Intestinal Permeability Study of Minoxidil: Assessment of Minoxidil as a

Article pubs.acs.org/molecularpharmaceutics

Intestinal Permeability Study of Minoxidil: Assessment of Minoxidil as a High Permeability Reference Drug for Biopharmaceutics Classification Makoto Ozawa,†,‡ Yasuhiro Tsume,† Moran Zur,§ Arik Dahan,§ and Gordon L. Amidon*,† †

College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109-1065, United States Pharmacokinetics & Safety Laboratory, Discovery Research, Pharmaceutical Research Center, Mochida Pharmaceutical Company Limited, 722 Uenohara, Jimba, Gotemba, Shizuoka 412-8524, Japan § Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel ‡

ABSTRACT: The purpose of this study was to evaluate minoxidil as a high permeability reference drug for Biopharmaceutics Classification System (BCS). The permeability of minoxidil was determined in in situ intestinal perfusion studies in rodents and permeability studies across Caco-2 cell monolayers. The permeability of minoxidil was compared with that of metoprolol, an FDA reference drug for BCS classification. In rat perfusion studies, the permeability of minoxidil was somewhat higher than that of metoprolol in the jejunum, while minoxidil showed lower permeability than metoprolol in the ileum. The permeability of minoxidil was independent of intestinal segment, while the permeability of metoprolol was region-dependent. Similarly, in mouse perfusion study, the jejunal permeability of minoxidil was 2.5-fold higher than that of metoprolol. Minoxidil and metoprolol showed similar permeability in Caco-2 study at apical pH of 6.5 and basolateral pH of 7.4. The permeability of minoxidil was independent of pH, while metoprolol showed pH-dependent transport in Caco-2 study. Minoxidil exhibited similar permeability in the absorptive direction (AP-BL) in comparison with secretory direction (BL-AP), while metoprolol had higher efflux ratio (ER > 2) at apical pH of 6.5 and basolateral pH of 7.4. No concentration-dependent transport was observed for either minoxidil or metoprolol transport in Caco-2 study. Verapamil did not alter the transport of either compounds across Caco-2 cell monolayers. The permeability of minoxidil was independent of both pH and intestinal segment in intestinal perfusion studies and Caco-2 studies. Caco-2 studies also showed no involvement of carrier mediated transport in the absorption process of minoxidil. These results suggest that minoxidil may be an acceptable reference drug for BCS high permeability classification. However, minoxidil exhibited higher jejunal permeability than metoprolol and thus to use minoxidil as a reference drug would raise the permeability criteria for BCS high permeability classification. KEYWORDS: minoxidil, metoprolol, intestinal permeability, Biopharmaceutics Classification System (BCS)

■

INTRODUCTION Since Amidon et al. introduced the Biopharmaceutics Classification System (BCS) in 1995,1 considerable attention has been given to this system for drug development. BCS is a scientific framework and classifies drugs into four groups based on two key parameters; permeability and solubility. BCS classes consist of the following groups: Class I (high solubility−high permeability), Class II (low solubility−high permeability), Class III (high solubility−low permeability), and Class IV (low solubility−low permeability).1 This system not only gives us scientific insights for the developability of a chemical entity, it also has had significant impact on the regulatory processes required to ensure the quality of pharmaceutical products. The United States Food and Drug Administration (FDA) and the European Medicine Agency (EMA), as well as many other drug regulatory agencies worldwide, have adopted the BCS © XXXX American Chemical Society

classification allowing waivers of clinical bioequivalence studies.2,3 The FDA utilizes the BCS as a scientific approach to permit a biowaiver of clinical study for immediate release (IR) solid oral dosage forms of Class I drugs.2 The EMA guideline for BE study issued in 2010 extends their discussion of a biowaiver to Class III drugs that exhibit very rapid dissolution (>85% within 15 min).3 Thus, the BCS-based biowaiver has been recognized as a time and cost saving tool for the pharmaceutical industries throughout the process of drug discovery and development.4−6 Received: August 11, 2014 Revised: November 20, 2014 Accepted: November 25, 2014

A

dx.doi.org/10.1021/mp500553b | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

Article

When more than 90%2 (or 85% following to EMA3) of an orally administered drug is absorbed, the drug is considered highly permeable. To ensure the extent of drug absorption (fraction absorbed) in human, the guidelines indicated mass balance studies or systemic bioavailability studies. In addition, the guidelines describe several methods as alternatives to demonstrate high permeability and thus fraction absorbed in human. These methods include in vivo perfusion studies in human, in vivo/in situ intestinal perfusion studies in animals, in vitro permeability studies using human or animal intestinal tissues, and in vitro permeability experiments across a monolayer of cultured human epithelial cells.2 Among these methods, in situ intestinal perfusion study and in vitro permeability study across a monolayer of cultured epithelial cells have been widely used for the determination of test drug absorption in human in several academic and industrial settings.7−9 When the permeability of a test drug is equal to or higher than that of an appropriate reference compound with high permeability, the drug is safely classified as a highly permeable drug.2 The FDA lists several reference drugs as highly permeable to aid classifying test drugs into BCS high or low permeability.2 Metoprolol (Table 1) shows compete intestinal absorption in human, as was demonstrated by pharmacokinetic and mass

administration of minoxidil, 9.8% of minoxidil was found unmetabolized in urine sample, suggesting the main elimination mechanism was metabolism followed by urinary excretion. Minoxidil showed rapid absorption with Tmax of 1 h in patients with hypertension25 and 0.5 h in healthy volunteers,28 suggesting a rapid absorption of minoxidil from the proximal jejunum, similarly to metoprolol. Therefore, the upper site of the small intestine is considered the main absorptive site for minoxidil. Hence, minoxidil resembles metoprolol in several aspects including absorptive site and clearance mechanism; minoxidil and metoprolol are well absorbed drugs and extensively metabolized. These observations imply that minoxidil may be suitable as a high permeability reference drug based on BCS definition. The purpose of this study was to evaluate if minoxidil can be a reference high permeability drug for BCS classification. In this study, the effective permeability (Peff) of minoxidil was investigated in the in situ intestinal perfusion system in rat and mouse, including segmental-dependent absorption throughout the small intestine. Metoprolol, a FDA highpermeability reference drug, was coperfused with minoxidil for the direct comparison of their permeability values. The apparent permeability of minoxidil was also demonstrated using Caco-2 cell monolayers, including concentration-dependent transport and apical (AP) to basolateral (BL) vs BL-AP transport.

