Lipolysis of the semi-solid self-emulsifying excipient Gelucire® 44/14 by digestive lipases

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Abstract

Gelucire® 44/14 is a semi-solid self-emulsifying excipient used for the oral delivery of poorly water-soluble drugs. It is composed of C8-C18 acylglycerols and PEG-32 esters, all of which are potential substrates for digestive lipases. Here we studied the lipolysis of Gelucire® 44/14 by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases. Human pancreatic lipase (HPL), the main lipase involved in the digestion of triacylglycerols, did not show any significant activity on Gelucire® 44/14 or on either of its individual compounds, C8-C18 acylglycerols and PEG-32 esters. Other pancreatic lipases such as human pancreatic lipase-related protein 2 (HPLRP2) showed low activity on Gelucire® 44/14 although the highest activity of HPLRP2 was that observed on the C8-C18 acylglycerol fraction, which accounts for 20% (w/w) of Gelucire® 44/14. In addition, HPLRP2 showed low activities on the PEG-32 esters, whether these were tested individually or mixed together. Carboxyl ester hydrolase (CEH) showed high activity on Gelucire® 44/14, and the highest activities of CEH were those recorded on the total PEG-32 ester fraction and on each individual PEG-32 ester, except for PEG-32 monostearate. The highest activity of all the enzymes tested was that of dog gastric lipase (DGL) on Gelucire® 44/14, although DGL showed low activity on the PEG-32 ester fraction and on each individual PEG-32 ester. We compared the lipolysis of Gelucire® 44/14 with that of Labrasol®, another self-emulsifying excipient, which is liquid at room temperature. Human pancreatic juice showed similar rates of activity on both Gelucire® 44/14 and Labrasol®. This finding means that these excipients are hydrolyzed in vivo during pancreatic digestion, mainly by CEH in the case of Gelucire® 44/14 and by both HPLRP2 and CEH in that of Labrasol®, whereas HPL showed very low activities on each of these two excipients. This is the first time the effects of PEG and acyl chain length on the lipolytic activity of digestive lipases on PEG esters have been investigated.

Introduction

One of the main challenges facing the pharmaceutical industry at present is how to improve the oral bioavailability of poorly water-soluble drugs using innovative formulations. Among the latest methods developed, SEDDS (Self Emulsifying Drug Delivery Systems), SMEDDS (Self MicroeEmulsifying Drug Delivery Systems) and recently, SNEDDS (Self NanoEmulsifying Drug Delivery Systems) are efficient formulations which improve the oral bioavailability of poorly water-soluble active substances [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. SEDDS and SMEDDS are isotropic mixtures of oil, surfactant, co-surfactant, and drug that form oil-in-water emulsions and microemulsions, respectively, under gentle stirring [11], [12].

The enhanced oral bioavailability of poorly water-soluble drugs administered in a lipid-based drug delivery system is probably due to both the pre-dissolved state of the drug present in the system itself and the ability of these formulations and their digestion products to interact with endogenous biliary amphiphilic molecules and dietary lipids in the gastrointestinal tract [13], [14]. This suggests that the lipolysis of lipid-based formulations plays a decisive role in the absorption of poorly water-soluble drugs [15].

Gelucire® 44/14 and Labrasol® are macrogolglycerides which are able to form microemulsions in the gastrointestinal fluids [16]. These excipients are already being used in commercial formulations of Fenofibrate (LIPIREX-Gé® in France, CIL® and FENOFIBRAT AZU® in Germany), Ibuprofen (SOLUFEN® in Europe) and Piroxicam (PIROFLAM® in Germany). Gelucire® 44/14 is a mixture of C8 to C18 mono-, di-, and triacylglycerols, C8 to C18 mono- and diesters of PEG-32, and free PEG-32. This macrogolglyceride is obtained by performing polyglycolysis of medium and long chain triacylglycerols with PEG-32 (molecular weight = 1500). The main fatty acid involved here is lauric acid. Gelucire® 44/14 is able to increase the bioavailability of various drugs, such as DMP 323 (HIV protease inhibitor) [17], α-tocopherol [18], nifedipine [19], 17-β estradiol [20], ontazolast [21], solvent green 3 [22], propranolol [23] and piroxicam [24]. Labrasol® is a mixture of PEG-8 caprylocaproyl acylglycerols, which is obtained by performing polyglycolysis of medium chain triacylglycerols with PEG-8 (molecular weight = 400). Labrasol® is composed of C8-C10 mono-, di-, and triacylglycerols, C8-C10 mono- and diesters of PEG-8, and free PEG-8. The main fatty acids present are caprylic and capric acids. However, how exactly these excipients enhance the bioavailability of poorly water-soluble drugs is not yet fully understood.

