Toward the Establishment of Standardized In Vitro Tests for Lipid-Based Formulations, Part 1: Method Parameterization and Comparison of In Vitro Digestion Profiles Across a Range of Representative Formulations
2012; Elsevier BV; Volume: 101; Issue: 9 Linguagem: Inglês
10.1002/jps.23205
ISSN1520-6017
AutoresHywel D. Williams, Philip Sassene, Karen Kleberg, Jean-Claude Bakala-N’Goma, Marilyn Calderone, Vincent Jannin, Annabel Igonin, Anette Partheil, Delphine Marchaud, Eduardo Jule, Jan Vertommen, Mario Maio, Ross Blundell, Hassan Benameur, Frédéric Carrière, Anette Müllertz, Christopher J. H. Porter, Colin W. Pouton,
Tópico(s)Antibiotics Pharmacokinetics and Efficacy
ResumoThe Lipid Formulation Classification System Consortium is an industry–academia collaboration, established to develop standardized in vitro methods for the assessment of lipid-based formulations (LBFs). In this first publication, baseline conditions for the conduct of digestion tests are suggested and a series of eight model LBFs are described to probe test performance across different formulation types. Digestion experiments were performed in vitro using a pH-stat apparatus and danazol employed as a model poorly water-soluble drug. LBF digestion (rate and extent) and drug solubilization patterns on digestion were examined. To evaluate cross-site reproducibility, experiments were conducted at two sites and highly consistent results were obtained. In a further refinement, bench-top centrifugation was explored as a higher throughput approach to separation of the products of digestion (and compared with ultracentrifugation), and conditions under which this method was acceptable were defined. Drug solubilization was highly dependent on LBF composition, but poorly correlated with simple performance indicators such as dispersion efficiency, confirming the utility of the digestion model as a means of formulation differentiation. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association The Lipid Formulation Classification System Consortium is an industry–academia collaboration, established to develop standardized in vitro methods for the assessment of lipid-based formulations (LBFs). In this first publication, baseline conditions for the conduct of digestion tests are suggested and a series of eight model LBFs are described to probe test performance across different formulation types. Digestion experiments were performed in vitro using a pH-stat apparatus and danazol employed as a model poorly water-soluble drug. LBF digestion (rate and extent) and drug solubilization patterns on digestion were examined. To evaluate cross-site reproducibility, experiments were conducted at two sites and highly consistent results were obtained. In a further refinement, bench-top centrifugation was explored as a higher throughput approach to separation of the products of digestion (and compared with ultracentrifugation), and conditions under which this method was acceptable were defined. Drug solubilization was highly dependent on LBF composition, but poorly correlated with simple performance indicators such as dispersion efficiency, confirming the utility of the digestion model as a means of formulation differentiation. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association INTRODUCTIONThe issue of low aqueous drug solubility continues to hinder the robust testing of new chemical entities during drug discovery and development. Of the many formulation strategies that have been used to address the obstacles associated with low solubility, lipid-based formulations (LBFs) have generated significant interest.1.Hauss DJ. Enhancing the bioavailability of poorly water-soluble drugs. Informa Healthcare, New York2007Google Scholar The composition of LBFs can vary widely, although common design features include the presence of molecularly dispersed drug within a blend of various polar and nonpolar oils with/without surfactant and cosolvent.2.Pouton CW. Lipid formulations for oral administration of drugs: Non-emulsifying, self-emulsifying and "self-microemulsifying" drug delivery systems.Eur J Pharm Sci. 2000; 11: S93-S98Crossref PubMed Scopus (1015) Google Scholar,3.Pouton C.W. Porter C.J.H. Formulation of lipid-based delivery systems for oral administration: Materials, methods and strategies.Adv Drug Deliv Rev. 2008; 60: 625-637Crossref