The AAPS Journal, Vol. 10, No. 2, June 2008 ( # 2008)
`DOI: 10.1208/s12248-008-9035-6
`
`Review Article
`Themed Issue: Monocarboxylate Transporters in Drug Disposition
`Guest Editor: Marilyn Morris
`
`Overview of the Proton-coupled MCT (SLC16A) Family of Transporters:
`Characterization, Function and Role in the Transport of the Drug of Abuse
`γ-Hydroxybutyric Acid
`
`Marilyn E. Morris1,2 and Melanie A. Felmlee1
`
`Received 4 March 2008; accepted 1 April 2008; published online 4 June 2008
`Abstract. The transport of monocarboxylates, such as lactate and pyruvate, is mediated by the SLC16A
`family of proton-linked membrane transport proteins known as monocarboxylate transporters (MCTs).
`Fourteen MCT-related genes have been identified in mammals and of these seven MCTs have been
`functionally characterized. Despite their sequence homology, only MCT1–4 have been demonstrated to
`be proton-dependent transporters of monocarboxylic acids. MCT6, MCT8 and MCT10 have been
`demonstrated to transport diuretics, thyroid hormones and aromatic amino acids, respectively. MCT1–4
`vary in their regulation, tissue distribution and substrate/inhibitor specificity with MCT1 being the most
`extensively characterized isoform. Emerging evidence suggests that in addition to endogenous substrates,
`MCTs are involved in the transport of pharmaceutical agents, including γ-hydroxybuytrate (GHB), 3-
`hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins), salicylic acid, and bumetanide.
`MCTs are expressed in a wide range of tissues including the liver, intestine, kidney and brain, and as such
`they have the potential to impact a number of processes contributing to the disposition of xenobiotic
`substrates. GHB has been extensively studied as a pharmaceutical substrate of MCTs; the renal clearance
`of GHB is dose-dependent with saturation of MCT-mediated reabsorption at high doses. Concomitant
`administration of GHB and L-lactate to rats results in an approximately two-fold increase in GHB renal
`clearance suggesting that inhibition of MCT1-mediated reabsorption of GHB may be an effective
`strategy for increasing renal and total GHB elimination in overdose situations. Further studies are
`required to more clearly define the role of MCTs on drug disposition and the potential for MCT-
`mediated detoxification strategies in GHB overdose.
`
`KEY WORDS: butyrate; gamma-hydroxybutyrate; lactate; monocarboxylate transporters; SLC16A.
`
`INTRODUCTION
`
`Monocarboxylic acids play a major physiological role in
`that they represent an energy source for all cells in the body.
`Of these compounds, lactate is critically important as it is the
`end product of glycolysis and intracellular accumulation of
`lactate results in the inhibition of glycolysis. Furthermore,
`lactate can be oxidized in the brain and red skeletal muscle to
`fuel cellular respiration. As such, the transport of lactate and
`other monocarboxylic acids both into and out of cells is vital
`for cellular function.
`Two transporter families have been identified that
`facilitate this need:
`the proton-coupled monocarboxylate
`transporters (MCTs) and the sodium-coupled monocarbox-
`ylate transporters (SMCTs). MCTs (SLC16A) were first
`identified in the mid-nineties and to date 14 members of this
`
`1 Department of Pharmaceutical Sciences, School of Pharmacy and
`Pharmaceutical Sciences, University at Buffalo, State University of
`New York, Amherst, New York 14260, USA.
`2 To whom correspondence should be addressed. (e-mail: memorris
`@buffalo.edu)
`
`family have been identified through sequence homology
`(1,2). Currently, seven isoforms have been functionally
`characterized and it has been demonstrated that not all
`members function as proton-coupled transporters and that a
`wide variety of endogenous and exogenous compounds are
`substrates, including lactate, pyruvate, butyrate, γ-hydroxy-
`butyrate, bumetanide, and simvastatin acid (3–6). In contrast,
`the SMCT family contains only two members, SLC5A8 and
`SLC5A12, which were identified within the past 5 years
`(7–9). SMCTs have strikingly similar substrate specificities
`transporting short-chain monocarboxylates and sodium ions
`with ratios between 4:1 and 2:1 (Na:substrate) (9). These two
`distinct transporter families are further differentiated by their
`respective tissue distributions: SMCTs demonstrate a more
`restricted distribution (primarily kidney and intestine) while
`MCTs show a more ubiquitous distribution (4,9).
