鲑鱼降钙素喷鼻剂的人体生物等效性研究

目的:研究国产和进口鲑鱼降钙素鼻喷剂的生物等效性。方法:采用双周期自身随机交叉设计。用放射免疫法(RIA)测定22例健康志愿者喷鼻给药后的血药浓度。结果:国产制剂及进口鼻喷剂实测平均血药峰浓度C_(max)分别为(64.72±9.68)ng·L~(-1)和(65.61±8.46)ng·L~(-1);实测平均达峰时间t_(max)分别为(0.27±0.05)h和(0.27±0.05)h;国产制剂及进口制剂t_(1/2)(ke)分别为(0.68±0.23)h和(0.69±0.24)h;血药浓度-时间曲线下面积AUC_(0-8)h平均值分别为(61.70±15.53)ng·L~(-1)·h和(64.89±15.12)ng·L~(-1)·h;以进口制剂为参比制剂,试验制剂鲑鱼降钙素鼻喷剂的相对生物利用度为(95.95±15.21)%。结论:国产与进口鲑鱼降钙素鼻喷剂生物等效。     

鲑降钙素鼻喷雾剂的研制

制备了用于治疗骨质疏松症的鲑降钙素鼻喷雾剂，采用正交试验法，以加速实验后产品的含量为指标对处方进行优化，以HPLC法测定含量。结果显示制剂的稳定性达到实验设计要求。2－8℃贮存稳定性好。     

Adsorption of Salmon Calcitonin to PLGA Microspheres

Purpose. The interaction of salmon calcitonin (sCT) and poly (d,l-lactide-co-glycolide) was detected during preparation and evaluation of microspheres. The purpose of this study was to quantitate the extent and nature of the interaction. Methods. Blank microspheres were prepared by an aqueous emulsification solvent extraction technique. Adsorption studies were carried out at six concentrations of sCT and three concentrations of microspheres. Adsorption isotherms were constructed using the Langmuir and Freundlich treatments. Results. Adsorption at 1 mg/ml sCT concentration resulted in almost complete depletion of the peptide from the adsorption medium with the time to reach maximum adsorption decreasing with increasing microsphere concentration. At sCT concentrations below 100 µg/ml, a true equilibrium occurred in 1 hour or less while at higher concentrations (up to 350 µg/ml), a transient equilibrium was reached in 1 to 2 hours, followed by further adsorption of the peptide. The adsorption followed the Langmuir isotherm at concentrations below 200 µg/ml, indicating formation of a monolayer. Multilayer interaction, described by the Freundlich isotherm, occurred at higher concentrations and resulted in complete depletion of sCT from the adsorption medium. The affinity constant during monolayer formation was 0.09 and the plateau surface concentration was 5.1 µg/mg. The multilayer peptide-peptide adsorption showed a lower affinity (0.025) but higher capacity (24 µg/mg) than the monolayer peptide-polymer adsorption. Conclusions. The results show that poly (d,l-lactide-co-glycolide) microspheres have a high adsorption capacity for sCT which must be considered in formulating a controlled delivery product of this peptide.     

Drug Reservoir Composition and Transport of Salmon Calcitonin in Transdermal Iontophoresis

Purpose. The aim of the work was to study iontophoretic transdermal administration of salmon calcitonin (sCt) in rabbits, with particular attention to drug reservoir composition. A dry sCt disc, to be dissolved on the application site, was used for preparing the reservoir for transdermal iontophoresis. As a reference drug reservoir, a pad wetted with drug solution was used. Methods. Experiments were done in rabbits depositing 100 IU of salmon calcitonin on skin and applying anodal iontophoresis. Serum calcium concentration was measured during iontophoresis, passive diffusion and after i.v. administration. Parameters such as pH value and reservoir type were examined. Results. Transdermal iontophoresis of sCt elicited a decrease in the serum calcium level, whereas, in the absence of electric current, no significant fall was measured. Using the reservoir prepared from drug solution, anodal iontophoresis at pH 4.2 was more effective than at pH 7.4, probably due to higher sCt net positive charge. Using the reservoir prepared from dry disc, similar kinetics and extent of drug effect were observed at both pH values. The reservoir prepared from solid drug deposit concentrated sCt next to the skin. Conclusions. Anodal iontophoresis for transdermal calcitonin administration shows therapeutical applicability. The type of reservoir is an important parameter affecting sCt transdermal iontophoresis.     

