Temperature and pH-sensitive Polymers for Human Calcitonin Delivery

Purpose. Stimuli-sensitive polymers are suitable candidates for oral peptide drug delivery vehicles since they will prevent gastric degradation in the stomach while providing a controlled release of a peptide drug such as calcitonin later. The purpose of this study was to fabricate polymeric beads from pH/temperature sensitive linear terpolymers (poly(N-isopropylacrylamide-co-butylmethacrylate-co-acrylic acid)) and load them with a peptide drug, human calcitonin, which was dissolved in aqueous phase. Methods. The polymeric beads were formed by solubilizing a cold, aqueous solution of temperature sensitive polymer with human calcitonin. This solution was added dropwise into an oil bath kept at a temperature above the LCST of a polymer, precipitating polymer and entrapping the peptide. The quantity and the physical state of the peptide were analyzed by reverse-phase HPLC, CD and FTIR and its biological activity after loading was determined in vivo. Results. The loading efficiency and stability of human calcitonin into the polymeric beads was studied as a function of pH and ionic strength of the loading buffer and temperature of the oil bath. Final optimal loading conditions were 20 mM glycine/HCl buffer, pH 3.0 containing 0.15 M NaCl as a dissolution medium and 23°C as the oil bath temperature. Loading and release of human calcitonin were also studied as a function of acrylic acid content in the terpolymers. As the acrylic acid content increased from 0 to 10 mol %, the loading efficiency and stability of calcitonin improved significantly. The same trend was observed for the quantity of released calcitonin. In vivo biological activity of the released hormone was preserved. Conclusions. The results showed that the beads made of the polymers with high content of acrylic acid (most hydrophilic) provided better loading, stability and release of human calcitonin. The designed beads represent a new potential system for oral delivery of calcitonin and other peptides.     

Pulmonary Delivery of Salmon Calcitonin Dry Powders Containing Absorption Enhancers in Rats

Purpose. To evaluate the effects of absorption enhancers in dry powders and in liquids, pulmonary absorption of salmon calcitonin (sCT) in various formulations was measured. Methods. The dry powder of sCT was prepared by a freeze-drying method with a jet mill. After intratracheal administration of sCT dry powder and liquid (solution) preparations to rats, plasma sCT levels and calcium levels were measured. Results. After intratracheal administration without absorption enhancers, sCT in the dry powder and in the liquid were absorbed nearly to the same degree. Absorption enhancers (oleic acid, lecithin, citric acid, taurocholic acid, dimethyl-β-cyclodextrin, octyl-β-D-glucoside) were much more effective in the dry powder than in the solution. The reason may be that the enhancers added to the dry powder dissolved at high concentrations in a trace volume of the fluid lining the alveolar epithelium. Conclusions. The present results suggest that the pulmonary absorption of peptides and proteins can be greatly improved by formulating them into dry powders with smaller amounts of enhancers than in liquid dosage forms.     

Transdermal Delivery of Interferon Alpha-2B using Microporation and Iontophoresis in Hairless Rats

Purpose To demonstrate transdermal delivery of interferon alpha-2b (IFNα2b) in hairless rats through aqueous microchannels (micropores) created in the skin and enhanced by iontophoresis. Materials and Methods The Altea Therapeutics PassPort™ System was configured to form an array of micropores (2.0 cm²; 72 micropores/cm²) on the rat abdomen. The transdermal patch (Iomed TransQ1-GS-hydrogel) was saturated with an IFNα2b solution (600 μg/ml) and applied for 4 h. Delivery was evaluated with and without cathodic iontophoresis (0.1 mA/cm²). Intravenous delivery (0.4 μg/100 g body weight) was performed to support pharmacokinetic calculations. Results IFNα2b was not delivered through intact skin by itself (passive delivery) or during iontophoresis. However, passive delivery through micropores was achieved in vivo in rats. A dose of 397 ± 67 ng was delivered over 6 h, with steady state serum concentrations reaching a plateau at 1 h post-patch application. These levels dropped rapidly after patch removal, and returned to baseline within 2 h of patch removal. Iontophoresis-enhanced delivery through micropores resulted in a two-fold increase in the dose delivered (722 ± 169 ng) in the hairless rat. Conclusions In vivo delivery of IFNα2b was demonstrated through micropores created in the outer layer of the skin. Iontophoresis enhanced delivery through microporated skin in hairless rats.     

