Chemical Pathways of Peptide Degradation. VII. Solid State Chemical Instability of an Aspartyl Residue in a Model Hexapeptide

The chemical stability of an Asp-hexapeptide (Val-Tyr-Pro-Asp-Gly-Ala) in lyophilized formulations was evaluated as a function of multiple formulation variables–specifically pH of the bulk solution, temperature, moisture content, and type of bulking agent (amorphous vs. crystalline). The disappearance of the starting Asp-hexapeptide in the solid state conformed to pseudo-first-order reversible kinetics. This type of degradation profile was accounted for by the product distribution. The factorial experimental design of this study allowed statistical analysis of the effects of individual formulation variable (main effects) as well as those of two-factor interactions on the degradation of the Asp-hexapeptide. Analysis of Variance (ANOVA) calculations of the main effects indicated that while the influence of pH of the starting solution was not statistically significant, residual moisture level, temperature, and, especially, type of bulking agent had a significant impact on the solid state chemical reactivity of the hexapeptide. Furthermore, depending on which type of excipient was used in the lyophilized formulations, residual moisture level and temperature could be important stability variables. These types of factorial experiments have proven to be useful in the rapid identification of significant formulation variables in a given system and, consequently, in optimization of formulations.     

Chemical Pathways of Peptide Degradation. II. Kinetics of Deamidation of an Asparaginyl Residue in a Model Hexapeptide

Deamidation of Asn residues is a major chemical pathway of degradation of peptides and proteins. To understand better the external factors that influence deamidation, we studied the degradation of the hexapeptide Val–Tyr–Pro–Asn–Gly–Ala, a fragment of adrenocorticotropic hormone, by HPLC. The deamidation of this model peptide showed marked dependence on pH, temperature, and buffer composition. In the pH range 5 to 12, the peptide deamidated exclusively via a cyclic imide intermediate with the formation of both the Asp- and the isoAsp-hexapeptides. Buffer catalysis was also observed in the pH range of 7 to 11. However, at acidic pH's, the pathway of deamidation involved direct hydrolysis of the amide side chain of Asn residue to produce only the Asp-hexapeptide.     

In a Model of Neuroinflammation Designed to Mimic Delirium,Quetiapine Reduces Cortisol Secretion and Preserves Reversal Learning in the Attentional Set Shifting Task

Quetiapine, an atypical antipsychotic medication has lacked pre-clinical validation for its purported benefits in the treatment of delirium. This laboratory investigation examined the effects of quetiapine on the attentional set shifting task (ASST), a measure of cognitive flexibility and executive functioning, in a rodent model of lipopolysaccharide (LPS) mediated neuroinflammation. 19 Sprague Dawley female rats were randomly selected to receive intraperitoneal placebo (N = 5), LPS and placebo (N = 7) or LPS and quetiapine (n = 7) and performed the ASST. We measured trials to criterion, errors, non-locomotion episodes and latency to criterion, serum cortisol and tumor necrosis factor alpha (TNF-α) levels. TNF-α levels were not different between groups at 24 h. Cortisol levels in the LPS + Quetiapine group were reduced compared to LPS + Placebo (P < 0.001) and did not differ from the placebo group (P = 0.15). Analysis between LPS + Quetiapine and LPS + Placebo treated rats demonstrated improvement in the compound discrimination reversal (CD Rev1) (P = 0.016) and the intra-dimensional reversal (ID Rev2) (P = 0.007) discriminations on trials to criterion. LPS + Quetiapine treated rats had fewer errors than LPS + Placebo treated animals in the compound discrimination (CD) (P = 0.007), CD Rev1 (P = 0.005), ID Rev2 (P < 0.001) discriminations. There was no difference in non-locomotion frequency or latency to criterion between the three groups in all discriminations (P > 0.0167). We demonstrated preserved reversal learning, no effect on attentional set shifting and normalized cortisol levels in quetiapine-treated rats in this neuroinflammatory model of delirium. This suggests that quetiapine’s beneficial effects in delirium may be related to the preservation of reversal learning and potential downstream effects related to reduction in cortisol production.

