ACTAUNIVERSITATISUPSALIENSISUPPSALA2006

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 39

Drug Dissolution underPhysiologically Relevant ConditionsIn Vitro and In Vivo

EVA PERSSON

ISSN 1651-6192ISBN 91-554-6684-2urn:nbn:se:uu:diva-7195

Papers discussed

This thesis is based on the following papers, which will be referred to in the text by the Roman numerals assigned below.

I E.M. Persson., L. Löfgren., G. Hansson., B. Abrahamsson., H. Lennernäs., B. Abrahamsson., and R.G. Nilsson. Simultaneous assessment of lipid classes and bile acids in human intestinal fluid by solid phase extraction and HPLC methods. Submitted

II. E.M. Persson., R.G. Nilsson., G.I. Hansson., L.J. Löfgren., F. Libäck., L. Knutson., B. Abrahamsson., and H. Lennernäs. A clinical single-pass perfusion investigation of the dynamic in vivo secretory response to a dietary meal in human proximal small intestine. Pharm Res 23(4):742-751 (2006). Reprouced with kind permission of Springer Science and Business Media.

III. E.M. Persson, A. Gustafsson, A.S. Carlsson, R.G. Nilsson, L. Knutson, P. Forsell, G. Hanisch, H. Lennernäs, and B. Abrahamsson. The effects of food on the dissolution of poorly soluble drugs in human and in model small intestinal fluids. Pharm Res. 22(12):2141-2151 (2005). Reprouced with kind permission of Springer Science and Business Media.

IV. E.M. Persson, A. Nordén, P. Forsell, L. Knutson, C. Öhgren, S. Forssén, H. Lennernäs, and B. Abrahamsson. Improved understanding of the effect of food on drug absorption and bioavailability for lipophilic compounds from the use of an intestinal pig perfusion model. Manuscript

Papers not included in the thesis:

E. Bergman., P. Forsell., A. Tevell., E.M. Persson, M. Hedeland., U. Bondesson., L. Knutson., and H. Lennernäs. Biliary secretion of Rosuvastatin and bile acids in hu-mans during the absorption phase. Eur J Pharm Sci. In press.

E. Bergman., P. Forsell., E.M. Persson., L. Knutson., and H. Lennernas. The effect of the jejunal pH environment on the precipitation and pharmacokinetics of a gefit-inib using the Loc-I-Gut technique. Manuscript.

L. Kalantzi., E.M. Persson., B. Polentarutti., B. Abrahamsson., K. Goumas., J.B. Dressman., and C. Reppas. Canine intestinal contents vs. simulated media for the assessment of solubility of two weak bases in the human small intestinal contents. Pharm Res. 23(6):1373-1381 (2006).

Contents

Introduction.....................................................................................................9Biopharmaceutical Classification System ................................................10Dissolution ...............................................................................................11Basic physiology and morphology of the gastrointestinal tract ...............12

The intestinal epithelium .....................................................................13Intestinal pH ........................................................................................15Motility in the GI tract .........................................................................15Bile.......................................................................................................16

Classification of lipids..............................................................................18Lipid digestion model...............................................................................19

Inhibition of lipid digestion .................................................................21Analytical methods for the determination of lipid contents in intestinal fluids.........................................................................................................21

Sample preparation ..............................................................................21High throughput liquid chromatography of lipid classes.....................22

Effect of food on dissolution ....................................................................22Solubilization.......................................................................................23Lymphatic uptake ................................................................................24Transporters .........................................................................................24

Measurement of dissolution rates in vitro ................................................26Rotating disc ........................................................................................26Design of dissolution media ................................................................27

Methods for studying absorption of drugs ...............................................28In vitro models.....................................................................................28In vivo perfusion models .....................................................................29

Aims of this thesis.........................................................................................30

Methods ........................................................................................................31Investigational drugs and radiolabelled markers......................................31Intestinal fluids.........................................................................................32

Collection of intestinal fluids...............................................................32Characterisation of intestinal fluids .....................................................34

In vitro dissolution ...................................................................................35In situ single pass jejunal perfusion experiments .....................................36

Calculation of partition coefficients between water and the lipid phase in simulated fed intestinal fluid ....................................................................37Analytical methods...................................................................................37Perfusate data analysis .............................................................................37Statistical analysis ....................................................................................38

Results and discussion ..................................................................................39Characterisation of intestinal fluids..........................................................39Dissolution in intestinal fluids..................................................................44Influence of food on absorption on poorly soluble drugs – in vivo perfusion...................................................................................................46

Conclusions...................................................................................................49

Acknowledgements.......................................................................................51

References.....................................................................................................53

Abbreviations

BA Bile acids BCS Biopharmaceutical classification system CA Cholic acid CDCA Chenodeoxycholic acid CE Cholesterol ester CMC Critical micelle concentration DCA Deoxycholic acid DG Diglyceride DIF Dog intestinal fluid DPG Diphosphatidylglycerol ELS Evaporative light scattering fabs Fraction absorbed FaSSIF Fasted simulated small intestinal fluid FeSSIF Fed simulated small intestinal fluid FFA Free fatty acids GCA Glycocholic acid GCDCA Glycochenodeoxycholic acid GDCA Glycodeoxycholic acid Hacc Number of hydrogen acceptors Hdon Number of hydrogen donors HIF Human intestinal fluid

Hm Change in melting entropy HPLC High throughput liquid chromatography IF Intestinal fluid LCA Lithocholic acid LOD Limit of detection LOQ Limit of quantification LPC Lyso-phosphatidylcholine MAG Monoglycerides MMC Migrating motor complex Mw Molecular weight NL Neutral lipids PA Phosphatidic acid PC Phosphatidylcholine PE Phosphatidylethanol amine Peff Effective jejunal permeability

PG Phosphatidylglycerol P-gp P-glycoprotein PI Phosphatidylinositol PL Phospholipids PS Phosphatidylserine Saq Aqueous solubility SEM Standard error of the mean SM Sphingomyelin SPE Solid phase extraction TCA Taurocholic acid TCDCA Taurochenodeoxycholic acid TDCA Taurodeoxycholic acid TG Triglyceride TLC Thin layer chromatography Tm Melting point UC Unesterified cholesterol UWL unstirred water layer

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Introduction

Bioavailability is defined by the Food and Drug Administration as the rate and extent to which the active moiety is absorbed from a drug product and become available at the site of action (1). A schematic illustration of the steps involved in the release and absorption of a drug taken as an oral solid dosage form is presented in Figure 1.

Liver Gut lumen

Solid dosage form

DisintegrationDissolution

Drug in solution

Precipitation

degradation Adsorption Complexation EG

fa

EH

(Metabolsim, biliary excretion)

Systemic Circulation

Gut wall Portal vein

Tran

sit

Enzymatic/Chemical

(metabolism)

Figure 1. Processes that may influence the bioavailability of orally administered drugs.

The oral bioavailability can be divided into three major determinants, according to the following equation:

HGa E1E1fF (1)

where fa is the fraction of the dose that is absorbed across the apical cell membrane of the enterocyte and EG and EH are the extraction of the drug over the gut and liver, respectively. The fa may be limited by all the reactions that may happen in the lumen and at the apical membrane. This include the dissolution of the drug in the gastrointestinal (GI) tract, since in order to be absorbed in the GI, a drug has to be dissolved. This can be a problem with poorly water-soluble substances, for which the dissolution often limits the absorption after oral administration. Most of the new substances in drug development today are highly lipophilic, and the solubility and dissolution rates in gastric and intestinal fluids (IF) are therefore often critical for the oral bioavailability.

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Biopharmaceutical Classification SystemThe Biopharmaceutical Classification System (BCS), which was proposedby Amidon et al. in 1995, classifies drugs into four different groups,depending on their solubility and permeability (Table 1) (2). The intention ofthe system was to set up a theoretical basis for correlating the in vitrodissolution profiles with in vivo bioavailability of drugs. Drugs are assignedas highly soluble if the dose can be dissolved in 250 mL (the volume in thestomach) over the entire physiological pH range (1.0-7.5) (3). A drug isclassified as highly permeable if the fraction absorbed is <90% (fromsolution).

Table 1. The Biopharmceutical Classification System (2).Class Solubility PermeabilityI High HighII Low HighIII High LowIV Low Low

The BCS is based on a simple absorption model, in which the intestine is acylindrical tube where absorption occurs; particles are spheres of the samesize; there are no reactions (i.e., there is no metabolism) in the intestine;solubility is independent of the particle size and the intestinal pH gradient;and no aggregation occurs. Amidon et al. (2) have demonstrated that the keyparameters controlling drug absorption are three dimensionless numbers: anAbsorption Number, An; a Dissolution Number, Dn; and a Dose Number,Do; representing the fundamental processes of membrane permeation, drugdissolution and dose, respectively (2):

sCVM

NumberDoseDo 00 (1)

Dissres ttNumbernDissolutioDn (2)

reseff tRP

NumberAbsorptionAn (3)

where M0 is the dose of drug administered, V0 is the initial gastric volume(~250 ml), Cs is the saturation solubility, tres is the mean residence time(~180 min), tdiss is the time required for a drug particle to dissolve, Peff is theeffective permeability, and R is the radius of the intestinal segment.For Class II drugs, limits are imposed on the absorption by the solubility

(Do) or dissolution rate (Dn), either in general or on a regional basis within

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the GI tract, leading to incomplete absorption and bioavailability, despite the high membrane permeability (An). These drugs may be particularly sensitive to factors that influence the GI transit time (4). The limitations to absorption can be either equilibrium or kinetic in nature. For drugs with a high Do, the dose of the drug is too high in relation to the solubility of the drug to be dissolved in the fluid available in the GI tract. In the case of drugs with Dn<1, the dissolution process is too slow to have been finished before the drug has passed by its intestinal absorption window (5). For these drugs, good in vitro/in vivo correlation is expected if the in vitro dissolution rate is similar to the in vivo dissolution rate. In general, anything that increases the rate and extent of the in vivo dissolution of Class II drugs will also increase the bioavailability of that compound. Thus, administration with a fatty meal is believed to increase the solubility and thereby the bioavailability of a Class II drug (6).

DissolutionThe dissolution of a solid substance can be described in two steps. In the first of these, molecules are released from the surface to the surrounding dissolution media. This creates a saturated layer, called the stagnant layer, adjacent to the solid surface. Thereafter, the drug diffuses into the bulk of the solvent from regions of high drug concentration to regions of low drug concentration. The rate of drug dissolution at a specific time can be described with the Noyes-Whitney’s equation (7):

VX

CDAdtDx d

s (4)

where Dx/dt is the dissolution rate, A is the surface area of the particle available for dissolution, D is the diffusion rate constant, is the thickness of the stagnant layer surrounding the particle, Cs is the saturation solubility of the drug, Xd is the amount dissolved of drug at time t and V is the volume of the dissolution media. The dissolution rate is influenced both by the physicochemical properties of the substance and by the prevailing physiological conditions in the GI tract (Table 2), which varies between the fasted and fed state as well as within and between subjects. Formulation strategies intended to alter these properties have been employed to increase the dissolution rate of low soluble drugs. These include micronisation, nano-suspensions, lipid-formulations, microemulsions, and the use of complexing agents such as cyclodextrins (8).

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Table 2. List of the physicochemical and the physiological properties that can influence drug dissolution in the GI tract (9).

