1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

DMPK31_proof ■ 2 February 2015 ■ 1/6

Drug Metabolism and Pharmacokinetics xxx (2015) 1e6

Contents lists available at ScienceDirect

Drug Metabolism and Pharmacokinetics journal homepage: http://www.journals.elsevier.com/drug-metabolism-andpharmacokinetics

Regular article

Effects of Cremophor EL on the absorption of orally administered saquinavir and fexofenadine in healthy subjects Q3

Atsuko Tomaru a, Mariko Takeda-Morishita a, c, *, Kazuya Maeda b, Hirokazu Banba a, Kozo Takayama a, Yuji Kumagai d, Hiroyuki Kusuhara b, Yuichi Sugiyama b, e a

Department of Pharmaceutics, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo, 142-8501, Japan Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan Faculty of Pharmaceutical Sciences, Kobe Gakuin University, 1-1-3 Minatojima, Chuo-ku, Kobe, Hyogo, 650-8586, Japan d Clinical Trial Center, Kitasato East Hospital, 2-1-1 Azamizodai, Minami-ku, Sagamihara, Kanagawa, 252-0380, Japan e Sugiyama Laboratory, RIKEN Innovation Center, RIKEN Research Cluster for Innovation, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan b c

Q1

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 December 2014 Accepted 15 January 2015 Available online xxx

Modulation of CYP3A and/or P-gp function by several excipients has been reported. However, relatively few studies have investigated their effects in humans. Therefore, the aim of this clinical study was to clarify the effects of Cremophor EL on the inhibition of CYP3A and P-gp in the human small intestine. Eight healthy Japanese subjects received an oral dose of saquinavir (2 mg, substrate of P-gp/CYP3A) or fexofenadine (50 mg, substrate of P-gp) without or with Cremophor EL (720 mg and 1440 mg). Significant increases in Cmax (1.3-fold) and AUC0e24 (1.6-fold) were observed for fexofenadine when administered with 1440 mg of Cremophor EL. In contrast, a significant decrease was observed for saquinavir when administered with 720 mg of Cremophor EL. The equilibrium dialysis experiment was performed to investigate the micellar interaction between Cremophor EL and drugs. The equilibrium dialysis study showed that saquinavir was far extensively entrapped into the micelles. The reduced concentration of free saquinavir by entrapping in micelles was considered to cause the reduction of systemic exposure for saquinavir. In conclusion, this clinical study suggests that Cremophor EL at least inhibits P-gp in the human small intestine.

Keywords: Saquinavir Fexofenadine Cremophor EL P-gp Oral absorption

Copyright © 2015, The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

1. Introduction The low or variable bioavailability of a drug is considered to be one of the reasons to terminate its development as an oral pharmaceutical product. Low oral bioavailability of drugs can be caused by the drug's low solubility, low intrinsic membrane permeability, high first-pass effect including metabolic enzymes and efflux transporters expressed in the apical membrane of small intestine, or combinations thereof. Therefore, it is worth clarifying the reason of the low or variable bioavailability to overcome them. Recently, it has been reported that several excipients can modulate the activities of efflux transporters, such as p-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance associated protein 2 (MRP2) [1e3]. Excipients are extensively used in human pharmaceutical formulations, therefore, it is important to

* Corresponding author. E-mail address: [email protected] (M. Takeda-Morishita).

know that detailed characteristics of excipients as inhibitors of efflux transporters and/or metabolic enzymes. Several clinical studies have also evaluated the inhibitory effects of excipients on the oral absorption of drugs [4e7]. In most of these clinical studies, d-a-tocopheryl polyethylene glycol 1000 succinate (VE-TPGS) was used as the inhibitor of efflux transporters, and the AUCs of talinolol and cyclosporine increased 1.4- and 1.6-fold, respectively, after their coadministration with VE-TPGS [8]. Regarding excipients other than VE-TPGS, Martin-Facklam et al. reported that the AUC0e∞ for saquinavir (SQV) increased 5-fold when it was administered with 5000 mg of Cremophor EL [9]. Cremophor EL, a polyethoxyethylated castor oil, is a non-ionic excipient, and is used as the solvent for SandimmunⓇ (cyclosporine) and TaxolⓇ (paclitaxel). SQV is an HIV-1 protease inhibitor and its oral bioavailability in humans is about 4% [10]. The poor bioavailability of SQV is attributed to the extensive first-pass effect in the liver, and pre-systemic metabolism and active efflux in the small intestine because it is a substrate of CYP3A and P-gp. Martin-Facklam et al. demonstrated

http://dx.doi.org/10.1016/j.dmpk.2015.01.002 1347-4367/Copyright © 2015, The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Tomaru A, et al., Effects of Cremophor EL on the absorption of orally administered saquinavir and fexofenadine in healthy subjects, Drug Metabolism and Pharmacokinetics (2015), http://dx.doi.org/10.1016/j.dmpk.2015.01.002