Graphical Abstract

     

大鼠创伤性休克后血浆内皮素与降钙素基因相关肽水平的变化

目的 探讨创伤性休克对大鼠血浆内皮素(endothelin,ET)、降钙素基因相关肽(calcitonin-gene related peptide,CGRP)水平及动脉血气分析的影响.方法 采用后肢创伤法建立创伤性休克大鼠模型,测定创伤前后及复苏后1、3、5、1 2h血浆ET、CGRP及动脉血气分析水平.结果 在休克末及复苏后1、3、5、12h,大鼠血浆ET、CGRP水平及动脉血二氧化碳分压(PCO2)、氧分压(PO2)及氧饱和度(SO2)发生明显变化.血浆ET含量分别为(122±27),(123±28),(139±36),(149±38)和(141±32)ng/L;PCO2水平分别为(5.0±0.4),(5.2±0.5),(5.3±0.4),(6.0±0.4)and(7.0±0.4)kPa,PO2水平分别为(1 2.2±0.8),(11.7±0.6),(9.5±0.6),(8.1±0.8)和(5.8±0.7)kPa,S02分别为(90.2±1.3),(95.0±1.4),(93.4±1.4),(90.6±1.6)和(79.3±4.2)%,复苏后1、3、5、12h,大鼠血浆CGRP水平分别为(190±30),(193±32),(221±40)and(220±38)ng/L.结论 创伤性休克血浆ET、CGRP水平明显升高且伴随着PCO2、PO2和SO2的变化.ET和CGRP在创伤性休克中起着重要的作用.     

Chemical Pathways of Peptide Degradation. IV. Pathways,Kinetics, and Mechanism of Degradation of an Aspartyl Residue in a Model Hexapeptide

In this study the hexapeptide Val-Tyr-Pro-Asp-Gly-Ala (Asp-hexapeptide) was used as a model to investigate the kinetics of aspartate degradation in aqueous solution. The apparent rate of degradation of the Asp-hexapeptide was determined as a function of pH, buffer concentration, and temperature. At very acidic pH levels (0.3, 1.1, 1.5, 2.0, and 3.0), the apparent rate of degradation followed pseudo-first-order kinetics. In this pH region, the Asp-hexapeptide predominantly underwent specific acid-catalyzed hydrolysis of the Asp-Gly amide bond (Asp-X hydrolysis) to form a tetrapeptide (Val-Tyr-Pro-Asp) and a dipeptide (Gly-Ala). In addition, parallel formation of a cyclic imide intermediate could be observed, although no isoAsp-hexapeptide was detected. At pH 4.0 and 5.0, the Asp-hexapeptide simultaneously isomerized via the cyclic imide to form the iso-Asp-hexapeptide and underwent Asp-X hydrolysis to produce the cleavage products. The pH-rate profiles (pH 0.3–5.0) for the Asp-X hydrolysis and the formation of cyclic imide revealed that the degree of ionization of the carboxylic acid side chain of Asp residue significantly altered the rate of reaction, with the ionized form being more reactive than the unionized form. Little or no buffer catalysis was observed for either pathway. Solvent isotope experiments were used to probe the mechanism of the Asp-X hydrolysis reaction. At pH values above 6.0, the apparent rate of degradation of the Asp-hexapeptide followed pseudo-first-order reversible kinetics, with the isoAsp-hexapeptide being the only observed product (isomerization). Above pH 8.0, the isomerization kinetics were found to be independent of pH and buffer concentration. The kinetics of degradation of Asp-hexapeptide (Val-Tyr-Pro-Asp-Gly-Ala) and Asn-hexapeptide (Val-Tyr-Pro-Asn-Gly-Ala) were compared to determine the relative instability of the Asp and Asn residues and to understand the mechanism of formation of cyclic imide at near neutral to basic pH.     

Peptide sweeteners. 6. Structural studies on the C-terminal amino acid of L-aspartyl dipeptide sweeteners

Stereochemical and structural aspects of the variations in the C-terminal residue of L-aspartyl-L-phenylalanine methyl ester have been investigated. Novel configurational analogues such as L-aspartyl-D-alanine benzyl ester and L-aspartyl-D-alpha-aminobutyric acid benzyl ester were found to be sweet. In addition, chiral and achiral alpha, alpha-dialkylglycine and alpha-aminocycloalkanecarboxylic acids were incorporated into the dipeptides. The L-aspartic acid based dipeptide derivatives of alpha-aminoisobutyric acid methyl ester, alpha-aminocyclopropanecarboxylic acid methyl ester, alpha-aminocyclobutanecarboxylic acid methyl ester, and alpha-aminocyclopentanecarboxylic acid methyl ester are sweet. Dipeptides with alpha-aminocyclohexanecarboxylic acid methyl ester and alpha-aminocycloheptanecarboxylic acid methyl ester are bitter, whereas the analogues with alpha-aminocyclooctanecarboxylic acid methyl ester, alpha, alpha-diethylglycine methyl ester, and alpha-aminoisobutyric acid benzyl ester are tasteless. Aspects on chirality and effective volume of the C-terminal residue are discussed and correlated with taste.     