`In addition, unlike SMCTs, some members of the MCT
`family have been demonstrated to transport exogenous
`compounds including drugs. The impact of MCT substrate/
`inhibitor specificity and tissue distribution needs to be further
`examined with respect to drug substrates, and the overall
`influence of MCTs on drug disposition. The present review
`
`311
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`1550-7416/08/0200-0311/0 # 2008 American Association of Pharmaceutical Scientists
`
`Ranbaxy Ex. 1025
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`
`

`
`312
`
`Morris and Felmlee
`
`focuses on the proton-coupled MCTs and aims to summarize
`our current understanding of their structure, function and
`regulation as well as their role in drug disposition using γ-
`hydroxybutyrate (GHB; a known MCT substrate) (10–12) as
`a specific example.
`
`STRUCTURE, FUNCTION AND REGULATION
`OF MONOCARBOXYLATE TRANSPORTERS
`
`The uptake of monocarboxylates was first demonstrated
`to be transporter-mediated in erythrocytes (13,14). Subse-
`quently, the existence of a family of monocarboxylate trans-
`porters was proposed following the characterization of lactate
`transport in a variety of cell types (13,15,16). To date, 14
`members of the MCT family have been identified through
`screening of genomic and expressed sequence tag (EST)
`databases (4). Hydropathy plots have predicted that MCTs
`have 12 transmembrane domains with the N- and C-termini
`located in the cytoplasm (2,4). The transmembrane domains
`(TMDs) are highly conserved between isoforms with the
`greatest sequence variations observed in the C-terminus and
`the large intracellular loop between TMDs 6 and 7, which has
`a range of 29–105 amino acid residues (2). This observed
`variability is common to transporters with 12 TMDs and it is
`thought that these sequence variations are related to sub-
`strate specificity or regulation of transport activity (2,17).
`Human tissue distribution of all currently identified isoforms
`has been investigated and is summarized in Table I. A
`number of recent reviews and articles have examined the
`tissue specific localization and physiological functions of MCT
`isoforms in both humans and rodents (18–25). Regulation of
`MCTs has been demonstrated to occur via transcriptional,
`translational and post-transcriptional mechanisms (26–28).
`These regulatory pathways appear to be age- and tissue-
`dependent, which further complicates the understanding of
`these pathways (27,28). Some MCTs require an ancillary
`protein (see Table I) which can be involved in cellular
`localization (29) or protein–protein interactions (30); howev-
`er, the role of these accessory proteins in overall transporter
`function is not yet completely understood (29).
`Functional characterization of MCT isoforms has been
`extended to seven isoforms (MCT1–4, 6, 8, 10) with the seven
`remaining MCT family members being classified as orphan
`MCTs (MCT5, 7, 9, 11–14). Table II provides a summary of
`currently identified substrates and inhibitors of functionally
`characterized MCT isoforms from humans and rats. Our current
`understanding indicates that the transport mechanism varies
`between MCT isoforms and that not all MCT isoforms transport
`monocarboxylates (e.g. MCT8). The following sections aim to
`provide an overview of our current understanding of individual
`MCT isoforms with respect to unique structural features,
`substrate/inhibitor specificity and regulation.
`
`MCT1
`
`MCT1 was first identified in Chinese hamster ovary cells
`when altered mevalonate transport resulting from a single
`point mutation was detected (15). Subsequently, human, rat
`and mouse homologues were cloned and functionally charac-
`terized (16,31–34). Tissue distribution of MCT1 is ubiquitous
`(Table I); however, localization within specific tissues varies.
`
`in the retinal pigment epithelium (RPE),
`For example,
`expression is restricted to the apical membrane (2,17).
`Transport kinetics have been thoroughly explored using
`lactate for this isoform and have demonstrated that
`it
`functions as a proton-dependent cotransporter/exchanger
`(13,35). Transport occurs by ordered sequential binding with
`association of a proton followed by lactate binding. The
`complex is translocated across the membrane and the lactate
`and proton are released sequentially. Since the transporter
`functions as an exchanger, transport can occur bidirectionally;
`however,
`it
`is primarily responsible for the uptake of
`substrates (17).