Factor Physicochemical properties Physiological properties Surface area of drug (A) Particle size, wettability Surfactants in gastric juice

and bile Diffusion coefficient of the drug (D)

Molecular size Viscosity of luminal contents

Stagnant layer thickness ( )

Motility patterns and flow rate

Solubility (Cs) Hydrophilicity, crystal structure, solubilization

pH, buffer capacity, bile and food composition

Amount of drug already dissolved (Xd)

Permeability

Volume of solvent available (V)

Secretion, co-administered fluids

According to this equation, any change in the nature of the dissolution media that will effectively increase the solubility should, in theory, increase the dissolution rate of the drug. Thus, for substances with Dn<1, it is crucial to use dissolution media that closely resemble the conditions in the GI tract to receive good in vitro in vivo correlation. Inclusion of surfactant in the media for this purpose has however, resulted in a decreased dissolution rate despite an increased solubility. This might imply that the solubilization of low soluble drug substances in such media is probably more complex than described by the equation due to the distribution into the micelles and the dependency of removal of the drug substance by absorption due to the low aqueous solubility.

Basic physiology and morphology of the gastrointestinal tractThe oral route is the most common and convenient one for drug delivery. This means that the physiology of the intestinal tract is important in the dissolution and absorption of drugs. Physiological factors affecting drug dissolution and absorption include the GI motility, gastric emptying, surface pH of the mucosa, the unstirred water layer, intestinal blood and lymph flow, bile salts, and colonic microflora. The GI tract consists of three major anatomical regions: the stomach, the small intestine and the colon. Owing to its large surface, the small intestine, which is divided into three regions, the duodenum, jejunum and ileum, is the most important site for drug absorption.

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The intestinal epithelium The length of the small intestine is about 3.5 meter (10). Due to the presence of the foldings of Kerckring, as well as the villi and microvilli the area available for absorption is greatly increased. The permeability differs along the crypt villus axis as the crypt appears to be more easily penetrated (11,12). The high permeability drugs are mainly absorbed at the tips of the villi (13), where most of the functionally active transport protein systems and metabolising enzymes are expressed. The low permeability compounds may diffuse down the length of the crypt-villus axis to be absorbed over a larger surface area (14-16). The cell population in the intestinal epithelial is, up to 90% composed of enterocytes, which constitute the most important anatomical barrier against drug absorption. The apical side of the enterocytes is composed of densely packed microvilli, about 1 µm in length and 0.1 µm in diameter and the lateral membranes are connected through tight junctions at the apical side of each epithelial cell. Beneath the enterocytes is the lamina propia, composed of connective tissue fibers, lymphocytes, plasma cells, macrophages and occasional mast cells. After passing through the enterocyte and lamina propria, a drug molecule enters into the blood or the lymphatic circulations or both. The relatively slow rate of lymphatic flow renders the lymphatic system less important in drug absorption. Adjacent to the mucosa is an unstirred water layer (UWL) of water, mucus and glyco-calyx, created by incomplete mixing of the luminal contents near the intestinal mucosal surface (17). It is believed that the UWL may contribute to the major resistance to intestinal absorption for rapidly permeating solutes (Peff = 2 x 10-4 cm/s) (18). However, the resistance of the UWL to intestinal absorption is probably lower in vivo, owing to more efficient motility and stirring, decreasing the thickness of the layer (19-22).

Figure 2. Schematic drawing of an intestinal epithelium. The arrows indicate the four different drug transport routes; 1) Transcellular passive diffusion through membranes; 2) paracellular transport; 3) carrier mediated transport; 4) Endocytosis (16) (Reprinted from Artursson et al (16) with permission from Elsevier).

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Absorption across membranes Drugs can be absorbed in two ways, either through the cell (transcellular transport) or between the cells (paracellular absorption) as illustrated in Figure 2. Transcellular absorption can be divided into passive diffusion across the cell, carrier-mediated transport and endocytosis. The transport of a drug across an epithelium often involves several processes. Passive diffusion is the major absorption process for most drugs and refers to movement of a solute along its concentration and electrical gradient. According to Fick’s first law of diffusion, drug molecules diffuse from a region of high drug concentration to a region of low drug concentration according to Equation 5:

21 CCAPdtdQ

(5)

where dQ/dt is the diffusion rate; P is the permeability coefficient; A is the surface area of membrane; and C1-C2 is the difference between the concentrations of drug in the GI tract and in the plasma. The drug entering the blood will be continuously transported away, thus sink conditions will prevail. The expression can therefore be simplified to make it depend on the concentration of the apical side (C1) alone. However, the permeability is not only dependent on the physicochemical properties of the compounds, but also on the properties of the epithelial membrane. The apical membrane of the enterocyte is considered to be the rate-limiting step in passive diffusion, since it is thicker and more rigid than the basolateral one (23,24). The permeability across the membrane can be described by the simplified equation:

DKP (6)

where K is the lipid water partition coefficient, is the thickness of the membrane and D is the membrane diffusion coefficient. Thus, lipophilic substances may diffuse more easily through the membrane. Permeation of ionized compounds by this route relies on the equilibrium between unionized and ionized species. The rate is also lower for larger molecules.

Carrier-mediated transport can be divided into active transport and facilitated diffusion. Active carrier-mediated transport, refers to an energy-consuming process, which transports compounds against a concentration gradient. Transport proteins, which facilitate absorption of nutrients, such as amino acids, oligo peptides, monosaccharides, and bile acids, are located in the apical membrane of enterocytes in the small intestine. These transporters may also transport drugs with similar structure to the normal substrates (24-27). However, the transporters are often selective for one or a couple of

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compounds and have a saturable capacity (28) Active transport may also transport compounds out of the enterocyte and into the lumen, and thereby restrict the absorption of drugs (29-31). Facilitated diffusion is transport of a compound along the concentration gradient, without the input of energy.

Paracellular transport is the primary form of transport for the absorption of polar or charged molecules of low weight. The rate-limiting step for paracellular absorption is the tight junctions, which separate the apical and basolateral membranes (32,33). It is believed that the drugs are absorbed by passive diffusion, but it appears that cationic drugs are more readily trans-ported than anionic and neutral compounds (34,35). As the paracellular pores account for only 0.1 % of the intestinal surface (36), drugs transported by this route are often incompletely absorbed. Furthermore, it has been suggested that paracellular transport is of minor importance in the upper part of the small intestine in humans for drugs with a molecular weight above 200 (14,37).

Intestinal pH The pH of the gastric fluid is highly acidic, exhibiting a median vlaue of 1.7 (38), but can vary between 0.8 and 7.2 (5,39,40). In the presence of food, the pH is buffered to less acidic values with a median of 5.0 (38) and depends on the type and volume of the meal ingested. The pH is returned to the more acidic values after about two to three hours after food intake (41). Intestinal pH are considerably higher than gastric pH due to the neutralisation of the incoming acid by bicarbonate ions secreted from the pancreas (42) and increases along the small intestine from 5.5 to 7.5 (5,43). The contents of the stomach after food intake lower the pH in the intestine to a value between 5.1 and 5.4 (38). Next to the mucosa in the small intestine is an acidic microlayer, with a thickness of approximately 20 µm and a 0.5 units lower pH than the bulk phase (44). The acidity of this microlayer was found to be more pronounced in the duodenum than in the distal small intestine (45). Bacterial degradation of unabsorbed carbohydrates lowers the pH in the colon to a value between 5.5 and 7 (46).

Motility in the GI tract Since the absorption of drug molecules from the stomach is generally minimal compared to that of the small intestine, gastric emptying is an important physiological event, which might influence both the amount and rate of the uptake of drug substances from the intestine. In the fasted state, alternating cycles of activity known as the Migrating Motor Complex (MMC) empty the GI tract. Irregular contractions followed by regular contractions of high amplitude push any residual contents distally and further down the alimentary canal (47,48). In the presence of food, the MMC

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is replaced by irregular contractions, which mix the intestinal content and move it down towards the colon. In addition, the activities of the stomach are delayed, which results in a longer residence time for particulates (> 2 mm). The discharge of particles with sizes smaller than 2 mm has been shown to occur even when the stomach is in the digestive mode. (48). The emptying of liquids from the stomach is a rapid and continuous process that is unaffected by food (47,48). The intestinal transit time has been reported to be approximately similar in the fasted and fed state, with mean axial rates of 1.5 and 1.3 cm/min, respectively (49). The volumes in the fasted state stomach and intestine is 20-30 and 120-350 ml, respectively (50,51). Under fed conditions, the volume of the upper intestinal contents is highly variable, measuring up to 1600 ml (52).

BileBile plays a significant role in the solubilization of lipid digestion products and low water-soluble drugs. Proteins and lipids in the diet stimulate the secretion of bile into the small intestine via secretin and cholecystokinin secretion (53-55). After food intake, the gallbladder is emptied to 75 %, with the peak flow occurring approximately 30 minutes after ingestion of a meal. A threshold value of 10 g of fat is required for gallbladder contraction. For maximal contraction, 25 g is required (56). However, the gallbladder also empties during fasting following the movements of the MMC in the duodenum. Each cycle takes about 120 min and ends with the emptying of the gallbladder (57). The major components of bile, are the bile acids (115 g/l). Other important constituents are phosphatidylcholine (34 g/l), cholesterol (6.3 g/l) and free fatty acids (24 g/l). The bile also contains small amounts of bile pigments, proteins and inorganic ions and cations (58,59). Bile acids (BA) are produced in the liver from cholesterol (UC), which is first converted to cholic acid (CA) or chenodeoxycholic acid (CDCA). These then combine with glycine or taurine to form glyco- and tauro-conjugated BA (Figure 3). This renders the molecule more water-soluble and also lowers its pKa from approximately 6 to 3.7 (glycine conjugated) or 1.5 (taurine conjugated). This is important in reducing the precipitation of BA in the relatively acidic environment of the duodenum (60), were the taurine conjugated BA will be ionized and the glycine conjugates will exist as a mixture of ionized and unionized molecules (51,61).The ratio of glycine-conjugated to taurine-conjugated BA is about 3 (62,63). In human bile, 50 % of the BA is CA and 30 % are CDCA, which are trihydroxy and dihydroxy BA, respectively (64). The salts of these acids, mainly sodium salts, are after storage in the gallbladder, where they are concentrated during fasting conditions by reabsorption of water and electrolytes, secreted with the bile into the duodenum. Typical concentrations of BA in the fasted intestine are 4-6 mM compared with post-prandial concentrations of 10-20 mM (63).

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Secreted BA are primarily reabsorbed through transporters located in the terminal ileum and recirculated to the liver and, to a lesser extent, by passive diffusion in the jejunum (65-69). Approximately one quarter of the conjugated BA reaching the ileum have amino acid moieties removed by the action of the bacterial enzymes (67,70). The size of the BA pool is about 3.5 g, and it is recirculated four to five times every day, losing only 0.2 g/day in faeces.

X3

X2X1

COR

glycine conjugates taurine conjugates R = -NH-CH2-COOH R = -NH-CH2-CH2-SO3H

CHOLIC ACIDX1, X2, X3 = OH

CHENODEOXYCHOLIC ACIDX1, X2 = OH, X3 = H

DEOXYCHOLIC ACIDX1, X3 = OH, X2 = H

LITHOCHOLIC ACIDX1 = OH, X2, X3 = H

Figure 3:Molecular structure of bile acids. Modified from Davenport (51).

