Lymphatic Absorption Is a Significant Contributor to the Subcutaneous Bioavailability of Insulin in a Sheep Model

Purpose: This study was conducted to explore the role of the peripheral lymphatics in insulin absorption following subcutaneous (SC) administration using a sheep model that allows continuous collection of peripheral lymph and simultaneous assessment of systemic bioavailability. Methods: In a parallel group design, soluble human insulin (0.5 IU/kg) was administered by bolus SC injection into the interdigital space of the hind leg of non-cannulated control sheep, and sheep in which the efferent popliteal lymph duct was cannulated. A separate group received a bolus IV injection (0.15 IU/kg). Blood was sampled from all animals, and lymph was collected continuously over 12 h post-dosing. Samples were assayed for insulin by ELISA. Results: The SC bioavailability of insulin in control sheep was 31.5 ± 3.2%, which was significantly higher than when the peripheral lymph was continuously collected (18.4 ± 1.7%). In the lymph-cannulated animals, 17.3 ± 1.0% of the dose was collected in peripheral lymph. Conclusions: Based on the direct measurement of insulin in regional lymph and on the decrease in the systemic bioavailability when regional lymph was continuously collected, the results demonstrate that lymphatic absorption contributed significantly to the overall insulin bioavailability following SC administration to sheep.     

健康志愿者单次皮下注射重组人胰高血糖素类多肽-1(7-36)[rhGLP-1(7-36)]耐受性研究

目的：在中国健康成年志愿者中评价单次皮下注射重组人胰高血糖素类多肽-1（7-36）[rhGLP-1（7-36）]的安全性、耐受性。方法：根据GCP设计试验方案，并获得伦理委员会批准。受试者须自愿签署知情同意书。选择42名18～50岁健康成人，将受试者随机分至0．10～0．45mg7个剂量组，每组6名，男女各半，分别接受单次皮下注射rhGLP-1（7-36），进行临床和实验室检查，考察药物耐受性。结果：单次皮下注射rhGLP-1（7-36）耐受性试验中，各组受试者各项指标测定值均在正常范围内，条件均衡，具较好可比性。因剂量至0．30mg时，不良事件（恶心、呕吐）在该组发生率超过50％，故于该剂量组试验完成后终止了下一剂量组试验，仅有4个剂量组共24名健康受试者完成了本试验。24例受试者完成的4个剂量组耐受性试验中，给药后实验室检查未见有临床意义的改变。试验中出现10例（共15例次）可能与药物有关的不良反应，如头晕、恶心、呕吐等，但均可耐受，且为一过性反应，于给药后1h内自行消失。其中，不良反应主要发生在0.25、0．30mg组（共7例12例次），而低剂量组（0．10、0．20mg）仅有3例发生轻度不良反应。15例次不良反应中，头晕、恶心有10例次，呕吐有5例次；整个试验过程未见严重不良事件。结论：24名中国健康成年受试者分别单次皮下注射rhGLP-1（7-36），最大剂量至0．20mg，比较安全、耐受性较好，为最大耐受剂量。而单次给药剂量至0．25mg或0．30mg则不良反应发生率较高，最大耐受剂量为0．20mg。建议单次用药剂量不宜超过0．20mg。在Ⅱ期临床试验中需严密观察恶心、呕吐这些与药物可能有关的不良反应及其发生机制。     

Effect of Molecular Weight on the Lymphatic Absorption of Water-Soluble Compounds Following Subcutaneous Administration

The lymphatic absorption of four water-soluble compounds with different molecular weights (MW) was determined by measuring their cumulative recovery in lymph draining from the site of s.c. administration in sheep. The cumulative recoveries (% of dose, mean ± SD; N = 3) were 4.0 ± 1.5 (5-fluoro-2-deoxyuridine, MW 246.2), 21.0 ± 7.1 (inulin, MW 5200), 38.6 ± 6.7 (cytochrome c, MW 12,300), and 59.5 ± 7.7 [human recombinant interferon (rIFN) alpha-2a, MW 19,000], respectively. Our data show that in the investigated MW range, there is a linear relationship between the molecular weight and the proportion of the dose absorbed lymphatically. An increase in molecular weight results in an increased lymphatic absorption. Molecules with MW > 16,000 are absorbed mainly by the lymphatics which drain the application site. The knowledge gained in this investigation may help to improve the mode of administration and therapeutic efficacy of endogenous proteins whose targets are lymphoid cells (e.g., interferons, interleukins). Practical implications for the clinical use of such proteins are discussed.     