55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

DMPK31_proof ■ 2 February 2015 ■ 2/6

2

A. Tomaru et al. / Drug Metabolism and Pharmacokinetics xxx (2015) 1e6

that Cremophor EL increased the AUC0e∞ for SQV without affecting its elimination half-life [9]. This suggests that Cremophor EL primarily acts as a modulator of the absorption process by inhibiting P-gp and/or CYP3A. Moreover, the logP of SQV is 4.4, and SQV has poor aqueous solubility [11]. Therefore, Cremophor EL may increase the solubility of SQV in the lumen of intestine, and apparently facilitate its absorption from the gastrointestinal mucosa. However, the effects of Cremophor EL on CYP3A and P-gp have not been clarified in humans. The purpose of this study is to clarify the effect of Cremophor EL on the inhibition of CYP3A and P-gp in humans. In this study, SQV and fexofenadine (FEX) were used as the substrates of P-gp/CYP3A and P-gp, respectively. In the clinical study, each drug was administered as a solution to minimize the effect of Cremophor EL on its solubility. Therefore, the effect of Cremophor EL on drug solubility was negligible. Furthermore, an equilibrium dialysis experiment was performed to confirm the results of the clinical study. 2. Materials and methods 2.1. Materials Saquinavir mesylate was purchased from Sequoia Research Products (Pangbourne, UK). Fexofenadine hydrochloride, fexofenadine-d6 and saquinavir-d9 were purchased from Tronto Research Chemicals Inc. (Ontario, Canada). Cremophor EL was purchased from SigmaeAldrich Co. (St Louis, MO, USA). Gelatin capsules were purchased from Matsuya Corporation (Osaka, Japan). All other chemicals were of analytical grade and are commercially available. 2.2. Subjects Eight healthy male volunteers, ranging from 20 to 40 years and in body weight from 55 to 69 kg, who gave their informed consent, were enrolled in the study. All subjects were confirmed to be healthy with a physical examination and routine clinical testing, with no history of significant medical illness or hypersensitivity to any drug. This study was approved by the Institutional Review Board at Kitasato East Hospital and the University of Tokyo. The clinical study was conducted in accordance with the Declaration of Helsinki and current Japanese ethical guidelines for clinical research and registered in the UMIN Clinical Trials Registry at www. umin.ac.jp/ctr/index.htm (UMIN000004621). 2.3. Clinical study design This study was a single-centre, open-label three-phase crossover study. The subjects received 50 mg of FEX or 2 mg of SQV in period I. After a seven-day wash out period, the subjects received 50 mg of FEX or 2 mg of SQV immediately after administration of 720 mg of Cremophor EL (period II). After another seven-day wash out period, the subjects received 50 mg of FEX or 2 mg of SQV immediately after 1440 mg of Cremophor EL administration (period

III). The dosing regimen is shown in Fig. 1. SQV and FEX were dissolved in water for injection, then made 0.2 mg base/mL and 5 mg base/mL solutions, respectively. Cremophor EL was filled in gelatin capsules (26.1  9.53 mm). The subjects were not allowed food or beverages containing alcohol from 10 h before the administration of the drug until 4 h after its administration. Food and beverages containing grapefruits, orange, or apple were excluded from seven days before the study commenced until its completion. Blood samples (7 mL) were collected by direct venipuncture (sodium heparin anticoagulant) before dosing and 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 24 h after dosing. The blood samples were placed immediately on ice and centrifuged at 3000 rpm for 10 min. The plasma samples were stored at 80  C until analysis.

