International Journal of Pharmaceutics 368 (2009) 116–122
`
`Contents lists available at ScienceDirect
`
`International Journal of Pharmaceutics
`
`j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / i j p h a r m
`
`Estimation of effective intestinal membrane permeability
`considering bile micelle solubilisation
`Kiyohiko Sugano∗
`
`Global Research & Development, Sandwich Laboratories, Research Formulation, Pfizer Inc., CT13 9NJ, Sandwich, Kent, UK
`
`a r t i c l e
`
`i n f o
`
`a b s t r a c t
`
`Article history:
`Received 8 August 2008
`Received in revised form 1 October 2008
`Accepted 3 October 2008
`Available online 15 October 2008
`
`Keywords:
`Oral absorption
`Low solubility
`Bile micelles
`Unstirred water layer
`Simulation
`
`1. Introduction
`
`In this study, the calculation method of effective intestinal membrane permeability (Peff) for bile micelle
`solubilised drugs was investigated. The intestinal membrane permeation is the tandem process of
`unstirred water layer (UWL, superimposes to the mucus layer) and epithelial cell membrane perme-
`ation. In most cases of lipophilic compounds, UWL permeation is the rate-limiting step. Four scenarios of
`UWL permeation were investigated: (A) only free drug permeates the UWL by self-diffusion, (B) both free
`drug and micelle bound drug permeate through the UWL by self-diffusion, (C) water convection carries
`the drug in addition to (B), and (D) both free drug and bile micelle bound drug permeate through the UWL
`by self-diffusion with the diffusion coefficient of the free monomer. Using danazol as a model drug, the
`simulation results of the four scenarios were compared with the observed fraction of a dose absorbed
`(Fa%) in fasted and fed state humans (fasted: 11–25%, fed: 44–72%). Scenario (A) largely underestimated
`the fraction of a dose absorbed (2% and 2% for fasted and fed, respectively). Scenarios (B) and (C) predicted
`the Fa% appropriately (B: 8% and 43%, C: 17% and 60%). Scenario (D) overestimated the Fa% (62% and 99%).
`The relationship between octanol–water partition coefficient and Peff was also investigated.
`© 2008 Elsevier B.V. All rights reserved.
`
`Computational oral absorption simulation is expected to be an
`effective tool in drug discovery and development (Jones et al., 2006;
`Kuentz et al., 2006; Parrott and Lave, 2002; Takano et al., 2006;
`Sugano et al., 2007). In the current drug discovery paradigm, the
`number of low solubility compounds is increasing (Lipinski, 2000;
`Sugano et al., 2007). Therefore, oral absorption simulation for low
`solubility compound is currently extensively investigated. In the
`case of low solubility compounds, bile micelle solubilisation in the
`small intestine plays an important role for oral absorption.
`However, there are discrepancies between the theories and
`the experimental observations for bile micelle solubilised drugs:
`(1) in several reports of oral absorption simulation for low sol-
`ubility drugs, the total concentration in bile micelle media (free
`drug + micelle bound drug) and membrane permeability of free
`monomer (e.g., obtained from Caco-2 cell assay) were simul-
`taneously used for simulation, resulting in appropriate in vivo
`prediction (corresponding to scenario D described below) (Jones
`et al., 2006; Takano et al., 2006). However, in theory, when the per-
`meability value of free monomer is used, the concentration of free
`
`∗ Tel.: +44 1304 644338.
`E-mail address: Kiyohiko.Sugano@pfizer.com.
`
`0378-5173/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
`doi:10.1016/j.ijpharm.2008.10.001
`
`molecule (free fraction) should be used instead of the solubility in
`bile micelle media. (2) It is well known that many low solubility
`compounds undergo the positive food effect, due to the increase
`of solubility by bile micelles (Gu et al., 2007). However, if only free
`monomer molecules are available for permeation, there should be
`no positive food effect, since solubilisation by bile micelles does not
`increase the free monomer concentration. Therefore, it is of great
`interest to investigate the reason for these discrepancies.
`The intestinal membrane permeation is the tandem process
`of unstirred water layer (UWL) and epithelial cellular membrane
`permeation (Fig. 1). The unstirred water layer superimposes to
`the mucus layer. In most cases of lipophilic compounds (the
`octanol–water distribution coefficient (log Doct) > 2–3), cellular
`membrane permeation is very rapid. Therefore, UWL permeation
`would be the rate-limiting step for lipophilic compounds (Avdeef
`et al., 2007; Fagerholm and Lennernaes, 1995; Lennernaes, 2007a;
`Obata et al., 2005; Sugano et al., 2003; Youdim et al., 2003).
