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Effect of Milk on the Pharmacokinetics of Oseltamivir in Healthy Volunteers
Effect of Milk on the Pharmacokinetics of Oseltamivir in Healthy Volunteers KAORI MORIMOTO,1 KOZUE KISHIMURA,1 TAKAAKI NAGAMI,1 NAO KODAMA,1 YOICHIRO OGAMA,2 MIDORI YOKOYAMA,2 SHINYA TODA,2 TAKESHI CHIYODA,2 RIEKO SHIMADA,3 AKIHIRO INANO,3 TAKASHI KANO,1 IKUMI TAMAI,4 TAKUO OGIHARA1 1
Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan
2
Medical Company LTA, Sumida Hospital, Tokyo, Japan
3
Neues Corporation, Fukuoka, Japan
4
Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
Received 18 February 2011; revised 11 April 2011; accepted 27 April 2011 Published online 23 May 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22627 ABSTRACT: We previously showed that oseltamivir, a prodrug of the influenza virus neuraminidase inhibitor Ro 64-0802, is a substrate of proton-coupled oligopeptide transporter (PEPT1), and its intestinal absorption in rats is markedly inhibited by administration with milk. To investigate the importance of PEPT1 for oseltamivir absorption in humans, and the characteristics of the drug–milk interaction, a crossover clinical study was conducted in healthy volunteers, who received 75 mg of oseltamivir with 400 mL of water or milk. Milk significantly reduced the maximum plasma concentration (Cmax ) and the area under the plasma concentration–time curve from 0 to 2 h (AUC0–2 ) of both oseltamivir and Ro 64-0802 (oseltamivir, 68.9% and 34.5%; Ro 64-0802, 69.5% and 14.2%, respectively, vs. water), but had no significant effect on the apparent terminal half-life (t1/2 ) or AUC0–∞ . Urinary recovery of oseltamivir and Ro 64-0802 was significantly reduced to 77.5% of the control by milk. The early reduction of oseltamivir absorption might be through the PEPT1 inhibition by milk peptides. However, the extent of interaction in humans was limited as compared with that in rats, possibly because of species difference in the PEPT1 expression and its contribution. This might be the first report suggesting the clinical drug–food interaction via PEPT1. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3854–3861, 2011 Keywords: intestinal absorption; bioavailability; food interactions; peptide transporters; pharmacokinetics
INTRODUCTION Dietary constituents may influence drug response in various ways, ranging from loss of efficacy to toxicity. In particular, interactions that modify drug absorption may be of clinical importance because drug effects are dependent on the concentration of the active species at the site of action. The absorption process itself may be influenced by drug-metabolizing enzymes as well as by influx and efflux transporters Additional Supporting Information may be found in the online version of this article. Supporting Information Correspondence to: Takuo Ogihara (Telephone: +81-27-3521180; Fax: +81-27-352-1118; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 100, 3854–3861 (2011) © 2011 Wiley-Liss, Inc. and the American Pharmacists Association
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expressed in the small intestine. For example, it is well known that dietary constituents of grapefruit juice exhibit clinically important interactions with drugs by inhibiting intestinal CYP3A, P-glycoprotein, and organic anion transporting polypeptides.1–3 Proton-coupled oligopeptide transporter PEPT1 (gene name SLC15A1), localized at the brush-border membranes of the small intestine, serves as an uptake system for di/tripeptides generated by the digestion of dietary proteins. It also transports orally administered peptidomimetic drugs, including $-lactam antibiotics such as cephalexin and cefixime, an antineoplastic drug bestatin, an antiviral drug valacyclovir and so on.4 These drugs are widely used to treat adults, children, and infants. However, some aged patients take medicines with milk to avoid
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gastrointestinal irritation. Also, infants generally drink significant amounts of milk, and parents sometimes mask the taste of unpalatable drugs by mixing them with milk or dairy products (e.g., ice cream) in order to make them more acceptable to infants. Therefore, there is a possibility that di and tripeptides generated by digestion of milk proteins may inhibit PEPT1-mediated drug absorption at therapeutic doses, resulting in subtherapeutic concentrations in plasma. Especially in the case of milk,5 proteins are rapidly broken down to large polypeptides in the stomach. Further breakdown to free amino acids and small peptides with two to six amino acid residues also occurs rapidly, mediated by pancreatic enzymes. Disappearance of several amino acids in protein meal from intestinal lumen is faster than the breakdown of the protein to free amino acids. Seventy-five percent of the net absorption of amino acids derived from milk proteins occurs before the arrival at the lower jejunum in humans. Dipeptidase activity in the proximal duodenum is low, but increases sharply in the distal part of the duodenum. Therefore, it seems likely that a large part of the amino acids derived from milk proteins is rapidly absorbed in the upper small intestine in the form of small peptides. Indeed it has been reported that the rates of the breakdown to free amino acids of protein meal in intestinal lumen was not rapid enough for the absorption rates of glycine, threonine, serine, the imino acids, or the dicarboxylic amino acids in humans.5 And large amount of small peptides were detected in the upper intestine 30 min after milk intake.5 Oseltamivir is an ester-type prodrug of the neuraminidase inhibitor [3R,4R,5S]-4-acetamido-5amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid (Ro 64-0802), which targets influenza viruses. After oral administration in humans, oseltamivir is rapidly and extensively converted to its active metabolite, Ro 64-0802, by hepatic carboxylesterase.6 At 100 mg oral dose, the bioavailability of Ro 64-0802 estimated from urinary recovery is more than 63%.7 We found that oseltamivir was effectively taken up by PEPT1 in vitro and that oseltamivir absorption in rats was reduced to one-fifth by coadministration of 125 mM glycylsarcosine, a typical inhibitor of PEPT1, and was reduced to two-fifths when administered with milk.8 These results suggest that small peptides derived from milk proteins significantly inhibit PEPT1-mediated intestinal absorption of oseltamivir, at least in rats. Therefore, it is important to examine whether the same mechanism operates in the intestinal absorption of this drug in humans, in order to ensure therapeutic efficacy of the drug. Moreover, because oseltamivir has been suggested to be associated with neuropsychiatric side effects,9 several groups, including ours, have been studying DOI 10.1002/jps
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the mechanisms of interindividual variability in the brain distribution of oseltamivir and Ro 64-0802 in animals and humans.10–12 Because Ro 64-0802 can hardly penetrate the blood–brain barrier, brain distribution of intact oseltamivir might be an important factor.10,11 If milk inhibits the intestinal absorption of oseltamivir in humans, and if the information for infants in the drug’s package insert is based on the meal condition, unweaned infants with poor appetite might be at risk of being exposed to high plasma levels of this drug. The purpose of this study was to assess the clinical relevance of the effect of intestinal PEPT1 inhibition by milk on the pharmacokinetics of oseltamivir by means of a crossover clinical study in healthy volunteers. In addition, we discuss species difference between rats and humans in PEPT1-mediated drug— food interaction.