Table 1. Physicochemical Properties of Metoprolol and Minoxidil

■

MATERIALS AND METHODS Chemicals. Minoxidil, metoprolol, verapamil, phenol red, calcium chloride, magnesium chloride, sodium dihydrogen phosphate, D-glucose, sodium chloride, 2-morpholinoethanesulfonic acid (MES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, MO). Potassium chloride, high performance liquid chromatography (HPLC) grade acetonitrile and physiological saline solution were obtained from Fisher Scientific, Inc. (Pittsburgh, PA). Cell culture reagents were from Life Technologies (Grand Island, NY), and cell culture supplies were from Corning Inc. (Corning, NY). All other chemicals were of analytical grade or higher. In Situ Intestinal Perfusion Studies in Rats and Mice. All animal experiments were conducted using protocols approved by the University of Michigan Committee of Use and Care of Animals (UCUCA). The animals were housed and handled according to the University of Michigan Unit for Laboratory Animal Medicine guidelines. Male Sprague−Dawley rats (Charles River, IN) weighing 500−550 g and male C57BL/ 6N mice (Charles River, IN) weighing 25−30 g were used for all perfusion studies. Prior to the perfusion studies, rats and mice were fasted overnight (12−18 h) with free access to water. Animals were randomly assigned to the different experimental group. The procedures for the in situ intestinal perfusion study were according to previous publications.14,29 Briefly, rats and mice were anesthetized with an intramuscular injection of 5 mg/kg xylazine and 80−90 mg/kg ketamine. After they were placed on a heated surface maintained at 37 °C (Harvard Apparatus Inc., Holliston, MA), the abdomen was opened by a midline incisions for rats and mice. For in situ intestinal perfusion study in rats, the proximal jejunal segment (∼3 cm average distance of the inlet from the ligament of Treitz) and the distal ileal segment (∼3 cm average distance of the outlet from the

balance study in humans.13 For metoprolol, 96% of the oral dose was excreted in the urine as the parent compound or its metabolites within 72 h after oral administration of radiolabeled metoprolol.13 The absorptive site of metoprolol in human intestine was also investigated by an intubation technique in human subjects.14,15 Metoprolol has been widely used as a high permeability reference drug in order to classify drugs into BCS high or low permeability class in several permeability studies.16−19 However, some drugs, whose permeabilities are lower than that of metoprolol, exhibit complete absorption. Therefore, metoprolol is considered an overly conservative high permeability reference drug.20−22 The oral dosage forms of minoxidil have been used for the treatment of hypertension (Table 1).23,24 Despite its moderate lipophilicity,12 minoxidil shows good oral absorption in human25 and animals.26,27 Gottlieb et al. conducted a pharmacokinetic study of minoxidil in patients with hypertension.25 The authors reported that over 80% of orally administered radiolabeled minoxidil and its metabolites were recovered from urine within 24 h after oral administration. The remainder was accumulated in urine over the subsequent 4 days. Totally, 97% of radioactivity was recovered from urine and only 3% of that was found in feces. After oral B



Article

General Procedure for Caco-2 Permeability Studies. The transport buffers (pH 6.5 and pH 7.4) were identical to the buffers used in in situ perfusion studies. Drug concentration used in the permeability studies were determined based on the HDS, 80 μg/mL for minoxidil and 400 μg/mL for metoprolol. On the day of experiment, DMEM was removed, and the monolayers were rinsed and incubated with blank MES or HEPES buffer for 15 min. Then, blank buffer was removed from both AP and BL sides. For AP-BL direction studies, 0.5 mL of the drug solution buffer prepared with MES or HEPES buffer was added to the AP side and 1.5 mL of fresh HEPES buffer was added to the BL side. For BL-AP direction studies, 1.5 mL of the drug solution buffer prepared with HEPES buffer was added to the BL side and 0.5 mL of fresh MES of HEPES buffer was added to the AP side. Throughout the experiment, the plates were kept at 37 °C. For AP-BL direction studies, 0.1 mL of the buffer in the BL side was drawn at predefined time points (15, 30, 45, 60, 90, and 120 min) and replaced by the same volume of fresh HEPES buffer to keep the buffer volume in the BL side. For BL-AP direction studies, 0.1 mL of the buffer in the AP side was collected at the same time point and replaced by the same volume of fresh MES or HEPES buffer. For both AP-BL and BL-AP direction studies, samples were taken from a donor side before and after experiments to determine the concentration of the drugs in the donor compartment. The drug concentration under the sink condition was immediately determined by HPLC. Buffer Preparation and Permeability Study Investigating pH Dependency Using Caco-2 Cell Monolayers. To investigate pH dependency in the transport of minoxidil and metoprolol, two drug solutions were prepared using MES and HEPES buffer containing both minoxidil and metoprolol. Drug concentration was the same as described in the “General Procedure for Caco-2 Permeability Studies” section. For AP-BL direction study, 0.5 mL of drug solution prepared with MES buffer or HEPES buffer was added to the AP side and 1.5 mL of blank HEPES buffer was added to the BL side. For BL-AP direction study, 1.5 mL of drug solution prepared with HEPES buffer was added to the BL side and 0.5 mL of blank MES or HEPES buffer was added to the AP side. Buffer Preparation and Permeability Study Investigating the Involvement of P-Glycoprotein in the Transport of Minoxidil Using Caco-2 Cell Monolayers. For this study verapamil was used as a P-gp inhibitor. HEPES buffer was used for both AP and BL sides to avoid the effect of pH on the transport of test compounds. Drug concentration was the same as described in “General Procedure for Caco-2 Permeability Studies” section. Verapamil concentration was set to 0.1 mM (45.5 μg/mL). For AP-BL direction study, 0.5 mL of drug solution with or without verapamil was added to the AP side and 1.5 mL of blank HEPES buffer was added to the BL side. For BL-AP direction study, 1.5 mL of drug solution with or without verapamil was added to the BL side and 0.5 mL of blank HEPES buffer was added to the AP side. Buffer Preparation and Permeability Study Investigating the Involvement of Carrier Mediated Process in the Transport of Minoxidil Using Caco-2 Cell Monolayers. Transport experiments were conducted at 80, 8, and 0.8 μg/mL for minoxidil and 400, 40, and 4 μg/mL for metoprolol. Drug solution was prepared with MES buffer, and only AP-BL direction study was performed. To the AP side, 0.5 mL of drug