`Previously, it was suggested that bile micelles can permeate
`through the UWL by self-diffusion, and therefore, bile micelles work
`as the carrier of a compound (Amidon et al., 1982; Li et al., 1996).
`Micelles from self-emulsifying drug delivery system (SEDDS) for-
`mulation were also suggested to permeate through the UWL (Araya
`et al., 2006; Porter et al., 2007). In addition, it is well known that
`nano-scale particles (ca <500 nm) can permeate through the mucus
`layer (Norris and Sinko, 1997; Sanders et al., 2000). The effect of
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`117
`
`The human small intestine has a fold and villi structure. The UWL
`is on the top of the villi structure. Therefore, Peff can be described
`as
`
`1 F
`
`E
`
`(cid:3)
`
`=
`
`1
`Peff
`
`1
`Pep,eff
`
`+
`
`1
`PUWL,eff
`
`(cid:2)
`(cid:2)
`
`=
`
`1
`Acc VE fmonoPep
`
`+
`
`(2)
`
`(3)
`
`(4)
`
`1 F
`
`E
`
`(cid:3)
`
`1
`PUWL,eff
`where Pep,eff is the effective epithelial cellular membrane per-
`meability (=Acc× VE× fmono × Pep), Pep is the epithelial cellular
`membrane permeability of free monomer, fmono is the fraction of
`free monomer, PUWL,eff is the effective UWL permeability, Acc is
`the accessibility to the villi surface, VE is the surface expansion
`by villi structure (VE = 10 in humans) (DeSesso and Jacobson, 2001;
`Oliver et al., 1998), and FE is the surface expansion by fold structure
`(FE = 3 in humans) (DeSesso and Jacobson, 2001). Acc depends on
`Pep, fmono, the effective diffusion coefficient in the UWL (DUWL,eff)
`and the permeability by water convection (PWC). Acc was calculated
`according to the reference (Oliver et al., 1998). In this equation, it
`was assumed that only free monomer molecules can permeate the
`epithelial membrane.
`PUWL,eff of the above four scenarios A–D can be represented as
`Eqs. (3)–(6), respectively:
`PUWL,eff = fmono DUWL,mono
`heff
`PUWL,eff = DUWL,eff
`= 1
`heff
`heff
`PUWL,eff = DUWL,eff
`heff
`
`Fig. 1. Schematic presentation of intestinal membrane permeation. Both free
`monomer molecule and micelle bound molecule can permeate the unstirred water
`layer. The equilibrium between the monomer and micelle bound molecule are rapid
`and free monomer fraction permeate the epithelial membrane.
`
`water convection on UWL permeability has been suggested in addi-
`tion to self-diffusion (Nilsson et al., 1994; Pappenheimer, 2001).
`However, UWL permeation of a bile micelle bound drug has not
`been considered in oral absorption simulation.
`In this study, four scenarios of UWL permeation were considered
`(Fig. 1).
`
`(A) Only free monomer molecule (free fraction) permeates the
`UWL by self-diffusion.
`(B) Both free monomer molecules and bile micelle bound
`molecules permeate across the UWL by self-diffusion.
`(C) (B) plus convection accompanying water absorption (water
`convection).
`(D) Both free drug and bile micelle bound drug permeate through
`the UWL by self-diffusion with the diffusion coefficient of the
`free monomer.
`
`The predicted values of the fraction of a dose absorbed (Fa%) by
`the four scenarios were compared with the clinical oral absorption
`data for danazol, a typical low solubility compound.
`
`2. Theory
`
`2.1. Peff equation
`
`In the case of a low solubility compound, solubility in the intesti-
`nal fluid is often increased by bile micelles. In this study, the
`effective concentration for permeation in the intestinal fluid (Ceff) is
`defined as the sum of the concentrations of free molecules and the
`bile micelle bound molecules (Sugano et al., 2007). The absorption
`rate (dXabs/dt) can be described as
`dXabs
`= SAsi Peff Ceff = SAsi
`Peff Ceff Vsi = ka Xeff
`Vsi
`dt
`where SAsi is the effective surface area of the small intestine, Peff is
`the effective intestinal membrane permeability, Vsi is the effective
`volume of the intestinal fluid, ka is the absorption rate constant
`(=SAsi/Vsi × Peff), and Xeff is the drug amount which is available for
`permeation (Ceff × Vsi).
