Probing drug solubilization patterns in the gastrointestinal tract after administration of lipid‐based delivery systems: A phase diagram approach

Probing Drug Solubilization Patterns in the Gastrointestinal Tract after Administration of Lipid-Based Delivery Systems: A Phase Diagram Approach GREG A. KOSSENA,1 WILLIAM N. CHARMAN,1 BEN J. BOYD,2 DAVE E. DUNSTAN,3 CHRISTOPHER J.H. PORTER1 1

Department of Pharmaceutics, Victorian College of Pharmacy, Monash University (Parkville Campus), 381 Royal Pde, Parkville, 3052, Australia 2

DBL Australia, Mayne Pharma, 2/27 Laser Drive, Rowville, 3178, Australia

3

Department of Chemical Engineering, University of Melbourne, Parkville, 3052, Australia

Received 2 June 2003; revised 29 July 2003; accepted 14 August 2003

ABSTRACT: The formation of lyotropic phases resulting from the digestion of formulation lipids has a pronounced impact on the intestinal solubilization and resultant bioavailability of poorly water-soluble drugs. In this study, phase diagrams were produced to determine the phase behavior of the digestion products of common formulation lipids (C8:0, C12:0, and C18:1) under model physiological conditions. Pseudoternary phase diagrams were constructed using varying proportions of SEIF (Simulated Endogenous Intestinal Fluid) and fatty acid (FA) and monoglyceride (MG) (as representative exogenous lipid digestion products). A change from liquid crystal to colloidal liquid (containing mixed micelles and vesicles) was observed with decreasing FA/MG concentrations. The solubilization enhancement ratio (SER) afforded by these phases for a series of poorly water-soluble compounds (hydrocortisone and hydrocortisone esters, clogP ¼ 1.4 to 5.2) was measured relative to the intrinsic solubility in buffer. Large increases in SER were observed in both lamellar (10–2000 fold) and cubic (10– 30,000 fold) liquid crystal phases. Positive correlations were observed between the solubilization benefit provided by each phase and drug lipophilicity (r2
0.9). These phase/solubility trends assist in our understanding of the mechanism by which poorly water-soluble drugs are trafficked across the intestinal colloidal species that form during the digestion of lipid-based drug delivery systems. ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:332–348, 2004

Keywords: drug solubilization; lipid-based formulations; poorly water-soluble drugs; phase diagram; solubilization enhancement ratio

INTRODUCTION The digestion of common formulation lipids, such as triglycerides (TG) and diglycerides (DG) by lipase enzymes in the gastrointestinal (GI) tract results in the production of increasingly amphi-

Correspondence to: Christopher J.H. Porter (Telephone: þ 61 3 99039649; Fax: þ61 3 99039583; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 93, 332–348 (2004) ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association

332

philic digestion products such as monoglyceride (MG) and fatty acid (FA).1 Association of these species with both themselves and endogenous, biliary derived colloidal components [such as bile salts (BS), phosphatidylcholine (PC), and cholesterol (Chol)] subsequently provides an intestinal environment with a high solubilization capacity for poorly water-soluble drugs, and may lead to enhanced oral bioavailability for this class of compound.2 The effect of simple BS micelles or mixed BS/PC micellar species on the solubilization of drugs has

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

PROBING DRUG-SOLUBILIZATION PATTERNS IN THE GASTROINTESTINAL TRACT

been widely studied.3–5 Although these studies provide insight into the capacity of endogenous, biliary derived lipids (primarily BS and PC) to enhance drug solubility within the gastrointestinal tract (GIT), they rarely provide information as to the additional solubility contributions provided by the presence of the digestion products derived from formulation lipids (FA and MG). These additional components are expected to markedly change the nature of the endogenous colloidal components, and as a result, to concurrently alter the solubilization profile of coadministered poorly water-soluble drugs. In a previous study, we have shown that the presence of formulation-derived lipids can have a more significant impact on drug solubilization than endogenous biliary derived components alone and that drug solubility enhancements of up to 10-fold can be achieved when moving from a purely endogenous colloidal system to one containing incorporated digestion products.6 It is apparent that to produce systems representative of the intestinal contents after the administration of lipid-based formulations, there is a need to include both endogenous (biliaryderived) and exogenous (formulation-derived) lipids. In attempting to simulate intestinal fluid, selection of the most appropriate components and estimation of the representative concentrations at which each of these components should be employed is a key consideration.7 Historically, the use of biorelevant intestinal media has been reserved for the area of dissolution testing.8 Typically, such media use a single BS for reasons of simplicity, cost, and adequacy for the purposes of dissolution studies. However, to model the colloidal intestinal phases more accurately, a mixture containing the range of BS present in human bile at the average biliary ratios is appropriate, especially as each BS has a different pKa and molecular aggregation number and therefore behaves differently in terms of colloidal association.9 When addressing the issue of phospholipid inclusion, although the major phospholipid component in human bile is PC, this is a substrate for phospholipase and nonspecific lipases released from the pancreas.10 Hence, the use of the digested form of PC, lyso-phosphatidylcholine (L-PC) (a significantly more polar material) is warranted if digested formulation lipids (FA and MG) are included. Cholesterol, a counterpart of BS and PC within the bile, has also been shown to drastically affect the colloidal environment,11 and should therefore be included in such simulated media.

333

In this study, endogenous lipid concentrations were selected that are representative of the concentrations measured in aspirated human duodenal fluid obtained from fasting subjects, and are present in the ratios typically present in human bile.2,12–14 This endogenous media, which represents the materials likely to be present in the fasted intestine, was then combined with varying proportions of exogenous (formulation-derived) lipids to construct pseudoternary phase diagrams to characterize the likely range of colloidal and phase structures that might be expected to be encountered during the digestion of a lipidic formulation. Phase diagrams describing combinations of endogenous, biliary-derived lipids with varying amounts of FA and MG derived from three exogenous (formulation-derived) lipids were constructed. The exogenous lipids were based on C8 (caprylic) and C12 (lauric) FA (which span the range of fatty acid chain lengths commonly seen in medium chain formulation lipids) and C18:1 (oleic) FA (the most common component present in long chain lipid formulations, such as soybean oil). The phases and colloidal structures formed were identified, isolated, and assessed for their capacity to enhance the solubility of a series of hydrocortisone esters. From the resulting data, clear positive trends were identified between drug lipophilicity and solubilization enhancement in each phase. Major solubility differences were observed between higher order liquid crystal phases and the more dilute (in terms of exogenous lipid) colloidal liquid regions. These changes in drug solubility (although measured under equilibrium conditions) provide information pertinent to a better understanding of the factors which dictate the potential concentration gradients likely to be observed as a drug is shuttled from a digesting TG droplet to the aqueous colloidal region present in the bulk of the intestinal lumen. These data confirm the importance of considering digestion-related phase transitions in the design and evaluation of lipid-based drug delivery systems.