2.4. Determination of SQV and FEX concentrations in human plasma The human plasma samples were pretreated as follows to determine the SQV concentrations. Each plasma sample (200 mL) was pipetted into a test tube and spiked with 10 mL of the internal standard (saquinavir-d9 100 pg/mL) and 500 mL of deionized water. The tube was shaken and the sample loaded onto an OASIS HLB 96well plate (Nihon Waters K.K., Tokyo, Japan). Each extraction column was preconditioned with 1 mL of methanol and 1 mL of deionized water. After the sample was loaded, the column was washed with 1 mL of deionized water and the sample was eluted with 200 mL of methanol. The mobile phase was added to this elution and vortex mixed, and then 20 mL of the mixture was injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS). To determine the FEX concentrations, the human plasma samples were pretreated as follows. Each plasma sample (20 mL) was pipetted into a test tube and spiked with 20 mL of the internal standard (fexofenadine-d6 1 ng/mL) and 150 mL of methanol. The tube was shaken and centrifuged at 12,000  g for 2 min. After centrifugation, the supernatant was filtrated with Ultrafree-MC (0.45 mm, polyvinylidene difluoride; Millipore Co., Billerica, MA, USA). The mobile phase was added to this filtered solution and vortex mixed, and then 10 mL of the mixture was injected into the LC-MS/MS. The plasma concentrations of SQV and FEX were analyzed by LC-MS/MS in turbo ion spray, positive ion mode, equipped with a Prominence LC system (Shimadzu Co., Kyoto, Japan) coupled to an API5000 mass spectrometer (AB SCIEX, San Jose, CA, USA). The mass transitions were m/z 671.4 to 570.3 (SQV), 680.4 to 570.3 (SQV-d6), 502.3 to 466.2 (FEX), and 508.3 to 472.1 (FEX-d9). Chromatographic separation was performed at 40  C on a CAPCELL PAK C18 MG column (4.6  35 mm, 5 mm; Shiseido Co., Ltd, Tokyo, Japan) under gradient conditions at a flow rate of 0.5 mL/ min. The mobile phase was 10 mM ammonium formate/formic acid (1000:1,v/v) as solvent A and methanol as solvent B. The following linear gradient steps were used: (A:B) 0e2 min, 50:50 / 10:90; 2e4 min, 10:90 / 10:90, and then return to the initial conditions, followed by column equilibration. The data were acquired with Analyst ver. 1.4.2 (AB SCIEX). Calibration ranges were 1e100 pg/mL for SQV and 5e5000 pg/mL for FEX. The accuracy was 1.86%e

Fig. 1. Dosing regimen for the clinical study.

Please cite this article in press as: Tomaru A, et al., Effects of Cremophor EL on the absorption of orally administered saquinavir and fexofenadine in healthy subjects, Drug Metabolism and Pharmacokinetics (2015), http://dx.doi.org/10.1016/j.dmpk.2015.01.002

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

DMPK31_proof ■ 2 February 2015 ■ 3/6

A. Tomaru et al. / Drug Metabolism and Pharmacokinetics xxx (2015) 1e6

3

8.76% for SQV and 15.5%e2.55% for FEX. The precision was less than 10.8% for SQV and 20.2% for FEX. All clinical samples were measured within 3 freeze/thaw cycles.

under the plasma concentration versus time curve from time 0 to the last measurable concentration (AUC0-t) was calculated with the linear trapezoidal rule.

2.5. Equilibrium dialysis experiment

2.7. Statistical analysis

SQV and FEX were dissolved in water at concentrations of 0.04 mg base/mL and 1 mg base/mL, respectively. Phosphate buffer (pH6.7) was placed in a 200 mL bottle as the outer fluid. A cellulose tube (Spectra/Por® 6 Dialysis Membrane with a 1000 molecularweight cut-off, Spectrum Laboratory Inc., CA, Canada) containing 10 mL of SQV or FEX solution with or without Cremophor EL was ligated at both ends and placed into the outer fluid. This bottle was placed in a shaking incubator with stirring at 37  C. At specific time intervals, 1 mL of the outer fluid was sampled to analyze the concentration of the drug and 1 mL of fresh buffer was added to the bottle. The concentrations of SQV and FEX in the outer fluid were determined by HPLC with ultraviolet detection (HPLC-UV), as described below. HPLC was performed with a Prominence LC system (Shimadzu Co.), equipped with a Hypersil Gold column (3.0  50 mm, 5 mm; Thermo Fisher Scientific K.K., Tokyo, Japan). The mobile phases were 60 mM Na2HPO4 in 35% acetonitrile for SQV and 3 mM phosphate buffer (pH6) in 35% acetonitrile for FEX. The flow rate was 0.4 mL/min at 40  C and the elution was monitored at wavelengths of 254 nm for SQV and 220 nm for FEX. The entrapped ratio of drugs into the micelles of Cremophor EL was calculated with the following equation. The entrapped ratio of drugs into micelles (%) ¼ [1  (drug concentration in outer fluid with Cremophor EL/drug concentration in outer fluid without Cremophor EL)]  100.