`
`(1)
`
`(fmono DUWL,mono + fmic DUWL,mic)
`
`(fmono DUWL,mono+fmic DUWL,mic)
`+ PWC,eff = 1
`heff
`+ (fmono PWC,mono + fmic PWC,mic) (5)
`
`PUWL,eff = DUWL,mono
`heff
`where heff is the effective thickness of the UWL, fmic is the frac-
`tion of micelle bound molecule (1− fmono), DUWL,mono and DUWL,mic
`are the diffusion coefficients of free monomer and micelle bound
`molecules in the UWL, respectively, and PWC,mono and PWC,mic are
`the permeability of free monomer and micelle bound molecules by
`water convection, respectively.
`
`(6)
`
`2.1.1. Calculation of intrinsic epithelial membrane permeability
`Pep for undissociated molecular species was estimated from
`the relationship between to the octanol–water partition coefficient
`(Poct) and the Caco-2 intrinsic membrane permeability of undisso-
`ciated molecular species (Pcaco,int) as
`Pep = Pcaco,int = APoct
`B
`(7)
`where A and B are 2.36× 10−6 and 1.10 respectively (Fig. 2) (Avdeef
`et al., 2005). Pep of danazol (log Poct = 4.2) (Glomme et al., 2006) was
`calculated to be 0.098 cm/s.
`
`2.1.2. Calculation of free monomer fraction
`The fmono can be calculated as
`fmono = Sbuffer
`Smicelles
`where Smicelles is the solubility in biorelevant media with bile
`micelles and Sbuffer is the solubility in the buffer without bile
`micelles. The measured solubility values in the fasted state and
`fed simulated intestinal fluid (FaSSIF and FeSSIF, respectively) were
`used for simulation (Galia et al., 1998; Vertzoni et al., 2004). FaSSIF
`
`(8)
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`K. Sugano / International Journal of Pharmaceutics 368 (2009) 116–122
`
`Eq. (11) does not consider the fold structure. If the fold struc-
`ture is considered, the mucus thickness on the villi is ca. 300 ␮m.
`This value is in good agreement with the thickness of the mucus
`layer obtained by direct measurements (170–480 ␮m in the small
`intestine) (Atuma et al., 2001). In this study, 300 ␮m was used as
`heff.
`
`2.1.5. Estimation of UWL permeability by water convection
`Lennernas et al. reported that the water absorption rate was
`1.3± 3.0, 2.6± 2.2, and 3.7± 3.5 mL/h/cm for each experiment
`(Fagerholm et al., 1999; Lennernaes et al., 1994; Nilsson et al.,
`1994). Lambert et al. (1997) reported the water absorption was
`3.3 mL/h/cm) in the jejunum and much higher in the duode-
`num. On average, the water absorption is 2.7 mL/h/cm/GI length
`in humans, which corresponds to 0.69× 10−4 cm/s (based on the
`intestinal tube radius (rGI) of 1.75 cm, 1 cm GI length = 11.0 cm2).
`This value is based on the smooth tube surface area same as Peff
`calculation. Considering the 3-fold expansion by the fold structure,
`PWC,eff = 0.23× 10−4 cm/s was used for UWL permeability calcu-
`lation in this study. This value was used for the permeability
`by water convection for both monomer and micelle bound drug
`(PWC,eff = PWC,mono = PWC,mic).
`
`3. Oral absorption simulation
`
`Fig. 2. Log Poct vs Caco-2 intrinsic permeability. Data obtained from Avdeef et al.
`(2005) Pcaco,int is the permeability of undissociated species in Caco-2 assay Pcaco,int =
`−6 × P1.1
`2.36 × 10
`oct .
`
`and FeSSIF consist of taurocholic acid (TC) and egg lecithin (EL):
`TC/EL = 3 mM/0.75 mM for FaSSIF and TC/EL = 15 mM/3.75 mM for
`FeSSIF. The solubility values of danazol in buffer, FaSSIF and FeSSIF
`are 0.00021, 0.018 and 0.047 mg/mL, respectively (Okazaki et al.,
`2008). The fmono was calculated to be 0.012 and 0.0045 in FaSSIF
`and FeSSIF, respectively. Previously, Glomme et al. (2006) proposed
`an equation to estimate Smicelles of TC/EL micelles from log Poct for
`undissociable compounds
`1 + Cbile
`Smicelles = Sint
`10log Kbm
`Cwater
`log Kbm = 0.75 log Poct + 2.27
`where Sint is the intrinsic solubility, Kbm is the bile micelle–water
`partition coefficient, Cbile is the concentration of taurocholic acid,
`and Cwater is the concentration of water (55.55 M).
