Elsevier

Biochemical Engineering Journal

Volume 41, Issue 2, 1 September 2008, Pages 106-110
Biochemical Engineering Journal

Design and in vivo evaluation of solid-in-oil suspension for oral delivery of human growth hormone

https://doi.org/10.1016/j.bej.2008.04.001Get rights and content

Abstract

Solid-in-oil (S/O) suspension containing human growth hormone (hGH), in which hGH was complexed with an edible surfactant, was designed and validated for oral administration of hGH. To optimize the formulation procedures, protein release behavior from the S/O suspensions under physiological conditions was first investigated with horseradish peroxidase (HRP) as a model protein. Evaluation of functional integrity of HRP released from the HRP–surfactant complex suggested that the solubilization process partly impaired the specific activity of HRP, however, the addition of trehalose in the formulation process regained up to about 50% of the biological activity. On the basis of the data collected with HRP, a surfactant–hGH complex prepared under optimized conditions was suspended in soybean oil to formulate S/O suspensions. After oral administration of the S/O suspension to male New Zealand rabbits, we detected hGH in the serum with 3.3% bioavailability, suggesting that hGH can be orally delivered to the systemic circulation by the present formulation.

Introduction

Synthetic human growth hormone (hGH) is a polypeptide comprising 191 amino acids (22 kDa) with two internal disulfide bridges identical to that of the major component of human pituitary growth hormone [1], and has been manufactured by recombinant DNA technology. Therapeutically, hGH is employed in children to treat growth retardation, for example, short stature due to insufficient growth hormone secretion, Turner's syndrome or chronic renal insufficiency. In adults, it is used as a treatment for growth hormone deficiency and for management of HIV-related wasting and cachexia [2].

In most cases, hGH is administrated by injection. However, several disadvantages, such as low patient compliance, possibility of infection and pain during repeated administration by injection or implant, have prompted the discovery of a non-invasive way to administrate hGH. Several reports have focused on parenteral administration of hGH, except for injection. For example, administration via lung has recently been demonstrated [2]. Although the administration through the nose has its problems, such as damage to the nasal mucosa, due to repeated administration. Therefore, efforts towards the development of oral delivery systems have been intensified because of ease of administration and greater patient compliance and acceptability [3]. However, the oral route has yet to be adopted for pharmaceutical macromolecular substances, due to absorption barriers and the extensive enzymatic degradation in the gastrointestinal tract [4].

As far as insulin (5.8 kDa) is concerned, a number of strategies have been proposed to overcome these barriers, including the use of enteric-coated pellets [5], chitosan nanoparticles [6], hydrogels [7], and liposomes [8]. By contrast, there have been very few reports on the development of oral delivery of proteins with relatively high molecular weight, such as hGH. We have previously reported the feasibility of solid-in-oil (S/O) suspensions for the oral delivery of hydrophilic drugs such as diclofenac sodium [9] and insulin [10], which are stably entrapped in the oil phase by complex formation with an edible lipophilic surfactant.

In this paper, we investigated the potential of S/O suspensions for oral delivery of hGH. We first optimized the formulation processes with horseradish peroxidase (HRP) as a model protein, and examined the protein release behavior in vitro. Finally, S/O suspension containing hGH prepared under the optimized conditions was validated in in vivo experiments.

Section snippets

Materials

Recombinant hGH, HRP, pullulan, trehalose and soybean oil were purchased from Wako Pure Chemical Industries (Osaka, Japan). Sucrose was purchased from Kishida Chemical (Osaka, Japan). Sucrose fatty acid esters, sucrose laurate (L-195), sucrose oleate (O-170) and sucrose erucate (ER-290), used as surfactants were kindly provided by Mitsubishi–Kagaku Foods (Tokyo, Japan). According to the manufacturer's information, the first capital alphabets in the abbreviations stand for fatty acids used for

Protein release behavior from surfactant–protein complexes

S/O suspensions are oil-based formulations that are capable of encapsulating hydrophilic drugs into an oil phase by complex formation of edible lipophilic surfactants and target drugs. We have previously found that S/O suspensions can deliver insulin to the systemic circulation if soybean oil is employed as the base. In that case, surfactant–insulin complex is released in the intestinal fluid, upon degradation of the oil phase by intestinal lipases [10]. The absorption of surfactant–protein

Conclusion

In the present study, we characterized and improved a new type of pharmaceutical protein formulation, the S/O suspension, from the standpoint of oral protein delivery. With proper combination of surfactant and additives, the protein release rate was controllable, while retaining functional integrity. It was revealed that trehalose appeared to be effective in stabilizing the proteins in its complex form with surfactants, even after suspending in soybean oil. Finally, we succeeded in improving

Acknowledgments

This work was supported in part by a Grant-in-Aid for the Global COE Program, “Science for Future Molecular Systems” from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M.G.).

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