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.     

Investigation of Drug Delivery by Iontophoresis in a Surgical Wound Utilizing Microdialysis

PURPOSE: This study investigated the penetration of lidocaine around and through a sutured incision following the application of iontophoretic and passive patches in the CD Hairless rat. MATERIALS AND METHODS: Concentrations in localized areas (suture, dermis, subcutaneous, and vascular) were determined using microdialysis sampling followed by analysis using liquid chromatography with UV detection. RESULTS: Iontophoresis significantly enhanced the dermal penetration of lidocaine. In an intact skin model, dermal concentrations were 40 times greater following iontophoretic delivery compared to passive delivery. In a sutured incision model, iontophoresis enhanced localized concentrations in the dermis, suture, and subcutaneous regions by 6-, 15-, and 20-fold, respectively. Iontophoretic delivery to a region containing a sutured incision was focused to the incision resulting in a greater increase in the suture concentration and in the subcutaneous region directly below the incision. CONCLUSIONS: The four microdialysis probe design was successful in the determination of localized drug penetration in a sutured incision model. Iontophoresis enhanced skin penetration and allowed for site specific delivery when applied to a sutured incision.     

Transdermal Delivery of Heparin Using Pulsed Current Iontophoresis

Purpose In clinical practice heparin has to be administered by injection with obvious disadvantages; thus, transdermal delivery by electrically assisted methods have been studied. In this study we evaluated the efficacy of a Food and Drug Administration-approved pulsed current iontophoresis system in delivering heparin through living rat skin. Methods Fluorescent and radioactive heparin as well as a commercial heparin preparation were delivered through rat skin via a pulsed current iontophoresis system. Results Pulsed current iontophoresis allowed fluorescent heparin to cross the stratum corneum localizing in epidermis and dermis. Unfractionated, high-, and low molecular weight fraction pools, obtained by fractionating [³⁵S]-unfractionated heparin on a molecular weight sieve, were then separately tested. Pulsed current iontophoresis elicited the transdermal delivery of low molecular weight heparin, but not that of high molecular weight heparin. Finally, pulsed current iontophoresis of an unfractionated pharmaceutical heparin preparation significantly decreased plasmatic factor Xa activity. Conclusions We hypothesize that this technique could be used to administer low molecular weight heparin in a cost-efficient and safe manner without the need for syringes and needles.     

Effect of Charge and Molecular Weight on Transdermal Peptide Delivery by Iontophoresis

Purpose The study was conducted to investigate the impact of charge and molecular weight (MW) on the iontophoretic delivery of a series of dipeptides. Methods Constant current iontophoresis of lysine and 10 variously charged lysine- and tyrosine-containing dipeptides was performed in vitro. Results Increasing MW was compensated by additional charge; for example, Lys (MW = 147 Da, +1) and H-Lys-Lys-OH (MW = 275 Da, +2) had equivalent steady-state fluxes of 225 ± 48 and 218 ± 40 nmol cm⁻² h⁻¹, respectively. For peptides with similar MW, e.g., H-Tyr-d-Arg-OH (MW = 337 Da, +1) and H-Tyr-d-Arg-NH₂ (MW = 336 Da, +2), the higher valence ion displayed greater flux (150 ± 26 vs. 237 ± 35 nmol cm⁻² h⁻¹). Hydrolysis of dipeptides with unblocked N-terminal residues, after passage through the stratum corneum, suggested the involvement of aminopeptidases. The iontophoretic flux of zwitterionic dipeptides was less than that of acetaminophen and dependent on pH. Conclusions For the series of dipeptides studied, flux is linearly correlated to the charge/MW ratio. Data for zwitterionic peptides indicate that they do not behave as neutral (“charge-less”) molecules, but that their iontophoretic transport is dependent on the relative extents of ionization of the constituent ionizable groups, which may also be affected by neighboring amino acids.     

