(T1130-04-26) Investigation of a Novel Iron Chelator: A Pharmacokinetic and Pharmacodynamic Study

Purpose: Iron is biologically essential, but excess iron is potentially toxic due to iron’s ability to react with hydrogen peroxides and turn them into toxic radicals (Fig. 1a). Secondary iron overload occurs from repeated transfusions or prolonged iron therapy typically in patients with impaired erythropoiesis for e.g., thalassemia, sickle cell anemia (Fig. 1b). Increased iron stores are linked to well established risk factors for heart failure, hepatic cirrhosis, arthritis, diabetes, and age-related neurodegenerative diseases. As there is no known active excretion pathway for iron, disposal of extra iron from the body is the primary therapeutic goal of treating patients with iron overload. Iron chelation therapy represents a critically important strategy to remove excess iron, but the existing FDA-approved iron chelators (deferoxamine, deferasirox, and deferiprone) have displayed poor pharmacokinetics, incomplete efficacy, and significant adverse effects (Fig. 1c). Therefore, there is an unmet need for a new therapeutic strategy using safer chelators that can overcome these concerns. Hinokitiol, a naturally occurring tropolone derivative, isolated from essential oil of the Chamaecyparis taiwanesis tree is a newly characterized lipophilic iron chelator. The objectives of our study were 1) to investigate the absorption, distribution, metabolism, and excretion in rodents after intravenous and oral administration and 2) to evaluate the efficacy of hinokitiol in alleviating aberrant iron accumulation using an animal model of dietary iron overload.

Methods: To evaluate single-dose pharmacokinetics, hinokitiol was administered as 50 mg/Kg orally to CD-1 mice and samples were taken for up to 24 hours. The samples were analyzed via derivatization using a LC-MS/MS method. Compartmental PK analysis was performed with Phoenix WinNonlin^â. For mass balance study, Fisher rats were dosed orally (50 mg/Kg) to collect urine and feces for 24 hours using metabolic cages. For Pharmacodynamic study, CD-1 mice were fed high iron diet (10,000 ppm) for a week and were dosed with 10 mg/Kg hinokitiol intra-peritoneally once a day for one week (Fig. 3a). Animals were sacrificed after the last dose and tissues were analyzed for non-heme iron and ferritin.

Results: Derivatisation of hinokitiol with dabsyl chloride eluted a stable conjugate with a retention time of 3.1 min and a limit of detection of ~3 ng/mL, allowing for analysis of samples up to 24 h (Fig. 2a). Hinokitiol disposition was best described using a three-compartment model with a rapid initial disappearance from plasma within 0.3 h and followed by a steady disappearance up to 24 h. The absorption rate constant (k_a) is 114.66 h^-1 indicative that hinokitiol is rapidly absorbed, reaching peak blood levels (C_max) in less than 5 minutes after an oral dose. Hinokitiol exhibited favorable distribution kinetics, with a volume of distribution (Vd) of ~6 L/Kg, which correlates with its lipophilicity, suggesting that it can readily enter the intracellular compartment, chelate iron and exit as an iron complex. Additionally, hinokitiol distribution in liver, primary organ for iron accumulation, was ~2-fold higher than plasma, indicating that it can rapidly chelate liver iron (Fig. 2c). Hinokitiol was found to have extensive binding to plasma proteins (~98%). A decreased fraction of protein binding was observed with increased concentrations, indicating a saturation in hinokitiol-protein binding (Fig. 2d). Interestingly, it displayed a terminal half-life (t_1/2𝛄) of 16 h which can overcome the current need for cumbersome dosing schemes (e.g., 8–12-hour daily infusion of Deferoxamine). Moreover, it was observed that 40% of unchanged drug was eliminated in urine and feces (Fig. 2e) with the possibility that most of it gets eliminated via metabolism, suggesting that regular systemic exposure is unlikely to put patients at risk for excessive iron chelation and unintended intracellular toxicity. Further studies are currently underway to investigate dose escalation, bioavailability, metabolism, and biliary excretion. In addition to the favorable pharmacokinetics, non-heme iron analysis in dietary iron overload mice model revealed that hinokitiol significantly decreased serum and liver iron levels by 40% (p < 0.0001) and 46% (p < 0.0001), respectively (Fig. 3e and 3g). Hinokitiol restored serum iron levels close to normal levels. Furthermore, levels of ferritin, the major iron storage protein, in serum and liver were decreased by 46% (p < 0.001) and 35% (p < 0.001), respectively as compared to saline-control (Fig. 3f and 3h). The tissues are currently being analyzed for markers of oxidative stress and histopathological analysis to determine safety of hinokitiol.

Conclusion: Hinokitiol displays a rapid distribution, long half-life with a potential enterohepatic recirculation and ameliorates abnormal iron buildup possibly via biliary excretion holding promise to be used clinically in treatment of iron-overload condition.

Secondary Iron Overload

Pharmacokinetics of Hinokitiol

PD and therapeutic efficacy