`While initial studies focused on the transport of lactate
`by MCT1, subsequent studies revealed that the substrate
`specificity of MCT1 was much less specific than initially
`thought (2,4,35). Substrate and inhibitor affinities are detailed
`in Table II. Transport of lactate was shown to be stereo-
`selective with MCT1 having a greater affinity for L-lactate
`than L-lactate (35). Uptake of butyrate by intestinal epithelia
`cells is highly dependent on MCT1 expression; alterations in
`MCT1 levels results in altered uptake of butyrate which is the
`primary energy source for these cells (36,37). Interestingly,
`XP13512 (a gabapentin prodrug) was specifically designed to
`be a substrate for MCT1 in the intestine to improve the
`bioavailability of gabapentin (38,39). In addition to the
`transport of short-chain monocarboxylic acids, MCT1 was
`demonstrated to transport branched oxo-acids with a greater
`affinity than lactate (35). The higher affinity of these acids for
`MCT1 supports previous studies demonstrating their inhibi-
`tory potential towards lactate transport. Inhibitors of MCT1
`fall
`into three broad categories: (1) bulky or aromatic
`monocarboxylates which act as competitive inhibitors (e.g.
`phenyl-pyruvate and α-cyano-4-hydroxycinnamate (CHC));
`(2) amphiphilic compounds with divergent structures (e.g.
`quercetin and phloretin); and (3) some 4,4’-substituted
`stilbene-2,2’-disulphonates (e.g. DIDS) (4). Other isoforms
`can be distinguished from MCT1 based on the inhibitory
`potential of these compounds (Table II).
`Relatively few studies have been conducted to assess the
`regulation of MCTs. Studies have indicated that altered
`physiological conditions and the presence of xenobiotics
`may alter the regulation of MCTs,
`in addition to altered
`expression at different developmental stages (40–42). MCT1
`expression undergoes transcriptional, post-transcriptional and
`post-translational regulation and appears to be regulated in a
`tissue-specific manner (26–28). In colonic epithelium, expo-
`sure to butyrate resulted in a concentration- and time-
`dependent increase in MCT1 mRNA, protein expression
`and a corresponding increase in butyrate transport (43).
`These data suggest the possibility of altered transcriptional
`regulation; however, the authors further demonstrated in-
`creased transcript stability indicating additional post-tran-
`scriptional regulation mechanisms (43). High concentrations
`of lactate have also been demonstrated to increase MCT1
`mRNA and protein levels in L6 cells (44). In contrast,
`treatment with testosterone resulted in increased skeletal
`muscle MCT1 protein expression and lactate transport in the
`absence of mRNA changes suggesting the importance of
`post-transcriptional regulation (27). These results indicate
`that careful experimental design is required to assess the
`induction potential of exogenous compounds with respect to
`
`

`
`SLC16A Transport Family
`
`313
`
`(4)
`(4)
`
`(4)
`
`(4)
`
`Orphan
`Orphan
`
`Orphan
`
`Orphan
`
`exchanger
`
`(4,70)
`
`Facilitateddiffusion/
`
`membrane
`
`Basolateral
`
`(4)
`
`(4,105)
`(4)
`
`(3,4)
`(4,19)
`
`Orphan
`
`Orphan
`Orphan
`
`diffusion
`Facilitated
`Orphan
`
`(4,19,62,103,104)
`
`H+cotransporter
`
`CD147
`
`(4,19,60–62,66)
`
`H+cotransporter
`
`CD147
`
`(4,29,51,53)
`
`(4,19)
`
`H+cotransporter
`
