High BCS Permeability Class Boundary - ACS Publications

Apr 3, 2014 - labetalol as the permeability class reference drug. Labetalol,s. BCS solubility class was determined, and its physicochemical properties...
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The Low/High BCS Permeability Class Boundary: Physicochemical Comparison of Metoprolol and Labetalol Moran Zur,† Marisa Gasparini,† Omri Wolk,† Gordon L. Amidon,‡ and Arik Dahan*,† †

Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel ‡ Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 481409 ABSTRACT: Although recognized as overly conservative, metoprolol is currently the common low/high BCS permeability class boundary reference compound, while labetalol was suggested as a potential alternative. The purpose of this study was to identify the various characteristics that the optimal marker should exhibit, and to investigate the suitability of labetalol as the permeability class reference drug. Labetalol’s BCS solubility class was determined, and its physicochemical properties and intestinal permeability were thoroughly investigated, both in vitro and in vivo in rats, considering the complexity of the whole of the small intestine. Labetalol was found to be unequivocally a high-solubility compound. In the pH range throughout the small intestine (6.5−7.5), labetalol exhibited pH-dependent permeability, with higher permeability at higher pH values. While in vitro octanol−buffer partitioning (Log D) values of labetalol were significantly higher than those of metoprolol, the opposite was evident in the in vitro PAMPA permeability assay. The results of the in vivo perfusion studies in rats lay between the two contradictory in vitro studies; metoprolol was shown to have moderately higher rat intestinal permeability than labetalol. Theoretical distribution of the ionic species of the drugs was in corroboration with the experimental in vitro and the in vivo data. We propose three characteristics that the optimal permeability class reference drug should exhibit: (1) fraction dose absorbed in the range of 90%; (2) the optimal marker drug should be absorbed largely via passive transcellular permeability, with no/negligible carrier-mediated active intestinal transport (influx or efflux); and (3) the optimal marker drug should preferably be nonionizable. The data presented in this paper demonstrate that neither metoprolol nor labetalol can be regarded as optimal low/high-permeability class boundary standard. While metoprolol is too conservative due to its complete absorption, labetalol has been shown to be a substrate for P-gp-mediated efflux transport, and both drugs exhibit significant segmentaldependent permeability along the gastrointestinal tract. Nevertheless, the use of metoprolol as the marker compound does not carry a risk of bioinequivalence: Peff value similar to or higher than metoprolol safely indicates high-permeability classification. On the other hand, a more careful data analysis is needed if labetalol is used as the reference compound. KEYWORDS: Biopharmaceutics Classification System (BCS), biowaiver, intestinal permeability, labetalol, metoprolol, reference standard, solubility

1. INTRODUCTION Throughout the past two decades, the Biopharmaceutics Classification System (BCS) has become an increasingly important tool in drug product regulation worldwide, by presenting a new paradigm in bioequivalence.1−3 Bioequivalence (BE) is the critical step that connects the physical drug product with the clinical properties claimed on its label, ensuring continuing quality of the innovative products and the generic products. Before the presentation of the BCS, the BE standard was solely empirical, depending on in vivo bioavailability (BA) studies, i.e., plasma levels, AUC, and Cmax. By revealing the fundamental parameters dictating the in vivo oral drug absorption process, the BCS is able to ensure BE by mechanistic tools, rather than empirical observation; if two drug products that contain the same active pharmaceutical ingredient (API) have a similar GI concentration−time profile © 2014 American Chemical Society

under all luminal conditions, then a similar rate and extent of absorption are ensured for these products, i.e., they are bioequivalent. Thus, BE can be guaranteed on the basis of in vitro dissolution tests that provide the mechanistic proof for similar bioavailability, rather than empirical in vivo human studies.4−6 This is the regulatory waiver of in vivo BE, based on the scientific and mechanistic rationale provided by the BCS. Initially, waivers of in vivo BE were accepted only for scale-up and postapproval changes (SUPAC), but later, the biowaiver principle was extended to the approval of new generic drug products, thus avoiding unnecessary human experiments and Received: Revised: Accepted: Published: 1707

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that of metoprolol safely indicates high-permeability classification.