Each value is expressed as a mean ± standard deviation (SD). Statistical significance was assessed with a paired t test in the clinical study. A p value of less than 0.05 was considered significant. 3. Results 3.1. Clinical study No clinically undesirable signs and symptoms possibly attributable to the administration of SQV, FEX, or Cremophor EL were observed during the study. All subjects successfully completed the study according to the protocol. The plasma concentrationetime profiles are shown in Figs. 2 and 3. The pharmacokinetic parameters are shown in Tables 1 and 2. The Cmax and AUC0e24 for SQV were significantly lower in the group treated with 720 mg of Cremophor EL than in the control group. There was no significant difference in the Cmax or AUC0e24 between the control group and the group treated with 1440 mg Cremophor EL. In contrast, both the Cmax and AUC0e24 for FEX increased in a dose-dependent manner and these increases were significant in the group treated with 1440 mg of Cremophor EL (1.6fold in Cmax and 1.3-fold in AUC0e24). 3.2. Equilibrium dialysis experiment

The maximum plasma concentration (Cmax) and the time reach to Cmax (tmax) were obtained directly from the raw data. The area

The micellar interaction between Cremophor EL and SQV or FEX was examined. The theoretical concentrations of SQV and FEX without Cremophor EL in the outer fluid were calculated as 2.27 and 0.12 mM, respectively. The drug recovery against the theoretical concentration was calculated as approximately 100% for SQV and

Fig. 2. Effects of Cremophor EL on the systemic exposure to SQV following oral administration to healthy subjects. SQV (2 mg) was given orally to healthy male subjects without or with Cremophor EL (Period I: without Cremophor EL, Period II: with 720 mg Cremophor EL, Period III: with 1440 mg Cremophor EL). Plasma concentrations of SQV were determined at designated times. Each point represents the mean ± SD (n ¼ 8).

Fig. 3. Effects of Cremophor EL on the systemic exposure to FEX following oral administration to healthy subjects. FEX (50 mg) was given orally to healthy male subjects without or with Cremophor EL (Period I: without Cremophor EL, Period II: with 720 mg Cremophor EL, Period III: with 1440 mg Cremophor EL). Plasma concentrations of FEX were determined at designated times. Each point represents the mean ± SD (n ¼ 8).

2.6. Pharmacokinetic calculations

Please cite this article in press as: Tomaru A, et al., Effects of Cremophor EL on the absorption of orally administered saquinavir and fexofenadine in healthy subjects, Drug Metabolism and Pharmacokinetics (2015), http://dx.doi.org/10.1016/j.dmpk.2015.01.002

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

DMPK31_proof ■ 2 February 2015 ■ 4/6

4

A. Tomaru et al. / Drug Metabolism and Pharmacokinetics xxx (2015) 1e6

Table 1 Pharmacokinetic parameters of SQV given orally with or without Cremophor EL in clinical study. Period

Tmax (hr)

Cmax (pg/mL)

AUC0e24 (pg$h/mL)

I II III

0.5 0.5 0.5

58.8 ± 22.8 31.0 ± 6.4** 51.4 ± 16.2

90.1 ± 24.7 63.3 ± 33.3* 80.6 ± 38.5

Data represents the mean ± SD (n ¼ 8) *: p < 0.05, **p < 0.01. Period I: without Cremophor EL, Period II: with 720 mg Cremophor EL, Period III: with 1440 mg Cremophor EL.

