The effects of excipients and particle engineering on the biophysical stability and aerosol performance of parathyroid hormone (1-34) prepared as a dry powder for inhalation

Abstract

Pulmonary delivery of therapeutic peptides and proteins has many advantages including high relative bioavailability, rapid systemic absorption and onset of action and a non-invasive mode of administration which improves patient compliance. In this study, we investigated the effect of spray-drying (SD) and spray freeze-drying processes on the stability and aerosol performance of parathyroid hormone (PTH) (1-34) microparticles. In this study, the stabilisation effect of trehalose (a non-reducing sugar) and Brij 97 (a non-ionic surfactant) on spray-dried PTH particles was assessed using analytical techniques including circular dichroism (CD), fluorescence spectroscopy, modulated differential scanning calorimetry and an in vitro bioactivity assay. Physical characterisation also included electron microscopy, tap density measurement and laser light diffraction. The aerosol aerodynamic performance of the formulations was assessed using the Andersen cascade impactor. Based on these studies, a formulation for spray freeze-drying was selected and the effects of the two particle engineering techniques on the biophysical stability and aerosol performance of the resulting powders was determined. CD, fluorescence spectroscopy and bioactivity data suggest that trehalose when used alone as a stabilising excipient produces a superior stabilising effect than when used in combination with a non-ionic surfactant. This highlights the utility of CD and fluorescence spectroscopy studies for the prediction of protein bioactivity post-processing. Therefore, a method and formulation suitable for the preparation of PTH as a dry powder was developed based on spray-drying PTH with trehalose as a stabiliser with the bioactivity of SD PTH containing trehalose being equivalent to that of unprocessed PTH.

Fig. 1

Scanning electron micrographs of PTH (1-34) microparticles. a Unprocessed PTH, b spray-dried PTH/trehalose 1:5, c spray-dried neat PTH, d spray-dried PTH/trehalose/Brij 97 (1:5:0.058), e spray-dried neat trehalose and f spray freeze-dried PTH/trehalose (1:5)

Fig. 2

CD spectra (far UV region) of reconstituted PTH microparticles in double-distilled deionized water. From top to bottom: spray-dried PTH/trehalose/Brij 97, spray-dried neat PTH, unprocessed PTH, spray freeze-dried PTH and spray-dried PTH/trehalose

Fig. 3

a Fluorescence spectra comparing the tertiary structures of (from top to bottom) spray-dried PTH/trehalose, unprocessed PTH and spray-dried neat PTH and spray-dried PTH/trehalose/Brij 97. b Fluorescence spectra comparing the tertiary structures of (from top to bottom) spray-dried PTH/trehalose, unprocessed PTH and spray freeze-dried PTH/trehalose

Fig. 4

The in vitro stimulation of cAMP synthesis by the different PTH formulations (mean ± SD; n = 5)

Fig. 5

MDSC thermal profiles of a spray freeze-dried PTH/trehalose, b spray-dried neat PTH, c unprocessed PTH, d spray-dried neat trehalose, e spray-dried PTH/trehalose/Brij and f spray-dried PTH/trehalose