`exchanger
`
`H+cotransporter
`
`Ref.
`
`Transportmechanism
`
`EMBIGIN
`
`CD147
`
`Apicalandbasolateral
`
`protein
`
`Accessory
`
`Subcellularlocation
`
`membrane
`
`Basolateral
`
`(RPE)
`membrane
`
`Basolateral
`
`membrane
`
`Basolateral
`
`membranes
`
`choroidplexus
`lung,pancreas,RPE,
`
`Kidney
`
`plexus
`pancreas,RPE,choroid
`
`Skin,lung,ovary,breast,
`
`liver,placenta
`muscle,heart,
`
`adrenal,retina
`breast,brain,kidney,
`Endometrium,testis,ovary,
`
`placenta
`
`pancreas,prostate,lung
`intestine,brain,heart,
`
`intestine,lung,heart
`kidney,placenta,small
`cells,tumors,RPE,brain
`
`spleen,pancreas
`muscle,heart,brain,
`
`Brain,heart,ovary,breast,
`Breast,bonemarrow
`
`2q36.3
`17p13.1
`
`10q23.3
`
`BN000146
`BN000145
`00152779
`
`ENSG000
`
`MCT14SLC16A14
`MCT13SLC16A13
`
`MCT12SLC16A12
`
`17p13.2
`
`NM_153357
`
`MCT11SLC16A11
`
`Intestine,kidney,skeletal
`
`6q21-q22
`
`NM_018593
`
`TAT1
`
`MCT10SLC16A10
`
`Liver,brain,kidney,heart,
`Pancreas,brain,muscle
`
`10q21.2
`
`Xq13.2
`17q24.2
`
`BN000144
`
`NM_006517
`NM_004694
`
`(*MCT7)
`XPCT
`(*MCT6)
`
`MCT9SLC16A9
`
`MCT8SLC16A2
`MCT7SLC16A6
`
`Kidney,muscle,placenta,
`Placenta,intestine,colon
`
`17q25.1
`1p13.3
`
`NM_004695
`NM_004696
`
`(*MCT5)
`(*MCT4)
`
`MCT6SLC16A5
`MCT5SLC16A4
`
`Whitemuscle,whiteblood
`
`aorta,placenta,kidney
`(RPE),choroidsplexus,
`Retinalpigmentepithelium
`
`17q25.3
`
`NM_004207
`
`(*MCT3)
`
`MCT4SLC16A3
`
`22q13.1
`
`NM_013356
`
`REMP
`
`MCT3SLC16A8
`
`Testis,liver,kidney,skeletal
`
`12q14.1
`
`NM_004731
`
`Ubiquitous
`
`1p13.2
`
`NM_003051
`
`MCT2SLC16A7
`
`MCT1SLC16A1
`
`Tissuedistribution
`
`locus
`Humangene
`
`accessionID
`Sequence
`
`(*former)Name
`Alternate
`
`name
`
`UniGene
`
`MCT
`
`TableI.TheHumanSLC16ATransporterFamily
`
`

`
`314
`
`Morris and Felmlee
`
`Table II. Comparison of Substrates and Inhibitors for Various MCT Isoforms in Humans and Rats
`
`Species Isoform
`
`Expression System
`
`Substrate
`
`Km (mM)
`
`Inhibitor
`
`References
`
`(17,35,38,43,53)
`
`Kia or IC50b
`(μM)
`
`28a
`n.a.
`425a
`n.a.
`0.620b
`
`Human MCT1
`
`Xenopus oocytes
`
`MCT2
`
`Xenopus oocytes
`
`Lactate
`Pyruvate
`Acetoacetate
`α-Ketoisovalerate
`α-oxoisohexanoate
`α-oxoisovalerate
`Butyrate
`XP13512
`Pyruvate
`
`MCT3
`MCT4
`
`MCT6
`
`MCT8
`
`Rat
`
`MCT1
`
`ARPE-19 cells
`Xenopus oocytes
`
`Lactate
`L-lactate
`D-lactate
`Pyruvate
`D-β-hydroxybutyrate
`Acetoacetate
`α-ketobutyrate
`α-ketoisocaproate
`α-ketoisovalerate
`Bumetanide
`Nateglinide
`Prostaglandin F2α
`COS1 and JEG3 cells T3
`T4
`Lactate
`GHB
`
`Xenopus oocytes
`
`Xenopus oocytes
`
`MCT2
`
`MDA-MB231
`Xenopus oocytes
`
`MCT4
`
`Xenopus oocytes
`
`MCT8
`
`Xenopus oocytes
`
`MCT10
`
`Xenopus oocytes
`
`γ-hydroxybutyrate
`Lactate
`Pyruvate
`
`L-lactate
`Pyruvate
`2-oxoisohexanoate
`Acetoacetate
`β-hydroxybutyrate
`T3
`T4
`L-Tryptophan
`L-Tyrosine
`L-Phenylalanine
`L-DOPA
`
`3.5–6
`1.8–2.5
`5.5
`1.3
`0.67
`1.25
`9
`0.22
`0.025
`
`n.a.
`28
`519
`153
`130
`216
`57
`95
`113
`0.084
`n.a.
`n.a.
`n.a.
`n.a.
`3.5
`4.6
`
`4.6
`0.74
`n.a.
`
`34
`36.3
`13
`31
`65
`n.a.
`n.a.
`3.8
`2.6
`7.0
`6.4
`
`Phloretin
`Quercetin
`CHC
`pCMBS
`XP13512
`
`CHC
`L-Lactate
`GHB
`
`pCMBS
`CHC
`Phloretin
`NPPB
`Fluvastatin
`Atorvastatin
`Lovastatin
`Simvastatin
`Furosemide
`Azosemide
`
`Phloretin
`Quercetin
`Benzbromaron
`CHC
`
`Phloretin
`Quercetin
`Benzbromaron
`CHC
`CHC
`pCMBS
`
`n.a.
