable compared with the other powders examined. To improve insulin stability, a combination of insulin powder and citric acid powder was prepared (MIC Mix). MIC Mix showed hypoglycemic activity comparable to MIC0.2 SD while the insulin stability was much better than that of MIC SD. Next, dry insulin powders with mannitol were prepared with supercritical carbon dioxide (SCF); the powder thus prepared reduced blood glucose level rapidly and was more effective than that prepared by spray drying. Chitosan-pDNA complex powders as a pulmonary gene delivery system were also prepared with SCF and their in vivo activity was evaluated. The addition of chitosan suppressed the degradation of pCMV-Luc during preparation and increased the storage stability. The luciferase activity in mouse lung was evaluated after pulmonary administration of the powders. The chitosan-pDNA powder with an N/P ratio=5 increased the luciferase activity to 27 times that of the pCMV-Luc solution. These results suggest that gene powder with chitosan is a useful pulmonary gene delivery system.     

A Comparison of the Pulmonary Bioavailability of Powder and Liquid Aerosol Formulations of Salmon Calcitonin

PURPOSE: To compare the pulmonary pharmacokinetics and relative bioavailability of salmon calcitonin delivered as aqueous droplets, pH 6.6 and pH 4.8 with that of a spray dried powder in healthy volunteers. METHODS: Spray dried powders (1.6 microm [GSD 2.1]) containing 5% by wt. sCal, 6.25% human serum albumin, 73.55% mannitol and 15% citric acid/sodium citrate were prepared using a Buchi model 190 spray drier. Aqueous solutions were prepared by dissolving the spray dried powder at a sCal concentration of 1.25 mg/ml, pH was adjusted using 21 mM sodium hydroxide. Aerosols were delivered as part of a 4 way cross-over study to 16 healthy volunteers. The Nektar pulmonary delivery device was used to deliver the dry powder aerosol. A Salter nebulizer controlled by a Rosenthal dosimeter was used to deliver the aqueous aerosols. Miacalcin injection was used as the subcutaneous control. Dose delivered to the lung was estimated by gamma scintigraphy. Plasma concentrations of sCal were measured using a radioimmunoassay. RESULTS: Aerosol size distributions were matched, 3.3 microm MMAD and approximately 2.2 GSD. Inhaled flow rates were similar, although not equal, 5.8 and approximately 9.8 l/min respectively for dry powder and liquid inhalations. Lung doses of sCal ranged from 53 to 88 microgm, peripheral lung doses from 25 to 51 microgm. Pharmacokinetic profiles and lung bioavailability relative to subcutaneous injection for all formulations were similar (not statistically significantly different p > 0.05), relative lung bioavailability ranged from 11% to 18%, estimates of relative bioavailability based on peripheral lung dose ranged from 20% to 33%. CONCLUSION: The study showed no difference in pharmacokinetic profiles between the various aerosol dosage forms. pH of the aqueous solutions did not affect kinetics or relative bioavailability.     

Improvement of insulin absorption from intratracheally administrated dry powder prepared by supercritical carbon dioxide process

The purpose of this study was to improve insulin absorption from dry powder after administration in lung without an absorption enhancer. The dry powders, with mannitol as a carrier, were prepared with or without an absorption enhancer (citric acid) by supercritical carbon dioxide (SCF) and spray drying (SD) processes. Insulin powder was precipitated from dimethyl sulfoxide and aqueous solutions by dispersing the insulin solutions from parallel and V-type nozzles, respectively, into supercritical carbon dioxide, which is an antisolvent for insulin. In vitro aerosol performance was evaluated with a cascade impactor. Insulin powder containing citric acid prepared by the SCF method (MIC SCF) showed improved inhalation performance compared with insulin powder prepared by the SD process, although the particle size of the former powder was larger than that in powders prepared by SD. Insulin absorption was estimated from the change in plasma glucose level. The blood glucose level after administration of the insulin powder without citric acid prepared by the SCF process (MI SCF) decreased rapidly, and a significant difference was observed for areas under the curve of change in plasma glucose concentration versus time (AUCs) between MI SCF and the insulin powder without citric acid prepared by the SD process (MI SD). These results suggest that the SCF technique would be useful to prepare dry powders suitable for inhalation.     