胰高血糖素样肽-1及相关降糖药物的研究进展

胰高血糖素样肽-1（glucagon-like peptide-1,GLP-1）是近几年糖尿病治疗药物研究的热点之一,具备多重降血糖作用。它的两大类药物：GLP-1类似物和二肽基肽酶-Ⅳ抑制剂作为新的降糖药物相继完成临床研究,并在糖尿病治疗中发挥越来越重要的作用。本文就GLP-1的结构、药理作用以及两类相关降糖药物的临床应用作一概述。     

前胰高血糖素样肽-1对非糖尿病小鼠降糖作用的实验研究

摘要目的：观察胰高血糖素样肽-1（glucagon—likepeptide-1，GLP-1）的前体药物前胰高血糖素样肽-1（Pro-GLP-1）对非糖尿病小鼠的血糖和血清胰岛素水平的影响。方法：C57BL／6小鼠皮下注射不同剂量Pro-GLP-1，不同时间间隔取血测定血糖及血清胰岛素水平。结果：非糖尿病小鼠注射Pro-GLP-1后，血糖血清和胰岛素水平均无明显改变；给予糖负荷后的小鼠Pro—GLP-1呈剂量依赖性降低血糖浓度，同时升高血清胰岛素水平。等剂量Pro-GLP-1的降糖作用较GLP-1作用强。于第一次糖负荷后150min给予第二次糖负荷，Pro—GLP-1仍能发挥其降糖作用。结论：Pro—GLP-1通过葡萄糖依赖方式促进胰岛素分泌，剂量依赖性降低过高血糖，不降低正常血糖，其降糖作用可以维持3h以上。     

基于胰高血糖素样肽-1的多重受体激动剂的研究进展

胰高血糖素样肽-1(GLP-1)受体激动剂因其独特的作用优势,在降糖及减重领域的市场份额越来越大。但由于肥胖及2型糖尿病等异质性及并发性的病理学特点,以及能量平衡调节的复杂性,其减重效果无法达到预期标准。以GLP-1为基础的单分子多重受体激动剂有望将几种胃肠激素的有益作用整合到同一个分子中,以改善代谢紊乱。在总结市场上各种GLP-1类似物的分子结构及临床表现的基础上,着重介绍各类以GLP-1为基础的单分子多靶向激动剂的研究进展,为治疗肥胖和糖尿病提供新的思路。

     

胰高血糖素样肽-1——阿尔采末病治疗新策略

胰高血糖素样肽-1(GLP-1)是一种由30个氨基酸组成的肠肽,结合于GLP-1R,并与cAMP第二信使途径相偶联。神经系统GLP-1 R的激活在神经可塑性及神经细胞的存活中起着重要作用。GLP-1可以诱导神经元轴突的生长,抵御体外培养神经细胞的兴奋性死亡和氧化损伤。GLP-1及其天然类似物exend in-4均可以降低小鼠脑中内源性Aβ的水平及神经元β前体蛋白(βAPP)的水平。因此,以GLP-1或其相关肽来实施治疗可以影响到AD相关的多个治疗靶点。该文对GLP-1治疗阿尔采末病(AD)的潜力进行探讨。     

Modeling of Subcutaneous Absorption Kinetics of Infusion Solutions in the Elderly Using Technetium