MATERIALS AND METHODS Study Design A randomized, open-label, two-period, crossover clinical pharmacokinetic study was conducted. Six healthy male Japanese volunteers aged 21–25 were enrolled. An evaluation before the study showed that the subjects had unremarkable medical history, and normal findings on physical examination and standard clinical laboratory tests (general hematology, blood chemistry). The study was conducted in compliance with the Declaration of Helsinki. All individuals gave written informed consent for the study, which had been approved by the institutional review board of Sumida Hospital, Tokyo, Japan. Study Protocol Subjects received a 75-mg capsule of oseltamivir (as 98 mg phosphate salt, Chugai Pharmaceutical Company, Ltd., Tokyo, Japan) with either 400 mL of water or milk. Each subject randomly received the two treatments with an interval of 7 days. Concomitant medication or taking of health foods or supplements was prohibited, along with an intake of St. John’s wort, certain fruit juices (grapefruit, orange, and apple), and any drinks containing alcohol or caffeine. Smoking was also prohibited for a week before the study and throughout its duration. Venous blood (7 mL) samples were collected just before and at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, and 24 h after the administration of oseltamivir. Heparinized winged needles were used for anticoagulation. Plasma samples were obtained by immediate centrifugation of blood, and were stored at –80◦ C until assay. Urine was collected during the periods of 0–3, 3–6, 6–12, and 12–24 h postdosing to determine renal elimination of oseltamivir and Ro 64-0802. For each JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011
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collection, the volume was recorded and two aliquots of approximately 10 mL were stored at –80◦ C until analysis. Analytical Method The concentrations of oseltamivir and Ro 64-0802 in plasma and urine were measured by using a highperformance liquid chromatography–tandem mass spectrometry system consisting of an API3000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, California) with electrospray ionization in the positive mode. The multiple reaction monitor was set at 313–225 m/z for oseltamivir and 285—197 m/z for Ro 64-0802. The samples and standards for oseltamivir and Ro 64-0802 were prepared by solid-phase extraction. Briefly, aliquots of plasma (100 :L) or urine (100 :L), which had been diluted 40–100 times with water, were 10-fold diluted with 5 mM ammonium acetate buffer (pH 3.5), and 1 mL aliquots were subjected to solidTM phase extraction using InertSep MPC (mixed-phase cation; GL Science Inc., Tokyo, Japan). After application of a sample, the column was rinsed with 4 mL of methanol, followed with 4 mL of 90% methanol. Then the analytes were eluted with 4 mL of methanol containing 10% 50 mM ammonium acetate (pH 9.0). The eluate was evaporated to dryness under 50◦ C with nitrogen gas-blowing. The residue was dissolved in 100 :L of 20% acetonitrile containing 0.05% formic acid for analysis. Chromatographic conditions for oseltamivir and Ro 64-0802 were the same as previously described, with some modifications.8 Aliquots (10 :L) of samples were injected into a high-performance liquid chromatography system (HP1100 system, Hewlett Packard, Waldbronn, Germany) equipped with a Capcell pak C18 MGII column (50 × 2.0 mm i.d., 3 :m; Shiseido Company, Tokyo, Japan) and a guard column (C18 MGII S-3, Shiseido Company) using isocratic elution at
0.2 mL/min with 20% acetonitrile containing 0.05% formic acid. The method was validated in the concentration range 1.0–500 ng/mL, in plasma or urine. Calibration curves, with 1/x2 weighting, were linear over the validated range. Analysis Pharmacokinetic parameters (area under the plasma drug concentration–time curve from time 0 h extrapolated to infinity, AUC0–∞ , and apparent half life, t1/2 ) for oseltamivir and Ro64-0802 were estimated R Winvia noncompartmental analysis using Phoenix Nonlin6.1 (Pharsight, Mountain View, California). The maximum plasma concentration (Cmax ), the time to reach Cmax (tmax ), and the plasma concentration at 12 h after administration (C12 ) were obtained directly from the observed data. AUC from time 0 to 2 h (AUC0–2 ), from 2 to 4 h (AUC2–4 ), and from 0 to 24 h (AUC0–24 ) were obtained by employing the trapezoidal formula. The pharmacokinetic parameters of the two treatments were compared by using a paired t-test. Differences were considered statistically significant at P < 0.05.
RESULTS Plasma Concentration Figure 1 shows the plasma concentration–time profiles of oseltamivir and Ro 64-0802 after a single oral dose of 75 mg oseltamivir (as 98 mg phosphate salt) with either 400 mL of water or 400 mL of milk. The pharmacokinetic parameters of oseltamivir and Ro 64-0802 are summarized in Table 1. In the case of coadministration with water, Cmax was 38.2 ng/ mL at 1.4 h after administration, whereas AUC0–2 , AUC2–4 , AUC0–24 , and AUC0–∞ were 47.6, 32.8, 109, and 114 ng·h/mL, respectively. In the case of administration with milk, Cmax of oseltamivir was
Figure 1. Plasma concentration–time profiles of (a) oseltamivir and (b) Ro 64-0802 after single oral dose of 75 mg oseltamivir to six human volunteers with either 400 mL of water (open circles) or 400 mL of milk (closed circles). Data are expressed as means ± SD. Asterisks indicate a significant difference between treatments; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011
DOI 10.1002/jps
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Table 1. Plasma and Urinary Pharmacokinetic Parameters of Oseltamivir and Ro 64-0802 After Single Oral Administration of 75 mg Oseltamivir with 400 mL Water or 400 mL Milk Oseltamivir
Plasma concentration Cmax (ng/mL) C12 (ng/mL) tmax (h) t1/2 (h) AUC0–2 (ng·h/mL) AUC2–4 (ng·h/mL) AUC0–24 (ng·h/mL) AUC0–∞ (ng·h/mL) % Of dose (0–3) % Of dose (0–24) V0–24 (L) Urinary recovery % (Oseltamivir + Ro 64-0802)
Ro 64-0802
Water
Milk
Milk/Water (%)
Water
Milk
Milk/Water (%)
38.2 ± 7.3 0.55 ± 0.30 1.4 ± 1.4 11.6 ± 5.3 47.6 ± 12.1 32.8 ± 11.6 109 ± 25 114 ± 25 2.33 ± 1.00 4.03 ± 1.03 2.30 ± 0.75 76.9 ± ± 13.0
26.3 ± 3.2∗∗ 1.08 ± 0.39∗ 2.7 ± 0.5 7.4 ± 3.1 16.4 ± 4.7∗∗∗ 43.5 ± 7.6 107 ± 25 111 ± 24 1.22 ± 0.38∗ 4.1 ± 1 ± 1.24 2.29 ± 0.45 59.6 ± 9.1∗
68.8 196 193 63.8 34.5 133 98.2 97.4 52.4 102 99.6 77.5
255 ± 36 88.3±20.0 4.0 ± 0.6 5.9 ± 0.8 123 ± 58.0 390 ± 94.0 2463 ± 300 2647 ± 361 6.76 ± 3.62 72.8 ± 12.4
177 ± 27∗∗ 86.7 ± 14.0 5.7 ± 1.2∗ 6.5 ± 1.2 17.5 ± 5.1∗∗ 172 ± 39.5∗∗ 1979 ± 310∗ 2215 ± 334 1.40 ± 0.58∗ 55.5 ± 8.14∗
69.4 98.2 143 110 14.2 44.1 80.3 83.7 20.7 76.2
Data were expressed as means ± SD. Cmax , peak plasma drug concentration; C12 , plasma drug concentration at 12 h; tmax , time to reach Cmax ; t1/2 , elimination half-life; AUC0–2 , AUC2–4 , and AUC0–24 , area under the plasma drug concentration–time curve from time 0 to 2 h, from 2 to 4 h, and from 0 to 24 h, respectively; AUC0–∞ , AUC from time 0 h extrapolated to infinity; V0–24 , volume of urine from time 0 to 24 h. Asterisks indicate significant difference from water group; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.
