Organic Ion Transporters

1
Organic Ion Transporters Involved in Diseases
and Toxicity Ikumi Tamai, Ph.D.,
Professor, Tokyo University of Science Department
of Molecular Biopharmaceutics Chiba, Japan
2

• There are a variety of transporters and they are
  classified into two types
• (1) Drug Transporters
• Have broad substrate selectivity.
• Substrates may be classified into anionic,
  cationic or neutral.
• Are involved to determine PK of drugs.
• Can be factors that cause alterations of PK.
• Can be ysed as the tools useful for novel DDS or
  
• controlling PK .
• (2) Physiological Transporters
• Have specific substrates.Substrates/Inhibitors
  are predicted from chemical structures.
• Are involved in toxicity/adverse effect of drugs.
  
• Are involed to disease and could be a novel drug
  targets.

3
Examples of Drug/Physiological Transporters
Amino acid transporters, LAT SLC7A5 LAT1 (BBB,
Placenta, L-DOPA) SLC7A8 LAT2 (many tissues,
basic and neutral amino acids) Bile acid
transporter, SLC10A1 NTCP(Liver, bile
acids) SLC10A2 ASBT(ileum, bile acids) Peptide
transporters, PEP SLC15A1/2 PEPT1/2(gut/kidney)
(oligopeptides, ß-lactams) Monocarboxylate
transporters, MCT SLC16A1 MCT1?7(gut, BBB etc.,
?SLC16A7 lactate, benzoate) Organic anion
transporting polypeptides, OATP SLCO2A1
PGT(lung et al., PG) SLCO1A2 OATP-A, OATP
(Brain, anions) SLCO1B1 OATP-C, LST1 (Liver
specific) SLCO1B3 OATP-8(liver specific) SLCO2B1
OATP-B(liver, gut etc.) SLCO3A1 OATP-D(many
tissues) SLCO4A1 OATP-E(many tissues) SLCO1C1
OATP-F(?) SLCO4C1 OATP-R (Kidney, digoxin,
thyroid hormones)
Organic ion transporters, OCT, OCTN, OAT SLC22A1
OCT1(liver, cations, TEA, MPP ) SLC22A2
OCT2(kidney, TEA, dopamine) SLC22A3
OCT3(placenta, brain) SLC22A4 OCTN1(kidney,
blood cell, cations) SLC22A5 OCTN2(kidney
etc., carnitine) SLC22A6 OAT1(kidney, PAH,
) SLC22A7 OAT2(liver, PAH, MTX, cAMP) SLC22A8
OAT3(kidney, PCG, cimetidine) SLC22A
OAT4(placenta, PAH, ochratoxin A) SLC22A
OAT5(liver, ?) SLC22A12 URAT1(kidney, uric
acid) Nucleoside transporter, CNT, ENT SLC28A1,2
CNT1,2(many tissues, nucleoside) concentrative
transporters (active) SLC29A1,2 ENT1,2(many
tissues, nucleoside) equilibrium transporters
(facilitative) ABC ATP-dependent
transporters ABCA1 Cholesterol ABCB1
P-glycoprotein(many tissues) ABCC1 MRP1(many
tissues, anionic?) ABCC2 MRP2(liver, gut etc.,
anions) ABCC3 MRP3( liver, gut etc., anions
) ABCC4 MRP4(lung etc. antiviral drugs,
anions) ABCG2 BCRP(placenta, liver etc.
anions)
4
OATP-R (SLCO4C1)
Oatp-E (slco4a1)
rGST-1 (slco6b1)
OATP-E (SLCO4A1)
Oatp4c1 (slco4c1)
OATP-J (SLCO5A1)
GST (SLCO6B1)
Oatp-D (slco3A1)
rGST-2 (slco6c1)
OATP-D (SLCO3A1)
oatp4/rlst-1 (slco1b2)
rPGT (slco2A1)
OATP-C (SLCO1B1)
PGT (SLCO2A1)
OATP8 (SLCO1B3)
OATP-F (SLCO1C1)
moat1 (slco2B1)
oatp3 (slco1a5)
oatp2 (slco1a4)
OATP-B (SLCO2B1)
OATP-A (SLCO1A2)
Oatp5 (slco1a6)
OAT-K2 (slco1a3)
oatp1 (slco1a6)
OAT-K1 (slco1a3)
Organic Anion Transporting Polypeptides, OATPs
(SLCO)
5
Rat oatp
Human OATP
D
E
8
9
1
2
3
4
A
B
C
OATP-A
100
34
44
36
32
42
67
73
72
42
34
OATP-B
100
35
36
34
35
35
33
34
35
77
OATP-C
100
37
31
80
44
46
46
64
36
OATP-D
100
36
38
36
37
38
37
35
OATP-E
100
34
35
35
34
31
32
OATP8
100
46
45
46
66
33
oatp1
100
77
80
43
34
oatp2
100
82
44
33
oatp3
100
44
33
oatp4
100
33
oatp9
100
Amino-Acid Sequence Identities of OATP
Superfamily between Human and Rat
Species Difference in OATP
Human Transporter Studies are Desirable
6
Human OATPs (SLCO) Family
HUGO Name Tissues/Substrates SLCO2A1 PGT
lung etc. prostaglandins SLCO1A2
OATP-A brain (BBB?) etc. steroid
conjugates, BSP SLC1B1 OATP-C liver
sepcific LST-1 bile acids, bilirubin,
anionic drugs pravastatin, PCG, DPDPE,
BSP SLCO1B3 OATP-8 liver specific Steroid
conjugates, N-methylquinidine digoxin, BSP,
CCK8 SLCO2B1 OATP-B liver, gut, placenta
etc. steroid conjugates, anionic
drugs fexofenadine, BSP SLCO3A1 OATP-D
heart, testis etc. prostaglandins SLCO4A1
OATP-E placenta etc. thyroid
hormones SLCO1C1 OATP-F brain etc. thyroid
hormones SLCO4C1 OATP-R kidney digoxin,
thyroid hormones
7
Substrate Selectivity of Human OATPs
OATP-A
C
D
E
F
8
B
R
Taurocholate ? ? ? ? DHEAS ? ? ?
? E217ßG ? ? ? ? ? E13S ? ? ? ? ? ?
? T3, T4 ? ? ? ? ? LTC4 ?
? PGE2 ? ? ? ? BQ123 ? ?
? DPDPE ? ? ? BSP ? ? ? ?
? Bilirubin ? Conjugated Bilirubin
? ? CCK-8 ? PCG ?
? ? ? Digoxin ? ? Fexofenadine ?
N-Methylquinine ? Pravastatin
? Methotrexate ? ?
8
OATP-C Is Important for Hepatic Uptake of
Anionic Drugs
Intestine
Portal Vein
Liver
Systemic Circulation
OATP-B
OATP-A,C?
Metabolism
MRP2
Important for Hepatic Uptake of Anions
Bile
Tissues
9
(No Transcript)
10
Troglitazone Sulfate 10 µM pH 7.4, 120 min
Mean S.E.M. Plt0.05 (vs. no injection)
Cell to Medium Ratio (µL/oocyte)
Uptake of Troglitazone Sulfate by Xenopus
Oocytes Expressed with OATP-C, OATP8 and OATP-B
Nozawa et al., DMD, 32, 291-294 (2004)
11
Mean S.E.M. (n68) Substracted by Uptake of
No-Injected Oocytes Plt0.05 (vs. Control)
3HEstrone-3-sulfate 9.2 nM, pH 7.4
OATP-C
OATP8
30 min
120 min
Control