Intranasal bioavailability of insulin powder formulations: effect of permeation enhancer-to-protein ratio.

The intranasal administration of powder formulations containing insulin and the permeation enhancer sodium tauro-24,25-dihydrofusidate (STDHF) were investigated in the sheep model. Both the hypoglycemic response and the serum insulin levels increased as the mole ratio of STDHF to insulin was increased from 0 to 16.8. In vitro dissolution rates of the powders and the rapid tmax (approximately 5 min) observed after intranasal administration suggest that the absorption of insulin is not dissolution limited. The bioavailabilities (F) of the powder formulations ranged from 2.9 to 37.8%. In comparison, the F values for a solution formulation with a STDHF:insulin ratio of 8.4 administered as either drops or spray were 15.7 and 37.4%, respectively. The permeation enhancer STDHF increases mucosal permeability and reduces the average molecular weight of the insulin species.     

Characterization of human insulin microcrystals and their absorption enhancement by protease inhibitors in rat lungs

Pulmonary route appears to be an attractive alternative as a non-invasive systemic delivery for peptide and protein drugs. An appropriate formulation, however, is important for increasing their bioavailability in lung. In this study, the human insulin microcrystals were produced. The particle size analysis and scanning electron microscopy (SEM) showed that the microcrystals were uniform and had a monodispersed size distribution (mean diameter = 0.95 microm) for pulmonary delivery. The physicochemical properties of the microcrystals developed were similar to those of the commercial crystalline powder in powder X-ray diffraction (XRD) and differential scanning calorimetry (DSC) analyses. The percentage of high molecular weight proteins (%HMWP), the percentage of other insulin related compounds (%OIRC) and the percentage of A-21 desamido insulin (%D) of the microcrystals were very low. In addition, the cytotoxicity of microcrystals developed and protease inhibitors (aprotinin, bacitracin and soybean-trypsin inhibitor) was investigated, and the enhancement of insulin absorption in the presence of these protease inhibitors at various concentrations was studied. The cell viability of A549 was over 80% at various concentrations of aprotinin and soybean-trypsin inhibitor, except for bacitracin (below 60%). The percent of decrease in blood glucose (D%) was 42.68+/-1.62% after intratracheal instillation of insulin microcrystals (5 U/kg). An enhancement of hypoglycemic effect with protease inhibitors was also found. Soybean-trypsin inhibitor (48.86+/-3.24% at 10 mg/ml; 55.78+/-0.71% at 5 mg/ml; 51.49+/-5.27% at 1 mg/ml) and aprotinin (52.57+/-8.78% at 10 mg/ml; 51.97+/-1.98% at 5 mg/ml; 56.90+/-3.42% at 1 mg/ml) were effective for absorption enhancement. These findings suggest that the use of insulin microcrystals and protease inhibitors would be useful to improve the hypoglycemic effect in pulmonary route.     

Niosomes with sorbitan monoester as a carrier for vaginal delivery of insulin: studies in rats

To prepare and investigate the potential of the niosomes vaginal delivery system for systemic treatment of insulin is the goal of this study. Two kinds of vesicles with Span 40 and Span 60 were prepared by lipid phase evaporation methods with sonication. The niosomal entrapment efficiency was determined by column chromatography. The particle size and morphology of the vesicles also were evaluated. The results showed optimized niosomes prepared in this study had niosomal entrapment efficiency 26.68 +/- 1.41% for Span 40 and 28.82 +/- 1.35% for Span 60, respectively. The particle sizes of Span 40 niosomes and Span 60 niosomes were 242.5 +/- 20.5 nm and 259.7 +/- 33.8 nm, respectively. There were no significant differences in appearance between the two types of vesicles. The hypoglycemic effects, and insulin concentrations after vaginal administration of insulin vesicles to rats were investigated. Compared with subcutaneous administration of insulin solution, the relative pharmacological bioavailability and the relative bioavailability of vaginal administration of insulin vesicles were determined. Compared with subcutaneous administration of insulin solution, the relative pharmacological bioavailability and the relative bioavailability of insulin-Span 60 vesicles group were 8.43% and 9.61%, and insulin-Span 40 niosomes were 9.11% and 10.03% (p > 0.05). Span 60 and Span 40 niosomes were both higher than blank Span 40, Span 60 vesicles, and free insulin physical mixture groups (p < 0.05). The results indicates insulin-Span 60, Span 40 niosomes had an enhancing effect on vaginal delivery of insulin. Although the factors controlling the process for penetration of a portion of vaginally administrated niosomes into bloodstream from vaginal tract is still not fully understood, our results demonstrated that after encapsulation in niosomes of definite type, insulin became an active and efficiently therapeutic agent when administrated vaginally and might be a good carrier for vaginal delivery of protein drugs.     

