Absorption Enhancing Effect of Labrasol on the Intestinal Absorption of Insulin in Rats

The oral absorption enhancing effect of Labrasol? has been studied in rats using insulin as a model peptide/protein drug. Insulin solution was prepared by dissolving insulin in pH 7.4 buffer followed by the addition of Labrasol. The insulin concentration was 50.0 IU/ml. The test insulin/Labrasol solution was administered to the jejunum, ileum and ascending colon of rats at 10.0 IU/kg. After administration, blood samples were collected for 5 h and serum glucose levels and insulin levels were measured. In another group of rats, insulin solution was injected intravenously at 1.0 IU/kg, and both serum glucose and insulin levels were measured. The pharmacological availability of insulin from Labrasol solution was found to be 3.9, 8.9 and 9.1% following jejunal, ileal and colonic administrations, respectively, by comparing the serum glucose level vs. time profiles obtained after intestinal and i.v. administrations. By comparing the serum insulin levels vs. time profiles, the bioavailability of insulin was found to be 0.25 and 0.20% for intra-ileum and colonic administrations, respectively. The hypoglycemic effect of insulin after intra-ileum administration showed a dose-dependency in the insulin dose range from 10.0 to 1.0 IU/kg. These results suggest the absorption enhancing effect of Labrasol on the intestinal absorption of insulin in rats.     

Absorption enhancing effect of labrasol on the intestinal absorption of insulin in rats

The oral absorption enhancing effect of Labrasol has been studied in rats using insulin as a model peptide/protein drug. Insulin solution was prepared by dissolving insulin in pH 7.4 buffer followed by the addition of Labrasol. The insulin concentration was 50.0 IU/ml. The test insulin/Labrasol solution was administered to the jejunum, ileum and ascending colon of rats at 10.0 IU/kg. After administration, blood samples were collected for 5 h and serum glucose levels and insulin levels were measured. In another group of rats, insulin solution was injected intravenously at 1.0 IU/kg, and both serum glucose and insulin levels were measured. The pharmacological availability of insulin from Labrasol solution was found to be 3.9, 8.9 and 9.1% following jejunal, ileal and colonic administrations, respectively, by comparing the serum glucose level vs. time profiles obtained after intestinal and i.v. administrations. By comparing the serum insulin levels vs. time profiles, the bioavailability of insulin was found to be 0.25 and 0.20% for intra-ileum and colonic administrations, respectively. The hypoglycemic effect of insulin after intra-ileum administration showed a dose-dependency in the insulin dose range from 10.0 to 1.0 IU/kg. These results suggest the absorption enhancing effect of Labrasol on the intestinal absorption of insulin in rats.     

壳聚糖及其衍生物包覆脂质体对胰岛素肠道吸收的影响

目的考察壳聚糖及其衍生物包覆脂质体对胰岛素肠道吸收的影响。方法采用逆相蒸发制备胰岛素脂质体；采用在体肠灌流法研究壳聚糖及其衍生物包覆胰岛素脂质体的肠道吸收；用酶-苯酚法测定血糖值；用放射免疫法测定血清和肠组织中胰岛素含量。结果壳聚糖(CH)、壳聚糖-EDTA轭合物(CEC)包覆胰岛素脂质体和CH-CEC双层包覆胰岛素脂质体的最佳吸收部位均集中在十二指肠，胰岛素溶液的最佳吸收部位在结肠，而未包覆胰岛素脂质体和N-三甲基壳聚糖盐酸盐(TMC)包覆胰岛素脂质体的最佳吸收部位尚不能确定。在各肠段中，以CH-CEC双层包覆胰岛素脂质体的吸收最佳。结论壳聚糖及其衍生物包覆脂质体能促进胰岛素经肠道吸收，并可提高其在肠道中的稳定性。     