ligament of cecum) of approximately 10 cm were carefully exposed and cannulated on two ends with flexible PVC tubing (2.29 mm i.d., Fisher Scientific Inc., Pittsburgh, PA). For in situ intestinal perfusion study in mice, the proximal jejunal segment of approximately 10 cm was carefully exposed and cannulated on two ends with flexible PVC tubing. Care was taken to avoid disturbing the circulatory system, and the exposed segment was kept moist with 37 °C normal saline solution. All solutions were kept at 37 °C using water bath. The isolated segment of intestine was rinsed with normal saline to clean out any residual debris. Perfusion buffers were prepared with 5 mM MES, pH 6.5, or 5 mM HEPES, pH 7.4. Both buffers contain 1 mM calcium chloride, 0.5 mM magnesium chloride, 145 mM sodium chloride, 1 mM sodium dihydrogen phosphate, 3 mM potassium chloride, and 5 mM D-glucose. Drug concentrations in the perfusate were prepared based on the highest dose strength (HDS) of the drug product dissolved in 250 mL of water. At the start of the study, the perfusate containing minoxidil (80 μg/mL), metoprolol (400 μg/mL), and phenol red (60 μg/mL) was perfused through the intestinal segment (Watson Marlow Pumps 323S, Watson−Marlow Bredel Inc., Wilmington, MA), at flow rates of 0.2 and 0.1 mL/min for rats and mice, respectively. Phenol red was added to the perfusate as a nonabsorbable marker for water efflux measurement. Metoprolol was coperfused with minoxidil as a reference drug to determine the permeability of minoxidil. The perfusate was first perfused for 30 min to ensure steady state condition and samples were collected for 5 or 10 min intervals in rats and mice, respectively, for 1 h. The length of intestinal segment used for perfusion was measured at the end of the experiment. The drug concentrations in all samples were determined by HPLC. Net Water Efflux Measurement. Net water efflux in the perfusion study was determined by measuring phenol red, a nonabsorbable and nonmetabolized marker. The measured Cout/Cin ratio was corrected for water transport according to eq 1: ′ Cout C C in,phenol red = out C in′ C in Cout,phenol red

(1)

where Cin and Cout are the concentrations of minoxidil or metoprolol in the inlet and outlet samples, respectively. Cin,phenol red and Cout,phenol red are the concentrations of phenol red in the inlet and outlet samples, respectively. Caco-2 Cell Culture. Caco-2 cells (passage 39−40) from American Type Culture Collection (Rockville, MD) were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum. Cells were cultured in an atmosphere of 5% CO2 and 90% relative humidity at 37 °C. Caco-2 cells were seeded on collagen coated PTFE membrane inserts with 0.4 μm pore size and 12 mm diameter (12-well Transwell, Corning Inc., Corning, NY). The cells on the insets were cultured for 21−24 days. Assessment of tight junction formation was performed by measuring the transepithelial electrical resistance (TEER) using Millicell-ERS epithelial Voltohmmeter (Millipore Co., Bedford, MA). The permeability studies were conducted with the monolayers that developed TEER > 300 Ω × cm2 following 21−24 days in cell culture. C



Article

solution was added and 1.5 mL of blank HEPES buffer was added to the BL side. Calculation of the Effective and Apparent Permeability (Peff and Papp). The effective permeability (Peff, cm/ sec) through rats and mice small intestinal wall in the perfusion study was calculated assuming the plug flow model expressed in eq 2: Peff =

′ /C in′ ) −Q ·ln(Cout 2πRL

Table 2. Gradient Profiles for HPLC Analysis for Samples of Caco-2 Permeability Studies (A) and Perfusion Studies (B) A

(2)

where Q is the perfusion flow rate (0.2 and 0.1 mL/min for rats and mice, respectively), C′out/C′in is the ratio of the outlet and inlet concentration for the drug, which has been adjusted for water flux, R is the radius of the small intestine (set to 0.2 and 0.1 cm for rats and mice, respectively), and L (cm) is the length of the intestinal segment. The apparent permeability (Papp, cm/sec) across Caco-2 cell monolayer was calculated using eq 3:

Papp =

dQ 1 dt C0A

% of B

time

% of B

0−1.5 min 1.5−5.5 min 5.5−6.5 min 6.5−7.5 min 7.5−8.5 min 8.5−11 min

15% B 15−60% B 60−80% B 80% B 80−15% B 15% B

0−1.5 min 1.5−5.5 min 5.5−6.5 min 6.5−10 min

15% B 15−75% B 75−15% B 15% B

standard deviation (SD). The independent t test and one-way ANOVA (analysis of variance) were used to assess differences for two groups and multiple comparisons, respectively. Differences were considered statistically significant at p < 0.05.

