鲑鱼降钙素前体多肽在毕赤酵母中的分泌表达

将鲑鱼降钙素前体多肽（sCTGly）基因克隆到表达载体pPIC9K上,然后转化毕赤酵母营养缺陷型菌株GS115,利用酿酒酵母分泌信号肽α-factor将目的产物分泌到发酵上清液中.经组氨酸缺陷筛选、G418高拷贝筛选及摇瓶表达筛选,获得较高水平表达菌株GS115-sCTGly.酶联免疫反应检测上清液中酶的活性,表明sCTGly产量在10mg/L以上.通过5L发酵罐发酵条件优化,表达水平提高到200mg/L上.     

Glucagon as a target for the treatment of Type 2 diabetes

Glucagon is the key counter-regulatory hormone that opposes the action of insulin. In states of relative hypoglycaemia, glucagon acts to increase blood glucose by stimulating hepatic glycogen breakdown and gluconeogenesis to achieve euglycaemia. Type 2 diabetes is characterised by inappropriate regulation of hepatic glucose production, which is due, at least in part, to an imbalance in the bihormonal relationship between plasma levels of glucagon and insulin. The glucose-lowering effects of glucagon peptide antagonists and antiglucagon neutralising antibodies first demonstrated the potential of glucagon receptor (GCGR) antagonism as a treatment for hyperglycaemia. In recent years, the development of GCGR antisense oligonucleotides and small molecular weight GCGR antagonists have been pursued as possible therapeu-tic agents to target glucagon action as a treatment for Type 2 diabe-tes.     

Glucagon: Its evolving role in the management of hypoglycemia

More than 10% of the United States population has diabetes, characterized by hyperglycemia. Insulin and other agents used to treat diabetes predispose people to hypoglycemia, which can be life threatening. Glucagon is an emergency medication that can save lives by quickly raising glucose in people who are unconscious or unable to consume glucose due to severe hypoglycemia. Although glucagon has been commercially available since 1960, earlier formulations required reconstitution of a dry powder with diluent immediately prior to injection, due to lack of long-term stability once reconstituted. Glucagon has been underutilized due to the lack of confidence or ability to administer in emergency situations. More recently, new formulations including a nasal powder glucagon and liquid-stable glucagon have become available. This article discusses the evidence surrounding new glucagon formulations compared with the original glucagon emergency kit including ease of use, efficacy, and safety with a focus on important patient counseling points and relevant clinical information on hypoglycemia.     

Effects of Non-Covalent Self-Association on the Subcutaneous Absorption of a Therapeutic Peptide

Purpose. To utilize an acylated peptide as a model system to investigate the relationships among solution peptide conformation, non-covalent self-association, subcutaneous absorption and bioavailability under pharmaceutically relevant solution formulation conditions. Methods. CD spectroscopy, FTIR spectroscopy, equilibrium sedimentation, dynamic light scattering, and size exclusion chromatography were employed to characterize the effects of octanoylation on conformation and self-association of the 31 amino acid peptide derivative des-amino-histidine(7) arginine(26) human glucagon-like peptide (7-37)-OH (IP(7)R(26)GLP-1). Hyperglycemic clamp studies were performed to compare the bioavailability, pharmacokinetics, and pharmacodynamics of solution formulations of oct-IP(7)R(26)GLP-l administered subcutaneously to normal dogs. Results. Octanoylation of IP(7)R(26)GLP-1 was shown to confer the propensity for a major solvent-induced conformational transition with an accompanying solvent- and temperature-dependent self-association behavior. Formulations were characterized that give rise to remarkably different pharmacodynamics and pharmacokinetics that correlate with distinct peptide conformational and self-association states. These states correspond to: (i) a minimally associated -helical form (apparent molecular weight = 14 kDa), (ii) a highly associated, predominantly -sheet form (effective molecular diameter 20 nm), and (iii) an unusually large, micelle-like soluble -sheet aggregate (effective molecular diameter 50 nm). Conclusions. Bioavailability and pharmacokinetics of a self-associating peptide can be influenced by aggregate size and the ease of disruption of the non-covalent intermolecular interactions at the subcutaneous site. Hydrophobic aggregation mediated by seemingly innocuous solution formulation conditions can have a dramatic effect on the subcutaneous bioavailability and pharmacokinetics of a therapeutic peptide and in the extreme, can totally preclude its absorption. A size exclusion chromatographic method is identified that distinguishes subcutaneously bioavailable aggregated oct-IP(7)R(26)GLP-1 from non-bioavailable aggregated oct-IP(7)R(26)GLP-1.     

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.     

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.     