Absorption kinetics of solutes given with the subcutaneous administration of fluids is ill-defined. The gamma emitter, technitium pertechnetate, enabled estimates of absorption rate to be estimated independently using two approaches. In the first approach, the counts remaining at the site were estimated by imaging above the subcutaneous administration site, whereas in the second approach, the plasma technetium concentration-time profiles were monitored up to 8 hr after technetium administration. Boluses of technetium pertechnetate were given both intravenously and subcutaneously on separate occasions with a multiple dosing regimen using three doses on each occasion. The disposition of technetium after iv administration was best described by biexponential kinetics with a V_ss of 0.30±0.11 L/kg and a clearance of 30.0±13.1 ml/min. The subcutaneous absorption kinetics was best described as a single exponential process with a half-life of 18.l6±3.97min by image analysis and a half-life of 11.58±2.48 min using plasma technetium time data. The bioavailability of technetium by the subcutaneous route was estimated to be 0.96±0.12. The absorption half-life showed no consistent change with the duration of the subcutaneous infusion. The amount remaining at the absorption site with time was similar when analyzed using image analysis, and plasma concentrations assuming multiexponential disposition kinetics and a first-order absorption process. Profiles of fraction remaining at the absorption site generated by deconvolution analysis, image analysis, and assumption of a constant first-order absorption process were similar. Slowing of absorption from the subcutaneous administration site is apparent after the last bolus dose in three of the subjects and can be associated with the stopping of the infusion. In a fourth subject, the retention of technetium at the subcutaneous site is more consistent with accumulation of technetium near the absorption site as a result of systemic recirculation.     

胰高血糖素样肽1与糖尿病的治疗

胰高血糖素样肽1(glucagon-like peptide-1,GLP-1)是由肠道L细胞分泌的一种重要的肠促胰岛素激素,其在体内的主要生理作用有刺激胰岛素的分泌和释放、抑制胰高血糖素的分泌、促进胰腺β细胞的增殖并抑制其凋亡、抑制胃的排空、促进饱食感的产生等。GLP-1对糖尿病和肥胖具有很好的治疗前景。由于GLP-1在体内很快被二肽酰基肽酶Ⅳ降解,血浆半衰期很短,因而限制了其临床应用。现已发现了促进GLP-1分泌的物质,开发了多种GLP-1的衍生物和二肽酰基肽酶Ⅳ抑制剂,为开发新型糖尿病治疗药物开辟了新的天地。     

胰高血糖素样肽-1在药物成瘾中的作用及机制

胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)是一种肠降血糖素,具有调节葡萄糖稳态和食欲的作用,与2型糖尿病和肥胖症的发生发展有关。GLP-1主要通过调节胰岛素的分泌和激活GLP-1受体(GLP-1R)而起作用。最近研究发现GLP-1与可卡因、尼古丁、酒精和苯丙胺类等所致的成瘾行为有关。该文主要对GLP-1在药物成瘾中的作用及机制进行综述。     

人胰高血糖素样肽-1类似物基因治疗对糖尿病大鼠机体代谢的影响

目的观察人胰高血糖素样肽-1(hGLP-1)类似物基因(2×Val2-hGLP-1)对糖尿病大鼠模型机体代谢的影响。方法建立糖尿病大鼠模型,尾静脉注射携带hGLP-1类似物基因的重组表达载体(pIRES2-EGFP/Val2-hGLP-1),观察30d,期间测定相关指标。结果治疗后糖尿病大鼠体重、进食及饮水量改善明显(P<0.01)。治疗15d后,血糖水平由(27.06±1.73)mmol/L降至(17.57±2.17)mmol/L(P<0.01),血清胰岛素水平由(5.53±0.79)mIU/L升高至(9.88±0.96)mIU/L(P<0.01)。总胆固醇水平降低(P<0.05或P<0.01)。hGLP-1蛋白在胰腺、肝、肾、肌肉组织中表达。结论 hGLP-1类似物基因可导入糖尿病大鼠体内并成功表达,基因治疗降低大鼠血糖,血脂水平,改善大鼠的机体代谢。     