■

RESULTS Effective Permeability in the Intestinal Perfusion Model in Rats and Mice. Peff values of minoxidil and metoprolol in intestinal perfusion studies in rat are shown in Figure 1. The permeability of metoprolol showed intestinal

(3)

where dQ/dt is the steady state appearant rate of the drug on the receiver side, C0 is the initial concentration of the drug in the donor side, and A is the growth area of Transwell for Caco2 cells (1.12 cm2). The efflux ratio (ER) was calculated by the ratio of Papp in the secretory (BL-AP) to the absorptive (AP-BL) direction according to eq 4:

ER =

B

time

Papp(BL − AP) Papp(AP − BL)

(4)

Determination of Octanol−Buffer Partition Coefficients. Experimental octanol−buffer partition coefficients, log D, for minoxidil (vs metoprolol) at pH 6.5, 7.0, and 7.5 were determined using the traditional shake-flask method. Briefly, solutions of minoxidil or metoprolol were prepared in octanolsaturated phosphate buffers with pH values of 6.5, 7.0, and 7.5. These aqueous solutions were then equilibrated at room temperature with an equivalent volume of buffer saturated octanol for 48 h. The octanol and aqueous phases were then separated by centrifugation, and the drug concentration in the aqueous phase was determined by UPLC. The drug concentration in the octanol phase was obtained by mass balance. From these data, the apparent octanol/buffer partition coefficient was determined. Analytical Methods. The analysis of minoxidil and metoprolol in Caco-2 samples and in the perfusion buffer with phenol red was performed using HP1100 series HPLC system equipped with photodiode array UV−vis detector (Agilent Technologies, Santa Clara, CA). Separation was carried out using ZORBAX eclipse XDB-C18 column (4.6 × 150 mm, 3.5 μm, Agilent Technologies). The mobile phase consisted of A, water containing 0.1% trifluoroacetic acid (TFA), and B, acetonitrile containing 0.1% TFA. Gradient profile for the analysis is shown in Table 2. Flow rate was set to 1.0 mL/min at room temperature, and 20 μL of sample was injected into HPLC to determine drug concentration. The wavelengths were 280, 230, and 423 nm for minoxidil, metoprolol, and phenol red, respectively. Statistical Analysis. All animal experiments were performed in quadruplicate, and all Caco-2 experiments were performed in triplicate. The data are presented as mean ±

Figure 1. Effective permeability (Peff, cm/sec) of minoxidil and metoprolol in the proximal jejunum at pH 6.5 and the distal ileum at pH 7.4 by in situ intestinal perfusion study in rat. Drug concentrations adopted for this perfusion study were 80 μg/mL for minoxidil and 400 μg/mL for metoprolol, respectively. Data are presented as mean ± SD; n = 4 (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

segment-dependent values, with significantly higher permeability in the ileum than in the jejunum (1.2 ± 0.2 × 10−5 cm/ sec in jejunum, pH 6.5, vs 3.3 ± 0.7 × 10−5 cm/sec in ileum, pH 7.4). However, the permeability of minoxidil was similar regardless of intestinal segments (2.2 ± 0.7 × 10−5 cm/sec in jejunum, pH 6.5, vs 1.7 ± 0.6 × 10−5 cm/sec in ileum, pH 7.4). The permeability of minoxidil was significantly higher than that of metoprolol in the jejunum, whereas the permeability of metoprolol was significantly higher than that of minoxidil in the ileum. Peff values of minoxidil and metoprolol in mouse perfusion studies at pH 6.5 and pH 7.4 are presented in Figure 2. Minoxidil showed 2.5-fold and 2.3-fold higher permeability than metoprolol in both pH conditions (4.0 ± 1.0 × 10−5 cm/ sec vs 1.6 ± 0.4 × 10−5 cm/sec in pH 6.5 and 3.5 ± 1.2 × 10−5 cm/sec vs 1.5 ± 0.6 × 10−5 cm/sec in pH 7.4). In both conditions, minoxidil exhibited significantly higher permeability than metoprolol. D



Article

The effect of verapamil on the bidirectional permeability of minoxidil and metoprolol was also investigated (Figure 4). The

Figure 2. Effective permeability (Peff, cm/sec) of minoxidil and metoprolol in the jejunum at pH 6.5 and pH 7.4 by in situ intestinal perfusion study in mouse. Drug concentrations adopted in this perfusion study were 80 μg/mL for minoxidil and 400 μg/mL for metoprolol, respectively. Data are presented as mean ± SD; n = 4 (*, P < 0.05; **, P < 0.01).

Figure 4. Apparent permeability (Papp, cm/sec) of minoxidil (8 μg/ mL) and metoprolol (40 μg/mL) in in vitro permeability study across Caco-2 cell monolayers in the existence of 0.1 mM verapamil. Data for absorptive direction (AP-BL) and secretory direction (BL-AP) are shown. Data are presented as mean ± SD; n = 3 (**, P < 0.01; ***, P < 0.001).

Apparent Permeability in the Transport Studies across Caco-2 Cell Monolayers. Papp values of minoxidil and metoprolol in absorptive (AP-BL) and secretory (BL-AP) directions are shown in Figure 3 at AP/BL pH 6.5/7.4 and pH

permeability of minoxidil with or without verapamil was 0.8 ± 0.3 × 10−5 and 0.7 ± 0.2 × 10−5 cm/sec for AP-BL direction, and 0.9 ± 0.4 × 10−5 and 0.9 ± 0.2 × 10−5 cm/sec for BL-AP direction (ER = 1.1 with verapamil, 1.4 without verapamil). The permeability of metoprolol with or without verapamil was 2.2 ± 0.1 × 10−5 and 1.9 ± 0.2 × 10−5 cm/sec for AP-BL direction, and 2.4 ± 0.2 × 10−5 and 2.7 ± 0.2 × 10−5 cm/sec for BL-AP direction (ER = 1.1 with verapamil, 1.4 without verapamil). Therefore, verapamil had no effects on the permeability of metoprolol and minoxidil in both AP-BL and BL-AP directions, indicating no involvement of P-gp in the transport of both drugs across Caco-2 cell monolayers. This was further supported by concentration-dependent transport test of minoxidil and metoprolol presented in Figure 5. Both minoxidil and metoprolol presented constant permeability across the different concentrations, indicating no involvement of carrier mediated transport across Caco-2 cell monolayers.

