Solute Carrier Family 22a is Involved in Drug Transfer into Milk in Mice

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Organic Cation Transporter/Solute Carrier Family 22a is Involved in Drug Transfer into Milk in Mice NAOKI ITO,1 KOUSEI ITO,2 YUKI IKEBUCHI,3 TOMOKO KITO,4 HIROSHI MIYATA,3 YU TOYODA,3 TAPPEI TAKADA,3 AKIHIRO HISAKA,5 MASASHI HONMA,3 AKIRA OKA,1 HIROYUKI KUSUHARA,4 HIROSHI SUZUKI3 1

Department of Pediatrics, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan 2 Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chuo-ku, Chiba 260-8675, Japan 3 Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan 4 Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan 5 Pharmacology and Pharmacokinetics, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan Received 20 April 2014; revised 7 July 2014; accepted 28 July 2014 Published online 29 August 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24138

ABSTRACT: Drug transfer into milk is a general concern during lactation. So far, breast cancer resistance protein (Bcrp) is the only transporter known to be involved in this process, whereas participation of other transporters remains unclear. We investigated the importance of organic cation transporter (Oct) in drug transfer into milk in mice. The mammary glands of lactating versus nonlactating FVB strain mice revealed elevated mRNA levels of Oct1 and Bcrp, whereas Oct2 and Oct3 mRNA levels were decreased. Specific uptake of cimetidine, acyclovir, metformin, and terbutaline was observed in human embryonic kidney 293 cells transfected with murine Oct1 or Oct2. The milk-to-plasma concentration ratio (M/P) values of cimetidine and acyclovir were significantly decreased in Bcrp knockout and Oct1/2 double-knockout (DKO) mice compared with control FVB mice, whereas the M/P values of terbutaline and metformin were significantly decreased in Oct1/2 C 2014 Wiley DKO mice alone. These are the first to suggest that Oct1 might be involved in secretory transfer of substrate drugs into milk.  Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3342–3348, 2014 Keywords: drug transfer into milk; lactation; organic cation transporter/solute carrier 22a; pediatric; mammary transport/secretion; disposition

INTRODUCTION The benefits of breastfeeding are widely accepted and predicated on the results of extensive epidemiological research.1–3 Importantly, approximately 90% of women take some form of medication during their first week postpartum,4,5 and, therefore, a general concern is the avoidance of drug transfer into milk and toxicity in breast-fed infants. However, only a few clinical studies have actually investigated drug transfer into milk, necessitating the use of prediction methods to address this crucial issue.6–8 In principal, these methods consider transcellular transport across the mammary epithelium, drug binding to proteins in the milk and plasma, and drug partition into fat globules in milk. Abbreviations used: Abcg2, ATP-binding cassette transporter g2; BCA, bicinchoninic acid; Bcrp, breast cancer resistance protein; DKO, double-knockout; HEK, human embryonic kidney; KO, knockout; KHBS, Krebs–Henseleit buffer solution; LC–MS/MS, liquid chromatography–tandem mass spectroscopy; M/P, milk-to-plasma concentration ratio; M/Punbound , unbound milk-to-plasma concentration ratio; Oct, organic cation transporter; qPCR, quantitative polymerase chain reaction; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; Mate, multidrug and toxin extrusion protein; Slc, solute carrier; TEA, tetraethylammonium; UPLC, ultra-performance liquid chromatography. Correspondence to: Kousei Ito (Telephone: +81-43-226-2886; Fax: +81-43-2262886; E-mail: [email protected]) This article contains supplementary material available from the authors upon request or via the Internet at http://wileylibrary.com. Journal of Pharmaceutical Sciences, Vol. 103, 3342–3348 (2014)