Preparation of unilamellar fusogenic liposomes using the Sendai virus

We describe a method to prepare unilamellar fusogenic liposomes (FL) that can deliver their content directly into tissue cells and cancer cells of living animals as well as into cultured animal cells. The method consists of three important steps: 1) preparation of unilamellar liposomes encapsulating the molecules to be delivered: 2) preparation of the Sendai virus using fertilized chicken eggs; and 3) preparation of FLs by fusing UV-inactivated Sendai virus particles and naked liposomes. Purified FLs are stable at 4 degrees C for 3 weeks and can be stored indefinitely below -70 degrees C without loss of activity. The FL-mediated delivery is applicable to cells originating from various tissues, including those of humans, monkeys, cows, dogs, mice, rats, hamsters, and rabbits. However, human primary T cells primary B cells, and B cell lines are not susceptible to delivery. FLs are best suited to an examination of the biological activity of cytokines in the tissue cells of living animals. We also describe the possibility of developing FL-based hybrid vectors, which would mimic the functions of viruses and chromosomes.     

Bioadhesive Chitosan-Coated Cationic Nanoliposomes With Improved Insulin Encapsulation and Prolonged Oral Hypoglycemic Effect in Diabetic Mice

Oral administration of insulin is hampered by the lack of carriers that can efficiently achieve high encapsulation, avoid gastric degradation, overcome mucosal barriers, and prolong the hypoglycemic effect. Chitosan (CS)-coated insulin-loaded cationic liposomes have been developed and optimized for improved oral delivery. Liposomes were prepared cationic to improve insulin encapsulation. CS was selected as a mucoadhesive coat to prolong the system's residence and absorption. The performance of CS-coated liposomes compared with uncoated liposomes was examined in vitro, ex vivo, and in vivo in streptozotocin-induced diabetic mice. Free uncoated liposomes showed high positive zeta potential of +58.8 ± 2.2 mV that reduced (+29.9 ± 1.4 mV) after insulin encapsulation, confirming the obtained high entrapment efficiency of 87.5 ± 0.6%. CS-coated liposomes showed nanosize of 439.0 ± 12.3 nm and zeta potential of +60.5 ± 1.9 mV. In vitro insulin release was limited to 18.9 ± 0.35% in simulated gastric fluid, whereas in simulated intestinal fluid, 73.33 ± 0.68% was released after 48 h from CS-coated liposomes. Ex vivo intestinal mucoadhesion showed increased tissue residence of CS-coated liposomes compared with uncoated liposomes. A striking reduction in the glucose level was observed 1 h after oral administration of CS-coated liposomes and maintained up to 8 h (p <0.01 vs. insulin solution or uncoated liposomes) within the normal value 129.29 ± 3.15 mg/dL. In conclusion, CS-coated insulin-loaded cationic liposomes improved loading efficiency with promising prolonged pharmacological effect.     

Pharmacokinetics of a C5a receptor antagonist in the rat after different sites of enteral administration

Pharmacokinetics of the orally active, cyclic peptide complement factor C5a receptor antagonist, AcF-[OP(d-Cha)WR] (PMX53) were determined in the rat. Biliary excretion of the unchanged drug was a major route of elimination after intravenous administration, but not following oral administration. Portal and peripheral blood levels of PMX53 were determined after oral administration or direct injection into the ileum, colon or local administration into the rectum. PMX53 was rapidly absorbed from mucosal sites, with peak plasma levels occurring as early as 5 min post-administration. Early portal blood levels were consistently higher than peripheral levels following ileal, colonic and rectal administration, but not after oral dosing. The results suggest that hepatic elimination occurs rapidly with higher (≥100 ng/ml) peripheral blood levels of the drug. Combination of PMX53 with the excipient chitosan resulted in significantly higher peripheral levels of the drug following ileal and colonic application, but not with buccal or oral administration. Buccal administration resulted in a similar plasma pharmacokinetic profile to oral administration. These results suggest that PMX53 is rapidly absorbed across mucosal membranes in the rat, and that administration using excipients such as chitosan may offer a method of increasing bioavailability.     