Figure 3. Apparent permeability (Papp, cm/sec) of minoxidil (80 μg/ mL) and metoprolol (400 μg/mL) in in vitro permeability study across Caco-2 cell monolayers. Data for absorptive direction (AP-BL) and secretory direction (BL-AP) are shown. Data are presented as mean ± SD; n = 3 (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

7.4/7.4. Minoxidil exhibited similar membrane permeability across Caco-2 monolayers regardless of pH condition and direction; 1.1 ± 0.8 × 10−5 cm/sec for AP-BL direction and 0.9 ± 0.1 × 10−5 cm/sec for BL-AP direction (ER = 0.8), respectively, at AP/BL pH 6.5/7.4, and 1.1 ± 0.1 × 10−5 cm/ sec for AP-BL direction and 1.3 ± 0.2 × 10−5 cm/sec for BL-AP direction (ER = 1.2), respectively, at AP/BL pH 7.4/7.4. Metoprolol showed direction-dependent permeability at AP/ BL pH 6.5/7.4 (1.3 ± 0.6 × 10−5 cm/sec for AP-BL direction and 3.1 ± 0.6 × 10−5 cm/sec for BL-AP direction, ER = 2.5). The permeability of metoprolol for BL-AP direction (2.5 ± 0.1 × 10−5 cm/sec) was significantly higher than that for AP-BL direction 2.1 ± 0.2 × 10−5 cm/sec) at AP/BL pH 7.4/7.4 (ER = 1.2), but it was considered similar permeability because ER was close to 1.0. These results clearly showed that the permeability of metoprolol was pH-dependent while that of minoxidil was constant regardless of pH.

Figure 5. Apparent permeability (Papp, cm/sec) of minoxidil (0.8, 8, and 80 μg/mL) and metoprolol (4, 40, and 400 μg/mL) in in vitro permeability study across Caco-2 cell monolayers. Experiment was performed only for AP-BL direction at AP/BL pH 6.5/7.4. Data are presented as mean ± SD; n = 3. E



Article

Octanol−Buffer Partition Coefficients. The log D values for minoxidil vs metoprolol at the three pH values 6.5, 7.0, and 7.5, representing the conditions throughout the small intestine, are presented in Figure 6. It can be seen that, while for

pH/segmental transport and exhibited higher permeability than sotalol both in the jejunum and in the ileum. Yet, the permeability of sotalol in the distal ileum was higher than that of metoprolol in the proximal jejunum, suggesting that the main absorptive site of sotalol was the lower small intestine. This observation is supported by the fact that Tmax of metoprolol and sotalol were ∼113 and ∼4 h,31 respectively. Dahan and co-workers concluded that a well-absorbed drug should show high permeability, not necessarily in the jejunum, but somewhere in the intestine. Fairstein and co-workers reported that pseudoephedrine showed segmental-dependent transport in an intestinal perfusion study in rat.32 The permeability of pseudoephedrine was lower than that of metoprolol in each intestinal segment, while the permeability of pseudoephedrine in the distal ileum was higher than that of metoprolol in the proximal jejunum, allowing its complete absorption.32 On the basis of these observations, the permeability of metoprolol is considered to be somewhat too high, and the pH/segment-dependent permeability of metoprolol often makes it difficult to interpret the results of several permeability studies. In this report, the permeability of minoxidil was investigated and compared to metoprolol to assess the suitability of minoxidil as a reference drug in several permeability studies. Intestinal perfusion studies in rat and mouse and the permeability studies across Caco-2 cell monolayers were conducted to determine the permeability of minoxidil. The Peff value of a test drug in an intestinal perfusion study in rat was often used to predict drug absorption in human.7 However, in situ mouse intestinal perfusion model was used to investigate the intestinal permeability along with carrier mediated processes.33−35 Escribano et al. reported the usefulness of mouse intestinal perfusion study to predict drug absorption in human.36 They investigated five model compounds, which FDA proposed as reference drugs, and displayed a good correlation between fraction absorbed in human and the permeability study in mice. They also found that the permeability values in an intestinal perfusion study in mouse were closer to the values in human than those in rat.36 Mouse model generally requires a lower amount of drug than rat model. They proposed to use a mouse model to predict gastrointestinal absorption of orally administered drugs in human. We have previously reported the feasibility of labetalol as a new reference drug in the perfusion study in mice.17 The results of the intestinal perfusion studies in rat and mouse revealed that the permeability of minoxidil in the jejunum at pH 6.5 was somewhat higher than that of metoprolol. This result supports previous findings that minoxidil is absorbed rapidly in human25 and animals.26 In an intestinal perfusion study in rat, the permeability of minoxidil in the ileum, pH 7.4, was similar to that in the jejunum, pH 6.5, while metoprolol showed significantly higher permeability in the ileum than the jejunal permeability. The permeability of minoxidil was constant regardless of pH and intestinal segment, while metoprolol showed different permeability relative to pH and intestinal segment. The Caco-2 permeability study also showed that the permeability of minoxidil was pH independent and that the permeability of metoprolol was pH dependent. In corroboration with these observations, the octanol-buffer partitioning studies at the small intestinal pH range (6.5−7.5) also indicated constant permeability for minoxidil and pH-dependent permeability for

Figure 6. Octanol-buffer partition coefficients, log D, for minoxidil vs metoprolol at the three pH values 6.5, 7.0, and 7.5. Data are presented as the mean ± SD; n = 5 in each experimental group.

metoprolol a clear pH dependent octanol−buffer partition coefficient was found across the investigated pH range, with higher partitioning at higher pH, minoxidil exhibited constant octanol−buffer partition coefficient throughout the small intestinal pH range. At any investigated pH, the octanol− buffer partition coefficient of minoxidil was significantly higher than that of metoprolol; metoprolol’s log D values at pH 6.5− 7.5 were negative and ranged between −0.7 to −0.2, whereas minoxidil exhibited a constant and positive (0.8) log D value at these pHs.