Niosomes with Sorbitan Monoester as a Carrier for Vaginal Delivery of Insulin: Studies in Rats

To prepare and investigate the potential of the niosomes vaginal delivery system for systemic treatment of insulin is the goal of this study. Two kinds of vesicles with Span 40 and Span 60 were prepared by lipid phase evaporation methods with sonication. The niosomal entrapment efficiency was determined by column chromatography. The particle size and morphology of the vesicles also were evaluated. The results showed optimized niosomes prepared in this study had niosomal entrapment efficiency 26.68 ± 1.41% for Span 40 and 28.82 ± 1.35% for Span 60, respectively. The particle sizes of Span 40 niosomes and Span 60 niosomes were 242.5 ± 20.5 nm and 259.7 ± 33.8 nm, respectively. There were no significant differences in appearance between the two types of vesicles. The hypoglycemic effects, and insulin concentrations after vaginal administration of insulin vesicles to rats were investigated. Compared with subcutaneous administration of insulin solution, the relative pharmacological bioavailability and the relative bioavailability of vaginal administration of insulin vesicles were determined. Compared with subcutaneous administration of insulin solution, the relative pharmacological bioavailability and the relative bioavailability of insulin-Span 60 vesicles group were 8.43% and 9.61%, and insulin-Span 40 niosomes were 9.11% and 10.03% (p > 0.05). Span 60 and Span 40 niosomes were both higher than blank Span 40, Span 60 vesicles, and free insulin physical mixture groups (p < 0.05). The results indicates insulin-Span 60, Span 40 niosomes had an enhancing effect on vaginal delivery of insulin. Although the factors controlling the process for penetration of a portion of vaginally administrated niosomes into bloodstream from vaginal tract is still not fully understood, our results demonstrated that after encapsulation in niosomes of definite type, insulin became an active and efficiently therapeutic agent when administrated vaginally and might be a good carrier for vaginal delivery of protein drugs.     

胰岛素吸入粉雾剂的体外沉降及大鼠体内吸收促进剂的药效学评价

目的采用喷雾干燥法制备胰岛素吸入粉雾剂，对吸入粉雾剂体外沉降性质进行考察，并对其吸收促进剂在大鼠体内的药效学进行初步研究。方法采用中国药典(2000版)附录XH的装置测定粉末的体外有效部位沉积量，以葡糖氧化酶法（GOD-PAP法）测定大鼠的血糖浓度来评价其降血糖效果。结果喷雾干燥法制得的胰岛素吸收粉雾剂沉积量在各湿度下均大于40％，在气流量≥18 L·min^-1的情况下其有效部位沉积量变化不大。8 mmol·L^-1/dose牛黄胆酸钠［PA=59.91%,C_nadir=(33±6)%］和10 mmol·L^-1/dose去氧胆酸钠［PA=47.46%，C _nadir=(32±7)%］对胰岛素肺部吸收促进作用明显。而1%辛酸钠、1%十二烷基硫酸钠、250 μg/dose卵磷脂和10 mmol·L^-1/dose EDTA并未显示明显效果。结论制得的胰岛素吸收粉雾剂沉积量受湿度影响小，环境湿度依赖性和吸气流量依赖性小。8 mmol·L^-1/dose牛黄胆酸钠和10 mmol·L^-1/dose去氧胆酸钠可有效促进胰岛素干粉吸入剂的降血糖效果。     