reducing cost and time of developing generic IR oral drug products.7,8 Up to now, the FDA has implemented the BCS system to allow waiver of in vivo BA/BE testing of IR solid dosage forms for class 1, high-solubility, high-permeability drugs.9 The solubility classification is based on the highest dose strength in an IR product. According to the current FDA guidance,9 a drug substance is considered highly soluble if the highest strength is soluble in 250 mL or less of aqueous media throughout the pH range of 1.0−7.5. A drug substance is considered highly permeable if the extent of intestinal absorption is determined to be 90% or higher. Otherwise, the drug substance is considered poorly permeable. The permeability classification is based either directly on the extent of intestinal absorption of a drug substance in humans determined by mass balance or in comparison to an intravenous reference dose, or indirectly on the measurements of the rate of mass transfer across the human intestinal membrane. Alternatively, animal or in vitro models that predict human intestinal absorption, e.g., intestinal rat perfusion models or epithelial cell culture models, can be used. When evaluating intestinal permeability (Peff) data, one must first define the borderline for the low/high-permeability class membership; the completely absorbed β-blocker metoprolol is currently the acceptable and widely used marker for this purpose.4,10,11 However, metoprolol is a conservative reference standard, as it is completely absorbed in humans based on the urinary excretion of the unchanged compound and its total radioactive metabolites after oral and iv administration in healthy volunteers.12 Alternatively, the mixed α/β adrenergic blocker labetalol was proposed to be used as the low/highpermeability class reference,13−15 which was accepted by the FDA. As such, the FDA has classified drugs whose permeability values are higher than labetalol’s, but lower than metoprolol’s, as high-permeability drugs.16 Recently, Incecayir et al. have studied labetalol, and questioned its suitability as a potential BCS high-permeability reference standard, mainly because it is a substrate for P-gp mediated efflux transport.13 The purpose of the present study was to assess labetalol as a potential high-permeability reference standard for the application of BCS. We have determined the solubility class of labetalol, and thoroughly investigated its physicochemical properties and intestinal permeability, both in vitro and in vivo in rats, taking into consideration the complexity of the whole of the small intestine. The results were compared with metoprolol. We then performed a thorough theoretical physicochemical analysis of labetalol, to further clarify the mechanistic explanation behind the experimental data. Overall, this work aims to identify the various characteristics that the optimal marker should exhibit; we propose the following three characteristics for the optimal BCS permeability class reference drug: (1) fraction dose absorbed in the range of 90%; (2) the optimal marker drug should be absorbed largely via passive transcellular permeability, with no/negligible carrier-mediated active intestinal transport (influx or efflux); and (3) the optimal marker drug should preferably be nonionizable. In assessing the suitability of labetalol and metoprolol as high-permeability marker compounds for BCS application, the data demonstrate that neither metoprolol nor labetalol can be regarded as optimal low/high-permeability class boundary standard. However, the use of metoprolol as the marker compound does not carry a risk of bioinequivalence, as Peff value similar to or higher than

2. MATERIALS AND METHODS 2.1. Materials. Labetalol hydrochloride, metoprolol tartrate, phenol red, potassium chloride, potassium phosphate monobasic, sodium chloride, octanol, hexadecane, and trifluoroacetic acid (TFA) were purchased from Sigma Chemical Co. (St. Louis, MO). Acetonitrile, methanol, and water (Merck KGaA, Darmstadt, Germany) were ultraperformance liquid chromatography (UPLC) grade. All other chemicals were of analytical reagent grade. 2.2. Solubility Studies. The solubility class membership of labetalol was determined according to the FDA’s BCS guidance for industry.9 The equilibrium solubility of the drug was determined at both 37 °C and room temperature (25 °C), in phosphate buffer at pH 7.5, acetate buffer at pH 4.0, and maleate buffer at pH 1.0, using the shake-flask method, as previously reported.17−20 Briefly, excess amounts of labetalol were added to glass vials containing the different buffers. Solution pH was verified after addition of the drug to the buffer. The vials were tightly closed and placed in a shaking (100 rpm) water bath at 37 or 25 °C. Establishment of equilibrium was confirmed by comparison of 48 and 72 h samples. Prior to sampling, the vials were centrifuged (10,000 rpm for 10 min) and the supernatant was carefully withdrawn, filtered, and immediately assayed by UPLC. 2.3. Determination of Octanol−Buffer Partition Coefficients. Experimental octanol−buffer partition coefficients, Log D, for labetalol (vs metoprolol) at pH 6.5, 7.0, and 7.5 were determined using the traditional shake-flask method.21−24 Briefly, solutions of labetalol or metoprolol were prepared in octanol-saturated 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. 2.4. Parallel Artificial Membrane Permeability Assay. Permeability studies through artificial membrane were carried out in the precoated PAMPA assay (BD Gentest), according to the manufacturer’s instructions, with the addition of tracking the transport rate.25−27 Briefly, three solutions of labetalol or metoprolol were prepared with different ratios of potassium phosphate monobasic and sodium phosphate dibasic, to give pH values of 6.5, 7.0, and 7.5. Osmolarity (290 mOsm/L) and ionic strength were similar in all buffers. The donor wells were filled with the different labetalol solutions (200 μL), the receiver wells were filled with blank buffers (300 μL), and the PAMPA sandwich was incubated at room temperature. Receiver plates were collected hourly for 5 h.28,29 Apparent permeability coefficient (Papp) values were calculated from the linear plot of drug accumulated in the acceptor side vs time using the equation Papp =