用大鼠血钙法测定降钙素生物效价的探讨

本文报道了用大白鼠血钙法测定降钙素生物效价的方法。选定适当的大鼠血钙测定方法，测定了大白鼠的正常血钙值。探讨了不同给药途径及各种实验条件、剂量和反应的关系等。测定了４批降钙素注射液的喷鼻剂的生物效价。     

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.     

Topical Iontophoresis of Valaciclovir Hydrochloride Improves Cutaneous Aciclovir Delivery

Purpose To investigate the topical iontophoresis of valaciclovir (VCV) as a means to improve cutaneous aciclovir (ACV) delivery.Methods ACV and VCV electrotransport experiments were conducted using excised porcine skin in vitro.Results While the charged nature of the prodrug, VCV, enabled it to be more efficiently iontophoresed into the skin than the parent molecule, ACV, only the latter was detectable in the receptor chamber, suggesting that VCV was enzymatically cleaved into the active metabolite during skin transit. Iontophoresis of VCV was significantly more efficient than that of ACV; the cumulative permeation of ACV after 1, 2 and 3 h of VCV iontophoresis at 0.5 mA cm⁻² and using an aqueous 2 mM (∼0.06%) formulation was 20 ± 10, 104 ± 47 and 194 ± 82 μg cm⁻², respectively (cf. non-quantifiable levels, 0.1 and 1.0 ± 0.7 μg cm⁻² after ACV iontophoresis).Conclusions These delivery rates provide ample room to reduce either current density or the duration of current application. Preliminary in vitro data serve to emphasize the potential of VCV iontophoresis to improve the topical therapy of cutaneous herpes simplex infections and merit further investigation to demonstrate clinical efficacy.     

Iontophoresis of Nafarelin Across Human Skin in Vitro

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.     

Transdermal Delivery of Sumatriptan Succinate Using Iontophoresis and Dissolving Microneedles

This study focuses on the in vitro transdermal transport of sumatriptan succinate using combined iontophoresis and dissolving polymeric microneedle arrays. Permeation experiments were performed to evaluate the effects of formulation parameters on drug release from polyvinylpyrrolidone systems under mild electrical current (≤500 μA/cm²). The preparations consisted of hydrophilic, positively charged molecules encapsulated in a water-soluble and biocompatible polymeric material. Current densities of 100, 300, and 500 μA/cm² were applied during a 6-h period using silver/silver chloride electrodes. The circular array consisted of 600 needles and occupied a 0.785 cm² area. Tests, carried out with Franz diffusion cells and skin of Göttingen minipigs, showed that small decreases in the polymer concentration led to negligible lag times and marked increases in the cumulative amount of drug permeated in 6 h (Q_6h) and in the flux (J_ss). At 500 μA/cm², Q_6h and J_ss nearly doubled for a microneedle loaded with 5% (w/w) sumatriptan and 20% (w/w) PVP (lag time = 0 min; Q_6h = 2888 μg/cm²; J_ss = 490 μg/cm²/h) relative to a system loaded with 5% (w/w) drug and 30% (w/w) PVP (lag time = 36 min; Q_6h = 1437 μg/cm²; J_ss = 266 μg/cm²/h).     