MATERIALS AND METHODS Materials Sodium glycocholate (GC), sodium glycodeoxycholate (GDC), sodium glycochenodeoxycholate (GCDC), sodium taurocholate (TC), sodium JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

334

KOSSENA ET AL.

taurodeoxycholate (TDC), and sodium taurochenodeoxycholate (TCDC) (all >97% pure by TLC), egg lyso-phosphatidylcholine, cholesterol, caprylic acid, monocaprylin, monolaurin, oleic acid, monoolein, tricaprylin, and triolein (all >99% pure) were purchased from Sigma Chemicals (St. Louis, MO). Lauric Acid (>99% pure) was from Fluka AG (Buchs, Switzerland). Commercial partially digested glycerides (principally MG and DG) Capmul1 MCM C8 (>95% C8, 3% C10, consisting 68% MG, 27% DG, 3% TG, as determined by GC) and Capmul1 GMO 50 (>80% C18:1, 10% C18:2, consisting 56% MG, 38% DG, 6% TG, by GC) were generously donated by Abitec Corp (Janesville, WI). Sodium chloride, sodium azide, sodium hydroxide, sodium di-hydrogen phosphate and di-sodium hydrogen phosphate (Ajax Chemicals, Australia) and all other chemicals were of AR quality or higher. Methanol was of ChromAR1 HPLC grade (Selby-Biolab, Australia). Water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA). Hydrocortisone (HC) and the 21-acetate, 17-butyrate, 17-valerate, and 21-caprylate esters were obtained from Sigma Chemicals (St. Louis, MO) and used as received.

Table 1. Composition of Simulated Endogenous Intestinal Fluid (SEIF) Concentrations (mM)

Component Bile Salts (4 mM)a,b

Lyso-phosphatidylcholineb Cholesterolb NaH2PO4.H2Oc Na2HPO4  2H2Oc NaN3 NaCl

GC GDC GCDC TC TDC TCDC

1 0.7 1 0.5 0.3 0.5 1 0.25 18 12 6 98

a Bile salts and the ratios at which they were present were selected based on the relative prevalence of the six major species in human bile (from refs. 17 and 19). Bile salts present as sodium salts of the following conjugates; GC ¼ glycocholate, GDC ¼ glycodeoxycholate, GCDC ¼ glycochenodeoxycholate, TC ¼ taurocholate, TDC ¼ taurodeoxycholate, TCDC ¼ taurochenodeoxycholate. b Total bile salt, lyso-phosphatidylcholine, and cholesterol fasted levels adapted from refs. 12, 14, 15, and 16, and fasted ratios adapted from refs. 2 and 14. c 30 mM phosphate buffer to average pH of 6.50, adapted from refs. 12 and 15.

Simulated Endogenous Intestinal Fluid (SEIF) The concentration of components in simulated endogenous intestinal fluid (4 mM total BS, 1 mM L-PC and 0.25 mM Chol), and are representative of human duodenal fluid in the fasted state, were chosen using values obtained from human intestinal aspirates12,14 –16 and the known ratios (16:4:1)2,13 of these components within human bile (see Table 1). The total BS concentration was made up of the average proportions of the six most prevalent BS found in human bile (GC, GDC, GCDC, TC, TDC, and TCDC) (see Table 1).17–19 During intestinal lipolysis, in addition to the digestion of TG and DG to FA and MG, PC is also hydrolyzed to L-PC.20 In an attempt to simulate conditions post digestion and prior to absorption, L-PC was therefore used in these phase studies. While the hydrolysis of PC to L-PC liberates one free FA unit per molecule, the inclusion of this minimal amount of additional FA in SEIF was considered insignificant when compared to the addition of exogenous FA from formulation sources. A pH of 6.50 ( 0.01) was selected, as this is the average pH at the intestinal region near the ligament of Trietz (being the point of greatest lipid digestion).8,12,15 Sodium azide at 6 mM was included as an antimicrobial.21 NaCl was added to JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

provide a total Naþ concentration of 150 mM and the overall ionic strength was 139 mM.15 Before use, the formed SEIF was vortexed and sonicated for 20 min (Bransonic1 220, SK, Shelton, CO) before sealing under nitrogen and equilibrating for 48 h at 378C.

Construction of Pseudoternary Phase Diagrams To identify the likely phases formed post digestion of formulation-derived lipid, typical exogenous digestion products (FA and MG) were combined with SEIF at varying percentages (% w/w) at 378C under nitrogen and maintained at pH 6.5 (adjusted with solid sodium hydroxide to minimize volume changes). The total exogenous lipid concentration was limited to a maximum of 50% w/w (because unworkable solid systems were produced above this level). The MG composition was restricted to no more than 50 mol % of the FA composition, in accord with the maximum stoichiometric ratios resulting from TG hydrolysis. These combinations were mixed to homogeneity and left to equilibrate for 7 days under N2 in airtight Pyrex glass screw-cap tubes.19 Tubes

PROBING DRUG-SOLUBILIZATION PATTERNS IN THE GASTROINTESTINAL TRACT

were stored in a 450  350  350 mm glass water tank filled with water maintained at 378C. Either side of the tank was lined with perpendicular linear polarized light filters and a light source was placed at the rear of the tank. This allowed continual viewing of the phase tubes for birefringence, a characteristic of liquid crystal behavior.22 After equilibration, representative samples were taken for analysis using crosspolarizing light microscopy (CPLM). Crosspolarizing Light Microscopy (CPLM) Samples of FA and MG in SEIF that showed birefringence in the equilibration tank were transferred onto 1-cm welled microscope slides to allow identification of the morphology of the possible subtypes of liquid crystalline phases present.23,24 Microscope slides were covered with a coverslip and the edges sealed with clear nail polish to prevent evaporative water loss. Slides were viewed at 378C using a temperature controlled hot stage microscope (Zeiss Axiolab E microscope, HFS 91 hot stage with TP 93 temperature programmer (Linkam, Surrey, UK) and Zeiss MC-80 35 mm camera (Zeiss, Oberkochen, Germany). Photon Correlation Spectroscopy (PCS) Samples within the liquid (L1) phase region of the phase diagram and which were not identified as macroemulsions under the light microscope (droplets >1 mm) were selected for PCS studies. Analysis was performed using either a Malvern 4700 equipped with a 10-mW Ar ion laser operating at 488 nm, or a Malvern Zetasizer 3000 equipped with a 5 mW He-Ne laser at 633 nm (Malvern Instruments Ltd., Malvern, UK). The higher powered Malvern 4700 was used for the smaller sized (1–100 nm) and more dilute systems, while the Zetasizer 3000 was used to analyze the larger sized, more concentrated systems. Analysis of samples in the overlap region (50–100 nm) by both instruments was internally consistent. Analysis of the correlation function, measured at diameter ¼ 908 and at 378C, was carried out using the CONTIN1 algorithm for both instruments. The viscosity of the aqueous medium at 378C was assumed to be 0.692 cp. Diffusion coefficients were converted into hydrodynamic radii using the Stokes-Einstein equation.25 Samples were analyzed without dilution to prevent concentration dependent modifications of