The BAs have hydrophilic OH-groups that can align on one side of the lipophilic stem, making them amphiphilic (71). This gives them two important functions in the intestinal tract. First, they act as detergents on fat particles in the chyme, decreasing the surface tension of the particles and allowing further emulsification. Second, the BA help in the absorption of free fatty acids (FFA), monoglycerides (MG), UC and other lipids from the intestinal tract by forming micelles into which these lipids can be incorporated. The lipids are transported in this form to the mucosa, where they are absorbed. Drugs can also be incorporated into the micelles, and are believed to be transported in the same way.

The critical micelle concentration (CMC) values of the free and the conjugated BA are in the range of 0.6 to 14 mM, depending on the BA, the amount of NaCl present in the solution, and the pH and temperature of the medium (73). The CMC of the BAs falls in the order: unconjugated > glycine conjugated > taurine conjugated (74). Small BA micelles (primary micelles) are formed by trihydroxy BA and by dihydroxy BA at low salt concentrations. Larger BA micelles (called secondary micelles) are only formed by dihydroxy BA in the presence of high counter ion concentrations, it has been suggested that these are formed by aggregation of primary micelles. The aggregation number in the primary and the secondary BA micelles are 2 to 8 and 12 to 100, respectively (71,75) (Figure 6). These

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simple micelles have mean hydrodynamic radii of 1.0-1.5 nm (74,61). Phospholipids (PL) present in gallbladder bile, mainly as phosphatidyl-choline (PC), may create micelles together with BA. According to Hammad and Müller (76), sodium glycocholate is able to solubilise PC (the major BA and PL in bile, respectively) up to a molar ratio of glycocholate:PC of 1.7:1. The shape of these mixed micelles is dependent on the ratio of BA to PC. BA rich micelles are spherical with a radius of approximately 3 nm, while that of PC-rich micelles are approximately 15 nm (Figure 4) (77). Schersten (55) and Isaksson (78) have reported the molar ratio of BA to PC to be about 2:1 to 5:1, which is above the molar ratio (1.7:1) reported for coexistence of simple BA micelles and mixed micelles (79-80). Only mixed micelles are present at lower ratios.

b) a)

Figure 4. Organisation of micelles containing bile salts and phospholipids. a) Spherical micelle, b) Disc-shaped micelle. Modified from Müller (72). Reprinted with the kind permission from Müller, 1981 © 1981 American Chemical Society.

Classification of lipids Polar lipids are categorised according to their interaction with water, as soluble amphiphiles, insoluble swelling amphiphiles or insoluble non-swelling amphiphiles (Figure 5) (59). Only the soluble amphiphiles, which include BA and lyso-phosphatidylcholine (LPC), are able to form micelles on their own. They possess a high capacity to solubilize insoluble amphiphiles. Sodium salts of FFA also form micelles above their cmc, but prior to that they form an intermediate liquid crystalline phase. MG, ionized FFA and PL belong to the class of insoluble swelling amphiphiles. These form liquid crystalline structures in water, with the non-polar groups of the lipid molecules facing each other and the water molecules sandwiched between the polar groups. Among the polar lipids are insoluble non-swelling amphiphiles, including triglycerides (TG), diglycerides (DG) and non-ionized long-chain FFA and UC. When these are added to water, a thin film (monolayer) of lipid is formed (59).

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Air

Water

Lipid soluble portion of molecule

Water soluble portion of molecule

b) e) d) c)

Figure 5. Classification of lipids based on their ability to interact with water. Modi-fied from Carey and Small (59).

Lipid digestion model The digestion of lipids in the GI tract is very complex and will only be explained briefly here. For further details the reader is directed to the previous reviews (77;81-83). The absorption of dietary lipids in the human is an efficient process; over 90 % occur in the duodenum and proximal jejunum. TG in the diet is enzymatically degraded by lipases in the stomach and the small intestine to its more polar products, FFA and MG, before being absorbed into the enterocytes. Lipase cleaves the two ester bonds of the TG molecule, producing first a molecule of DG and one FFA, and then two molecules of FFA and one of MG. The digestion is initiated in the stomach by the action of lingual lipase from the salivary glands and gastric lipase secreted from chief cells in the stomach, and then it continues in the upper small intestine with a colipase/lipase complex. The optimal pH range for lingual and gastric lipase is 3-6, with medium chain TG being hydrolyzed at a faster rate than long chain TG (84,85). The grinding of the stomach creates an emulsion of lipid droplets (0.5 µm), which is secreted to the small intestine. When entering the duodenum, the lipid droplets are covered by amphiphilic lipids, like PL, MG, proteins and ionized FFA, and after secretion of bile, BAs also join the surface. The presence of the fatty chyme in the duodenum stimulates secretion of bile and pancreatic juice. Biliary lipids adsorb to the surface of the TG and DG emulsion entering from the stomach, thereby stabilising the system and reducing the droplet size (77). Biliary secreted PL, UC and BA coexist as a micellar phase in duodenum. Pancreatic juice contains phospholipase A2, that hydrolyzes PC into LPC and FFA and pancreatic carboxyl ester hydrolase that hydrolyzes the ester linkages of a variety of both water-soluble and water insoluble lipids, such as cholesterol esters (CE). TG hydrolysis in the duodenum occurs primarily under the action of a pancreatic lipase/colipase complex, which binds to the surface of emulsified TG droplets (86). Liberated FFA and MG further promote the binding of the lipase/colipase complex to the emulsified surface (87,88). As a result of this, the rate of emulsification will increase when lipolytic products are formed.

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Figure 6: Accumulation of lipolytic products in a multilamellar liquid chrystalline phase at the surface of the lipid droplet and the transition from the multilamellar phase to vesicles and finally mixed micelles as the ratio of lipolytic products to bile salts increase. Reprinted from Rigler et al. (89) with permission from J Lipid Res.

As the pancreatic lipase-mediated hydrolysis proceeds, the lipolytic products first accumulate at the surface of the emulsion and form a multilamellar liquid crystalline phase, which, in the presence of unsaturated BA micelles is dispersed into multilamellar vesicles (77,90-92). Provided that sufficient BA micelles are present, the multilamellar vesicles will turn into unilamellar vesicles and, owing to the continuous secretion of bile, a two-phase system (Figure 6) is formed where the unilamellar vesicles are in equilibrium with mixed micelles. Eventually, when the lipolytic product:BA ratio increases, only mixed micelles will remain (Figure 5) (89,93,94). Solubilization of FFA and MG in BA micelles increases their luminal solubility (100-1000-fold) as well as facilitating their passage through the UWL, thereby enhancing the quantitative aspects of absorption (95). Although the specific mechanisms of absorption of the lipid digestion products have not been elucidated, experimental data suggest that no uptake of intact micelles takes place (96). Lipids are thought to be absorbed from a monomolecular inter-micellar phase (95). The dissociation of monomolecular lipid from the mixed micellar phase prior to absorption may be stimulated by the microclimate of lower pH associated with the intestinal absorptive site, resulting in more protonated FFA (44,97). Because of this, only the free drug, which is in

21

equilibrium with the drug solubilised in micelles, can be absorbed (65). The uptake of FFA is believed to be a carrier mediated process (98).

Inhibition of lipid digestion Lipases from the pancreas (pancreatic lipase, carboxyl ester lipase, and phos-pholipase A2) can be inhibited by changes in the temperature or pH, or by covalent binding to the active site (99). Lipase inactivation caused by heat or change in the pH is non-specific ways of inhibition compared to the covalent binding to the active site. Heating, the classical method, promotes artefactural lipolysis during the time taken for the samples to reach 70˚ C (99). In addition, phospholipase A2, appears to be more heat stable than the other lipsases (100). Lowering the pH in the IF is effective, but might result in precipitation of BA and hydrolysis of glycerols and PL. Both heating and lowering the pH may lead to disruption of the colloidal phases if the samples are centrifugated (99). It is possible to inhibit lipolytic enzymes by covalently binding different reagents to the amino acid residues in or near the active site (101). This kind of inhibition is very effective and does not cause phase disruption. Orlistat, also known as tetrahydrolipstatin, is a potent (IC50=0.36 µM; 102), specific and irreversible inhibitor of gastric, pancreatic lipases and carboxylester hydrolase while it has no effect on other hydrolases (103,104). 4-bromo-benzene-boronic acid is also an inhibitor of gastric, pancreatic lipase and carboxylester hydrolase, but does not show complete inhibition (99). Both orlistat and 4-bromo-benzene-boronic acid bind covalently to the serine residue in the active site and thereby promote inhibition (105,106).

Analytical methods for the determination of lipid contents in intestinal fluids

Sample preparation The aim of a chromatographic analysis of lipids is the complete resolution of lipid classes and molecular species for the ultimate purpose of full identification and quantitation of all components. Since IFs contain complex mixtures of lipids differing in molecular mass, degree of saturation and in the number and kind of functional group, no single analytical method is capable of identifying and quantitating all lipid species. Modern lipid analysis therefore involves multiple extraction and analytical steps. Conventionally, thin layer chromatography (TLC) has been used to provide efficient separation of lipids. However, TLC produces low lipid recoveries,

22

is time-consuming and may result in oxidation of poly-unsaturated fatty acids due to prolonged exposure to air. Because of this, TLC is almost completely abandoned for the use of solid phase extraction (SPE). The mechanism for solid phase extraction is similar to that of high performance liquid chromatography (HPLC), with the difference that HPLC utilize a continuous flow of mobile phase, whereas the analyte in SPE is first adsorbed and then eluted in a two step fashion. The SPE methods presented for lipid separation are optimized either for BA (107) or for PL and neutral lipids (NL) (108-116) and are often combined with a previous liquid-liquid extraction, such as the Folch extraction (117). However, there are no methods presented for simultaneous separation of BA, PL and NL.

High throughput liquid chromatography of lipid classes In recent years, the application of HPLC in conjunction with evaporative light-scattering (ELS) detection for lipid analysis has become important and can be a very useful technique for lipid class analysis (118,119). Numerous applications of the use of these techniques in combination for the separation and quantification of lipid classes have been reported (120-134) with typical detection limits around 10-50 µM. HPLC is also advantageous in the assay of BA in biological materials, since it can perform direct analyses of the different naturally occurring classes of these compounds without prior removal of the conjugating moiety. In addition, it provides a simple and affordable detection system for analysis of high concentration samples like bile and faeces. Several investigations have demonstrated that satisfactory reversed phase HPLC separation with UV detection of unconjugated (135-137), glycine- and taurine-conjugated bile acids (138-143) occurs in the eluent pH range 3.0-5.0. Scalia (1988) showed that by varying the buffer molarity in the HPLC solvent, simultaneous baseline resolution of nearly all bile acids could be obtained (143).

Effect of food on dissolution Great increases in bioavailability have been seen after administration of low soluble drugs such as halofantrine (144), griseofulvin (145,146) and danazol (147-149) together with lipid-based formulations or food (Table 3). Possible reasons for the increased bioavailability of drugs when administered together with food are increased drug solubility and dissolution, lymphatic uptake (150-152), decreased first pass metabolism (153) and/or inhibition of efflux transporters in the apical membrane (154) (Figure 7).