dQ /dt A ·C0

where dQ/dt is the steady-state appearance rate of labetalol (or metoprolol) on the receiver side, C0 is the initial concentration 1708

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of the drug in the donor side (250 μM in all experiments), and A is the membrane surface area (0.048 cm2). Linear regression was carried out to obtain the steady-state appearance rate of the drugs on the receiver side. 2.5. Rat Intestinal Perfusions. The in situ effective permeability coefficient (Peff) of labetalol vs metoprolol was determined using the single-pass rat intestinal perfusion model. All animal experiments were conducted using protocols approved by the Ben-Gurion University of the Negev Animal Use and Care Committee (Protocol IL-60-11-2010). The animals (male Wistar rats weighing 270−300 g, Harlan, Israel) were housed and handled according to the Ben-Gurion University of the Negev Unit for Laboratory Animal Medicine Guidelines. The experimental procedure followed previous reports.30−33 Briefly, anesthetized rats were placed on a heated (37 °C) surface (Harvard Apparatus Inc., Holliston, MA), and a midline abdominal incision of 3 cm was made. To account for the complexity of the whole of the small intestine, permeability was determined in three different 10 cm segments: a proximal jejunal segment (starting 2 cm below the ligament of Treitz), a mid-small intestinal segment (isolated between the end of the upper and the beginning of the lower segments), and a distal ileal segment (ending 2 cm above the cecum). Each intestinal segment (approximately 10 cm) was cannulated on two ends and was rinsed with blank perfusion buffer. All solutions were incubated in a 37 °C water bath. Three perfusion buffers containing labetalol or metoprolol and phenol red (a nonabsorbable marker for water flux measurements) were prepared with different ratios of potassium phosphate monobasic and sodium phosphate dibasic, to give pH values of 6.5, 7.0, and 7.5, while osmolarity (290 mOsm/L) and ionic strength were similar in all buffers. The permeability in each intestinal segment was measured at the pH that corresponds to the physiological pH of that region: (1) proximal jejunum, pH 6.5; (2) mid-small intestine, pH 7.0; and (3) distal ileum, pH 7.5.5,34 The perfusion buffer was first perfused for 1 h, to ensure steady-state conditions, followed by additional 1 h of perfusion with samples taken every 10 min. The pH of the collected samples was measured at the outlet, to verify that there was no pH change throughout the perfusion. All samples were immediately assayed by UPLC. At the end of the experiment, the length of each perfused intestinal segment was accurately measured. The effective permeability (Peff) through the rat gut wall was determined according to the following equation: Peff =

for labetalol, metoprolol, and phenol red were 303, 275, and 400 nm and 3.5, 3.0, and 4.2 min, respectively. Injection volumes for all UPLC analyses ranged from 2 to 50 μL. 2.7. Physicochemical Analysis. The theoretical distribution of ionic species for labetalol was calculated based on the Henderson−Hasselbalch equation, using pKaacidic and pKabasic values of 7.4 and 9.4, respectively.37−39 For each ionic species a separate equation depicting its molar fraction from the total amount vs pH was derived. Based on Henderson−Hasselbalch equation, the ratios between the cation, anion, zwitterion, and neutral forms of labetalol can be expressed as acidic Z+N = 10(pH − pKa ) C

and basic A = 10(pH − pKa ) Z+N

where A, C, N, and Z are the anion, cation, neutral, and zwitterion forms of labetalol. From these expressions, equations depicting the theoretical distribution of each species vs pH were derived: Cation: C 1 = acidic basic (pH − p K ) a total × (1 + 10(pH − pKa )) 1 + 10

