P r o g r a m N u m b e r : AAPS/M1321

In vitro Systems to Assess the Potency of Selected Uptake Transporter Inhibitors Blair Miezeiewski, Jessica Hewlig, Libin Li, Joseph Rager, Sid Bhoopathy, Ismael J. Hidalgo, and Qing Wang Absorption Systems, Exton, PA

IntroductionOrganic anion transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3), which are localized on the sinusoidal (basolateral) membrane of hepatocytes, exhibit broad substrate selectivity. Numerous drugs are substrates of OATP1B1 and OATP1B3, for example, atorvastatin, pravastatin, pitavastatin, rosuvastatin, and rifampicin. In addition to drugs, OATPs transports endogenous substances such as bile acids, thyroid hormones, steroid sulfates, glucuronide conjugates and peptides [1]. OATP1B1 and OATP1B3 have relevance to hepatic drug uptake, drug disposition, and drug-drug interactions (DDIs). Both the FDA and EMA require that drug interaction liability involving inhibition of these two transporters be evaluated in vitro for all drug candidates because inhibition of OATP function by a drug may lead to pharmacokinetic alterations and an increase in the propensity for transporter-mediated DDIs [2,3]. The in vitro data drives decisions regarding OATP-based clinical DDI trials [2]. In the current study, atorvastain was chosen as the probe substrate for both OATP1B1 and OATP1B3. First, the K

m and V

max of atorvastatin were determined; secondly, the IC

50 values of selected inhibitors toward OATP1B1 and

OATP1B3 were determined using different experimental conditions.

MethodsCell Culture. Human embryonic kidney epithelial cells (HEK293) transfected with individual uptake transporters (OATP1B1, OATP1B3) were used to characterize the Michaelis-Menten parameters and inhibition profiles. The cells were maintained in DMEM supplemented with 10% FBS, non-essential amino acids, penicillin, streptomycin, and sodium pyruvate in a humidified incubator (37°C, 5% CO

2). The culture medium was changed three times weekly, and cell growth was observed

by light microscopy. When the cells became confluent, they were harvested by trypsinization, seeded onto plates for uptake studies, and kept in a humidified incubator (37°C, 5% CO

2).

Uptake Studies. The probe substrate solution was prepared by diluting the probe substrate stock in assay buffer (Hanks’ balanced salt solution with 10 mM HEPES and 15 mM D-glucose [HBSSg]). The final concentration of atorvastatin was 0.15 μM. Two different conditions were applied: pre-incubation with inhibitor for 15 minutes, or pre-incubation with buffer alone. The inhibition assay consisted of incubation with probe substrate and the test compound together for 2 minutes; the cells were rinsed twice with ice-cold HBSSg and lysed with 75% acetonitrile containing internal standard; and the lysates were collected for analysis of probe substrate concentration.

LC-MS/MS. A PE Sciex API3000 triple quadrupole mass spectrometer was used in the multiple reaction monitoring mode using a turbo ionspray interface, following reverse-phase chromatography on a Keystone Hypersil BDS C18 column. Total run time was 3.5 min.

Data AnalysisThe uptake parameters of each probe substrate were determined using equation (1): (1)

where NIR is the net influx rate (pmol/min/mg protein) at substrate concentration S, obtained by subtracting the flux rate in vector control (VC) cells from that in transporter-transfected cells; K

m is the Michaelis-Menten constant (μM); and NIR(max) is the maximal net influx rate (pmol/min/mg protein). IC

50 values of test

compounds (TCs) were determined using the following equations:

(2) % Activity remaining (Y) = (3)

IC50

values were determined by nonlinear regression using GraphPad Prism (version 5.0), with the minimum value of Y constrained to 0. X is log (nominal concentration of TC). Y

L is the lowest % activity remaining and Y

H is the highest (vehicle control).

ResultsThe uptake of atorvastain in OATP1B1- and OATP1B3-transfected cells could be modeled with the Michaelis-Menten equation; the K

m values were 0.30 and

2.01 μM, respectively (Figure 1). In general, pre-incubation prior to the inhibition assay resulted in a left-shift of the IC50

curve, i.e., an increase in inhibitory potency.