`
`(cid:4)
`
`(cid:5)
`
`(9)
`
`(10)
`
`2.1.3. Diffusion coefficients of free monomer and bile micelles in
`UWL
`Dmono of danazol was set to be 8× 10−6 cm2/s (Okazaki et al.,
`2008). The diffusion coefficients of bile micelles in FaSSIF and FeSSIF
`were 0.12× 10−6 cm2/s and 1.05× 10−6 cm2/s, respectively (the bile
`micelle diameters were 54.4 nm and 6.3 nm, respectively) (Okazaki
`et al., 2008). It was reported that the mucus layer only slightly
`affected the diffusion coefficient of bile micelles at TC > 10–20 mM
`range, whereas mucus layer increases the diffusion coefficient more
`than 3-fold at TC < 10–20 mM range (Li et al., 1996). Therefore, for
`fasted state, 0.36× 10−6 cm2/s was used, whereas the same value
`(1.05× 10−6 cm2/s) as in the buffer was used for fed state (Li et al.,
`1996).
`
`2.1.4. Thickness of the unstirred water layer
`The apparent thickness of the UWL (happ) has been calculated
`from Peff and DUWL,eff for UWL limited permeation compounds
`using Eq. (2) assuming Pep,eff (cid:3) PUWL,eff and FE = 1, as
`happ = DUWL,eff
`Peff
`Lennernas et al. reported happ to be ca. 100 ␮m in humans
`(Fagerholm and Lennernaes, 1995; Lennernaes, 2007a). However,
`
`(11)
`
`(cid:2)
`(cid:2)
`
`=
`
`1 −
`
`(cid:4)
`(cid:4)
`
`Peff
`
`(12)
`
`1 + SVR PeffTsi
`7
`
`100
`
`(13)
`
`A compartment transit absorption model consisting of nine
`compartments (1 for the stomach, 7 for the small intestine, and
`1 for the colon) was used for oral absorption simulation (Haruta et
`al., 1998; Jinno et al., 2006; Yu and Amidon, 1999). Dissolution and
`absorption in the stomach and colon were neglected in the same
`way as in previous reports (Jones et al., 2006; Kuentz et al., 2006;
`Parrott and Lave, 2002; Takano et al., 2006). Dissolution in the small
`intestine was simulated by the Nernst–Brunner equation (NBE) as
`previously reported (Okazaki et al., 2008). Log-normal distribution
`of particle size was assumed with d50 = 4.46 ␮m (Sunesen et al.,
`2005b) and standard deviation of 0.693 log unit. Spherical particle
`shape was assumed. The dissolution of danazol was reported to be
`simulated appropriately by this method (Okazaki et al., 2008).
`Transfer of particles was simulated by assigning 100 virtual par-
`ticles to each particle size bin. In this way, the transfer of particles
`and the reduction of particle radius by dissolution could be simul-
`taneously taken into account. First-order kinetics with T1/2 = 10 min
`and T1/2 = 30 min were used for stomach emptying in fasted state
`and fed state, respectively (Sugito et al., 1990). The total intestinal
`transit time was set to be 3.5 h (T1/2 = 21 min for each small intestine
`compartment) (Yu, 1999).
`Peff was converted to absorption rate constant (ka) as
`ka = SAsi
`Peff = SVR Peff = 2DF
`Vsi
`rGI
`where SVR is the surface/volume ratio (=SAsi/Vsi), and DF is the
`degree of flatness of the intestinal tube (DF = 1 for a cylindrical tube)
`(Chiou, 1994). SVR can be obtained from the relationship between
`Fa% and Peff using low permeability-high solubility drugs, assuming
`that colonic absorption is negligible (Fagerholm et al., 1997; Sutton
`et al., 2006) and the mean transit time through the small intestine
`(Tsi) is 3.5 h. Fa% can be expressed as Eq. (13) by assuming the seven-
`compartment model (Yu et al., 1996)
`1 + kaTsi
`Fa% =
`1 −
`7
`
`(cid:5)−7
`
`(cid:3)
`(cid:5)−7
`
`100
`
`(cid:3)
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`K. Sugano / International Journal of Pharmaceutics 368 (2009) 116–122
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`119
`
`Eq. (13) was fitted to the Fa–Peff relationship previously reported
`(Lennernaes, 2007a). SVR for human was obtained to be 2.2 and this
`value was used in this study.