口腔粘膜内酶对胰岛素口腔吸收的影响

目的　研究口腔粘膜内酶对胰岛素口腔吸收的影响。方法　用三氯醋酸沉淀法,考察胰岛素在仓鼠口腔粘膜匀浆物中不同条件下的降解情况。在胰岛素口腔喷雾剂中加入酶抑制剂(杆菌肽、屈来赛多)和去氧胆酸钠,考察了大鼠经口腔喷入胰岛素后的血糖降低情况。结果　小肠粘膜细胞内酶活性远高于口腔粘膜细胞中的酶的活性。杆菌肽、屈来赛多和去氧胆酸钠均能抑制口腔粘膜内胰岛素的降解。在相同浓度下,去氧胆酸钠的酶抑制作用比杆菌肽弱,但比屈来赛多强。胰岛素在正常仓鼠口腔内的降解显著大于糖尿病仓鼠。胰岛素溶液中加入杆菌肽、屈来赛多及去氧胆酸钠后,给正常大鼠口腔喷雾给药,药理相对生物利用度分别有不同程度的提高。只含胰岛素的喷雾液(胰岛素空白液) ,喷雾液中加入屈来赛多(0.1% ) ,加入杆菌肽(0.5% ) ,去氧胆酸钠(1% ) ,与sc胰岛素比较药理相对生物利用度分别为2.89% ,4.84% ,7.52 %和9.60%。结论　口腔粘膜中的酶可以限制胰岛素在口腔粘膜的吸收     

口腔粘膜内酶对胰岛素口腔吸收的影响

目的：研究口腔粘膜内酶对胰岛素口腔吸收的影响。方法：采用三氯醋酸沉淀法，考察胰岛素在仓鼠口腔粘膜匀浆物中不同条件下的降解情况，在胰岛素口腔喷雾剂中加入酶抑制剂（杆菌肽，屈来赛多）和去氧胆酸钠，考察了大鼠经口腔喷入胰岛素后的血糖降低情况。结果：小肠粘膜细胞内酶活性远高于口腔粘膜细胞中的酶的活性。杆菌肽，屈来赛多和去氧胆酸钠均能抑制口腔粘膜内胰岛素的降解，在相同浓度下，去氧胆酸钠的酶抑制作用比杆菌肽弱，但比屈来赛多强，胰岛素在正常仓鼠口腔内的降解显著大于糖尿病仓鼠。胰岛素溶液中加入杆菌肽，屈来赛多及去氧胆酸钠后，给正常大鼠口腔喷雾给药，与皮下注射胰岛素比较，药理相对生物利用率分别有不同程度的提高，只含胰岛素的喷雾液（胰岛素空白液），药理相对生物利用度为2．89％，喷雾液中加入屈来赛多（0．1％），药理相对生物利用率为4．84％，加入杆菌肽（0．5％），药理相对生物利用度为6．60％，加入去氧胆酸钠（1％），药理相对生物利用度为9．60％，结论：口腔粘膜中的酶可以限制胰岛素在口腔粘膜的吸收，为了提高胰岛素口腔吸收的生物利用度，应该尽量降低口腔粘膜中的酶对胰岛素的降解。     

Absorption Enhancement of Intranasally Administered Insulin by Sodium Taurodihydrofusidate (STDHF) in Rabbits and Rats

The enhancement of nasal insulin absorption by sodium taurodihydrofusidate (STDHF) was studied in rabbits and rats. Using identical nasal formulations remarkable interspecies differences were observed. The fusidate derivative at 1% (w/v) enhanced nasal insulin bioavailability from 0.9 to 5.2% and from 0.3 to 18.0% in rabbits and rats, respectively. In both species the insulin formulations with STDHF resulted in strong hypoglycemic responses. Coadministration with the trypsin inhibitor aprotinin tended further to increase insulin bioavailability in rats and decrease insulin bioavailability in rabbits; however, these aprotinin effects were not statistically significant. Addition of the aminopeptidase inhibitor bacitracin to the STDHF containing formulation did not have any effect on insulin bioavailability in rats. Hence, STDHF is a potent enhancer of nasal insulin absorption, probably both by facilitating insulin transport through the nasal mucosa and possibly also by inhibiting enzymatic degradation. Further, interspecies differences and, experimental animal conditions can greatly affect nasal drug absorption.     