335

the species present. Mode peak values of the populations present were used from the premanipulated CONTIN1 results. Polydispersity indices were recorded with maximum accuracy limits set at 0.7 for the multicomponent systems studied. Light scattering cells were cleaned by soaking overnight in nitric acid and rinsing with steam, while excluding dust particles. Accuracy of the PCS apparatus was established using polystyrene polymer standards (Nanosphere1, Duke Scientific Corp, Palo Alto, CA). Model Poorly Water-Soluble Drugs The steroidal anti-inflammatory hydrocortisone (HC) and its related esters were selected for study to examine the impact of changes to compound lipophilicity on solubilization in the absence of significant changes to chemical structure and functionality. HC and the 21-acetate, 17-butyrate, 17-valerate, and 21-caprylate esters are shown in Figure 1. Calculated logP values were determined using ACD labs software v.4.55 (ACD labs, Toronto, Canada). Assessment of Solid State Properties of HC Ester Series Differences in solid-state properties (specifically DHf and melting point) for each compound were analyzed by differential scanning calorimetry (DSC), performed on a Perkin-Elmer DSC 7, with DX thermal analysis controller (Pyris1 software, v3.81). Melting point was also assessed visually using a hot stage microscope. HPLC Assays for Poorly Water-Soluble Drugs HC and the related esters were assayed using a 3.9  150 mm Waters Symmetry1 RP-18 (5 mm) analytical column (Waters Corp., Milford, MA) and 15  3 mm Brownlee RP-18 (7 mm) guard column (Alltech Associates Inc., Deerfield, IL). The HPLC system consisted of a Waters 510 pump, 717 autosampler and 486 tuneable detector (Waters Corp., Milford, MA), together with a Shimadzu CR6A Chromatopac integrator (Shimadzu Corp., Kyoto, Japan). The mobile phase consisted of Milli-Q water (Millipore, Bedford, MA) and varying proportions of methanol (Mallinckrodt, Paris, KY) as shown in Table 2. Samples for all assays were diluted at least twofold in the mobile phase prior to assay and JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

336

KOSSENA ET AL.

concentrations, made up in lipid matrices matching the solubility samples, were accurate and precise to within  5%. Selection of Phases for Equilibrium Solubility Study Phases identified in the phase diagrams were selected for solubility studies on the basis of significant differences in physical properties (such as colloidal size, turbidity, birefringence, and microscopic texture). Solubility studies were restricted to liquid samples or those containing relatively low viscosity liquid crystal phases. The more concentrated, rigid liquid crystal phases prevented attainment of accurate solubility values due to difficulties in reproducible sampling. The phases selected for solubility assessment were from the physiologically relevant FA:MG 2:1 molar ratio axis (being the ratio expected from TG digestion) and are listed in Table 3 (and crossreferenced by letters A–K in Fig. 4. Comparative solubilities in both pure TG and the commercially available blends of DG/MG were also obtained for the C8 and C18:1 systems. Drug solubility in C12 TG and DG/MG was not possible, because TG and DG derivatives of this chain length were wax-like solids at 378C. Equilibrium Drug Solubility Studies

Figure 1. Structures of the five probe compounds used in this study, Hydrocortisone (clogP ¼ 1.43), Hydrocortisone Acetate (1.98), Hydrocortisone Butyrate (2.81), Hydrocortisone Valerate (3.34), and Hydrocortisone Caprylate (5.16).

the injection volume was 40 mL. Absorbances forall assays were measured at the HC lmax of 247 nm. All assays were validated in terms of accuracy and precision using standard methodologies.26 Quality control samples (n ¼ 6) at three JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

After preparation and equilibration, 2 mL of each phase was dispensed into 4 mL borosilicate glass culture tubes containing excess drug. Experiments were conducted in triplicate and tubes stored at 378C and mixed regularly. Tubes were sampled every 24 h over a period of 72 h, during which time equilibrium solubility was attained in all cases (defined when two consecutive sample times gave drug concentrations which varied by less than 5%). Samples were centrifuged at 3500 rpm for 30 min at 378C (Beckman GS-6R centrifuge, Beckman Instruments, Fullerton, CA) prior to sampling to pellet any unsolubilized drug. This low-speed centrifugation step did not result in separation of the dispersed lipidic phases. Solutions were inspected (both macro- and microscopically) with respect to consistency and color throughout the duration of the studies. To enable relative comparison of solubility enhancement, the solubilization enhancement ratio (or SER, eq. 1) was calculated from the drug solubility in the phase of interest, divided by the aqueous solubility of the drug in 30 mM phosphate

PROBING DRUG-SOLUBILIZATION PATTERNS IN THE GASTROINTESTINAL TRACT

337

Table 2. Details of the HPLC Assays Used to Determine Concentrations of Poorly Water-Soluble Compounds

Compound HC HC HC HC HC

Mobile Phase 55% 58% 65% 68% 80%

Acetate Butyrate Valerate Caprylate

MeOH:45% MeOH:42% MeOH:35% MeOH:32% MeOH:20%

Flow Rate (mL/min)

Retention Time (min)

Calibration Range (mg/mL)

LOQ (mg/mL)

0.6 1 1 1 1

8.0 6.0 5.5 6.1 6.5

0.05–10.0 0.05–10.0 0.05–10.0 0.05–10.0 0.05–5.0

0.05 0.05 0.05 0.05 0.05

H2O H2O H2O H2O H2O

buffer, pH 6.5.3 SER ¼

Solubilityphase Solubilitybuffer

ð1Þ

RESULTS Construction of Pseudoternary Phase Diagrams As expected, phase diagrams for all three lipid groups (Fig. 2a–c) showed distinct aqueous colloidal liquid (L1) regions in areas containing a high proportion of SEIF. The size of the L1 region reduced as the chain length of the exogenous lipid increased (Fig. 2a–c). These liquid regions were expected to contain the highly dispersed colloidal species present within the intestinal fluid and to consist of coexisting mixed lipid micelles and vesicles.27 Phase analysis of the C8 system (Fig. 2a) at relatively low levels of dilution (i.e., at higher levels of exogenous lipid) revealed the existence of a La (lamellar liquid crystal) phase. Upon further dilution in SEIF, a phase change to L1 was observed, where the L1 phase contained the more disperse colloidal particles (micellar and vesicular species) that have previously been observed in the bulk of the intestine (i.e., distant from the surface of the TG droplet).12 In comparison, the C12 system (Fig. 2b), while similarly classified as a ‘‘medium chain’’ lipid (and which also produced an La to L1 phase change) interestingly revealed the presence of a small region of cubic (C) liquid crystal phase at FA:MG molar ratios of 4:1 and at approximately 40% w/w FA. The long chain C18:1 system (Fig. 2c) produced more complex phase behavior. At higher exogenous lipid concentrations a phase dependence on the FA:MG ratio was observed, with coexistence of L1 and an oily liquid (L2) phase at higher FA:MG ratios. At lower FA:MG ratios (less than 20:1), a phase change of L2 to C was observed with any

excess FA soap crystals taken up into the C phase. The coexistence of the C with L1 suggests that the C phase may be quite stable to dilution in the intestinal fluid over a large dilution range. However, at very high levels of dilution (97.5– 99.0% w/w SEIF), a total conversion to the existence of a single L1 phase was observed where the aqueous colloidal species were saturated with C18:1 FA/MG. Crosspolarizing Light Microscopy (CPLM) The liquid crystal and emulsified species present at higher exogenous lipid concentrations were assessed under the light microscope with and without cross polarizing filters. Birefringent lamellar liquid crystal regions were identified by characteristic textures such as oily streaks and the presence of Maltese crosses (Fig. 3a and b).24 Nonbirefringent cubic liquid crystal phases appeared both macro- and microscopically as thick viscous isotropic gels (Fig. 3c). Cubic phases were predominantly a feature of samples containing higher levels of exogenous MG in the C18:1 system, but were also unexpectedly observed in the C12 system. Liquid emulsion regions observed in the C18:1 system, when separated by centrifugation, revealed a L2 supernatant and L1 infranatant. Photon Correlation Spectroscopy (PCS) To complement the phase solubility data, particle size data for the species present in the L1 phase formed on addition of varying quantities of the most physiologically relevant 2:1 ratio of FA to MG to SEIF were obtained and are presented in Table 4. Samples used to obtain particle size data, and which were identical to the samples used for phase solubility analysis, are referenced in Table 4 by letter, and the same nomenclature is used in Table 3 and Figures 4 and 5. PCS analysis confirmed the existence of two major particle species within SEIF itself, and in the L1 liquid JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