 C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

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Most transcellular transport mechanisms assume that only the unionized, unbound form of the drug can traverse the apical/basolateral membranes of the mammary epithelium by passive diffusion. Under this assumption, the unbound milk-toplasma concentration ratio (M/Punbound ) can be calculated by using the Henderson–Hasselbalch equation and the drug pKa, a plasma pH of 7.4, and a milk pH of 6.8–7.2. This prediction is generally sound, but there are exceptions. For example, acyclovir, cimetidine, and nitrofurantoin are secreted at higher than predicted rates in human subjects9–11 and experimental animals,12,13 suggesting that transporter-mediated transport might be involved in the case of these exceptional drugs. Jonker and colleagues14–16 first reported the induction of breast cancer resistance protein (Bcrp)/ATP-binding cassette transporter g2 (Abcg2) on the apical membrane of mammary epithelial cells during lactation, and attenuated transfer of Bcrp substrates (acyclovir, cimetidine, nitrofurantoin) was subsequently shown in Bcrp knockout (KO) mice. In addition to Bcrp, the expression levels of peptide transporter/solute carrier (Slc) 15a, concentrative nucleoside transporter/Slc28a, and organic cation transporter (Oct)/Slc22a family members are all upregulated in the mammary gland during lactation.12,17–20 However, Bcrp is the only established transporter clearly shown to contribute to drug transfer into milk. To gain further insight into drug transport in the mammary gland, we recently determined the M/Punbound values for

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27 clinical drugs in mice, and compared these values with the predicted values based on passive diffusion/pH partitioning.21 Certain known Bcrp substrates (e.g., cimetidine and acyclovir) and terbutaline (not a known Bcrp substrate) were secreted into milk at rates that surpassed the predicted values by more than threefold [(observed M/Punbound )/(predicted M/Punbound ) = 4.5, 3.7, and 4.8 for cimetidine, acyclovir, and terbutaline, respectively].21 Because cimetidine and acyclovir are also Oct family substrates,22–24 and Oct mRNA expression levels are augmented in the mammary gland during lactation,17,20 we hypothesized that Oct family members might participate together with Bcrp in drug transfer into milk. Here, we compared the milk-to-plasma concentration ratio (M/P) values of cimetidine and acyclovir among Bcrp KO, Oct1/Oct2 double-knockout (DKO), and wild-type FVB mice. Metformin, a known substrate of murine Oct1/2,25 was included as a positive control. Moreover, clindamycin and verapamil were included as negative controls because their M/Punbound values are similar to those predicted by the pH-partitioning theory21 and because they are not known substrates of Bcrp or Oct transporters.

MATERIALS AND METHODS Drugs Acyclovir was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Clindamycin hydrochloride, terbutaline hemisulfate salt, and (±)-verapamil hydrochloride were obtained from Sigma–Aldrich (St. Louis, Missouri). Cimetidine was obtained from Nacalai Tesque (Kyoto, Japan). Metformin was purchased from Alexis Biochemicals (San Diego, California). All other reagents were of analytical grade, except where otherwise noted. Animals

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Norcross, Georgia). Reverse transcription was performed by using ReverTra Ace reverse transcriptase (Toyobo Life Sciences, Osaka, Japan), followed by quantitative PCR (qPCR) analysis with SYBR GreenERTM qPCR Supermix Universal ready-to-use cocktail (Life Technologies Inc., Carlsbad, California) and an Eco Real-Time PCR system (Illumina Inc., San Diego, California). PCR primers were as follows: Bcrp forward, 5 -CATCAGCCTCGGTATTCCATand reverse, 5 -AATCCGCAGGGTTGTTGTAG-3 ; 3 ,  Oct1 forward, 5 -CAGGTTTGGCCGTAAGCTCT-3 , and Oct2 reverse, 5 -GCAACATGGATGTATAGTCTGGG-3 ; forward, 5 -TGCTGGACCTGTTTCAGTCAT-3 , and reverse, 5 -CTGTCTGCTAGGTAACCAATGC-3 ; Oct3 forward, 5 -CTATGCAGCGGACAGATATGG-3 , and reverse, 5 -AGCGGAAAATCACAAACACAGAA-3 ; and $-actin forward, 5 -CCCGAAGGAAAACTGACAGC-3 , and reverse, 5 GTGGTGGTGAAGCTGTAGCC-3 . R

Implantation of Micro-Osmotic Pumps Single dosing was used for the administration of drugs (acyclovir, cimetidine, clindamycin, metformin, terbutaline, verapamil) to lactating female mice. Dimethyl sulfoxide (50%) was used as the drug solvent according to the manufacturers’ instructions. Drug doses were determined according to the clinical dose (mg/kg•day) and the detection limit for each compound (Supplementary Table 1). Lactating mice received inhaled ether anesthesia when their pups were 14 days old, followed by intraperitoneal implantation of an Alzet model 1003D microosmotic pump (Durect Corporation, Cupertino, California) containing acyclovir, cimetidine, clindamycin, metformin, terbutaline, or verapamil, as reported previously.21 The pumps had a reservoir volume of 97 ± 7 :L, and the flow rate was maintained at a constant rate of 0.95 ± 0.02 :L/h over a period of 72 h. After implantation of the pumps, the mothers were returned to their home cages with their pups and fed ad libitum. All mice survived the surgical procedure and appeared to be lactating normally, and all pups thrived throughout the course of the experiment. R