■

DISCUSSION The definition of high permeability drug by FDA and EMA is based on the extent of drug absorption in humans.2,3 In other words, a clinical study in human subjects is the best method to classify the permeability of test drugs. However, several surrogate methods such as in situ intestinal perfusion study and the permeability study across cultured cell monolayers are widely adopted to determine the membrane permeability of test drugs. In these permeability methods, a low and a high permeability model drugs should be used as a reference drug to classify the permeability of a test drug. Metoprolol has been widely used as a high-permeable reference drug for BCS classification but several reports demonstrated that some drugs showed almost complete absorption despite their lower permeability than metoprolol.20−22 Yang et al. examined the permeability values of several β-blockers using Caco-2 cell monolayers and displayed good correlation between absorption in human and the permeability in Caco-2 with the exception of sotalol.21 The permeability of sotalol was more than 50-fold lower than that of metoprolol even though sotalol absorption in human was 95%.21 Dahan and co-workers revealed that the permeability of sotalol was low in the rat jejunum at pH 6.5, and this observation agreed with the results of the Caco-2 permeability study.30 However, sotalol showed pH/segmental-dependent transport, representing higher permeability in the lower segment of the rat small intestine. Metoprolol also showed F



Article

metoprolol (Figure 6). The significantly higher log D obtained for minoxidil vs metoprolol throughout the small intestinal pH range is harder to interpret since the partitioning of the unionized form, log P, of metoprolol is insignificantly higher than that of minoxidil (2.2 vs 0.6 respectively). However, these octanol-buffer partitioning studies may explain the higher rat and mouse jejunal Peff obtained for minoxidil vs metoprolol (Figures 1 and 2). The intestinal perfusion study in mouse with HEPES buffer at pH 7.4 in the jejunum, an unphysiological condition, was performed to investigate the effect of pH on the transport of minoxidil in the jejunum. The result showed that the permeabilities of minoxidil and metoprolol at pH 7.4 were similar to those at pH 6.5. It seemed that pH of HEPES buffer was lowered during the passage of the small intestine. Incecayir et al. performed rat intestinal perfusion study with alternate pH condition17 and revealed that there were no significant differences in the permeability of metoprolol and labetalol between physiological pH condition and the unphysiological pH condition. While the rat and mouse jejunal permeability of minoxidil was somewhat higher than that of metoprolol, similar permeability was observed in the Caco-2 system for these drugs. This lack of correlation between the in vivo and the in vitro results may be related to the permeability mechanism of these two drugs. Minoxidil’s low molecular weight, 209.2 g/ mol, suggests that paracellular transport through tight junctions may play a role in the intestinal permeability of minoxidil, while paracellular transport is not observed for metoprolol. The tight junctions in cultured Caco-2 cell monolayers are known to be tighter than their actual in vivo pore size. In this case, the paracellularly transported component of minoxidil’s absorption may be hampered in Caco-2 studies in comparison to rat/ mouse experiments, which will lead to the observed lack of in vitro−in vivo correlation, and the higher permeability in animal studies than in Caco-2 experiments. In the context of our search for an optimal Peff marker compound, one may argue that the optimal Peff reference compound should show transcellular absorption since paracellular absorption may add considerable position dependence and variability, and substantially complicate the use of cell culture methods. This analysis points out that using metoprolol, rather that minoxidil, as the BCS permeability reference compound may be preferred. The FDA requires the characterization of efflux and influx transporters in the permeability methods,2 and it is ideal that reference drugs show no carrier mediated transport in their absorption or exsorption processes. In this study, no effect of verapamil on the transport of minoxidil across Caco-2 cell monolayers was observed (Figure 4), indicating no involvement of P-gp mediated efflux process in the transport of minoxidil. This was also supported by the study showing the absence of concentration-dependent transport of minoxidil using Caco-2 cell monolayers (Figure 5). While minoxidil has been proposed as a potential reference standard with a reported lower Caco-2 permeability than metoprolol,37 we did not see that in our studies (Figure 3). In fact metoprolol and minoxidil exhibited similar permeability (∼1 × 10−5 cm/sec) in our Caco-2 system. Minoxidil and metoprolol are both high permeability drugs based on mass balance fraction absorbed studies. In both cases urinary excretion of radio labeled material was more than 90%. The difference observed in these results for Caco-2 permeability may be due to the variably of cell passage number and growing conditions, especially as it effects the tight junctions in the

Caco-2 monolayer. Thus, we may summarize the absorption characteristics of minoxidil and metoprolol in Table 3. Table 3. Transport Characteristics of Metoprolol and Minoxidil characteristic intestinal segment dependent (jejunum, ilium,and colon) pH dependence carrier mediated (including exporter) paracellular transport human permeability

