Biodistribution of Agmatine to Brain and Spinal Cord after Systemic Delivery

Abstract

Agmatine, an endogenous polyamine, has been shown to reduce chronic pain behaviors in animal models and in patients. This reduction is due to inhibition of the GluN2B subunit of the N-methyl-D-aspartate receptor (NMDAR) in the central nervous system (CNS). The mechanism of action requires central activity, but the extent to which agmatine crosses biologic barriers such as the blood-brain barrier (BBB) and intestinal epithelium is incompletely understood. Determination of agmatine distribution is limited by analytical protocols with low sensitivity and/or inefficient preparation. This study validated a novel bioanalytical protocol using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) for quantification of agmatine in rat biologic matrices. These protocols were then used to determine the plasma pharmacokinetics of agmatine and the extent of distribution to the CNS. Precision and accuracy of the protocol met US Food and Drug Administration (FDA) standards in surrogate matrix as well as in corrected concentrations in appropriate matrices. The protocol also adequately withstood stability and dilution conditions. Upon application of this protocol to pharmacokinetic study, intravenous agmatine showed a half-life in plasma ranging between 18.9 and 14.9 minutes. Oral administration led to a prolonged plasma half-life (74.4-117 minutes), suggesting flip-flop kinetics, with bioavailability determined to be 29%-35%. Intravenous administration led to a rapid increase in agmatine concentration in brain but a delayed distribution and lower concentrations in spinal cord. However, half-life of agmatine in both tissues is substantially longer than in plasma. These data suggest that agmatine adequately crosses biologic barriers in rat and that brain and spinal cord pharmacokinetics can be functionally distinct. SIGNIFICANCE STATEMENT: Agmatine has been shown to be an effective nonopioid therapy for chronic pain, a significantly unmet medical necessity. Here, using a novel bioanalytical protocol for quantification of agmatine, we present the plasma pharmacokinetics and the first report of agmatine oral bioavailability as well as variable pharmacokinetics across different central nervous system tissues. These data provide a distributional rationale for the pharmacological effects of agmatine as well as new evidence for kinetic differences between brain and spinal cord.

Fig. 1.

Chromatograms of agmatine and ¹³C₄-agmatine in various matrices. Peaks of (A) agmatine at LLOQ (5 ng/ml) in 5% BSA; (B) agmatine at LLOQ in rat plasma; (C) agmatine at LLOQ in rat CNS homogenate; (D) blank 5% BSA; (E) blank rat plasma; (F) blank rat CNS homogenate; (G) ¹³C₄-agmatine in 5% BSA; (H) ¹³C₄-agmatine in rat plasma; (I) ¹³C₄-agmatine in rat CNS homogenate; and (J) blank mobile phase (97% H₂O, 3% acetonitrile, 0.1% formic acid). AA, automatic area; AH, automatic height; RT, retention time; SN, signal to noise.

Fig. 2.

Plasma pharmacokinetics of agmatine after intravenous and oral administration in Sprague-Dawley rat. (A) Plasma concentration-time profiles after a single i.v. dose via jugular catheter of 3 or 30 mg/kg agmatine sulfate in Sprague-Dawley rat (n = 10 and 9, respectively). (B) Plasma concentration-time profiles after a single oral dose via oral gavage of 100 or 300 mg/kg agmatine sulfate in Sprague-Dawley rat (n = 6 and 8, respectively). Data are presented as mean ± S.E.M.

Fig. 3.

Distribution of agmatine to CNS regions after intravenous administration. (A) Concentration-time profiles after a single i.v. dose of 100 mg/kg agmatine sulfate via tail-vein injection in Sprague-Dawley rat (n = 6). (B) Tissue-to-plasma concentration ratio over time in spinal cord and brain. Data are presented as mean ± S.E.M.