PubMed Scopus (656) Google Scholar As the drug is presented to the gastrointestinal (GI) tract in solution, although in an oily liquid, the use of LBFs circumvents the limitations to solubility introduced by solute–solute interactions in the crystalline solid.4.Wassvik C.M. Holmen A.G. Bergstrom C.A.S. Zamora I. Artursson P. Contribution of solid-state properties to the aqueous solubility of drugs.Eur J Pharm Sci. 2006; 29: 294-305Crossref PubMed Scopus (112) Google Scholar LBFs also promote drug solubilization in the GI fluids via the provision of surfactants and lipids (and their digestion products) that collectively supplement the inherent solubilization capacity of the endogenous GI fluids (i.e., that provided by bile salts, phospholipids, and cholesterol secreted in bile).Unlike many traditional formulations, the physical and chemical nature of most LBFs is dramatically changed after oral administration by the interaction with biliary and pancreatic secretions in the small intestine, in a process analogous to the digestion of food-based lipids.5.Porter C.J.H. Trevaskis N.L. Charman W.N. Lipids and lipid-based formulations: Optimizing the oral delivery of lipophilic drugs.Nat Rev Drug Discover. 2007; 6: 231-248Crossref PubMed Scopus (1397) Google Scholar The major mechanism of chemical change is that of lipid digestion. Lipid digestion is mediated by pancreatic lipases and esterases that are secreted into the upper small intestine in response to the ingestion of exogenous lipid, and to a lesser extent by acid-stable lipases in the stomach.6.Bakala N'Goma J.C. Amara S. Dridi K. Jannin V. Carriere F. Understanding lipid digestion in the GI tract for effective drug delivery.Ther Deliv. 2012; 3: 105-124Crossref PubMed Scopus (126) Google Scholar The lipid digestion products generated are subsequently solubilized by bile salt–phospholipid–cholesterol-mixed micelles secreted in bile, resulting in the formation of a range of colloidal species in the GI fluids. The resulting colloidal structures support the solubilization of exogenously administered lipids and coadministered poorly water-soluble drugs. LBFs, therefore, exploit the lipid digestion and absorption cascade, such that drug incorporated into the administered lipid vehicle is transferred into the colloidal phases produced during lipid digestion and these species shuttle digestion products and drug from the lipid substrate to the intestinal wall for absorption.5.Porter C.J.H. Trevaskis N.L. Charman W.N. Lipids and lipid-based formulations: Optimizing the oral delivery of lipophilic drugs.Nat Rev Drug Discover. 2007; 6: 231-248Crossref PubMed Scopus (1397) Google Scholar,7.Carey M.C. Small D.M. Bliss C.M. Lipid digestion and absorption.Annu Rev Physiol. 1983; 45: 651-677Crossref PubMed Scopus (625) Google Scholar, 8.Hofmann A.F. Borgstrom B. The intraluminal phase of fat digestion in man: The lipid content of the micellar and oil phases of intestinal content obtained during fat digestion and absorption.J Clin Invest. 1964; 43: 247-257Crossref PubMed Scopus (244) Google Scholar, 9.Hofmann A.F. Borgstrom B. Physico-chemical state of lipids in intestinal content during their digestion and absorption.Fed Proc. 1962; 21: 43-50PubMed Google Scholar, 10.Hofmann AF. Function of bile salts in fat absorption—Solvent properties of dilute micellar solutions of conjugated bile salts.Biochem J. 1963; 89: 57-68Crossref PubMed Scopus (195) Google Scholar Current understanding suggests that a critical aspect of this process is the avoidance of drug precipitation during processing of lipid formulations because regeneration of the solid state results in a reversion to a situation consistent with administration of a crystalline drug suspension (where dissolution rate is typically a limitation to drug absorption for poorly water-soluble drugs). One caveat to this overarching suggestion is the recent realization that drug precipitation from LBFs may, for some compounds, result in the amorphous drug being formed.11.Sassene P.J. Knopp M.M. Hesselkilde J.Z. Koradia V. Larsen A. Rades T. Mullertz A. Precipitation of a poorly soluble model drug during in vitro lipolysis: Characterization and dissolution of the precipitate.J Pharm Sci. 2010; 99: 4982-4991Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar In this case, the process of redissolution of