Effectiveness of submicron-sized, chitosan-coated liposomes in oral administration of peptide drugs

The mucoadhesive behavior of chitosan-coated liposomes in the intestinal tract of the rat was examined to elucidate their particle size effects on the absorption of an entrapped drug, calcitonin. The intestine was removed from rats after oral administration of liposomes containing a fluorescent dye, and its various parts were observed with confocal laser scanning microscopy. Penetration of submicron-sized liposomes (ssLip) or chitosan-coated ssLip (ssCS-Lip) into the mucosa was observed, while such behavior was not observed for the multilamellar liposomes, even when coated with chitosan (CS-Lip). The retentive property of ssCS-Lip was confirmed by measuring the amount of dye in each part of the intestine. The pharmacologic effects of calcitonin-loaded liposomes of different particle size were measured after oral administration in rats. The pharmacologic effect of oral administration of ssLip coated with chitosan was detected up to 120 h after administration. The extensive pharmacologic effect of ssCS-Lip was attributed to their prolonged retention in the intestinal mucosa, partly owing to their penetrative property into the intestinal mucosa. The chitosan-coated ssLip, with their higher retentive property in the intestinal tract, are much more effective than ssLip and CS-Lip in improving the enteral absorption of peptide drugs.     

Absorption of lefradafiban from different sites of the gastrointestinal tract

AIMS: Fibrinogen receptor antagonists show a close relationship between plasma concentrations and inhibitory effect. Optimal efficacy at an acceptable bleeding risk requires low inter- and intrasubject variability on low peak trough fluctuation in receptor occupancy and therefore also of plasma concentrations. Therefore, the enteral absorption of lefradafiban, an orally available fibrinogen receptor antagonist prodrug, was investigated after local administrations to different sites of the gastrointestinal tract in order to investigate the feasibility of an oral extended release formulation. METHODS: Twelve healthy male subjects received in a randomised, open-labelled, four-period crossover trial four consecutive administrations of lefradafiban: 1. orally; 2. administration into the jejunum, 3. administration into the lower jejunum/ileum (300 cm distally to the teeth), and 4. administration into the lumen of the sigmoid region (30 cm proximally to the anus). Local intestinal administrations were performed through a gastrointestinal tube. RESULTS: Compared with oral administration, ratios [mean (two-sided 90% confidence intervals)] of maximum drug plasma concentrations and AUC(0,24 h) of fradafiban were 1.05 (0.80, 1.39) and 1.06 (0.85,1.31) after jejunal, 0.98 (0.75,1.30) and 0.98 (0.79,1.21) after ileal, 0.52 (0.39,0.69) and 0.68 (0.55,0.85) after colonic administration. Urinary excretion of fradafiban was about 16% of the dose after oral, jejunal and ileal applications whereas after rectal administration about 11% were excreted. CONCLUSIONS: Lefradafiban is absorbed throughout the entire gastrointestinal tract. Therefore, an extended release formulation seems to be feasible with regard to bioavailability.     

Improvement of large intestinal absorption of insulin by chemical modification with palmitic acid in rats.

The intestinal absorption of 125I-labelled palmitoyl insulin was examined following administration into in-situ closed large intestinal loops of rats. When mono- and dipalmitoyl insulins (Palins-1 and Palins-2, respectively) were administered in polyoxyethylene hydrogenated castor oil (HCO 60) micellar system into intestinal loops, a marked increase in plasma radioactivity and a corresponding disappearance of residual radioactivity in the intestinal lumen were observed in the following rank order: Palins-2 greater than Palins-1 greater than native insulin. In addition, the derivatives were more stable than native insulin in the mucosal tissue homogenates of the large intestine. These results suggest that chemical modification of insulin with palmitic acid may not only increase the lipophilicity of insulin but also reduce its degradation, resulting in the increased transfer of insulin across the large intestinal mucous membrane. The linoleic acid-HCO 60 mixed micelles system did not have a significant effect on the large intestinal absorption of radioactivity associated with the lipophilic insulin analogues.     