significantly decreased to 68.9% (26.3 ng/mL) as compared with dosing with water. AUC0–2 of oseltamivir (16.4 ng·h/mL) was significantly reduced to 34.5% by milk, although there was no significant difference of AUC0–24 , AUC0–∞ , or AUC2–4 between the treatments. The half life (t1/2 ) of oseltamivir was not different between treatments, suggesting that milk did not affect the elimination or metabolism of oseltamivir. When oseltamivir was administered with water, Cmax of Ro64-0802 was 255 ng/mL at 4.0 h after administration, whereas AUC0–2 , AUC2–4 , AUC0–24 , and AUC0–∞ were 123, 390, 2463, and 2647 ng·h/mL, respectively. The Cmax of Ro 64-0802 was reduced to 69.5% when oseltamivir was administered with milk. AUC0–2 , AUC2–4 , and AUC0–24 were also significantly reduced to 14.2%, 44.1%, and 80.4% by milk. The tmax values of oseltamivir and Ro 64-0802 were nonsignificantly and significantly increased, respectively, in the case of administration with milk. Plasma concentration of Ro 64-0802 at 12 h after administration (C12 ) was not significantly different between the treatments. When individual plasma concentration–time curves of oseltamivir administered with water and milk were compared, a second peak was observed only in the case of water, in three of six subjects (#3, #4, and #6; Fig. 2). The first peak disappeared in the case of administration with milk, in two of the three subjects (#3 and #6). In the other three subjects with no second-peak phenomenon (#1,#2, and #5), tmax was delayed by milk from 1 to 2 h after administration, which is similar to the time of the second peak observed in #3, #4 and #6 (around 2 h after administration). Milk also delayed tmax and reduced Cmax of Ro 64-0802. DOI 10.1002/jps
Urinary Excretion When oseltamivir was administered with water, urinary excretion of unchanged drug up to 3 and 24 h after administration amounted to 2.33% and 4.03% of the dose, respectively. In agreement with that, the effect of milk on AUC was observed only from 0 to 2 h after administration; milk produced a significant reduction in the urinary excretion of oseltamivir (52.4% of control) in the period from 0 to 3 h after administration (Fig. 3 and Table 1). When oseltamivir was administered with water, urinary excretion of Ro 64-0802 up to 3 and 24 h after the administration amounted to 6.76% and 72.8% of dose (equivalent to oseltamivir), respectively (Table 1). Milk significantly reduced urinary excretion of Ro 64-0802 from 0 to 3 h (20.7% of control) and from 0 to 24 h (76.2% of control) after administration. The total absorbed fraction of oseltamivir, which was estimated from the urinary recoveries of oseltamivir and Ro64-0802, was significantly reduced to 77.5% after dosing of oseltamivir and milk concurrently. There was no difference in the volume of urine between treatments.
DISCUSSION Our clinical study showed that milk reduced the total absorption of oseltamivir, estimated by adding the urinary recovery of oseltamivir and Ro 64-0802, to 77.5% of the control. However, the plasma concentration of Ro 64-0802 at 12 h after administration (C12 ), an index of anti-influenza virus effect,13 was not significantly different between the two treatments. This suggests that no dose adjustment is required when oseltamivir is administered with milk. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011
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Figure 2. Individual plasma concentration–time profiles of oseltamivir after single oral dose of 75 mg oseltamivir to six human volunteers, with either 400 mL of water (open circles) or 400 mL of milk (closed circles).
Although the effects of milk in humans were not prominent when the total absorbed fractions were compared, there was a large difference when the AUC of oseltamivir from time 0 to 2 h and the urinary excretion in the very early period were compared between treatments (Table 1). These results suggest that the period during which oseltamivir absorption is inhib-
ited by milk is restricted to a few hours immediately postdosing in humans. We previously demonstrated that oseltamivir is a substrate of human PEPT1 in vitro and that oseltamivir absorption in rats is greatly reduced not only by milk, but also by coadministration of glycylsarcosine, a typical inhibitor of PEPT1, and by
Figure 3. Urinary excretion of (a) oseltamivir and (b) Ro 64-0802 after a single oral dose of 75 mg oseltamivir to six human volunteers with either 400 mL of water (open circles) or 400 mL of milk (closed circles). Data are expressed as means ± SD. Asterisks indicate a significant difference between treatments; ∗ p < 0.05, ∗∗ p < 0.01. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011
DOI 10.1002/jps
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Table 2. Pharmacokinetic Parameters After Single Oral Administration of Oseltamivir [30 mg/(10 mL/kg)] in Water, Milk, or 125 mM Gly–Sar Solution in Rats
Cmax (:g/mL) tmax (h) AUC0−1 (:g·h/mL) AUC1−2 (:g·h/mL) AUC0−6 (:g·h/mL)
Water (Control)
Milk
1.53 ± 0.35 0.5 ± 0.0 1.10 ± 0.21 0.643 ± 0.050 2.49 ± 0.12
0.31 ± (20.3) 0.7 ± (140) 0.237 ± (21.5) 0.230 ± (35.8) 0.942 ± (37.8)
+ 125 mM Gly–Sar 0.081 ± 0.00∗ 6.0 ± 0.0∗ 0.065 ± 0.001∗ 0.075 ± 0.002∗ 0.449 ± 0.006∗
(5.2) (1200) (5.9) (11.7) (18.0)
Data are expressed as means ± SD. Values in parenthesis are percentage versus water control. Cmax , peak plasma drug concentration; tmax , time to reach Cmax ; t1/2 , elimination half-life; AUC0–1 , AUC1–2 , AUC0–6 , area under the plasma drug concentration–time curve from time 0 to 1 h, from 1 to 2 h, and from 0 to 6 h, respectively. Asterisks indicate significant difference from water group; ∗ p < 0.05.