23

Table 3. Examples of increased postprandial oral bioavailability in humans, and the likely basis for the increased absorption, modified from Charman et a.l (155).Comound Increase in bioavailability Basis for food effect References Griseofulvin 3-5 fold Solubilization 156-158 Danazol 4-fold Solubilization 147,159

-Tocopheryl nicotinate

28-fold Solubilization, Lymph transport

160,161

Halofantrine 3-fold Solubilization, Possible lymph transport

162,163

Atovaquone 3-5 fold Solubilization 164 Retinoic acid derivatives

2-5 fold Likely solubilization Possible lymph transport

165,166

SolubilizationThe solubility of low soluble drugs has been examined in simulated IFs and found to increase with increasing BA concentration, in amounts corresponding to the levels found during fed conditions (156,157,159,167-169). The solubility of drug substances is dependent on the chemical nature of the drug substance as well as the composition of the BA media. Data obtained in simulated intestinal media have shown that no solubilization was obtained for drugs with log P < 2, whereas more lipophilic compounds were increasingly solubilized at increased lipophilicity (170). The number of hydroxyl groups has been shown to be of importance for the extent of solubilization. Dihydroxy BAs are more discriminating than trihydroxy BA, with regard to drug hydrophobicity. Addition of PC to BA solutions creates mixed micelles and may further improve the solubility (171,172). Hammad and Muller (76) found that some drug substances showed higher solubility in pure BA media, whereas others showed higher solubility in BA media containing PL.

In vitro lipolysis models have been developed in recent years to study the solubility of low soluble drugs during the degradation of dietary lipids (173-176). These models have demonstrated the importance of dietary lipids, such as MG and FFA in the solubilization of lipophilic drugs (168,173). Further studies conducted using in vitro lipolysis models have shown that the length of the fatty acid chain on the TG does not matter for the solubilization capacity (174,175,177). Inclusion of medium-chain TG in media containing BA, PL and FFA results in formation of vesicles, micelles and mixed micelles, while long-chain triglycerides in the same media resulted in vesicles and mixed micelles. The drug in vesicle to micelle ratio was shown to increase with increasing logP value of the drugs and increasing concentrations of MG and FFA in BS/PC systems (168). Depending on the physico-chemical properties of the drug substance, incorporation in micelles can take place in the core of the micelles, in the palisade layer or by adsorption at the surface (157). Poorly soluble drug substances are

24

solubilized in the apolar core of the micelles where the mixed micelles have a larger solubilization capacity compared to simple BA micelles.

Despite the many studies showing an increased solubility of low soluble drugs, studies have also been performed showing that despite an increased solublity, the dissolution rate may decrease in the presence of surfactants (159,178-180).

Lymphatic uptake The majority of orally administered drugs gain access to the systemic circulation by absorption into the portal blood. However, for some extremely lipophilic drugs (log P > 5, solubility in TG > 50 mg/ml), transport via the intestinal lymphatics provides an access route to the systemic circulation, which avoids hepatic first-pass metabolism (181). Compounds absorbed via the intestinal lymph are generally transported in association with the TG lipid core of intestinal lipoproteins formed in the enterocyte after re-esterification of FFA and MG. Drug transport via the lymphatics, there-fore requires co-administration of lipid to stimulate lipoprotein formation (182). Recent studies have also shown that the composition and size of the lymph lipid precursor pool in the enterocyte is major determinants for lymphatic drug transport (183). As a general guide, FFA of chain length less than 12 carbons (10 % of dietary lipid) are absorbed primarily be means of the portal blood, whereas FFA with chain lengths greater than 12 carbons are re-esterified and transported via intestinal lymph (184-187). In addition, lipids with increasing degrees of unsaturation appear to produce larger size lymph lipoproteins and preferentially promote lymphatic lipid transport (188-193). Lipid formulations and pro-drug strategies have been applied to boost lymphatic transport (150-152).

TransportersIt has recently been demonstrated that most of the Class II substances are substrates for CYP 3A4 metabolism and P-glycoprotein (P-gp), an efflux transporter in the enterocytes (154). Inhibition of P-gp by FFA or other lipids present in the diet have therefore been suggested as a possible mechanism for increased bioavailability of Class II substances when administered with food. Inhibition of P-gp may result in a decreased CYP 3A4 metabolism, and thereby further increase the bioavailability (154). Studies in Caco-2 cells have shown that both taurocholate (TCA) and FFA as well as surfactants such as tocopheryl polyethylene glycol succinate and polyethyleneglycol increase the apical to basolateral transport and decrease the basolateral to apical transport of low soluble drugs (194-199). In addition, the absorptive transport for cyclosporine has been shown to increase and the secretory to decrease in a concentration dependent manor when diluted fasted HIF was

25

used as medium in the Caco-2 model (195). High concentrations of dietary lipids and BAs have been shown to damage the cell integrity of the Caco-2 cell monolayer, and it has therefore been difficult to study the impact of dietary lipids on P-gp inhibition in the concentrations prevailing of these lipids in the postprandial intestine (194). No studies have been performed in vivo verifying the role of P-gp for increased drug bioavailability when administered together with food. BA have also been observed to affect the permeability of drugs by altering the barrier function of the cell membrane (200-203), the mucus layer (204-206) or the paracellular route (207-210).

Figure 7. Schematic diagram of intestinal drug absorption after administration together with food or in lipid-based formulations. The drug is able to partition between the phases formed upon lipid digestion and thereby renders a higher solubility in the intestinal lumen. Only free drug is absorbed. In addition, food may impact the absorption by (A) Increased membrane fluidity facilitating transcellular absorption; (B) Opening of tight junctions to allow paracellular transport; (C) Inhibition of P-gp and or CYP450 to increase intracellular concentration and residence time and (D) Stimulation of lipoprotein/chylomicron production. ABL, aqueous boundary layer; D, Drug; D-, ionized drug substance; FA, fatty acid; MG, monoglyceride; LCFA, long chain fatty acid; TG, triglyceride; TJ; tight junction. Modified from O’Driscoll (211). Reprented with permission from Elsevier.

26

Measurement of dissolution rates in vitro

Rotating disc Dissolution testing is an important tool to forecast the in vivo performance of drugs and their formulations. There are many methods used to determine the rate of release of drugs into solution from tablets and capsules, because such release may have an important effect on the therapeutic efficiency. Some of the commonly used methods are flow-through methods, the rotating basket method, the paddle method and rotating or static disc methods. To determine the dissolution rate in Paper III in this thesis, a rotating disc method was used. In this method the planar surface area, from which dissolution can occur, is assumed to remain constant. The amount of substance dissolved per unit time and unit surface can therefore be determined.

The rate of dissolution, G, can be considered to be governed by two processes, the tendency to dissolve, k1, and the tendency of dissolved drug to re-enter the disc, k2c, as given by:

ckkG 21 (7)

where c is the time average concentration of solute in the absolute vicinity of the disc. If the conditions are arranged so that the disc will always be in contact with fresh solvent and the hydrodynamic boundary layer = 0, the intrinsic rate of dissolution is achieved. The intrinsic dissolution rate is the maximum rate of mass transfer and can be calculated from the following expression for G at laminar flow, developed by Nicklasson et al. (212):

r1ckG 1 (8)

where r is the distance of the centre of the disc support to the outer edge of the tablet and is the rotational speed of the disc. According to this equation, G is inversely proportional to r . For large values of r and/or ,G will approach the intrinsic rate of dissolution k1. Experimentally, k1 is obtained by measuring G as a function of r and and extrapolating to (r )-1 = 0 in a plot of G versus 1/r . This means essentially, that if r is sufficiently large, the concentration gradient governing the diffusion flow becomes so large that the Fickian flow is no longer the rate-determining step (213,214). Placing the disc eccentrically will expose it to fresh dissolution media all the time. When placing the disc centrically, this has to be compen-sated for by replacing r with in Equation 8.

27

The intrinsic dissolution rate is a rate phenomenon instead of an equilibrium phenomenon, so it might be expected to correlate more closely with in vivo drug dissolution dynamics than solubility. Because of this it has been suggested that intrinsic dissolution rate should be used to classify drugs instead of solubility, setting the phase boundary at 0.1 mg/cm2/min.

Design of dissolution media For immediate release dosage forms, Class II drugs have been shown to be more dependent on the choice of dissolution medium than Class I drugs (215). Galia et al. (215) showed that a simple mild aqueous dissolution medium is sufficient for Class I drugs to provide a meaningful in vitro/invivo correlation, while the choice of medium for Class II drugs was depend-ent on whether the drug was ionizable or not and whether it was a simulation of the fasted or fed state. Thus, for the latter, dissolution media need to closely represent the prevailing conditions in the gastrointestinal tract, to achieve a meaningful in vitro/in vivo correlation. The complexity of human intestinal fluids (HIF) has been studied in both fasted and fed state (Table 4).

Table 4. Characterisation of HIF collected in the fasted and fed state (mean ± SD). Fasted HIF Fed HIF BA (mM)

Taurocholate (%) Glycocholate (%) Taurodeoxycholate (%) Glycodeoxycholate (%) Taurochenodeoxycholate (%) Glycochenodeoxycholate (%)

2.1a-2.9b, 1.52 1.77c

12-18b,d

35-49b,d

5-6b,d

11-18b,d

5-12b,d

12-19b,d

9.3 0.75e, 15 8.8 (5.8-37)f,9.9 0.2 (2.8-23)g, 14.5 (2.3-17)h

Phosphatidylcholine (mM) <0.2c 0.07 0.04e, 4.8 1.8 (2.3-9.4)f

6.3 1.0i

Cholesterol (mM) 1.5 0.12e

Monoglycerides (mM) 5.4 4.5 (1.3-18)f,3.9 1.7 (1.4-6.8)i, 11 3.9g

Free fatty acids (mM) 23.5e 28.3 4.1e

Proteins (g/l) 2.31 0.2b 16.1 1.2e

pH 6.63 0.23b 5.1-5.4j

Surface tension (mN/m) 33.7 2.8c

a Lindahl et al. (216), b Kostewicz et al. (217), c Pedersen et al. (172), d Hoffmann (70), e Mansbach et al. (91), f Armand et al. (218), h Fausa (62), i Rauterau et al. (64), j Dressman et al. (38)

Though HIF is the preferred medium, it is expensive and difficult to obtain. Dog intestinal fluid (DIF) and simulated IFs, based on buffers to which BA and PL are added, has been studied as alternatives to HIF (5,9,171,172,215,219,217,220), showed that the dissolution rate of low soluble drugs is often better in media containing surfactants than in simple

28

aqueous buffers because of the increased wetting and micellar solubilization. In fact, for neutral compounds the concentration of solubilisers in the medium seemed to be the major determinant for dissolution (215). The two most widely used simulated intestinal fluids are FaSSIF and FeSSIF, representing fasted and fed state conditions in the upper jejunum, respectively (Table 5). The concentrations in the simulated IF were chosen to represent the concentrations in vivo. It has been shown, however, that the in vitro dissolution rate of low soluble drugs in these media does not always correlate with the dissolution rate in aspirated gastric fluids (217,219) in the fasted state. FeSSIF is considered to be a reasonable starting point for the assessment of the impact of food on drug dissolution in the small intestine. However, it does not account for the presence of ingested lipids in the intestine and may lead to underestimates of dissolution for highly lipophilic drugs (221).