`For the intestinal fluid volume (Vsi), 600 mL (8.6 mL/kg) has
`been reported for oral absorption simulation simulation (Takano
`et al., 2006; Yu, 1999). On the other hand, Schiller et al. (2005)
`reported that the total volume of water pocket measured by water-
`sensitive magnetic resonance imaging is 105 mL in the human small
`intestine. However, this value might not be the real total volume
`available along the intestine, since only the free volume in the
`water pocket was counted (Lennernaes, 2007b). Biopharmaceutical
`classification system employs 250 mL (Yu et al., 2002). The same
`volume was also used to simulate the dose-dependent absorp-
`tion of Gabapentin by active transport (Madan et al., 2005). Other
`reports also suggest smaller fluid volume than 600 mL (Macheras
`et al., 1990; Masaoka et al., 2006). In this study, the intestinal fluid
`volume was assumed to be 250 mL (36 mL in each compartment).
`Numeric integration was performed by 4th Runge–Kutta
`method with integration interval of 0.5 min. The simulation pro-
`gramme was developed in-house utilizing Microsoft Excel Visual
`Basic Application (Microsoft Corporation, Redmont, WA).
`
`4. Results and discussion
`
`4.1. Fa% prediction for danazol
`
`in humans were
`The observed Fa% values of danazol
`obtained as follows (Table 1). Sunesen et al. (2005b) reported
`that the absolute bioavailability (BA%abs) of a danazol capsule
`(d50 = 4.46 ␮m) was 11% in fasted state, and the relative bioavailabil-
`ity against fed state (BA%rel = AUC(fasted)/AUC(fed)) was 25%. Since
`BA%abs < Fa% < BA%rel, it is appropriate to estimate Fa% = 11–25%
`(The BA%rel was calculated from the AUCs of oral administration.
`Therefore, the first pass effect was cancelled out. However, oral
`absorption in the fed state could be incomplete and the AUC for
`complete absorption could be larger. Therefore, Fa% is smaller than
`BA%rel.). In another report from Charman et al. (1993) BA%rel of
`a capsule dosed in the fasted state against the emulsion forma-
`tion in the fed state was 23%. If i.v. data from Sunesen et al. was
`used, BA%abs was 19%. Therefore, it would be appropriate to esti-
`mate Fa%solid = 19–23%. Overall, Fa% of a danazol capsule in fasted
`state humans would be 11–25%. Fa% in the fed stated was estimated
`in the same way to be 44–72%. The Fa% of emulsion formation in
`the fed state was assumed to be 100%, because of the non-existence
`of dose independency in this formulation.
`
`Table 1
`PK data of danazol in humansa.
`
`Formulation
`
`Dose (mg)
`
`Fasted/fed
`
`Solida
`Solida
`i.v.a
`
`Solidd
`Solidd
`Emulsiond
`Emulsiond,f
`
`Emulsiond
`Emulsiond,f
`Emulsiond
`
`100
`100
`50
`
`100
`100
`100
`100
`
`50
`100
`200
`
`Fasted
`Fed
`
`Fasted
`Fed
`Fasted
`Fed
`
`Fed
`Fed
`Fed
`
`AUC (ng/(h−1 mL−1))
`120 ± 60
`469 ± 164
`531 ± 104
`204 ± 125
`639 ± 259
`779 ± 189
`844 ± 194
`296 ± 78
`695 ± 171
`1415 ± 303
`
`BA%rel
`BA%abs
`11 14b, 25c
`44
`–
`
`19e
`60e
`73e
`83e
`
`56e
`65e
`67e
`
`23b
`72b
`88b
`–
`
`–
`–
`–
`
`a Data from Sunesen et al. (2005b).
`b Relative BA% against AUC of the emulsion formulation in the fed data.
`c Relative BA% against AUC of the fed data of the same study.
`d Data from Charman et al. (1993).
`e Calculated based on the i.v. data from Sunesen et al. (2005b).