Troglitazone
10


M-1
10


1

M-2
10
1


M-3
10


1
Pioglitazone


10
Rosiglitazone

10
0
20
40
60
80
100
120
0
20
40
60
80
100
120
(µM)
3HEstrone-3-sulfate Uptake ( of Control)
Inhibitory Effects of Several Compounds on Uptake
of 3HEstrone-3-sulfate by Xenopus Oocytes
Expressed with OATP-C and OATP8
Nozawa et al., DMD, 32, 291-294 (2004)
12
Portal Vein
Liver
Troglitazone
Troglitazone
Conjugation
M-1
M-1
?
Bile Canaliculus
BSEP
Oral Administration
MRP2
M-1
BCRP
Deconjugation
Troglitazone
Faeces
Enterohepatic Circulation
Reabsorption
?
Intestine
M-1
Troglitazone
Portal Vein
Scheme of Enterohepatic Circulation of
Troglitazone and Its Major Metabolite, M1
13
Liver
Irinotecan
?
Conjugation
CE
Irinotecan SN-38 SN-38G
SN-38
P-gp
UGT
Bile Canaliculus
OATP8
SN-38G
Irinotecan
OATP-B
SN-38G
Toxicity?
BCRP
Deconjugation
Irinotecan
Gastrointestinal Toxicity
SN-38
Cancer Cells
SN-38
Irinotecan
Reabsorption
CE
Intestine
Pharmacological Effect
SN-38
SN-38
BCRP
Metabolism and Disposition of Irinotecan, SN-38
and SN-38G
14
Uptake SN38, CPT-11, SN38-glucuronide 10 µM pH
7.4 25?