不同吸收促进剂对大鼠口腔胰岛素吸收作用的影响

目的 :研究胰岛素的口腔吸收及吸收促进剂、酶抑制剂对药物吸收的影响。方法 :以血糖水平为指标 ,考察各种吸收促进剂和酶抑制剂经糖尿病模型的大鼠口腔给药后对胰岛素溶液 (insulinsolution ,INS SOL)和胰岛素贴片 (insulinpatch ,INS PAT)降血糖作用的影响 ,以皮下注射为对照 ,计算不同条件下INS SOL和INS PAT的药理生物利用度。结果 :不加吸收促进剂的条件下 ,32U·kg-1的INS SOL和10U·kg-1的INS PAT经口腔给药后的生物利用度较低 (0 .77%和 1.82 % )。 3%去氧胆酸钠和 5 %壬苯醇醚均能显著增加INS SOL和INS PAT的降血糖作用 (P <0 .0 5 )。其中 3%去氧胆酸钠作用最明显 ,INS SOL和INS PAT的生物利用度增加了近 3倍(2 .83%和 7.2 6 % )。结论 :胰岛素可通过口腔吸收 ,适当的吸收促进剂对其吸收具有显著的促进作用。     

胰岛素肺部给药对大鼠的降血糖作用

目的:研究胰岛素溶液(INS SOL)经正常大鼠肺部给药后的降血糖作用。方法:以血糖水平为指标,考察各种吸收促进剂以及酶抑制剂经正常大鼠肺部给药后对INS SOL降血糖作用的影响。同时比较了INS SOL在两种pH条件下的降血糖作用,并以皮下注射为对照,计算不同条件下INS SOL的药理生物利用度(pharmacologicalbioavailability,PBA)。结果:05u·kg^-1的INS SOL经肺部给药后即有明显的降血糖作用,在不加吸收促进剂的条件下的PA为260%。辛酸钠、胆酸钠、苄泽35、苄泽78和酶抑制剂杆菌肽均显著地增加INS SOL的降血糖作用。INS SOL在pH3时的降血糖效果比pH7时有显著的提高。结论:INS SOL经肺部给药后有显著的降血糖效果。     

Proteolytic enzymes as a limitation for pulmonary absorption of insulin: in vitro and in vivo investigations

In vitro biodegradation of insulin in lung cytosol and subcellular pellets of normal and diabetic rats was investigated. Rat lung was homogenized and subcellular fractions were isolated by ultracentrifugation. Degradations of [125I]-insulin after incubation with lung cytosol or subcellular pellets was determined using the trichloroacetic acid method. The results show that insulin is highly degraded in cytosol and subcellular pellets. Cytosolic insulin degradation was strongly inhibited by bacitracin or sodium cholate. The degradation of insulin in the lung cytosol from diabetic rats was significantly less than from normal rat. The lung protease activity reached a maximum at pH 7.4. Enzyme inhibitors like bacitracin and sodium cholate noticeably enhanced the relative pharmacological bioavailability of insulin when given intratracheally with insulin to normal rats. Acidic insulin solutions (pH 3.0) had more pronounced hypoglycaemic effects than neutrol solution (pH 7.0). These in vitro and in vivo results suggest that the proteolytic enzymes in the lung limit pulmonary delivery of insulin. The coadministration of protease inhibitors would be a useful approach for improving the pulmonary absorption of insulin.     