`f Different cohort.
`
`Fig. 3. Predicted concentration–time profile in the small intestine for danazol at
`100 mg dose in fasted state humans. The numbers in the figure correspond to each
`small intestinal compartment (from left to right, compartment 1–7, proximal to
`distal, respectively).
`
`The Pep value of danazol, 0.098 cm/s, was obtained from the rela-
`tionship between Caco-2 intrinsic permeability and log Poct (Fig. 2).
`In an in vitro system using plate wells, the apparent permeabil-
`ity is usually less than 50× 10−6 cm/s since it is interfered with
`by the thick UWL. In an in vitro system, this UWL can be ca.
`2000–4000 ␮m depending on the agitation conditions (Avdeef et
`al., 2004; Fujikawa et al., 2007; Youdim et al., 2003). In the case of
`dissociable compounds, the intrinsic permeability can be obtained
`from the pH-permeability profile and pKa data (Avdeef et al., 2005;
`Sugano, 2007). However, in the case of undissociable compounds
`such as danazol, this method can no be applied. Therefore, Pep value
`for danazol was estimated by Eq. (7) using log Poct. Even though this
`was an estimated value, the estimation error had little effect on the
`prediction results, since the predicted Pep,eff of danazol is much
`higher than UWL permeability which is less than 10× 10−4 cm/s.
`The insensitivity of Fa% (less than 3% difference) at the Pep value of
`>0.01 cm/s was confirmed (data not shown).
`Log-normal distribution of particle size was assumed since only
`the d50 data was available. However, in the case of solubility limited
`absorption, such as in the case of danazol capsules, the estimation
`error of the dissolution rate has little effect on the Fa% prediction
`(Sugano et al., 2007; Takano et al., 2006; Yu, 1999).
`The SVR value obtained from the Peff–Fa% relationship was 2.2.
`This value is higher than the theoretical value for a cylindrical tube
`(2/rGI = 1.3, rGI = 1.5 cm in humans) (Yu, 1999), suggesting that the
`intestinal tube is rather flat (DF = 1.7) (Chiou, 1994).
`In all simulation conditions, the calculated concentration in the
`small intestine reached the saturated solubility (Fig. 3). Although
`the mean Tsi was set to be 3.5 h, the predicted concentration in the
`6th and 7th compartments remained to be the saturated solubility
`until ca. 5 h, because a portion of the undissolved particles remain in
`the small intestine (Sugito et al., 1990) and maintain the saturated
`solubility.
`Predicted Peff and Fa% in scenarios A–D are shown in Table 2.
`Scenario A largely underestimated Fa% values. On the other hand,
`scenarios B and C resulted in good prediction. Scenario D largely
`overestimated Fa%.
`The Fa%–time profiles predicted by scenarios B and C were also in
`good agreement with the previous results of Sunesen et al. (2005a)
`(Fig. 4).
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`K. Sugano / International Journal of Pharmaceutics 368 (2009) 116–122
`
`Fasted/fed
`
`Scenario
`
`Table 2
`Simulation results for Fa% of danazol in humans (dose = 100 mg).
`Predicted Peff (×10−4 cm/s)
`0.07
`0.44
`1.1
`8.0
`
`Fasted
`
`A (Eq. (3))
`B (Eq. (4))
`C (Eq. (5))
`D (Eq. (6))
`
`Fed
`
`A (Eq. (3))
`B (Eq. (4))
`C (Eq. (5))
`D (Eq. (6))
`
`0.04
`1.0
`1.6
`8.0
`
`Predicted Fa%
`
`2
`8
`17
`62
`
`2
`43
`60
`99
`
`more appropriate compared to scenarios A and D based on the large
`prediction error (Table 2). This was in good agreement with the
`previously proposed mechanism (Amidon et al., 1982; Araya et al.,
`2006; Li et al., 1996; Porter et al., 2007). However, more accurate
`estimation for the intestinal fluid volume, the UWL thickness, the
`diffusion coefficient of bile micelles in the mucus layer, the flatness
`of the intestinal tube, and the solubility in the intestinal fluid would
`be required to draw a conclusion about scenarios B and C from a
`simulation predictability aspect. In this study, danazol was used as
`a model drug, since all clinical pharmacokinetic data (solid dosage,
`p.o. solution and i.v.), particle size, solubility and physicochemi-
`cal properties were available in the literature. The same conclusion
`was obtained for felodipine and danazol in dogs (manuscript under
`preparation).
