Geotrichum sp．SYBC WU-3脂肪酶的双水相萃取和酶学性质

初步研究双水相体系对Geotrichum sp．SYBC WU-3脂肪酶的萃取分离效果,选用PEC4000／NaH2 PO4作为戍相系统进行系统研究,考察影响脂肪酶萃取的各种因素（如PEG相对分子质量及质量分数、NaH2PO4质量浓度、pH）,并采用正交实验进一步优化实验条件,确定双水相萃取体系为PEG质量分数为30％、NaH2PO4质量分数为20％、体系pH为6,在此条件下Geotrichum sp．SYBC WU-3脂肪酶经硫酸铵沉淀和双水相萃取两步纯化的纯化倍数达到最大,较Geotrichum sp．SYBC WU-3脂肪酶粗酶纯化了22倍。Geotrichum sp．SYBC WU-3脂肪酶纯酶为低温碱性脂肪酶,最适反应温度为15oC,最适pH为9．5,相对分子质量为3．58×10^4。     

Influence of the alkyl-substituted silane precursor on sol–gel encapsulated lipase activity

Lipase from Candida rugosa was encapsulated within a chemically inert sol–gel support prepared by polycondensation of three precursor types (tetraethoxysilane (TEOS), methyltrimethoxysilane (MTMS) and polydimethylsilane (PDMS)) in the presence and absence of polyethylene glycol (PEG) and polyvinyl alcohol (PVA) as additives. Silica and their derivatives were characterised with regard to mean pore diameter, specific surface area, pore size distribution (BET method), weight loss upon heating thermogramivemetric analysis (TGA), chemical composition Fourier transform infrared spectroscopy (FT-IR), and catalytic activities. Immobilisation yields based on the recovered lipase activity vary from 3.02 to 31.98% and the highest efficiency was attained when lipase was encapsulated using TEOS in the presence of the PEG. Further information was obtained by testing the derivatives in esterification reactions and a different reactivity profile was found. Better performance was obtained with derivatives containing lipase encapsulated within gels prepared with MTMS as precursor in the presence of PEG. This lipase preparation exhibits increased esterification activity (155 μmol g⁻¹ min⁻¹), up to of three times greater than that prepared with TEOS (52 μmol g⁻¹ min⁻¹), and almost twice that prepared with MTMS/PDMS (89 μmol g⁻¹ min⁻¹) as precursors.     

Enhanced activity of Candida rugosa lipase modified by polyethylene glycol derivatives

The derivatives of polyethylene glycol (PEG) were prepared by reacting PEG with propylene oxide to enhance its hydrophobicity and introduce a branched structure. The PEG derivatives were activated with cyanuric chloride and used to modify the lipase fromCandida rugosa. The maximum specific activity of lipase modified with the PEG derivatives was about 2-fold of that modified with PEG for the esterification of oleic acid and lauryl alcohol in hexane.     

Lipase immobilization on magnetic microspheres via spacer arms: Effect of steric hindrance on the activity

Poly(styrene-acrylic acid) magnetic microspheres with an average diameter of 2 μm were successfully prepared and used as carriers to immobilize lipase. Lipase immobilized on microspheres with no spacer arm exhibited low activities, which were attributed to steric hindrance on the lipase conformation. To avoid steric effects, ethylenediamine and poly(ethylene glycol) (PEG) 400/800/4000 were utilized as spacer arms to bind the lipase to the microspheres. The immobilized lipase activities were improved using PEG 800/4000 as a spacer arm. Furthermore, the influence of enzyme loading on lipase activity was investigated, and the results indicated that enzyme overloading could exert steric effect on lipase activity. The degree of PEG modification was demonstrated to affect lipase activity because excess PEG on the surface of microspheres could interact with lipase due to its mobility, consequently reducing lipase activity.     