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.     

Degradation of Synthetic Salmon Calcitonin in Aqueous Solution

Pharmaceutical Research -     

Enhancement of Nasal Salmon Calcitonin Absorption by Lauroylcarnitine Chloride in Rats

Purpose. We investigated optimum formulation characteristics in the nasal absorption of salmon calcitonin (sCT) by incorporation of acylcarnitines. Methods. Nasal sCT formulations were administered to anesthetized rats. Plasma calcium level was measured and pharmacological bioavailability (P.bioav) was calculated. Results. Nasal sCT absorption was significantly enhanced by carnitines with acyl groups of 12 or more carbon atoms. Enhancement by lauroylcarnitine chloride (LCC) was observed at its critical micelle concentration and reached a plateau at the concentration of 0.1%. Optimal absorption was achieved at a molar ratio of LCC to sCT of 5:1. Enhancement was not influenced by osmolarity and maximum enhancement was obtained at pHs 3.1 and 4.0. Conclusions. The 12-carbon LCC was the strongest enhancer among acylcarnitines. Micelle formation played a key role in this enhancement effect.     

鲑鱼降钙素合成中二硫键形成方法的探讨

在鲑鱼降钙素的合成中，采用空气氧化、二甲亚砜－三氟乙酸氧化、二四亚砜－水氧化和铁氰化钾氧化四种方法，对二硫键的形成方法进行了比较，实验结果表明用铁氰化钾法氧化时间短，氧化率高。     

鲑鱼降钙素促进老年转子间骨折愈合的疗效观察

目的:观察鲑鱼降钙素促进老年转子间骨折愈合的疗效。方法:将40例老年患者随机分为2组:治疗组20例,肌肉注射鲑鱼降钙素;对照组20例,在相同的部位肌肉注射等量注射用生理盐水。自术后第1天开始,2组均连续用药3个月。用药后的第1、3个月复查X线片,观察骨痂生长情况。治疗前和治疗后3个月检测患者腰椎(L4)骨密度(BMD)及血清钙、磷、碱性磷酸酶等浓度。结果:所有患者均得到随访。术后第1、3个月X线评估发现,治疗组比同时期对照组的骨痂生成量多。治疗后3个月,治疗组腰椎(L4)BMD明显高于对照组(P<0.05),治疗组血钙浓度明显低于对照组(P<0.05),2组血磷浓度无明显差异(P>0.05),治疗组碱性磷酸酶明显高于对照组(P<0.05)。治疗组1例出现恶心、呕吐等胃肠道症状,2例出现面颈部潮红。结论:鲑鱼降钙素能显著促进老年转子间骨折的愈合。     

HPLC法和生物测定法测定鲑鱼降钙素肠溶胶囊含量的比较

目的:采用高效液相色谱(HPLC)法测定鲑鱼降钙素肠溶胶囊中降钙素的含量,并与生物测定法进行比较。方法:HPLC法色谱柱为依利特ODS2 C18,流动相为0.4%四丁基氢氧化铵水溶液(pH 3.8)-乙腈(334∶166,V/V),流速为1 ml/min,检测波长为220 nm;生物测定法参照《中国药典》相关方法进行。结果:HPLC法降钙素检测质量浓度线性范围为5²00μg/ml(r=0.999 3),辅料对主药测定无干扰,精密度试验RSD<5%,平均回收率为96.5%,含量测定结果为99.96%;生物测定法可信限率为28.84%,虽符合要求(<45%),但含量测定结果为111.62%,估计与制剂中含吸收促进剂有关。结论:与生物测定法相比,HPLC法测定鲑鱼降钙素肠溶胶囊含量,操作简便、成本低、重复性好,结果更为可靠。     