90% for FEX. As shown in Fig. 4, the entrapped ratio of drugs into micelles (%) for FEX with Cremophor EL reached at a plateau at around 36 h, whereas it has not reached at a plateau within 48 h for SQV. However, there is a big difference of concentrations in the outer fluid in the presence and in absence of Cremophor EL, it may not be very important whether the SQV concentration reached at a plateau or not. The ratio of entrapped of drugs into Cremophor EL micelles (%) at 48 h was 90.2e92.8% for SQV and 19.0e24.0% for FEX (Table 3). 4. Discussion The purpose of this study is to clarify the effects of Cremophor EL on the intestinal absorption and the mechanism underlying its improvement of the bioavailability of various orally administered drugs. Generally, the maximum inhibitory effect can be evaluated in the linear region, in which the metabolism of the drug and the membrane permeability are not saturated. In this study, assuming that the apparent volume of the intestine is 2.8e11 L, which is the reasonable intestinal volume for prediction of the drugedrug interaction by using in vitro Ki value [12], the intestinal concentrations of FEX and SQV were calculated to be about 0.008e0.03 nM and 0.27e1.06 mM, respectively. The Km values for FEX was 150 mM and SQV was 20 mM as substrate of P-gp [13,14], therefore, their intestinal concentrations were thought to be very low compared with their Km values. In addition, the Km values for SQV as substrate of CYP3A was 0.61 mM and the intestinal concentrations was thought to be around Km values [15]. SQV dose was selected as 2 mg in the present clinical study, because our prediction suggested that 2 mg dose might be necessary to analyze quantitatively the plasma concentrationetime profile of SQV. Although the dose of SQV is higher than that of FEX in this study, the maximum inhibitory effects of Cremophor EL could be assessed because the estimated intestinal concentration was under the linear region. A dose-dependent effect of Cremophor EL on the absorption of orally administered FEX was observed and the Cmax and AUC0e24 for FEX increased significantly after its coadministration with 1440 mg of Cremophor EL. These results suggest that Cremophor EL inhibited P-gp and thus enhanced the bioavailability of orally administered FEX. However, when we consider that the Fa$Fg of FEX is from 0.31 to 0.48 in humans [12,16], the estimated maximum increase in

Table 2 Pharmacokinetic parameters of FEX given orally with or without Cremophor EL in clinical study. Period

Tmax (hr)

Cmax (ng/mL)

AUC0e24 (ng$h/mL)

I II III

1.0 1.0 1.5

0.195 ± 0.0840 0.240 ± 0.0881 0.302 ± 0.123**

1.26 ± 0.312 1.34 ± 0.456 1.61 ± 0.600*

Data represents the mean ± SD (n ¼ 8) *: p < 0.05, **p < 0.01. Period I: without Cremophor EL, Period II: with 720 mg Cremophor EL, Period III: with 1440 mg Cremophor EL.

Fig. 4. SQV (A) and FEX (B) concentrations in the outer fluid after incubation up to 48 h in the equiblibrium dialisys experiment. Each data represents the mean ± SD (n ¼ 4).

AUC was about two to three-fold when P-gp is fully inhibited by Cremophor EL in the small intestine. Therefore, a higher dose of Cremophor EL is required to inhibit the function of P-gp completely. However, a maximum dose of Cremophor EL used in commercial drugs is 1440 mg [17]. For this reason, it was difficult to use a dose of more than 1440 mg in this clinical study. On the contrary to the results of FEX in the clinical study, the AUC0e24 for SQV was significantly reduced when SQV was coadministered with 720 mg of Cremophor EL, whereas the AUC0e24 for SQV coadministered with 1440 mg of Cremophor EL was almost the same as the control. In general, excipients such as Cremophor EL form micelles at concentrations above the critical micelle concentrations (cmc) and hydrophobic compounds like SQV are entrapped

Table 3 Micellar interaction between Cremophor EL and SQV or FEX. Cremophor EL (mg)

720 1440

Ratio of entrapped of drugs into Cremophor EL micelles (%) at 48 h FEX

SQV

19.0 ± 2.3 24.0 ± 3.8

90.2 ± 0.9 92.8 ± 0.5

Data represents the mean ± SD (n ¼ 4).