Effects of Fatty Acids and Iontophoresis on the Delivery of Midodrine Hydrochloride and the Structure of Human Skin

Purpose. The purpose of this work was to investigate if fatty acids can increase the iontophoretic delivery of midodrine hydrochloride through human dermatomed skin and to observe the effects of iontophoresis and fatty acids on skin using SEM. Methods. After prehydration for 1 h, human dermatomed skin was treated with 0-0.3 M fatty acids (oleic acid, linoleic acid, decanoic acid, and lauric acid) in propylene glycol (PG) for 1 h. Then the fatty acid solution was replaced by 1% midodrine hydrochloride aqueous solution, and 0.1 mA/cm² constant current was applied. Samples were taken over 24 h and analyzed by HPLC. After the treatments outlined above, the epidermis was separated, fixed with glutaraldehyde, and dehydrated for SEM. Results. SEM studies revealed that only 1 h of treatment with fatty acids opened up the tightly compact stratum corneum cell layer, and the permeation study showed a significant increase of the permeability of skin to midodrine hydrochloride after fatty acid treatment. Conclusions. Using 5% oleic acid pretreatment, with the electrical current offset at 0.1 mA/cm², the daily delivery of midodrine hydrochloride can provide an adequate clinical application. The enhancement of passive and iontophoretic delivery by fatty acids may be occurring through the same mechanism.     

Localization of a FITC-Labeled Phosphorothioate Oligodeoxynucleotide in the Skin After Topical Delivery by Iontophoresis and Electroporation

Purpose. The aim of this study was to verify the hypothesis that the application of high voltage to the skin enhances both stratum corneum and keratinocyte permeability. Therefore, the transport of FITC labelled phosphorothioate oligonucleotides (FITC-PS) administered by passive diffusion, iontophoresis or electroporation was localized. Methods. Fluorescent microscopy and laser scanning confocal microscopy were used to visualize the FITC-PS transport at the tissue and cell level respectively in hairless rat skin after electroporation (5 × (200 V 500 ms) or iontophoresis (same amount of charges transferred). Results. FITC-PS did not penetrate the viable skin by passive diffusion. Molecular transport in the skin upon electroporation or iontophoresis was localized and implied mainly hair follicles for iontophoresis. In the stratum corneum, the pathways for FITC-PS transport were more transcellular during electroporation and paracellular during iontophoresis. FITC-PS were detected in the nucleus of the keratinocytes a few minutes after pulsing. In contrast, iontophoresis did not lead to an uptake of the oligomer. Conclusions. The internalization of FITC-PS in the keratinocytes after electroporation confirms the hypothesis and suggests that electroporation, which allows both efficient topical delivery and rapid cellular uptake of the oligonucleotides, might be useful for antisense therapy of epidermal diseases.     

Buccal Transmucosal Delivery of Calcitonin in Rabbits Using Thin-Film Composites

Purpose. Salmon Calcitonin (sCT) is used to treat hypercalcemia resulting from Paget's disease and osteoporosis. sCT is available either in a sterile injectable form or nasal spray. Alternative and more cost-effective dosage forms for the delivery of calcitonin are needed. We sought to deliver sCT transmucosally using a previously reported mucoadhesive bilayer thin-film composite (TFC) via the buccal route. Methods. Forty micrograms of salmon calcitonin (200-IU) was loaded on preformed TFCs. In vitro release of sCT from TFCs was monitored in phosphate-buffered saline (10 mM, pH 7.4) at 37°C. Female New Zealand White rabbits (n = 6) were dosed with 40 g of sCT either by injection via the ear vein or by applying sCT-loaded TFCs directly on the buccal pouch. Blood was collected at various times, and the plasma sCT and calcium concentrations were quantified. WinNonlin® was used to determine the relevant pharmacokinetic parameters. Results. In vitro, over 80% of sCT was released from the TFCs within 240 min. Super Case-II transport was indicated as the primary release mechanism. Rabbits injected intravenously had C _max, Cls, Vss, and AUC_0-inf values of 75.1 ± 6.5 ng/mL, 20.7 ± 3.3 mL/min, 637 ± 141 mL, and 1925 ± 237 ng*min/mL, respectively. Rabbits dosed via the buccal route had C _max, Cls, and AUC_{0-400 min} values of 4.6 ± 1.6 ng/mL, 22.0 ± 5.9 mL/min, and 842.9 ± 209.7 ng*min/mL, respectively. The relative bioavailability for rabbits treated with the TFCs was 43.8 ± 10.9% with a CV of 24.9%. The reductions in plasma calcium levels after administration of sCT by both the intravenous and buccal route were comparable. Conclusions. The TFCs effectively delivered therapeutically efficacious amounts of sCT across the buccal mucosa in rabbits.     

