This white paper provides updated International Transporter Consortium (ITC) recommendations on transporters that are important in drug development following the 3rd ITC workshop. New additions ...include prospective evaluation of organic cation transporter 1 (OCT1) and retrospective evaluation of organic anion transporting polypeptide (OATP)2B1 because of their important roles in drug absorption, disposition, and effects. For the first time, the ITC underscores the importance of transporters involved in drug‐induced vitamin deficiency (THTR2) and those involved in the disposition of biomarkers of organ function (OAT2 and bile acid transporters).
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
We previously reported the unbound reversible (IC
) and time-dependent (K
) inhibition potencies of cannabidiol (CBD), delta-9-tetrahydrocannabinol (THC), and THC metabolites 11-hydroxy THC (11-OH ...THC) and 11-nor-9-carboxy-delta-9-THC (11-COOH THC) against the major cytochrome P450 (P450) enzymes (1A2, 2C9, 2C19, 2D6, and 3A). Here, using human liver microsomes, we determined the CYP2A6, 2B6, and 2C8 IC
values of the aforementioned cannabinoids and the IC
and K
of the circulating CBD metabolites 7-hydroxy CBD (7-OH CBD) and 7-carboxy CBD (7-COOH CBD), against all the P450s listed above. The IC
of CBD, 7-OH CBD, THC, and 11-OH THC against CYP2B6 was 0.05, 0.34, 0.40, and 0.32
M, respectively, and against CYP2C8 was 0.28, 1.02, 0.67, and 3.66
M, respectively. 7-COOH CBD, but not 11-COOH THC, was a weak inhibitor of CYP2B6 and 2C8. All tested cannabinoids except 11-COOH THC were weak inhibitors of CYP2A6. 7-OH CBD inhibited all P450s examined (IC
<2.5
M) except CYP1A2 and inactivated CYP2C19 and CYP3A, with inactivation efficiencies (k
/K
) of 0.10 and 0.14 minutes
M
, respectively. Using several different static models, we predicted the following maximum pharmacokinetic interactions (affected P450 probe drug and area under the plasma concentration-time curve ratio) between oral CBD (700 mg) and drugs predominantly metabolized by CYP3A (midazolam, 14.8) > 2C9 (diclofenac, 9.6) > 2C19 (omeprazole, 7.3) > 1A2 (theophylline, 4.0) > 2B6 (ticlopidine, 2.2) > 2D6 (dextromethorphan, 2.1) > 2C8 (repaglinide, 1.6). Oral (130 mg) or inhaled (75 mg) THC was predicted to precipitate interactions with drugs predominately metabolized by CYP2C9 (diclofenac, 6.6 or 2.3, respectively) > 3A (midazolam, 1.8) > 1A2 (theophylline, 1.4). In vivo drug interaction studies are warranted to verify these predictions. SIGNIFICANCE STATEMENT: This study, combined with our previous findings, provides for the first time a comprehensive analysis of the potential for cannabidiol, delta-9-tetrahydrocannabinol, and their metabolites to inhibit cytochrome P450 enzymes in a reversible or time-dependent manner. These analyses enabled us to predict the potential of these cannabinoids to produce drug interactions in vivo at clinical or recreational doses.
(-)-Δ
-tetrahydrocannabinol (THC) is the primary pharmacological active constituent of cannabis. 11-hydroxy-THC (11-OH-THC) and 11-
-9-carboxy-THC (THC-COOH) are respectively the active and nonactive ...circulating metabolites of THC in humans. While previous animal studies reported that THC could be a substrate of mouse P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), we have shown,
, that only THC-COOH is a weak substrate of human BCRP, but not of P-gp. To confirm these findings and to investigate the role of P-gp and/or Bcrp in the maternal-fetal disposition of THC and its metabolites, we administrated 3 mg/kg of THC retro-orbitally to FVB wild-type (WT),
,
, or
/
pregnant mice on gestation day 18 and estimated the area under the concentration-time curve (AUC) of the cannabinoids in the maternal plasma, maternal brain, placenta, and fetus, as well as the tissue/maternal plasma AUC geometric mean ratios (GMRs) using a pooled data bootstrap approach. We found that the dose-normalized maternal plasma AUCs of THC in
and
/
mice, and the placenta-to-maternal plasma AUC GMR of THC in
mice were 279%, 271%, and 167% of those in WT mice, respectively. Surprisingly, the tissue-to-maternal plasma AUC GMRs of THC and its major metabolites in the maternal brain, placenta, or fetus in
,
or
/
mice were 28-78% of those in WT mice. This study revealed that P-gp and Bcrp do not play a role in limiting maternal brain and fetal exposure to THC and its major metabolites in pregnant mice. SIGNIFICANCE STATEMENT: This study systematically investigated whether P-gp and/or Bcrp in pregnant mice can alter the disposition of THC, 11-OH-THC, and THC-COOH. Surprisingly, except for Bcrp, which limits placental (but not fetal) exposure to THC, we found that
,
, and/or
/
significantly decreased exposure to THC and/or its metabolites in maternal brain, placenta, or fetus. The mechanistic basis for this decrease is unclear and needs further investigation. If replicated in humans, P-gp- or BCRP-based drug-cannabinoid interactions are not of concern.