鲑鱼降钙素喷鼻剂在中国健康志愿者人体生物等效性研究

目的研究国产与进口鲑鱼降钙素喷鼻剂的生物等效性。方法选择18-40岁健康成年男性24名,用随机交叉实验设计,用放射免疫法(RIA)测定健康志愿者分别用国产(每喷100U,每次2喷)与进口(每喷200U,每次1喷)鲑鱼降钙素喷鼻剂喷鼻给药200U后各时血药浓度。结果国产及进口鲑鱼降钙素喷鼻剂的主要药代动力学参数:tmax为0.28±0.06和0.26±0.02h;Cmax为0.07±0.01和O.07±0.01μg·L-1;AUC0-8h为0.07±0.01,0.07±0.01μg.h·L-1。以进口鲑鱼降钙素喷鼻剂为对照,国产喷鼻剂的人体相对生物利用度为(100.3±11.8)％。结论国产与进口鲑鱼降钙素喷鼻剂生物等效。     

鲑鱼降钙素(考克)治疗骨质疏松性骨痛的疗效评价

目的:评价鲑鱼降钙素(商品名考克)对骨质疏松性骨痛的疗效.方法:采用多中心自身对照研究,入选210例骨质疏松及骨量丢失伴有骨痛的患者,给予鲑鱼降钙素50 IU·d-1肌内注射,治疗2周,观察治疗前后骨质疏松性骨痛的变化.结果:199例患者完成治疗,自我评定及医生评分显示不同部位骨痛症状均消失或明显减轻,总有效率均在80%以上(P＜0.005),其中无效者多为合并腰椎间盘突出等其他原因引起的疼痛.有31.43%(66/210)患者出现不良反应,多为恶心、脸红、头晕等,其中5.23%因反应严重或恐惧退出治疗.结论:鲑鱼降钙素治疗骨质疏松性骨痛快速、有效.     

注射用鲑鱼降钙素的人体生物利用度

目的:评价注射用鲑鱼降钙素人体生物利用度和生物等效性.方法:男性健康志愿者20名,采用自身交叉方式单剂量注射鲑鱼降钙素与密钙息20 μg,以放射免疫法测定其血药浓度.结果:试验制剂注射用鲑鱼降钙素与参比制剂密钙息的主要药动学参数:Cmax分别为(154.5±35.2)ng·L-1和(153.8±28.1)ng·L-1;Tmax为(0.52±0.19) h和(0.48±0.14) h;AUC0～10为(344.6±46.8)ng·h·L-1和(339.4±40.6)ng·h·L-1,AUC0～∞为(356.4±48.3)ng·h·L-1和(350.5±41.7) ng·h·L-1.两种制剂的主要药动学参数差异无显著性(P>0.05).注射用鲑鱼降钙素对密钙息的相对生物利用度为(102.0±7.6)%.结论:两种制剂具有生物等效性.     

Biopharmaceutical Approaches for Developing and Assessing Oral Peptide Delivery Strategies and Systems: In Vitro Permeability and In Vivo Oral Absorption of Salmon Calcitonin

Purpose. To evaluate a biopharmaceutical approach for selecting formulation additives and establishing the performance specifications of an oral peptide delivery system using sCT as a model peptide. Methods. The effect of formulation additives on sCT effective permeability and transepithelial electrical resistance (TEER) was evaluated in side-by-side diffusion chambers using rat intestinal segments. Baseline regional oral absorption of sCT was evaluated in an Intestinal and Vascular Access Port (IVAP) dog model by administration directly into the duodenum, ileum, and colon by means of surgically implanted, chronic catheters. The effect of varying the input rate and volume of the administered solution on the extent of sCT absorption was also evaluated. Citric acid (CA) was utilized in all studies to cause a transient reduction in local pH. In vitro samples and plasma samples were analyzed by radioimmunoassay (RIA). Two oral delivery systems were prepared based on the results of the in vitro and IVAP studies, and evaluated in normal dogs. Results. Maximal permeability enhancement of sCT was observed using taurodeoxycholate (TDC) or lauroyl carnitine (LC) in vitro. Ileal absorption of sCT was higher than in other regions of the intestine. Low volume and bolus input of solution formulations was selected as the optimal condition for the IVAP studies since larger volumes or slower input rates resulted in significantly lower sCT bioavailability (BA). Much lower BA of sCT was observed when CA was not used in the formulation. The absolute oral bioavailability (mean ± SD) in dogs for the control (sCT + CA) and two proprietary sCT delivery systems was 0.30% ± 0.05%, 1.10 ± 0.18%, and 1.31 ± 0.56%, respectively. Conclusions. These studies demonstrate the utility of in vitro evaluation and controlled in vivo studies for developing oral peptide delivery strategies. Formulation additives were selected, the optimal intestinal region for delivery identified, and the optimal release kinetics of additives and actives from the delivery system were characterized. These methods were successfully used for devising delivery strategies and fabricating and evaluating oral sCT delivery systems in animals. Based on these studies, sCT delivery systems have been fabricated and tested in humans with favorable results.     

