Development of low cost pulmonary surfactants composed of a mixture of lipids or lipids–peptides using higher aliphatic alcohol or soy lecithin

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Abstract

The artificial pulmonary surfactant composition in the present study is characterized by a lipid mixture system composed of higher aliphatic alcohol, egg yolk phosphatidylcholine (egg PC), soy lecithin and higher aliphatic acid as the major components or a peptide–lipid mixture system composed of a combination of the lipid mixture system to which a peptide is added. Three peptides with amphiphilic surface-staying, membrane spanning, and both properties were designed and synthesized. The evaluation of pulmonary surfactant assay was performed by a hysteresis curve drawn upon the measurement for the surface tension–area curve with the Wilhelmy surface tensometer in vitro and the recovery of lung compliance for the pulmonary surfactant-deficient rat models in vivo. Lipid-mixture systems composed of octadecanol or soy lecithins containing no peptide were favorable hysteresis curves as compared with commercially available Surfacten®, but were not prominent. The peptide–lipid mixture systems composed of a combination of the lipid mixture of alkyl alcohol or soy lecithin to which peptides designed were added were desirable hysteresis curves similar to Surfacten® and amphiphilic Hel 13-5 peptide–lipids mixture systems were much more effective than the lipid mixture system. Particularly, the recovery of lung compliance treated with hydrogenated soy lecithin–fractionated soy lecithin PC70–palmitic acid–peptide Hel 13-5 (40:40:17.5:2.5, w/w) was comparable to that with Surfacten®. Because the artificial pulmonary surfactant compositions of this study can be prepared at lower costs, they are useful for the treatment of respiratory distress syndrome and acute respiratory distress syndrome as well as for inflammatory pulmonary diseases, dyspnea caused by asthma, etc.

Introduction

The pulmonary surfactant is a lipid–protein complex produced by the alveolar cells and secreted there from, which is a substance essential for the maintenance of life and which plays a role for the pulmonary function by reducing the surface tension of the alveoli [1]. Fujuwara et al. have reported about effect of the artificial pulmonary surfactant on human respiratory distress syndrome (RDS) in the newborn infant and succeeded for the first time in the world in the development of an agent for curing RDS (“Surfacten®”) [2], which is prepared by extracting the active pulmonary surfactant components from the bovine lung. The incidence of acute respiratory distress syndrome (ARDS) in adults is also reported that the administration of large amounts of the artificial pulmonary surfactant at the early stage of ARDS can improve the pulmonary functions and minimizing the damages of the lung reducing the mortality up to 20% [3].

It is also known that the pulmonary surfactant substance is secreted from the bronchus, and the substance is considered to play a role as an expectorant by preventing the block of the peripheral airway. The pulmonary surfactant is expected to apply to various diseases that require improvements in respiratory disorders because, for example, the inhalation of the pulmonary surfactant can relieve a fit of allergy-induced asthma [4], [5]. Thus, there is an increasing necessity of the pulmonary surfactant for the application not only to RDS and ARDS but also to inflammatory pulmonary diseases.

From the above, many medical doctors involved in the treatment of respiratory diseases have pointed out the possibilities of application of the pulmonary surfactant to many different kinds of pulmonary diseases; however, the pulmonary surfactant is very expensive that the application of Surfacten® to RDS only is currently covered by the health insurance in Japan. A further issue is that Surfacten® is a bovine protein preparation so that it still has the possibility of infection due to its antigenicity and unknown antigenicity and the problem with bovine spongiform encephalopathy (BSE) for example still remains unsolved. In this sense, the development of an artificial pulmonary surfactant that can be prepared at lower costs and has no side effects has been demanded.

Relating to this, the artificial synthetic surfactant (non-animal-derived pulmonary surfactant) has been also developed [6], which includes two categories: one is protein (or peptide)-free synthetic surfactants (Exosurf®, etc.) and the other is protein (peptide)-containing synthetic surfactants [7]. The peptide-containing surfactant that is currently under clinically available is a lipid–peptide complex (Surfaxin®) composed of 21 amino acids (KL4) [8], [9]. More specifically, it is composed of phospholipids dipalmitoyl-l-α-phosphatidylcholine (DPPC) and PG, a synthetic peptide KL4 having a particular amino sequence, and an aliphatic acid [10]. However, because these surfactants are still expensive, the application to various lung diseases is limited.

