Design and evaluation of poly(dl-lactic-co-glycolic acid) nanocomposite particles containing salmon calcitonin for inhalation

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Abstract

Salmon calcitonin, for the treatment of calcium homeostasis and bone remodeling, was used as a model peptide drug and adsorbed on the surface of biodegradable polymeric poly(dl-lactic-co-glycolic acid) (PLGA) nanospheres. Subsequently, the nanospheres were treated using lyophilizer and loaded onto inhalable carrier using MechanofusionTM to obtain nanocomposite particles suitable for inhalation. The physicochemical properties and in vitro inhalation properties of the nanocomposite particles were investigated. The pulmonary distribution and pharmacological effect were also evaluated in male Wistar rats. The results showed that the drug loading efficiency of salmon calcitonin on PLGA nanospheres were exceeding 96% (w/w). Inhalation efficiency of the lyophilized PLGA nanospheres was largely improved after they were loaded on the surface of inhalable carrier. Over 50% (w/w) of the lyophilized PLGA nanospheres could be deposited in the alveoli section after intratracheal administration to male Wistar rats, while a rapid elimination rate of the lyophilized nanospheres from the lung was found in pulmonary distribution study. The in vivo pharmacological study showed that the nanocomposite particles exhibited superior hypocalcemic action over salmon calcitonion solution and the lyophilized nanospheres. It suggested that the MechanofusionTM technique can impart improved inhalation properties to the lyophilized nanospheres for pulmonary delivery of therapeutic peptide drugs.

Introduction

With the development of biotechnology and genetic engineering during last several decades, peptide and protein drugs have become more and more important therapeutic agents to improve the quality of life of patients (Davis, 1999). However, due to the low absorption rate and instability in gastrointestinal tract, the administration of these active agents was mainly limited to parenteral routes. Among many non-invasive administration routes the lungs have shown to be the most promising alternative to injection to deliver biopharmaceuticals such as peptide and protein drugs to obtain systemic absorption (Codrons et al., 2003, Gonda, 2000, Smith, 1997).

Currently, three types of aerosol formulations and devices are available: nebulizers (jet or ultrasonic), (pressurized) metered dose inhalers (pMDIs) and dry powder inhalers (DPIs). Dry powder inhalers have initially found their application in inhalation therapy as a CFC-free alternative for the older MDIs. Nowadays they seem to have a much larger potential than other devices (Ashurst et al., 2000, Malcolmson and Embleton, 1998), because a higher lung deposition can be attained through DPIs and also because they are more suitable for delivery of labile active substances like therapeutic peptides and proteins due to its dry dosage form (Patton, 1996). For the preparation of dry peptide or protein-contained powders for inhalation the main techniques include lyophilization, milling, spray-drying, spray freeze-drying, co-precipitation and super critical fluid technology (Zijlstra et al., 2004). In these, lyophilization is the most commonly used technique and considered to be the mildest technique to treat these labile bio-macromolecular drugs (Wang, 2000). However a subsequent milling process is often required to improve the powder characteristics when lyophilization is used as the technique to dry peptide and protein drugs. Moreover the micronized lyophilized powder need to be further loaded onto inhalable carriers like lactose to aid in handling and to impart aerodynamic benefits to the formulation.

The objective of this study is to report a new particulate design technique in which lyophilization, milling and dry coating process were combined to obtain a nanocomposite particles of biopharmaceuticals suitable for inhalation. Salmon calcitonin, a polypeptide of 32 amino acids was employed as a model drug. The main biological effects of salmon calcitonin are to inhibit bone resorption, increase the urinary excretion of calcium and inhibit the absorption of calcium in the gastrointestinal tract. It is currently formulated as a sterile solution for intramuscular or subcutaneous injection in the management of several bone-related diseases including Paget’s disease, hypercalcemia and osteoporosis (Lee et al., 1991). Considering its high potency and high cost, the drug was firstly incorporated into nano-carriers, i.e. biodegradable polymeric poly(dl-lactic-co-glycolic acid (PLGA) nanospheres by adsorption method and then lyophilization was used to transform them into dry powder. Nano-carriers were used because they can provide many features to therapeutics, e.g. controlled release property, improved stability and enhanced absorption of the drug. Subsequently, the lyophilized nanospheres were composed on inhalable lactose (Pharmatose325MTM) using MechanofusionTM to obtain nanocomposite particles. The adsorption mechanism and release behaviors of the PLGA nanospheres were studied using HPLC method. The lyophilized nanospheres and nanocomposite particles were characterized with regard to their physicochemical properties and in vitro inhalation properties. Furthermore the pulmonary distribution, pulmonary clearance and pharmacological effect of the nanocomposite particles were evaluated in male Wistar rats.

Section snippets

Chemicals and reagents

Salmon calcitonin was supplied from EMD Biosciences, Inc., USA. Pharmatose325MTM, with a mean diameter of 50 μm, was obtained from DMV International, Netherland. Coumarin-6, which was employed as a marker molecule to investigate the inhalation properties, was supplied by ICN Biomedicals, USA. PLGA with an average molecular weight of 20 kDa and a copolymer ratio of lactide to glycolie of 75:25 and polyvinylalcohol (PVA 403, degree of polymerization = 300, 80% hydrolyzation degree) were obtained from

Adsorption of salmon calcitonin on PLGA nanospheres

The drug loading efficiency and average diameter of PLGA nanospheres produced in this study are listed in Table 1. The results showed that more than 96.7% w/w (corresponding to 0.1% w/w drug loading) of salmon calcitonin was loaded onto the PLGA nanospheres, which is much higher than the drug loading efficiency of other peptides onto PLGA nanospheres prepared by the same emulsion solvent diffusion method reported in our previous research papers (Kawashima et al., 1999). The average particle

Conclusions

The biodegradable polymeric PLGA nanospheres prepared using emulsion solvent diffusion method in water were employed as nano-carriers, to which salmon calcitonin was incorporated by adsorption method, and a subsequent lyophilization process was conducted to produce dry powder. To improve the handling property of the lyophilized powder, MechanofusionTM, a dry coating processor was employed to load the lyophilized nanospheres onto the surface of inhalable carrier to obtain nanocomposite

Acknowledgement

This work was supported by NEDO (New Energy and Industrial Technology Development Organization).

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Current address: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark.

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Current address: School of Pharmacy, Aichi Gakuin University, 1–100 Kusumoto, Chikusaku, Nagoya 464-8650, Japan.

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