Anion: A 1 = basic acidic (p K − pH) total × (1 + 10(pKa − pH)) 1 + 10 a

Neutral: N 1 = basic (pH − pK aacidic) total × (1 + 10(pH − pKa )) 1 + 10 1 × acidic (pK abasic − pH) × (1 + 10(pKa − pH)) 1 + 10

Zwitterion: Z 1 = acidic basic (p K − pH) a total + 10(pH − pKa ) 1 + 10 1 − basic (pH − pK aacidic) × (1 + 10(pH − pKa )) 1 + 10 1 × acidic (pK abasic − pH) × (1 + 10(pKa − pH)) 1 + 10

−Q ·ln(C′out /C′in ) 2πRL

where Q is the perfusion buffer flow rate (0.2 mL/min) and C′out/C′in is the ratio of the outlet and the inlet concentration of drug that has been adjusted for water transport.31,35,36 R is the radius of the intestinal segment (set to 0.2 cm), and L is the length of the perfused intestinal segment. 2.6. Ultraperformance Liquid Chromatography. UPLC analyses were performed on a Waters (Milford, MA) Acquity UPLC H-class system equipped with photodiode array detector and Empower software. The simultaneous determination of labetalol, metoprolol, and phenol red was achieved using a Waters Acquity UPLC BEH C18 1.7 μm 2.1 × 100 mm column. The mobile phase consisted of 90:10 going to 15:85 (v/v) water:acetonitrile (both with 0.1% TFA) over 6 min (flow rate, 0.5 mL/min). The detection wavelengths and retention times

2.8. Statistical Analysis. Log D determinations were n = 5, and all other in vitro experiments were replicated with n = 4. All animal experiments were replicated with n = 6. Values are expressed as means ± standard deviation (SD). To determine statistically significant differences among the experimental groups, the nonparametric Kruskal−Wallis test was used for multiple comparisons and the two-tailed nonparametric Mann−Whitney U test for two-group comparison where appropriate. A p value of less than 0.05 was termed significant.

3. RESULTS 3.1. Solubility. The solubility of labetalol in the three pH values of 1.0, 4.0, and 7.5, at both 37 °C and room temperature, is presented in Table 1. The data indicate that labetalol is 1709

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Table 1. Solubility (mg/mL) of Labetalol in the Three pH Values 1.0, 4.0, and 7.5, at 37 °C and at Room Temperature (25 °C)a

a

temp (°C)

pH 1.0

pH 4.0

pH 7.5

37 25

6.2 ± 0.1 3.9 ± 0.7

18.8 ± 0.5 14.1 ± 0.3

15.0 ± 0.5 10.1 ± 0.1

Data presented as mean ± SD; n = 5.

unequivocally a high-solubility compound; taking 400 mg as the highest single-unit dose strength (M) and 250 mL as the initial volume of water (V0), according to the equation D0 = M/V0/Cs the minimal dose number (D0) at 37 °C is 0.25, indicating a BCS high-solubility class membership. 3.2. Permeability. The Log D values for labetalol 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 1. It can be seen that, for both labetalol and metoprolol, Figure 2. The transport flux of labetalol and metoprolol at the three pH values 6.5, 7.0, and 7.5 in the precoated PAMPA experiments (BD Gentest). Data are presented as the mean ± SD; n = 4 in each experimental group.

Figure 1. The octanol−buffer partition coefficients, Log D, for labetalol 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.