Please cite this article in press as: Tomaru A, et al., Effects of Cremophor EL on the absorption of orally administered saquinavir and fexofenadine in healthy subjects, Drug Metabolism and Pharmacokinetics (2015), http://dx.doi.org/10.1016/j.dmpk.2015.01.002

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

DMPK31_proof ■ 2 February 2015 ■ 5/6

A. Tomaru et al. / Drug Metabolism and Pharmacokinetics xxx (2015) 1e6

in the micelles. As a result, the free concentrations of these hydrophobic compounds decrease, reducing their absorption. In fact, concentrations of the excipients Tween 80, Pluronic P85 and Cremophor EL above their cmc values have been shown to cause significant reductions in the cellular accumulation of P-gp substrates, corresponding to reductions in the substrate-free fraction [18,19]. Bardelmeijer et al. investigated the effect of Cremophor EL on the bioavailability of orally administered paclitaxel using Mdr1ab knockout mice. In their study, paclitaxel was administered to the Mdr1a/1b knockout mice with either the conventional amount of Cremophor EL (controls) or sevenfold greater amount (test group). As a result, the AUC for paclitaxel was reduced in the test group, and this result suggested that Cremophor EL entrapped paclitaxel in the gastrointestinal lumen by incorporating it into micelles, thus reducing the absorption of the drug [20]. Furthermore, Zastre et al. compared the micelle association and Caco-2 cellular accumulation of two structurally homologous P-gp substrates using diblock copolymers, the relatively hydrophobic rhodamine-6 glucronide and the hydrophilic rhodamine 123, at excipient concentrations above and below the cmc [21]. The accumulation of rhodamine 123 was enhanced below the excipient cmc, but there was a concentrationdependent reduction in its accumulation above the excipient cmc. These observations suggest that it is important that excipients exist as monomers in the small intestine, and that the cmc is a key factor in determining the inhibitory effect. In our clinical study, the intestinal concentration of Cremophor EL after 720 mg was administered was calculated to be 26e102 mM when the intestinal volume was assumed to be 2.8e11 L 12. Because the cmc of Cremophor EL is 50 mM [22], micelles may have formed in the small intestine. Therefore, the sequestration of the substrate within micelles would have reduced the concentration of the substrate available for cellular accumulation, causing both the Cmax and AUC0e24 of the drug to decrease. To confirm this hypothesis, an equilibrium dialysis experiment was performed and the % of FEX and SQV entrapped into micelles were compared. At first, the concentrations of SQV and FEX were set at the same concentration as that estimated by calculation from apparent intestinal volume and dosage used in the clinical study. However, SQV was precipitated in the cellulose tube during the incubation. In fact, the concentrations of substrates in the small intestine were thought to be lower than those in this equilibrium dialysis study because it was diluted by large volume of intestinal fluid. Therefore, the equilibrium dialysis experiment was conducted at the concentration reduced by one-fifth of the dose of the clinical study. As a result, the entrapped ratio of SQV into the micelles of Cremophor EL was about fivefold higher than that of FEX, and it was clearly shown that SQV was far extensively entrapped in the micelles. Based on these results, it was considered that the reduced concentration of free SQV in the small intestine caused the reduction in the AUC0e24 of SQV relative to that of the control in our clinical study. The AUC0e24 for SQV coadministered with 1440 mg of Cremophor EL was almost the same as the control. From the results of the equilibrium dialysis experiment, the entrapped ratios of SQV into micelles of Cremophor EL 720 mg and Cremophor 1440 mg were the same. Therefore, it is estimated that the concentration of free SQV in the small intestine when coadministered with 1440 mg of Cremophor EL is the same as that with 720 mg of Cremophor EL. In addition, the amount of monomer in the small intestine may be high when coadministered with 1440 mg of Cremophor EL compared to 720 mg of Cremophor EL, because not all excipient form micelles above the cmc. Thus, the inhibition of CYP3A/P-gp by the monomer resulted in the increase of absorption and became comparable to the control. In addition, Martin et al. investigated an effect of excipients on cytochrome P450 activity, and IC50 of Cremophor EL against the