`n.a.
`n.a.
`
`21a
`991a
`41a
`240a
`32b
`32b
`44b
`79b
`46b
`21b
`
`28b
`14b
`22b
`425b
`
`14b
`5b
`9b
`24b
`350b
`n.a.
`
`(53)
`
`(21)
`(64,65)
`
`(3)
`
`(72)
`
`(12,35,58)
`
`(12)
`(58)
`
`(104)
`
`(71)
`
`(70)
`
`N-bromoacetyl-T3
`Bromosulfophthalein
`
`n.a.
`n.a.
`
`CHC α-Cyano-4-hydroxycinnamate, NPPB 5-nitro-2-(3-phenylpropylamino)benzoate, pCMBS p-chloromercuribenzenesulphonic acid, n.a.
`transporter kinetic parameters were not determined
`The superscripts are used with the data in the same column of the table to indicate if the values are IC50 or Ki values
`
`MCT1 and multiple regulation pathways appear to be
`involved in its regulation. The MCT1 5’-flanking and 3’
`untranslated regions were recently cloned and a variety of
`transcription factor binding sites were identified (26). In
`addition, increased MCT1 expression and activity have been
`reported in human neuroblastoma and melanoma cell lines
`resulting from low extracellular pH (41,45). Inhibition and
`silencing of MCT1 in neuroblastoma and glioma cell lines
`resulted in increased cellular pH leading to apoptotic cell
`death suggesting that MCT1 may represent a novel chemo-
`
`therapeutic target (41,46,47). Additional studies need to
`address the potential for varied physiological states and
`xenobiotics to alter MCT1 (or other isoforms) regulation, as
`this may impact the disposition of both endogenous and
`exogenous MCT substrates.
`MCT1 is further regulated by its association with the cell
`surface glycoprotein CD147, which has a single transmem-
`brane domain with the C-terminus located in the cytosol
`(48,49). Topology studies suggest that one MCT1 molecule
`interacts with a single CD147 molecule with subsequent
`
`

`
`SLC16A Transport Family
`
`315
`
`dimerization with another MCT1/CD147 pair.(49). The initial
`association of CD147 and MCT1 is required for the translo-
`cation of MCT1 to the plasma membrane (48). Furthermore,
`studies indicate that covalent modification of CD147 results
`in inhibition of lactate transport as is seen with pCMBS-
`mediated inhibition of
`transport (48,50). In addition to
`MCT1, CD147 functions as an ancillary protein for MCT4
`but not MCT2 (48).
`
`MCT2
`
`MCT2 was initially isolated and functionally character-
`ized from a Syrian hamster liver library (51) with subsequent
`identification of homologues in rat (52) and human (53). In
`humans, expression of MCT2 is more restricted than MCT1
`(Table I), with the greatest expression observed in the testis
`(53). In addition, species differences have been observed in
`the tissue distribution of MCT2. For example, rodents express
`higher levels of MCT2 in the liver, while MCT2 protein
`expression is not detectable in human liver (53). Brain MCT2
`expression and cellular localization also appears to be highly
`species dependent (53–55). This variability in tissue expres-
`sion may be a result of species differences in gene regulation.
`In both rodents and humans, MCT2 splice variants have been
`detected in a species and tissue-dependent manner suggesting
`that transcriptional and post-transcriptional regulation path-
`ways play an important role in the tissue specificity of this
`isoform (52,53,55,56). Similar to MCT1, MCT2 requires an
`accessory protein for translocation to the plasma membrane.
`However, MCT2 requires gp70 (EMBIGIN), not CD147 (29).
`In addition, tissue specific post-translational regulation of
`MCT2 has recently been demonstrated in the mouse brain
`with the association of MCT2 and the scaffolding protein
`Delphilin which results in colocalization of MCT2 with δ-
`glutamate receptors (30,57). Further studies on the species-
`and tissue-specific regulation are required to identify the
`complex pathways involved in MCT2 regulation.
`MCT2 has remarkably similar substrate specificity to
`MCT1. However,
`in contrast
`to the observed substrate
`affinities of MCT1, MCT2 was demonstrated to be a high
`affinity pyruvate transporter in humans (Km=25 μM) which
`concurs with results obtained using hamster and rat MCT2
`(Table II) (51,58). Furthermore, MCT2 is inhibited by
`phloretin and CHC, but not by the organomercurial thiol
`reagent pCMBS, which distinguishes it from MCT1 (4). It is
`thought that this difference in inhibitor sensitivity results from
`the requirement of MCT1 and MCT2 for different accessory
`proteins (4).
