(M1030-09-54) Quantitative Analysis of levo-alpha-acetyl-methadol (LAAM) in Rabbit Plasma for A Pharmacokinetic Study of Multiple Dosage Forms

Purpose: According to the CDC, more than 80% of overdose deaths involve opioids. Having alternative prevention and therapeutics is critical to alleviate the opioid epidemic. Levo-alpha-acetyl-methadol (LAAM) was previously used for treating opioid dependence. LAAM was pulled from the European market in 2001 due to reports of proarrhythmic side-effects and Roxane Laboratories discontinued LAAM in 2003. Given the increasing opioid epidemic, the return of LAAM to the market could help in the fight against opioid use disorder (OUD). Therefore, studies are in-process to develop alternative dosage forms for LAAM. In these studies, pharmacokinetic studies were performed to compare at least three dosage forms in rabbits. In order to determine the plasma concentrations of LAAM and its metabolites (norLAAM and dinorLAAM), we developed and validated a method to accommodate rabbit plasma samples according to the FDA bioanalytical method validation guidance (May 2018). Following the method validation, samples were analyzed for the pharmacokinetic studies.

Methods: Levo-alpha-acetyl-methadol (LAAM) method optimization included the use of a rabbit plasma, instrument conditions (Liquid Chromatography Tandem Mass Spectrometry- LC-MS/MS), linearity, carryover, and sample preparation. A 200 µL sample aliquot, 50 µL stable isotope labeled internal standard for each analyte, and 150 µL of water was added to a 96-well plate. After vortex mixing, these samples were then transferred to a supported liquid extraction plate (SLE+) and equilibrated for 5 min. Extraction solvent was then added twice (450 µL x 2 of 95:5 dichloromethane:isopropanol); vacuum was applied after each addition.  Extracts are then evaporated under a nitrogen stream at 50°C and then reconstituted with mobile phase. Samples are transferred to a 1 mL 96-well plate for injection (10 µL) into the LC-MS/MS. Samples were separated using 1D chromatography with column diversion on a Waters Acquity HSS T3 2.1x50 mm 1.8 um column under isocratic conditions with 55:45 H₂O:(ACN:MeOH) w/ 0.1% formic acid. A post- elution wash is executed from 4-5 min with 40:20:20:20 ACN:MeOH:IPA:H₂O, and the total run time is 7 min. A Waters TQ-S Micro was used with positive electrospray and selected reaction monitoring (SRM) transitions for LAAM, norLAAM, dinorLAAM, and their respective internal standards.

Results: Following optimization, the method was validated with a linear range from 0.200 to 500 ng/mL in rabbit plasma. Accuracy and precision runs (n =6) were acceptable. In addition, recovery indicated 69% for LAAM, 84% for norLAAM, and 80% for dinorLAAM in rabbit plasma. Stability studies showed acceptable results for post-preparative, and refrigerator stability (24 h). A summary of the validation data is shown in Table 1.

Conclusion: Following optimization, this method was validated to quantitatively measure LAAM and its metabolites in rabbit plasma for multiple pharmacokinetic studies following various dosage forms. Having this validated method will allow us to finalize optimal PK studies and selection of LAAM dosage forms to transition to clinical studies.

References: [1] Chiu, H., Tsai, I., Lu, Y., Lin, C., & Kuo, C. (2017). Determination of l-a-acetylmethadol (LAAM), norLAAM, and dinorLAAM in Clinical and In Vitro Samples Using Liquid Chromatography with Electrospray Ionization and Tandem Mass Spectrometry. Journal of Pharmaceutical Sciences, vol. 92, no. 1, January 2003.

Acknowledgments: This work was supported by an NIH/NIDA HEAL initiative grant (1UG3DA048768).

Table 1. Summary of the validation results for LAAM, dinorLAAM, and norLAAM in rabbit plasma.