Bcrp KO mice (male and female, FVB. 129P2-Abcg2tm1Ahs N7) and Oct1/2 DKO mice (male and female, FVB. 129P2Slc22a1tm1Ahs Slc22a2tm1Ahs N7) were purchased from Taconic Farms (Hudson, New York). Control wild-type FVB mice (8 weeks of age) were purchased from CLEA Japan (Tokyo, Japan). Mating was initiated at 9 weeks of age and confirmed by the presence of vaginal plugs, whereas pregnancy was confirmed by weight gain following coitus on gestational day 7. During pregnancy, parturition, and lactation, the mothers were individually pair-housed with their pups. All animals were treated humanely and maintained under standard conditions, with a reverse dark-light cycle in a room kept at 25◦ C with 50% relative humidity. A standard rodent chow diet (CMF diet, Oriental Yeast Company, Ltd., Tokyo, Japan) and water were provided ad libitum. The litter size was standardized to eight pups (four males and four females) at 4 days postpartum. The research protocol adhered to the “Principles of Laboratory Animal Care” (National Institutes of Health publication #85-23, revised in 1985) and was approved by the Animal Studies Committee of the University of Tokyo (Tokyo, Japan).

Milk was collected from nursing mice at 60 h after pump implantation as reported previously.21 Because pups were separated from mother mice 8 h before milking to accumulate milk in the sinus, and only tracer amount of milk (∼20 :L/nipple) was collected, steady state M/P value might be minimally affected by milk flow rate but mostly determined by secretion and reuptake clearance across the mammary epithelia. Immediately after milk collection (total 200 :L from 10 nipples), blood samples were drawn from the jugular vein and separated into red blood cells and plasma. Milk and plasma samples were frozen and stored at −80◦ C until use. The protein content of the milk was measured by using the bicinchoninic acid (BCA) assay (Pierce BCA Protein Assay Kit; Thermo Fisher Scientific, Rockford, Illinois), and the lipid content of the milk was evaluated by creamatocrit measurement.26

Messenger RNA Expression of Transporters in the Mammary Gland

Measurement of Drug Concentrations in Milk and Plasma

Mammary gland tissue was manually isolated after removal of hair and surrounding muscle as much as possible. Total RNA was extracted from mammary gland tissue by using the RNA-Solv Reagent system (Omega Bio-Tek Inc.,

Drug concentrations in the milk and plasma were measured as reported previously.21 Briefly, an aliquot (50 :L) of thawed milk or plasma was mixed with acetonitrile (500 :L) containing 500 nM carbamazepine as an internal standard, vortexed, and

