Expansion of Knowledge on OCT1 Variant Activity In Vitro and In Vivo Using Oct1/2-/- Mice

Abstract

The role of organic cation transporter 1 (OCT1) in humans is gaining attention as data emerges regarding its role in physiology, drug exposure, and drug response. OCT1 variants with decreased in vitro function correlate well with altered exposure of multiple OCT1 substrates in variant carriers. In the current research, we investigate mechanisms behind activity of OCT1 variants in vitro by generating cell lines expressing known OCT1 variants and quantifying membrane OCT1 protein expression with corresponding OCT1 activity and kinetics. Oct knockout mice have provided additional insight into the role of Oct1 in the liver and have reproduced effects of altered OCT1 activity observed in the clinic. To assess the complex effect of Oct1 depletion on pharmacokinetics of prodrug proguanil and its active moiety cycloguanil, both of which are OCT1 substrates, Oct1/2^-/- mice were used. Decreased membrane expression of OCT1 was demonstrated for all variant cell lines, although activity was substrate-dependent, as reported previously. Lack of change in activity for OCT1*2 resulted in increased intrinsic activity per pmol of OCT1 protein, particularly for sumatriptan but also for proguanil and cycloguanil. Similar to that reported in humans with decreased OCT1 function, systemic exposure of proguanil was minimally affected in Oct1/2^-/- mice. However, proguanil liver partitioning and exposure decreased. Cycloguanil exposure decreased following proguanil administration in Oct1/2^-/- mice, as did the systemic metabolite:parent ratio. When administered directly, systemic exposure of cycloguanil decreased slightly; however liver partitioning and exposure were decreased in Oct1/2^-/- mice. Unexpectedly, following proguanil administration, the metabolite ratio in the liver changed only minimally, and liver partitioning of cycloguanil was affected in Oct1/2^-/- mice to a lesser extent following proguanil administration than direct administration of cycloguanil. In conclusion, these in vitro and in vivo data offer additional complexity in understanding mechanisms of OCT1 variant activity as well as the effects of these variants in vivo. From cell lines, it is apparent that intrinsic activity is not directly related to OCT1 membrane expression. Additionally, in situations with a more complicated role of OCT1 in drug pharmacokinetics there is difficulty translating in vivo impact simply from intrinsic activity from cellular data.

Keywords: drug transport; knockout mice; metabolite kinetics; organic cation transporter 1 (OCT1); pharmacogenetics; pharmacokinetics; targeted proteomics.

FIGURE 1

Uptake of OCT1 substrates in HEK cells expressing wildtype (*1) and variant OCT1 protein. Uptake was assessed over 1 or 2 min, depending on substrate, in triplicate, at substrate concentration of 3 μM, with the exception of metformin, which was assessed at 22 µM. Data are presented as fold uptake compared to OCT1*1 (mean ± SD). Data in column (A) represent uptake prior to normalization for absolute OCT1 membrane protein expression. Data in column (B) represent uptake after normalization for absolute OCT1 membrane protein expression measured in cells expressing each variant.

FIGURE 2

Kinetics of sumatriptan (A) and fenoterol (B) in HEK cells expressing OCT1*1 or OCT1*2. Uptake was assessed over 1 min, in triplicate, at substrate concentration of 3 µM. Data are presented as mean ± SD.

FIGURE 3

Pharmacokinetics of proguanil/cycloguanil in wildtype (WT) and Oct1/2 ^−/− mice following IV administration of proguanil. Shown are dose-normalized blood (A) and liver (B) concentrations for mice (n = 5 or 5/timepoint) administered proguanil 2, 10 or 30 mg/kg. Filled symbols represent wildtype mice and open symbols represent knockout mice. Circles represent concentrations of proguanil and triangles represent concentrations of cycloguanil (C and D, E) Liver Kp and M:P values determined from tissues collected at 0.75 and 2 h (2 mg/kg), 1.5 and 4 h (10 mg/kg) or 8 h (30 mg/kg) post-dose. Data presented as mean ± SD. *p < 0.05 using student's t-test, compared to WT. **p < 0.01 using student’s t-test, compared to WT. ***p < 0.001 using student’s t-test, compared to WT. Kp = tissue:plasma partition coefficient. M:P = metabolite:parent ratio.

FIGURE 4

Pharmacokinetics of cycloguanil in wildtype (WT) and Oct1/2 ^−/− mice following IV administration of cycloguanil. Shown are blood (A) and liver (B) concentrations for mice (n = 5 or 4–5/timepoint) administered cycloguanil 2 mg/kg. Filled symbols represent wildtype mice and open symbols represent knockout mice (C) Tissues were collected at 0.75, 2-, 4- and 8 h post-dose. Data presented as mean ± SD. **p < 0.01 using student’s t-test, compared to WT. ***p < 0.001 using student’s t-test, compared to WT. Kp = tissue:plasma partition coefficient.

FIGURE 5

Partitioning of proguanil (A and B, C) and cycloguanil metabolite:parent ratio (D and E, F) in tissues other than liver in wildtype (WT) and Oct1/2 ^−/− mice following IV administration of proguanil. Mice (n = 5/timepoint) were administered proguanil 2, 10 or 30 mg/kg and tissues collected at 0.75 and 2 h (2 mg/kg), 1.5 and 4 h (10 mg/kg) or 8 h (30 mg/kg) post-dose. Data presented as mean ± SD. **p < 0.01 using student’s t-test, compared to WT. Kp = tissue:plasma partition coefficient. M:P = metabolite:parent ratio.

FIGURE 6

Partitioning of cycloguanil in tissues other than liver (A and B, C) in wildtype (WT) and Oct1/2 ^−/− mice following IV administration of cycloguanil. Mice (n = 4–5/timepoint) were administered 2 mg/kg cycloguanil and sacrificed 0.75, 2, 4, or 8 h post-dose. Data presented as mean ± SD. *p < 0.05 using student’s t-test, compared to WT. ***p < 0.001 using student’s t-test, compared to WT. Kp = tissue:plasma partition coefficient.