Figure 3. The apparent permeability (Papp; cm/s) obtained for labetalol and metoprolol at the three pH values 6.5, 7.0, and 7.5 in the precoated PAMPA experiments (BD Gentest). Data are presented as the mean ± SD; n = 4 in each experimental group.

a clear pH-dependent octanol−buffer partition coefficient was found across the investigated pH range, with higher partitioning at higher pH. At any given pH, the octanol−buffer partition coefficient of labetalol was higher than that of metoprolol; metoprolol’s Log D values at pH 6.5−7.5 were negative and ranged between −0.7 and −0.2, whereas Log D values of labetalol at these pHs were positive and ranged between 0.5 and 1.1. Due to the similar underlying mechanism, octanol−buffer partitioning has been widely used as a surrogate for passive intestinal permeability.40−43 Hence, the significantly higher Log D values may indicate an advantage for labetalol over metoprolol from the passive permeability point of view. The partition coefficient of the un-ionized form, log P, of labetalol is also considerably higher than that of metoprolol: 3.144 vs 2.2,45 respectively. However, the opposite trend was found in the PAMPA permeability assay. The accumulated amount transported vs time plots of labetalol and metoprolol in the precoated PAMPA assay, at the three pH values 6.5, 7.0, and 7.5, are presented in Figure 2, and the corresponding Papp values are presented in Figure 3. It can be seen that for both drugs the pH-dependent permeability observed in the Log D experiments was obtained also in the

PAMPA assay, with higher permeability at higher pH. However, in contrast to the Log D comparison, metoprolol exhibited significantly higher permeability than labetalol in the PAMPA experiments. This result is in corroboration with the polar surface area (PSA) value of the two drugs; metoprolol has significantly lower PSA than labetalol, 50.7 vs 95.6 respectively,46 which may indicate an advantage for metoprolol over labetalol from the passive permeability point of view. Likewise, metoprolol has lower total hydrogen bond count (sum of donors and acceptors) than labetalol (6 vs 9 respectively). The contradictory picture obtained throughout the in vitro comparison between labetalol and metoprolol may be clarified with in vivo experiments. Intestinal perfusion studies in rats have been shown to quantitatively predict the fraction of dose absorbed in vivo in humans.2,47−50 The effective permeability coefficient (Peff) values of labetalol vs metoprolol determined using the single-pass rat intestinal perfusion (SPIP) model, in the three small intestinal segments the proximal jejunum (pH 6.5), the mid-small intestine (pH 7.0), and the distal ileum (pH 7.5), are presented in Figure 4. It can be seen that a clear 1710

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Figure 4. Effective permeability values (Peff; cm/s) obtained for labetalol vs metoprolol after in situ single pass perfusion to the rat proximal jejunum at pH 6.5, the mid-small intestine at pH 7.0, and the distal ileum at pH 7.5. Data are presented as the mean ± SD; n = 6 in each experimental group; *p < 0.05.

Figure 6. The theoretical distribution of the ionic species of labetalol vs pH, calculated according to the equations listed in Materials and Methods.

segmental/pH-dependent permeability was obtained for both drugs, similarly to all other experimental methods tested. This experimental method captures the full complexity and the influence of the various physicochemical/biochemical/physiological parameters on drug absorption. Indeed, the SPIP results lay between the two contradictory in vitro studies; metoprolol has higher rat intestinal permeability than labetalol, however the gap is moderate. In the proximal jejunum at pH 6.5, for instance, the Peff values of metoprolol and labetalol were 4.0 and 2.7 (×10−5 cm/s) respectively. This result agrees well with many cell culture permeability studies: Yang et al. reported Caco-2 P app values of 36.8 and 22.6 (×10 −6 cm/s) respectively;39 Thiel-Demby et al. reported MDCK Papp values of 410 and 201 (nm/s) respectively;14 Incecayir et al. reported Caco-2 Papp values of 9.4 and 5.9 (×10−6 cm/s) respectively;13 Yazdanian et al. reported Caco-2 Papp values of 23.7 and 9.3 (×10−6 cm/s) respectively;51 and Varma et al. reported MDCK Papp values of 25.5 and 13.0 (×10−6 cm/s) respectively.52 3.3. Physicochemical Analysis. The molecular structure of labetalol, with indication of the two ionizable centers, the acidic phenolic group, and the basic secondary amine group, is presented in Figure 5, and the theoretical distribution of the

behavior similar to that of a simple basic compound in the GI tract, with increased passive permeability as the drug travels along the small intestine and the local pH rises. This analysis is in corroboration with the in vitro and the in vivo data presented in this paper (Figures 1−4). As for metoprolol, the basic secondary amine is the only ionizable center, and hence the fraction un-ionized ( f u) of metoprolol is negligible at low pH, and gradually increases as the pH rises, resulting in the classic sigmoidal shape. Overall, in the physiological pH range throughout the gastrointestinal tract, both compounds present similar ionization pattern, which explains the in vitro and the in vivo data presented in this paper (Figures 1−4).