Peptide aggregates: a novel model system to study self-assembly of peptides

Ordered aggregates of Val-Leu-Pro-Phe, tetrapeptide 1 , have been found in aqueous solutions. Evidence for the formation of aggregates for the above peptide was obtained by conductometric, pH metric, UV and fluorescence spectroscopic techniques. Values of critical micelle concentration (CMC) for the above peptide obtained by these methods are in good agreement with each other. The formation of organized aggregates of the peptide is favoured upon increasing the temperature (viz. the process of aggregation is endothermic). The aggregation number has been determined at different temperatures. Values of ΔG°_mΔH°_mΔS°_m and ΔC°_p have also been estimated. Binding studies with the 8-anilinonaphthyl sulfonic acid (ANS) and pyrene indicate that the interior of the aggregate is nonpolar. There are two processes with regard to the change of thermodynamical parameters like ΔG°_mΔH°_mΔS°_mΔC°_p aggregation number (N). In the first process (from 5°C to 40°C) the driving force for aggregation seems to be the positive entropy because of water release due to intermolecular association of ionic moieties. The second process (from 40°C and above) is due to intramolecular ionic interaction. The chemical shifts of the amide protons of the peptide have been presented in the light of inter- and intramolecular hydrogen-bond formation, and forces implicated in aggregation for both the first and second processes. © Munksgaard 1995.     

Stabilizing Peptide Fusion for Solving the Stability and Solubility Problems of Therapeutic Proteins

Purpose Protein aggregation is a major stability problem of therapeutic proteins. We investigated whether a novel stabilizing peptide [acidic tail of synuclein (ATS) peptide] could be generally used to make a more stable and soluble form of therapeutic proteins, particularly those having solubility or aggregation problems. Methods We produced ATS fusion proteins by fusing the stabilizing peptide to three representative therapeutic proteins, and then compared the stress-induced aggregation profiles, thermostability, and solubility of them. We also compared the in vivo stability of these ATS fusion proteins by studying their pharmacokinetics in rats. Results The human growth hormone–ATS (hGH–ATS) and granulocyte colony-stimulating factor–ATS (G-CSF–ATS) fusion proteins were fully functional as determined by cell proliferation assay, and the ATS fusion proteins seemed to be very resistant to agitation, freeze/thaw, and heat stresses. The introduction of the ATS peptide significantly increased the storage and thermal stabilities of hGH and G-CSF. The human leptin–ATS fusion protein also seemed to be very resistant to aggregation induced by agitation, freeze/thaw, and heat stresses. Furthermore, the ATS peptide greatly increased the solubility of the fusion proteins. Finally, pharmacokinetic studies in rats revealed that the ATS fusion proteins are also more stable in vivo. Conclusion Our data demonstrate that a more stable and soluble form of therapeutic proteins can be produced by fusing the ATS peptide. E. N. Lee and Y. M. Kim equally contributed to this work.     

Exendin-4对非糖尿病小鼠的血糖和糖耐量的影响

目的:观察exendin4对非糖尿病小鼠的血糖和糖耐量的影响。方法:非糖尿病小鼠分为exendin4(Exe4)各剂量组和生理盐水(NS)组。腹腔内注射Exe40.1μg·g-1,观察注射1h后、连续注射10d期间和停药后2wk内血糖,并于停药后20d行腹腔葡萄糖耐量试验(IPGTT)。另外,非糖尿病小鼠每日注射不同剂量Exe40.1,0.2,0.4μg·g-1×3d,观察每日血糖。结果:非糖尿病小鼠注射Exe40.1μg·g-11h后血糖为(6.2±s1.3)mmol·L-1,低于NS组(10.6±1.1)mmol·L-1,P<0.01。连续注射Exe4期间的平均血糖以及停药后2wk内的平均血糖与NS组比较,均无明显差异,P>0.05;且IPGTT中2组各点血糖差异无显著意义,不同剂量Exe4注射组小鼠的3d平均血糖与NS组比较也无明显差别。结论:Exe4对非糖尿病小鼠的血糖具有即刻降低作用,但是每日1次腹腔内注射对非糖尿病小鼠的血糖和糖耐量无明显影响。     