Blank buffer Blank SEIF A ¼ 0.025% FA, 0.019% MG, 99.956% SEIF B ¼ 0.25% FA, 0.19% MG, 99.56% SEIF C ¼ 1.25% FA, 0.94% MG, 97.81% SEIF D ¼ 2.0% FA, 1.5% MG, 96.5% SEIF E ¼ 5.0% FA, 3.8% MG, 91.2% SEIF MG/DG TG F ¼ 0.010% FA, 0.007% MG, 99.983% SEIF G ¼ 0.25% FA, 0.17% MG, 99.58% SEIF H ¼ 5.0% FA, 3.4% MG, 91.6% SEIF I ¼ 0.010% FA, 0.006% MG, 99.984% SEIF J ¼ 0.25% FA, 0.16% MG, 99.59% SEIF K ¼ 7.5% FA, 4.7% MG, 87.8% SEIFa K ¼ 7.5% FA, 4.7% MG, 87.8% SEIFa MG/DG TG

Nil Endogenous C8

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

L1 L1 L1 L1 L1 L1 La L2 L2 L1 L1 La L1 L1 L1 C L2 L2

Phase

HC Acetate 6.1  0.1 8.2  0.1 8.5  0.1 9.5  0.2 25.1  0.2 41.1  2.0 355.4  18.0 747.6  30.6 146.2  8.4 9.5  0.2 11.2  0.3 33.8  3.0 8.7  0.2 9.9  0.5 8.3  0.4 652.4  4.7 192.8  1.8 29.3  1.3

HC 279.2  7.1 351.2  6.2 399.1  5.3 446.4  3.8 946.1  11.3 1200.6  25.3 3384.9  138.4 7737.6  257.1 404.2  14.3 427.4  6.7 477.0  3.6 1273.5  77.1 393.8  3.1 444.6  13.9 354.6  15.3 6341.9  165.4 2249.1  112.7 93.0  1.6

56.8  0.6 79.5  0.6 84.4  2.2 117.4  1.9 680.1  5.0 1176.1  16.7 4197.9  98.2 32194.4  379.5 1898.0  53.9 96.4  0.6 160.9  15.0 1276.2  24.6 100.9  2.5 176.5  4.3 79.0  7.1 31375.3  455.9 8419.3  226.7 420.0  17.3

HC Butyrate

31.9  2.3 54.7  1.5 53.0  1.7 88.0  0.4 770.8  8.5 1526.6  16.6 4201.0  31.0 45426.6  336.3 3700.3  188.7 59.6  0.5 134.5  2.6 1769.7  52.9 62.5  1.1 169.0  3.8 55.7  1.2 39626.3  1190.0 15009.6  303.5 925.1  16.4

HC Valerate

0.7  0.0 3.0  0.1 2.9  0.0 7.6  0.0 156.5  0.9 377.9  7.0 1844.3  76.8 111124.6  3725.2 15260.3  319.3 3.5  0.0 22.7  0.2 623.0  18.3 4.9  0.1 58.8  1.2 14.8  0.5 24476.0  884.2 26243.8  568.0 3109.3  66.4

HC Caprylate

Samples A–K are crossreferenced to the binary phase diagrams shown in Figure 4, the solubility data in Figure 5 and with the PCS data in Table 4. Hydrocortisone (HC) and related ester solubilities are shown as a mean  SD (n ¼ 3). a The L1 aqueous colloidal liquid phase and cubic liquid crystal phase of the c18:1 system were in coexistence.

C18:1

C12

System

Lipid Type

Drug Solubility (mg/mL)

Table 3. Details of the Systems Used in the Solubility Studies, Including Blank Buffer and SEIF (Containing No Exogenous Lipid) and the Phases Present on the 2:1 FA/MG Molar Ratio Axis of the Phase Diagram for Each Chain Length Lipid (C8, C12, and C18:1)

338 KOSSENA ET AL.

PROBING DRUG-SOLUBILIZATION PATTERNS IN THE GASTROINTESTINAL TRACT

339

Figure 3. Representative crosspolarized light micrographs at 378C of (a) the brightly birefringent oily streak texture (magnification 20) and (b) Maltese crossappearance (magnification 32) of the lamellar liquid crystal phase (La) observed, for example, in both C8 and C12 systems and (c) nonbirefringent viscous gel texture (magnification 10), characteristic of the cubic liquid crystal (C) observed in both C12 and C18:1 systems.

Figure 2. Phase diagrams of the regions of interest for C8 (a), C12 (b), and C18:1 (c) lipid systems. L1 denotes an aqueous colloidal liquid, L2 denotes an oily liquid, La denotes a lamellar liquid crystal, C denotes cubic liquid crystal, and Xstals denotes incorporated fatty acid soap crystals. The black dots depict samples where phase data was obtained. Data was not obtained at MG:FA ratios greater than 1:2 in accord with the maximum stoichiometric ratios resulting from TG hydrolysis. This area has therefore been left blank on the partial phase diagrams depicted. The total exogenous lipid concentration was also limited to a maximum of 50% w/w (as unworkable solids were produced above this level).

regions containing SEIF and relatively low concentrations of exogenous lipids. These two colloidal particles (on the basis of size difference) likely represent a mixed micelle and a larger vesicle species. After a certain percentage of exogenous lipid was added (0.1–2.5% w/w FA), a distinctive change (increase) was observed in species particle size for all three chain length lipid groups, presumably reflecting the production of larger multilamellar vesicle species of FA/MG. This increase in size correlated well with the onset of visual turbidity (Fig. 4a–c). Phase Behavior of Dilute Aqueous Region In the C8 system (Fig. 4a) when moving from the regions of highest lipid concentration to the most dilute (100% SEIF), for all three exogenous lipid JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

340

KOSSENA ET AL.

compositions (FA alone, FA:MG 2:1 and FA:MG 3:1) a change from La (e.g., sample E, in the 2:1 FA:MG composition) to a FA/MG rich micelle ( 5 nm) and vesicle ( 70–500 nm) coexisting

region (e.g., sample D) within the L1 phase was observed. These species were present up to the point at which the system changed to become optically clear. Unlike the other two chain length systems, the optically clear system present at lower FA/MG lipid loads ( <2.5% w/w FA) consisted of a single micellar species ( 4 nm, e.g., samples B and C) and coincided with a drop in signal intensity from the PCS. Eventually, as the ratio of FA/MG to SEIF dropped below a threshold, the coexisting micelles ( 2 nm) and vesicles ( 40–80 nm) present in SEIF became evident (presumably swollen with FA/MG, e.g., sample A). The C12 system (Fig. 4b) behaved similarly to that of C8, where for all three exogenous compositions, a change from La (e.g., sample H) to coexisting FA/MG rich micelles ( 4 nm) and vesicles ( 60–130 nm, e.g., sample G) was observed. These species persisted until the change to an optically clear system consisting of the micelles ( 3 nm) and vesicles ( 30–40 nm) present in SEIF was observed (also presumably swollen with FA/MG, e.g., sample F). In the C18:1 system (Fig. 4c) the coexistence of the aqueous colloidal liquid (termed L1(c)) with L2 or C (e.g., sample K) was largely dependent on the presence of MG. On loss of the concentrated L2 or C phase the presence of coexisting FA/MG rich micelles ( 5 nm) and vesicles ( 70–160 nm) were again observed (e.g., sample J). At much lower concentrations than those observed for the medium chain lipid systems, optical clarity was achieved and the system reverted to the swollen coexisting micelles ( 3 nm) and vesicles ( 40– 60 nm) present in SEIF (e.g., sample I). Solid State Properties of HC Ester Series Table 5 shows the observed solid state properties of HC and the related esters as determined by