coadministration of casein, one of the principal proteins of milk.8 As previously mentioned, milk proteins are rapidly broken down to free amino acids or small peptides; so it was considered that the inhibition of intestinal absorption of oseltamivir by milk in humans could be explained by the inhibition of PEPT1mediated drug absorption by large amounts of small peptides derived from milk proteins. There is a marked species difference in the extent of inhibition between rats and humans (Table 1 and Table 2). The AUC of oseltamivir in humans was reduced only at 0–2 h postdosing of milk, whereas that in rats was strongly reduced throughout the experimental period. These differences might be at least in part due to the regional difference in smallintestinal luminal expression of PEPT1 between humans and rats.14 PEPT1 is localized predominantly in the duodenum, with sharply decreasing expression in lower regions in humans, whereas PEPT1 mRNA is uniformly expressed throughout the gastrointestinal tract in rats. Therefore, human PEPT1-mediated absorption of oseltamivir may occur predominantly in the duodenum, which would explain why the interaction with milk was observed only in the early period postdosing (approximately 95% of the dose passes through the duodenum within 40 min).15,16 In contrast, PEPT1 expressed throughout the intestine in rats may make a significant contribution to oseltamivir absorption. The assumption for the inhibitory time window on PEPT1-mediated absorption in humans can be supported by the previous report. In the 1970s, McCracken et al.17 reported that Cmax and AUC of cephalexin, benzylpenicillin, and phenoxymethylpenicillin among several antibiotics studied were reduced 40%–60% in infants and children given milk and drugs concurrently. Later studies revealed that the former two drugs are substrates of PEPT1.18,19 As we found in the case of oseltamivir, the reduction in the plasma levels of these drugs was limited to an early time window.17 Several other possibilities should also be considered. There might be differences in gastric emptying time, solubility or dissolved state of oseltamivir, and/ DOI 10.1002/jps
or elution rate of the drug from the capsule in the presence of milk and water. However, these possibilities seem unlikely for the following reasons: It has been reported that the tmax of erythromycin or amoxicillin was not changed by dosing with milk in humans,17 suggesting that milk does not alter the gastric emptying time. In this study, we tested the dissolution rate and solubility of an oseltamivir capsule with 400 mL of acidified water or milk. This medicine is completely eluted and dissolved into both solvents within 15 min (Supplementary Fig. 1). In addition, milk coagulation occurs only in unweaned infants, owing to the presence of a specific enzyme in the stomach.5 Therefore, oseltamivir completely dissolved in either water or milk is likely to reach the duodenum at the same rate following administration. Moreover, milk-equivalent calcium ions did not alter the ultraviolet spectra of oseltamivir in both water and acidified water (Supplementary Fig. 2). It was indicated that oseltamivir did not form insoluble complex with calcium ions, although it is known that certain drugs like quinolones form insoluble complex with calcium ions. Further, because oseltamivir has a low octanol–water partition coefficient at pH 7 (0.54), its absorption should not be affected by micelle formation or the coexistence of milk fat. When we examined the individual plasma concentration–time profiles of oseltamivir in humans, we noticed a double-peak phenomenon, which occurred only when oseltamivir was administered with water (Fig. 2). Previous reports on the clinical pharmacokinetics of oseltamivir have also noted the doublepeak phenomenon, which is considered to be due to enterohepatic circulation.20,21 In this study, the first peak specifically disappeared when oseltamivir was administered with milk. The tmax of the first peak of oseltamivir was around 0.5 h after the administration. Because the transit time of the duodenum in humans is considered to be 40 min,15,16 it is suggested that the disappearance of the first peak of oseltamivir reflects specific inhibition of PEPT1mediated absorption in the duodenum. In contrast, no double-peak phenomenon was observed in rats, which JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011
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express PEPT1 uniformly throughout the small intestine. The second peak observed in humans may reflect absorption through pathways other than PEPT1. Another transporter(s) may be involved in oseltamivir absorption because one-third of the active transport of oseltamivir was not inhibited by excess dipeptides in Caco-2 cells.8 The cause of the double-peak phenomenon is not fully understood, but several possible explanations have been proposed: biphasic gastric emptying,22 enterohepatic ciculation,23 and two different absorption sites.24 The last one may be related to regional differences in expression of the specific transporter(s) as previously speculated in the case of P-glycoprotein.25 Interestingly, oseltamivir absorption in subject #4, whose first peak was apparently smaller than other subjects, was not inhibited by milk. If the first peak is assumed to represent the PEPT1-mediated absorption of oseltamivir, there may be considerable interindividual variability in this process in humans. It has been reported that genetic polymorphisms,18 feeding, and hormonal status26 may be associated with interindividual differences in PEPT1-mediated absorption. The reduction in the AUC of Ro 64-0802 by milk ingestion was more notable than that of oseltamivir. This suggests that milk inhibited the oseltamivir conversion to Ro64-0802, catalyzed by hepatic carboxylesterase1 (CES1). Milk contains 3%–4% of triglycerides consisting of various fatty acids. Because a fatty acid palmitate is one of the endogenous substrates of CES127 and one of the most abundant constituent of milk fat, it is speculated that rapidly absorbed fatty acids including palmitate inhibited the oseltamivir conversion to Ro 64-0802.
CONCLUSION Our results indicate that milk inhibited the intestinal absorption of oseltamivir in humans. However, the extent of interaction in humans was limited as compared with that in rats, possibly because of species difference in the regional expression of PEPT1 in the small intestine and/or differences in the contribution of other absorption mechanisms. Among PEPT1 substrates, it seems likely that compounds whose absorption would be largely completed within the transit time of the duodenum may show marked inhibition of their absorption by milk in humans. This information might be useful not only for clinical medication, but also for drug development.
ACKNOWLEDGMENTS This study was supported by a grant from the Research Foundation for Pharmaceutical Sciences, and by government grants-in-aid for scientific research. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011