(-)-Δ
9
-tetrahydrocannabinol (THC) is the primary pharmacological active constituent of cannabis. 11-hydroxy-THC (11-OH-THC) and 11-
nor
-9-carboxy-THC (THC-COOH) are respectively the active and ...nonactive circulating metabolites of THC in humans. While previous animal studies reported that THC could be a substrate of mouse P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), we have shown,
in vitro
, that only THC-COOH is a weak substrate of human BCRP, but not of P-gp. To confirm these findings and to investigate the role of P-gp and/or Bcrp in the maternal-fetal disposition of THC and its metabolites, we administrated 3 mg/kg of THC retro-orbitally to FVB wild-type (WT),
P-gp
−/−
,
Bcrp
−/−
, or
P-gp
−/−
/
Bcrp
−/−
pregnant mice on gestation day 18 and estimated the area under the concentration-time curve (AUC) of the cannabinoids in the maternal plasma, maternal brain, placenta, and fetus, as well as the tissue/maternal plasma AUC geometric mean ratios (GMRs) using a pooled data bootstrap approach. We found that the dose-normalized maternal plasma AUCs of THC in
P-gp
−/−
and
P-gp
−/−
/
Bcrp
−/−
mice, and the placenta-to-maternal plasma AUC GMR of THC in
Bcrp
−/−
mice were 279%, 271%, and 167% of those in WT mice, respectively. Surprisingly, the tissue-to-maternal plasma AUC GMRs of THC and its major metabolites in the maternal brain, placenta, or fetus in
P-gp
−/−
,
Bcrp
−/−
or
P-gp
−/−
/
Bcrp
−/−
mice were 28–78% of those in WT mice. This study revealed that P-gp and Bcrp do not play a role in limiting maternal brain and fetal exposure to THC and its major metabolites in pregnant mice.
(-)-Δ
-Tetrahydrocannabinol (THC) is the primary psychoactive constituent of cannabis. In humans, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THC-COOH) are psychoactive and nonpsychoactive ...circulating metabolites of THC, respectively. Whether these cannabinoids are substrates or inhibitors of human P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP) is unknown. Previous animal studies suggest that THC and its metabolites could be substrates of these transporters. Therefore, we performed Transwell, cellular accumulation, and vesicular transport assays, at pharmacologically relevant concentrations of these cannabinoids, using Madin-Darby canine kidney (MDCK) II cells or plasma membrane vesicles overexpressing human P-gp or BCRP. Neither THC nor 11-OH-THC was found to be a substrate or inhibitor of P-gp or BCRP. The efflux ratio of THC-COOH in MDCKII-BCRP cells was 1.6, which was significantly decreased to 1.0 by the BCRP inhibitor Ko143. Likewise, cellular accumulation of THC-COOH was significantly increased 1.6-fold in the presence versus absence of Ko143. THC-COOH also significantly inhibited BCRP-mediated transport of Lucifer yellow, a BCRP substrate; however, THC-COOH was neither a substrate nor an inhibitor of P-gp. Collectively, these results indicate that THC and 11-OH-THC are not substrates or inhibitors (at pharmacologically relevant concentrations) of either P-gp or BCRP. THC-COOH is a weak substrate and inhibitor of BCRP, but not of P-gp. Accordingly, we predict that P-gp/BCRP will not modulate the disposition of these cannabinoids in humans. In addition, use of these cannabinoids will not result in P-gp- or BCRP-based drug interactions. SIGNIFICANCE STATEMENT: This study systematically investigated whether Δ
-tetrahydrocannabinol (THC) and its major metabolites, 11-hydroxy-THC and 11-nor-9-carboxy-THC, are substrates and/or inhibitors of human P-gp and BCRP at pharmacologically relevant concentrations. The results obtained are highly valuable for mechanistic understanding and prediction of the roles of P-gp and BCRP in determining the human pharmacokinetics, tissue distribution, and drug interactions of cannabinoids.