胰高血糖素样肽-1对糖尿病心血管病变的影响

目的:综述近年胰高血糖素样肽1及其类似物的研究进展,为糖尿病心血管病变的临床防治提出新的思路。方法:通过检索近10余年的相关文献,总结胰高血糖素样肽1及其类似物对糖尿病心血管病的作用及其相关机制。结果结论:目前研究提示胰高血糖素样肽1除了可以有效降低血糖以外,还通过多种机制影响心脏和血管的功能,此类活性物质以其独特的作用机制,为糖尿病心血管病变的治疗掀开新的一页。     

Enhanced protection of Ins-1 β cells from apoptosis under hypoxia by delivery of DNA encoding secretion signal peptide-linked exendin-4

In this study, we developed an expression system of exendin-4, a glucagon-like peptide (GLP-1) analog, using a secretion signal peptide (SP) to facilitate exendin-4 secretion. For delivery of the exendin-4 expression system, high-molecular-weight polyethylenimine (25 kDa, PEI25k), low-molecular-weight polyethylenimine (2 kDa, PEI2k), and polyamidoamine (PAMAM) dendrimers were evaluated as gene carriers to Ins-1 β cells. As a result, PEI25k showed the highest transfection efficiency. For the construction of the exendin-4 expression vector, DNA coding the SP sequence was inserted upstream of the exendin-4 cDNA, resulting in the construction of pβ-SP-Ex-4. Transfection assay showed that the secretion level of exendin-4 increased in the pβ-SP-Ex-4 transfected cells, compared with the pβ-Ex-4 transfected cells. To identify the β-cell protection effect of pβ-SP-Ex-4 delivery, the Ins-1 β cells were transfected with pβ-SP-Ex-4 or pβ-Ex-4 and incubated under normoxia or hypoxia. An MTT assay showed that the pβ-SP-Ex-4 transfected cells had higher β-cell viability than the pβ-Ex-4 transfected cells under hypoxia. In addition, the pβ-SP-Ex-4 transfected cells exhibited lower caspase-3 activity than the pβ-Ex-4 transfected cells. Therefore, PEI25k/pβ-SP-Ex-4 complex may be useful to protect isolated β cells from apoptosis during transplantation.     

Studies on the Subcutaneous Absorption in Mice VII: Absorption of 3H2O and 14C-sucrose from Non-buffered Solutions at Different pH Values

The possible influence on the subcutaneous absorption process of the pH of the injected unbuffered solutions was examined in normal and oestradiol pretreated mice. Eighty μl of 0.9 % NaCl, pH 4.60, 5.70, and 8.90, containing ³H₂O and ¹⁴C-sucrose in tracer concentrations were injected. In the oestradiol pretreated mice, the clearance of the tracer substance was significantly faster at pH 4.60 than at pH 8.90. Addition of hyaluronidase to the solutions abolished these differences. The results point to the connective tissue ground substance as the medium capable of modifying the absorption process according to slight pH differences of the injected solutions.     

Impact of Regional Intestinal pH Modulation on Absorption of Peptide Drugs: Oral Absorption Studies of Salmon Calcitonin in Beagle Dogs