Figure 4. Binary phase diagrams for the most dilute region of the (a) C8, (b) C12, and (c) C18:1 systems along three fixed exogenous lipid compositions, FA only, FA:MG molar ratio 3:1, and FA:MG 2:1 (the latter being the most physiologically representative, and the likely stoichiometric ratio resulting from TG digestion). Samples for the system obtained using 2:1 FA:MG (samples A–K) were subsequently progressed into phase solubility studies and particle size studies. The horizontal axes were truncated to 4% w/w FA for visual clarity; however, samples E, H, and K contained 5, 5, and 7.5% w/w FA, respectively. The vertical dashed line indicates the line of visual turbidity. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

PROBING DRUG-SOLUBILIZATION PATTERNS IN THE GASTROINTESTINAL TRACT

341

Table 4. PCS Data for Samples Taken from the L1 Liquid Phase Regions (Mean  SD, n ¼ 10) Obtained on Addition of Differing Quantities of FA/MG to SEIF System SEIF C8 A

B

C D C12

F

G

C18:1

I

J

Lipid Concentrations

Rh Micelle (nm)

Rh Vesicle (nm)

Appearance

100% SEIF 0.010% FA, 0.007% MG, 99.983% SEIF 0.025% FA, 0.019% MG, 99.956% SEIF 0.050% FA, 0.038% MG, 99.912% SEIF 0.100% FA, 0.076% MG, 99.824% SEIF 0.250% FA, 0.189% MG, 99.561% SEIF 0.500% FA, 0.378% MG, 99.122% SEIF 1.000% FA, 0.757% MG, 98.243% SEIF 1.250% FA, 0.946% MG, 97.804% SEIF 1.500% FA, 1.135% MG, 97.365% SEIF 2.000% FA, 1.514% MG, 96.486% SEIF 2.250% FA, 1.703% MG, 96.047% SEIF 0.0010% FA, 0.0007% MG, 99.9983% SEIF 0.0025% FA, 0.0017% MG, 99.9958% SEIF 0.0050% FA, 0.0034% MG, 99.9916% SEIF 0.010% FA, 0.007% MG, 99.983% SEIF 0.025% FA, 0.017% MG, 99.958% SEIF 0.050% FA, 0.034% MG, 99.916% SEIF 0.100% FA, 0.068% MG, 99.832% SEIF 0.250% FA, 0.171% MG, 99.579% SEIF 0.500% FA, 0.342% MG, 99.158% SEIF 1.000% FA, 0.685% MG, 98.315% SEIF 0.0010% FA, 0.0006% MG, 99.9984% SEIF 0.0025% FA, 0.0016% MG, 99.9959% SEIF 0.0050% FA, 0.0032% MG, 99.9918% SEIF 0.010% FA, 0.006% MG, 99.984% SEIF 0.025% FA, 0.016% MG, 99.959% SEIF 0.050% FA, 0.032% MG, 99.918% SEIF 0.100% FA, 0.063% MG, 99.837% SEIF 0.250% FA, 0.158% MG, 99.592% SEIF

2.3  0.1 3.0  0.1 2.6  0.1 2.5  0.1 2.5  0.3 3.0  0.6 3.2  0.2 5.1  0.4 6.9  1.3 10.0  0.3 5.2  0.3 4.8  0.4 2.4  0.1 2.6  0.1 3.2  0.4 2.7  0.2 2.8  0.1 3.0  0.3 3.6  0.6 5.8  0.6 5.1  0.2 3.5  0.4 3.4  0.0 3.0  0.1 2.7  0.3 2.5  0.2 4.6  0.4 6.8  0.9 4.5  0.5 5.4  0.1

38.1  3.5 51.1  0.9 54.9  5.3 64.5  11.8 78.5  14.2 np np np np np 502.6  106.1 483.8  128.8 42.8  0.2 45.6  1.7 43.9  1.3 46.9  2.6 65.8  0.5 76.7  6.5 114.8  31.2 74.9  4.7 74.9  4.0 130.6  10.2 56.8  0.6 57.2  1.3 58.0  2.8 58.3  3.6 62.8  2.8 85.0  3.7 90.3  2.7 101.5  2.9

Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Turbid Turbid Clear Clear Clear Clear Clear Clear Turbid Turbid Turbid Turbid Clear Clear Clear Clear Clear Clear Turbid Turbid

Population Particle Radii (nm) from Premanipulated CONTIN1 results. Samples (A–J) are crossreferenced to the binary phase diagrams shown in Figure 4 and the solubility data in Figure 5 and depict the compositions used in the solubility studies summarized in Table 3. Particle size data was not obtained for samples E, H, and K because at these higher lipid levels either La or C phases were present, which were not amenable to accurate particle size analysis. np ¼ not present.

DSC and hot stage microscopy. As expected, melting point decreased with increasing length of the ester chain. A slightly higher DHf value for the acetate ester was seen and was reflected in its lower relative aqueous solubility. Equilibrium Solubility Studies of Hydrocortisone Compounds in Selected Phases All compounds in each phase studied, reached equilibrium within the 72-h period of the solubility studies. Equilibrium solubility was defined here as the value reached when further changes to solubility were less than 5% over a 24-h period. This does not rule out the possibility of continual changes to solubility over extended periods of

time; however, stability concerns for both the lipids and esters employed in this study precluded solubility assessment over longer time periods. The hydrolytic stability of the HC esters was assessed over the 72-h period of the solubility study, with less than 5% being hydrolyzed to HC in all cases. Phases and excess solid drug remained visually consistent throughout the course of the study. The solubilities of HC and its related esters in the selected phases are given in Table 3. All solubility data obtained (Table 3) were converted to a solubilization enhancement ratio (SER) to avoid differences in absolute solubility related to solid-state properties, and these data are illustrated in Figures 5a–c. Both CPLM and PCS (for liquid JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

342

KOSSENA ET AL.

phases) methods were used to reanalyze the phases after the duration of the solubility studies, and indicated that no observable physical changes had occurred in the phase medium. Moving from right to left along the x-axis of the C8 system (Fig. 5a), trends in solubility enhancement can be observed when passing from conditions representative of the TG rich formulation

lipid droplet, through the DG/MG and La liquid crystal regions that may be present on the droplet surface, through to the dispersed L1 species and SEIF (the most dilute in exogenous lipid). In the C8 system, the solubility benefits of TG are improved by digestion to DG/MG, and this benefit gradually decreases upon change to La and dilution through the different species present in the L1 phase. A gradual decrease in the solubility benefit of the C12 La phase is also observed upon dilution through the L1 species (Fig. 5b). In the case of C18:1 (Fig. 5c), the solubilization benefit of the TG is greatly improved by the partial digestion to DG/MG, and this is further maintained (and in most cases improved) by the formation of the cubic liquid crystal phase species. A gradual decrease is then observed upon dilution through the different L1 species.