A High Throughput Protein Formulation Platform: Case Study of Salmon Calcitonin

Purpose  The feasibility of using high throughput spectroscopy for characterization and selection of physically stable protein formulations was studied. Materials and Methods  A hundred aqueous formulations of salmon calcitonin (sCT) were prepared using 20 buffer compositions. The solutions had pH values between 2.5 and 10.5. The stability of the sCT formulations was analyzed over 1 week by the following assays: (1) protein concentration, (2) volume control by measuring pathlength, (3) turbidity (absorbance at 350 nm), (4) intrinsic tyrosine fluorescence, (5) 1-anilino-naphthalene-8-sulfonate (ANS) fluorescence, (6) Nile Red fluorescence. Addition of the dyes (Nile Red and ANS) was used to study protein conformational changes. Results  After 1 day, 27 out of the 100 formulations of salmon calcitonin were stable. After 7 days, 12 stable sCT formulations remained. The best salmon calcitonin formulation was in 10 mM sodium acetate buffer with pH values between 3.5 and 5.5. Conclusions  The findings are in accordance with the sCT formulations that were patented and used commercially. This can be considered as a proof of concept for the high throughput protein formulation platform. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Bioavailability of Intranasal Salmon Calcitonin in Healthy Volunteers with and Without a Permeation Enhancer

Serum levels of radioimmunoactive salmon calcitonin (sCT) were determined in 10 healthy volunteers after intranasal administration (IN) of 100-, 205-, and 450-IU of sCT with 0.5% sodium tauro-24,25-dihydrofusidate (STDHF), a 200-IU commercial IN formulation, and a 100-IU intramuscular (IM) formulation. Relative to the IM dose, the bioavailabilities of the IN formulations containing 0.5% STDHF were 3.9, 7.9, and 7.4%, respectively. The 200-IU commercial formulation resulted in serum levels above the limit of detection in only 5 of 10 patients, with an average bioavailability of 1.6%.     

鲑鱼降钙素对老年性骨质疏松症骨密度和骨生化指标的影响

目的 :探讨鲑鱼降钙素治疗老年性骨质疏松症的疗效。方法 :120例老年性骨质疏松症患者随机分为试验组和对照组。对照组服用钙尔奇 -D和维生素AD ,治疗组在对照组治疗基础上加用鲑鱼降钙素。结果 :治疗6个月后 ,试验组腰椎L3～4、股骨颈部的骨密度较治疗前明显升高 (P<0 05) ,Wards区骨密度无显著性改变 ,而对照组各部位骨密度较用药前无明显改变 (P>0 05) ;试验组骨痛总有效率为92 31 % ,对照组为30 91 % (P<0 01) ;试验组血碱性磷酸酶、骨钙素较治疗前明显下降 (P<0 05) ,对照组用药前、后无明显改变。结论 :鲑鱼降钙素与钙剂联合治疗老年性骨质疏松症 ,能有效缓解骨痛 ,提高患者骨量的作用。     

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.     

鲑鱼降钙素联合护理干预治疗老年糖尿病合并骨质疏松症的疗效观察

目的 探讨鲑鱼降钙素联合护理干预对老年糖尿病合并骨质疏松症患者的临床疗效.方法 选取我科2012年2月-2013年5月的2型糖尿病合并骨质疏松患者106例,随机分为观察组和对照组各53例,对照组给予服钙尔奇D治疗,观察组在此基础上加用鲑鱼降钙素及护理干预.比较两组干预前后骨痛程度及股骨颈骨密度（FN BMD）、腰2~腰4骨密度（L2~4 BMD）的变化.结果 两组治疗前FN BMD和L2~4 BMD比较差异无统计学意义（P＞0.05）.观察组治疗后FN BMD和L2~4 BMD均高于对照组,差异有统计学意义（P＜0.05）.观察组骨痛改善总有效率88.7%高于对照组的34.0%,差异有统计学意义（P＜0.05）.结论 鲑鱼降钙素联合康复干预能明显改善老年糖尿病合并骨质疏松症骨痛症状,提高骨密度.     