It is needed to form a stable single molecular membrane and a bimolecular membrane in order to consider the mechanism of transcription of the pulmonary surfactant activity. Further, in order to prevent a collapse of the lung at the time of the compression of the lung, the presence of the phospholipids [11], particularly DPPC, is considered to be essential. Further, a small amount of PG is also considered to be need. However, the conventional artificial pulmonary surfactant composition using DPPC and PG has the demerits that the scope of application is limited because DPPC and PG are so expensive that the resulting artificial pulmonary surfactant composition results in an expensive one. Therefore, we considered in the present study the uses of an alternative material for the DPPC and PG. As an alternative material of PC, a phospholipid such as, for example, egg yolk phosphatidylcholine (egg PC) extracted and purified from relatively less expensive egg yolk lecithin or soy lecithin. Fractional soy lecithin from crude soy oils is commercially available at lower costs. As an alternative material to be used for the DPPC having a saturated alkyl chain, there may be used, for example, a saturated higher alcohol (e.g., n-1-octadecanol, OD) and hydrogenated lecithin prepared by hydrogenating the unsaturated aliphatic chain of the fractional soy lecithin. As the acidic component, there may be used a higher aliphatic acid (e.g., palmitic acid (PA), stearic acid, etc.) which has been frequently used as the lipid for the conventional artificial pulmonary surfactant. In addition, a neutral lipid (e.g., triacylglycerol, cholesterol (Ch), etc.) may also be used.

On the other hand, the proteins contained in the artificial pulmonary surfactant composition are considered to play a catalyzing action in order to permit the phospholipids to smoothly achieve the transduction between the single molecular membrane and the bimolecular membrane [12]. Systematic review of clinical trial has also shown that non-protein-containing synthetic surfactants are less effective than animal-derived or peptide-containing products. Therefore the peptides are added to the above-mentioned lipid mixture system as needed. As preferred examples of the artificial pulmonary surfactant peptides to be used for the present study are shown in Fig. 1.

The artificial pulmonary surfactant compositions according to the present study were prepared by admixing the above lipids with each other or the above lipids with the peptide or peptides at a predetermined rate. The artificial pulmonary surfactant compositions were assessed for their surfactant activity by using a surface tension–area diagram. For comparative purposes, the measurements were conducted as a control for Exosurf® composed of the lipid system only and containing no peptide, which is to be used as a medicine for treating respiratory disorders, Surfaxin® containing DPPC as a major ingredient and a synthetic peptide consisting of lysine (K) and leucine (L), and Surfacten®.

Section snippets

Materials

As materials there were used the following: egg PC, a phospholipid purified from egg yolk (Avanti Polar Lipids, Inc.); egg PG (Sigma); egg yolk lecithin; and other lipids as well as reagents (Wako Pure Chemical Industries, Japan). Soy lecithin: hydrogenated soy lecithin (“SLP White H”), fractionated soy lecithin 70 (fractional lecithin SLP-PC70), hydrogenated soy lecithin 70 H (prepared by hydrogenating soy lecithin SLP-PC70) were purchased from True Lecithin Mfg. Co., Ltd. (Mie, Japan). The

Designed peptides

The pulmonary surfactant is composed of a complex of lipids and proteins. The proteins comprise four kinds of surfactant proteins (SPs), i.e., SP-A, SP-B, SP-C and SP-D, which amount to approximately 5% of the total weight [12]. The pulmonary surfactant proteins, SP-B and SP-C, which are of significance to the pulmonary surfactant activity, are different from each other in the mode of action to the membrane [12], [14]. The surfactant protein SP-B lies on the membrane surface by partially

Discussion

The pulmonary surfactant present in the alveoli is composed of approximately 10% of proteins and lipid components consisting mainly of phospholipids [11], [19]. The pulmonary surfactant contains the lipids, particularly a neutral phospholipid, i.e., PC which amounts to 80.5% of the total lipids; while PG which is an acidic lipid amounts to 9.1%, phosphatidylinositol (PI) to 2.6% and cholesterol to 7.3% [11]. In particular, it is to be noted that DPPC composed of saturated alkyl groups amounts

Acknowledgements

We thank Mr. Shinobu Maekawa, Muromachi Chemical Co., Ltd., for helpful technical assistance. This work was supported by the grant of Research and Development (2004–2005), Fukuoka Industry Technology Foundation.

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