REFERENCES 1. Dresser GK, Bailey DG, Leake BF, Schwarz UI, Dawson PA, Freeman DJ, Kim RB. 2002. Fruit juices inhibit organic anion transporting polypeptide-mediated drug uptake to decrease the oral availability of fexofenadine. Clin Pharmacol Ther 71:11–20. 2. Dresser GK, Spence JD, Bailey DG. 2000. Pharmacokinetic–pharmacodynamic consequences and clinical relevance of cytochrome P450 3A4 inhibition. Clin Pharmacokinet 38:41–57. 3. de Castro WV, Mertens-Talcott S, Derendorf H, Butterweck V. 2007. Grapefruit juice–drug interactions: Grapefruit juice and its components inhibit P-glycoprotein (ABCB1) mediated transport of talinolol in Caco-2 cells. J Pharm Sci 96:2808– 17. 4. Terada T, Inui K. 2004. Peptide transporters: Structure, function, regulation, and application for drug delivery. Curr Drug Metab 5:85–94. 5. Nixon SE, Mawer GE. 1970. The digestion and absorption of protein in man. 2. The form in which digested protein is absorbed. Br J Nutr 24:241–258. 6. Shi D, Yang J, Yang D, LeCluyse EL, Black C, You L, Akhlaghi F, Yan B. 2006. Anti-influenza prodrug oseltamivir is activated by carboxylesterase human carboxylesterase 1, and the activation is inhibited by antiplatelet agent clopidogrel. J Exp Pharmacol Ther 319:1477–1484. 7. Massarella JW, He GZ, Dorr A, Nieforth K, Ward P, Brown A. 2000. The pharmacokinetics and tolerability of the oral neuraminidase inhibitor oseltamivir (Ro 64-0796/GS4104) in healthy adult and elderly volunteers. J Clin Pharmacol 40:836–843. 8. Ogihara T, Kano T, Wagatsuma T, Wada S, Yabuuchi H, Enomoto S, Morimoto K, Shirasaka Y, Kobayashi S, Tamai I. 2009. Oseltamivir (Tamiflu) is a substrate of peptide transporter 1. Drug Metab Dispos 37:1676–1681. 9. Kitching A, Roche A, Balasegaram S, Heathcock R, Maguire H. 2009. Oseltamivir adherence and side effects among children in three London schools affected by influenza A(H1N1)v, May 2009: An internet-based cross-sectional survey. Eurosurveill 14:1–4. 10. Morimoto K, Nakakariya M, Shirasaka Y, Kakinuma C, Fujita T, Tamai I, Ogihara T. 2008. Oseltamivir (Tamiflu) efflux transport at the blood–brain barrier via P-glycoprotein. Drug Metab Dispos 36:6–9. 11. Ose A, Kusuhara H, Yamatsugu K, Kanai M, Shibasaki M, Fujita T, Yamamoto A, Sugiyama Y. 2008. P-glycoprotein restricts the penetration of oseltamivir across the blood–brain barrier. Drug Metab Dispos 36:427–34. 12. Yang D, Pearce RE, Wang H, Gaedigk R, Wan YY, Yan B. 2009. Human carboxylesterase HCE1 and HCE2: Ontogenic expression, interindividual variability, and differential hydrolysis of oseltamivir, aspirin, deltamethrin, and permethrin. Biochem Pharmacol 77:238–247. 13. Basic product information of Tamiflu capsule. 19th revision. Tokyo, Japan: Chugai Pharmaceutical Company, Ltd. 14. Herrera-Ruiz D, Wang Q, Cook TJ, Knipp GT. 2001. Spatial expression patterns of peptide transporters in the human and rat gastrointestinal tracts, Caco-2 in vitro cell culture model, and multiple human tissues. AAPS PharmSci 3(1):E9. ¨ 15. Schwarz R, Kaspar A, Seelig J, Kunnecke B. 2002. Gastrointestinal transit times in mice and humans measured with 27 Al and 19 F nuclear magnetic resonance. Magn Reson Med 48:255–261. 16. Oberle RL, Chen TS, Lloyd C, Barnett JL, Owyang C, Meyer J, Amidon GL. 1990. The influence of the interdigestive migrating myoelectric complex on the gastric emptying of liquids. Gastroenterology 99:1275–1282. DOI 10.1002/jps
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17. McCracken GH Jr, Ginsburg CM, Clarsen JC, Thomas ML. 1978. Pharmacologic evaluation of orally administered antibiotics in infants and children: Effect of feeding on bioavailability. Pediatrics 62:738–743. 18. Anderle P, Nielsen CU, Pinsonneault J, Krog PL, Brodin B, Sad´ee. 2006. Genetic variants of human dipeptide transporter PEPT1. J Pharmacol Exp Ther 316:636–646. 19. Bhardwaj RK, Herrera-Ruiz D, Sinko PJ, Gudmundsson OS, Knipp G. 2005. Delineation of human peptide transporter 1 (hPepT1)-mediated uptake and transport of substrates with varying transporter affinities utilizing stably transfected hPepT1/Madin–Darby canine kidney clones and Caco-2 cells. J Pharmacol Exp Ther 314:1093–100. 20. Schentag JJ, Hill G, Chu T, Rayner CR. 2007. Similarity in pharmacokinetics of oseltamivir and oseltamivir carboxylate in Japanese and Caucasian subjects. J Clin Pharmacol 47:689–96. 21. Snell P, Dave N, Wilson K, Rowell L, Weil A, Galitz L, Robson R. 2005. Lack of effect of moderate hepatic impairment on the pharmacokinetics of oral oseltamivir and its metabolite oseltamivir carboxylate. Br J Clin Pharmacol 59:598–601.
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22. Oberle RL, Amidon GL. 1987. The influence of variable gastric emptying and intestinal transit rates on the plasma level curve of cimetidine; an explanation for the double peak phenomenon. J Pharmacokinet Biopharm 15:529–544. 23. Pedersen PV, Miller R. 1980.Pharmacokinetics of dixycycline reabsorption. J Pharm Sci 69:204–207. 24. Wagner JG. 1984. Unusual pharmacokinetics. In Pharmacokinetics, a modern view;Benet LZ. Levy G, Ferraiolo BL, Eds.. New York: Plenum Press, pp 173–189. 25. Ogihara T, Kamiya M, Ozawa M, Fujita T, Yamamoto A, Yamashita S Ohnishi S, Isomura Y. 2006. What kinds of substrates show P-glycoprotein-dependent intestinal absorption? Comparison of verapamil with vinblastine. Drug Metab Pharmacokinet 21:238–244. 26. Gilbert ER, Wong EA, Webb KE. 2008. Peptide absorption and utilization: Implication for animal nutrition and health. J Anim Sci 86:2135–2155. 27. Bencharit S, Edwards CC, Morton CL, Howard-Williams EL, Kuhn P, Potter PM, Redinbo MR. 2006. Multisite promiscuity in the processing of endogenous substrates by human carboxylesterase 1. J Mol Biol 63:201–214.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011
Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan
2
Medical Company LTA, Sumida Hospital, Tokyo, Japan
3
Neues Corporation, Fukuoka, Japan
4
Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
Received 18 February 2011; revised 11 April 2011; accepted 27 April 2011 Published online 23 May 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22627 ABSTRACT: We previously showed that oseltamivir, a prodrug of the influenza virus neuraminidase inhibitor Ro 64-0802, is a substrate of proton-coupled oligopeptide transporter (PEPT1), and its intestinal absorption in rats is markedly inhibited by administration with milk. To investigate the importance of PEPT1 for oseltamivir absorption in humans, and the characteristics of the drug–milk interaction, a crossover clinical study was conducted in healthy volunteers, who received 75 mg of oseltamivir with 400 mL of water or milk. Milk significantly reduced the maximum plasma concentration (Cmax ) and the area under the plasma concentration–time curve from 0 to 2 h (AUC0–2 ) of both oseltamivir and Ro 64-0802 (oseltamivir, 68.9% and 34.5%; Ro 64-0802, 69.5% and 14.2%, respectively, vs. water), but had no significant effect on the apparent terminal half-life (t1/2 ) or AUC0–∞ . Urinary recovery of oseltamivir and Ro 64-0802 was significantly reduced to 77.5% of the control by milk. The early reduction of oseltamivir absorption might be through the PEPT1 inhibition by milk peptides. However, the extent of interaction in humans was limited as compared with that in rats, possibly because of species difference in the PEPT1 expression and its contribution. This might be the first report suggesting the clinical drug–food interaction via PEPT1. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3854–3861, 2011 Keywords: intestinal absorption; bioavailability; food interactions; peptide transporters; pharmacokinetics
INTRODUCTION Dietary constituents may influence drug response in various ways, ranging from loss of efficacy to toxicity. In particular, interactions that modify drug absorption may be of clinical importance because drug effects are dependent on the concentration of the active species at the site of action. The absorption process itself may be influenced by drug-metabolizing enzymes as well as by influx and efflux transporters Additional Supporting Information may be found in the online version of this article. Supporting Information Correspondence to: Takuo Ogihara (Telephone: +81-27-3521180; Fax: +81-27-352-1118; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 100, 3854–3861 (2011) © 2011 Wiley-Liss, Inc. and the American Pharmacists Association
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expressed in the small intestine. For example, it is well known that dietary constituents of grapefruit juice exhibit clinically important interactions with drugs by inhibiting intestinal CYP3A, P-glycoprotein, and organic anion transporting polypeptides.1–3 Proton-coupled oligopeptide transporter PEPT1 (gene name SLC15A1), localized at the brush-border membranes of the small intestine, serves as an uptake system for di/tripeptides generated by the digestion of dietary proteins. It also transports orally administered peptidomimetic drugs, including $-lactam antibiotics such as cephalexin and cefixime, an antineoplastic drug bestatin, an antiviral drug valacyclovir and so on.4 These drugs are widely used to treat adults, children, and infants. However, some aged patients take medicines with milk to avoid