HPLC法测定鲑鱼降钙素注射液的含量

目的　建立一种用于鲑鱼降钙素注射液含量测定的HPLC方法。方法　采用C18色谱柱 ,以二种不同比例的乙腈 -四甲基胺 -磷酸盐 (pH =3 0 )混合液为流动相 ,进行二元梯度程序洗脱 ,流速为 1 0mL·min- 1 ,检测波长为 2 2 0nm ;对本方法的选择性、准确性、精密度、检测限和定量限等进行了认证。结果　本方法的线性范围为 3- 30 μg·mL- 1 (r =0 9997,n =3) ,平均回收率为 99 82 % (RSD =0 18% ,n =9) ,精密度为 0 2 2 % (n =6 )。结论　本法选择性好、准确度高 ,可用于鲑鱼降钙素注射液的含量测定     

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.     

Oral absorption enhancement of salmon calcitonin by using both N-trimethyl chitosan chloride and oligoarginines-modified liposomes as the carriers

Abstract

In this study both N-trimethyl chitosan chloride (TMC) and oligoarginine (Arg8) were utilized to modify liposomes as the multifunctional carriers (TMC-Arg8-Lips) for enhancing the oral absorption of salmon calcitonin. Two permeation enhancers with positive charges were sequentially adsorbed on the liposomal surface with negative charges by electrostatic interaction. Instead of salmon calcitonin, fluorescein isothiocyanate dextran (FD4) was loaded in TMC-Arg8-Lips for Caco-2 cell permeation test in vitro and penetration examination in rat intestinal tract in vivo. The results showed that the apparent permeability coefficient (P_app) of TMC-Arg8-Lips containing FD4 were 7.0-, 4.4-, 1.8- and 1.4-folds higher than FD4 solution, FD4-TMC solution, non-modified liposomes (Non-Lips) and TMC modified liposomes (TMC-Lips), respectively. A strong fluorescence was observed by confocal laser scanning microscope (CLSM) at rat intestinal wall isolated in different times after the FD4 loaded carriers were intragastrically administrated. Furthermore, the images revealed that TMC-Arg8-Lips could penetrate deeply inside the mucosal membrane. The pharmacodynamic study indicated that TMC-Arg8-Lips containing calcitonin were more efficient in enhancing the absorption and prolonging the reduction of blood calcemia in rats. The area above the plasma calcium concentration–time curve (AAC) of TMC-Arg8-Lips containing calcitonin was increased by more than 16.6- and 1.6-fold when compared to Non-Lips and TMC-Lips, respectively.     

Proteolysis of Human Calcitonin in Excised Bovine Nasal Mucosa: Elucidation of the Metabolic Pathway by Liquid Secondary lonization Mass Spectrometry (LSIMS) and Matrix Assisted Laser Desorption lonization Mass Spectrometry (MALDI)

Purpose. Two calcitonins, i.e. human calcitonin (hCT) and, for comparison, salmon calcitonin (sCT), were chosen as peptide models to investigate nasal mucosal metabolism. Methods. The susceptibility of hCT and sCT to nasal mucosal enzymes was assessed by in-and-out reflection kinetics experiments in an in vitro model based on the use of freshly excised bovine nasal mucosa, with the mucosal surface of the mucosa facing the peptide solution. The kinetics of CT degradation in the bulk solution was monitored by HPLC. Peptide sequences of the main nasal metabolites of hCT were analyzed by using both liquid secondary ionization mass spectrometry (LSIMS), following HPLC fractionation of the metabolites, and matrix-assisted laser desorption ionization mass (MALDI) spectrometry. For sCT, the molecular weights of two major metabolites were determined by LC-MS with electrospray ionization. Results. Both CTs were readily metabolized by nasal mucosal enzymes. In the concentration range studied metabolic rates were higher with hCT than with sCT. Presence of endopeptidase activities in the nasal mucosa was crucial, cleaving both calcitonins in the central domain of the molecules. Conclusions. Typically, initial metabolic cleavage of hCT in nasal mucosa is due to both chymotryptic- and tryptic-like endopeptidases. The subsequent metabolic break-down follows the sequential pattern of aminopeptidase activity. Tryptic endopeptidase activity is characteristic of nasal sCT cleavage.     