`
`MCT3
`
`MCT3 is believed to have the most restricted distribution
`of any MCT with expression in the basolateral membrane of
`the RPE and the choroid plexus in humans, rodents and
`chickens (21,59,60). However, recent studies demonstrated
`MCT3 expression in vascular smooth muscle cell lines (61),
`human aorta (61), human kidney (62) and human intestinal
`Caco-2 cells (unpublished), suggesting that MCT3 mRNA
`may be more widely distributed than originally thought.
`Furthermore, decreased MCT3 mRNA and protein expres-
`sion was observed with increasing severity of atherosclerosis
`
`which concurs with changes in smooth muscle cells charac-
`teristic of
`this disease state (61). The authors further
`demonstrated that DNA methylation of the MCT3 gene
`likely contributed to the observed expression changes (61).
`Chicken MCT3 has been demonstrated to transport
`lactate in a yeast expression system (Km=6 mM) and
`demonstrates a profound resistance to prototypical MCT
`inhibitors (60). Additional
`information on human MCT3
`substrates or inhibitors is not present in the literature nor is
`there detailed information regarding the regulation of MCT3.
`
`MCT4
`
`MCT4 demonstrates remarkable similarities to MCT1
`with respect to tissue distribution, regulation and substrate/
`inhibitor specificity (Tables I and II). The principal difference
`between these isoforms lies in their tissue specific localization
`and substrate affinities. In contrast
`to MCT1, MCT4 is
`predominantly expressed in highly glycolytic cells such as
`white muscle and white blood cells suggesting that
`its
`physiological function is lactate efflux (17,63). MCT4 and
`CD147 expression were induced in MDA-MB231 cells (a
`highly invasive breast cancer cell
`line) supporting the
`metabolic switch to highly glycolytic cells in metastasis and
`the corresponding increase in lactate efflux (42). MCT4
`localization at the plasma membrane was dependent on
`CD147 expression, which is consistent with results obtained
`for MCT1 (42). The role of MCT4 in lactate efflux is further
`supported by its high expression in the placenta where it is
`involved in the transfer of lactate into the maternal circula-
`tion (4). While there is a great degree of overlap in the
`substrate specificity of MCT1 and MCT4, these two isoforms
`differ in their substrate affinities with MCT4 having lower
`affinities for a range of monocarboxylates (64). In contrast to
`other MCTs, lactate transport via MCT4 is inhibited by a
`range of statin drugs which may play a role in cytotoxicities
`observed with statin administration (65).
`
`MCT6
`
`MCT6 was first identified by Price et al. in 1998 (66)
`through genomic and EST database screening. Northern blot
`analysis was used to determine the tissue distribution of
`MCT6 (Table I) with expression being predominantly in the
`kidneys (66).
`In contrast to other members of the MCT family, MCT6
`does not transport short-chain monocarboxylates or amino
`acids; rather, all substrates identified to date are pharmaceu-
`tical agents (Table II) (3). Murakami et al. (3) demonstrated
`that bumetanide uptake is mediated by MCT6 in a pH- and
`membrane potential-, but not proton-dependent manner
`suggesting that it may be net charge dependent. Furthermore,
`uptake of bumetanide was inhibited by probenecid and
`several thiazides, but not inhibited by lactate or succinic acid
`(3). This suggests that a carboxylic moiety is not essential for
`MCT6 affinity, as was anticipated based on results obtained
`with other MCT isoforms (3). MCT6 mRNA expression has
`been demonstrated along the entire length of the human
`intestine with the highest expression levels observed in the
`stomach (66,67). This expression pattern suggests that MCT6
`may play an important role in the intestinal absorption of
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`Morris and Felmlee
`
`xenobiotics. Further studies are required to determine the
`physiological role of MCT6 as well as its role in drug
`disposition.
`
`MCT8 and MCT10
`
`MCT8 was identified during studies on X-chromosome
`inactivation, and was previously known as X-linked PEST-
`containing transporter due to the presence of a PEST domain
`in the N-terminus of the protein (2,68). The gene encoding
`human MCT8 (hSLC16A2) contains two translation start
`sites either of which would result in a functional protein; it is
`currently unknown if these sites encode different MCT8
`isoforms, and whether this would alter the isoforms function
`or regulation (69). Interestingly,
`in other species studied,
`SLC16A2 contains only a single start site that corresponds to
`the second site in the human gene (69). Further genomic
`analyses revealed a remarkable homology (52% amino acid
`sequence identity) (69) between MCT8 and the T-type amino
`acid transporter-1, now known as MCT10. MCT10 contains a
`PEST domain within its N-terminus, a structural feature that
`is present in only MCT8 and MCT10, which is thought to
`result in rapid protein degradation (69).