5

CYP3A4 activity was reported as 306 mM [23]. The estimated intestinal concentration of Cremophor EL in our clinical study was lower than the IC50 value even at 1440 mg of Cremophor EL (52e204 mM). Therefore, the inhibition effect of Cremophor EL on the CYP3A activity might not be observed clearly in our study. By contrast, IC50 of Cremophor EL against P-gp has been reported as 0.009% (approximately 9 mM) [24]. In addition, IC50 of a related Cremophor RH40 has been reported as 0.3% for CYP3A and 0.03% for P-gp 6. In this way, the inhibition effect of Cremophor EL may be more potent against p-gp than CYP3A. Martin-Facklam et al. reported that the AUC0e∞ of SQV was increased 1.4-fold by its coadministration with 1000 mg of Cremophor EL in their clinical study. However, no significant increase in the AUC0e24 was observed at almost the same dose of Cremophor EL in our clinical study. This discrepancy in the effect of Cremophor EL on the systemic exposure to SQV is attributed to a difference in the method of administration and dose in the two clinical studies. In the study of Martin-Facklam et al., 600 mg of SQV was administered by mixing the contents of Invirase® capsules with the appropriate amount of Cremophor EL and dispensing the mixture in hard gelatin capsules. However, in our study, we set the low dose of SQV to dissolve completely and SQV was administered after its complete dissolution in water. When drugs are administered as solid formulations, they are absorbed after the processes of collapse, dissolution, and dispersion in the small intestine. Excipients may solubilize poorly soluble drugs like SQV, increasing their absorption in this way. An oral bioavailability and FaFg values of SQV have been reported as about 4% 10 and 0.114 13, respectively. In addition, SQV has very low solubility (logP is 4.4) 11, therefore, solubilization is also the important factor for increasing the absorption of SQV. Thus, the increase in the AUC0e∞ for SQV observed in the clinical study of Martin-Facklam et al. was also attributable to the improved solubility of SQV, in addition to the inhibitory effects of Cremophor EL on P-gp. In conclusion, our results suggest that Cremophor EL at least inhibits P-gp in the small intestine in humans to some extent, resulting in an increase in the bioavailability of orally administered P-gp substrate drugs. Therefore, excipients may be effective for increasing poor bioavailability of P-gp substrate drugs. However, for compounds which are easily entrapped into micelle, only a dose of excipient below its cmc in the gastrointestinal tract may achieve an effective inhibitory effect. Acknowledgments This study is a part of a research project for the “Establishment of Evolutional Drug Development with the Use of Microdose Clinical Trials” sponsored by the New Energy and Industrial Technology Development Organization (NEDO). We thank Takeshi Okuzono and his colleague (Sekisui Medical Co., Ltd.) for an analysis of clinical sample. References [1] Yu L, Bridgers A, Polli J, Vickers A, Long S, Roy A, et al. Vitamin E-TPGS increases absorption flux of an HIV protease inhibitor by enhancing its solubility and permeability. Pharm Res 1999;16(12):1812e7. [2] Hugger ED, Novak BL, Burton PS, Audus KL, Borchardt RT. A comparison of commonly used polyethoxylated pharmaceutical excipients on their ability to inhibit P-glycoprotein activity in vitro. J Pharm Sci 2002;91(9):1991e2002. [3] Rege BD, Kao JPY, Polli JE. Effects of nonionic surfactants on membrane transporters in Caco-2 cell monolayers. Eur J Pharm Sci 2002;16:237e46. [4] Boudreaux JP, Hayes DH, Mizrahi S, Maggiore P, Blazek J, Dick D. Use of watersoluble liquid vitamin E to enhance cyclosporine absorption in children after liver transplant. Transpl Proc 1993;25:1875e81. [5] Sokol RJ, Narkewicz MR, Smith D, Karrer FM, Kam I, Johnson KE. Improvement of cyclosporin absorption in children after liver transplantation by means of water-soluble vitamin E. Lancet 1991;338:212e4.

Please cite this article in press as: Tomaru A, et al., Effects of Cremophor EL on the absorption of orally administered saquinavir and fexofenadine in healthy subjects, Drug Metabolism and Pharmacokinetics (2015), http://dx.doi.org/10.1016/j.dmpk.2015.01.002

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 Q2 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