We therefore developed an in vitro method for studying the lipolysis of lipid-based excipients under conditions simulating the environment of the gastrointestinal tract. This method sorts out the lipases liable to hydrolyze the lipid-based excipient [25]. It also takes into consideration the gastric digestion step, which is generally overlooked in classic bioavailability and/or solubility studies. In vivo, gastric digestion is necessary to stimulate the bile and pancreatic juice secretion processes, as well as to trigger the action of pancreatic lipase. Porcine pancreatin has often been used as a model for human pancreatic juice in classic lipolysis and/or solubility studies on poorly water-soluble drugs [26], [27]. It is generally assumed that the lipolytic activity of pancreatic juice is due to the action of classic pancreatic lipase and its cofactor, colipase. However, although classic pancreatic lipase is the main lipase involved in the lipolysis of dietary triacylglycerols [28], [29], other lipolytic enzymes produced by the pancreas can act on the various esters present in lipid-based excipients such as PEG esters.

The aim of this study was therefore to identify the lipases capable of hydrolyzing Gelucire® 44/14. In vitro experiments were performed for this purpose using porcine pancreatic extracts, human pancreatic juice, recombinant human pancreatic lipase, human pancreatic lipase-related protein 2, dog gastric lipase, and purified bovine carboxyl ester hydrolase. The specificity of each digestive lipase towards the fractions of C8-C18 acylglycerols and PEG-32 esters of Gelucire® 44/14 was also investigated, and the specific activities of these lipases on various individual acylglycerols (from triacylglycerols to monoacylglycerols) and PEG-32 esters with several acyl chain lengths (mono- and diesters) were determined. We further investigated the effects of PEG length and the degree of substitution of PEG on the lipolytic activity of these lipases. Lastly, the results obtained with Gelucire® 44/14 were compared with those previously obtained upon the in vitro lipolysis of the liquid excipient Labrasol®.

Section snippets

Chemicals

Trilauroylglycerol (TC12, ≥ 97.0% GC), sodium taurodeoxycholate (NaTDC, 97% TLC), bovine serum albumin (BSA), and calcium chloride dihydrate (CaCl2, 2H2O; minimum 99%), were purchased from Sigma-Aldrich-Fluka Chimie (Saint-Quentin-Fallavier, France). Dilauroylglycerol (DC12), monolauroylglycerol (MC12), monopalmitoylglycerol (MC16), and monostearoylglycerol (MC18) were purchased from Larodan AB (Malmö, Sweden). Tris-(hydroxymethyl)-aminomethane (Tris) and sodium chloride (NaCl) were purchased

Lipolysis by porcine pancreatic extracts and human pancreatic juice

The highest specific activities of porcine pancreatic extracts and human pancreatic juice were recorded on Gelucire® 44/14 at pH 7.5 (Table 1). These specific activities were determined by performing back-titration of FFAs. The ratios between the activities deduced from FFAs back-titration and direct titration at pH 7.5, (3.6 in the case of PPE and 1.4 in that of HPJ: see Fig. 1A and B, respectively) were found, however, to differ significantly, which suggests that PPE and HPJ did not release

Identification of digestive lipases acting on Gelucire® 44/14

In studies on the lipolysis of lipid-based excipients, porcine pancreatin is generally used in vitro to mimic the secretion of human pancreatic enzymes [36], [37], and the lipolytic activity is often attributed to the classic pancreatic lipase which represents 8% of the total protein content of human pancreatic juice [38]. This lipase and its cofactor, colipase, form a complex which is mainly responsible for lipolysis of dietary lipids [28], [29]. However, we established that the human

Acknowledgments

We are grateful to Cecilia Eydoux and Alain De Caro for their help with the production and purification of rHPLRP2. Sylvie Fernandez's PhD research was supported by a CIFRE contract from Agence Nationale de la Recherche Technique (ANRT, France).

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