Materials and Methods

Materials.

Hydroxyzine, triprolidine, and diphenhydramine were obtained from Sigma-Aldrich (St. Louis, MO). Loratadine and desloratadine were extracted and purified from prescribed capsules with purity of >99%. Compounds A and B were synthesized at Pfizer Inc. (Groton, CT). All the other chemicals and reagents were the highest grade available from commercial sources.

Animals.

Male FVB (control) and mdr1a/b KO mice, 4 to 5 week of age (Taconic Farms, Germantown, NY), were housed in a group of 20 and 2, respectively, with free access to food and water and were maintained on a 12-h light/dark cycle.

Plasma-Brain Disposition of H1-Antagonists in WT and KO Mice.

Mice were administered each individual compound intravenously at 5 mg/kg through tail vein injection (<100 μl in less than 30 s). Blood and brain samples were harvested at 2, 5, 15, 45, 120, 240, 480, and 1440 min postdose (n = 3 mice/time point). Plasma samples were obtained by centrifuging the blood samples at 13,000 rpm for 2 min. Brain was rinsed with saline and blotted dry and weighed. Samples were stored at −20°C before analysis by liquid chromatography-mass spectrometry.

Quantitation of H1-Antagonists in Plasma and Brain.

Sample pretreatment

Loratadine and triprolidine plasma samples were extracted using liquid-liquid extraction. Briefly, to 100 μl of sample was added 10 μl of internal standard (i.s.). Compound A (Fig.1) and diphenhydramine were used as the i.s. for loratadine and triprolidine, respectively. The samples were extracted by adding methyl-t-butyl ether (500 μl) using Personal-550 Pipettor 96 Channels (Apricot Designs Inc., Monrovia, CA). An aliquot (350 μl) of the supernatant was transferred to a new set of tubes after mixing and centrifugation (3000 rpm × 15 min). The supernatant was evaporated to dryness with Evaporex 96 Channels (Apricot Designs Inc.) under nitrogen gas, and the residue was reconstituted with mobile phase [acetonitrile/water containing 0.1% acidic acid, 50:50 (v/v), 100 μl]. Cetirizine, diphenhydramine, hydroxyzine, and desloratadine were extracted via acetonitrile precipitation. Briefly, to 100 μl of sample was added 300 μl of acetonitrile containing i.s. (as shown in Fig. 1, compound B and A were used as i.s. for cetirizine and diphenhydraime, respectively, and loratadine was used as i.s. for hydroxyzine and desloratadine). The rest of the procedures was the same as for liquid-liquid extraction. The brains were homogenized after adding 4 volumes of saline. The resulting brain homogenate (100 μl) was used for analysis. The method for extraction of brain samples was same as described for the plasma samples using acetonitrile precipitation.

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Figure 1

Chemical structures of compounds A and B that were used as i.s. for quantitation.

LC-MS-MS instrumentation and conditions.

High-performance liquid chromatography-mass spectrometry consisted of a Hewlett Packard 1100 quaternary pump with membrane degasser (Hewlett Packard, Palo Alto, CA), a 215 liquid handler (Gilson Inc., Middleton, WI), and a 3000 mass spectrometer with a turbo ion spray interface (PerkinElmerSciex Instruments, Thornhill, ON, Canada). An aliquot of the reconstitute (20 μl) was injected to an Asahipak ODP C₁₈ column (2.6 i.d. × 20 mm; Keystone Scientific, Inc., Charlotte, NC). The analytes and i.s. were eluted with a mobile phase composed of solvent A (95% water/5% acetonitrile, containing 0.01% acetic acid) and B (5% water/95% acetonitrile, containing 0.01% acetic acid) under the following gradient: 0 to 0.20 min, 100% of solvent A; 0.20 to 0.30 min, linear gradient from 100% solvent A to 100% solvent B; and then keep at 100% of solvent B for up to 1.5 min. The analyte and i.s. were monitored based on the ion pair (parent and daughter) that is specific to each compound at a collision energy of positive 40 V. The ion pairs (parent and daughter) under the current mass spectrometry conditions for diphenhydramine, hydroxyzine, triprolidine, cetirizine, loratadine, and desloratadine were 255.3/136.1, 374.9/202.2, 278.4/208.2, 388.9/202.2, 383.0/338.1, and 310.8/259.2, respectively. The peak areas of the analyte and i.s. were obtained using MacQuan (PerkinElmerSciex Instruments). The limits of quantitation were 1 to 10 ng/ml for plasma and brain homogenate. Validation of the analytical procedure was carried out and the quality control samples provided values within 20% of the added value throughout calibration range of 1 ng/ml to 10 μg/ml.