In the United States, cannabis consumption is increasing. (‐)‐Δ9‐tetrahydrocannabinol (THC) is the primary pharmacological active constituent of cannabis, while 11‐hydroxy‐THC (11‐OH‐THC) and ...11‐nor‐9‐carboxy‐THC (THC‐COOH) are active and nonactive circulating metabolites of THC in humans, respectively. P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP) are ATP‐binding cassette efflux transporters important for drug disposition, including brain penetration and fetal exposure. Our previous in vitro transport study showed that only THC‐COOH is a weak substrate of human BCRP, while previous animal studies reported that THC and its metabolites could be substrates of the P‐gp and BCRP orthologs in mice and macaques. To further investigate the roles of P‐gp and BCRP in the maternal‐fetal disposition of THC and its metabolites, we administrated 3 mg/kg THC (retro‐orbitally) to FVB wild‐type (WT), P‐gp‐/‐, and Bcrp‐/‐ pregnant mice on gestational day 18, and determined maternal, placental, and fetal concentrations of THC and its metabolites by LC‐MS/MS. The AUCs of THC and its major metabolites were estimated using a pooled data bootstrap approach. Differences in tissue/maternal plasma AUC ratios between WT and P‐gp‐/‐ or Bcrp‐/‐ were compared via unpaired parametric t‐test, and differences with p values of < 0.05 were considered statistically significant. In Bcrp‐/‐ pregnant mice, the placenta/maternal plasma AUC ratio for THC was 188% (p < 0.05) of respective AUC ratio in WT pregnant mice. In P‐gp‐/‐ pregnant mice, the maternal brain/maternal plasma AUC ratio for THC, maternal brain/maternal plasma and fetus/maternal plasma AUC ratios for 11‐OH‐THC, and fetus/maternal plasma AUC ratio for THC‐COOH were 29%, 65%, 49%, and 54% (p < 0.05) of respective AUC ratio in WT pregnant mice. There were no significant differences in fetal exposure of THC in either P‐gp‐/‐ or Bcrp‐/‐ mice, and in fetal exposure of 11‐OH‐THC and THC‐COOH in Bcrp‐/‐ mice, compared to WT mice. In conclusion, this study revealed that P‐gp and Bcrp exerted differential impacts on maternal brain, placental, and fetal exposure to cannabinoids in pregnant mice. P‐gp and Bcrp do not seem to play a role in limiting fetal exposure to THC and its major metabolites; but could differentially alter maternal brain exposure to the cannabinoids.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Some women take medication during pregnancy to address a variety of clinical conditions. Because of ethical and logistical concerns, it is impossible to determine fetal drug exposure, and therefore ...fetal risk, during pregnancy. Hence, alternative approaches need to be developed to predict maternal-fetal drug exposure throughout pregnancy. To do so, we previously developed and verified a maternal-fetal physiologically based pharmacokinetic model, which can predict fetal exposure to drugs that passively cross the placenta. However, many drugs are actively transported by the placenta (e.g., human immunodeficiency virus protease inhibitors). To extend our maternal-fetal physiologically based pharmacokinetic model to these actively transported drugs, we determined the gestational age–dependent changes in the protein abundance of placental transporters. Total cellular membrane fractions from first trimester (T1;
n
= 15), second trimester (T2;
n
= 19), and term (
n
= 15) human placentae obtained from uncomplicated pregnancies were isolated by ultracentrifugation. Transporter protein abundance was determined by targeted quantitative proteomics using liquid chromatography tandem mass specrometry. We observed that breast cancer resistance protein and P-glycoprotein abundance significantly decreased from T1 to term by 55% and 69%, respectively (per gram of tissue). Organic anion–transporting polypeptide (OATP) 2B1 abundance significantly decreased from T1 to T2 by 32%. In contrast, organic cation transporter (OCT) 3 and organic anion transporter 4 abundance significantly increased with gestational age (2-fold from T1 to term, 1.6-fold from T2 to term). Serotonin transporter and norepinephrine transporter did not change with gestational age. The abundance of bile salt export pump, multidrug resistance-associated protein 1-5, Na
+
-taurocholate cotransporting polypeptide, OATP1B1, OATP1B3, OCTN1-2, concentrative nucleoside transporter 1-3, equilibrative nucleoside transporter 2, and multidrug and toxin extrusion 1 could not be quantified. These data can be incorporated into our maternal-fetal physiologically based pharmacokinetic model to predict fetal exposure to drugs that are actively transported across the placenta.