`Both MCT8 and MCT10 demonstrate a wide tissue
`distribution (Table I). The recent functional characterization
`of MCT8 and MCT10 revealed that monocarboxylates,
`including lactate and pyruvate, were not substrates for these
`transporters (69–71). MCT8 was demonstrated to actively
`transport the thyroid hormones, T3 and T4 (71,72), while
`MCT10 is involved in the transport of aromatic amines (70).
`The substrate specificity of MCT8 has further been confirmed
`by a linkage between mutations in MCT8 and Allan–
`Herndon–Dudley Syndrome which is associated with abnor-
`mally high levels of circulating T3 (73). Both isoforms have
`been demonstrated to transport their respective substrates in
`a proton- and Na+-independent manner (70), which is in contrast
`to other members of MCT family. Interestingly, MCT10-
`mediated transport of aromatic amines in the kidney has been
`demonstrated to occur in both directions thereby equalizing
`intra- and extracellular amino acid concentrations (69).
`
`Orphan MCTs
`
`Seven additional members of the MCT family (MCT5,
`MCT7, MCT9, AND MCT11–14) have been identified
`through searches of the human genomic and EST databases
`(4,66). Table I details the human tissue distribution of these
`MCT isoforms as determined by Northern blot analyses
`(4,66);
`limited data is available on the tissue-dependent
`protein expression of these isoforms (17). MCT5 protein
`expression has been demonstrated in the basolateral mem-
`brane of human colon and ileum with the greatest expression
`observed in the distal colon (19). It remains unclear whether
`monocarboxylates are substrates for these transporters.
`Riboflavin has been suggested as a substrate for MCT12
`based on its sequence homology to Mch5p, which is
`responsible for plasma membrane uptake of riboflavin in
`Saccharomyces cerevisiae (74). However, functional character-
`izations of the orphan MCTs have yet to be completed. Until
`recently, no information was available regarding the regula-
`
`tion of the orphan MCTs. Hirai et al. (75) demonstrated that
`MCT13 was induced by PPAR-α agonists in mouse liver and
`small
`intestine suggesting that
`this transporter may be
`involved in nutrient uptake. Further studies are required to
`elucidate the exact mechanism of induction via this pathway
`and the role of PPAR-α in the overall regulation of MCT13.
`
`ROLE OF MCTS IN DRUG DISPOSITION
`
`Studies examining MCTs have focused primarily on their
`identification and understanding their physiological role in
`lactate homeostasis as well as the transport of additional
`endogenous substances; however, emerging evidence sup-
`ports the further investigation of the impact of MCTs on drug
`disposition. For example, functional characterization of MCT6
`indicated that it was involved in the transport of bumetanide,
`and not endogenous monocarboxylates (3). Furthermore,
`GHB has been demonstrated to be both a substrate and
`inhibitor for a number of MCT isoforms (10–12,53).
`MCTs are expressed in a wide range of tissues, including
`the liver, kidney, intestine and brain (4). This localization has
`the potential to impact a number of processes contributing to
`the overall pharmacokinetics and distribution of therapeutic
`agents. Specifically, inhibition of renal reabsorption via MCTs
`results in increased renal clearance and decreased drug
`exposure. In addition, inhibition of MCT-mediated intestinal
`absorption may substantially decrease drug bioavailability.
`These alterations have the potential
`to adversely affect
`patient exposure and subsequent therapeutic outcomes. Few
`studies have been conducted assessing the contribution of
`MCT isoforms to overall drug disposition and the impact of
`MCT modulation on drug pharmacokinetics and disposition.
`The impact of MCT function on drug pharmacokinetics has
`been most extensively characterized for GHB (5,76). The aim
`of this section is to summarize work assessing the impact of
`MCTs on drug disposition specifically focusing on the role of
`MCTs in the renal clearance of GHB. Current studies on the
`impact of MCTs on the disposition of additional drugs will
`also be summarized.
`
`GHB
`
`GHB is a naturally occurring short-chain fatty acid
`formed from γ-aminobutyric acid (GABA) that is found in
`the mammalian brain, heart, liver and kidney (77). It acts
`potentially as a neuromodulator through binding to the
`GABA(B) receptor (78). In addition, GHB is formed from
`the precursors γ-butyrolactone and 1,4-butanediol (79).