Assessment of intestinal permeability in rats by permeation of inulin-fluorescein

The measurement of intestinal permeability is widely used to assess different aspects of mucosal barrier disorders and related disease states, and has been proposed for evaluation of disease activity. To provide a simple method for assessment of intestinal permeability, we examined the permeation of inulin-fluorescein (InFl) in rat models of small intestinal injury and colitis. Small intestinal or colonic inflammation was induced by either i.p. administration of indomethacin or rectal administration of trinitrobenzene sulfonic acid (TNBS), respectively. For monitoring of intestinal permeability, InFl was administered orally or rectally to rats with small intestinal or colonic inflammation, respectively, and its level in blood was determined by the fluorescence intensity in the plasma. In small intestinal injury, InFl reached its peak in plasma 3 h after oral administration, while in colitis the InFl peak was reached 1 h after rectal administration. The highest permeability was observed at 72 h or 12 h after induction of small intestinal or colonic inflammation, respectively. In small intestinal injury the InFl permeation, as measured by its plasma level prior to sacrifice, was in agreement with intestinal damage evaluated after sacrifice. In colitis, the permeability at 12 h after induction of the disease correlated well with mortality. These findings demonstrate that InFl can be used as a novel, safe and easy-to-use probe for the evaluation of gut permeation and follow-up of gastrointestinal injury.     

Changes in Intestinal Mucosal Permeability Caused by Nonprotein Thiol Loss in Rats

The barrier selectivity of the intestinal mucosal membrane permeability may be impaired in certain disease conditions. Membrane permeability was previously shown to be correlated with changes in nonprotein thiol in rat intestinal tissue by the everted sac method. In the present study, the mucosal effects of alloxan-induced diabetes and chronic alcohol administration to intact rats, as well as pre-treatment with diethyl maleate, ethanol, and salicylate, were investigated. In each case, a drop of mucosal nonprotein thiol was associated with an increased absorption of cefoxitin, cefmetazole, and phenol red, hydrophilic compounds that are poorly absorbed through intact membrane, and with a decreased absorption of L-phenylalanine. The effect of nonprotein thiol loss on rectal absorption of cefoxitin, cefmetazole, and phenol red was greater than that on the small intestinal absorption. The increase in phenol red absorption by diethyl maleate in the in vitro everted sac method correlated with Ca²⁺ release from the intestinal mucosa, which was induced by nonprotein thiol loss. Resistance to the effect of nonprotein thiol loss on Ca²⁺ homeostasis was greater in rat ileum than in rat colon (including rectum). The administration of cysteamine as an exogenous nonprotein thiol restored non-protein thiol levels in the mucosa along with the barrier function of the intestinal mucosa to the absorption of cefoxitin, cefmetazole, and phenol red. In contrast, the transport of L-phenylalanine in the small intestinal mucosa was not restored by cysteamine treatment.     

Enhanced enteral bioavailability of vancomycin using water-in-oil-in-water multiple emulsion incorporating highly purified unsaturated fatty acid

The aim of this study was to evaluate the potential of an emulsion incorporating unsaturated fatty acids to improve the mucosal absorption of poorly absorbed drugs from rat intestinal loops in situ, using a water-in-oil-in-water (W/O/W) multiple emulsion. Vancomycin hydrochloride (VCM) was used as a model drug with low oral bioavailability. The entrapment efficiency of VCM in the emulsion was approximately 60% and remained constant over storage for 1 month at 4 degrees C. The emulsion incorporating C18 unsaturated fatty acids or docosahexaenoic acid (DHA) markedly enhanced VCM absorption after colonic and rectal dosing. The effectiveness of DHA on VCM colonic absorption improvement was the same as that of oleic acid, and less than that of linoleic and linolenic acids. For rectal dosing, bioavailability was similar among various emulsions, in the range 40-50%. The effect of the emulsion incorporating oleic acid or DHA on improving VCM enteral bioavailability was not increased proportional to the incorporated amount. The electrical resistance of membranes was not changed by the incorporation of various fatty acids in emulsions. Our results indicated that W/O/W emulsions incorporating C18 unsaturated fatty acid or DHA were useful carriers for improving the absorption of poorly absorbable drugs via the intestinal tract without gross changes to tight junction function.     