DOI 10.1002/jps.24138

Collection of Milk

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deproteinized by centrifugation. An aliquot (450 :L) of each supernatant was condensed and dried by using a SpeedVac centrifugal concentrator (Thermo Fisher Scientific, Inc., Springfield, New Jersey). Desiccated milk samples were dissolved in 40% acetonitrile (200 :L) plus n-hexane (100 :L), vortexed, and centrifuged for 5 min at 20,000g to remove lipids. Plasma samples were dissolved in 40% acetonitrile (200 :L), vortexed, and centrifuged for 5 min at 20,000g. Liquid chromatography– tandem mass spectroscopy (LC–MS/MS) was performed by using an ultra-performance LC (UPLC) system and a Quattro Premier XE mass spectrometer (Waters, Milford, Massachusetts) with a 1.7 :m particle Acquity UPLCTM bridged ethyl hybrid C18 analytical column (2.1 mm × 100 mm; Waters). Detailed analytical conditions are described in Supplementary Table 1. Transport Assay of Drug Uptake In Vitro Human embryonic kidney (HEK) 293 cells stably transfected with murine Oct1, Oct2, or control (mock) vector27 were seeded into poly-L-lysine- and poly-L-ornithine-coated 12-well plates at a density of 4 × 105 cells per well at 72 h before performing the transport assay. The cell culture medium was replaced with fresh culture medium supplemented with 5 mM sodium butyrate at 24 h before the assay to induce Oct1 and Oct2 expression. The transport assay was then carried out as described previously.27 Briefly, the cells were washed twice and preincubated with Krebs–Henseleit buffer solution (KHBS) at 37◦ C for 15 min. Drug uptake was initiated in the presence or absence of the Oct substrate, tetraethylammonium (TEA, 5 mM), by replacing the preincubated KHBS with fresh KHBS containing each drug (10 :M) at 37◦ C. Uptake was terminated after 5 min by removal of the drug-containing KHBS and addition of fresh, ice-cold KHBS. The cells were then scraped into distilled water (800 :L) and sonicated. An aliquot of sample (500 :L) was added to acetonitrile (1000 :L) and centrifuged for 10 min at 20,000g for deproteinization. The supernatant was collected, and an aliquot of the sample was dried by using a centrifugal concentrator, dissolved in 40% acetonitrile, and subjected to LC–MS/MS analysis (see Supplementary Table 1 for detailed analytical conditions). The remainder of the supernatant was suspended in RIPA buffer (50 mM Tris–HCl, pH 7.4, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 150 mM NaCl, and 1% NP-40), and the protein concentration was determined via the BCA assay. Statistical Analysis Quantitative data are presented as the mean ± SEM. IBM SPSS Statistics software, version 19 (SPSS Inc., Chicago, Illinois) was used to perform all statistical analyses. Data were analyzed by using the one-way ANOVA followed by Dunnett’s post hoc test to identify significant differences between groups. In all cases, p < 0.05 was considered statistically significant. R

RESULTS Oct Family Members are Expressed During Lactation in Mice Messenger RNA expression levels of Oct1, Oct2, Oct3, and Bcrp were normalized by $-actin mRNA expression then evaluated before and after parturition in wild-type FVB strain mice. Oct1 mRNA was barely detected on gestational days 14 and 20, but was dramatically elevated throughout the lactation period (Fig. 1). We also confirmed that the mRNA expression of Bcrp Ito et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3342–3348, 2014

Figure 1. Messenger RNA expression profiles of drug transporters in the mammary gland of wild-type FVB strain mice. Mammary glands were surgically collected on gestational days 14 and 20 (G14 and G20) and lactation days 1, 4, 7, 10, 14, 18, and 21 (L1, L4, L7, L10, L14, L18, and L21). Oct1, Oct2, Oct3, and Bcrp mRNA expression levels are shown relative to $-actin mRNA levels in arbitrary units. Data are given as the mean ± SEM (n = 3).

increased after parturition and during lactation (Fig. 1), as reported previously.16 By contrast, Oct2 and Oct3 mRNAs were readily observed during gestation, but decreased after parturition and remained low from lactation day 1 through lactation day 21 (Fig. 1). Similar results were obtained when the mRNA expression was normalized by another internal control gene such as glyceraldehyde 3-phosphate dehydrogenase (Gapdh) (Supplementary Fig. 1). Moreover, mRNA expression of other Octs including multidrug and toxin extrusion protein DOI 10.1002/jps.24138

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Figure 2. In vitro uptake of drugs into Oct1- and Oct2-transfected HEK293 cells. Uptake of drugs (10 :M) into HEK293 cells stably transfected with murine Oct1, Oct2, or control (mock) vector. Uptake assays were performed for 5 min at 37◦ C. Closed and open columns represent drug uptake in the absence and presence of TEA (5 mM), a prototypical Oct1/2 substrate. Data are given as the mean ± SEM (n = 3; *p < 0.05 and **p < 0.01, one-way ANOVA followed by Dunnett’s post hoc test).

(Mate)1/Slc47a1 and Mate2/Slc47a2 were extremely low and not induced during lactation (data not shown). Acyclovir, Cimetidine, Terbutaline, and Metformin are Substrates of Oct1 and Oct2 To evaluate the Oct-mediated transport of the test drugs, in vitro uptake experiments were performed by using HEK293 cells stably transfected with murine Oct1 or Oct2. Figure 2 demonstrates that the uptake of acyclovir, cimetidine, terbutaline, and metformin (the positive control) by Oct1- or Oct2transfected HEK293 cells was significantly greater than uptake by mock (empty vector)-transfected HEK293 cells, and was inhibited by excess amounts of the prototypical Oct substrate, TEA. The uptake of clindamycin was also significantly enhanced in Oct2- versus mock-transfected HEK293 cells, but TEA did not affect clindamycin uptake. Neither Oct1- nor Oct2transfected cells transported verapamil at least in our experimental condition. These results are in keeping with the fact that clindamycin and verapamil are not known substrates of Oct1/2. Contribution of Oct1/2 to Drug Secretion into Milk We next examined whether the test compounds were secreted into the milk via Bcrp, the only transporter known to be involved in drug secretion into milk, or Oct1, the candidate transporter forming the focus of this study. Drug concentrations in plasma and milk are shown in Supplementary Table 2, and the DOI 10.1002/jps.24138