Estimation of Pharmacokinetic Parameters.

Noncompartmental model analysis was used to estimate the pharmacokinetic parameters (Gibaldi and Perrier, 1982) such as systemic clearance (CL, which was calculated based on the ratio between the dose and AUC_0-∞), volume of distribution at steady state (V_ss), and terminal half-life (t_1/2, which was calculated using a minimal of the last three concentration-time data). Only AUC_0-∞ was calculated for the brain concentration-time data for each compound. All calculations were based on the mean concentration-time data; each data point was the mean of three animals. Therefore, the pharmacokinetic parameter estimates are expressed only as mean. The brain partition of each H1-antagonist was estimated based on the brain-to-plasma AUC ratio, and the in vivo P-gp function was defined as the ratio of brain-to-plasma AUC ratio between KO and WT mice (Adachi et al., 2001).

Metabolism of Loratadine and Hydroxyzine in Vivo.

Cetirizine and desloaratadine, the major metabolites postdosing of hydroxyzine and loratadine, respectively, in mice were identified using LC-MS-MS with the aid of the standard of cetirizine and desloratadine. The plasma samples were pooled and were pretreated in the same way as for quantitation as described previously.

Transepithelial Transport of Two Sedating H1-Antagonists Triprolidine and Diphenhydramine and Two Nonsedating H1-Antagonists Cetirizine and Desloratadine in MDR1-MDCK Cells.

Cells obtained from the laboratory of Piet Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands) were seeded at a density of 2 × 10⁵ cell/ml on 96-well plates. Cells were incubated in a standard growth media at 37°C with 95% humidity and 5% CO₂ and maintained as described previously (Lala et al., 2000). For measurement of transepithelial transport of triprolidine, diphenhydramine, cetirizine, or desloratadine, each compound was applied to the donor side [either apical (A) or basolateral (B) side] at a concentration of 2 μM to initiate the transport study. Determining the amount of compound present at the receiving side after 5-h incubation at 37°C assessed the extent of permeation (P_app). Parallel studies were conducted in the parental MDCK cells to correct for non-MDR1-mediated transport. Additionally, known P-gp substrates quinidine (Kusuhara et al., 1997; Dagenais et al., 2001; Polli et al., 2001) and prazosin (Shapiro et al., 1999; Polli et al., 2001) were used as positive controls. All analyses were performed in triplicate. Quantitation of compounds was by modification of the methods described previously for brain homogenate. The P_app values were calculated for both A-to-B and B-to-A transport. The ratio of P_app values between B-to-A and A-to-B was defined as efflux ratio. The Student's t test was used to determine statistical significance in efflux ratio between the MDR1-MDCK and parental MDCK cells. Statistical significance was defined as ap value of less than 0.5.

Results

Plasma-Brain Disposition of Six H1-Antagonists in WT and KO Mice.

In general, the plasma concentration-time profiles were superimposible between the two strains of mice (Figs. 2a,3a, 4a, and5a). Consequently, no difference in systemic pharmacokinetic parameters between the two strains of mice was observed for H1-antagonists tested in the present study (Table1). The CL,V_ss, andt_1/2 values were comparable between KO and WT mice for all six H1-antagonists except cetirizine. The terminalt_1/2 for cetirizine for the KO mice was more than twice that for the WT mice. Among the six H1-antagonists, cetirizine has the smallest V_ss (1 l/kg) followed by diphenhydramine (1.3–1.6 l/kg), loratadine and triprolidine (∼2 l/kg), and hydroxyzine (2.4–3.7 l/kg). Likewise, cetirizine has the lowest CL (5.4–7.5 ml/min/kg), followed by desloratadine (22–23 ml/min/kg), diphenhydramine (62–66 ml/min/kg), hydroxyzine (70–80 ml/min/kg), loratadine (83–90 ml/min/kg), and triprolidine (119–134 ml/min/kg) (Table 1).