metoprolol

minoxidil

yes

no

yes no no yes

no no yes? no

Our results indicate that minoxidil and metoprolol may both be used as a high-permeability reference standard. However, appropriate care and validation must be given to the Caco-2 system since the Caco-2 system in a particular laboratory can depend on a number of variables and laboratory conditions. On the basis of our investigation, minoxidil may be an acceptable drug for the BCS high permeability reference. However, as shown in the intestinal perfusion study in the rat and mouse jejunum, minoxidil exhibited higher permeability than metoprolol. Adoption of minoxidil as a reference drug for BCS high/low permeability boundary may raise the criteria for a high permeability classification. In conclusion, the present study demonstrated that minoxidil exhibited high permeability in in situ intestinal perfusion studies and in vitro Caco-2 permeability studies. Absorption of minoxidil was independent of both pH and intestinal segment. Minoxidil showed no evidence of carrier mediated transport in its absorption process with Caco-2 permeability studies. However, minoxidil may potentially be partially absorbed through a transcellular process. Moreover, the permeability of minoxidil is somewhat higher than that of metoprolol in the rat and mouse jejunum. Thus, minoxidil can be a reference compound to classify a drug as high permeability using one of several permeability methods. However, adoption of minoxidil as a reference compound for high permeability BCS classification would raise the criteria for BCS high permeability classification. Hence, using metoprolol, rather that minoxidil, as the BCS permeability marker compound, may be preferred.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: 734-764-2464. Fax: 734-764-6282. E-mail: glamidon@ umich.edu. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Amidon, G. L.; Lennernäs, H.; Shah, V. P.; Crison, J. R. A Theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm. Res. 1995, 12 (3), 413−420. (2) U.S. Food and Drug Administration, Center for Drug Evaluation and Research (CDER). Guidance for Industry: Bioavailability and Bioequivalence Studies for Orally Administered Drug Products General Considerations, March 2003; Division of Drug Information Branch, HFD-240, Center for Drug Evaluation and Research, FDA: R ockville , MD. http:/ /www .f da.gov/downloads/Drugs/ G u i d a n c e C o m p l i a n c e R e g u l a t o r y I n f o r m a t i o n / G u i d a nc e s / UCM070124.pdf.

G



Article

(3) European Medicines Agency (EMEA) Committee for Medicinal Products for Human Use. Guideline on the Investigation of Bioequivalence; Doc. Ref.: CPMP/EWP/QWP/1401/98Rev.1/Corr; EMEA: London, January 20, 2010. http://www.emea.europa.eu/docs/ en_GB/docume nt_libr ar y/Scientifi c_guideline/2010/01/ WC500070039.pdf. (4) Cook, J.; Addicks, W.; Wu, Y. H. Application of the biopharmaceutical classification system in clinical drug developmentan industrial view. AAPS J. 2008, 10 (2), 306−310. (5) Ku, M. S. Use of the biopharmaceutical classification system in early drug development. AAPS J. 2008, 10 (1), 208−212. (6) Lennernäs, H.; Abrahamsson, B. The use of biopharmaceutic classification of drugs in drug discovery and development: current status and future extension. J. Pharm. Pharmacol. 2005, 57 (3), 273− 285. (7) Cao, X.; Gibbs, S. T.; Fang, L.; Miller, H. A.; Landowski, C. P.; Shin, H.-C.; Lennernäs, H.; Zhong, Y.; Amidon, G. L.; Yu, L. X.; Sun, D. Why is it challenging to predict intestinal drug absorption and oral bioavailability in human using rat model. Pharm. Res. 2006, 23 (8), 1675−1686. (8) Volpe, D. A.; Faustino, P. J.; Ciavarella, A. B.; Asafu-Adjaye, E. B.; Ellison, C. D.; Yu, L. X.; Hussain, A. S. Classification of drug permeability with a Caco-2 cell monolayer assay. Clin. Res. Regul. Aff. 2007, 24 (1), 39−47. (9) Volpe, D. A. Drug-permeability and transporter assays in Caco-2 and MDCK cell lines. Future Med. Chem. 2011, 3 (16), 2063−2077. (10) Shalaeva, M.; Kenseth, J.; Lombardo, F.; Bastin, A. Measurement of dissociation constants (pKa values) of organic compounds by multiplexed capillary electrophoresis using aqueous and cosolvent buffers. J. Pharm. Sci. 2008, 97, 2581−2606. (11) Henchoz, Y.; Guillarme, D.; Martel, S.; Rudaz, S.; Veuthey, J. L.; Carrupt, P. A. Fast log P determination by ultra-high-pressure liquid chromatography coupled with UV and mass spectrometry detections. Anal. Bioanal. Chem. 2009, 394, 1919−1930. (12) Pfizer Canada, Inc. Product Monograph, LONITEN. http:// www.pfizer.ca/en/our_products/products/monograph/192. (13) Regårdh, C. G.; Borg, K. O.; Johansson, R.; Johnsson, G.; Palmer, L. Pharmacokinetic studies on the selective beta1-receptor antagonist metoprolol in man. J. Pharmacokinet. Biopharm. 1974, 2 (4), 347−364. (14) Jobin, G.; Cortot, A.; Godbillon, J.; Duval, M.; Schoeller, J. P.; Hirtz, J.; Bernier, J. J. Investigation of drug absorption from the gastrointestinal tract of man. I. Metoprolol in the stomach, duodenum and jejunum. Br. J. Clin. Pharmacol. 1985, 19 (Suppl 2), 97S−105S. (15) Vidon, N.; Evard, D.; Godbillon, J.; Rongier, M.; Duval, M.; Schoeller, J. P.; Bernier, J. J.; Hirtz, J. Investigation of drug absorption from the gastrointestinal tract of man. II. Metoprolol in the jejunum and ileum. Br. J. Clin. Pharmacol. 1985, 19 (Suppl 2), 107S−112S. (16) Kim, J.-S.; Mitchell, S.; Kijek, P.; Tsume, Y.; Hilfinger, J.; Amidon, G. L. The suitability of an in situ perfusion model for permeability determinations: utility for BCS class I biowaiver requests. Mol. Pharmaceutics 2006, 3 (6), 686−694. (17) Incecayir, T.; Tsume, Y.; Amidon, G. L. Comparison of the permeability of metoprolol and labetalol in rat, mouse, and Caco-2 cells: use as a reference standard for BCS classification. Mol. Pharmaceutics 2013, 10 (3), 958−966. (18) Dahan, A.; Amidon, G. L. Segmental dependent transport of low permeability compounds along the small intestine due to Pglycoprotein: the role of efflux transport in the oral absorption of BCS class III drugs. Mol. Pharmaceutics 2009, 6 (1), 19−28. (19) Peternel, L.; Kristan, K.; Petruševska, M.; Rižner, T. L.; Legen, I. Suitability of isolated rat jejunum model for demonstration of complete absorption in humans for BCS-based biowaiver request. J. Pharm. Sci. 2012, 101 (4), 1436−1449. (20) Grès, M.-C.; Julian, B.; Bourrié, M.; Meunier, V.; Roques, C.; Berger, M.; Boulenc, X.; Berger, Y.; Fabre, G. Correlation between oral drug absorption in humans, and apparent drug permeability in TC-7 cells, a human epithelial intestinal cell line: comparison with the parental Caco-2 cell line. Pharm. Res. 1998, 15 (5), 726−733.