DISCUSSION In this study the physical phases that are likely to form during the digestion of formulation-derived TG lipids have been identified. These phases are expected to be involved in the trafficking of poorly water-soluble drugs from TG-based lipid delivery systems to the intestinal colloidal species from where absorption occurs. Solubility studies have assessed the relative solubilization benefits of these different phases for HC and a series of related esters, and these data have subsequently been used to suggest the potential role that these phases may play in dictating the distribution/ partitioning behavior of lipophilic drugs in the

Figure 5. Solubility profiles of the selected poorly water-soluble compounds for each lipid system as a function of the different physical phases present in the phase diagram (samples were selected from the phase diagram described in Fig. 4 along the most physiologically relevant FA:MG 2:1 composition and are labeled A–K), also included as a comparator is 100% TG (to reflect drug solubility in an undigested TG formulation) and the commercially available MG/DG blend (to reflect partially digested formulation components). Solubility results are presented as SER for each system, C8 (a), C12 (b), and C18:1 (c) for the compounds (*) HC, (*) HC acetate, (!) HC butyrate, (!) HC valerate, and (&) HC caprylate. SER data given as mean where n ¼ 3 and error bars show standard deviation. In many cases the error bars are concealed within the data points. TG or DG/MG data was not obtained for the C12 systems, as these are solid at 378C. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

PROBING DRUG-SOLUBILIZATION PATTERNS IN THE GASTROINTESTINAL TRACT

Table 5. Differential Scanning Calorimetry (DSC) and Microscopy Melting Point Data for Hydrocortisone (HC) and Related Esters Melting Point8C Compound

DSC

Microscopy

DHf (J/mol)

HC HC HC HC HC

223.1 219.5 213.5 174.8 110.3

217 224 200 170 117

3845 5034 2767 2955 3080

Acetate Butyrate Valerate Caprylate

intestine after administration of a lipid-based formulation. Phase Studies Comparison of the phase diagrams reported here to those published in the surfactant and lipid chemistry literature reveals similarities, most noticeably at higher exogenous lipid concentrations. Not surprisingly, the major differences pertain to the dilute areas of exogenous lipid that are principally manifest in changes to the size and colloidal nature of the L1 region. As expected, the ability of SEIF (as opposed to water, which is more commonly used as the aqueous phase in published phase diagrams) to incorporate a greater amount of exogenous lipid in the L1 region, via colloidal (micelle and vesicle) association, gives rise to this larger region. For the C8 lipid-based systems, previous studies by Fontell and Mandell described the phase behavior of C8 FA, ionized C8 FA, and water, and highlighted the presence of a large region of La phase,23,28 which is consistent with the current data obtained using more physiological aqueous media (i.e., inclusive of BS, L-PC, Chol) and including varying amounts of C8 MG. Limited phase studies have been performed with C12 FA; however the existence of La phases has been reported in C12 MG systems.29 Identification of the cubic phase region in the C12 system has not been reported previously in the literature, and provides an interesting focus for further investigation. Phase studies of the C18:1 FA, ionized C18:1 FA, and water system have previously shown more complex behavior and the presence of areas of two or more coexisting phases.30 The presence of swollen cubic phases (which are relatively stable to dilution) consisting of C18:1 FA/MG31 and C18:1 MG in water, has also been reported previously.31,32

343

Further characterization of the dilute L1 region (which is the phase most likely to reflect the intestinal phase from where drug absorption occurs) was performed to allow more effective correlation of drug solubility characteristics with the likely colloidal species present. Submicroscopic changes were assessed using PCS, revealing in general, the existence of larger (and most likely multilamellar) vesicular structures presumably swollen with exogenous (FA/MG) lipids, in coexistence with smaller micellar particles.31,32 In the case of both C12 and C18:1, this was observed at higher lipid levels in the L1 phase and under conditions of increasing dilution. Beyond the visual line of turbidity, a clear system containing the endogenous micelles and smaller (likely unilamellar) vesicles present in SEIF (swollen with a small quantity of exogenous lipid) remained. Surprisingly, a visually clear micelle-only region existed in the C8 system between the larger exogenous lipid vesicle phase and the endogenous SEIF colloidal species (Fig. 4a). A complete explanation for this observation is not apparent at this time; however, it is possible that the concentrations of ionized C8 FA present in solution may have resulted in the formation of C8 FA micelles (the CMC has previously been reported to be  360 mM,33 but this value may be lowered in the presence of unionized FA, BS, L-PC, and Chol). These FA micelles are capable of solubilizing L-PC and Chol components (which are primarily present as vesicles in SEIF) and may have resulted in the loss of the vesicular species at high C8 FA concentrations. At significantly higher lipid concentrations, the creation of a larger vesicular phase, consisting primarily of FA (both ionized and unionized  incorporated MG) was observed, which is consistent with the findings of Hargreaves and Deamer.34 In contrast, the solubilities and CMC of the longer chain C12 and C18:1 ionized FA are far lower in aqueous media33 than that of the C8 FA, and the ratio of unionized to ionized FA is higher. As a result, the concentration of ionized FA in solution is much lower than is the case for the C8 lipids. This may provide an explanation as to why the intermediate micellar species were not observed in the longer chain lipid systems. On the basis of the data obtained in these studies, the potential phase changes that may occur during dilution of the lipid digestion products that are produced on the surface of a digesting lipid droplet are summarized in Figure 6. Figure 6a shows the phase transitions that may JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

344

KOSSENA ET AL.

with the uptake of small amounts of intestinal fluid may result in the creation of an La phase under the relatively lipid rich conditions present on the droplet surface. In contrast, under increasingly dilute conditions, a change to the L1 phase is likely and as dilution increases, the L1 phase is expected to switch from a FA/MG-rich vesicle/ mixed micelle region (denoted L1(c) from Fig. 4), to a micelle only region (L1(b)), and finally to a region containing primarily the endogenous vesicles/ micelles present in SEIF (L1(a)). Figure 6b indicates that similar trends may be predicted for the C12 lipid, although in this case the micelle only region is absent. Figure 6c highlights that for the C18:1 system, the same transitional trends are likely, although in this case the presence of a C phase at high lipid concentrations (and therefore potentially on the TG droplet surface) is possible. Hydrocortisone (HC) and HC Ester Solubility in Model Lipid Digestion Phases

Figure 6. Schematic representation of the phase transitions potentially involved in the solubilization of a lipophilic drug in a digesting TG formulation droplet during dilution in intestinal fluid. The changes are depicted for the most physiologically relevant FA:MG 2:1 lipid composition for C8 (a), C12 (b), and C18:1 (c).

occur during the digestion of a C8 TG lipid droplet and the subsequent interaction of the digestion products formed with intestinal fluid. The data suggest that digestion of C8 TG to MG/DG, coupled JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

