Movement of Hydroxyurea Across Cell Membranes Is Mediated by Specific Solute Carrier (SLC) Transporters.

Hydroxyurea is the only FDA-approved drug for the treatment of sickle cell anemia (SCA) in adults. Hydroxyurea increases fetal hemoglobin, decreases hospitalizations and painful events, and reduces mortality. With an oral bioavailability of > 90%, hydroxyurea is rapidly absorbed and distributed throughout the body. Though hydroxyurea has proven to be effective in treating SCA, there is considerable inter-patient variability observed in the pharmacokinetics and pharmacodynamics of hydroxyurea. Currently, mechanisms involved in the absorption, distribution, and elimination of hydroxyurea remain unclear. Recently, key transmembrane proteins have been identified as drug transporters due to their ability to move a variety of xenobiotic substances across cell membranes. Drug transporters are widely distributed throughout the body, and most are specific to certain substrates. Solute carrier (SLC) transporters in particular have been to shown to significantly impact drug pharmacokinetics by influencing the absorption, distribution, and elimination of specific drugs. The present study was designed to identify SLC transporters that may influence the absorption, distribution, and/or elimination of hydroxyurea in patients with SCA.

In vitro studies using an equilibrium dialysis plate were performed to determine the amount of hydroxyurea that binds to human serum proteins. Transporter-mediated cellular uptake of hydroxyurea was determined in vitro by measuring [¹⁴C]-hydroxyurea accumulation in HEK293 cells and oocytes that overexpress organic anion transporters (OAT1-3), organic cation transporters (OCT1-3), organic cation/carnitine transporters (OCTN1-2), organic anion transporting polypeptides (OATP1A2/OATP1B1/OATP1B3), or vector control. LLC-PK1 cells that overexpress urea transporters A or B (UTA/UTB) were used to determine UTA/UTB mediated transcellular transport of hydroxyurea in transwell plates. The transport of [¹⁴C]-hydroxyurea from apical to basal or from basal to apical compartments was measured for the UTA/UTB overexpressing cells and compared to vector control. UTA and UTB mRNA expression was measured by real-time PCR of cDNA obtained from human tissue samples.

Protein binding assays showed that >76% of [¹⁴C]-hydroxyurea remained unbound to proteins in human serum containing hydroxyurea at concentrations ranging from 1.5μM to 500μM. The fraction of unbound hydroxyurea was similar using serum obtained from pediatric patients with SCA. In uptake studies, [¹⁴C]-hydroxyurea was a potent substrate for OATP1B3 with an approximately 2-fold increase in drug accumulation compared to control (p<0.001). In contrast, hydroxyurea was found to be a weak substrate for OCTN1, OCTN2, OATP1A2, and OATP1B1 with only a 1.3-fold increase in drug accumulation compared to control (p<0.04). Transcellular transport of hydroxyurea was increased 3- and 2-fold by UTA and UTB, respectively, compared to vector control demonstrating hydroxyurea to be a potent substrate for these transporters as well (p<0.02). When the urea transporter inhibitor dimethylurea was added, hydroxyurea transport by UTA and UTB-expressing cells was decreased to levels observed with the vector control. In real-time PCR assays, kidney, muscle, and small intestine were among human tissues with high expression of UTA mRNA, while prostate, brain, and bone marrow had high levels of UTB mRNA expression.

Cellular uptake of hydroxyurea is mediated by active transport via specific SLC transporters OATP1B3, UTA and UTB, which are expressed in liver, kidney, brain, intestine, and blood cells. Studies to further characterize hydroxyurea transporters should improve our understanding of the pharmacokinetic and pharmacodynamic profiles of hydroxyurea used in clinical practice for patients with SCA.

No relevant conflicts of interest to declare.