降钙素基因相关肽和内皮素在大鼠急性肾衰竭中的变化及作用

目的:研究降钙素基因相关肽(CGRP)对急性肾衰竭(ARF)的影响,探讨CGRP和内皮素-1(ET-1)在ARF中的变化和作用.方法:采用肌内注射甘油法复制ARF大鼠模型,将大鼠随机分成实验组、治疗组及对照组,动态观察各组动物肾功能指标,并用放射免疫方法检测大鼠不同时相点血浆CGRP、ET-1水平的变化.结果:治疗组大鼠肾功能明显减轻,实验组血浆CGRP水平明显降低(P＜0.05),血浆ET-1水平明显增高(P＜0.01).结论:血浆CGRP、ET-1含量测定可以作为反映肾功能状态和分析ARF病程发展与转归的敏感指标,CGRP对ARF时的肾脏有保护作用.     

降钙素基因相关肽和内皮素在大鼠急性肾衰竭中的变化及作用

目的:研究降钙素基因相关肽(CGRP)对急性肾衰竭(ARF)的影响,探讨CGRP和内皮素-1(ET-1)在ARF中的变化和作用.方法:采用肌内注射甘油法复制ARF大鼠模型,将大鼠随机分成实验组、治疗组及对照组,动态观察各组动物肾功能指标,并用放射免疫方法检测大鼠不同时相点血浆CGRP、ET-1水平的变化.结果:治疗组大鼠肾功能明显减轻,实验组血浆CGRP水平明显降低(P＜0.05),血浆ET-1水平明显增高(P＜0.01).结论:血浆CGRP、ET-1含量测定可以作为反映肾功能状态和分析ARF病程发展与转归的敏感指标,CGRP对ARF时的肾脏有保护作用.     

Design and Synthesis of Peptide YY Analogues with C-terminal Backbone Amide-to-Ester Modifications

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氯吡格雷对不稳定型心绞痛患者血浆内皮素和降钙素基因相关肽的影响

目的 探讨氯吡格雷对不稳定型心绞痛(UAP)患者血浆内皮素和降钙素基因相关肽的影响.方法 将100例UAP患者随机分为两组,分别用消心痛(50例)及氯吡格雷加消心痛(50例)联合治疗,采用放免法观察治疗前后患者血浆内皮素(ET)及降钙素基因相关肽(CGRP)含量,另选20名健康体检者作对照.结果 与健康志愿者相比,UAP患者血浆ET含量明显升高(P＜0.05),CGRP明显降低(P＜0.05).较消心痛治疗组相比,联合使用氯吡格雷治疗后患者ET明显下降(P＜0.01),CGRP明显升高(P＜0.05).结论 氯吡格雷能显著改善ET、CGRP代谢失衡状况,是治疗UAP的有效药物.     

多沙唑嗪治疗轻中度高血压的疗效及其对血浆内皮素和降钙素基因相关肽的影响

目的观察多沙唑嗪治疗轻、中度原发性高血压的疗效,探讨多沙唑嗪对血浆内皮素(ET)和降钙素基因相关肽(CGRP)的影响。方法采用随机、对照、双盲法,将轻、中度原发性高血压48例分为两组各24例,治疗组和对照组分别给予多沙唑嗪和特拉唑嗪,均为2mg,po,qd,共治疗8周。结果治疗组和对照组降血压有效率分别为835%,783%,差异无显著性(P>005)。治疗组总胆固醇亦明显下降(P<005)。两组治疗后ET浓度下降、CGRP水平升高。结论多沙唑嗪为一安全有效的降血压药物,在降血压的同时有降血脂作用;多沙唑嗪的降血压作用可能部分与其拮抗ET、升高CGRP的浓度有关。     

Esterase-Sensitive Cyclic Prodrugs of Peptides: Evaluation of a Phenylpropionic Acid Promoiety in a Model Hexapeptide