The solubilization enhancement for HC and the HC esters provided by each of the different phase regions relative to drug solubility in buffer was assessed in an attempt to evaluate the ability of the digesting lipidic species to maintain drug in solution during intestinal processing. With the exception of HC acetate, the measured solubilities in buffer were negatively correlated with the logP of the compound (i.e., the aqueous solubility of the drug reduced as a function of increasing logP). The deviation of HC acetate from this trend was likely related to its markedly different solid-state properties (i.e., high DHf) when compared with the other esters (Table 5). It should be noted, however, that while major differences in chemical structure were minimized by employing this series of related esters, it is impossible to rule out the potential impact of positional differences between the 17 or 21 substituted steroids in terms of drug solubilization patterns, because the hydrogen bonding properties of the two series are likely to be different.35 However, variations in intrinsic solubility across the ester series were effectively corrected for in these studies by the use of solubilization enhancement ratio as an indication of relative solubility enhancement (as opposed to absolute solubility). For all the phases analyzed, a clear trend was evident between increasing SER and increase in drug logP. This positive correlation existed for each phase studied, with good linear correlations (r2 > 0.9) observed between partition coefficient (P)

PROBING DRUG-SOLUBILIZATION PATTERNS IN THE GASTROINTESTINAL TRACT

and SER (see Fig. 7). An increase in slope of these plots was also seen with increasing lipid content suggesting that the increase in solubilization benefit seen with increasing drug lipophilicity was most prevalent in the most lipid-rich phases.

Figure 7. Correlations observed between SER and drug partition coefficient (P) are shown for C8 (a), C12 (b), and C18:1 (c) for each phase A to K highlighted in Figure 4. The slopes of these plots are as follows for each phase; A ¼ 0.13, B ¼ 0.23, C ¼ 0.51, D ¼ 0.58, E ¼ 0.58, F ¼ 0.14, G ¼ 0.35, H ¼ 0.64, I ¼ 0.19, J ¼ 0.48, and K ¼ 0.83 [K phase depicted is the coexisting cubic (C) phase].

345

The differences in solubilization enhancement provided by the different phases observed within each lipid group (i.e., C8, C12, or C18:1) were gradual with the largest difference in solubility being observed between the L1 and liquid crystal (La/C) phases. Assessing the different lipid groups (Table 3), differences in drug solubility in the lower exogenous lipid-containing regions (denoted as A, F, and I) were relatively small, confirming the likely dependence of the phase behavior (and solubility behavior) in this region on the endogenous biliary lipid content (which is essentially the same for each lipid type within the dilute FA/MG region). Upon passing the boundary at which the L1 phase is no longer principally a reflection of endogenous lipids ( 0.1–2.5% w/w FA, denoted as B, G, and J), more significant differences between lipid types are observed. Thus, for the lower logP compounds (HC and HC acetate) the C12 L1 phase surprisingly provides slightly greater solubility enhancement when compared to the corresponding L1 phase in the C18:1 or C8 systems. In contrast, for the higher logP compounds, the C18:1 L1 phase provided greater solubility enhancement than the C8 or C12 L1 phase. The increased solubility enhancement provided by the long chain lipids for the highly lipophilic drugs may be explained by the fact that the volume of the apolar region of the colloidal particle is proportional to the FA chain length of any incorporated lipid, and hence, the thermodynamics of interaction of the longer chain HC esters with the long chain FA containing colloidal particles is favored. When examining the liquid crystalline phases, the lipid group differences become more distinct. For all compounds, the greatest solubilization enhancement was provided by the C phase formed by the C18:1 system, closely followed by the La regions of the C8 systems, and was lowest in the C12 systems. Interestingly, in the coexisting two phase systems of C18:1 (denoted as K), the L1 phase solubilized approximately the same amount of drug as the blank SEIF systems (Fig. 8), whereas the C phase was capable of solubilizing considerably larger amounts. This suggests that the majority of the exogenous lipid resides in the liquid crystal matrix, and that this matrix therefore acts as a reservoir for drug. It is expected that upon dilution in intestinal fluid, this matrix will gradually rupture to release vesicular FA/MG species, eventually undertaking a full phase change to the diluted L1 phase (denoted as J) containing FA/ MG-based vesicular and micellar species. This L1 phase is further diluted to the point where the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

346

KOSSENA ET AL.

Figure 8. SER provided for each compound by the coexisting phases of the C18:1 system (FA:MG 2:1) at 7.5% FA, 4.7% MG, 87.7% SEIF (phase sample K), the L1 phase (unshaded) and C phase (gray shade), compared to the SER of the SEIF (black), (mean  SD, n ¼ 3). In some cases error bars have been obscured by the data.

endogenous biliary lipids dominate (denoted as I), providing endogenous vesicles and mixed micelles (i.e., essentially SEIF). Differences in the solubilization benefits of the C18:1 cubic phase versus the C8 La phase for drugs of lower logP (HC and acetate ester) were less pronounced than the differences observed with the higher logP drugs, where the cubic phase provided substantial solubility enhancement. Implications of Drug Solubility Data for Drug Trafficking in the Gastrointestinal Tract (GIT) For lipophilic, poorly water-soluble drugs, oral bioavailability is commonly limited by solubility in the intestinal lumen and slow and incomplete dissolution from solid dose forms during transit down the GIT. Lipid-based dose forms typically enhance oral bioavailability for this class of compound, and while the exact mechanism of this enhancement is not well described, the primary rationale is that dosing as a lipidic solution provides two major advantages. First, administration as a solution (albeit a lipid solution) reduces the energy input required to overcome the crystal lattice energy of a solid dose form. Second, the digestion and dispersion of a lipidic vehicle results in the production of a dispersed lipidic microenvironment (micelles/vesicles/emulsion droplets) that persists during gastrointestinal transit, and provides a solubilization sink for JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

the drug, precluding precipitation. In contrast, solid dispersions or cosolvent systems, for example, lose their solubilization benefits on dilution leading to drug precipitation and reduced bioavailability. However, although these general tenets hold true, there is relatively little data available in the literature describing the impact of digestion and dispersion/dilution of lipidic dose forms on drug solubilization capacity. The data reported here describe in some detail the nature of the different physical phases that may form on interaction of the lipid digestion products of different chain length lipids (C8, C12, and C18) with intestinal fluid. The relative solubilization capacities of these different phases for a series of HC esters have subsequently been assessed. At high lipid concentrations (e.g., under the conditions that might be expected to form on the surface of a digesting droplet), long chain lipids (C18) form cubic liquid crystalline phases, which have extremely high solubilizing capacities (greater even than the parent TG or pure lipid digestion products such as FA or MG). In contrast, the solubilization capacities of the lamellar structures formed at relatively high concentrations of C12 or C8 FA/ MG, are typically less highly solubilizing, and may therefore more readily lead to drug precipitation on dispersion of a TG formulation. For all compounds, the solubility advantages imparted by the long chain lipids were also retained on dilution to the L1 phase (with the exception of HC and HC acetate where C12 systems provided slightly improved solubility). Taken together, these observations may help to explain the oral bioavailability benefits often observed when using lipidic formulations based on long chain lipids when compared with their medium chain equivalents. Parenthetically, while the phase behavior and solubility information reported here give an indication of the species that may form on digestion and dispersion of lipidic dose forms and the solubilization capacity of the phases formed, it is important to note that the data was collected under equilibrium conditions, whereas the digestion and dispersion of lipidic dose forms is clearly a dynamic process. The overall likelihood of drug precipitation on intestinal processing, therefore, will depend not only on phases formed and their equilibrium solubilization capacity, but also the rate at which the phases form and the partitioning/ trafficking of lipophilic drug molecules from relatively undispersed lipidic ‘‘reservoirs’’ through to the more dispersed micellar and vesicular

PROBING DRUG-SOLUBILIZATION PATTERNS IN THE GASTROINTESTINAL TRACT

phases. Studies are on going in an attempt to provide this dynamic data using in vitro models of lipid digestion.