Oral bioavailability of pentachlorophenol from soils of varying characteristics using a rat model

Evidence accumulated during the last two decades suggests that only a fraction of any chemical in soil is available to organisms, and soil-related effects on bioavailability should be considered in optimizing soil remediation cleanup levels. In the current study, the absolute and relative bioavailabilities of pentachlorophenol (PCP) from freshly spiked and environmentally aged soils varying in organic carbon content, clay content, and pH were examined using a rat model. PCP is a broad-spectrum biocide widely used as a wood preservative, and thus is ubiquitous in the environment. Soils and corn oil containing equivalent levels of PCP were administered to male Sprague-Dawley rats by gavage at 2 dose levels: 100 and 200 microg/kg body weight. Equivalent doses were also given intravenously. The areas under the plasma concentration of PCP versus time curves were calculated, and absolute and relative bioavailabilities of PCP from each soil were determined. At a dose of 100 microg/kg body weight, the absolute bioavailabilities of PCP across soils ranged from 36% to 65%, and the relative bioavailabilities ranged from 48% to 82%. At the higher dose level (200 microg/kg body weight), absolute and relative bioavailability ranges were somewhat higher at 46% to 77% and 52% to 87%, respectively. All soils decreased absolute PCP bioavailability significantly at both dose levels and relative bioavailability at the lower PCP dose level. At the higher dose level, only one of the two field-contaminated soils decreased the relative PCP bioavailability. The data indicate that PCP-soil interactions do significantly decrease the oral bioavailability of PCP from soil, but no obvious correlation was observed between soil properties and bioavailabilities.     

胰岛素粉雾剂肺部给药对大鼠的降血糖作用

目的:观察胰岛素吸入粉雾剂肺部给药后的降血糖效果.方法:胰岛素与合适辅料制成的干粉经大鼠肺气管切口吹入肺中,测定随后7小时的血糖浓度,以血糖曲线上面积(AAC)为指标对其药效进行评价.结果:吸入胰岛素剂量为20,10,5和2.5U/kg时,最低血糖浓度可分别降至给药前的6.5％,16.6％,24.6％和57.0％;剂量为5 U/kg吸入给药的AAC值和5U/kg皮下注射给药的AAC值相近;AAC值与对数剂量间存在线性关系.结论:胰岛素肺部给药的降血糖效果明显且作用迅速.     

Investigation of lectin-modified insulin liposomes as carriers for oral administration

The aim of this study was to design and characterize lectin-modified liposomes containing insulin and to evaluate the potential of these modified colloidal carriers for oral administration of peptide and protein drugs. Wheat germ agglutinin (WGA), tomato lectin (TL), or Ulex europaeus agglutinin 1 (UEA1) were conjugated by coupling their amino groups to carbodiimide-activated carboxylic groups of N-glutaryl-phosphatidylethanolamine (N-glut-PE). Insulin liposomes dispersions were prepared by the reverse-phase evaporation technique and modified with the lectin-N-glut-PE conjugates. Lectin-modified liposomes were characterized according to particles size, zeta potential and entrapment efficiency. The hypoglycemic effect indicated by pharmacological bioavailability of insulin liposomes modified with WGA, TL and UEA1 were 21.40, 16.71 and 8.38% in diabetic mice as comparison with abdominal cavity injection of insulin, respectively. After oral administration of the insulin liposomes modified with WGA, TL and UEA1 to rats, the relative pharmacological bioavailabilities were 8.47, 7.29 and 4.85%, the relative bioavailability were 9.12, 7.89 and 5.37% in comparison with subcutaneous injection of insulin, respectively. In the two cases, no remarkable hypoglycemic effects were observed with the conventional insulin liposomes. These results confirmed that lectin-modified liposomes promote the oral absorption of insulin due to the specific-site combination on GI cell membrane.     