DMPK31_proof ■ 2 February 2015 ■ 6/6

6

A. Tomaru et al. / Drug Metabolism and Pharmacokinetics xxx (2015) 1e6

[6] Wandel C, Kim RB, Stein CM. “Inactive” excipients such as Cremophor can affect in vivo drug disposition. Clin Pharmacol Ther 2003;73(5):394e6. [7] Japanese pharmaceutical excipients directory 2007. [8] Drewe J, Beglinger C, Kissel T. The absorption site of cyclosporin in the human gastrointestinal tract. Br J Clin Pharmacol 1992;33:39e43. [9] Bogman K, Zysset Y, Degen L, Hopfgartner G, Gutmann H, Alsenz J, et al. Pglycoprotein and surfactants: effect on intestinal talinolol absorption. Clin Pharmacol Ther 2005;77(1):24e32. [10] Martin-Facklam M, Burhenne J, Ding R, Fricker R, Mikus G, Walter-Sack I, et al. Dose-dependent increase of saquinavir bioavailability by the pharmaceutic aid Cremophor EL. Br J Clin Pharmacol 2002;53:576e81. [11] Lin JH. Human immunodeficiency virus protease inhibitors from drug design to clinical studies. Adv Drug Deliv Rev 1997;27:215e33. [12] Park S, Sinko PJ. P-glycoprotein and mutlidrug resistance-associated proteins limit the brain uptake of saquinavir in mice. J Pharmacol Exp Ther 2005;312(3):1249e56. [13] Tachibana T, Kato M, Watanabe T, Mitsui T, Sugiyama Y. Method for predicting the risk of drug-drug interactions involving inhibition of intestinal CYP3A4 and P-glycoprotein. Xenobiotica 2009;39(6):430e43. €s H. Transport characteristics of [14] Petri N, Tannergren C, Rungstand D, Lennerna fexofenadine in the Caco-2 cell model. Pharm Res 2004;21(8):1398e404. [15] Troutman MD, Thakker DR. Novel experimental parameters to quantify the modulation of absorptive and secretory transport of compounds by P-glycoprotein in cell culture models of intestinal epithelium. Pharm Res 2003;20(8): 1210e24. [16] Eagling VA, Wiltshire H, Whitcombe IW, Back DJ. CYP3A4-mediated hepatic metabolism of the HIV-1protease inhibitor saquinavir in vitro. Xenobiotica 2002;32(1):1e17.

[17] Ieiri I, Tsunemitsu S, Maeda K, Ando Y, Izumi N, Kimura M, et al. Mechanism of pharmacokinetic enhancement between ritonavir and saquinavir; micro/ small dosing tests using midazolam (CYP3A4), fexofenadine (p-glycoprotein), and pravastatin (OATP1B1) as probe drugs. J Clin Pharmacol 2013;53(6): 654e61. [18] Nerurkar MM, Ho NFH, Burton PS, Vidmar TJ, Borchardt RT. Mechanistic roles of neutral surfactants on concurrent polarized and passive membrane transport of a model peptide in Caco-2 cells. J Pharm Sci 1997;86(7):813e21. [19] Batrakova EV, Han H, Alakhov VY, Miller DW, Kabanov AV. Effects of pluronic block copolymers on drug absorption in Caco-2 cell monolayers. Pharm Res 1998;15(6):850e5.  MM, Schellens JHM, Beijnen JH, van [20] Bardelmeijer HA, Ouwehand M, Malingre Tellingen O. Entrapment by Cremophor EL decreases the absorption of paclitaxel from the gut. Cancer Chemother Pharmacol 2002;49:119e25. [21] Zastre JA, Jackson JK, Wong W, Burt HM. P-glycoprotein efflux inhibition by amphiphilic diblok copolymers: relationship between copolymer concentration and substrate hydrophobicity. Mol Pharm 2008;5(4):643e53. [22] Yamagata T, Kusuhara H, Morishita M, Takayama K, Benameur H, Sugiyama Y. Effect of excipients on breast cancer resistance protein substrate uptake activity. J Control Release 2007;124:1e5. [23] Martin P, Giardiello M, McDonald TO, Rannard SP, Owen A. Mediation of in vitro cytochrome P450 activity by common pharmaceutical excipients. Mol Pharm 2013;10:2739e48. [24] Bogman K, Erne-Brand F, Alsenz J, Drewe J. The role of surfactants in the reversal of active transport mediated by multidrug resistance proteins. J Pharm Sci 2003;92(6):1250e61.

Please cite this article in press as: Tomaru A, et al., Effects of Cremophor EL on the absorption of orally administered saquinavir and fexofenadine in healthy subjects, Drug Metabolism and Pharmacokinetics (2015), http://dx.doi.org/10.1016/j.dmpk.2015.01.002

23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

Effects of Cremophor EL on the absorption of orally administered saquinavir and fexofenadine in healthy subjects.

Modulation of CYP3A and/or P-gp function by several excipients has been reported. However, relatively few studies have investigated their effects in h...
578KB Sizes 0 Downloads 7 Views