4. DISCUSSION Before choosing the low/high-permeability class boundary standard, we must define the various characteristics that the optimal marker should exhibit. First and foremost, the fraction dose absorbed, Fabs, of the marker drug should be in the range of 90%, indicating on one hand high-permeability but on the other hand not being too conservative. Then, minimal variability in the marker drug’s absorption and permeability is essential. To ensure this minimal variability, no/negligible carrier-mediated active intestinal transport (influx or efflux) of the marker drug is required, since concentration-dependent permeability can be expected due to active absorption processes. Moreover, the transporters’ expression intensity and pattern are dynamic and likely to differ between subjects and increase absorption variability. The optimal low/highpermeability class boundary standard, hence, should be passively absorbed. Moreover, the optimal Peff marker compound should be largely absorbed transcellularly, since paracellular permeation may add considerable variability, and substantially limit the use of cell culture methods (e.g., Caco-2) since they are well-known to poorly predict paracellular permeability. In addition, fairly constant permeability throughout the small intestine should also be considered as an advantage, since segmental-dependent permeability makes it more complex to relate the permeability value to the overall intestinal absorption. The pH change throughout the small intestine may lead to significant variations in the fraction ionized of acids/bases, and since ionization state directly affects

Figure 5. The molecular structure of labetalol, with indication of the two ionizable centers, the acidic phenolic group and the basic secondary amine group, and their pKa values.

ionic species of labetalol vs pH is presented in Figure 6. It can be seen that the distribution of labetalol species that are more likely to be passively absorbed, i.e., the zwitterion and the neutral species, reaches a maximum at the isoelectric point (pH 8.4). Since this pH value is beyond the physiological pH range throughout the gastrointestinal tract, labetalol presents a 1711

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and an additional 20% is absorbed from the following 30 cm of the jejunum, leading to absorption of 80% of the dose from the upper 50 cm of the small intestine.55 It follows, hence, that metoprolol’s Peff value at pH 7.5 (ileum) is not likely to be physiologically relevant, in terms of mass of drug absorbed, for an IR dosage form; rather, the permeability at pH 6.5, the average pH of the human jejunum (and the pH used in human jejunal permeability studies), would govern metoprolol’s in vivo intestinal absorption from an IR dosage form, and this permeability value allows the complete absorption of metoprolol. Hence, for metoprolol, the Peff−Fabs correlation can be safely established. The lack of comparable data for labetalol’s absorption prevents the establishment of the Peff− Fabs correlation for this drug. When carrier-mediated intestinal permeability, influx or efflux, contributes to the drug absorption process, the expression levels of the relevant transporters along the intestine may lead to a segmental-dependent permeability phenomenon. For instance, the expression level of the efflux transporter P-gp along the rat small intestine was shown to be regionaldependent, increasing from the proximal to the distal segments.33,56,57 A similar trend, although with higher variability, has been shown in humans as well.58,59 On the other hand, the expression of the efflux transporter MRP2 throughout the rat small intestine has been shown to follow an opposite gradient, decreasing from the proximal to the distal segments.33,57 These segmental-dependent expression trends of the various transporters may result in high variability of the permeability, both within and between subjects, which emphasizes the need for the marker compound to be passively absorbed. This discussion indicates that neither metoprolol nor labetalol can be regarded as optimal low/high-permeability class boundary standard. Yet, the use of metoprolol as the marker compound does not carry a risk of bioinequivalence: if a compound has a Peff value similar to or higher than that of metoprolol, then it can be safely considered as highpermeability. On the other hand, a more careful data analysis is needed if labetalol is used as the reference compound, as was also recently concluded by Incecayir et al.13 Indeed, labetalol exhibits high in situ rat intestinal permeability at the highest clinical dose, ensuring its high-permeability classification. However, in cell culture experimental methods, for instance, P-gp may be overly expressed, resulting in lower permeability value for labetalol, and in this case, a compound exhibiting similar/higher Papp than labetalol must be further examined before a high-permeability classification may be safely approved, especially in light of labetalol’s expected concentrationdependent permeability.