Improved bioavailability of inhibitors of Trypanosoma cruzi trans-sialidase: PEGylation of lactose analogs with multiarm polyethyleneglycol

The trans-sialidase of Trypanosoma cruzi (TcTS) catalyzes the transfer of sialic acid from host glycoconjugates to terminal β-galactopyranosides in the mucins of the parasite. During infection, the enzyme is actively shed by the parasite to the bloodstream inducing hematological alterations. Lactitol prevents cell apoptosis caused by the TcTS, although it is rapidly eliminated from the circulatory system. Linear polyethyleneglycol (PEG) conjugates of lactose analogs were prepared but their clearance from blood was still quite fast. With the aim of improving their circulating half-lives in vivo, we now synthesized covalent conjugates of eight-arm PEG. The star-shape of these conjugates allows an increase in the molecular weight together with the loading of the active sugar. Two approaches were used for PEGylation of disaccharide derivatives containing β-d-Galp as the non-reducing unit. (1) Amide formation between benzyl β-d-galactopyranosyl-(1→6)-2-amino-2-deoxy-α-d-glucopyranoside and a succinimide-activated PEG. (2) Conjugation of lactobionolactone with amino end-functionalized PEG. Two 8-arm PEG derivatives (20 and 40?kDa) were used for each sugar. Substitution of all arms was proved by (1)H nuclear magnetic resonance (NMR) spectroscopy. The bioavailability of the conjugates in mice plasma was considerably improved with respect to the 5?kDa linear PEG conjugates retaining their inhibitory properties.     

A Polymeric Prodrug of 5-Fluorouracil-1-Acetic Acid Using a Multi-Hydroxyl Polyethylene Glycol Derivative as the Drug Carrier

Purpose

Macromolecular prodrugs obtained by covalently conjugating small molecular drugs with polymeric carriers were proven to accomplish controlled and sustained release of the therapeutic agents in vitro and in vivo. Polyethylene glycol (PEG) has been extensively used due to its low toxicity, low immunogenicity and high biocompatibility. However, for linear PEG macromolecules, the number of available hydroxyl groups for drug coupling does not change with the length of polymeric chain, which limits the application of PEG for drug conjugation purposes. To increase the drug loading and prolong the retention time of 5-fluorouracil (5-Fu), a macromolecular prodrug of 5-Fu, 5-fluorouracil-1 acid-PAE derivative (5-FA-PAE) was synthesized and tested for the antitumor activity in vivo.

Methods

PEG with a molecular weight of 38 kDa was selected to synthesize the multi-hydroxyl polyethylene glycol derivative (PAE) through an addition reaction. 5-fluorouracil-1 acetic acid (5-FA), a 5-Fu derivative was coupled with PEG derivatives via ester bond to form a macromolecular prodrug, 5-FA-PAE. The in vitro drug release, pharmacokinetics, in vivo distribution and antitumor effect of the prodrug were investigated, respectively.

Results

The PEG-based prodrug obtained in this study possessed an exceedingly high 5-FA loading efficiency of 10.58%, much higher than the maximum drug loading efficiency of unmodified PEG with the same molecular weight, which was 0.98% theoretically. Furthermore, 5-FA-PAE exhibited suitable sustained release in tumors.

Conclusion

This study provides a new approach for the development of the delivery to tumors of anticancer agents with PEG derivatives.     

Long functionalized poly(ethylene glycol)s of defined molecular weight: synthesis and application in solid-phase synthesis of conjugates

A concise synthesis of long-chain poly(ethylene glycol) (PEG) of defined molecular weight up to 29 ethyleneoxy units is described. These PEG diols were converted in a two-step synthesis into Fmoc-protected PEG amino acids, suitable as long linkers and compatible with solid-phase peptide synthesis. Long PEG chains (MW > 1000) can be readily synthesized with this method, which has the advantage of defined single molecular weight products over the comparable commercial polymers. The application of these PEG linkers to the synthesis of peptide-PEG-folate conjugates on a solid support was investigated. A method for the solid support synthesis of the targeting component of the conjugate, folic acid-cysteine, was developed, resulting in improved yields with respect to literature methods. The assembly of the peptide, PEG linker, and targeting group on solid support resulted in the synthesis of a conjugate of defined molecular weight and structure.     

