Pharmacokinetics and Pharmacodynamics of Strategically Substituted Agmatines

Abstract

Chronic pain remains a major issue affecting patients, with current pharmacotherapy limited to drugs with low efficacy, negative side effects, and/or social stigma. Thus, there is a critical need to develop novel pharmacotherapies that are effective in reducing chronic pain while being safe for long-term use. Agmatine has been extensively shown to reduce chronic pain behaviors in animal models with no cardiac, motor, or neurological adverse effects1. This reduction is due to antagonism of the N-methyl-D-aspartate (NMDA) receptor, specifically at the GluN2B subunit2-4. This specificity allows agmatine to modulate the biological changes underlying chronic pain without the negative side effects observed with complete inhibition of the NMDA receptor. However, agmatine is a polar small molecule and does not efficiently diffuse passively across biological membranes. It has been shown to cross the intestinal epithelium and blood-brain barrier (BBB)5,6, but these distributions are limited, requiring high doses to generating quantifiable disposition. Additionally, the barrier in drug appearance in the CNS necessitates high doses (30-300 mg/kg in animal models) to achieve pharmacological effects. Furthermore, although agmatine has a long half-life in the CNS, its short elimination half-life in plasma restricts efficacy as a systemic therapeutic7. Therefore, our team and collaborators have designed and synthesized a series of agmatine-based prodrugs, the Strategically Substituted Agmatines (SSAs), which are designed to have increased lipophilicity, prolonged plasma half-lives, and equivalent pharmacological activity to agmatine. The central hypothesis of this thesis research is that the SSAs reduce pain behaviors in preclinical models of chronic pain in a manner comparable to agmatine while exhibiting improved pharmacokinetic parameters in rat. From this hypothesis, my objective has been to answer several questions: How can these compounds be measured in plasma and the central nervous system (CNS)? Do strategic substitutions improve pharmacokinetic parameters over agmatine? Do the SSAs distribute more readily to the CNS? Are the SSAs metabolized to agmatine within the CNS? And do the SSAs show improved potency over agmatine following oral administration? To answer these questions, my goals have been to develop techniques to quantify agmatine and the SSAs in complex tissues, determine the pharmacokinetic parameters of agmatine and the SSAs after IV and oral administration, investigate the prodrug activity of the SSAs, and explore the efficacy on chronic pain of these compounds as oral therapeutics. I accomplished these goals by completing the following specific aims: Specific Aim 1: Develop and Validate Analytical Methods to Accurately Quantify Agmatine and the SSAs in Complex MatricesUsing HPLC-MS/MS, a series of tissue preparation methods and chromatographic techniques were developed and validated according to FDA guidance8 to determine the concentration of agmatine, SSA1, SSA2, SSA3, and SSA4 in rat plasma, and quantify agmatine and SSA3 in rat brain and spinal cord. Specific Aim 2: Determine the Plasma Pharmacokinetic Parameters of Agmatine and the SSAs in Rat Following Intravenous and Oral DeliveryI hypothesized that the lipophilic substitutions of the SSAs would increase plasma half-life and volume of distribution over agmatine, as well as improve the oral bioavailability over agmatine. Sprague-Dawley rats with surgically implanted catheters received IV or oral agmatine, SSA1, SSA2, SSA3, or SSA4. Serial blood samples were collected, and plasma was analyzed using HPLC-MS/MS. These plasma concentrations were used to generate individual pharmacokinetic profiles of each drug in individual rats. Specific Aim 3: Assess the Tissue Distribution Profiles of Agmatine and SSA3 in RatI hypothesized that SSA3 would show increased CNS distribution across all tissues compared to agmatine. Furthermore, I hypothesized that SSA3 would be metabolized to agmatine within the CNS. Following IV administration of agmatine or SSA3 via tail-vein in Sprague-Dawley rat, multiple tissues from within the CNS were collected and heat-treated, along with plasma. Agmatine and SSA3 content were assessed using HPLC-MS/MS in each tissue, from which I determined distribution and pharmacokinetic profiles of each compound within individual tissues, such as spinal cord, cortex, ventral tegmental area (VTA), and nucleus accumbens (NAcc). Agmatine content was also assessed in CNS tissues following IV SSA3 and SSA4 to estimate prodrug activity. Specific Aim 4: Evaluate the Efficacy of Agmatine and the SSAs on NMDA-Evoked Responses and Chronic Pain Following Systemic Administration Intrathecal NMDA evokes characteristic behaviors in mice, including biting/scratching and tail-flick hyperalgesia. I hypothesized that the SSAs, due to their increased lipophilicity, would have increased systemic activity at lower doses than agmatine in the NMDA-evoked behavioral model. Additionally, I hypothesized that increased activity at lower doses would be observed in reversal of tactile hypersensitivity in inflammatory pain. I assessed changes in NMDA-evoked behaviors following subcutaneous and oral administration of agmatine and the SSAs to determine the CNS effects of these compounds from non-central delivery. Furthermore, these compounds were tested after oral administration for reduction of tactile hypersensitivity following inflammatory injury via CFA administration.