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gastrointestinal irritation. Also, infants generally drink significant amounts of milk, and parents sometimes mask the taste of unpalatable drugs by mixing them with milk or dairy products (e.g., ice cream) in order to make them more acceptable to infants. Therefore, there is a possibility that di and tripeptides generated by digestion of milk proteins may inhibit PEPT1-mediated drug absorption at therapeutic doses, resulting in subtherapeutic concentrations in plasma. Especially in the case of milk,5 proteins are rapidly broken down to large polypeptides in the stomach. Further breakdown to free amino acids and small peptides with two to six amino acid residues also occurs rapidly, mediated by pancreatic enzymes. Disappearance of several amino acids in protein meal from intestinal lumen is faster than the breakdown of the protein to free amino acids. Seventy-five percent of the net absorption of amino acids derived from milk proteins occurs before the arrival at the lower jejunum in humans. Dipeptidase activity in the proximal duodenum is low, but increases sharply in the distal part of the duodenum. Therefore, it seems likely that a large part of the amino acids derived from milk proteins is rapidly absorbed in the upper small intestine in the form of small peptides. Indeed it has been reported that the rates of the breakdown to free amino acids of protein meal in intestinal lumen was not rapid enough for the absorption rates of glycine, threonine, serine, the imino acids, or the dicarboxylic amino acids in humans.5 And large amount of small peptides were detected in the upper intestine 30 min after milk intake.5 Oseltamivir is an ester-type prodrug of the neuraminidase inhibitor [3R,4R,5S]-4-acetamido-5amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid (Ro 64-0802), which targets influenza viruses. After oral administration in humans, oseltamivir is rapidly and extensively converted to its active metabolite, Ro 64-0802, by hepatic carboxylesterase.6 At 100 mg oral dose, the bioavailability of Ro 64-0802 estimated from urinary recovery is more than 63%.7 We found that oseltamivir was effectively taken up by PEPT1 in vitro and that oseltamivir absorption in rats was reduced to one-fifth by coadministration of 125 mM glycylsarcosine, a typical inhibitor of PEPT1, and was reduced to two-fifths when administered with milk.8 These results suggest that small peptides derived from milk proteins significantly inhibit PEPT1-mediated intestinal absorption of oseltamivir, at least in rats. Therefore, it is important to examine whether the same mechanism operates in the intestinal absorption of this drug in humans, in order to ensure therapeutic efficacy of the drug. Moreover, because oseltamivir has been suggested to be associated with neuropsychiatric side effects,9 several groups, including ours, have been studying DOI 10.1002/jps
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the mechanisms of interindividual variability in the brain distribution of oseltamivir and Ro 64-0802 in animals and humans.10–12 Because Ro 64-0802 can hardly penetrate the blood–brain barrier, brain distribution of intact oseltamivir might be an important factor.10,11 If milk inhibits the intestinal absorption of oseltamivir in humans, and if the information for infants in the drug’s package insert is based on the meal condition, unweaned infants with poor appetite might be at risk of being exposed to high plasma levels of this drug. The purpose of this study was to assess the clinical relevance of the effect of intestinal PEPT1 inhibition by milk on the pharmacokinetics of oseltamivir by means of a crossover clinical study in healthy volunteers. In addition, we discuss species difference between rats and humans in PEPT1-mediated drug— food interaction.
MATERIALS AND METHODS Study Design A randomized, open-label, two-period, crossover clinical pharmacokinetic study was conducted. Six healthy male Japanese volunteers aged 21–25 were enrolled. An evaluation before the study showed that the subjects had unremarkable medical history, and normal findings on physical examination and standard clinical laboratory tests (general hematology, blood chemistry). The study was conducted in compliance with the Declaration of Helsinki. All individuals gave written informed consent for the study, which had been approved by the institutional review board of Sumida Hospital, Tokyo, Japan. Study Protocol Subjects received a 75-mg capsule of oseltamivir (as 98 mg phosphate salt, Chugai Pharmaceutical Company, Ltd., Tokyo, Japan) with either 400 mL of water or milk. Each subject randomly received the two treatments with an interval of 7 days. Concomitant medication or taking of health foods or supplements was prohibited, along with an intake of St. John’s wort, certain fruit juices (grapefruit, orange, and apple), and any drinks containing alcohol or caffeine. Smoking was also prohibited for a week before the study and throughout its duration. Venous blood (7 mL) samples were collected just before and at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, and 24 h after the administration of oseltamivir. Heparinized winged needles were used for anticoagulation. Plasma samples were obtained by immediate centrifugation of blood, and were stored at –80◦ C until assay. Urine was collected during the periods of 0–3, 3–6, 6–12, and 12–24 h postdosing to determine renal elimination of oseltamivir and Ro 64-0802. For each JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011
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collection, the volume was recorded and two aliquots of approximately 10 mL were stored at –80◦ C until analysis. Analytical Method The concentrations of oseltamivir and Ro 64-0802 in plasma and urine were measured by using a highperformance liquid chromatography–tandem mass spectrometry system consisting of an API3000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, California) with electrospray ionization in the positive mode. The multiple reaction monitor was set at 313–225 m/z for oseltamivir and 285—197 m/z for Ro 64-0802. The samples and standards for oseltamivir and Ro 64-0802 were prepared by solid-phase extraction. Briefly, aliquots of plasma (100 :L) or urine (100 :L), which had been diluted 40–100 times with water, were 10-fold diluted with 5 mM ammonium acetate buffer (pH 3.5), and 1 mL aliquots were subjected to solidTM phase extraction using InertSep MPC (mixed-phase cation; GL Science Inc., Tokyo, Japan). After application of a sample, the column was rinsed with 4 mL of methanol, followed with 4 mL of 90% methanol. Then the analytes were eluted with 4 mL of methanol containing 10% 50 mM ammonium acetate (pH 9.0). The eluate was evaporated to dryness under 50◦ C with nitrogen gas-blowing. The residue was dissolved in 100 :L of 20% acetonitrile containing 0.05% formic acid for analysis. Chromatographic conditions for oseltamivir and Ro 64-0802 were the same as previously described, with some modifications.8 Aliquots (10 :L) of samples were injected into a high-performance liquid chromatography system (HP1100 system, Hewlett Packard, Waldbronn, Germany) equipped with a Capcell pak C18 MGII column (50 × 2.0 mm i.d., 3 :m; Shiseido Company, Tokyo, Japan) and a guard column (C18 MGII S-3, Shiseido Company) using isocratic elution at
0.2 mL/min with 20% acetonitrile containing 0.05% formic acid. The method was validated in the concentration range 1.0–500 ng/mL, in plasma or urine. Calibration curves, with 1/x2 weighting, were linear over the validated range. Analysis Pharmacokinetic parameters (area under the plasma drug concentration–time curve from time 0 h extrapolated to infinity, AUC0–∞ , and apparent half life, t1/2 ) for oseltamivir and Ro64-0802 were estimated R Winvia noncompartmental analysis using Phoenix Nonlin6.1 (Pharsight, Mountain View, California). The maximum plasma concentration (Cmax ), the time to reach Cmax (tmax ), and the plasma concentration at 12 h after administration (C12 ) were obtained directly from the observed data. AUC from time 0 to 2 h (AUC0–2 ), from 2 to 4 h (AUC2–4 ), and from 0 to 24 h (AUC0–24 ) were obtained by employing the trapezoidal formula. The pharmacokinetic parameters of the two treatments were compared by using a paired t-test. Differences were considered statistically significant at P < 0.05.