Purification and in situ immobilization of lipase from of a mutant of Trichosporon laibacchii using aqueous two-phase systems

The crude intracellular lipase (cell homogenate) from Trichosporon laibacchii was subjected to partial purification by aqueous two-phase system (ATPS) and then in situ immobilization by directly adding diatomites as carrier to the top PEG-rich phase of ATPS. A partition study of lipase in the ATPS formed by polyethylene glycol–potassium phosphate has been performed. The influence of system parameters such as molecular weight of PEG, system phase composition and system pH on the partitioning behaviour of lipase was evaluated. The ATPS consisting of PEG 4000 (12%) and potassium phosphate (K₂HPO₄, 13%) resulted in partition of lipase to the PEG-rich phase with partition coefficient 7.61, activity recovery 80.4%, and purification factor of 5.84 at pH of 7.0 and 2.0% NaCl. Moreover, the in situ immobilization of lipase in PEG phase resulted in a highest immobilized lipase activity of 1114.6 U g^?1. The above results show that this novel lipase immobilization procedure which couples ATPS extract and enzyme immobilization is cost-effective as well as time-saving. It could be potentially useful technique for the purification and immobilization of lipase.     

Synthesis of polyethylene glycol (PEG) derivatives and PEGylated-peptide biopolymer conjugates

We synthesized a library of 50 poly(ethylene glycol) (PEG) derivatives to expand the extent of conjugation with biologically active molecules (biopolymers, peptides, drugs, etc.) and biomaterial substrates. The formation of PEG derivatives was confirmed with HPLC, (1)H and (13)C NMR. PEG derivatives were polymerized into networks in order to study the role of PEG and terminal functional groups in modulating the hydrophilicity of biomaterials and cell-biomaterial interaction. The resulting surface hydrophilicity and the number of adhered fibroblasts were primarily dependent on the PEG concentration with the molecular weight and the terminal functional group of PEG derivatives being less important. One of PEG derivatives, PEG-bis-glutarate, was utilized to link peptide sequences to gelatin backbone in the formation of novel biomedical hydrogels. PEG-peptide conjugates were characterized by mass spectroscopy. PEG-peptide modified gelatins were characterized by gel permeation chromatography.     

Characterization of sol–gel encapsulated lipase using tetraethoxysilane as precursor

Lipase from Candida rugosa was encapsulated within a chemically inert sol–gel support prepared by polycondensation of the precursor tetraethoxysilane (TEOS) in the presence of polyethylene glycol (PEG) as additive. The properties of silica and their derivatives with regard to mean pore diameter, specific surface area, mean pore size, weight loss upon heating (thermogravimetric analysis, TGA) and ²⁹Si and ¹³C NMR are reported. The pH optimum shifted from 7.8 to 6.7 and optimum temperature jumped from 36 to 60 °C upon enzyme encapsulation. Encapsulated lipase in presence of PEG (EN-PEG) exhibited higher stability in the range of 37–45 °C, but from 50 to 65 °C the EN-PEG was inactivated after seven cycles. Hydrolytic activity during long-term storage at room temperature decreased to 50% after 94 days. High diffusional resistance was observed for large oil concentration reducing hydrolytic effectiveness by 60% in the case of the encapsulated lipase. NMR, pore size and specific surface area data suggested an active participation of the lipase enzyme during gelling of the silica matrix. This lead to reduction of available Si–OH groups, larger pores and smaller surface area. Larger pores increase substrate diffusion that correlates well with higher hydrolytic activity of the TEOS–PEG sol–gel matrix encapsulated enzyme in comparison with other sol–gel supports.     

New, highly efficient formulation of diclofenac for the topical, transdermal administration in ultradeformable drug carriers, Transfersomes

Unmodified and polyethylene glycol (PEG) modified neutral and negatively charged liposomes were prepared by freeze-thaw and extrusion followed by chromatographic purification. The effects of PEG molecular weight (PEG 550, 2000, 5000), PEG loading (0-15 mol%), and liposome surface charge on fibrinogen adsorption were quantified using radiolabeling techniques. All adsorption isotherms increased monotonically over the concentration range 0-3 mg/ml and adsorption levels were low. Negatively charged liposomes adsorbed significantly more fibrinogen than neutral liposomes. PEG modification had no effect on fibrinogen adsorption to neutral liposomes. An inverse relationship was found between PEG loading of negatively charged liposomes and fibrinogen adsorption. PEGs of all three molecular weights at a loading of 5 mol% reduced fibrinogen adsorption to negatively charged liposomes. Protein adsorption from diluted plasma (10% normal strength) to four different liposome types (neutral, PEG-neutral, negatively charged, and PEG-negatively charged) was investigated using gel electrophoresis and immunoblotting. The profiles of adsorbed proteins were similar on all four liposome types, but distinctly different from the profile of plasma itself, indicating a partitioning effect of the lipid surfaces. alpha2-macroglobulin and fibronectin were significantly enriched on the liposomes whereas albumin, transferrin, and fibrinogen were depleted compared to plasma. Apolipoprotein AI was a major component of the adsorbed protein layers. The blot of complement protein C3 adsorbed on the liposomes suggested that the complement system was activated.     