120
Mean S.E.M. (n4) Plt0.05 (vs. Mock)
OATP-C

SN-38
80

Mock
Cell to Medium Ratio (µL/mg protein)
Mock
OATP-C
40
Irinotecan
Mock
OATP-C
SN-38G
0
0
10
20
30
40
Time (min)
Time Courses of Irinotecan, SN-38, and SN-38G by
HEK293 Cells Expressing OATP-C
Nozawa et al., DMD, in press.
15
Single Nucleotide Polymorphisms (SNPs) in OATP-C
Human Liver
Troglitazone sulfate SN-38 (Irinotecan
metabolite) Pravastatin Estrone-3-sulfate Estradio
l-17ß-glucuronide
Variants
Alteration of Plasma/Liver Concentraion
16
A388G, Asn130Asp
T521C, Val174Ala
A
T
OATP-C1a (Wild)
G
OATP-C1b
C
OATP-C5
G
C
OATP-C15
Haplotype
In vivo Study
In vitro Study
Number of Alleles in Japanese (n534)
Transport Activity
(Plasma Conc. of Pravastatin)
Allele
Position
E13S
E217ßG
OATP-C1a
Wild
188 (35.2)
OATP-C1b
N130D
?
?
287 (53.7)
??
OATP-C5
?
?
?
4 (0.7)
V174A
?
OATP-C15
N130D/V174A
55 (10.3)


17
SN-38
14CPravastatin

140
140

120
120
120 min, 60 µM
60 min, 10 µM
100
100
80
80
60
60
40
40
20
20
Uptake Activity ( of OATP-1a)
0
0
1a
1b
5
15
1a
1b
5
15
3HEstrone-3-sulfate
14CEstradiol-17ß-glucuronide