ConclusionPre-incubation with a test compound can influence the apparent potency of inhibition at OATP1B1 and OATP1B3. As a consequence, it can be difficult to predict the risk of in vivo DDIs mediated by uptake transporters based on in vitro data. These results highlight the pitfalls associated with the interpretation and/or comparison of results from different laboratories using slightly different in vitro assay conditions.

S K

S RNINIR

m

(max)

+

×=

100 NIR

NIR

TC No

TC ×⎟⎟⎠

⎞⎜⎜⎝

⎛

slope Hill)XIC(logLH

L50101

YYYY

×−+

−+=

S K

S RNINIR

m

(max)

+

×=

100 NIR

NIR

TC No

TC ×⎟⎟⎠

⎞⎜⎜⎝

⎛

slope Hill)XIC(logLH

L50101

YYYY

×−+

−+=

S K

S RNINIR

m

(max)

+

×=

100 NIR

NIR

TC No

TC ×⎟⎟⎠

⎞⎜⎜⎝

⎛

slope Hill)XIC(logLH

L50101

YYYY

×−+

−+=

1. Klaassen CD and Aleksunes LM. Pharmacological Reviews 2010; 62: 1-96. 2. US FDA, Draft guidance for industry. Drug interaction studies—Study design, data analysis, implications for dosing, and labeling recommendations, February 2012.3. European Medicines Agency, Guideline on the investigation of drug interactions, June 2012.

ResultsFigure 1. Michaelis-Menten Characterization of OATP1B1 and OATP1B3

Figure 2. Inhibition of atorvastatin uptake by rifampicin (A), rifamycin SV (B), and ritonavir (C) in OATP1B1- and OATP1B3-transfected HEK cells. The concentration of the probe substrate atorvastatin was 0.15 μM, below the K

m for each transporter.

Table 1. IC

50 values of selected inhibitors towards OATP1B1 and OATP1B3.

Conditions InhibitorsIC

50 ( μ M )

OATP1B1 OATP1B3

Pre-incubation with inhibitor

Rifampicin 0.673 0.0282

Rifamycin SV 0.212 0.0070

Ritonavir 1.15 0.170

No pre-incubation

Rifampicin 1.35 0.504

Rifamycin SV 0.358 0.249

Ritonavir 0.858 0.732

Ne

t F

lux

Ra

te(p

mo

l/m

g p

rote

in/m

in)

Atorvastatin (µM)

20

15

10

5

00 1 2 3 4 5 6

OATP1B1OATP1B3

OATP1B1

Km 0.3035 2.007

OATP1B3

Ac

tiv

ity

Re

ma

inin

g (

%)

Log [Rifampicin, M]

OATP1B1_rifampicinA.150

100

50

0-10 -8 -6 -4

Ac

tiv

ity

Re

ma

inin

g (

%)

Log [Rifampicin, M]

OATP1B3_rifampicin150

100

50

0-10 -8 -6 -4

No pre-incubationPre-incubation

No pre-incubationPre-incubation

Ac

tiv

ity

Re

ma

inin

g (

%)

Log [rifamycin SV, M]

OATP1B1_rifamycin SVB.150

100

50

0-10 -8 -6 -4

Ac

tiv

ity

Re

ma

inin

g (

%)

Log [rifamycin SV, M]

OATP1B3_rifamycin SV150

100

50

0-10 -8 -6 -4

No pre-incubationPre-incubation

No pre-incubationPre-incubation

Ac

tiv

ity

Re

ma

inin

g (

%)

Log [Ritonavir, M]

OATP1B1_ritonavirC.150

100

50

0-12 -10 -8 -6 -4

Ac

tiv

ity

Re

ma

inin

g (

%)

Log [Ritonavir, M]

OATP1B3_ritonavir150

100

50

0-12 -10 -8 -6 -4

No pre-incubationPre-incubation

No pre-incubationPre-incubation