Purpose. To evaluate a cyclic phenylpropionic acid prodrug of a model hexapeptide (H-Trp-Ala-Gly-Gly-Asp-Ala-OH) as a novel approach to enhance the membrane permeation of a peptide and stabilize it to metabolism. Methods. Conversion to the linear hexapeptide was studied at 37°C in HBSS, pH 7.4, and in various biological milieus having measurable esterase activities. Transport and metabolism characteristics were assessed using the Caco-2 cell culture model. Results. In aqueous buffered solution, pH 7.4, the cyclic prodrug degraded quantitatively (t _1/2 = 1795 ± 289 min) to the linear hexapeptide and the lactone. Substantially faster degradation of the cyclic prodrug was observed in 90% human plasma (t _1/2 = 508 ± 24 min), and in homogenates of Caco-2 cells (t _1/2 = 940 ± 13 min), the rat intestinal mucosa (t _1/2 = 1286 ± 32 min), and rat liver (t _1/2 = 840 ± 42 min). Pretreatment of these biological media with paraoxon significantly decreased the degradation rate of the prodrug. When applied to the apical side of Caco-2 cell monolayers, the cyclic prodrug was significantly more stable than the hexapeptide and at least 71-fold more able to permeate (P_app = 1.21 ± 0.12 × 10^–7 cm/s) than was the parent peptide (P_app 0.17 × 10^–8 cm/s). In the presence of 0.1 mM palmitoyl-DL-carnitine, the transport rate of the cyclic prodrug (P_app = 2.19 × 10^–6 cm/s) was 1250-fold greater than that of the linear hexapeptide. Conclusions. Preparation of a cyclic peptide using a phenylpropionic acid promoiety reduced the lability of the peptide to peptidase metabolism and substantially increased its permeation through biological membranes. In various biological media the parent peptide was released from the prodrug by an apparent esterase-catalyzed reaction, sensitive to paraoxon inhibition.     

骨形成肽羧基端衍生物G35I对成骨细胞和破骨细胞的体外影响

目的：探讨骨形成肽羧基端衍生物G35I在体外对成骨细胞和破骨细胞的影响。方法：利用体外人成骨细胞和骨髓来源大鼠破骨细胞培养和RT—PCR方法进行研究。结果：(1)G35I在体外可以刺激人胚胎长骨来源的成骨细胞的增生。(2)G35I在10-7mol／L可以刺激骨髓来源的破骨细胞样细胞的诱导分化形成，而在其他浓度(10^-8-10^-11mol／L)此作用不明显。(3)G35I在10^-7mol／L条件下可以抑制成骨细胞骨保护素的表达，且在72h作用最明显。结论：骨形成肽羧基端衍生物G35I在不同浓度时对骨代谢的两种功能细胞均有一定作用。     

Diltiazem Facilitates Endothelin Clearance from the Blood Stream to Reduce Toxic Elevation of Plasma Endothelin Level in Rodents*

Abstract— Pretreatment with diltiazem at a dose of 2 mg kg^?1 intravenously protected against sudden death induced by intravenous administration of endothelin-1 (ET-1, 5 nmol kg^?1), with an apparent decrease in the plasma immunoreactive-ET-1 (IR-ET-1) in mice. These effects, which were also observed in anaesthetized rats, disappeared in rats with bilateral ligation of the renal arteries. In the latter, the exogenous ET-1-induced elevation of plasma IR-ET-1 tended to be higher than that in the sham-operated controls. Furthermore, in anaesthetized rats, diltiazem inhibited ET-1-induced decreases in renal blood flow and increased renal accumulation of IR-ET-1. These results indicate that part of the clearance of ET-1 from the bloodstream occurs in the kidney, and that diltiazem enhances the elimination of the peptide, presumably by improvement in the renal circulation, this action leading to alleviation of the toxic effects of ET-1.     

Stopping Guidelines for Harm in a Study Designed to Establish the Safety of a Marketed Drug

In a study designed to establish the safety of a marketed drug, interim analyses performed to detect harm can protect trial participants and the wider public before the final analysis occurs. Monitoring for harm within a safety study is different from monitoring for benefit, so techniques commonly used in an efficacy study of an experimental drug may not apply. We propose potentially more suitable techniques in this setting, including a novel spending function and conditional power. These techniques have reasonable operating characteristics in a simulation. The appropriate technique to implement will depend on circumstances of specific to the individual safety study.     

比索洛尔对纤溶活性和内皮血管活性物质的影响

目的观察比索洛尔治疗原发性高血压(EH)时对纤溶活性、内皮素(ET)、一氧化氮(NO)、降钙素基因相关肽(CGRP)的影响。方法42例健康体检者作为正常对照组,66例EH患者用比索洛尔治疗8周,观察用药前后血组织型纤溶酶原激活物(t-PA)及其抑制物(PAI)活性,ET、NO、CGRP浓度的变化。结果EH患者t-PA活性、CGRP和NO浓度明显低于正常对照组(P<0.01),而PA I活性、ET浓度明显高于正常对照组(P<0.01),比索洛尔治疗后,血t-PA活性、NO、CGRP浓度均较治疗前显著升高(P<0.01),血ET浓度显著下降(P<0.01)。结论EH患者纤溶活性和内皮功能异常,比索洛尔可改善EH患者纤溶活性和内皮功能。     