precipitated drug is expected to be faster than if the drug were to precipitate in the crystalline form.The basic mechanisms by which LBFs promote drug absorption are therefore reasonably well developed. The specific determinants of in vivo performance, however, are not fully understood, and robust approaches to probe in vivo performance using in vitro tests remain poorly defined. The determinants of in vitro and in vivo performance are likely to include (1) the capacity of the formulation to maintain solvent capacity on dilution and digestion, (2) the rate of formulation digestion [which dictates the rate and quantity of lipid (or surfactant) digestion products and drug that partitions from the oil reservoir into the intestinal milieu], and (3) the solubilization capacity of the localized GI environment when enriched with the products of the digested formulation.12.Larsen A. Holm R. Pedersen M.L. Mullertz A. Lipid-based formulations for danazol containing a digestible surfactant, labrafil M2125CS: In vivo bioavailability and dynamic in vitro lipolysis.Pharm Res. 2008; 25: 2769-2777Crossref PubMed Scopus (93) Google Scholar, 13.Kaukonen A.M. Boyd B.J. Charman W.N. Porter C.J.H. Drug solubilization behavior during in vitro digestion of suspension formulations of poorly water-soluble drugs in triglyceride lipids.Pharm Res. 2004; 21: 254-260Crossref PubMed Scopus (103) Google Scholar, 14.Kaukonen A.M. Boyd B.J. Porter C.J.H. Charman W.N. Drug solubilization behavior during in vitro digestion of simple triglyceride lipid solution formulations.Pharm Res. 2004; 21: 245-253Crossref PubMed Scopus (173) Google Scholar, 15.Humberstone A.J. Charman W.N. Lipid-based vehicles for the oral delivery of poorly water soluble drugs.Adv Drug Deliv Rev. 1997; 25: 103-128Crossref Scopus (240) Google Scholar These determinants are applicable to a wide range of LBF as digestion will inevitably impact on the solubilization capacity of all formulations that contain digestible lipids or surfactants. Digestion of poorly dispersed lipid-rich LBF (such as simple lipid solutions) is typically beneficial to drug absorption because digestion leads to the generation of more amphiphilic lipid digestion products that are more readily incorporated into bile salt–phospholipid-mixed micelles. Digestion therefore catalyzes the in situ assembly of highly dispersed colloidal phases with high drug solubilization capacities. In contrast, self-emulsifying formulations and formulations with high proportions of surfactant and cosolvent do not require digestion to reduce particle size because initial dispersion of the formulation commonly leads to the production of nanometer-sized colloidal droplets. Formulation digestion remains unavoidable, however, and may be detrimental to absorption if digestion leads to a decrease in solubilization capacity and drug precipitation.In an attempt to better understand the performance of a wide range of LBFs, in vitro lipid digestion models have emerged as a possible mechanism by which the complex series of in vivo interactions that underpin utility may be modeled and predicted in vitro.16.Porter C.J.H. Kaukonen A.M. Boyd B.J. Edwards G.A. Charman W.N. Susceptibility to lipase-mediated digestion reduces the oral bioavailability of danazol after administration as a medium-chain lipid-based microemulsion formulation.Pharm Res. 2004; 21: 1405-1412Crossref PubMed Scopus (230) Google Scholar, 17.Porter C.J.H. Kaukonen A.M. Taillardat-Bertschinger A. Boyd B.J. O'Connor J.M. Edwards G.A. Charman W.N. Use of in vitro lipid digestion data to explain the in vivo performance of triglyceride-based oral lipid formulations of poorly water-soluble drugs: Studies with halofantrine.J Pharm Sci. 2004; 93: 1110-1121Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 18.Dahan A. Hoffman A. Use of a dynamic in vitro lipolysis model to rationalize oral formulation development for poor water soluble drugs: Correlation with in vivo data and the relationship to intra-enterocyte processes in rats.Pharm Res. 2006; 23: 2165-2174Crossref PubMed Scopus (174) Google Scholar, 19.Cuine J.F. Charman W.N. Pouton C.W. Edwards G.A. Porter C.J.H. Increasing the proportional content of surfactant (Cremophor EL) relative to lipid in self-emulsifying lipid-based formulations of danazol reduces oral bioavailability in beagle dogs.Pharm Res. 2007; 24: 748-757Crossref PubMed Scopus (140) Google Scholar, 20.Cuine J.F. McEvoy C.L. Charman W.N. Pouton C.W. Edwards G.A. Benameur H. Porter C.J.H. Evaluation of the impact of surfactant digestion on the bioavailability of danazol after oral administration of lipidic self-emulsifying formulations to dogs.J Pharm Sci. 2008; 97: 995-1012Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 21.Di Maio S. Carrier R.L. Gastrointestinal contents in fasted state and post-lipid ingestion: In vivo measurements and in vitro models for studying oral drug delivery.J Control Release. 