Authors: Charles Hay, University of Arkansas for Medical Sciences (Main Author, Presenting Author); Laura Ewing, University of Arkansas for Medical Sciences; Michael Hambuchen, University of Arkansas for Medical Sciences; Sinthia Alam, University of Arkansas for Medical Sciences; Paris Margaritis, Children's Hospital of Philadelphia; S. Owens, University of Arkansas for Medical Sciences; Eric Peterson, University of Arkansas for Medical Sciences
Presenting Author: Charles Hay
Purpose: The current available treatments for methamphetamine (METH) addiction consist solely of behavioral approaches, as there are currently no FDA-approved pharmacological therapies available. Our laboratory is utilizing scFvs, derived from antibodies capable of high affinity binding to METH, and adeno-associated virus technology (AAV) to develop potential treatments. We have previously shown that these METH binding antibodies can be expressed for at least 212 days in mice with a peak expression of 100.7 &[micro]/ml. These anti-METH antibodies are also capable of binding to amphetamine (AMP), an active and important metabolite of METH. The goal of this project was to verify that two AAV-scFv constructs (AAV-scFv6H4 and AAV-scFv7F9), when expressed in mice, possess similar ex vivo affinity to METH and AMP as the antibodies from which they were derived. Finally, we determined AAV-scFvs’ ability to reduce both METH and AMP concentrations in brain and sequestered these drugs in the serum.
Methods: BALB/C mice, n=8, were administered 1x1012 vector copies/mouse of either one of two AAV-scFv constructs, AAV-scFv6H4 or AAV-scFv7F9. Serum samples taken from the mice were used to determine the IC50's of expressed constructs to METH and AMP via competitive binding assays. For PKPD studies, mice were allowed to reach peak AAV-scFv expression before administering either 0.56 mg/kg (ip) or 1.0 mg/kg (sc) METH. Mice were sacrificed and serum and brains collected at 30, 60 and 120 min post METH administration. Brains and serum were analyzed for METH and AMP concentrations using LC-MS/MS.
Results: Both AAV-scFv constructs show high affinity binding to METH 8.4 nM for AAV-scFv6H4 and 22.1 nM for AAV-scFv7F9, with similar results as the in vitro generated standards. These constructs also showed, albeit lower, affinity to AMP with 2.1 μM in AAV-scFv6H4 and 48.8 μM in AAV-scFv7F9. In PKPD studies, METH and AMP concentrations were 33% lower in brain tissue at 30 and 60 min, increasing to 50% and 68% respectively after 120 min. Further, the AAV-scFv therapies were able to sequester METH in the serum – 1.6 fold higher than sham mice at 30 min post METH administration, and 5.3 fold at 120 min.
Conclusion: This data suggests that both of our AAV-scFv anti-METH therapies have the potential to reduce the rewarding effects of METH as well as the metabolite AMP, and thus provide long term protection. Supported by NIDA R01 DA036600 & NIGMS T32 GM106999
See attached abstract pdf for images.