低频脉冲电磁场联合鲑鱼降钙素治疗原发性骨质疏松症

目的观察低频脉冲电磁场联合鲑鱼降钙素治疗对原发性骨质疏松症的疗效。方法将119例患者分为两组,一组采用低频脉冲电磁场联合鲑鱼降钙素治疗,一组采用鲑鱼降钙素治疗。结果 4周后疼痛缓解出现差异,低频脉冲电磁场联合鲑鱼降钙素治疗优于鲑鱼降钙素治疗组,6个月后,有67例复查骨密度,治疗组平均骨密度增长10.29%,两组比较差异有统计学意义。结论低频脉冲电磁场联合鲑鱼降钙素治疗原发性骨质疏松症,二者有明显协同作用,对缓解骨痛,提高骨密度,优于单一治疗。     

Therapeutic drug monitoring of atazanavir: surveillance of pharmacotherapy in the clinic

BACKGROUND: Therapeutic failure with antiretroviral therapy (ART) is a substantial issue where viral rebound, viral resistance and drug-related toxicity remain serious concerns. Drug exposure-response relationships have been described for the protease inhibitors, pharmacokinetic variability is substantial for this class of drugs and drug interactions can also alter ART exposure. Given this background we established a therapeutic drug monitoring (TDM) service to monitor atazanavir (ATV) plasma concentrations early after the therapy was made available to treatment-experienced people infected with HIV who were managed in a clinical setting. METHODS: This was a prospective observational study which evaluated plasma samples from 110 highly treatment-experienced people with HIV using TDM and applied pharmacokinetic analysis over a five month period. RESULTS: ATV trough concentrations exhibited substantial intersubject variability (<25-2108 microg l(-1)). A substantial number of subjects (50%,13/26) who received ATV400 mg daily had low exposure to ATV. Serum bilirubin concentrations correlated significantly with higher ATV trough concentrations (rho = 0.803; P < 0.001) and 55% (29/53) of subjects who received ATV300/100 mg RTV daily had plasma concentrations above a proposed target concentration associated with elevated bilirubin concentrations. This study confirmed low ATV exposure in eight subjects with HIV receiving ATV 400 mg daily. Reasons for low ATV exposure in this cohort include administration of interacting drugs, including a possible interaction with ritonavir, fluticasone and ATV, impaired ATV absorption secondary to suspected achlorhydria and potential interactions with colchicine and nandrolone. Viral load remained undetectable in most of these subjects with low ATV exposure. CONCLUSIONS: TDM and targeted pharmacokinetic studies should be viewed as fundamental tools in the development and clinical application of ART, to improve pharmacotherapy for people with HIV.     

醋柳总黄酮防治冠心病药理基础研究

醋柳总黄酮对动物在大剂量(400mg/kg)一次口服、连续3～7日小量用药(100～200mg/kg/d)或腹腔给药(400mg/kg)时均可增加心肌营养血流量,降低心肌耗氧量,提高动物耐缺氧的能力,对抗垂体后叶素所致的急性缺血性心肌损伤。此外,本品还能降低血清胆固醇,抑制血小板聚集,这些药理作用可能是本品临床上治疗冠心病有较好疗效的药理基础。醋柳总黄酮的毒性实验表明,本品无明显毒性,也没有致畸和致突变作用,是一种安全而有效的药物。     

Therapeutic patents for the treatment of pre-eclampsia

Pre-eclampsia (PE) is a pregnancy-specific syndrome that is a principal cause of maternal morbidity and mortality, accounting for almost 15% of pregnancy-associated deaths. In its mild form, PE most commonly presents with the features of maternal hypertension and proteinuria but can swiftly and unpredictably become severe with many extensive and life-threatening complications. The diverse symptoms of PE have made it a difficult disease not only to define, but also to identify a causative agent for the symptoms. It has therefore proved difficult to develop specific drugs that can be used to manage the condition. This review examines the patent literature to reveal current findings that exhibit the potential to target the effects of PE with the aim of either preventing or altering the course of this life-threatening disease of pregnancy.     

前列安颗粒急性毒性与主要药效学研究

目的考察前列安颗粒的急性毒性,验证其疗效,为临床安全用药提供依据。方法分别进行前列安颗粒急性毒性实验和主要药效学实验。考察前列安颗粒对1.5%角叉菜胶所致大鼠非细菌性前列腺炎的影响,对二甲苯所致小鼠耳肿胀的影响,0.6%醋酸所致小鼠腹腔毛细血管通透性的影响。结果急性毒性实验LD50无法测出,最大给药剂量为96 g&#8226;kg 1&#8226;d 1,相当于成人临床用量224.3倍。药效学实验表明,前列安颗粒可抑制角叉菜胶所致大鼠前列腺非细菌性炎症,增加前列腺组织内卵磷脂小体密度,并可明显抑制棉球肉芽肿形成;对二甲苯所致小鼠炎症均有明显抑制作用;可明显抑制0.6%醋酸所致小鼠腹腔毛细血管通透性增加。结论前列安颗粒临床用药安全,具有明显抑制前列腺炎作用。