RESULTS Plasma Concentration Figure 1 shows the plasma concentration–time profiles of oseltamivir and Ro 64-0802 after a single oral dose of 75 mg oseltamivir (as 98 mg phosphate salt) with either 400 mL of water or 400 mL of milk. The pharmacokinetic parameters of oseltamivir and Ro 64-0802 are summarized in Table 1. In the case of coadministration with water, Cmax was 38.2 ng/ mL at 1.4 h after administration, whereas AUC0–2 , AUC2–4 , AUC0–24 , and AUC0–∞ were 47.6, 32.8, 109, and 114 ng·h/mL, respectively. In the case of administration with milk, Cmax of oseltamivir was
Figure 1. Plasma concentration–time profiles of (a) oseltamivir and (b) Ro 64-0802 after single oral dose of 75 mg oseltamivir to six human volunteers with either 400 mL of water (open circles) or 400 mL of milk (closed circles). Data are expressed as means ± SD. Asterisks indicate a significant difference between treatments; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011
DOI 10.1002/jps
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Table 1. Plasma and Urinary Pharmacokinetic Parameters of Oseltamivir and Ro 64-0802 After Single Oral Administration of 75 mg Oseltamivir with 400 mL Water or 400 mL Milk Oseltamivir
Plasma concentration Cmax (ng/mL) C12 (ng/mL) tmax (h) t1/2 (h) AUC0–2 (ng·h/mL) AUC2–4 (ng·h/mL) AUC0–24 (ng·h/mL) AUC0–∞ (ng·h/mL) % Of dose (0–3) % Of dose (0–24) V0–24 (L) Urinary recovery % (Oseltamivir + Ro 64-0802)
Ro 64-0802
Water
Milk
Milk/Water (%)
Water
Milk
Milk/Water (%)
38.2 ± 7.3 0.55 ± 0.30 1.4 ± 1.4 11.6 ± 5.3 47.6 ± 12.1 32.8 ± 11.6 109 ± 25 114 ± 25 2.33 ± 1.00 4.03 ± 1.03 2.30 ± 0.75 76.9 ± ± 13.0
26.3 ± 3.2∗∗ 1.08 ± 0.39∗ 2.7 ± 0.5 7.4 ± 3.1 16.4 ± 4.7∗∗∗ 43.5 ± 7.6 107 ± 25 111 ± 24 1.22 ± 0.38∗ 4.1 ± 1 ± 1.24 2.29 ± 0.45 59.6 ± 9.1∗
68.8 196 193 63.8 34.5 133 98.2 97.4 52.4 102 99.6 77.5
255 ± 36 88.3±20.0 4.0 ± 0.6 5.9 ± 0.8 123 ± 58.0 390 ± 94.0 2463 ± 300 2647 ± 361 6.76 ± 3.62 72.8 ± 12.4
177 ± 27∗∗ 86.7 ± 14.0 5.7 ± 1.2∗ 6.5 ± 1.2 17.5 ± 5.1∗∗ 172 ± 39.5∗∗ 1979 ± 310∗ 2215 ± 334 1.40 ± 0.58∗ 55.5 ± 8.14∗
69.4 98.2 143 110 14.2 44.1 80.3 83.7 20.7 76.2
Data were expressed as means ± SD. Cmax , peak plasma drug concentration; C12 , plasma drug concentration at 12 h; tmax , time to reach Cmax ; t1/2 , elimination half-life; AUC0–2 , AUC2–4 , and AUC0–24 , area under the plasma drug concentration–time curve from time 0 to 2 h, from 2 to 4 h, and from 0 to 24 h, respectively; AUC0–∞ , AUC from time 0 h extrapolated to infinity; V0–24 , volume of urine from time 0 to 24 h. Asterisks indicate significant difference from water group; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.
significantly decreased to 68.9% (26.3 ng/mL) as compared with dosing with water. AUC0–2 of oseltamivir (16.4 ng·h/mL) was significantly reduced to 34.5% by milk, although there was no significant difference of AUC0–24 , AUC0–∞ , or AUC2–4 between the treatments. The half life (t1/2 ) of oseltamivir was not different between treatments, suggesting that milk did not affect the elimination or metabolism of oseltamivir. When oseltamivir was administered with water, Cmax of Ro64-0802 was 255 ng/mL at 4.0 h after administration, whereas AUC0–2 , AUC2–4 , AUC0–24 , and AUC0–∞ were 123, 390, 2463, and 2647 ng·h/mL, respectively. The Cmax of Ro 64-0802 was reduced to 69.5% when oseltamivir was administered with milk. AUC0–2 , AUC2–4 , and AUC0–24 were also significantly reduced to 14.2%, 44.1%, and 80.4% by milk. The tmax values of oseltamivir and Ro 64-0802 were nonsignificantly and significantly increased, respectively, in the case of administration with milk. Plasma concentration of Ro 64-0802 at 12 h after administration (C12 ) was not significantly different between the treatments. When individual plasma concentration–time curves of oseltamivir administered with water and milk were compared, a second peak was observed only in the case of water, in three of six subjects (#3, #4, and #6; Fig. 2). The first peak disappeared in the case of administration with milk, in two of the three subjects (#3 and #6). In the other three subjects with no second-peak phenomenon (#1,#2, and #5), tmax was delayed by milk from 1 to 2 h after administration, which is similar to the time of the second peak observed in #3, #4 and #6 (around 2 h after administration). Milk also delayed tmax and reduced Cmax of Ro 64-0802. DOI 10.1002/jps
Urinary Excretion When oseltamivir was administered with water, urinary excretion of unchanged drug up to 3 and 24 h after administration amounted to 2.33% and 4.03% of the dose, respectively. In agreement with that, the effect of milk on AUC was observed only from 0 to 2 h after administration; milk produced a significant reduction in the urinary excretion of oseltamivir (52.4% of control) in the period from 0 to 3 h after administration (Fig. 3 and Table 1). When oseltamivir was administered with water, urinary excretion of Ro 64-0802 up to 3 and 24 h after the administration amounted to 6.76% and 72.8% of dose (equivalent to oseltamivir), respectively (Table 1). Milk significantly reduced urinary excretion of Ro 64-0802 from 0 to 3 h (20.7% of control) and from 0 to 24 h (76.2% of control) after administration. The total absorbed fraction of oseltamivir, which was estimated from the urinary recoveries of oseltamivir and Ro64-0802, was significantly reduced to 77.5% after dosing of oseltamivir and milk concurrently. There was no difference in the volume of urine between treatments.
DISCUSSION Our clinical study showed that milk reduced the total absorption of oseltamivir, estimated by adding the urinary recovery of oseltamivir and Ro 64-0802, to 77.5% of the control. However, the plasma concentration of Ro 64-0802 at 12 h after administration (C12 ), an index of anti-influenza virus effect,13 was not significantly different between the two treatments. This suggests that no dose adjustment is required when oseltamivir is administered with milk. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011
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Figure 2. Individual plasma concentration–time profiles of oseltamivir after single oral dose of 75 mg oseltamivir to six human volunteers, with either 400 mL of water (open circles) or 400 mL of milk (closed circles).