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Figure 2

Plasma (a) and brain (b) concentration-time profiles of diphenhydramine after a 5-mg/kg i.v. administration to KO (open symbols) and WT (solid symbols) mice.

Data are presented as mean ± S.D. (n = 3).

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Figure 3

Plasma (a) and brain (b) concentration-time profiles of triprolidine after a 5-mg/kg i.v. administration to KO (open symbols) and WT (solid symbols) mice.

Data are presented as mean ± S.D. (n = 3).

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Figure 4

Plasma (a) and brain (b) concentration-time profiles of hydroxyzine (I) and its active metabolite cetirizine (II and III) after a 5-mg/kg i.v. administration of hydroxyzine (I and II) and cetirizine (III) to KO (open symbols) and WT (solid symbols) mice.

Data are presented as mean ± S.D. (n = 3).

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Figure 5

Plasma (a) and brain (b) concentration-time profiles of loratadine (I) and its active metabolite desloratadine (II and III) after a 5-mg/kg i.v. administration of loratadine (I and II) and desloratadine (III) to KO (open symbols) and WT (solid symbols) mice.

Data are presented as mean ± S.D. (n = 3).

Similar to the fact that comparable plasma concentration-time data were observed between the two strains of mice, comparable brain concentration-time profiles between the KO and WT were also observed for the three sedating H1-antagonists diphenhydramine, triprolidine, and hydroxyzine (Figs. 2b, 3b, and 4I.b). Furthermore, these sedating H1-antagonists showed great and rapid brain penetration with peak brain concentration at 2 min postdose (Figs. 2b, 3b, and 4I.b). The brain-to-plasma AUC ratios were equally high for the KO and WT mice (Table 2). Consequently, the ratio of brain-to-plasma AUC ratio between the two strains of mice was approximately 1 for the sedating H1-antagonists (Table 2).

In contrast to the sedating H1-antagonists, the nonsedating H1-antagonists exhibited different brain disposition between the KO and WT mice. The brain concentration-time profiles for cetirizine, loratadine, and desloratadine are shown in Figs. 4, II-III.b, 5I.b, and Fig. 5III.b, respectively. In general, for the nonsedating H1-antagonists, the brain-to-plasma AUC ratio is much higher in KO than WT mice because of the much higher brain AUC but comparable plasma AUC in KO compared with WT mice (Table 2). For cetirizine, brain concentration-time profile in KO mice differed greatly from that of WT postdosing either cetirizine (Fig. 4III.b) or its precursor hydroxyzine (Fig. 4II.b). After dosing hydroxyzine, KO mice showed slightly higher plasma cetirizine AUC than WT mice (418 versus 287 μg/ml × min). However, KO mice had a brain AUC of 67 μg/ml × min, whereas WT mice had a brain level of below the limit of quantitation of the current assay (1 ng/ml) (Fig. 4II.b). After dosing cetirizine, the brain C_max value was 0.09 and 0.28 μg/ml for WT and KO mice, respectively. The brain-to-plasma AUC ratio was 4-fold higher in KO than in WT mice (Table 2).

Likewise, loratadine also had a higher brain-to-plasma AUC ratio in KO compared with WT mice (2.8 versus 1.4). The brain concentration of loratadine peaked at 2 min postdose and had aC_max value of 14.7 and 6.9 μg/ml for KO and WT mice, respectively. However, it cleared from the brain very efficiently with more rapid clearance from brain in WT than KO mice (Fig. 5I.b). The active metabolite of loratadine, desloratadine, was also determined in plasma and brain tissue postdosing of loratadine. No significant difference in the formation of desloratadine in the two strains of mice was observed in plasma (Fig. 5II.a). The AUC was 25 and 30 μg/ml × min for WT and KO, respectively. Additionally, no detectable brain concentration of desloratadine was observed postloratadine administration in either strain of mice. However, when desloratadine was dosed directly to mice, no brain concentration was detected in WT mice except at 5 min postdose, whereas a high level of desloratadine was found in KO mouse brains (Fig. 5III.b). The brain AUC of desloratadine in KO mice was 3548 μg/ml × min.