Table 4. Composition of fasted state simulated intestinal fluid (FaSSIF) and fed state simulated small intestinal fluid (FeSSIF) (215).FaSSIF FaSSIF pH 6.5 pH 5.0 Osmolality 270 ± 10 mOsm Osmolality 635 ± 10 mOsm Sodium taurocholate 3 mM Sodium taurocholate 15 mM Lecithin 0.75 mM Lecithin 3.75 mM KH2PO4 3.9 g Acetic acid 8.65 g KCl 7.7 g KCl 15.2 g NaOH, qs pH6.5 NaOH, qs pH5.0 Deionized water, qs 1 L Deionized water, qs 1 L

Methods for studying absorption of drugs

In vitro models Human in vivo permeabilities can be predicted using preclinical permeability models, such as in situ perfusion of rat jejunum, the Caco-2 model, and excised intestinal segments in the Ussing chamber. Absorption studies in cell lines involve the transport of drug over a cell monolayer grown on a porous permeable filter. Caco-2 cells derived from human colonic adenocarcinoma, are one of the most popular cell culture models, due to the morphological as well as functional similarities to intestinal enterocytes. The cells form tight junctions and express a variety of transporters and some enzymes. It has been shown that apparent permeability values obtained in Caco-2 models correlate well with drug absorption in humans (222,223), with the exception of hydrophilic compounds that are absorbed via the paracellular route (15). The absorption is often underestimated because the pores in the tight

29

junctions of the Caco-2 cells are smaller than in human. In addition, the variation in the expression of transporters may result in over- or underestimation of carrier-mediated drug transport. P-gp can be over-expressed, while other transporters are often under-expressed. A disadvantage with studying drug transport in cell lines is the potential to physically lose some of the compound through non-specific binding to the plastics and to the cell culture filters.

In vivo perfusion models Human intestinal perfusion techniques are divided into open, semiopen and double ballon methodologies and the calculation of the absorption parameters are based on the disappearance of the drug under investigation from the perfused segment (14). These techniques may be used to study drug metabolism in liver and intestine, in vivo dissolution of drugs, local pharmacological studies of drugs, nutrient absorption, biological mechanisms of different gastrointestinal diseases, food-drug and drug-drug interactions, transport mechanisms and intestinal secretion of drugs and endogenous compounds. In this thesis, the Loc-I-Gut method has been used for collection of IF. Recently the Loc-I-Gut technique was successfully employed in pigs, with simultaneous blood and bile sampling (224).

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Aims of this thesis

The main objectives of this thesis were to increase the understanding of the in vivo dissolution of low soluble drugs and thereby improve the ability to predict the in vivo solubility from the properties of a substance, to generate ideas for improved formulation principles for low soluble compounds and more relevant methods for testing the in vitro dissolution for use in formulation development. Different in vitro and in vivo methods have been used to pursue this goal. The specific goals were:

To develop a method for the separation and subsequent analysis of phospholipids, bile acids and neutral lipids, including free fatty acids, in human intestinal fluid (Paper I).

To investigate the gastrointestinal secretory and enzymatic responses to a liquid meal during in vivo perfusion of the proximal human jejunum using highly sensitive analytical methods (Papers I & II).

To study food induced effects on drug solubility and dissolution in human intestinal fluid (Paper III).

To investigate the in vivo relevance of FeSSIF and dog intestinal fluid, as alternatives to human intestinal fluid (Paper III).

To investigate the relative importance of mechanisms behind the effect of food on the intestinal absorption kinetics for low solubility compounds by applying a jejunal single-pass perfusion model in porcine. A special focus of the current study was to compare the importance of inhibition of apical efflux and micelle solubilisation (Paper IV).

31

Methods

Investigational drugs and radiolabelled markers The drugs used in the in vitro dissolution study presented in Paper III were felodipine (AstraZeneca R&D, Sweden), griseofulvin (Acros Organics, USA), danazol, cyclosporine (Sigma-Aldrich, Germany), probucol (ICN Biomedicals, Germany) and ubiquinone (ABCR Gmbh & Co, Germany) (Figure 8, Table 6). These were all low solubility drugs according to the Biopharmaceutical Classification System (BCS) (32). The chosen drugs were all aprotic to exclude pH related effects on intestinal drug dissolution.The enzyme inhibitor orlistat (Apin Chemicals, UK) was added (1 mg/ml) to fed state HIF and DIF, prior to conducting the ex vivo studies (Papers I-III), to inhibit the enzymatic degradation of lipids (225). In the perfusion experiments (Papers II & IV) 14C-labelled polyethylene glycol (14C-PEG 4000) (2.5 µCi/L, Amersham Pharmacia Biotech, Little Chalfont, England) was used as a nonabsorbable volume marker. Danazol and cyclosporine were used as model drugs and verapamil (500 mg/l) as a P-gp inhibitor in the porcine model (Paper IV). Cyclosporine is a substrate of both P-gp and CYP 3A4 (226-229), while danazol is only a substrate for CYP3A4.

a) b) c)

d) e) f)

Figure 8. Chemical structure of the model substances: (a) felodipine, (b) griseofulvin, (c) danazol, (d) cyclosporine, (e) ubiquinone and (f) probucol.

NH

Cl

Cl

O

O

O

O O

O

O

O

ClO

O NO

OH

HH

H

N

OH O

N

N

OO

NN

O

O

N

O

N O

N

O

N

O

N

O

N

O

O

O

O

O S S

OHOH

32

Table 6. Physicochemical parameters of the model substances including molecular weight (Mw), water solubility (Saq), lipophilicity (logP), melting point (Tm), change in melting entropy ( Hm) and the number of hydrogen acceptors (Hacc) and donors (Hdon).

aMithani et al. (170), bScholz 2002 (230), cGramatte (231), dJack (232), eexperimentally determined according to the solubility methodology described below, fcalculated data (ACD Labs Version 8.0),gdetermined with differential scanning calorimetry, hObtained by performing a Monte Carlo simulation (Biochemical and Organic Simulation System *Data missing

The BA used as standards in the analysis of the intestinal fluids (Papers I-III) were the unconjugated and glyco- and tauro conjugates of cholic, deoxycholic and chenodeoxycholic acid. Nine different PL were included in the standards for the analysis of the intestinal fluids (Papers I-III) as follows: PC, phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidic acid (PA), sphingomyelin (SM), phosphatidylinositol (PI), phosphatidylserine (PS) and LPC. For the NL analyses, UC, cholesteryloleate, dipalmitin and tripalmitin, monoolein and palmitic acid were used as standards (Papers I-III).

Intestinal fluids Collection of intestinal fluids Fed and fasted HIF was collected from 6 and 12 healthy volunteers, respectively, aged 24-40 years and weighing 66-86 kg (males) and 50-70 kg (females). The subjects had fasted overnight before a perfusion tube (Loc-I-Gut) was positioned in the proximal part of the jejunum by oral intubation (233,234). The perfusion tube was a 175 cm long (5.3 mm external diameter) multichannel polyvinyl tube with two inflatable balloons, and a tungsten weight at the tip (Figure 9).

Substance Danazol Felodipine Cyclosporine Griseofulvin Probucol Ubiquinone Mw (g/mol) 337 384 1202 352 516 863 Saq at 37 C (µg/ml) 1a 1b 7a 14c 0.006e 0.0007e

Log P 5a 5b 3a 2a 10d 21f

Tm ( C) 225g 144g 150g 220g 126g 48g

Hm (kJ/mol) 42g 31g 34g 52g 36g 119g

Hacc 5h 4h * 6h 2h * Hdon 1h 1h * 0h 0h *

33

Perfusate inlet

Perfusate outlet

Gastric drainage

Perfusate inlet

Perfusate outlet

Gastric drainage

Perfusate inlet

Perfusate outlet

Gastric drainage

Perfusate inlet

Perfusate outlet

Gastric drainage

Perfusate inlet

Perfusate outlet

Gastric drainage

Perfusate inlet

Perfusate outlet

Gastric drainage

Perfusate inlet

Perfusate outlet

Gastric drainage

Perfusate inlet

Perfusate outlet

Gastric drainage

Figure 9. Schematic drawing of the Loc-I-Gut® tube. The perfusate inlet and outlet holes were positioned in the jejunum. Only the distal balloon was inflated for the collection of fed intestinal fluid, to prevent fluid from continuing further down the gastrointestinal tract. A gastric drainage tube was used to avoid nausea.

For the collection of fed IF (Papers I-III), only the distal balloon was in-flated. This prevented fluid from continuing further down the intestinal tract and guaranteed complete sampling of the intestinal contents. Another tube was positioned in the stomach to drain gastric juice to avoid nausea during the intestinal perfusion experiment. The subjects were given a well-defined nutritional drink (NuTRIflex , Braun, Table 7) to simulate fed conditions. The drink was administered directly to the small intestine through the Loc-I-Gut tube. The nutritional drink contained partly metabolised TG and proteins similar to the degradation in the stomach prior to emptying into the small intestine. The amount of fat administered in the nutritional drink was low, corresponding to about ¼ parts of the amount given in a standard FDA breakfast meal. After rinsing the jejunal segment with isotonic saline (37 C), the nutritional liquid was continuously perfused through the intestinal segment for 90 minutes at a flow rate of 2 ml/min to simulate the gastric emptying rate. The perfusate leaving the jejunal segment, containing both endogenous lipids and lipids from the NuTRIflex, was quantitatively collected on ice at 10 minute intervals. The HIF was pooled, and stored at –70 C prior to performing the analyses (Papers I & II). The fed intestinal fluid obtained during bile excretion (indicated by increased BA concentrations, Paper II), for the period 20-60 min after the start of the perfusion, was pooled and used in the dissolution and solubility experiments (Paper III).

34

Table 7. The composition and amount of the major nutrient groups contained in a liquid meal exposed to a segment of the human proximal jejunum during a single-pass in vivo perfusion.

Nutrient Composition (g/l) Amount given (g)Nitrogen 5 0.8 Amino acids 32 6 Glucose 64 12 Lipids 40 7 Energy 3200kJ 576 kJ

Fasted state fluid (Paper III) was collected using the procedure described above, but with both balloons on the Loc-I-Gut tube inflated, the IF was collected by vacuum drainage without administration of fluid. DIF (Paper III) was collected from three male Labradors, aged 3.5-6.5 years, fistulated at mid jejunum. The dogs received 200 ml of the same nutritional liquid as the human subjects (NuTRIflex®) through an orogastric tube. The DIF was collected through the fistulae and centrifuged for 10 minutes at 4 C and 3000 rpm, to remove large particles. The supernatants were pooled and stored at -70 C prior to performing the experiments.Fed simulated small intestinal fluid (FeSSIF) (Paper III) was prepared according to the method presented by Galia and coworkers (215).

Characterisation of intestinal fluids DIF, HIF and the nutritional drink were characterised in terms of the pH, buffer capacity, surface tension, protein concentration and lipid content (Papers I-III). The buffer capacity, surface tension and pH measurements were performed at 37º C and all other measurements at room temperature. The surface tension was determined with the Wilhelmy plate method (Sigma 70, KSV Instruments Ltd., Finland). A bicinchoninic acid protein assay reagent kit (PIERDE, Rockford) was used to determine the total protein concentration. The buffer capacity was determined by titration.