Endothelin: a potential modulator of cerebral vasospasm

1-(5-Isoquinolinesulfonyl)-homopiperazine, HA1077, is a calcium antagonist with anti-vasospastic properties. This compound blocks intracellular actions of calcium in a variety of experiments. In the present study, we examined the effects of HA1077 on the vascular actions of endothelin, an endothelium-derived vasoactive peptide, in dogs in vitro and in vivo. Intracisternal injections of endothelin (0.01 nmol) produced a significant vasospasm, as measured by angiography, similar to that seen in the canine hemorrhage model. Infusion of HA1077 led to a significant dilatation of the spastic basilar artery in endothelin-treated dogs. The rank order of in vitro contractile activity in canine cerebral arteries was a stable thromboxane A₂ analog > endothelin > 5-hydroxytryptamine > prostaglandin F₂ > histamine > noradrenaline. HA1077 effectively antagonized the endothelin-induced contraction of canine basilar arterial strips in both calcium-containing and calcium-free medium. The present results indicate that HA1077 is an effective antagonist for endothelin in vitro and in vivo.     

Esterase-Sensitive Cyclic Prodrugs of Peptides: Evaluation of an Acyloxyalkoxy Promoiety in a Model Hexapeptide

Purpose. To evaluate a cyclic acyloxyalkoxycarbamate prodrug of a model hexapeptide (H-Trp-Ala-Gly-Gly-Asp-Ala-OH) as a novel approach to enhance the membrane permeation of the peptide and stabilize it to metabolism. Methods. Conversion to the linear hexapeptide was studied at 37°C in aqueous buffered solutions and in various biological milieus having measurable esterase activities. Transport and metabolism characteristics were assessed using the Caco-2 cell culture model. Results. In buffered solutions the cyclic prodrug degraded chemically to the linear hexapeptide in stoichiometric amounts. Maximum stability was observed between pH 3–4. In 90% human plasma (t _1/2 = 100 ± 4 min) and in homogenates of the rat intestinal mucosa (t _- = 136 ± 4 min) and rat liver (t _- = 65 ± 3 min), the cyclic prodrug disappeared faster than in buffered solution, pH 7.4 (t _- = 206 ± 11 min). Pretreatment of these media with paraoxon significantly decreased the degradation rate of the prodrug. When applied to the apical side of Caco-2 cell monolayers, the cyclic prodrug (t _- = 282 ± 25 min) was significantly more stable than the hexapeptide (t _- = 14 min) and at least 76-fold more able to permeate (P _app = 1.30 ± 0.15 × 10^–7 cm/ s) than the parent peptide (P _app 0.17 × 10^–8 cm/s). Conclusions. Preparation of a cyclic peptide using an acyloxyalkoxy promoiety reduced the lability of the peptide to peptidase metabolism and substantially increased its permeation through biological membranes. In various biological media the parent peptide was released from the prodrug by an apparent esterase-catalyzed reaction, sensitive to paraoxon inhibition.     

Conversion of the Synthetic Catalase Mimic Precursor TAA‐1 into the Active Catalase Mimic in Isolated Hepatocytes

In previous studies we reported on the catalase‐like activity and antioxidative properties of a non‐heme Fe(III)‐tetraaza[14]annulene complex, 5,4‐didehydro‐5,9,14,18‐tetraaza‐di(2,2‐dimethyl‐[5,6]benzo[1,3]dioxolo)[a,h]cyclotetradecene–Fe(III) chloride (TAA‐1/Fe). We proposed that intracellular application of the parent, iron‐free tetraaza[14]annulene ligand, TAA‐1, as precursor would allow antioxidative defense along two lines, i.e. by chelation of potentially toxic cellular iron ions and, subsequently, by catalase‐mimic activity. We here set out to establish whether the active catalase mimic is indeed formed intracellularly when cells are loaded with the ligand. When isolated rat hepatocytes were preloaded with TAA‐1, they were protected against iron‐induced cell injury and oxidative stress elicited by exposure to the membrane‐permeable iron complex Fe(III)/8‐hydroxyquinoline. After lysis of the cells, followed by ultrafiltration to remove endogenous catalase, the lysate exhibited catalase‐like activity, while lysates of control cells not treated with TAA‐1 showed no catalase‐like activity. By comparison with authentic TAA‐1/Fe, an intracellular formation of 2.0 ± 0.3 μm of the active catalase mimic in native hepatocytes exposed to TAA‐1 and of 6.5 ± 1.0 μm in hepatocytes exposed to both TAA‐1 and iron ions was estimated. The intracellular formation of the active catalase mimic thus renders TAA‐1 an attractive compound for protection against iron‐ and/or hydrogen peroxide‐dependent cell injuries.