the permeability of the drug through the intestinal membrane, the optimal marker drug should preferably be nonionizable. In light of these optimal characteristics, a comparison of the suitability of metoprolol vs labetalol as the low/highpermeability class boundary standard can now be made. Metoprolol has been demonstrated to have complete (100%) absorption,12 making it a too conservative marker compound, since drugs with lower intestinal permeability may also have Fabs above 85% or 90% and hence be eligible for a biowaiver according to the EMA or FDA, respectively. From this aspect, labetalol may be better than metoprolol as the permeability marker compound: it has lower intestinal permeability (Figure 4), but still enough to produce >90% absorption. It should be noted that mass balance or PK data supporting labetalol’s >90% Fabs are not publicly available, though stated as such in references.14,53 With respect to the involvement of active transporters in the overall absorption of the marker drug, metoprolol may be better than labetalol as the permeability marker compound. While labetalol was shown to be a substrate for P-gp mediated efflux transport,13,14,54 metoprolol’s intestinal permeability is passive and does not involve carrier-mediated absorption. It should be noted that the involvement of carrier-mediated transport presents additional difficulties other than increased variability of the drugs’ intestinal absorption. It is well-known that carrier-mediated intestinal absorption of drugs is poorly predicted using cell culture studies (Caco-2 or others).47 Cell culture models reliably measure the passive drug transport (both transcellular and paracellular), which is one of the limitations of these experiments. The requirement for 20 reference or validation compounds was made for these (among other) reasons, so these could be used to validate a case for high-permeability determination. Hence, if active transport contributes significantly to a permeability marker drug’s overall absorption, it will be more difficult to work with this permeability marker in cell culture based experimental methods. With respect to constant permeability throughout the small intestine, it is clear that both labetalol and metoprolol do not meet this criterion. In all experimental methods tested, both drugs exhibited segmental/pH-dependent permeability, with higher permeability at more distal segments and higher pH. Extensive scientific research has established the good correlation between human jejunal permeability (Peff) and the fraction of dose absorbed (Fabs) from an immediate-release, rapidly dissolving, dosage form,48−50 and drug regulatory authorities worldwide rely on this Peff−Fabs correlation. However, when the permeability is not constant and the drug presents different Peff values in different intestinal segments, it is not clear which of the different Peff values should be used for the correlation with Fabs. We have recently shown several cases in which low jejunal but higher ileal Peff allowed for high Fabs.5,8,22 This point highlights the importance of constant intestinal permeability for the low/high-permeability class boundary standard, and since both metoprolol and labetalol do not meet this requirement, it can be concluded that both of them cannot be regarded as optimal boundary markers. Nevertheless, in the case of metoprolol, human data is available to support a thorough analysis of its intestinal permeability, since an IR oral dose of metoprolol has been shown to be completely absorbed in the upper small intestine. Jobin et al. have used the intubation technique in humans to show that 60% of a metoprolol oral dose is absorbed from the duodenum,

5. CONCLUSIONS In conclusion, neither metoprolol nor labetalol can be regarded as an optimal low/high-permeability class boundary standard. While metoprolol is too conservative due to its complete absorption, labetalol has been shown to be a substrate for P-gpmediated efflux transport, and both drugs exhibit significant segmental-dependent permeability along the gastrointestinal tract. Nevertheless, the use of metoprolol as the marker compound does not carry a risk of bioinequivalence: Peff value similar to or higher than that of metoprolol safely indicates high-permeability classification. On the other hand, a more careful data analysis is needed if labetalol is used as the reference compound. 1712

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AUTHOR INFORMATION

Corresponding Author

*Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel. Tel: (+972)-86479483. Fax: (+972)-8-6479303. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED API, active pharmaceutical ingredient; BA, bioavailability; BCS, Biopharmaceutics Classification System; BE, bioequivalence; D0, dose number; Fabs, fraction of dose absorbed; IR, immediate release; Peff, effective permeability; SPIP, single-pass rat intestinal perfusion; UPLC, ultraperformance liquid chromatography

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