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.     

鲑鱼降钙素联合钙尔奇D治疗糖尿病伴骨质疏松的疗效观察

目的探讨鲑鱼降钙素联合钙尔奇D治疗糖尿病伴骨质疏松症患者的疗效及安全性。方法 16例糖尿病伴骨质疏松症患者用鲑鱼降钙素50IU1次/d,肌肉注射,同时服用钙尔奇D,600mg/d,连续治疗4个周。在治疗前、治疗后分别测量骨密度（BMD）、骨代谢指标骨钙素（BGP）,血钙、血磷、肝肾功能等,观察骨质疏松症腰背疼痛症状的治疗效果。结果治疗4个周后骨密度较治疗前明显升高,骨钙素显著高于治疗前,甲状旁腺激素和碱性磷酸酶二者显著减低,腰背疼痛等骨病症状减轻甚至消失。结论鲑鱼降钙素加钙尔奇D能够抑制骨吸收,促进骨形成,降低骨转换,并能迅速改善腰背疼痛症状,对糖尿病伴骨质疏松症治疗效果显著。     

Study on Pulmonary Delivery of Salmon Calcitonin in Rats: Effects of Protease Inhibitors and Absorption Enhancers

Effects of protease inhibitors and absorption enhancers on the absorption of salmon calcitonin (sCT) were evaluated after intratracheal coadministration to rats using the plasma Ca level as an index. Remarkable absorption enhancement could be attained with unsaturated fatty acids such as oleic acid and polyoxyethylene oleyl ether (absorption enhancers) and with chymostatin, bacitracin, potato carboxypeptidase inhibitor and phosphoramidon (protease inhibitors). sCT degrading enzymes had four times higher activity per total protein in membrane fraction of lung homogenates than the activity in cytosol fraction. These enzymes are thought to be serine proteases and metalloenzymes from the in vitro action profile of protease inhibitors. A good correlation between the in vitro activity of protease inhibitors and the in vivo enhancing effect on sCT activity suggested that membrane enzymes are responsible for the inactivation of sCT. Metabolic degradation and low permeability of sCT may be possible barriers to the absorption of sCT.     

鲑降钙素联合仙灵骨葆治疗绝经后妇女骨质疏松症所致骨痛疗效观察

目的:观察鲑降钙素联合仙灵骨葆治疗绝经后妇女骨质疏松症所致骨痛的疗效及安全性。方法:160例绝经后骨质疏松症所致骨痛妇女随机分为3组,治疗组鼻喷鲑降钙素500IU每日1次,并口服仙灵骨葆胶囊1.5g,每日2次;对照组Ⅰ鼻喷鲑降钙素500IU,每日1次;对照组Ⅱ口服仙灵骨葆胶囊1.5g,每日2次。3组均口服钙尔奇D,每日1片。以30d为1疗程,连续使用3个疗程。评定3组骨痛性质和程度变化,记录药品不良反应发生情况。结果:治疗组、对照组Ⅰ、对照组Ⅱ的总有效率分别为92.59%、69.81%、67.92%,治疗组总有效率与对照组Ⅰ、对照组Ⅱ比较差异有统计学意义(P<0.05);不良反应发生率分别为18.52%、16.98%、11.32%,3组不良反应发生率差异无统计学意义(P>0.05)。结论:鲑降钙素联合仙灵骨葆治疗绝经后妇女骨质疏松症所致骨痛疗效确切,能有效增加患者骨密度,缓解骨质疏松症所致骨痛,是一种安全、有效的方法。