A SPE method and HPLC with ELS detection were developed for each lipid class to enable the lipid content in the IFs to be determined. The SPE procedure was optimised according to Table 8. For quantitative recovery experiments, radioactive lipids were added to the IF prior to conducting the SPE. In a first step, BA and PL/NL were separated on a C18 SPE column. The solvent fractions containing PL and NL were evaporated under a stream of nitrogen at 40 C and redissolved in chloroform prior to loading on silica columns for separation, as described previously (235). For the analysis of PL and NL, the methods developed by Silversand and Haux (118) were used with slight modification of the compositions of the mobile phases, the

35

gradient and the column temperature, to obtain optimal resolution of critical pairs.

Table 8. Solvents used in the C18 SPE of lipid classes in HIF. Solvent Modification Eluted

lipidsActivation MeOHa - - Conditioning H

2O - -

Sample load - Addition of MeOH (0-40%) and HAcb (0-10%, pH 6-3.5)

-

Fraction 1 (F1) MeOH/H2O (15:85) - -

Fraction 2 (F2) MeOH/H2O (75:25) - BA

Fraction 3 (F3) MTBE3/MeOH Adjusting the amount of MTBEc

(25-100%)NL, PL

Fraction 4 (F4) MeOH/HAc (98:2) - NL, PL aMethanol, bAcetic acid, cMethyl tert-butyl ether

In vitro dissolution The solubility and dissolution rate of griseofulvin, felodipine, danazol and cyclosporine were studied in FeSSIF, in fasted and fed HIF and in fed DIF at 37 C (Paper III). The solubility of probucol and ubiquinone was only determined in fasted and fed HIF. An excess of drug substance was added to the different media. After mixing for 24 hours, the samples were centrifuged and the supernatant analysed for drug content. Solubility experiments with griseofulvin were performed in FeSSIF, both with and without orlistat, to verify that addition of orlistat to the fed IF did not affect the solubility of the model drugs. The solubility of danazol and cyclosporine was also determined in the perfusion solutions used in the porcine perfusion model (Paper IV) to enable the solubility to be compared with the amount absorbed from each media. The relationship between the solubility-ratio between fed and fasted HIF and the physico-chemical properties of the model substances displayed in Table 6 was established by partial least squares calculation using the software SIMCA-P version 10.5 (UMETRICS AB, Sweden).

The dissolution rates (Paper III) were determined using a rotating disc method (USP 28), which was scaled down in size (disc diameter 3 mm, 5 mL dissolution media). The experiments were performed at 1000 rpm for 20 min under sink conditions. The dissolution rate was calculated by linear regression from the initial linear phase of the amount dissolved plotted as a function of the time.

36

In situ single pass jejunal perfusion experiments The influence of P-gp inhibition and solubilisation in micelles on the bioavailability of danazol and cyclosporine when administered with food was studied in a porcine model using the Loc-I-Gut method (Paper IV). The tube was introduced through an incision in the duodenum and a 10 cm long segment was created by fixing ligatures around the jejunum and the Loc-I-Gut tube in fully anaesthetised pigs. The perfusion was similar to that in humans, with the exception that the animals were perfused for 120 min at a rate of 1 mL/min (234). Four different test formulations of cyclosporine and danazol were perfused through the pig intestine (Table 9). Nano-suspensions of the two model substances were administrated in isotonic fluid (water with addition of 2.6% glycerol) alone (Control) or with addition of either a P-gp inhibitor (verapamil, 0.5 mg/ml) (Treatment 1) or dietary and endogenous lipids (Treatment 2). The ratio and concentrations of the lipids in the fed state media were based on the results obtained in the characterisation of the fed HIF in Paper III. Verapamil was included in the media to see the effect of a P-gp inhibitor on P-gp mediated efflux of cyclosporine. The drug nanoparticle suspensions were made by adding the drugs to an aqueous solution with a small amount of water-soluble polymer (< 3 %) and surfactant (1 %) as stabilisers/dispersants, which was milled to cyclosporine and danazol nanoparticles of 650 and 150 nm, respectively. The drugs were also administered as saturated solutions in isotonic fluid containing dietary and endogenous lipids, to enable observations to be made of the impact of the dissolution of the drug (Treatment 3).

Table 9. Composition of the four different perfusion solutions administered in the porcine perfusion study.

Control Treatment 1 Treatment 2 Treatment 3 Cyclosporine (mg/ml) 1.25 1.25 1.25 0.94 Danazol (mg/ml) 3 - 3 0.16 Verapamil (mg/ml) - 0.5 - - 14C-PEG 4000 (nCi/ml) 0.13 0.13 0.13 0.13 Glycerol (mg/ml) 26 26 26 26 Sodium taurocholate (mM) - - 20 20 Phosphatidylcholine (mM) - - 6 6 Monoolein (mM) - - 4 4 Oleic acid (mM) - - 20 20 NaOH (mM) - - 20 20

The size distribution of colloidal structures present in the isotonic fluid containing dietary and endogenous lipids was investigated by dynamic light scattering (DLS) on the media alone as well as saturated with cyclosporine and danazol.

37

Calculation of partition coefficients between water and the lipid phase in simulated fed intestinal fluid The partition coefficients between water and the lipid phase for danazol and cyclosporine in the lipid containing media used in the porcine perfusion study (Paper IV) were calculated as described by Grisafe et al (236). In brief, a series of centrifugation tubes was prepared containing an excess of cyclosporine and danazol in buffer, with an increasing amount of the lipid phase being added to successive tubes. The tubes were equilibrated for 48 h and centrifuged at 2500 g for 15 min, the supernatant was analysed for cyclosporine and danazol. The total volume of the lipid phase was calculated by summing the volumes of the FFA, MG, PC and BA. The result was divided by the total volume of the sample to obtain the lipid fraction. By plotting the total solubility divided by the solubility in the isotonic fluid against the lipid fraction, a straight line was obtained. The slope of the line represented the partition coefficient between water and the lipid phase.

Analytical methods The concentration of griseofulvin, felodipine, probucol, ubiquinone, danazol and cyclosporine, in real and simulated intestinal fluids were quantified with liquid chromatography with UV or atmospheric pressure photo ionosation tandem mass spectrometry (Papers I-IV). Scintillation counting was used for the quantification of radiolabelled substances (Papers I, II & IV).

Perfusate data analysis When the perfusate (NuTRIflex) administered through the Loc-I-Gut tube in Paper I-III reached the intestinal lumen it was immediately diluted according to the well-mixed hydrodynamics (237). The concentrations of the lipidic components in the NuTRIflex solution upon entry in the jejunum were therefore calculated for each time interval under the assumption that dilution took place immediately upon entering the small intestine and that no degradation or absorption occurred, according to Equation 9:

intout,

inin vivot,

QQCC (9)

where Ct,in vivo is the concentration of each component upon dilution in the small intestine in the time interval t, Cin is the concentration of the

38

component in the NuTRIflex and Qout,t is the flow rate of the perfusateleaving the segment during the time interval t.The following calculations from the perfusion experiments carried out in

Paper IV were made from steady-state concentrations of the outlet jejunalperfusate. Each sample represents the mean concentration of the aliquotscollected for each 10-minute interval in the sampling period. The fraction ofthe drug absorbed in the segment during the perfusion (fabs) was calculatedfrom Equation 10:

outin

inoutabs PEGC

PEGC1f (10)

where Cin and Cout are the concentrations of the compound and PEGin andPEGout are the measured radioactivity of the [14C]-PEG 4000, entering andleaving the jejunal segment, respectively. The effective jejunal permeability(Peff) of each drug (Paper IV) was calculated according to a well-mixed tankmodel, as shown in Equation 11 (238):

rLQ

CCC

P in

out

outineff

2(11)

where the cylindrical area representing the jejunal segment (2 rL) was cal-culated using the intestinal radius (r = 1.75 cm) and the length (L = 10 cm)of the segment. The value of the intestinal radius was measured after theballoons were inflated. Peff is a directly determined parameter of intestinaltransport that can be used regardless of the mechanisms involved (1).

Statistical analysisAll data are presented as the mean ± one standard deviation (SD) unlessotherwise stated. When comparing the mean of two groups, the student’st-test for paired or unpaired data was used. For multiple comparisons, theANOVA general linear model or one way ANOVA was used followed byDunnet and Bonferroni’s multiple comparison test. A p-value of < 0.05 wasconsidered to be significant.The partial least squares method was used to establish the relationship

between the data for the physico-chemical descriptors (x) and the solubility-ratio between fed and fasted HIF (y) in Paper III. The partial least squarescalculation was performed using the software SIMCA-P version 10.5(UMETRICS AB, Sweden).

39

Results and discussion

Characterisation of intestinal fluids In Paper I, SPE and HPLC methods were developed to make possible the accurate quantification of the BA, PL and NL, including FFA present in IFs. The retention of lipids on the small disposable C18 columns was a function of lipid polarity and of solvent strength and polarity. Addition of 30% methanol and lowering of the pH to 5 in the intestinal fluid prior to the SPE was sufficient to break up the micelles in the intestinal fluid, for improved sorption of TG, FFA and PL. Addition of methanol to the water-wash further increased the recovery of TG and FFA, by increased wettability of the analytes. Varying the amount of methyl tert-butyl ether in F3, did not enable us to separate NL and PL, because of this, a second extraction step using Si columns was needed. The ultimate SPE method and the recovery of the radioactive lipid standards used is shown in Figure 10 and Table 10, respectively.

Table 10. Recoveries and purities of lipid classes isolated by SPE. The results are presented as means ± SD (n=9).

Lipid % Recovery Taurocholate 94.3 2.0 Glycocholate 96.4 2.0 Triglyceride 95.5 2.9 Free fatty acid 94.5 2.8 Cholesterol 95.9 3.5 Phosphatidylcholine 90.2 4.6 Lyso-phosphatidylcholine 97.1 2.5

40

Figure 10. Schematic illustration of the SPE method ultimately developed for HIF. BA were separated from PL and NL by use of a C18 column. A silica column was subsequently used for separation of PL and NL. Each fraction was analyzed for the different lipid species with HPLC and ELS detection.

The HPLC methods developed for the quantification of BA, PL and NL including FFA are shown in Figure 11 with the standard chromatograms. All lipids were base-line separated, except PA and PE, and nearly all eluted as single peaks, the exceptions being PG, LPC and DG. The limit of quantification for the lipids was between 0.02 and 0.1 mM, which was well below the actual values in the IF. The standard curves followed a 2polynomial fitting (r = 0.99). The coefficient of variance was below 20 % for low, medium and high concentrations (n = 6 for each concentration) and the accuracy varied between 7 and 20%. The interday and intraday variation was below 20 % (n = 6).