Sustained release of insulin from insoluble inhaled particles

Conventional slow‐acting insulin preparations for subcutaneous injection, e.g., suspensions of the complex with protamine and/or zinc, were reformulated as dry powders for inhalation and the insoluble aerosol tested for providing sustained insulin plasma levels. Large porous particles made of lactose, albumin, and dipalmitoylphosphatidylcholine, and incorporating insulin, protamine, and/or zinc chloride were prepared using spray‐drying. Integrity of insulin after spray‐drying and insulin insolubilization in spray‐dried particles was verified in vitro. The pharmacokinetic profile of the formulation delivered by inhalation and subcutaneous injection was assessed in vivo in the rat. The formulation process of insulin as dry powders did not alter insulin integrity and did not impede, in most cases, insulin insolubilization by protamine and/or zinc. Large porous insulin particles presented 7 μm mass mean geometric particle diameters, 0.1 g/cm³ bulk powder tap densities and theoretical aerodynamic diameters suitable for deep lung deposition (in the range of 2.2–2.5 μm). The dry powders exhibited 40% respirable fractions in the Andersen cascade impactor and 58–75% in the Aero‐Breather™. Insoluble inhaled insulin provided sustained insulin plasma levels for half a day, similar to injected insulin, and exhibited a bioavailability of 80.5% relative to subcutaneous injection of the same formulation. Drug Dev. Res. 48:178–185, 1999. ©1999 Wiley‐Liss, Inc.     

Effects of Various Protease Inhibitors on the Intestinal Absorption and Degradation of Insulin in Rats

The effects of protease inhibitors on the intestinal absorption of insulin were investigated in situ in closed small and large intestinal loops in rats, and the stability of insulin was examined in homoge-nates of the small and large intestine. The intestinal absorption of insulin was evaluated by its hypoglycemic effect. When insulin alone was administered into small or large intestinal loops, no marked hypoglycemic response was observed in either region. Of the coadministered protease inhibitors, soybean trypsin inhibitor (1.5, 10 mg/ml) marginally promoted insulin absorption from the large intestine, whereas aprotinin (10 mg/ml) did to a moderate degree. However, a significant hypoglycemic effect was obtained following large intestinal administration of insulin with 20 mM of Na-glycocholate, camostat mesilate and bacitracin, when compared with the controls. In contrast, we found little hypoglycemic effect following small intestinal coadministration of insulin with these protease inhibitors. In the stability experiment, bacitracin, camostat mesilate and Na-glycocholate were effective in reducing insulin degradation in both small and large intestinal homogenates. It was found that the reduction in the proteolytic rate of insulin was related to the decrease in plasma glucose concentration by these protease inhibitors in the large intestine. These findings suggest that coadministration of protease inhibitors would be useful for improving the large intestinal absorption of insulin.     

Pharmacokinetic study of niosome-loaded insulin in diabetic rats

Background and the purpose of the study

Encapsulation of human insulin in lipid vesicular systems such as niosomes was sought as a route to protect this protein against proteolytic enzymes and to improve its oral bioavailability. The purpose of this study was to assess the effect of insulin encapsulation in niosomes on oral bioavailability in diabetic rats.

Methods

Recombinant human insulin was entrapped in multilamellar niosomes composed of polyoxyethylene alkyl ether surfactants (Brij 52 and Brij 92) or sorbitan monostearate (Span 60) and cholesterol. The amount of insulin released in simulated intestinal fluid (SIF) and simulated gastric fluid (SGF) were measured at 37°C. The protection of entrapped insulin against pepsin, α-chymotrypsin and trypsin were evaluated in comparison with free insulin solution. Diabetes was induced by IP injection of streptozotocin (65 mg/kg) in male wistar rats and effects of orally administered niosomes and subcutaneously injected insulin on hypoglycemia and elevation of insulin levels in serum were compared.

Results and conclusion

The extent and rate of insulin release from Brij 92 and Span 60 vesicles were lower than that of Brij 52 niosomes (P<0.05). Vesicles protected insulin in comparison with free insulin solution against proteolytic enzymes (P<0.05) significantly. Animals treated with oral niosome-encapsulated insulin (100 IU/kg) showed decreased levels of blood glucose and elevated serum insulin, which in the case of Brij 92 niosomes, hypoglycemic effect was significant (P<0.05).Niosomes were also stable in solubilizing bile salt solutions and could effectively prolong the release of insulin in both SGF and SIF. Results of this study showed that niosomes may be utilized as oral carriers of insulin; however, to increase bioavailability of insulin, further studies on the protease inhibitor co-encapsulation in niosomal formulations might be helpful.