Metabolism of H1-Antagonists in Vivo.

The active metabolite for hydroxyzine and loratadine was cetirizine and desloratadine, respectively. This was confirmed with LC-MS-MS fragmentation of the metabolite compared with that of the corresponding standard (data not shown).

Transepithelial Transport of the Two Sedating H1-Antagonists Triprolidine and Diphenhydramine and the Two Nonsedating H1-Antagonists Cetirizine and Desloratadine in MDR1-MDCK and parental MDCK Cells.

Initial experiment showed that transporter was linear within a 5-h incubation time period (data not shown). Therefore, 5 h was used as the duration for the transporter study. As shown in Table 2, both cetirizine and desloratadine showed asymmetric permeability with an efflux ratio of 5.8 and 9.1, respectively. The efflux was primarily associated with MDR1 as indicated by the low efflux in the parental MDCK cells. In contrast, the efflux ratio for triprolidine and diphenhydramine was approximately 1 in either MDR1-MDCK or parental MDCK cells. The positive controls quinidine and prazosin showed an efflux ratio of 26.5 and 4.3 in MDR1-MDCK and 2.3 and 1.9 in parental MDCK cells, respectively (Table 2).

Discussion

It is well known that first generation H1-antagonists are sedating and the second generation H1-antagonists are either nonsedating or less sedating. Factors such as physical-chemical properties (mol. wt., log D, and ionization), and active transporters (uptake and efflux) at the blood-brain barrier (BBB) may determine the blood-brain translocation of xenobiotics. Difference in the physical-chemical properties may play a role in determining the extent of brain penetration and CNS exposure of these compounds. For example, sedating H1-antagonists in general have smaller mol. wt. (<350) compared with that of nonsedating H1-antagonists (340–502). However, mol. wt. alone cannot explain the difference in brain penetration. For example, desloratadine, the active metabolite of loratadine, has a comparably small mol. wt. (338.9) to that of hydroxyzine (347.9), yet no desloratadine versus ∼8 μg/ml hydroxyzine was observed in brain tissue of WT mice 2 min after the same i.v. dose. Few studies have been conducted to examine whether there is a difference in plasma-brain translocation of sedating versus nonsedating H1-antagonists and if there is a difference, whether the difference is due to certain active transporters at BBB.

One potential active transporter system at BBB is P-gp. P-gp has been shown to decrease brain penetration of a variety of compounds with diverse structures (Schinkel et al., 1994, 1997; Schinkel, 1998; Chen and Pollack, 1999). There has been some speculation about a potential link between nonsedation of second generation H1-antagonists and P-gp efflux nature (Timmerman, 1999, 2000). However, only a few studies have shown that nonsedating H1-antagonists are P-gp substrates, thus reducing their brain penetration, which may limit their CNS side effects. Fexofenadine is the only nonsedating H1-antagonist that has been shown to be a P-gp substrate in both in vitro such as MDR1-transfected systems and in vivo mdr1a KO mice (Cvetkovic et al., 1999; Soldner et al., 1999). The present study was conducted to investigate the plasma-brain translocation and the role of P-gp in brain penetration of sedating and nonsedating H1-antagonists using mdr1a/b KO mice. Additionally, we tested two sedating and two nonsedating H1-antagonists in in vitro MDR1-MDCK cells.

In general, the brain-to-plasma AUC ratio, which reflects brain penetration, is much higher for the sedating H1-antagonists (3.80–9.00) compared with the nonsedating ones (0.02–1.65) in WT mice whose brain endothelium contains P-gp. The significant reduction in brain penetration for nonsedating H1-antagonists compared with sedating ones in WT mice is consistent with the fact that P-gp acts as an efflux pump that extrudes its substrates. The partitioning of P-gp substrates to the brain in mdr1a/b KO mice should no longer be restricted by P-gp and more governed by their physical-chemical properties. Therefore, a ratio of brain-to-plasma AUC ratio of approximately 1 between KO and WT mice for sedating H1-antagonists diphenhydramine, triprolidine, or hydroxyzine was observed, suggesting nonsedating H1-antagonists are not P-gp substrates. In contrast, a 2.0-, 4.4-, and >14-fold higher brain-to-plasma AUC ratio was observed in KO compared with WT mice for nonsedating H1-antagonists loratadine, cetirizine, and desloratadine, respectively, indicating that nonsedating H1-antagonists are P-gp substrates.