I. C18 columnActivation: 2 mL MeOH Conditioning: 2 mL H2OSample load: Application of 1 ml HIF sample (700 µl HIF mixed with 300 µl MeOH and adjustment of the pH to 5 with HAc)F1: 2 mL H2O/MeOH (85:15) F2: 3 mL H2O/MeOH (25:75) (BA elute) F3: 7.5 mL MeOH/MTBE (25:75) (NL/PL elute) F4: 7.5 mL MeOH/HAc (98:2) (NL/PL elute)

II. Si columnActivation: 2 mL CHCl3

Sample load: F3 and F4 evaporated under nitrogen gas and redissolved in 1 mL CHCl3 (NL elute) F5: 5 mL CHCl3

F6: 7.5 mL MeOH/HAc (98:2) (PL elute)

HIF

Bile acids Phospholipids Neutral lipids

2 3,4

C18

Phospholipids

5 6

Si

Neutral lipids

41

LP C

P I

P C

P G

0 2 .5 5 7 .5 10 12 .5 15 17 .5 20 22 .5 25 27 .5 30 32 .5 35 37 .5 40 44-100

0

125

250

375

500

625

800m V

m in F low : 0 .25 m L /m in

B : 40 %

B : 85 %

B : 5 %

P EP A

D P G

S M

P S

b )

A : H exan :Iso -p ropano l:ace tic acid :trie thylam ine (82 :18 :0 .5 :0.014 ) B : Iso -p ropanol:H 2O :ace tic acid :trie thylam ine (85 :15 :0 .5 :0 .014 )

B : 5 %

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30-200

-100

0

1 00

2 00

3 00

4 00

5 00

6 00

7 00

8 00

9 00

100 0

110 0

120 0m V

m in

T C A G C A

T C D C A T D C A

G C D C A

C A

G D C A

LC A 3S N o r-D C A

C D C A

D C A

LC A

F low : 1 .0 m l/m in

B : 50 %

B : 100 %

B : 10 %

a )

B : 10 %

A : M e thano l:bu ffe r (am m on ium a ce ta te 15 m M , 0 .2% trie thyla m ine , 0 .5% fo rm ic ac id , pH 3 .15 (60 :40 ) B : M e thano l:bu ffe r (95 :5 )

0 5 10 15 20 25 30 35 40 45 50 55 60-100

200

400

600

800

1000

1200m V

m in

C E

F F A T G

U C

D G -1

D G -2 M G

F low : 0 .8 m L /m in

B : 5 %

B : 100 %

B : 0 %

c)

B : 0 %

A : H exane (99 :1 ) B : Isoh exan :Iso -p ropanol:ace tic acid (84 :15 :1 )

Figure 11. Standard chromatograms from the characterisation of fed HIF analysed by HPLC. a) BA was analysed using a gradient of methanol and ammonium acetate buffer (pH 2.8). 25 µl of the sample dissolved in methanol was injected onto a Zorbax Extend C18 Column (150*4.6 mm, 3.5 µm) (Agilent Technologies, USA). The column temperature was held at 40º C. b) PL was analysed using a gradient of hexane, 2-propanol, H2O, acetic acid and triethylamine. 10 µl of the sample was injected onto a YMC-Pack Diol column (250*2.1 mm, 5 µm) (YMC Inc, USA). The column temperature was held at 55º C. c) NL was analysed using a gradient of hexane, acetic acid and 2-propanol. 10 µl of the NL sample dissolved in hexane/HAc (99:1) was injected onto an Apex II Diol C18 column (150*4.6 mm, 5 µm) (Jones Chromatography, USA). The column temperature was held at 15º C. DCA, deoxycholic acid; CA; cholic acid; CDCA; chenodeoxycholic acid; GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid; LCA, lithocholic acid; LCA-3S, lithocholic acid 3-sulfate; TCA, taurocholic acid; TCDCA, taurochenode-oxycholic acid; TDCA, taurodeoxycholic acid.

42

In Paper III, characterization of fasting and fed HIF and DIF was performed to determine the pH, total protein concentration, bile secretion components, surface tension, buffer capacity and total nutritional lipid content (Table 11). The lipid composition was determined in pooled IFs (Paper III) as well as over time after administration of a nutritional drink (Paper II). The results showed that there was a significant increase in the concentration of BA, UC and PL, all of which are components of human bile, 20 min after the start of the perfusion of the nutritional drink (Figure 12, 13a). This indicates that there was a rapid in vivo response to the liquid meal, leading to a rapid release of bile from the gall bladder into the perfused segment. Peak levels were reached after 40 min. The ratio of BA to PL in fed HIF was 6:1 at the start of the perfusion and 1:1-2.7:1 after contraction of the gall bladder. This was in agreement with the ratio of BAs to PLs in artificial IFs (171,215). The overall BA concentration in the pooled fed HIF (20 and 60 min) was four times higher than in fasted state HIF, although it corresponded well with the concentration in fed DIF (Table 11) and was only half of that in FeSSIF. The relative proportion of the different BA was quite similar in fasted and fed HIF, with GCA being the most abundant. TCA, the second most abundant in HIF, was the major component in fed DIF and FeSSIF (Figure 12a). Thus, if these two are equally potent as dissolution enhancing agents, either one should be able to function as a model BA in any future artificial IF.

a)

0

0.5

1

1.5

2

2.5

0 20 40 60 80 100

Time (min)

Con

cent

ratio

n (m

M)

TCA GCA TCDCA

TDCA GCDCA GDCA

b)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 20 40 60 80 100

Time (min)

Con

cent

ratio

n (m

M)

PCLPCPC in the NuTRiFlex

Figure 12. Mean concentrations of a) BA and b) PL from intestinal secretions and NuTRIflex in HIF 10-90 min after the start of perfusion of a nutritional drink. The theoretical in vivo concentrations (Ct, in vivo) of the NuTRIflex components calculated according to Equation 9 are included in the figures as dotted lines. GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid; GDCA, glycodeoxycholic acid; PC, phosphatidylcholine; LPC, lyso-phosphatidylcholine; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; TDCA, taurodeoxycholic acid.

43

The total concentration of PL in the pooled fed HIF was much higher than in the fasted state HIF. The other fed state medias provided similar values to that obtained for the pooled fed HIF. LPC was the dominant PL in both the fed and fasted state HIF (Figure 11b), whereas PC, which is added to FeSSIF, was found in a higher concentration in fed DIF. For the improvement of artificial media, further studies are needed to determine whether there is any difference between these two PL in terms of their solubilization capacity of low solubility drugs with different chemical structure.

The concentration of NLs including FFA in the pooled fed HIF was higher than the concentration in fasted HIF and fed DIF (Table 11). Only FFA and UC were present in the fasted HIF. In contrast, fed HIF also contained tri-, di-, and monoglycerides as a result of the nutritional drink being administered (Figure 13). However, the TG present in the nutritional drink was highly metabolized, resulting in FFAs being the major NL in the fed state HIF. The composition of NL in fed DIF was similar to the one in fed HIF. FeSSIF lacks the addition of NLs. This is probably the main departure of artificial media from the real intestinal fluids. Addition of PC and lipolytic products to simulated intestinal fluids has been shown to have great impact on the solubility of low soluble drugs (168,171,172). Thus, to improve in vivo predictability, MG and FFA should be added to simulated IFs, in a ratio of approximately 1:6.

a)

0

2

4

6

8

10

12

14

16

18

0 20 40 60 80 100

Time (min)

Con

cent

ratio

n (m

M)

FFA TG

DG MG

FFA in the NuTRiFlex TG in the NutriFlex

b)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 20 40 60 80 100

Time (min)

Concentration (mM)

CE

UC

UC in the NutriFlex

Figure 13. Mean concentrations of a) neutral lipids including FFAs and b) cholesterols from endogenous secretions and NuTRIflex in intestinal fluids 10-90 min after the start of the perfusion with a nutritional drink (n = 6). The theoretical in vivo concentrations (Ct, in vivo) of the NuTRIflex components calculated according to Equation 9 are included in the figures as dotted lines. CE, cholesterol eater; CH, cholesterol; DG, diglycerides; FFA, free fatty acids; MG, monoglycerides; TG, triglycerides

44

No obvious differences in pH, total protein concentration, surface tension or buffer capacity between fasting and fed HIF or fed DIF were observed, that are believed to influence the dissolution of poorly soluble drugs (Table 11).

Table 11. Characterisation of fasted HIF, fed HIF (20-60 min) and fed DIF. The results given are the mean ± SD when multiple tests have been performed (n = 3). Fasted HIF Fed HIF Fed DIF NuTRIflex pH 7.5 6.1 5.8 5.4 Total protein conc. ±SD (mg/ml) 1±0.1 5 0.1 5±0.2 19 1Total bile salt conc. (mM) 2 0.2 8 0.1 8 0.2 0 Total neutral lipid* conc. (mM) 0.1±0.01 22±1 12±1 12±3 Total phospholipids conc. (mM) 0.2 0.07 3 0.3 2 0.2 3 0.3Surface tension ±SD (mN/m) 28±1 27 1 27±1 29 1Buffer capacity, base (mmol/l/pH unit) 2.8 13.2 14.1 11.8 Buffer capacity, acid (mmol/l/pH unit) 2.4 14.6 14.7 27.4 *Including fatty acids

Dissolution in intestinal fluids The solubility and dissolution rate of the model substances in fasted and fed HIF are shown in Figure 13. Both the solubility and the dissolution rate were significantly higher in the fed state HIF than in the fasted fluid (Figure 14) for all the substances. The increase in solubility varied between a factor of 3.5 and 30 and correlated only with logP and the aqueous solubility of the model compounds (Figures 14, 15). The increase is probably due to the higher concentration of BAs and PLs in the fed state IF. In addition, lipidic components of nutritional origin are only present in the fed state. Thus, the findings of Paper III support the importance of these factors for drug solubilization in the intestinal tract. They also confirm that increased local intestinal drug concentrations can be achieved for low solubility, non-ionic drugs after intake of food.

The increase in dissolution rate was between 2- and 7-fold (Figure 14). This relatively small increase in the dissolution rate compared to that of the saturation solubility confirms the in vivo relevance of the previous findings obtained in artificial media (179,180) and may be explained by slower diffusion of larger micelles and a slow transfer of the drug into emulsion lipid droplets formed in the fed state IFs. The lack of correlation between the solubility and the dissolution rate supports the idea that the Noyes-Whitney equation might have to be modified when more complex fluids are used, so that the difference between the free drug and the solubilized drug diffusion coefficients is taken into account (239). In addition, the relatively small increase in the dissolution rate compared to the solubility in fed intestinal fluids will also have consequences in the prediction of the effect of food on

45

drug bioavailability suggesting that food effect will be most pronounced for drugs with solubility limited absorption in the GI tract.

a)

0

50

100

150

200

250

300

350

400

450

Fasted HIF Fed HIF

Con

cent

ratio

n (µ

g/m

l) Cyclosporine

Danazol

Griseofulvin

Felodipin

Probucol

Ubiquinone

b)

0

2

4

6

8

10

12

Fasted HIF Fed HIF

Dis

solu

tion

rate

(µg/

cm2 /m

in)

Figure 14. (a) Solubilities (µg/ml) and (b) dissolution rates (µg/cm2/min) for the model substances in fasted and fed HIF (mean ± SD).

- 0.6- 0.5

- 0.4- 0.3

- 0.2- 0.1

00.1

0.20.3

0.4

logP

Saq

Tm Hm

Hacc

Hdon

Figure 15. Regression coefficients of the PLS-model showing the effect of the physical chemical descriptors and their interaction on the solubility enhancement brought about by food. The different descriptors were lipophilicity (logP), aqueous solubility (Saq), melting point (Tm), the change in melting entropy ( Hm), and the number of hydrogen acceptors (Hacc) and donors (Hdon). The response coefficients are expressed as scaled and centered regression coefficients.