Influence of PEG endgroup and molecular weight on its reactivity for lipase-catalyzed polyester synthesis

Polycondensations were performed at 70 degrees C in bulk using physically immobilized lipase B from Candida antarctica (CAL-B) as catalyst. Study of copolymerizations between sebacic acid and PEG diols of differing Mn values (200, 400, 600, 1000, 2000, and 10 000) showed that PEG 400 and 600 were most reactive (DP(avg) up to about 6). Increasing the PEG diol chain length from 600 to 1000, 2000, and 10 000 resulted in large decreases in copolymer DP(avg) values. PEG200 diacids (i.e., HOOC-(CH2)x-O-(CH2CH2O)n-(CH2)x-COOH) were successfully synthesized where x was 1, 4, 5, 7, 9, and 11. Study of copolymerizations of these diacids with 1,8-octanediol showed that, by introduction of a five-carbon methylene spacer (x = 5), remarkable increases in the reactivity of PEG200 diacids were achieved. In addition, introduction of this spacer was also effective for increasing the reactivity of PEG diacids of higher molecular weight (i.e., PEG400, 600, and 1000). This work verified the hypothesis that, by conversion of PEG chain ends to structures more closely resembling fatty acids, modified PEG building blocks are obtained that are better recognized as substrates by CAL-B during condensation reactions.     

Synthesis and self-assembly of well-defined poly(amino acid) end-capped poly(ethylene glycol) and poly(2-methyl-2-oxazoline)

Poly(ethylene glycol) (PEG) and poly(2-methyl-2-oxazoline) (PMOx) are water-soluble, biocompatible polymers with stealth hemolytic activities. Poly(amino acid) (PAA) end-capped PEG and PMOx were prepared using amino-terminated derivatives of PEG and PMOx as macroinitiators for the ring-opening polymerization of γ-benzyl protected l-glutamate N-carboxyanhydride and S-benzyloxycarbonyl protected l-cysteine N-carboxyanhydride, respectively, in the presence of urea, at room temperature. The molecular weight of the PAA moiety was kept between M(n) = 2200 and 3000 g mol(-1). PMOx was polymerized by cationic ring-opening polymerization resulting in molecular weights of M(n) = 5000 and 10,000 g mol(-1), and PEG was a commercial product with M(n) = 5000 g mol(-1). Here, we investigate the self-assembly of the resulting amphiphilic block copolymers in water and the effect of the chemical structure of the block copolymers on the solution properties of self-assembled nanostructures. The PEG-block-poly(amino acid), PEG-b-PAA, and PMOx-block-poly(amino acid), PMOx-b-PAA, block copolymers have a narrow and monomodal molecular weight distribution (PDI < 1.3). Their self-assembly in water was studied by dynamic light scattering and fluorescence spectroscopy. In aqueous solution, the block copolymers associate into particles with hydrodynamic radii (R(H)) ranging in size from R(H) 70 to 130 nm, depending on the block copolymer architecture and the polymer molecular weight. Larger R(H) and critical association concentration values were obtained for copolymers containing poly(S-benzyloxycarbonyl-l-cysteine) compared to their poly(γ-benzyl-L-glutamate) analogue. FTIR investigations revealed that the poly(γ-benzyl-L-glutamate) block adopts a helical conformation, while the poly(S-benzyloxycarbonyl-L-cysteine) block exists as β-sheet.     

Drug release properties of polyethylene-glycol-treated ciprofloxacin-Indion 234 complexes

