Biopharmaceutical characterisation of insulin and recombinant human growth hormone loaded lipid submicron particles produced by supercritical gas micro-atomisation

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Abstract

Homogeneous dispersions of insulin and recombinant human growth hormone (rh-GH) in tristearin/phosphatidylcholine/PEG mixtures (1.3:1.3:0.25:0.15 w/w ratio) were processed by supercritical carbon dioxide gas micro-atomisation to produce protein-loaded lipid particles. The process yielded spherical particles, with a 197 ± 94 nm mean diameter, and the insulin and rh-GH recovery in the final product was 57 ± 8% and 48 ± 5%, respectively. In vitro, the proteins were slowly released for about 70–80 h according to a diffusive mechanism. In vivo, the insulin and glucose profiles in plasma obtained by subcutaneous administration of a dose of particles containing 2 μg insulin to diabetic mice overlapped that obtained with 2 μg of insulin in solution. Administration of a dose of particles containing 5 μg insulin resulted in faster and longer glycaemia reduction. Oral administration of 20 and 50 μg insulin equivalent particles produced a significant hypoglycaemic effect. The glucose levels decreased since 2 h after administration, reaching about 50% and 70% glucose reduction in 1–2 h with the lower and higher dose, respectively. As compared to subcutaneous administration, the relative pharmacological bioavailability obtained with 20 and 50 μg equivalent insulin particles was 7.7% and 6.7%, respectively. Daily subcutaneous administration of 40 μg of rh-GH-loaded particles to hypophysectomised rats induced similar body weight increase as 40 μg rh-GH in solution. The daily oral administration of 400 μg rh-GH equivalent particles elicited a slight body weight increase, which corresponded to a relative pharmacological bioavailability of 3.4% compared to subcutaneous administration.

Introduction

Biotech drugs, namely proteins and oligonucleotides, are thought to become the main source of therapeutics in the near future (Tsuji and Tsutani, 2008). Nevertheless, the therapeutic exploitation of such molecules will rely on the possibility to develop suitable formulations that can satisfactorily overcome the intrinsic limitations to their use, namely physicochemical instability and low bioavailability (Orive et al., 2003).

As peptide drugs are often indicated for chronic conditions, the requirement for long term daily injections has obvious drawbacks (Nestor, 2007). Although the oral route offers the advantages of self-administration with high patient acceptance and compliance, poor membrane permeability hampers oral, as well as other non-invasive administration routes (Morishita and Peppas, 2006). To enable effective transmucosal protein delivery, attention has been focused on formulations which improve transmembrane absorption and prevent from degradation (Jorgensen et al., 2006).

Micro- and nano-sized systems (e.g., liposomes, lipid and polymeric nanoparticles, micelles, etc.) have been found to provide advantages over traditional formulations for protein delivery. The entrapment of protein drugs into these systems provides for (1) a way to hide and protect the proteins from degradation during storage and delivery and (2) sustained release (Almeida and Souto, 2007, Bilati et al., 2005, Mundargi et al., 2008).

Among particulate formulations, solid lipid nanoparticles have been successfully explored for drug delivery because they combine the benefits of liquid lipid-based colloidal systems (e.g., emulsions and liposomes) and solid systems (Joshi and Müller, 2009, Kluge et al., 2009, Mehnert and Mader, 2001). These products possess excellent tissue biocompatibility, biodegradability, composition flexibility and small size, making them suitable for a variety of applications. Furthermore, these formulations have been found to enhance the drug bioavailability after oral or local administration. On the other hand, solid lipid particle manufacturing techniques are not easily adaptable to protein processing as they operate under harsh conditions; namely, high temperature, pressure, and shear stress, and in a few cases, involve the use of organic chemicals, which are detrimental to protein stability. Moreover, often these techniques are difficult to scale-up for industrial production.

In recent years, much effort has been made to provide solutions which meet these existing manufacturing needs. In particular, techniques based on supercritical fluids have been developed to process polymer and lipid materials and produce particulate pharmaceutical. These techniques can be properly adapted to protein processing as they can avoid denaturation and degradation phenomena and may be exploited to produce pharmaceutical grade protein delivery system formulations (Caliceti et al., 2004, Davies et al., 2008, Okamoto and Danjo, 2008, Reverchon et al., 2008, Reverchon et al., 2009, Tandya et al., 2007).

Recently, we described a novel supercritical fluid gas micro-atomisation process for preparation of protein-loaded lipid particles (Salmaso et al., 2009). This technique is an improved version of “Particle from Gas Saturated Solution” (PGSS) developed to process materials melting under supercritical conditions. In our previous study, we demonstrated that the introduction of a co-axial air injection device in the typical PGSS equipment can yield submicron particles. Furthermore, a preliminary study performed with insulin as the protein model showed that the use of selected excipient mixtures and the optimisation of operating conditions resulted in high product yield, protein loading and preservation of biological protein activity.

Since we demonstrated that the gas micro-atomisation process was suitable for the fabrication of submicron protein-loaded lipid particles, we further investigated the biopharmaceutical and in vivo performance of these particles. Aimed at evaluating the general applicability of the manufacturing process to produce therapeutically efficient drug delivery systems, the study was performed using insulin and recombinant human growth hormone (rh-GH), two proteins of relevant pharmaceutical interest with significantly different physicochemical properties. Protein-loaded lipid particles were produced under optimised operative conditions and their biopharmaceutical properties were evaluated. In vivo investigations were undertaken using appropriate animal models to evaluate the pharmacokinetic and pharmacodynamic performance of these formulations after either subcutaneous or oral administration.

Section snippets

Materials

Tristearin, Tween 80, 5 kDa poly(ethylene glycol) (PEG 5000) and dimethylsulfoxide were obtained from Fluka (Buchs, Switzerland). Streptozotocin, glucose Trinder kit, glucose standard solution and bovine insulin (5.7 kDa) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Recombinant human growth hormone (rh-GH, 21 kDa) was a kind gift of Bio-Ker (Pula, Italy). Phosphatidylcholine (Epikuron 200) was donated by Degussa (Padova, Italy). Acetonitrile was from Merck (Darmstadt, Germany) and CO

Particle preparation and characterisation

The combination of tristerain, phosphatidylcholine, 5 kDa PEG and DMSO yielded homogeneous low melting temperature mixtures. The DSC thermograms of tristerain, PEG and the tristearin/PEG/phosphatidylcholine/DMSO mixture are reported in Fig. 1.

The thermogram of the lipid mixture reported in Fig. 1A shows the disappearance of the endotherm peak at 61.16 °C of PEG (Fig. 1B) and the thermal modification of tristerain (Fig. 1C). The endothermal peak corresponding to the α form at 50.78 °C disappeared

Discussion

Tristearin, phosphatidylcholine, and PEG were selected for preparation of lipid particles because these pharmaceutical excipients, approved by the main regulatory agencies either for oral or parenteral formulations, possess the appropriate physicochemical features for supercritical processing and can yield delivery systems with suitable biopharmaceutical properties (Chen, 2008, Hauss, 2007, Schulze and Winter, 2009, Spilimbergo et al., 2006). Preliminary DSC studies showed that the lipid

Conclusions

Supercritical techniques represent an emerging versatile opportunity for customised fabrication of formulations with tailored physicochemical and biopharmaceutical features. Nevertheless, many operative obstacles must be overcome to set up reliable and scalable processes to yield products with the required pharmaceutical properties.

The implementation of the supercritical PGSS process by introducing a peristaltic pump and co-axial air-flow assisted device allowed for setting up a new flexible

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