Protein expression of renal uptake and efflux transporters was quantified by quantitative targeted proteomics using the surrogate peptide approach. Renal uptake transporters assessed in this study ...included organic anion transporters (OAT1-OAT4), organic cation transporter 2 (OCT2), organic/carnitine cation transporters (OCTN1 and OCTN2), and sodium-glucose transporter 2 (SGLT2); efflux transporters included P-glycoprotein, breast cancer resistance protein, multidrug resistance proteins (MRP2 and MRP4), and multidrug and toxin extrusion proteins (MATE1 and MATE2-K). Total membrane was isolated from the cortex of human kidneys (N = 41). The isolated membranes were digested by trypsin and the digest was subjected to liquid chromatography-tandem mass spectrometry analysis. The mean expression of surrogate peptides was as follows (given with the standard deviation, in picomoles per milligram of total membrane protein): OAT1 (5.3 ± 1.9), OAT2 (0.9 ± 0.3), OAT3 (3.5 ± 1.6), OAT4 (0.5 ± 0.2), OCT2 (7.4 ± 2.8), OCTN1 (1.3 ± 0.6), OCTN2 (0.6 ± 0.2), P-glycoprotein (2.1 ± 0.8), MRP2 (1.4 ± 0.6), MRP4 (0.9 ± 0.6), MATE1 (5.1 ± 2.3), and SGLT2 (3.7 ± 1.8). Breast cancer resistance protein (BCRP) and MATE2-K proteins were detectable but were below the lower limit of quantification. Interestingly, the protein expression of OAT1 and OAT3 was significantly correlated (r > 0.8). A significant correlation was also observed between expression of multiple other drug transporters, such as OATs/OCT2 or OCTN1/OCTN2, and SGLT2/OCTNs, OCT, OATs, and MRP2. These renal transporter data should be useful in deriving in vitro to in vivo scaling factors to accurately predict renal clearance and kidney epithelial cell exposure to drugs or their metabolites.
We determined whether in vivo transporter-mediated hepatobiliary clearance (CL) and hepatic concentrations of rosuvastatin (RSV) in the rat could be predicted by transport activity in ...sandwich-cultured rat hepatocytes (SCRHs) and/or transporter-expressing cell lines scaled by differences in transporter protein expression between SCRHs, cell lines, and rat liver. The predicted hepatobiliary CLs and hepatic concentrations of RSV were compared with our previously published positron emission tomography imaging data. Sinusoidal uptake CL (Formula: see text
Formula: see text
Formula: see text
Formula: see text
Formula: see text
Formula: see text for Oatp protein expression. This is the first demonstration of the successful prediction of in vivo hepatobiliary CLs and hepatic concentrations of RSV using transporter-expressing cells and SCRHs.
Intestinal transporters are crucial determinants in the oral absorption of many drugs. We therefore studied the mRNA expression (N = 33) and absolute protein content (N = 10) of clinically relevant ...transporters in healthy epithelium of the duodenum, the proximal and distal jejunum and ileum, and the ascending, transversal, descending, and sigmoidal colon of six organ donors (24–54 years). In the small intestine, the abundance of nearly all studied proteins ranged between 0.2 and 1.6 pmol/mg with the exception of those of OCT3 (<0.1 pmol/mg) and PEPT1 (2.6–4.9 pmol/mg) that accounted for ∼50% of all measured transporters. OATP1A2 was not detected in any intestinal segment. ABCB1, ABCG2, PEPT1, and ASBT were significantly more abundant in jejunum and ileum than in colon. In contrast to this, the level of expression of ABCC2, ABCC3, and OCT3 was found to be highest in colon. Site-dependent differences in the levels of gene and protein expression were observed for ABCB1 and ASBT. Significant correlations between mRNA and protein levels have been found for ABCG2, ASBT, OCT3, and PEPT1 in the small intestine. Our data provide further physiological pieces of the puzzle required to predict intestinal drug absorption in humans.
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IJS, KILJ, NUK, PNG, UL, UM
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