The solubility in fed HIF was higher for all the substances (2-5 times) com-pared to FeSSIF, whereas the values for fed HIF and DIF corresponded well, as indicated by differences of less than 30% (Figure 16). This is probably due to the presence of dietary lipids in the real IFs, which are absent in FeSSIF. Regarding BAs, which are the other main factor for intestinal solubilization, the levels were higher in simulated compared to real fluids, but the compensatory effect of the BAs was insufficient to make up for the absence of NLs. This further supports the addition of lipids of dietary origin

46

in artificial media, to obtain more realistic in vivo solubility predictions. The dissolution rate in fed HIF was well-predicted by both the DIF and the FeSSIF, as shown by the differences between them and HIF being less than 30% (Figure 16). The reason for this minor difference in dissolution rate between the three media may be due to the solubilization of the drugs in larger colloidal structures in the media containing dietary lipids, which would result in slower diffusion rates in these media.

a)

0

50

100

150

200

250

300

350

400

450

500

Cyclosporine Danazol Griseofulvin Felodipin

Con

cent

ratio

n (µ

g/m

l)

b)

0

2

4

6

8

10

12

14

16

18

Cyclosporine Danazol Griseofulvin Felodipine

Dis

solu

tion

rate

(µg/

cm2 /m

in)

FeSSIF

HIF fed

DIF fed

Figure 16. (a) Solubilities (µg/ml) and (b) dissolution rates (µg/cm2/min) for the model substances in fed state HIF, DIF and FeSSIF (mean ± SD).

Influence of food on absorption on poorly soluble drugs – in vivo perfusion The Peff and fabs of cyclosporine, danazol and verapamil administered in the in vivo porcine perfusion study in Paper IV are displayed in Table 12. The results showed that there was a small increase in Peff following the administration of cyclosporine with verapamil compared to the control, while addition of lipids in the media decreased the Peff. This is an important finding since it does not support the recent hypothesis that these lipids should increase the bioavailability of poorly soluble drugs by inhibition of enterocyte located efflux mechanisms, such as P-gp (154). The present data also shows that the absorption of BCS class II drugs does not necessarily increase when administered with food, as proposed by Fleisher (6).

Both the Peff and fabs were higher for danazol when administered as a nano-suspension in lipid containing media than in the control, but due to the rapid dissolution of the nano-particles, the difference in absorption did not correspond to previous observed increases in bioavailability of danazol when administered together with food. Administration of danazol in the same

47

media, but as a solution, resulted in a decreased Peff and fabs. This further shows the importance of rapid dissolution of the drug nano-particles and might be due to a decrease in the thermodynamic activity when danazol was administered as a solution, since absorption of the drug might create an unsaturated solution. Amidon et al (240) have shown that the absorption can decrease in the presence of micelle forming lipids owing to a decreased thermodynamic activity.

The difference in the affect that administration of food (Treatment 2) had on danazol and cyclosporine in this study might be explained by the higher partitioning of cyclosporine (2.5 times) to the colloidal structures present in the lipid containing media. From the size distribution analysis of the media two different size distributions within the range 2-300 nm could be identi-fied, probably represented mixed micelles (Rh 3-20 nm) and vesicles (Rh 40-60 nm) and/or a liquid crystalline phase (59,241). Based on previous observations (168), it can be concluded that danazol probably partitioned primarily to the mixed micelles, and, to a lesser degree, to the vesicles in the lipid containing media in the present study. Despite a lower log P value, cyclosporine might partition to the vesicles to a greater extent, owing to its larger molecular size. This may provide cyclosporine with a slower diffusion in the intestinal segment. In addition, a slow partitioning from the vesicles and a rapid absorption of cyclosporine, might create a temporarily unsaturated aqueous phase along the perfused jejunal segment, and thereby a decrease in the thermodynamic activity.

Table 12. Mean S.D. of the effective jejunal permeability (Peff) and fraction of the drug absorbed in the segment (fabs) for cyclosporine, danazol and verapamil. *from ref (224)

aControl = cyclosporine and danazol nanoparticles, bTreatment 1 = cyclosporine nanoparticles+verapamil, cTreatment 2 = fed state nanoparticles of danazol and cyclosporine, dTreatment 3 = fed state, saturated solution of danazol and cyclosporine.

An important diversity between the media containing lipids and intake of solid dosage forms together with food, is that there is no digestion of the lipids administered in the perfusion as shown by the particle size determina-

Parameter Controla Treatment 1b Treatment 2c Treatment 3d

Cyclosporine Peff (10-4 cm/s) 0.63 0.05 1.01 0.09 0.11 0.14 0.34 0.08 fabs (%) 29 2 38 3 5 6 13 8 Danazol Peff (10-4 cm/s) 0.8 0.2 - 2.3 0.4 0.33 0.09 fabs (%) 45 9 - 59 3 17 5 Verapamil Peff (10-4 cm/s) 0.64 0.36* fabs (%) 17 7*

48

tion of the media collected before and after the 2 hour jejunal perfusion. Thus, only a minor transition between vesicles and micelles is likely to occur in the media. This might keep the drug in the colloidal structures and pre-venting them from partitioning into the water phase.

49

Conclusions

The general aims of the present project were to increase the understanding of the in vivo dissolution of low soluble drugs and, thereby, improve the possibility of predicting in vivo solubility from substance properties. An increased understanding of the in vivo limitations of drug solubility could also potentially generate ideas for improved formulation principles for poorly soluble compounds and, more relevant in vitro dissolution test methods could be used in formulation development. From the investigations presented in this thesis, it can be concluded that:

The lipid content in human intestinal fluids differs greatly from that in simulated intestinal fluids used in drug development, the major difference being the absence of dietary lipids in the artificial media. In addition, more studies are needed to investigate the importance of the degradation products of the lipids in the diet, since these are the lipids that are most commonly found in fed human intestinal fluids.

The intestinal solubility of aprotic drugs with a low water solubility is higher in the fed compared to the fasted state, as expected from increased levels of bile secretion and lipids and relates to the lipophilicity and aqueous solubility of the drugs. Although this is also true for the dissolution rate, it does not increase to the same extent. These findings need to be considered in the design of in vitro models and in the prediction of food effects on oral bioavail-ability of poorly soluble drugs.

The dog seems to be a good model for man with respect to dissolution in the small intestine after intake of a meal. FeSSIF offers a good means of performing dissolution studies, whereas it is less feasible for prediction of the intestinal saturation solubility in the fed state.

Solubilisation seems to be a more important factor than P-gp inhibition for food-related effects on the intestinal absorption kinetics of Class II drugs.

50

Drugs may be differently distributed between the colloidal phases present in the intestinal lumen after intake of food, thereby, rendering different solubility and dissolution patterns that strongly affect both the rate and extent of intestinal absorption, and the bioavailability of Class II drugs.

51

Acknowledgements

The studies included in this thesis were carried out at the Department of Pharmacy, Faculty of Pharmacy, Uppsala University, Uppsala University Hospital and Department of Preformulation and Biopharmaceutics, Astra-Zeneca R&D, Mölndal.

I wish to express my sincere gratitude to:

Professor Hans Lennernäs och Dr. Bertil Abrahamsson, för bra handledarskap stort engagemang och intressanta diskussioner under dessa lärorika och utvecklande år.

Professor Göran Alderborn för tillhandahållande av ändamålsenliga lokaler och god atmosfär under mina vistelser i Uppsala.

Eva Nises Ahlgren, Ulla Wästberg-Galik, Eva Lide och Harriet Pettersson för hjälp med allehanda praktikaliteter.

Docent Lars Knutson och Patrik Forsell, medförfattare, för gott samarbete och skickligt handlag med både grisar och människor.

Alla nya och gamla medlemmar i Absorptionsgruppen, för att ni tagit hand om mig under mina vistelser i Uppsala, samt för trevligt sällskap på konferenser. Speciellt tack till EbbaBergman och Elin Sjödin, för att ni sett till att jag fylla mitt schema i Uppsala med luncher, middagar och annat roligt.

Alla andra vid institutionen för farmaci, det är alltid lika roligt att vara i Uppsala.

Anders Carlsson, medförfattare och analytiker, med intresse för colon-material, för all hjälp med analyser av diverse möjliga och omöjliga substanser samt för alla uppmuntrande ord på vägen. Kommer aldrig glömma din insats på step-upen.

Dr. Ralf Nilsson, Dr. Lars Löfgren, och Dr Göran Hansson, medförfattare, för ert stora engagemang och för att ingenting är omöjligt. Diskussioner med er blir alltid intressanta. Samt övriga i lipidgruppen på AstraZeneca, för att ni välkomnat mig på erat labb och för att ni alltid visar lika stort intresse för mitt arbete och villlighet att hjälpa till.

Dr. Henrik Zacchari, för all analytisk hjälp av mycket svårlösliga substanser.

Dr. Erik Söderlind, vän och kollega, för att jag fått låna ditt öra under alla dessa år och för allt ditt stöd du givit i tunga stunder. Skulle kanske blivit klar fortare utan våra pratstunder, men det hade inte varit lika roligt.

52

Dr. Christer Tannergren, vän och kollega, för uppmuntrande ord och ett ständigt gott humör som smittar av sig. Tack också för hjälp med genomläsning av avhandlingen. Du kommer bli saknad.

Gunilla Hanisch, medförfattare, för alla givande diskussioner och uppmuntrande ord på vägen.

Fd biofarmaci-gruppen på AstraZeneca, för ert stora engagemang och alla uppmuntrande ord, samt för trevligt sällskap och att ni hittat på så roliga saker på på och utanför jobbet. Pernilla Åkesson, Britta Polentarutti för hjälp med samlande av hundtarmsaft och för att ni håller mig ajour med skvallret inom Astra. Marie Sjöberg, för din expertis inom upplös-ning. Lena Denstedt, för hjälp med allt administrativt arbete och för alla revliga pratstunder. Angela Ku, för hjälp med PLS analyser och underhållanande sällskap på labb.

Hela före detta Avd. för Biofarmaci och Preformulering för att ni är en så himla trevlig grupp av människor som alla på sitt sätt bidragit till att göra min tid på Astra, så rolig som möjligt.

Maria Lindqvist och Matti Ahlqvist för hjälp med fastfas-bestämningar.

Dr. Lennart Lindfors för intressanta diskussioner om studieupplägg och resultat.

Dr. Jan-Erik Löfroth, för värdefulla diskussioner om partikelstorleksmätning.

Dr. Lennart Svensson, för tyckande och tänkande om enzymkit.

Eva Emanuelsson, för din hjälpsamhet med logD bestämningar.

Suzanne Lidström for the splendid linguistic revision of the papers and the thesis.

Ann-Sofie Gustafsson, min examensarbetare, för ditt bidrag till resultaten i den här boken. Jag kunde inte fått en bättre ex-jobbare.

Sara och Björn Magnusson, samt era underbara barn, Oscar och Maja, för alla trevliga stunder och för alla roliga upptåg ni tagit med mig och Johan på, samt för att ni alltid ställer upp med vad som än behövs, från ett lyssnande öra till en gammal volvo.

Alla vänner som sett till att jag hållt tankarna borta från jobbet, när jag inte varit där.

Kurt och Lena, för att ni tagit hand om mig och låtit mig få bo hos er alla gånger jag varit i Uppsala. Tror aldrig att jag skulle hunna få samma service på ett hotell.

Min familj (Mamma, Pappa, Magnus, Frida & Brasse), för att ni alltid ställer upp och för att ni tror på mig och finns i närheten när det behövs. Ni är bäst.

Johan, för all din kärlek och stöd. Du är fantastisk.

53

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