Pharmacokinetics and Tissue Distribution of Hydroxyurea in a Mouse Model
Authors: Marahatta Anu , McElhinney Kathryn , Howard Thad A. , Ware Russell E. 
Published: October 2019

Abstract
Background: Hydroxyurea is currently the only US FDA-approved treatment for patients with sickle cell anemia (SCA), and 30 years of research have documented its safety and efficacy for this life-threatening disorder. However, there is surprisingly little known about the tissue distribution and drug clearance of hydroxyurea in humans, and the influence of drug pharmacokinetics (PK) on the hematological responses and toxicities of hydroxyurea treatment. In 2014, the National Heart Lung and Blood Institute published evidence-based guidelines that recommend wider use of hydroxyurea for adults and children with SCA, even offering therapy to infants. Accordingly, it is critically important to understand the PK parameters of hydroxyurea and to elucidate its biological tissue distribution, to better understand its benefits and risks for patients with SCA.
Methods: C57BL/6 mice, age 8 to 10 weeks, received a single 100 mg/kg dose of hydroxyurea by either intraperitoneal (IP) or oral gavage (OG) route, since this dosage causes myelosuppression and hematological toxicity when administered on a daily basis. After drug administration, plasma and tissue samples were collected serially at multiple time points, with samples collected and analyzed from ≥5 different mice at each time point. Hydroxyurea concentrations in plasma and tissues were analyzed by high performance liquid chromatography (HPLC); an internal standard (methylurea) and standard curve using the appropriate tissue homogenate were used to quantify hydroxyurea accurately to a threshold sensitivity of 5-10mM. Pharmacokinetic parameters were determined via non-compartmental analysis using Phoenix WinNonlin® (version 6.2.1).
Results: Both routes of hydroxyurea administration featured rapid drug absorption, distribution, and clearance (Table). The maximum plasma concentration of hydroxyurea was approximately 4-fold higher following IP dosing, but the apparent elimination half-life (t1/2) was similar for both dosing routes at 15 minutes for IP and 12 minutes for oral. Following IP administration, the highest tissue concentrations of hydroxyurea were observed after ~10 minutes, which was same as plasma, indicating very rapid tissue distribution and clearance. Hydroxyurea was most highly concentrated in the kidney and spleen, along with the lung and heart, possibly reflecting overall blood flow. Testes and brain had small amounts of hydroxyurea detected by either route, but the liver had almost no hydroxyurea detected. Hydroxyurea was completely eliminated from all organs within 2 hours.
Conclusion: Using a novel, accurate, and sensitive HPLC method to quantify hydroxyurea, these data provide important information on the PK parameters and tissue distribution profile of hydroxyurea when administered by either IP or OG route in a mouse model, and likely mimic the exposure of patients with SCA to this drug. The peak concentrations of hydroxyurea were highest in the kidney, possibly reflecting predominantly renal uptake and clearance. The small amounts of hydroxyurea detectable in brain and testes are encouraging regarding potential organ toxicities. Future experiments using repeated dosing and treatment of sickle mice should provide additional insights regarding the potential toxicities of hydroxyurea exposure.
Table 1. PK parameters following a single dose of hydroxyurea (100 mg/kg) by either IP or OG route. PK parameters IP route OG route Plasma PK parameters Cmax (ug/mL) 111.9 ± 10.6 28.2 ± 5.6 tmax (minutes) 9.0 ± 2.2 22.5 ± 8.6 t½ (minutes) 15.1 ± 3.7 12.1 ± 1.3 V (L/kg) 1.1 ± 0.3 2.2 ± 1.0 CL/F, mL/kg/min 57.9 ± 25.2 147.3 ± 8.1 MRT (minutes) 20.2 ± 8.4 27.3 ± 2.8 AUC (ug /mL*min) 1971 ± 773 767 ± 168 Maximum Tissue Concentration (ug/mL) Kidney 88.8 ± 47.8 27.4 ± 18.8 Spleen 73.2 ± 14.9 24.5 ± 10.0 Heart 35.7 ± 11.2 22.9 ± 4.9 Lung 52.0 ± 12.0 17.2 ± 12.2 Testes 19.5 ± 6.8 8.5 ± 5.4 Brain 6.7 ± 1.3 4.5 ± 2.4 Liver ND ND Cmax refers to the maximum plasma concentration, t½ refers to the apparently elimination half-life, tmax refers to peak concentration time, V is the volume of distribution, CL/F is the apparent clearance, MRT refers to mean residence time and AUC is the area-under-the-curve.ND = not detected

Disclosures
Off Label Use: Hydroxyurea use for children with sickle cell anemia.. Ware:Eli Lilly: Other: DSMB membership; Biomedomics: Research Funding; Bristol Myers Squibb: Research Funding; Bayer Pharmaceuticals: Consultancy.