The polyethylene glycol (PEG) treatment of ciprofloxacin-Indion 234 complex was aimed to retard rapid ion exchange drug release at gastric pH. Ciprofloxacin loading on Indion 234 was performed in a batch process, and the amount of K⁺ in Indion 234 displaced by drug with time was studied as equilibrium constant K_DM. Drug-resin complex (DRC) was treated with aqueous PEG solution (0.5%–2% wt/vol) of different molecular weights (MWs) for 2 to 30 minutes. The PEG-treated ciprofloxacin-Indion 234 complex was evaluated for particle size, water absorption time, and drug release at gastric pH. During drug loading on Indion 234, the equilibrium constant (K_DM) increased rapidly up to 20 minutes with efficient drug loading. Increased time of immersion of the drug resinate in PEG solutions significantly retained higher size particles upon dehydration. The larger DRC particles showed longer water absorption times owing to compromised hydrating power. The untreated DRC showed insignificant drug release in deionized water; while at gastric pH, ciprofloxacin release was complete in 90 minutes. A trend of increased residual particle size, proportionate increase in water absorption time, and hence the retardation of release with time of immersion was evident in PEG-treated DRC. The time of immersion of DRC in PEG-treated DRC. The time of immersion of DRC in PEG solution had predominant release retardant effect, while the effect of molecular weight of PEG was insignificant. Thus, PEG treatment of DRC successfully retards ciprofloxacin ion exchange release in acidic pH.     

Biosynthesis of poly(3-hydroxybutyrate) copolymers by Azotobacter chroococcum 7B: A precursor feeding strategy

A precursor feeding strategy for effective biopolymer producer strain Azotobacter chroococcum 7B was used to synthesize various poly(3-hydroxybutyrate) (PHB) copolymers. We performed experiments on biosynthesis of PHB copolymers by A. chroococcum 7B using various precursors: sucrose as the primary carbon source, various carboxylic acids and ethylene glycol (EG) derivatives [diethylene glycol (DEG), triethylene glycol (TEG), poly(ethylene glycol) (PEG) 300, PEG 400, PEG 1000] as additional carbon sources. We analyzed strain growth parameters including biomass and polymer yields as well as molecular weight and monomer composition of produced copolymers. We demonstrated that A. chroococcum 7B was able to synthesize copolymers using carboxylic acids with the length less than linear 6C, including poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (PHB-4MHV) using Y-shaped 6C 3-methylvaleric acid as precursor as well as EG-containing copolymers: PHB–DEG, PHB–TEG, PHB–PEG, and PHB–HV–PEG copolymers using short-chain PEGs (with n?≤?9) as precursors. It was shown that use of the additional carbon sources caused inhibition of cell growth, decrease in polymer yields, fall in polymer molecular weight, decrease in 3-hydroxyvalerate content in produced PHB–HV–PEG copolymer, and change in bacterial cells morphology that were depended on the nature of the precursors (carboxylic acids or EG derivatives) and the timing of its addition to the growth medium.     

Optimization of Pseudomonas cepacia lipase preparations for catalysis in organic solvents

The activity of different lipase (from Pseudomonas cepacia) forms, such as crude powder (crude PC), purified and lyophilized with PEG (PEG + PC), covalently linked to PEG (PEG-PC), cross-linked enzyme crystals (CLEC-PC), and immobilized in Sol-Gel-AK (Sol-Gel-AK-PC) was determined, at various water activities (aw), in carbon tetrachloride, benzene and 1,4-dioxane. The reaction of vinyl butyrate with 1-octanol was employed as a model and both transesterification (formation of 1-octyl butyrate) and hydrolysis (formation of butyric acid from vinyl butyrate) rates were determined. Both rates depended on the lipase form, solvent employed, and aw value. Hydrolysis rates always increased as a function of aw, while the optimum of aw for transesterification depended on the enzyme form and nature of the solvent. At proper aw, some lipase forms such as PEG + PC, PEG-PC, and Sol-Gel-AK-PC had a total activity in organic solvents (transesterification plus hydrolysis) which was close to (39 and 48%) or even higher than (130%) that displayed by the same amount of lipase protein in the hydrolysis of tributyrin-one of the substrates most commonly used as standard for the assay of lipase activity-in aqueous buffer. Instead, CLEC-PC and crude PC were much less active in organic solvents (2 and 12%) than in buffer. The results suggest that enzyme dispersion and/or proper enzyme conformation (favored by interaction with PEG or the hydrophobic Sol-Gel-AK matrix) are essential for the expression of high lipase activity in organic media.     