140

140
120 min, 20 nM
30 min, 9.2 nM
120
120
Mean S.E.M. (n5-10) Plt0.05
100
100
80
80
60
60
40
40
20
20
0
0
1a
1b
5
15
1a
1b
5
15
Uptake Activity of OATP-C Variants
Nozawa et al., DMD, in press (2005)
18
Uptake 3HEstrone-3-sulfate 12 nM pH 7.4 25?
OATP-C1a
OATP-C15
Mean S.E.M. (n4-10)
Relationship Between Uptake Activity and Injected
Dose of cRNA of OATP-C1a or OATP-C15
Nozawa et al., DMD in press (2005).
19
Plasma Concentration Time Curves of Pravastatin
in Three Genotype Groups with Respect to V174A
Variant
130D/D174A/A
Serum concentration (ng/mL)
130D/D174V/A
130D/D174V/V
Time (hr)
Nishizato et al., CPT, 73554-565 (2003)
20
Human Liver
Troglitazone sulfate SN-38 (Irinotecan
metabolite) Pravastatin Estrone-3-sulfate Estradio
l-17ß-glucuronide
OATP-C1a, 1b
OATP-C5
P-gp
OATP-C15
MRP2
Increased Plasma/Liver Concentraion
Unexpected Effects?
21
Physiological Roles of Organic Ion Transporter
Family SLC22A
Rheumatoid Arthritis, Crohn Disease, Adverse
Effect of Gefitinib
Hypouricaemia
Male Infertility
OCTN Family
Systemic Carnitine Deficiency
mOCTN1
rOCTN1
hURAT1
hOCTN1
mOCTN3
hOAT4
hOCTN2
Drug Transporters Organic Cation OCT, OCTN
Organic Anion OAT Physiological Transporters
Carnitine Transporter OCTN2 and OCTN3 Uric
Acid Transporter UART1 Extraneuronal Monoamine
Transporter (EMT) OCT3
mOCTN2
UST3
rOCTN2
hCT2(hOCT6)
hOAT3
hOCT3
hOAT1
hOCT1
hOAT2
hOCT2
OCT Family
OAT Family
Phylogenetic Tree of SLC22A Family Transporters
22
SLC22A4
Association of OCTN1 with Rheumatoid Arthritis
Tokuhiro S. et al. Nature Genetics, 35, 341-348
(2003)
23
Functional Variants of OCTN1 Genes Are
Associated with Crohn Disease
SLC22A4
Nature Genetics, 36, 471-475 (2004)
OCTN1 May Be Related to Autoimmune Diseases
24
OCTN1 (SLC22A4) and OAT2 (SLC22A7)
SLC22A4 (OCTN1)
1672C?T (L503F)
Rheumatoid Arthritis
Crohn disease
?
?
?
?
?
?
?
?
?
?
?
?
?
?
E1
E2
E3
E4
E5
E6
E7
E8
E9
E10
? Diarrhea and gastrointestinal disorders by
Genifitib.
? Gastrointestinal disorders only by Genifitib
E Exon
SNP B
SLC22A7 (OAT2)
SNP C (synonymous)
SNP A (rare)
3
5
E1
E2
E3
E4
E5
E6
E7
E8
E9
E10
Case-Control Linkage Disequilibrium Mapping Using
SNPs
25
OCTN2 (SLC22A5)
Foods (meat, fish, dairy products
etc.) Biosynthesis (Lys and Met)
Carnitine (Vitamin BT)
Fatty Acids (Blood)
Blood
Tissues Sk. Muscle Heart
Glomerular Filtration
Kidney
Mitochondria
Carnitine
Carnitine
Carnitine
Carnitine
Acyl-CoA
OCTN2
OCTN2
CPT1
Outer Membrane
Na
Na
Acylcarnitine
Tissue Distribution
Reabsorption
Inner Membrane
Acyl-CoA
ß-Oxidation
Acetyl-CoA
TCA cycle
OCTN2 is directly related to genetic
disease Primary Systemic Carnitine Deficiency
26
OCTN2 (SLC22A5)
Nezu et al., Nature Genetics 2191 (1999)
27
OCTN2 (SLC22A5)
Symptoms of Systemic Carnitine Deficiency
Muscule Weakness
Heart Disease
Male Infertility
28
Tamai et al., JBC, 27540064 (2000)
29
Epididymis
Carnitine
Spermatozoa
Key Nutrient for Maturation and Motility of
Spermatozoa
1,2)
3)
Acquisition of Motility and Fertilizing Ability
Concentration of Carnitine
Plasma 10-50 µM Epididymal Fluid 2-63
mM Spermatozoa 24 mM
Vas Deferens
4)
Kp of 3HCarnitine after Administration
Epididymis 31-54 Testis 2.4
Testis
Travel of Spermatozoa in Male Reproductive Tissues
How Carnitine Is Accumulated in Epididymis?
1) Casillas and Chaipayungpan, J Reprod Fertil,
56439-444 (1979) 2) Hinton et al., J Reprod
Fertil, 6159-64 (1981) 3) Jeulin and Lewin, Hum
Reprod Update, 287-102 (1996) 4) Bremer, Physiol
Rev, 631420-1480 (1983)
30
(No Transcript)
31
Sertoli Cells
Chamber
Apical
Matrigel
Basal
Tight Junction
Inulin
Inulin
Sertoli Cells
Filter Alone
Inulin Clearance
0.348 µL/min/cm2
0.082 µL/min/cm2
60
Filter Alone
50
14CInulin 20 µM 37 ?
Permeability(µL/cm2)
40
30
Sertoli Cells
20
10
0
0
30
60
90
120
Permeability of 14CInulin across Rat Sertoli
Cells Primary Cultured on MatrigelTM-Coated
Invasion Chamber
32
Matrigel
Na Dependence
Concentration Dependence
125
Mean S.E.M. (n3) Subtracted by Inulin
Space plt0.05 (vs. Control)
6
100
3HCarnitine
3HCarnitine 25.4 nM 30 min pH 7.4
Relative Uptake ( of Control)
4
3HCarnitine Uptake (µL/mg protein)
75