Although the effects of milk in humans were not prominent when the total absorbed fractions were compared, there was a large difference when the AUC of oseltamivir from time 0 to 2 h and the urinary excretion in the very early period were compared between treatments (Table 1). These results suggest that the period during which oseltamivir absorption is inhib-
ited by milk is restricted to a few hours immediately postdosing in humans. We previously demonstrated that oseltamivir is a substrate of human PEPT1 in vitro and that oseltamivir absorption in rats is greatly reduced not only by milk, but also by coadministration of glycylsarcosine, a typical inhibitor of PEPT1, and by
Figure 3. Urinary excretion of (a) oseltamivir and (b) Ro 64-0802 after a single oral dose of 75 mg oseltamivir to six human volunteers with either 400 mL of water (open circles) or 400 mL of milk (closed circles). Data are expressed as means ± SD. Asterisks indicate a significant difference between treatments; ∗ p < 0.05, ∗∗ p < 0.01. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011
DOI 10.1002/jps
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Table 2. Pharmacokinetic Parameters After Single Oral Administration of Oseltamivir [30 mg/(10 mL/kg)] in Water, Milk, or 125 mM Gly–Sar Solution in Rats
Cmax (:g/mL) tmax (h) AUC0−1 (:g·h/mL) AUC1−2 (:g·h/mL) AUC0−6 (:g·h/mL)
Water (Control)
Milk
1.53 ± 0.35 0.5 ± 0.0 1.10 ± 0.21 0.643 ± 0.050 2.49 ± 0.12
0.31 ± (20.3) 0.7 ± (140) 0.237 ± (21.5) 0.230 ± (35.8) 0.942 ± (37.8)
+ 125 mM Gly–Sar 0.081 ± 0.00∗ 6.0 ± 0.0∗ 0.065 ± 0.001∗ 0.075 ± 0.002∗ 0.449 ± 0.006∗
(5.2) (1200) (5.9) (11.7) (18.0)
Data are expressed as means ± SD. Values in parenthesis are percentage versus water control. Cmax , peak plasma drug concentration; tmax , time to reach Cmax ; t1/2 , elimination half-life; AUC0–1 , AUC1–2 , AUC0–6 , area under the plasma drug concentration–time curve from time 0 to 1 h, from 1 to 2 h, and from 0 to 6 h, respectively. Asterisks indicate significant difference from water group; ∗ p < 0.05.
coadministration of casein, one of the principal proteins of milk.8 As previously mentioned, milk proteins are rapidly broken down to free amino acids or small peptides; so it was considered that the inhibition of intestinal absorption of oseltamivir by milk in humans could be explained by the inhibition of PEPT1mediated drug absorption by large amounts of small peptides derived from milk proteins. There is a marked species difference in the extent of inhibition between rats and humans (Table 1 and Table 2). The AUC of oseltamivir in humans was reduced only at 0–2 h postdosing of milk, whereas that in rats was strongly reduced throughout the experimental period. These differences might be at least in part due to the regional difference in smallintestinal luminal expression of PEPT1 between humans and rats.14 PEPT1 is localized predominantly in the duodenum, with sharply decreasing expression in lower regions in humans, whereas PEPT1 mRNA is uniformly expressed throughout the gastrointestinal tract in rats. Therefore, human PEPT1-mediated absorption of oseltamivir may occur predominantly in the duodenum, which would explain why the interaction with milk was observed only in the early period postdosing (approximately 95% of the dose passes through the duodenum within 40 min).15,16 In contrast, PEPT1 expressed throughout the intestine in rats may make a significant contribution to oseltamivir absorption. The assumption for the inhibitory time window on PEPT1-mediated absorption in humans can be supported by the previous report. In the 1970s, McCracken et al.17 reported that Cmax and AUC of cephalexin, benzylpenicillin, and phenoxymethylpenicillin among several antibiotics studied were reduced 40%–60% in infants and children given milk and drugs concurrently. Later studies revealed that the former two drugs are substrates of PEPT1.18,19 As we found in the case of oseltamivir, the reduction in the plasma levels of these drugs was limited to an early time window.17 Several other possibilities should also be considered. There might be differences in gastric emptying time, solubility or dissolved state of oseltamivir, and/ DOI 10.1002/jps
or elution rate of the drug from the capsule in the presence of milk and water. However, these possibilities seem unlikely for the following reasons: It has been reported that the tmax of erythromycin or amoxicillin was not changed by dosing with milk in humans,17 suggesting that milk does not alter the gastric emptying time. In this study, we tested the dissolution rate and solubility of an oseltamivir capsule with 400 mL of acidified water or milk. This medicine is completely eluted and dissolved into both solvents within 15 min (Supplementary Fig. 1). In addition, milk coagulation occurs only in unweaned infants, owing to the presence of a specific enzyme in the stomach.5 Therefore, oseltamivir completely dissolved in either water or milk is likely to reach the duodenum at the same rate following administration. Moreover, milk-equivalent calcium ions did not alter the ultraviolet spectra of oseltamivir in both water and acidified water (Supplementary Fig. 2). It was indicated that oseltamivir did not form insoluble complex with calcium ions, although it is known that certain drugs like quinolones form insoluble complex with calcium ions. Further, because oseltamivir has a low octanol–water partition coefficient at pH 7 (0.54), its absorption should not be affected by micelle formation or the coexistence of milk fat. When we examined the individual plasma concentration–time profiles of oseltamivir in humans, we noticed a double-peak phenomenon, which occurred only when oseltamivir was administered with water (Fig. 2). Previous reports on the clinical pharmacokinetics of oseltamivir have also noted the doublepeak phenomenon, which is considered to be due to enterohepatic circulation.20,21 In this study, the first peak specifically disappeared when oseltamivir was administered with milk. The tmax of the first peak of oseltamivir was around 0.5 h after the administration. Because the transit time of the duodenum in humans is considered to be 40 min,15,16 it is suggested that the disappearance of the first peak of oseltamivir reflects specific inhibition of PEPT1mediated absorption in the duodenum. In contrast, no double-peak phenomenon was observed in rats, which JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011
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express PEPT1 uniformly throughout the small intestine. The second peak observed in humans may reflect absorption through pathways other than PEPT1. Another transporter(s) may be involved in oseltamivir absorption because one-third of the active transport of oseltamivir was not inhibited by excess dipeptides in Caco-2 cells.8 The cause of the double-peak phenomenon is not fully understood, but several possible explanations have been proposed: biphasic gastric emptying,22 enterohepatic ciculation,23 and two different absorption sites.24 The last one may be related to regional differences in expression of the specific transporter(s) as previously speculated in the case of P-glycoprotein.25 Interestingly, oseltamivir absorption in subject #4, whose first peak was apparently smaller than other subjects, was not inhibited by milk. If the first peak is assumed to represent the PEPT1-mediated absorption of oseltamivir, there may be considerable interindividual variability in this process in humans. It has been reported that genetic polymorphisms,18 feeding, and hormonal status26 may be associated with interindividual differences in PEPT1-mediated absorption. The reduction in the AUC of Ro 64-0802 by milk ingestion was more notable than that of oseltamivir. This suggests that milk inhibited the oseltamivir conversion to Ro64-0802, catalyzed by hepatic carboxylesterase1 (CES1). Milk contains 3%–4% of triglycerides consisting of various fatty acids. Because a fatty acid palmitate is one of the endogenous substrates of CES127 and one of the most abundant constituent of milk fat, it is speculated that rapidly absorbed fatty acids including palmitate inhibited the oseltamivir conversion to Ro 64-0802.
CONCLUSION Our results indicate that milk inhibited the intestinal absorption of oseltamivir in humans. However, the extent of interaction in humans was limited as compared with that in rats, possibly because of species difference in the regional expression of PEPT1 in the small intestine and/or differences in the contribution of other absorption mechanisms. Among PEPT1 substrates, it seems likely that compounds whose absorption would be largely completed within the transit time of the duodenum may show marked inhibition of their absorption by milk in humans. This information might be useful not only for clinical medication, but also for drug development.
ACKNOWLEDGMENTS This study was supported by a grant from the Research Foundation for Pharmaceutical Sciences, and by government grants-in-aid for scientific research. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 9, SEPTEMBER 2011
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