Effect of medium-chain glycerides on the intestinal absorption of phenol red: studies on mechanisms of the promoting effect

To elucidate the mechanisms of the promoting action of medium-chain glyceride (MCG) on the intestinal absorption of phenol red (PR), the effect of different MCG dose levels on PR absorption, the absorption kinetics of MCG itself, and the mucosal reduced glutathione (GSH) levels were investigated to determine whether or not they are related to the promoting action of MCG. The absorption of PR was found to be enhanced with an increase in MCG dose (as an emulsion in the range 0.5-4.0%). Urinary excretion of PR was reduced by absorbed MCG components, which also resulted in an increase in the AUC. Moment analysis of plasma PR concentrations after intraintestinal administration revealed a shorter mean absorption time (MAT) for the emulsion formulation than for the control. The mucosal GSH level was lowered by the administration of the MCG emulsion (2-10%) into the intestinal lumen. Pretreatment with a 4% MCG emulsion decreased GSH levels to one-half of the control. In these cases, the level of mucosal GSH seemed to remain constant to the end of the experiment after it had decreased during the initial period. On the other hand, results from the in situ loop or recirculation study showed that enhancement of PR absorption appeared in the earlier period and then gradually decreased. The time course of the PR absorption rate, estimated from plasma concentration data, was similar to that of the MCG absorption rate. These results suggest that the promotion of intestinal absorption is related to the absorption kinetics of MCG itself, but not directly to the level of mucosal GSH.     

Improvement of absolute bioavailability of normally poorly absorbed drugs: inducement of the intestinal absorption of streptomycin and gentamycin by lipid-bile salt mixed micelles in rat and rabbit

The effect of lipid—bile salt mixed micelles on the intestinal absorption of streptomycin and gentamycin was investigated in the loop of small and large intestine of rat. While bile salts micellar solutions did not enhance the absorption of aminoglycosides, their mixed micellar solutions including monoolein, oleic or lauric acid markedly enhanced their absorption. However, lecithin—bile salt mixed micellar solution did not affect the absorption. Pretreatment with mixed micellar solutions showed neither a direct action on the mucosal membrane nor a transient increase in permeability to aminoglycosides.Improvement of bioavailability in the rabbit was evaluated using various formulations and different routes of administration. In the rectal administration, not only mixed micellar solutions but also lyophilized mixed micelles powder improved bioavailability. The duodenal administration of mixed micellar solution, however, was not effective, indicating that enhanced absorption of drugs by mixed micelles may be more pronounced with rectal administration.     

体外扩散池法评价瑞巴派特经大鼠肠黏膜的透过特性

研究瑞巴派特经空肠、回肠和结肠黏膜的透过特性。制备大鼠不同区段肠黏膜样本，使用体外扩散池法，评价80 μmol·L^-1瑞巴派特经空肠、回肠和结肠黏膜的经时吸收方向(M-S)和分泌方向(S-M)的透过量和透过系数(P_app)，瑞巴派特在接受室中的浓度用HPLC法测定。结果表明，瑞巴派特经空肠和回肠的透过性高于结肠。比较M-S方向和S-M方向的透过性发现，药物在空肠和结肠经两个方向的透过性无显著差异，但经回肠透过时，分泌方向的透过性高于吸收方向的透过性(P<0.05)。瑞巴派特经大鼠肠黏膜的透过性存在区段差异。瑞巴派特可能不受肠黏膜P-糖蛋白转运体的调控。     

The dose-related hypoglycemic effects of insulin emulsions incorporating highly purified EPA and DHA

The dose-related pharmacological effects of insulin emulsion incorporating highly purified eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were investigated. Water-in-oil-in-water multiple emulsions (insulin dose, 0, 10, 25 and 50 IU/kg) incorporating 2% DHA or EPA were administered directly into the colonic and rectal loops in situ. Serum insulin levels rose and serum glucose levels decreased in an insulin dose-related fashion. The relationship of insulin dose and C(max) or AUC(insulin) was linear at the rectum, but a non-linear relationship was observed at the colon. The trend was more predominant in DHA. In the in vivo rectal absorption experiment using emulsions incorporating 2% DHA, 5 IU/kg of insulin emulsion produced a rapid, transitory increase in serum insulin levels and strong reduction of serum glucose levels. The pharmacological availability determined from the dose-response curve by s.c. administration of insulin reached 43.2+/-26.3% (mean+/-S.D.). Mucosal irritation caused by administration of emulsions incorporating 2% EPA or DHA was evaluated by a lactate dehydrogenase (LDH) release study, and compared with those of the emulsion incorporating 2% oleic or linolenic acid. Only when emulsion incorporating 2% oleic acid was applied in the intestine did significant LDH release into the mesenteric veins occur. Our results indicate that emulsion incorporating highly purified long-chain polyunsaturated fatty acid, especially DHA, has the potential of becoming the formulation for enteral delivery of insulin.     