Systemic Delivery of Cetrorelix to Rats by a New Aerosol Delivery System

Purpose. To study the pulmonary absorption and tolerability of various formulations of the decapeptide cetrorelix acetate in rats by a new aerosol delivery system (ASTA-ADS) for intratracheal application. Methods. Using the ASTA-ADS, cetrorelix liquid formulations (aqueous solutions for ultrasonic nebulization) were firstly selected and subsequently delivered as nebulized aerosol to orotracheally cannulated rats. The pharmacologic effect (decrease of testosterone serum level) of four cetrorelix formulations was determined in rats by enzyme linked immunosorbant assay, and pharmacokinetic data were determined after measurement of cetrorelix serum level by radioimmunoassay. Histological examination of the lung was performed at the end of the experiments, and in a supplementary experiment the respiratory parameters (resistance and compliance) of rats were monitored by a validated pulmonary monitoring system during the aerosol application of the same formulations. Results. After an exposure time of 5 min, the applied formulations reduced the testosterone concentration in serum to subnormal levels (1 ng/ml) over a period of 24 h. Comparing the plasma concentration after intratracheal aerosolization with data of intravenous administration, the mean calculated bioavailabilities for the four formulations using the corrected dose (delivered—exhaled amount) were between 48.4 ± 27.0% and 77.4 ± 44.0%. The histologic examination of the lungs revealed different tolerability of the various tested formulations ranging from locally intolerable to well tolerated. The measurement of the lung function parameters did not reveal any compound or formulation related changes. Conclusions. Our studies show that cetrorelix can be effectively administered as aerosol and that intratracheal aerosolization via the ASTA-ADS provides results that are well comparable to other application routes, as demonstrated by statistical comparison of the newly obtained data with previous results from intratracheal instillation of cetrorelix solutions in rats.     

基于透皮离子导入技术的戒烟药物金雀花碱的可控递送研究

目的 探索烟碱乙酰胆碱受体部分激动剂金雀花碱(cytisine,CTS)经阳极离子导入透过离体猪皮。方法 采用HPLC-PDA建立并验证CTS的分析方法。研究了电流密度、药物浓度和药物基质对CTS透皮离子导入的影响。采用标志物对乙酰氨基酚解析CTS离子导入过程中电迁移和电渗的贡献。结果 CTS从水溶液中被动透皮不佳,但在离子导入条件下CTS的透皮递送量显著增加,将电流密度从0.15 mA·cm^–2增加到0.5 mA·cm^–2可使离子导入CTS的稳态流量呈线性增加(J=452.8I+31.51,r=0.998 3)。在使用0.5 mA·cm^–2电流密度的条件下,给药池药物浓度的增加(2.5,5.0,10.0 mg·mL^–1)可使累积透皮递送量显著增加。共离子导入对乙酰氨基酚证实了电迁移是CTS的主要递送机制(˃90%)。CTS的传导效率良好(6.63%~8.82%)。递送效率,即递送药物占所给予的制剂中的药物的百分数较高(在0.5 mA·cm^–2时>40%)。CTS从离子导入贮库HEC水凝胶中递送时的累积透皮递送量为(1 551.94±322.19)μg×cm^–2,与从CTS溶液中递送时的累积透皮递送量无统计学差异。结论 体外数据表明,使用较小面积的凝胶贴片通过透皮离子导入可以方便地递送治疗剂量的CTS。