N-trimethyl chitosan-modified liposomes as carriers for oral delivery of salmon calcitonin

Therapeutic peptide and protein drugs have high specificity and activity in their functions but present challenges in their administration route, requiring development of new delivery systems to improve their bioavailability. The aim of this work was to investigate the role of N-trimethyl chitosan- (TMC-) coated liposomes in the oral administration of calcitonin. TMC with a degree of quaternization around 78% was synthesized and its mucoadhesive properties were examined in vitro using the mucin-particle method, which confirmed that TMC showed mucoadhesion comparable to that of chitosan. TMC-coated liposomes containing calcitonin were prepared and characterized as having a particle size of 262?nm, zeta potential of 35.8 mV and high entrapment efficiency (89.1%). The in vivo evaluation of mucoadhesion was carried out using confocal laser microscopy to observe the residence time and permeation extent after intragastric administration. The results showed that TMC-coated liposomes prolonged the residence time and increased the penetration effect of the liposomal system compared to non-coated liposomes. The study of pharmacological effects confirmed that TMC-coated liposomes increased the area above the blood calcium concentration-time curves (AAC) from 3.13?±?20.50 to 448.84?±?103.56 compared to the calcitonin solution. These results support the feasibility of TMC-coated liposomes as a new oral delivery system for peptide and protein drugs.     

鲑鱼降钙素柔性脂质体大鼠鼻腔给药的降血钙效应

本文采用薄膜分散-超声挤压过膜法制备鲑鱼降钙素柔性脂质体，对其形态学及粒径进行了考察。以大鼠为实验动物，鼻腔给药，采用OCPC比色法测定大鼠血清钙离子浓度，计算降血钙百分率F%和降血钙效应D%，与鲑鱼降钙素普通脂质体、鲑鱼降钙素水溶液比较，以此评价鲑鱼降钙素柔性脂质体经大鼠鼻腔给药后的降血钙效应和附加剂对降钙素柔性脂质体的降血钙效应的影响。另外还对柔性脂质体的纤毛毒性进行考察。结果表明鲑鱼降钙素水溶液仅轻微降低血钙浓度，鲑鱼降钙素普通脂质体能提高降血钙效果，鲑鱼降钙素柔性脂质体呈现了最强的降血钙效果，其降血钙效果与脂质体中附加的表面活性剂的用量和种类相关，用去氧胆酸钠修饰的脂质体的降血钙效果最好，并且附加的表面活性剂用量越高，降血钙效果越强。柔性脂质体可以显著地降低去氧胆酸钠的纤毛毒性。结果显示，柔性纳米脂质体能显著提高降钙素经鼻腔给药的降血钙效果，柔性脂质体是蛋白多肽类药物鼻腔给药的一种有效的载体。     

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.     

Design,synthesis, characterization and in‐vivo activity of a novel salmon calcitonin conjugate containing a novel PEG‐lipid moiety

Objectives The aim of the study was to explore (1) the synthesis of a novel poly(ethylene glycol) modified lipid (PEG‐lipid, PL) containing a chemically active tri‐block linker, ε‐maleimido lysine (Mal), and its conjugation with salmon calcitonin (sCT), and (2) the biophysical properties and activity of the resulting conjugate, Mal‐PL‐sCT, relative to the control, 2PEG‐Mal‐sCT, which comprises sCT conjugated with α‐palmitoyl‐N‐ε‐maleimido‐l ‐lysine at cysteine 1 and cysteine 7, and PEG moieties at lysine 11 and lysine 18 via a conventional stepwise method. Methods The PEG‐lipid was obtained by condensing palmitic acid derivative of ε‐maleimido lysine with methoxy poly(ethylene glycol) amine. Under reductive conditions, the PEG‐lipid readily reacted with sCT to yield the resultant compound, Mal‐PL‐sCT. Key findings Dynamic light scattering analyses suggested that Mal‐PL‐sCT and 2PEG‐Mal‐sCT exhibited robust helical structures with a high tendency to aggregate in water. Both compounds were more stable against intestinal degradation than sCT, although Mal‐PL‐sCT was less stable than 2PEG‐Mal‐sCT. However, 2PEG‐Mal‐sCT did not possess hypocalcaemic activity while Mal‐PL‐sCT retained the hypocalcaemic activity of sCT when it was subcutaneously injected in the rat model. Multiple functional groups may be conjugated to a peptide via a tri‐block linker without the risk of obliterating the intrinsic bioactivity of the peptide. Conclusions The resultant novel PEG‐lipid has a potential role to optimize protein and peptide delivery.     

In Vivo Evaluation of an Oral Salmon Calcitonin-Delivery System Based on a Thiolated Chitosan Carrier Matrix

PURPOSE: To develop and evaluate an oral delivery system for salmon calcitonin. METHODS: 2-Iminothiolane was covalently bound to chitosan in order to improve the mucoadhesive and cohesive properties of the polymer. The resulting chitosan-TBA conjugate (chitosan-4-thiobutylamidine conjugate) was homogenized with salmon calcitonin. mannitol, and a chitosan-Bowman-Birk inhibitor conjugate and a chitosan-elastatinal conjugate (6.75 + 0.25 + 1 + 1 + 1). Optionally 0.5% (m/m) reduced glutathione. used as permeation mediator, was added. Each mixture was compressed to 2 mg microtablets and enteric coated with a polymethacrylate. Biofeedback studies were performed in rats by oral administration of the delivery system and determination of the decrease in plasma calcium level as a function of time. RESULTS: Test formulations led to a significant (p < 0.005) decrease in the plasma calcium level of the dosed animals in comparison to control tablets being based on unmodified chitosan. The addition of glutathione in the tablets led to a further improvement in the oral bioavailability of salmon calcitonin with an earlier onset of action and a decrease in the calcium level of about 10% for at least 10 h. CONCLUSIONS: The combination of mucoadhesive thiolated chitosan, chitosan-enzyme-inhibitor conjugates and the permeation mediator glutathione seems to represent a promising strategy for the oral delivery of salmon calcitonin.     

Development and in vivo evaluation of a new oral nanoparticulate dosage form for leuprolide based on polyacrylic acid

It was the aim of this study to develop a nanoparticulate oral drug delivery system for leuprolide based on polyacrylic acid (PAA). In order to achieve formation of nanoparticles in a mild, aqueous environment, two different techniques were combined, namely hydrophobic ion pairing between leuprolide and sodium dodecyl sulphate in a first step, followed by encapsulation into nanoparticles gained by interpolymer complexation between polyacrylic acid and Pluronic F68. The obtained nanoparticles were characterized regarding particle size distribution, drug encapsulation efficiency and in vitro release profile. Additionally, the pharmacokinetic profiles of leuprolide after oral administration of PAA-nanoparticulate and PAA-control tablets to male Sprague-Dawley rats were assessed and compared. It could be shown, that hydrophobic ion pairing increased encapsulation efficacy of leuprolide and leads to a slowed drug release of nanoparticulate suspensions. Relative oral bioavailability of leuprolide could be increased by nanoparticulate tablets up to 4.2-fold. Results verify that the suggested approach is a promising strategy for the design of oral delivery systems for oral administration of peptide drugs.