荆豆凝集素修饰脂质体对小鼠口服吸收胰岛素的促进作用

目的研究荆豆凝集素(UEA1)修饰的胰岛素脂质体在小鼠胃肠道的吸收作用。方法采用碳二亚胺偶联法制备荆豆凝集素修饰的磷脂酰乙醇胺(PE)，将PE-UEA1参入胰岛素脂质体中制成凝集素修饰脂质体，并证实UEA1凝集活性不受影响。分别对正常小鼠及糖尿病模型小鼠灌胃给予350 u·kg^-1胰岛素的修饰脂质体溶液，用葡萄糖酶试剂盒测定小鼠血糖，并与同剂量普通胰岛素脂质体比较。结果对于正常小鼠，荆豆凝集素修饰脂质体在4 h使血糖降至初始水平的(84±15)%，8 h降至(78±11)%，12 h为(90±12)%。胰岛素普通脂质体几乎没有降糖作用，与生理盐水对照组相当。对于糖尿病模型小鼠，荆豆凝集素修饰脂质体在4 h使血糖降至初始水平的(73±7)%，8 h降至(74±9)%，12 h为(86±9)%。结论荆豆凝集素修饰的脂质体可能通过与M细胞表面特异性受体的结合促进大分子药物的胃肠吸收。     

Adjuvant effects of glyceryl esters of acetoacetic acid on rectal absorption of insulin and inulin in rabbits

The promoting effect of glyceryl esters of acetoacetic acid on the rectal absorption of insulin and inulin was studied. A decrease in the serum glucose level was observed in rabbits following the administration of an insulin suppository containing glyceryl-1,3-diacetoacetate (adjuvant II) or 1,2-isopropylideneglycerine-3-acetoacetate (adjuvant IV). The promoting effects of adjuvants II and IV on the rectal absorption of insulin and inulin were suppressed by the addition of calcium and magnesium to the suppository. This indicates that adjuvant interaction with the calcium and magnesium ion located in the rectal membrane is involved in the enhanced absorption of insulin and inulin. Adjuvant release from the suppository formulation in addition to adjuvant lipid solubility were found to be other important factors for enhanced absorption of insulin and inulin.     

西红柿凝集素修饰脂质体对小鼠口服吸收胰岛素的促进作用西红柿凝集素修饰脂质体对小鼠口服吸收胰岛素的促进作用

目的研究西红柿凝集素修饰的胰岛素脂质体在小鼠胃肠道吸收作用。方法采用碳二亚胺交联法制备西红柿凝集素(TL)修饰的磷脂酰乙醇胺(PE),将TL-PE掺入胰岛素脂质体中制成凝集素修饰脂质体并证实TL凝集活性不受影响。分别对正常小鼠及糖尿病模型小鼠灌胃给予350 u·kg^-1胰岛素的修饰脂质体溶液,用葡萄糖酶试剂盒测定小鼠血糖,并与同剂量普通胰岛素脂质体比较。结果正常小鼠中,西红柿凝集素修饰脂质体在4 h血糖降至初始水平的(85±5)%,8 h降至(54±11)%,12 h为(57±6)%。胰岛素普通脂质体几乎没有降糖作用,与生理盐水对照组相当。糖尿病模型小鼠中,西红柿凝集素修饰脂质体在4 h血糖降至初始水平的(38±13)%,8 h降至(50±15)%,12 h为(50±16)%。结论西红柿凝集素修饰的脂质体可能通过与细胞表面特异性受体的结合作用促进大分子药物的胃肠吸收。