`Therapeutically, GHB is approved in the US to treat
`narcolepsy (marketed as Xyrem®) (80) and in Europe for
`the treatment of alcohol withdrawal (81). However, abuse of
`GHB is widespread; it is used by body builders for its growth
`hormone releasing properties (82), by drug abusers as a
`recreational drug for its euphoric effects (83), and in drug-
`facilitated sexual assault due to its sedative/hypnotic effects
`(84). The increased abuse of GHB has lead to a rise in
`associated overdoses and fatalities (82). Adverse events
`associated with GHB overdose are principally characterized
`by central nervous system and respiratory depression as well
`as cardiovascular and gastrointestinal effects with symptoms
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`
`SLC16A Transport Family
`
`317
`
`including seizures, dizziness, nausea, vomiting and uncon-
`sciousness potentially leading to coma and death. (82)
`Currently, the treatment of GHB overdose is limited to
`supportive care; physostigmine and naloxone have been tried
`as antidotes with minimal success (79).
`GHB pharmacokinetics have been demonstrated to be
`nonlinear in humans (85–88) and rats (89,90), with total
`clearance decreasing as a function of increasing dose. Several
`mechanisms contribute to the observed nonlinear pharmaco-
`kinetics including capacity-limited metabolism (85,87,89,90),
`saturable absorption (91), and nonlinear renal clearance (6).
`While metabolic clearance represents the predominant elim-
`ination pathway for GHB (77), renal clearance becomes
`increasingly important
`in overdose situations with high
`urinary concentrations reported in humans (92,93). In con-
`trast
`to the observed changes in total clearance with
`increasing dose, renal clearance increases in a dose-depen-
`dent manner in rats (6). Furthermore, the fraction of GHB
`excreted in urine increases tenfold (3% to 30%) over the
`dose range of 108–208 mg/h per kilogram (6). These dose-
`dependent increases suggest the involvement of active renal
`reabsorption which is saturated at high concentrations.
`In vitro studies have characterized the renal transport
`mechanisms of GHB and elucidated the MCT isoforms
`contributing to GHB reuptake. Studies were conducted in
`rat kidney membrane vesicles, a human kidney cell line (HK-
`2 cells) and rat MCT1 transfected MDA-MB231 cells. Studies
`conducted in rat brush border (BBM) and basolateral (BLM)
`membrane vesicles isolated from rat kidney cortex character-
`ized the renal transport mechanism (12). GHB and L-lactate
`both undergo pH- and sodium-dependent uptake in BBM
`vesicles and pH-dependent uptake in BLM vesicles, suggest-
`ing the involvements of proton-dependent and sodium-
`dependent MCTs (12). MCT1 is expressed at both mem-
`branes, although there is greater expression at the BLM;
`MCT2 is expressed only at the BLM (12). HK-2 cells express
`MCT1, MCT2 and MCT4 at both the mRNA and protein
`level, which agrees with expression patterns in the human
`kidney cortex (62). GHB uptake in HK-2 cells was driven by
`a pH-gradient, and was inhibited by CHC suggesting that
`MCTs, but not SMCTs, were responsible for its uptake in HK-
`2 cells (11). Similar uptake parameters and similar inhibitory
`effects were observed for GHB and lactate suggesting
`transport by the same or similar transporters (11). Addition-
`ally, GHB uptake was inhibited by pCMB indicating that
`MCT2 may not be an important transporter in GHB uptake
`(11). Silencing RNA for MCT1, 2 and 4 in HK-2 cell studies
`suggested that GHB is predominantly transported by MCT1
`(10,11), among the proton-dependent MCTs. Further studies,
`conducted in MDA-MB231 cells (endogenous expression of
`MCT2 and MCT4) and MDA-MB231 cells transfected with
`rat MCT1, provided further evidence regarding the specific
`MCT isoforms involved in GHB renal uptake (10,12). GHB
`was found to be a substrate for MCT1, 2 and 4 (10,11).
`However, based on the expression patterns of MCTs in the
`kidney, MCT1 is likely the primary isoform responsible for
`GHB renal uptake.
`To further explore the influence of MCT1 on GHB renal
`reabsorption, studies were conducted to assess the modula-
`tion of MCT1-mediated GHB transport through the evalua-
`tion of potential inhibitors. Uptake of GHB in MDA-MB231
`
`cells was inhibited by the classic MCT inhibitors CHC,
`phloretin and pCMB with uptake being approximately 60%
`of control cells (10). In rat MCT1-transfe