PEG和DBBF修饰猪血红蛋白及其携氧性质

采用聚乙二醇 (PEG)修饰蛋白质可以增大蛋白质的分子量 ,改善其生物相容性和在生物体内的停留时间。而小分子交联修饰则可以稳定血红蛋白的高级结构 ,改善其对组织的递氧能力。比较了 4种方法活化的PEG衍生物对猪血红蛋白的修饰效率、修饰产物的携氧功能和稳定性等。PEG的分子量、轭合PEG的数量及变构效应物的存在与否都会影响修饰产物的性质 ;考察了双 3,5二溴水杨酸延胡索酸酯 (DBBF)修饰猪血红蛋白的反应条件以及修饰产物的物理特性和携氧能力 ,并进一步采用PEG和DBBF联合修饰猪血红蛋白。结果证明 ,联合修饰产物具有稳定的四聚体结构 ,分子量达 10 70 0 0 ,半饱和氧分压P50 在 3.33kPa左右 ,接近于生理条件下人体红细胞的P50 值。     

Biodistribution and Antitumor Effect of Adriamycin Encapsulated in Long-Circulating Liposomes Containing Amphipathic Polyethylene Glycol or Ganglioside GM1

Abstract

Biodistribution and antitumor effect of adriamycin (ADM) encapsulated in liposomes with reduced uptake by reticuloendothelial system (RES) and prolonged circulation time were investigated in mice. Two different types of long-circulating liposomes, ganglioside GMI (GMl)/distearoyl phosphatidyl choline (DSPC) /cholesterol (CH) (0.13:1:1, m/m) and amphipathic polyethylene glycol (PEG)/DSPC/CH (0.13:1:1, m/m) were used. They were sized to 180-200 nm in mean diameter. In the case of amphipathic PEG, distearoyl phosphatidylethanolamine (DSPE) derivatives of PEG with various molecular weight (1000-12000 in mean molecular weight) were used. ADM was encapsulated by transmembrane pH gradient method. GM~DSPC /CH     

Polyethylene glycol as a spacer for solid-phase enzyme immobilization

With the aim to improve the performance of enzyme bound to hydrophilic solid phases, their immobilization with polyethylene glycol (PEG) tether have been studied. Sweet potato β-amylase, which hydrolyses the high molecular weight substrate starch and β-galactosidase, which acts on low molecular weight substrates, were used as model enzymes and beaded thiol–agarose as solid phase. Several two step methods for the introduction of the tether using a bis-oxirane homobifunctional PEG as well as a heterobifunctional derivative with a hydroxysuccinimide ester and a maleimide group have been evaluated. Amino groups, native and de novo thiol groups in the enzymes were utilized for immobilization.

The best approach was found to be to first introduce the PEG derivative via one of its reactive groups to the enzyme. Subsequently the formed conjugate was bound to the solid phase by the remaining reactive group.

Attempts to first introduce the PEG tether into the solid phase were not successful.

A high degree of substitution with PEG chains on the enzyme leads to high immobilization yields for both β-amylase and β-galactosidase, but relatively lower gel-bound activity for the former enzyme which is acting on a high molecular weight substrate and thus more sensitive for steric shielding effects. With optimal degree of PEG substitution (which occurred at five times molar excess of the heterobifunctional reagent) the gel-bound activity of β-amylase was increased from 12% (for the derivative without tether) to 31%.     

Comparative study on digestive lipase activities on the self emulsifying excipient Labrasol, medium chain glycerides and PEG esters

Labrasol is a lipid-based self-emulsifying excipient used in the preparation of lipophilic drugs intended for oral delivery. It is mainly composed of PEG esters and glycerides with medium acyl chains, which are potential substrates for digestive lipases. The hydrolysis of Labrasol by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases was investigated in the present study. Classical human pancreatic lipase (HPL) and porcine pancreatic lipase, which are the main lipases involved in the digestion of dietary triglycerides, showed very low levels of activity on the entire Labrasol excipient as well as on separated fractions of glycerides and PEG esters. On the other hand, gastric lipase, pancreatic lipase-related protein 2 (PLRP2) and carboxyl ester hydrolase (CEH) showed high specific activities on Labrasol. These lipases were found to hydrolyze the main components of Labrasol (PEG esters and monoglycerides) used as individual substrates, whereas these esters were found to be poor substrates for HPL. The lipolytic activity of pancreatic extracts and human pancreatic juice on Labrasol(R) is therefore mainly due to the combined action of CEH and PLRP2. These two pancreatic enzymes, together with gastric lipase, are probably the main enzymes involved in the in vivo lipolysis of Labrasol taken orally.