The fact that nonsedating H1-antagonists cetirizine and desloratadine are P-gp substrates was further substantiated by the results after dosing their precursors hydroxyzine and loratadine, respectively. The formation of cetirizine from hydroxyzine did not differ with mouse strains as indicated by the similarity of systemic exposure of cetirizine posthydroxyzine administration. Yet, significant cetirizine was measured in the brains of KO but not WT mice. Likewise, formation of plasma desloratadine was comparable between KO and WT mice, even though there was no detectable brain concentration of desloratadine after dosing loratadine in WT or KO mice. This may be due to low plasma desloratadine concentration, and subsequently even lower brain concentration, which may be below the limit of quantitation of the current assay for desloratadine (1 ng/ml). It has been shown that cetirizine (Paakkari, 2002) and desloratadine (Clissold et al., 1989) are the active metabolite in humans for hydroxyzine and loratadine, respectively. The metabolism of loratadine to desloratadine in humans is mainly mediated by a combination of cytochrome P450 2D6 and 3A4 (Yumibe et al., 1996). In contrast, it is not known regarding the specific enzyme that is responsible for the formation of cetirizine from hydroxyzine (Paakkari, 2002). Regardless of the enzymes involved in the formation of active metabolite from hydroxyzine and loratadine, a comparable systemic exposure of the active metabolite for hydroxizine and loratadine between KO and WT mice suggests the similarity in these metabolizing enzymes between the two strains of mice.

Consistent with the in vivo mouse data, which strongly indicate that P-gp mediates the brain-to-plasma efflux of nonsedating but not sedating H1-antagonists, the in vitro MDR1-MDCK results also demonstrate a significant MDR1-associated efflux for the two nonsedating but not for the two sedating H1-antagonists. The MDR1-MDCK data suggest that nonsedating H1-antagonists are, whereas sedating H1-antagonists are not MDR1 substrates, consistent with results from the in vivo mouse study.

In contrast to our finding, Wang et al. (2001) showed that desloratadine failed to significantly increase in basal ATPase activity, suggesting that desloratadine is unlikely a P-gp substrate under the experimental conditions. Unlike the MDR1-MDCK cell assay that directly measures transport of potential P-gp substrates, ATPase assay is a generic readout on the release of inorganic phosphate and does not directly measure transport (Scarborough, 1995; Polli et al., 2001).Polli et al. (2001) demonstrated that there was no instance of a compound that was positive in the ATPase assay and negative in other assays, including transport assay using MDR1-MDCK cells. In contrast, the opposite was true. For example, daunorubicin showed a significant efflux in MDR1-MDCK cells with an efflux ratio of 14.2 but an ambiguous readout in the ATPase assay (Polli et al., 2001). Therefore, the ATPase assay may provide false negative results in the case of desloratadine. Although the clinical implications are unknown and worthy of further study, the present study provides the first direct evidence that desloratadine is a P-gp substrate using both mdr1a/b KO mice and human MDR1-expressed cells.

In summary, the present study has demonstrated for the first time that sedating H1-antagonists hydroxyzine, diphenhydramine, and triprolidine are not P-gp substrates; they show equal brain penetration betweenmdr1a/b KO and WT mice and equal bidirectional (A-to-B and B-to-A) permeability in MDR1-MDCK cells. In contrast, nonsedating H1-antagonists cetirizine, loratadine, and desloratadine are P-gp substrates; they exhibit significantly reduced brain exposure in WT compared with KO mice and significant MDR1-associated efflux in MDR1-MDCK cells. Our results at least partially explain the reduced or devoid CNS side effects for nonsedating H1-antagonists and suggest that optimization of affinity toward H1-receptor and P-gp may be a useful strategy to select H1-antagonists with desired potency and without potential CNS sedating effect.