(21) Yang, Y.; Faustino, P. J.; Volpe, D. A.; Ellison, C. D.; Lyon, R. C.; Yu, L. X. Biopharmaceutics classification of selected beta-blockers: solubility and permeability class membership. Mol. Pharmaceutics 2007, 4 (4), 608−614. (22) Dahan, A.; Lennernäs, H.; Amidon, G. L. The fraction dose absorbed, in humans, and high jejunal human permeability relationship. Mol. Pharmaceutics 2012, 9 (6), 1847−1851. (23) Lowenthal, D. T.; Affrime, M. B. Pharmacology and pharmacokinetics of minoxidil. J. Cardiovasc. Pharmacol. 1980, 2 (Suppl2), S93−106. (24) Meyers, R. S.; Siu, A. Pharmacotherapy review of chronic pediatric hypertension. Clin. Ther. 2011, 33 (10), 1331−1356. (25) Gottlieb, T. B.; Thomas, R. C.; Chidsey, C. A. Pharmacokinetic studies of minoxidil. Clin. Pharmacol. Ther. 1972, 13 (3), 436−441. (26) Thomas, R. C.; Hsi, R. S.; Harpootlian, H.; Judy, R. W. Metabolism of minoxidil, a new hypotensive agent I: absorption, distribution, and excretion following administration to rats, dogs, and monkeys. J. Pharm. Sci. 1975, 64 (8), 1360−1366. (27) Thomas, R. C.; Harpootlian, H. Metabolism of minoxidil, a new hypotensive agent II: biotransformation following oral administration to rats, dogs, and monkeys. J. Pharm. Sci. 1975, 64 (8), 1366−1371. (28) Fleishaker, J. C.; Andreadis, N. A.; Welshman, I. R.; Wright, C. E., III. The pharmacokinetics of 2.5- to 10-mg oral doses of minoxidil in healthy volunteers. J. Clin. Pharmacol. 1989, 29 (2), 162−167. (29) Dahan, A.; West, B. T.; Amidon, G. L. Segmental-dependent membrane permeability along the intestine following oral drug administration: Evaluation of a triple single-pass intestinal perfusion (TSPIP) approach in the rat. Eur. J. Pharm. Sci. 2009, 36 (2−3), 320− 329. (30) Dahan, A.; Miller, J. M.; Hilfinger, J. M.; Yamashita, S.; Yu, L. X.; Lennernäs, H.; Amidon, G. L. High-permeability criterion for BCS classification: segmental/pH dependent permeability considerations. Mol. Pharmaceutics 2010, 7 (5), 1827−1834. (31) Antonaccio, M. J.; Gomoll, A. Pharmacology, pharmacodynamics and pharmacokinetics of sotalol. Am. J. Cardiol. 1990, 65 (2), 12A−21A. (32) Fairstein, M.; Swissa, R.; Dahan, A. Regional-dependent intestinal permeability and BCS classification: elucidation of pHrelated complexity in rats using pseudoephedrine. AAPS J. 2013, 15 (2), 589−597. (33) Moss, A. M.; Endres, C. J.; Ruiz-Garcia, A.; Choi, D.-S.; Unadkat, J. D. Role of the equilibrative and concentrative nucleoside transporters in the intestinal absorption of the nucleoside drug, ribavirin, in wild-type and Ent1(−/−) mice. Mol. Pharmaceutics 2012, 9 (9), 2442−2449. (34) Holmstock, N.; Annaert, P.; Augustijns, P. Boosting of HIV protease inhibitors by ritonavir in the intestine: The relative role of cytochrome P450 and P-glycoprotein inhibition based on Caco-2 monolayers versus in situ intestinal perfusion in mice. Drug Metab. Dispos. 2012, 40 (8), 1473−1477. (35) Holmstock, N.; Mols, R.; Annaert, P.; Augustijns, P. In situ intestinal perfusion in knockout mice demonstrates inhibition of intestinal P-glycoprotein by ritonavir causing increased darunavir absorption. Drug Metab. Dispos. 2010, 38 (9), 1407−1410. (36) Escribano, E.; Sala, X. G.; Salamanca, J.; Navarro, C. R.; Regué, J. Q. Single-pass intestinal perfusion to establish the intestinal permeability of model drugs in mouse. Int. J. Pharm. 2012, 436 (1− 2), 472−477. (37) Guerra, F.; Wang, Q.; Huang, Y.; Cheng, H.; Jia, R.; Rager, J.; Kardos, P.; Weinstein, K. E.; Murray, L. J.; Bhoopathy, S.; Hidalgo, I. J. Comparison of Non-Clinical Intestinal Permeability Techniques (In Vitro, In Situ, and Ex Vivo) for BCS Classification, Absorption Systems, Inc. http://www.absorption.com/wp-content/uploads/ 2014/07/BCS_Permeability_Techniques.pdf.

H


Intestinal Permeability Study of Minoxidil: Assessment of Minoxidil as a

Recommend Documents