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Figure 3. Comparison of steady-state M/P values between wild-type FVB, Bcrp KO, and Oct1/2 DKO mice. Lactating mice were intraperitoneally implanted with micro-osmotic pumps containing the drug of interest, and milk and plasma samples were collected at 60 h after implantation. The M/P value for each drug is shown. Data are given as the mean ± SEM (n = 3–5; *p < 0.05 and **p < 0.01 vs. wild-type FVB mice, one-way ANOVA followed by Dunnett’s post hoc test).

calculated M/P values are shown in Figure 3. The M/P values of cimetidine and acyclovir, known Bcrp substrates, were significantly decreased in Bcrp KO mice, as reported previously.14 The other four compounds (clindamycin, verapamil, terbutaline, and metformin) were unaffected by Bcrp KO (Fig. 3). These results confirm that our experimental approach (i.e., continuous dosing from an intraperitoneally implanted micro-osmotic pump) can accurately assess the contribution of transporters to drug transfer into milk. Next, we examined the importance of Oct1 and/or Oct2 to drug transfer into milk by comparing M/P values between wildtype and Oct1/2 DKO mice. The M/P values of cimetidine, terbutaline, metformin and acyclovir were significantly decreased in Oct1/2 DKO vs. wild-type mice, whereas the M/P value of verapamil was significantly increased in the DKO mice. The M/P value of clindamycin was unaffected by Oct1/2 DKO (Fig. 3). These findings substantiate those of Figure 2 and suggest that cimetidine, terbutaline, metformin and acyclovir are all substrates of Oct1/2. M/P values are influenced not only by transepithelial transport, but also by drug binding to proteins and lipids in milk.21 To exclude potential secondary effects caused by differential protein and lipid contents in milk, we quantified these values for wild-type, Bcrp KO, and Oct1/2 DKO mice. Figure 4 shows that the protein and lipid levels were similar among the Ito et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3342–3348, 2014

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Figure 4. Comparison of lipid and protein contents in the milk of wild-type FVB, Bcrp KO, and Oct1/2 DKO mice. Milk samples from mother mice were used for the analysis of creamatocrit (a) and protein (b) content when the pups were 16 days old. No significant differences were observed among the three groups of mice. Data are given as the mean ± SEM (n = 10; one-way ANOVA followed by Dunnett’s post hoc test).

three groups of mice, indicating that the observed changes in the M/P values for the KO mice indeed resulted from reduced transepithelial drug transport. It is also possible that KO of the particular transporter induces or suppresses other transporter(s) to show compensatory effect. However, it was not the case as shown in Supplementary Figure 2. Oct1, Oct2, and Oct3 mRNA were not changed in Bcrp KO compared with wild-type, while Oct3 and Bcrp mRNA were not changed in Oct1/2 DKO compared with wild-type.

DISCUSSION Our previous work showed that the M/Punbound values of certain drugs were larger than predicted in the mouse.21 In such cases, we speculated that transporters facilitated drug transfer into milk from the maternal blood circulation. Notably, the mRNA expression of Oct1 gradually increases after parturition in the rat, reaching a level approximately 40-fold higher than that found on gestational day 20.17 Moreover, Oct1 and Oct3 mRNAs are found in the lactating rat mammary gland, but Oct2 mRNA is not.12 Consistent with these reports, we showed that the mRNA expression of murine Oct1 was gradually induced after parturition (Fig. 1), whereas the mRNA expression of Oct2 and Oct3 declined. We also demonstrated for the first time that the transfer of selected Oct1/2 substrates into the milk was mitigated in Oct1/2 DKO mice (Fig. 3). The precise membrane localization of Oct1 and Oct2 in the mouse mammary gland remains to be elucidated. However, the following observations suggest that these transporters are Ito et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3342–3348, 2014