2011; 151: 110-122Crossref PubMed Scopus (67) Google Scholar, 22.Larsen A.T. Sassene P. Mullertz A. In vitro lipolysis models as a tool for the characterization of oral lipid and surfactant based drug delivery systems.Int J Pharm. 2011; 417: 245-255Crossref PubMed Scopus (122) Google Scholar, 23.Hur S.J. Lim B.O. Decker E.A. McClements D.J. In vitro human digestion models for food applications.Food Chem. 2011; 125: 1-12Crossref Scopus (637) Google Scholar, 24.McClements D.J. Li Y. Review of in vitro digestion models for rapid screening of emulsion-based systems.Food Funct. 2010; 1: 32-59Crossref PubMed Scopus (331) Google Scholar In these models, the lipid formulation (containing incorporated drug) is dispersed in a digestion medium that is broadly representative of the contents of the upper small intestine, and digestion of the formulation is initiated by addition of a porcine-derived pancreatic extract containing pancreatic lipase and other pancreatic enzymes. Formulation digestion results in the liberation of fatty acid (FA) from either glyceride lipids or surfactant FA esters, and this leads to a drop in the pH of the digest. By conducting digests in a pH-stat titrator, the transient drop in pH is monitored and neutralized by equimolar titration of sodium hydroxide, thereby maintaining pH at a set point. Quantification of the rate of addition of base therefore provides an indirect measure of the rate and extent of lipid digestion, on the condition that FAs are ionized at the pH of the assay. Samples may be removed at intervals during the digestion test and mixed with a lipolysis inhibitor prior to separation by high-speed ultracentrifugation. Ultracentrifugation may result in the separation of three potentially distinct phases: a pellet phase containing precipitated drug; an aqueous colloid phase containing solubilized drug; and an oily phase containing a mixture of incompletely digested lipid, any phase-separated digestion products, and incorporated drug.The use of lipid digestion models to better understand formulation performance continues to inform the broader literature, but interpretation of data obtained across different laboratories is currently limited by variations in the experimental conditions employed such as the test pH; the volume of the test; and the concentrations of bile salt, calcium, and buffering agents employed. The current communication presents the first in a series of papers generated by a consortium of academic and industrial members [the Lipid Formulation Classification System (LFCS) Consortium] that aim to establish common and discriminating methods for the conduct of in vitro digestion tests.Proposed by Pouton,2.Pouton CW. Lipid formulations for oral administration of drugs: Non-emulsifying, self-emulsifying and "self-microemulsifying" drug delivery systems.Eur J Pharm Sci. 2000; 11: S93-S98Crossref PubMed Scopus (1015) Google Scholar the LFCS categorizes lipid formulations according to formulation properties including whether or not water-soluble excipients are included, the particle size of the formulation on dispersion, and the role of lipid digestion in formulation performance in vivo. The initial objective of the LFCS was to generate a framework to facilitate better understanding of the performance of differing formulation types and to aid comparison of data obtained for similar "classes" of formulation.2.Pouton CW. Lipid formulations for oral administration of drugs: Non-emulsifying, self-emulsifying and "self-microemulsifying" drug delivery systems.Eur J Pharm Sci. 2000; 11: S93-S98Crossref PubMed Scopus (1015) Google Scholar In attempting to apply the LFCS, however, it has become increasingly evident that this is not possible without some level of standardization of