`Takano et al. previously employed scenario D for fasted state, and
`over estimation was also found for most low solubility compounds
`(e.g., for danazol, predicted Fa = 40%). However, the extent of over-
`estimation in their study is less than that in this study (Predicted
`Fa = 62%). In their simulation, they assumed a single compartment
`for the small intestine and at 4 h all particles exited the small intes-
`tine at once, where as in this study it was suggested that a portion
`of particles remained in the small intestine for longer time than 4 h
`(Fig. 3).
`
`4.2. Log Poct − Peff profile
`Since both Pep and Kbm can be predicted by log Poct, log Poct − Peff
`profile can be drawn (Fig. 5). This figure is not precisely quantitative
`due to the rough estimation of Pep and Kbm by log Poct. For exam-
`ple, fmono of danazol calculated by Eqs. (9) and (10) were 0.066
`and 0.014 for FaSSIF and FeSSIF, respectively (experimental values
`were 0.012 and 0.0045). However, it would be beneficial to under-
`stand a general trend with a logarithmic scale. In log Poct < 2 range,
`as log Poct increased, Peff also increased. In this log Poct region, the
`epithelial membrane permeation would be the rate-limiting step.
`If bile micelle binding is not significant (corresponds to scenario
`D), Peff increases up to ca. 8× 10−4 cm/s at which the UWL limits
`permeability. However, if the bile micelle binding is significant as
`Eq. (10) predicts, as lipophilicity increased to over 2, Peff decreases
`due to the increase of micelle binding (decrease of fmono). Micelle
`binding decreases both PUWL,eff and Pep,eff by reducing the effective
`diffusion coefficient and the free monomer fraction, respectively.
`Peff value achieved a constant value of bile micelle permeation at
`log Poct > 4, because the power of the micelle partition equation (Eq.
`
`Fig. 4. Fa%–time profile predicted by scenarios B and C for danazol at 100 mg dose
`in fasted and fed state humans.
`
`Recently, Persson et al. (2008) measured the Peff of danazol
`dissolved in bile media using an intestinal perfusion model in
`pigs. Iso-osmotic solution was used to neglect water absorption.
`Therefore, the experimental condition corresponded to scenario B.
`The predicted Peff value by scenario B (=0.44× 10−4 cm/s) in this
`study was in good agreement with the experimental result of ca.
`0.4× 10−4 cm/s in Persson’s study.
`Even considering some uncertainty in input of physiological and
`compound parameters, scenarios B and C were suggested to be
`
`Fig. 5. log Poct vs Peff predicted by scenarios A–D for humans. (A) Fasted state, (B) fed state. TC = 3 mM and TC = 15 mM were used for the fasted state and fed state, respectively.
`Pep and Kbm were calculated from log Poct by Eqs. (7) and (10), respectively. Peff was calculated from Pep and Kbm by Eqs. (2)–(6). DUWL,mono was set to 8× 10−6 cm2/s.
`
`Nalox1235
`Nalox-1 Pharmaceuticals, LLC
`Page 5 of 7
`
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`

`K. Sugano / International Journal of Pharmaceutics 368 (2009) 116–122
`
`121
`
`(10), 0.75) is lower than that of the epithelial membrane perme-
`ation equation (Eq. (7), 1.10). In log Poct < 4 range, predicted Peff in
`the fed state is lower than that in the fasted state, due to smaller
`fmono in the fed state. On the other hand, in log Poct > 4 range, pre-
`dicted Peff in the fed state is higher than that in the fasted state, due
`to the higher diffusion coefficient of bile micelles in the fed state
`(Okazaki et al., 2008; Sugano et al., 2007).
`
`5. Conclusion
`
`In this study, a mathematical equation for Peff was introduced to
`simulate oral absorption from bile micelles. Estimation of UWL per-
`meability is the key for appropriate simulation. A similar approach
`can be applied to the UWL permeation of nano-scale formula-
`tions, such as SEDDS (Porter et al., 2007) and nano-milled particles
`(Jinno et al., 2006; Liversidge and Cundy, 1995). Application of the
`methodology for nano-milled particles is under investigation.
`
`Acknowledgements
`
`K.S. greatly appreciates Dr. Chris Craig of Bend research Inc. for
`his suggestions. K.S. also would like to thank Mike Cram for critically
`reviewing the manuscript.
`
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