probably expressed on the basolateral side of the membrane: (1) Our results indicate that Oct family members act as secretory transporters to transfer drugs from the blood into the milk. (2) Oct family members are known to transport organic cations in an electrogenic manner (i.e., from the external environment into the cell). (3) Oct family members are generally expressed on the basolateral side of the membrane in other cells and tissues, including the liver (hepatocytes) and the kidney (renal epithelial cells).28 Furthermore, the functional expression of Oct family members on the basolateral side of the membrane was implicated in an in vitro human mammary epithelial cell model generated to study the M/P values of therapeutic drugs.29 In this model, the transport of TEA across the trypsin-resistant epithelial cell monolayer was significantly higher in the basolateral-to-apical direction than in the apical-to-basolateral direction. Concomitantly, elevated mRNA expression levels of human OCT1 and OCT3 were detected in the cell monolayer.29 Notably, cimetidine and acyclovir are substrates of Bcrp as well as Oct1 and Oct2,22–24 and genetic depletion of any of these transporters attenuated cimetidine and acyclovir transfer into milk in the present study (Fig. 2). These observations again support the putative localization of Oct1/2 on the basolateral side of the mammary epithelial cell membrane, permitting substrate uptake from the blood into the cell. Our results also support the localization of Bcrp on the apical side of the membrane, as previously reported,16 permitting substrate secretion from the cell into the milk. Such cooperative vectorial transport is a novel finding, particularly in the mammary epithelium, although similar systems are well established in other polarized tissues, including the liver, kidney, and intestine.30,31 Induction of Oct1 mRNA during lactation implies some physiological relevance. One of the possible roles is secretion of important nutrient, such as vitamin, into milk as already demonstrate for Bcrp. Bcrp is induced during lactation in mammary gland and mediates the secretion of riboflavin (vitamin B2) into milk.32 In line with this, we just recently found that M/P of endogenous thiamin (vitamin B1) was dramatically reduced by 28-fold in Oct1/2 DKO compared with wild-type and that thiamin was a transport substrate of both Oct1 and Oct2.* Although we have not yet seen any growth retardation or abnormality such as beriberi in pups raised by Oct1/2 DKO, Oct1 might be important at least during lactation to supply thiamin to the pup via breast milk. Close examination of the pups raised by the nurse mouse fed with thiamin restriction diet would give clear answer. Induction of OCT1/3 mRNA in the human mammary epithelium during lactation was previously reported, where samples from the lactating mother were purified from cell pellet contaminants in breast milk, and control samples were purified from reduction mammoplasty tissue.20 It is possible that OCT1 facilitates transfer of its substrates into the milk from the maternal blood circulation during breastfeeding in humans, but this requires further investigation, especially given that the differences between observed and predicted M/Punbound values are less pronounced in humans versus mice. In humans, the observed M/Punbound values of cimetidine, terbutaline, metformin, and acyclovir were 4.51, 1.63, 0.52, and 1.64, respectively, whereas the predicted M/Punbound values were 1.43, 2.77, 2.78, and 0.89.33 Therefore, the contribution of OCT1-mediated transport to drug transfer into milk may be of diminished DOI 10.1002/jps.24138

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importance in humans. Additional pharmacogenomics studies focusing on OCT1 KO genotypes are required to determine the exact role of human OCT1.

CONCLUSIONS The current study demonstrated that Oct1/2 is involved in the secretion of substrate drugs into milk in mice. This finding substantially adds to our understanding of the mechanism of drug transfer into milk, and is expected to contribute to the development of better prediction methods. However, elucidation of the specific contribution of Oct1 versus Oct2 will require further studies that utilize single KO mice (i.e., Oct1 KO and Oct2 KO mice) rather than Oct1/2 DKO mice.

ACKNOWLEDGMENTS This work was supported by a research scholarship from the Japan Research Foundation for Clinical Pharmacology, and a Grant-in-Aid for Scientific Research on Innovative Areas HDPhysiology (Grant 22136015) from the Ministry of Education, Science and Culture of Japan.

NOTE * Kato K, Moriyama C, Ito N, Ikebuchi Y, Hachiuma K, Hagima N, Iwata K, Yamaguchi J, Ito K, Suzuki H, Sugiyama Y, Kusuhara H, unpublished results

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DOI 10.1002/jps.24138