Purpose. To investigate the relationship between the modulation of intestinal pH and the oral absorption properties of a model peptide drug, salmon calcitonin (sCT), in conscious beagle dogs. Methods. Studies were performed to characterize the disintegration of the formulation, intestinal pH changes, and the appearance of the peptide in the blood. Enteric-coated formulations containing sCT and various amounts of citric acid (CA) were tethered to a Heidelberg capsule (HC) and given orally to normal beagle dogs. Blood samples were collected and analyzed by radioimmunoassay (RIA). Intestinal pH was continuously monitored using the Heidelberg pH capsule (HC) system. The integrity of the HC-delivery system tether was verified by fluoroscopy. Results. The intra-individual variation in gastric emptying (GE) of the delivery system was large. There were also large inter-individual differences in the disintegration and absorption properties of the various formulations. However, the peak plasma concentrations of sCT were always observed when the intestinal pH declined. The average baseline intestinal pH was 6.1 ± 0.2 (mean ± SEM, n = 12). The intestinal pH reduction was 2.6 ± 0.4 (mean ± SEM, n = 12, ranged from 0.5 to 4.0 units from baseline). There was a good correlation between the time to reach the trough intestinal pH (t_pH,min) and time to reach the peak plasma concentration (t_conc,max) of sCT (t_conc,max = 0.95 × t_pH,min + 14.1, n = 11, r² = 0.91). Plasma C_max and area under the curve (AUC) increased with increasing amounts of CA in the formulations. Conclusions. The results of these studies demonstrate that the oral absorption properties of a model peptide drug, sCT, can be modulated by changing intestinal pH. sCT is a substrate for the pancreatic serine protease trypsin which has maximal activity at pH 5 to 6. Reducing intestinal pH presumably stabilizes sCT in the GI tract enabling greater absorption of the intact peptide.     

Studies on the Subcutaneous Absorption in Mice VIII: Influence of Connective Tissue Ground Substance on the Absorption of Subcutaneously Injected Depots of Sodium and Chloride

A standard volume of 80 μl of physiological saline or of pure water was injected subcutaneously in mice. In both cases the electrolyte composition of the depots was the same 15 minutes later, at which time the injected volumes had not been significantly reduced. The sodium concentration in the depot corresponded to that of the plasma, while the chloride concentration corresponded to approximately 50 per cent of that of the plasma. The missing anions could probably be accounted for by the non-diffusible polyanions of the connective tissue ground substance. Radioactive sodium ions were cleared significantly more slowly than radioactive chloride ions, these in turn being significantly slower than the radioactive water molecules, after the subcutaneous injection of physiological saline or water depots containing the radioactive ions in tracer concentrations.     

南极磷虾肽对去卵巢骨质疏松症小鼠肠钙吸收的改善作用研究

目的 研究南极磷虾肽(peptide from Antarctic krill,AKP)对双侧去卵巢骨质疏松症模型小鼠肠钙吸收的改善作用。方法 对9周龄雌性健康C57BL/6J小鼠进行双侧卵巢摘除手术,建立骨质疏松症模型,术后12周,随机分为假手术组(生理盐水)、模型对照组(生理盐水)、阳性对照组(阿伦磷酸钠,1 mg/kg.BW)、南极磷虾肽组(800 mg/kg.BW)。每天灌胃一次,连续灌胃90 d后,通过测定血清生化指标(PTH、1,25(OH)2D3、血钙及血磷)以及肠钙吸收通道基因Trpv6、CaBP-d9k、PMCA1b的mRNA表达水平,研究了南极磷虾肽对去卵巢骨质疏松症小鼠肠钙吸收的改善及作用机制;并且通过骨密度和骨钙骨磷含量的测定,进一步验证了南极磷虾肽对肠钙吸收的改善作用。结果 血清结果表明,AKP能够显著抑制血清中PTH的水平,增加血清中1,25(OH)2D3的含量,维持血清中钙、磷稳定;qRT-PCR结果表明,AKP能显著上调十二指肠中激素1,25(OH)2D3的受体VDR以及肠钙吸收通道关键基因Trpv6、CaBP-d9k、PMCA1b的mRNA表达水平,增强去卵巢骨质疏松小鼠的肠钙吸收功能;骨密度和骨钙、骨磷结果表明,AKP能够显著增加模型组小鼠的骨密度及骨钙、骨磷含量,进一步验证了AKP能够增强机体对钙离子的吸收,从而增加骨矿化作用,增加小鼠的骨量。结论 南极磷虾肽能够改善去卵巢骨质疏松症小鼠的肠钙吸收作用,增加去卵巢小鼠的骨矿含量。