the conditions employed to test formulations in vitro.3.Pouton C.W. Porter C.J.H. Formulation of lipid-based delivery systems for oral administration: Materials, methods and strategies.Adv Drug Deliv Rev. 2008; 60: 625-637Crossref PubMed Scopus (656) Google Scholar To address this need, the LFCS Consortium has been established to develop standardized methods for formulation assessment of LBFs using in vitro testing protocols. The Consortium comprises academic groups from Monash University, the University of Copenhagen, and the CNRS in Marseille and industrial collaborators organized into primary and associate Consortium members. During the first year, the primary industrial members were Capsugel, Sanofi, Gattefossé, and Merck Serono.This first publication from the consortium establishes a panel of eight LBFs that have been chosen to span the range of the four LFCS classes and identifies the experimental variables that are critical to the in vitro performance of these exemplar formulations. The conditions required to facilitate the generation of consistent data across different laboratories have been evaluated and the impact of variation in sampling methodology and sample analysis across different formulation types examined. Preliminary conclusions regarding the design of testing protocols are described and form the baseline conditions for a series of subsequent investigations of the impact of method variation on in vitro performance.MATERIALS AND METHODSMaterialsDetails of the lipidic excipients used within the LFCS Consortium can be found in Table 1. Danazol was kindly supplied by Sterling Pharmaceuticals (Sydney, Australia) and Indis (Aartselaar, Belgium). Preliminary studies revealed that the two different batches of this drug showed comparable solubility in pH 6.5 Tris-maleate buffer (0.7–1.2 µg/mL), and the values obtained were within the range of previously reported values.25.Pedersen B.L. Mullertz A. Brondsted H. Kristensen H.G. A comparison of the solubility of danazol in human and simulated gastrointestinal fluids.Pharm Res. 2000; 17: 891-894Crossref PubMed Scopus (128) Google Scholar Sodium taurodeoxycholate >95% (NaTDC), 4-bromophenylboronic acid, and the porcine pancreatin extract [P7545, 8× United States Pharmacopeia (USP) specifications activity] were all obtained from Sigma Chemical Company (St. Louis, Missouri). Phosphatidylcholine (PC) (Lipoid E PC S, ∼99.2% pure, from egg yolk) was obtained from Lipoid (Lipoid GmbH, Ludwigshafen, Germany). One mole per liter sodium hydroxide, which was diluted to obtain 0.2 and 0.6 M NaOH titration solutions, was purchased from Merck (Darmstadt, Germany). Water was obtained from a Milli-Q water purification system (Millipore, Bedford, Massachusetts). All other chemicals and solvents were of analytical purity or high-performance liquid chromatography (HPLC) grade.Table 1Details of the Lipidic Excipients Used in the Model LBFs Investigated by the LFCS Consortium in this StudyExcipientSourceDescription of BatchesTriglyceride:Corn oilSigma, St. Louis, MissouriLong-chain triglyceride consisting of 39.4%–62.0% linoleic acid (C18:2), 20.0%–42.2% oleic acid (C18:1), 8.6%–16.5% palmitic acid (C16), and 1.0%–3.3% stearic acid (C18)Captex 300Abitec Corporation, Columbus, OhioMedium-chain triglyceride mainly consisting of 65.7% caprylic acid (C8) and 33.7% capric acid (C10)Mixed glycerides:Maisine™ 35-1Gattefossé, St. Priest, FranceBlend of partially digested long-chain glycerides: 33.5% monoglyceride, 50.9% diglyceride, and 14.7% triglyceride, predominantly of linoleic acid (51.1%, C18:2) and oleic acid (32.8%, C18:1)Capmul MCMAbitec CorporationBlend of partially digested medium-chain glycerides: 60.7% monoglyceride, 33.1% diglyceride, and 4.4% triglyceride, predominantly of caprylic acid (82.8%, C8)Surfactants:Tween 85SigmaPolyoxyethylene sorbitan trioleate (HLB 11)Cremophor ELBASF Corporation, Washington, New JerseyPolyethoxylated castor oil (HLB 14–16)Cosolvents:Transcutol HPGattefosséDiethylene glycol monoethyl ether Open table in a new tab Selection of Exemplar LBFs for Investigation in the LFCS ConsortiumThe compositions of the eight lipid formulations investigated by the consortium are shown in Table 2. Formulations were chosen to span the four classes of the LFCS,2.Pouton CW. Lipid formulations for oral administration of drugs: Non-emulsifying, self-emulsifying and "self-microemulsifying" drug delivery systems.Eur J Pharm Sci. 2000; 11: S93-S98Crossref PubMed Scopus (1015) Google Scholar and contained either long-chain (LC) or medium-chain (MC) lipid excipients. Attempts were made to keep the lipid–surfactant ratio constant between formulations with a view to aiding interformulation comparison. For example, types II and IIIA formulations comprised an equal quantity of lipid and surfactant and only differed by the nature of surfactant (Table 2). Properties that varied across the formulation range included (1) the capacity to self-emulsify, (2) digestibility, (3) drug solvency in the formulation, and (4) particle size on dispersion. The disparate nature of the formulations was critical to the aims of the LFCS consortium, that is, to establish in vitro testing methods that are applicable across a range of lipid formulations. At this point, the Consortium has focused on liquid–lipid formulations. Solid and semisolid systems will be addressed in subsequent studies.Table 2Composition of the Exemplar LBFs Investigated by the LFCS Consortium in this Study and Physical Properties Following Dispersion in the Digestion MediumFormulation TypeComposition (% w/w)Appearance on DispersionaAppearance following dispersion of 1g LBF in 36mL digestion medium (37°C).Particle SizebMean particle size of the dispersed LBF following a 1 in 36 and 1 in 250 dilution in the digestion medium (37°C). Values are expressed as means (n = 3) ± 1 SD. (nm)PolydispersitybMean particle size of the dispersed LBF following a 1 in 36 and 1 in 250 dilution in the digestion medium (37°C). Values are expressed as means (n = 3) ± 1 SD.I-MC50.0% Captex 300Opaque emulsionn/acColloids formed on dispersion were too large to be measured accurately (i.e., polydispersity > 0.5).–50.0% Capmul MCMII-MC32.5% Captex 300Opaque emulsion1 in 36: n/acColloids formed on dispersion were too large to be measured accurately (i.e., polydispersity > 0.5).0.068 ± 0.0532.5% Capmul MCM1 in 250: 190.2 ± 2.735.0% Tween 85IIIA-MC32.5% Captex 300Ultrafine dispersion1 in 36: 29.1 ± 0.60.062 ± 0.0132.5% Capmul MCM1 in 250: 36.2 ± 0.90.088 ± 0.0235.0% Cremophor ELIIIB-MC25.0% Capmul MCMUltrafine dispersion1 in 36: 21.4 ± 0.50.111 ± 0.0175.0% Cremophor EL1 in 250: 14.0 ± 2.70.138 ± 0.1025.0% Transcutol HPI-LC50.0% corn oilCoarse emulsionn/acColloids formed on dispersion were too large to be measured accurately (i.e., polydispersity > 0.5).–50.0% Maisine™ 35-1II-LC32.5% corn oilCoarse–opaque emulsionn/acColloids formed on dispersion were too large to be measured accurately (i.e., polydispersity > 0.5).–32.5% Maisine™ 35-135.0% Tween 85IIIA-LC32.5% corn oilUltrafine dispersion1 in 36: 61.8 ± 0.40.197 ± 0.0132.5% Maisine™ 35-11 in 250: 58.4 ± 1.30.149 ± 0.0135.0% Cremophor ELIV50.0% Cremophor ELTransparent solution1 in 36: 13.1 ± 0.40.033 ± 0.0150.0% Transcutol HP1 in 250: 9.9 ± 0.50.111 ± 0.06LC, long chain; MC, medium chain.a Appearance following dispersion of 1 g LBF in 36 mL digestion medium (37°C).b Mean particle size of the dispersed LBF following a 1 in 36 and 1 in 250 dilution in the digestion medium (37°C). Values are expressed as means (n = 3) ± 1 SD.c Colloids formed on dispersion were too large to be measured accurately (i.e., polydispersity > 0.5). Open table in a new tab Determination of Danazol Equilibrium Solubility in the LBFsThe equilibrium solubility of danazol in each formulation was determined using a previously reported method.26.Khoo S.M. Humberstone A.J. Porter C.J.H. Edwards G.A. Charman W.N. Formulation design and bioavailability assessment of lipidic self-emulsifying formulations of halofantrine.Int J Pharm. 1998; 167: 155-164Crossref Scopus (362) Google Scholar In brief, danazol was added in excess to 4 mL borosilicate glass vials containing 2 g lipid formulation. Vials were allowed to equilibrate at 37°C with periodic vortex mixing to ensure that undissolved drug particles were suspended in the lipid slurry. At intervals, vials were centrifuged (Eppendorf 5408R centrifuge; Eppendorf AG, Hamburg, Germany) at 4000 rpm (2800g) and 37°C for 30 min. This separated samples into a solid pellet phase and a particle-free supernatant. Accurately weighed samples were removed from the supernatant, transferred to 5 mL volumetric flasks and made up to volume with chloroform–methanol (2:1, v/v). Aliquots (50–100 µL) were diluted >10-fold with methanol and analyzed for danazol content by HPLC (see the section HPLC Detection of Danazol below for details). Equilibrium solubility was defined as the value attained when consecutive solubility values differed by less than 5%. For danazol, this was achieved within 24 h for most LBFs and within 72 h for all formulations.Drug Incorporation into the LBFsFormulations were loaded with danazol at 80% of the saturated solubility of the drug in the formulation. The required mass of danazol was weighed directly into clean screw-top glass vials, and drug-free lipid formulation was added up to the target mass loading. Vials were sealed, vortex mixed, and incubated at 37°C for at least 24 h prior to testing. The danazol content in the formulation was verified (in triplicate) on the day of testing using sampling and analysis processes as described in the section Determination of Danazol Equilibrium Solubility in the LBFs above.In Vitro Evaluation of the LBFsDigestion ExperimentsStandardized equipment for performing in vitro digestion tests was identified based largely on earlier descriptions of the in vitro digestion test.27.Zangenberg N.H. Mullertz A. Kristensen H.G. Hovgaard L. A dynamic in vitro lipolysis model I. Controlling the rate of lipolysis by continuous addition of calcium.Eur J Pharm Sci. 2001; 14: 115-122Crossref PubMed Scopus (244) Google Scholar,28.Sek L. Porter C.J.H. Kaukonen A.M. Charman W.N. Evaluation of the in vitro digestion profiles of long and medium chain glycerides and the phase behaviour of their lipolytic products.J Pharm Pharmacol. 2002; 54: 29-41Crossref PubMed Scopus (251) Google Scholar The experimental setup employed by the LFCS Consortium consisted of a pH-stat apparatus (Metrohm® AG, Herisau, Switzerland), comprising a Titrando 802 propeller stirrer/804 Ti Stand combination, a glass pH electrode (iUnitrode), and two 800 Dosino dosing units coupled to 10 mL autoburettes (Metrohm® AG). The apparatus was connected to a PC and operated using Tiamo 2.0 software (Metrohm®).Initial Reference Conditions for In Vitro Digestion in the LFCS ConsortiumThe initial reference conditions for the in vitro digestion experiments were as follows. The digestion buffer (pH 6.5) contained 2 mM Tris-maleate, 1.4 mM CaCl2•2H2O, and 150 mM NaCl and was supplemented with 3 mM NaTDC and 0.75 mM PC. Concentrations of bile, phospholipid, calcium, and sodium chloride were chosen to reflect the typical concentrations present in the fasted small intestine.29.Persson E.M. Gustafsson A.S. Carlsson A.S. Nilsson R.G. Knutson L. Forsell P. Hanisch G. Lennernas H. Abrahamsson B. The effects of food on the dissolution of poorly soluble drugs in human and in model small intestinal fluids.Pharm Res. 2005; 22: 2141-2151Crossref PubMed Scopus (225) Google Scholar,30.Hansky J. Calcium content of duodenal juice.Am J Dig Dis. 1967; 12: 725-&Crossref PubMed Scopus (16) Google Scholar Preliminary studies (see Supplementary Material) were conducted to examine the importance of the source and type of bile salt utilized and compared the effect of using 3 mM of (1) NaTDC, (2) bovine bile extract, or (3) a mixture of synthetic bile salts (mimicking the typical bile salt secretion in the human gall bladder) on the rate and extent of digestion of LC lipids. These studies revealed little difference in the data obtained using the different bile salt preparations. Consequently, on the basis of cost and batch-to-batch consistency, a single commercially available source of NaTDC was considered appropriate for use during in vitro digestion testing. A concentration of Tris buffer (2 mM) was chosen to be sufficient to allow stabilization of pH during initial adjustment of digest pH, but sufficiently low to have a negligible effect on the downstream detection of titratable FA. Pancreatin from porcine pancreas containing pancreatic lipolytic enzymes [colipase-dependent pancreatic lipase and carboxyl ester hydrolase (CEH)] was prepared by the vortex mixing 1 g of pancreatin powder in 5 mL digestion buffer (i.e., free of bile salt and phospholipid) and approximately 17 µL of 5.0 M sodium hydroxide that adjusted the mixture pH to 6.5. The enzyme suspension was centrifuged [4000 rpm (2800g), 5°C; Eppendorf 5408R
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