REFERENCES 1. MacGregor KJ, Embleton JK, Lacy JE, Perry EA, Solomon LJ, Seager H, Pouton CW. 1997. Influence of lipolysis on drug absorption from the gastrointestinal tract. Adv Drug Del Rev 25:33–46. 2. Hay DW, Cahalane MJ, Timofeyeva N, Carey MC. 1993. Molecular species of lecithins in human gallbladder bile. J Lipid Res 34:759–767. 3. Naylor LJ, Bakatselou V, Dressman JB. 1993. Comparison of the mechanism of dissolution of hydrocortisone in simple and mixed micelle systems. Pharm Res 10:865–870. 4. Mithani SD, Bakatselou V, TenHoor CN, Dressman JB. 1996. Estimation of the increase in solubility of drugs as a function of bile salt concentration. Pharm Res 13:163–167. 5. Cai X, Grant DJW, Wiedmann TS. 1997. Analysis of the solubilization of steroids by bile salt micelles. J Pharm Sci 86:372–377. 6. Kossena GA, Boyd BJ, Porter CJH, Charman WN. 2003. Separation and characterization of the colloidal phases produced on digestion of common formulation lipids and assessment of their impact on the apparent solubility of selected poorly water soluble drugs. J Pharm Sci 92:634–648. 7. Porter CJH, Charman WN. 2001. In vitro assessment of oral lipid based formulations. Adv Drug Del Rev 50:S127–S147. 8. Dressman JB, Amidon GL, Reppas C, Shah VP. 1998. Dissolution testing as a prognostic tool for oral drug absorption: Immediate release dosage forms. Pharm Res 15:11–22. 9. Carey MC, Small DM. 1972. Micelle formation by bile salts. Arch Intern Med 130:506–527. 10. Robinson N. 1961. Review article: Lysolecithin. J Pharm Pharmacol 13:321–354. 11. Cohen DE, Carey MC. 1990. Rapid (1 hour) high performance gel filtration chromatography resolves coexisting simple micelles, mixed micelles, and vesicles in bile. J Lipid Res 31:2103–2112. 12. Hernell O, Staggers JE, Carey MC. 1990. Physicalchemical behavior of dietary and biliary lipids during intestinal digestion and absorption. 2. Phase analysis and aggregation states of luminal lipids during duodenal fat digestion in healthy adult human beings. Biochemistry 29:2041–2056. 13. Schersten T. 1973. Formation of lithogenic bile in man. Digestion 9:540–553. 14. Duane WC, Ginsberg RL, Bennion LJ. 1976. Effects of fasting on bile acid metabolism and biliary lipid composition in man. J Lipid Res 17:211–219.

347

15. Lindahl A, Ungell A, Knutson L, Lennernas H. 1997. Characterisation of fluids from the stomach and proximal jejunum in men and women. Pharm Res 14:497–502. 16. Tangerman A, van Schaik A, van der Hock EW. 1986. Analysis of conjugated and unconjugated bile acids in serum and jejunal fluid of normal subjects. Clin Chim Acta 159:123–132. 17. Hofmann AF. 1976. The enterohepatic circulation of bile acids in man. Adv Intern Med 21:501–534. 18. Stella VJ, Martodihardjo S, Terada K, Rao VM. 1998. Some relationships between the physical properties of various 3-acyloxymethyl prodrugs of phenytoin to structure: Potential in vivo performance implications [published erratum appears in J Pharm Sci 1999 88(10):1100]. J Pharm Sci 87: 1235–1241. 19. Staggers JE, Hernell O, Stafford RJ, Carey MC. 1990. Physical-chemical behavior of dietary and biliary lipids during intestinal digestion and absorption. 1. Phase behavior and aggregation states of model lipid systems patterned after aqueous duodenal contents of healthy adult human beings. Biochemistry 29:2028–2040. 20. Mansbach CM, Cohen RS, Leff PB. 1975. Isolation and properties of the mixed micelles present in intestinal content during fat digestion in man. J Clin Invest 56:781–791. 21. Pedersen BL, Brondsted H, Lennernas H, Christensen FN, Mullertz A, Kristensen HG. 2000. Dissolution of hydrocortisone in human and simulated intestinal fluids. Pharm Res 17:183– 189. 22. Laughlin RG. 1976. An expedient technique for determining solubility phase boundaries in surfactant–water systems. J Colloid Interface Sci 55: 239–241. 23. Ekwall P, Mandell L, Fontell K. 1969. Solubilization in micelles and mesophases and the transition from normal to reversed structures. Mol Cryst Liq Cryst 8:157–213. 24. Rosevear FB. 1968. Liquid crystals: The mesomorphic phases of surfactant compositions. J Soc Cosmet Chem 19:581–594. 25. Berne B, Pecora R, editors. 1976. Dynamic light scattering with applications to chemistry, biology and physics. New York: John Wiley & Sons, p 376. 26. Shah VP, Midha KK, Dighe S, McGilvery IJ, Skelly JP, Yacobi A, Layloff T, Viswanathan CT, Cook CE, McDowall RD, Pittman KA, Spector S. 1992. Analytical methods validation: Bioavailability, bioequivalence and pharmacokinetic studies. Pharm Res 9:588–592. 27. Carey MC, Small DM, Bliss CM. 1983. Lipid digestion and absorption. Annu Rev Physiol 45:651–677. 28. Fontell K, Mandell L. 1993. Phase equilibria and phase structure in the ternary systems sodium or

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

348

29.

30.

31.

32.

KOSSENA ET AL.

potassium octanoate–octanoic acid–water. Colloid Polym Sci 271:974–991. Kanesaka I, Shimizu K. 2000. Infrared intensity and morphology of 1-monolaurin–water systems. Spectrochim Acta Part A 56:523–529. Small DM. 1968. A classification of biological lipids based upon their interaction in aqueous systems. J Am Oil Chem Soc 45:108–119. Borne J, Nylander T, Khan A. 2001. Phase behavior and aggregate formation for the aqueous monoolein system mixed with sodium oleate and oleic acid. Langmuir 17:7742–7751. Pitzalis P, Monduzzi M, Krog N, Larsson H, Ljusberg-Wahren H, Nylander T. 2000. Character-

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 2, FEBRUARY 2004

ization of the liquid-crystalline phases in the glycerol monooleate/diglycerol monooleate/water system. Langmuir 16:6358–6365. 33. Mukerjee P, Mysels KJ, editors. 1970. Critical micelle concentrations of aqueous surfactants. Washington, DC: National Bureau of Standards, pp 88–170. 34. Hargreaves WR, Deamer DW. 1978. Liposomes from ionic, single-chain amphiphiles. Biochemistry 17:3759–3768. 35. Anderson BD, Marra MT. 1999. Chemical and related factors controlling lipid solubility. Bulletı´n Technique Gattefosse´ 92:11–18.