50
2

25




0
0
Li
NMG
Control
0.01
mM
0.1
1
10
Na (Control)
Non-Labeled Carnitine
Sodium and Concentration Dependence of
Basolateral Uptake of 3HCarnitine in Primary
Cultured Rat Sertoli Cells
33
Germ Cells
ß-oxidation
?
Sertoli Cells
Testicular Toxicity ?
Carnitine
OCTN2
Blood
Carnitine
Drug
Roles of Carnitine and OCTN2 in Sertoli and Germ
Cells
34
Epididymal Lumen
Carnitine
2-63 mM
1)
10- 50 µM
?
?
2)
2)
Km 927 µM
3)
Blood Vessel
Epithelial Cell
Spermatozoa
Carnitine Transport in Epididymal Epithelial Cells
1) Jeulin and Lewin, Hum Reprod Update, 287-102
(1996) 2) Yeung et al., Biol Reprod, 23294-304
(1980) 3) James et al., FEBS Lett, 12653-56
(1981)
35
Epididymides of 35 Days Old Male SD Rats

• 0.25 Trypsin Treatment for 30 min
• 0.1 Collagenase Treatment for 1 hr

Seeding of Isolated Epididymal Cells
Floated Cell (Epithelial Cell)
For 10 hr at 32 C
Attached Cell (Fibroblast)
Reseeding of Floated Epididymal Epithelial Cells
For 4 Days at 32 C
Culture Medium Minimum Essential Medium
10FBS 5?-Dihidrotestosterone
Transport Experiment RT-PCR Analysis
Kierszenbaum et al. Proc Natl Acad Sci USA,
781675-1679 (1981) Leung et al. Am J Physiol,
280C1076-C1083 (2001)
Method in Primary Culture of Rat Epididymal
Epithelial Cells
36
Suspension
Time Course
Na Dependence
2
3HCarnitine 38 nM pH 7.4, 32.5 C
3HCarnitine 61 nM pH 7.4, 32.5 C
1.5
Mean S.E.M.(n 3-4)
3HCarnitine Uptake (µL/mg protein)
P lt 0.05 (vs. Na)
3HCarnitine Uptake (µL/mg/30 min)
1
Mean S.E.M. (n 3-4)


0.5

20 mM Unlabeled Carnitine
4? (1 min)
0
Na
Li
K
Choline
Time (min)
Uptake of 3HCarnitine by Primary Cultured Rat
Epididymal Epithelial Cells in Suspension
37
4
Mean S.E.M. (n3-4)
30 min after i.v. Dose
p lt 0.05 (vs. Wild )
3HCarnitine 2.7 ng / Head
3
Wild
Kp
2
jvs
1



0
Testis
Epididymis
Heart
jvs Juvenile Visceral Steatosis
Decreased 3HCarnitine Distribution in
OCTN2-Deficient jvs Mice after i.v.
Administration
38
(No Transcript)
39
Carnitine
Acetylcarnitine
100
400
3HAcetylcarnitine (12.5 nM) Uptake 37?, pH7.4
3HCarnitine (12.5 nM) Uptake 37?, pH7.4
80
Na
Na
300
3HCarnitine Uptake (µL/mg protein)
3HAcetylcarnitine Uptake (µL/mg protein)
60
Mean S.E.M. (n3-4)
200
40
100
20
-Na (NMG)
-Na (NMG)
Mean S.E.M. (n3-4)
0
0
10
20
30
10
20
30
0
0
Time Course of 3HCarnitine or
3HAcetylcarnitine Uptake by Mouse Spermatozoa
40
(No Transcript)
41
Physiological Roles and Relationship with
Diseases of SLC22A
Rheumatoid Arthritis, Crohn Disease, Adverse
Effect of Gefitinib
Hypouricaemia
Male Infertility
OCTN Family
Systemic Carnitine Deficiency
mOCTN1
rOCTN1
hURAT1
hOCTN1
mOCTN3
hOAT4
hOCTN2
Estimated Number of Transporters is SLC
Family gt 300 ABC Family ? 48
mOCTN2
UST3
rOCTN2
hCT2(hOCT6)
hOAT3
hOCT3
Abnormality in Transporters Are Related to
Diseases
hOAT1
hOCT1
hOAT2
hOCT2
OCT Family
OAT Family